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Synergy and resistance mechanisms in R115777 and PS-341 models of myeloma and leukemia

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Title:
Synergy and resistance mechanisms in R115777 and PS-341 models of myeloma and leukemia
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Book
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English
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Buzzeo, Robert William
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University of South Florida
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Subjects / Keywords:
Tipifarnib
8226/R5
Velcade
Farnesyl transferase
Proteasome
Dissertations, Academic -- Biology -- Masters -- USF   ( lcsh )
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non-fiction   ( marcgt )

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Abstract:
ABSTRACT: The farnesyl transferase inhibitor R115777 (Zarnestra, Tipifarnib) has been found to have clinical activity in diverse hematopoietic tumors. Clinical efficacy, however, does not correlate with Ras mutation status or inhibition of farnesyl transferase. To further elucidate the mechanisms by which R115777 induces apoptosis and to investigate drug resistance, we have identified and characterized a R115777-resistant human myeloma cell line. 8226/R5 cells were found to be at least 50 times more resistant to R115777 compared with the parent cell line 8226/S. 8226/R5 cells were insensitive to a diverse group of antitumor agents including PS-341 (Bortezomib, Velcade). Comparison of gene expression profiles between resistant and sensitive cells revealed expression changes in several genes involved in myeloma survival and drug resistance. Identification and characterization of the 8226/R5 cell line helped us evaluate and confirm that the Akt tumor survival pathway plays an important role in Tipifarnib induced apoptosis and resistance in myeloma cells. Additionally, 8226/R5 cells helped to evaluate other molecules exhibiting synergistic cell death. In this study, we investigated the activity of R115777 combined with Bortezomib in microenvironment models of multiple myeloma and AML. The combination proved to be synergistic in multiple myeloma and AML cell lines treated in suspension culture. Even in tumor cells relatively resistant to Tipifarnib, combined activity was maintained. Of importance, activation of the endoplasmic reticulum stress response was enhanced and correlated with apoptosis and reversal of CAM-DR. Our study provides the preclinical rationale for trials testing the Tipifarnib and Bortezomib combination in patients with multiple myeloma and AML.
Thesis:
Thesis (M.S.)--University of South Florida, 2009.
Bibliography:
Includes bibliographical references.
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Statement of Responsibility:
by Robert William Buzzeo.
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Title from PDF of title page.
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Document formatted into pages; contains 98 pages.

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ABSTRACT: The farnesyl transferase inhibitor R115777 (Zarnestra, Tipifarnib) has been found to have clinical activity in diverse hematopoietic tumors. Clinical efficacy, however, does not correlate with Ras mutation status or inhibition of farnesyl transferase. To further elucidate the mechanisms by which R115777 induces apoptosis and to investigate drug resistance, we have identified and characterized a R115777-resistant human myeloma cell line. 8226/R5 cells were found to be at least 50 times more resistant to R115777 compared with the parent cell line 8226/S. 8226/R5 cells were insensitive to a diverse group of antitumor agents including PS-341 (Bortezomib, Velcade). Comparison of gene expression profiles between resistant and sensitive cells revealed expression changes in several genes involved in myeloma survival and drug resistance. Identification and characterization of the 8226/R5 cell line helped us evaluate and confirm that the Akt tumor survival pathway plays an important role in Tipifarnib induced apoptosis and resistance in myeloma cells. Additionally, 8226/R5 cells helped to evaluate other molecules exhibiting synergistic cell death. In this study, we investigated the activity of R115777 combined with Bortezomib in microenvironment models of multiple myeloma and AML. The combination proved to be synergistic in multiple myeloma and AML cell lines treated in suspension culture. Even in tumor cells relatively resistant to Tipifarnib, combined activity was maintained. Of importance, activation of the endoplasmic reticulum stress response was enhanced and correlated with apoptosis and reversal of CAM-DR. Our study provides the preclinical rationale for trials testing the Tipifarnib and Bortezomib combination in patients with multiple myeloma and AML.
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Synergy and Resistance M e chanisms in R115777 and PS 341 Models of Myeloma and L eukemia b y Robert William Buzzeo A thesis submitted in partial fulfillment of the requirements for the degree of Masters of Science Department of Biology College of Arts and Sciences University of South Florida Major Professor: Patrick Bradshaw, Ph.D. Richard Pollenz, Ph.D. Kristina Schmidt, Ph.D. Date of Approval: June 25, 2009 Keywords: Tipifarnib, 8226/R5, Velcade, Farnesyl Transferase, Proteasome Copyright 2009, Robert Buzzeo

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Dedication Dedicated to my family for their support and R.C. & Seamore for teaching me the true meaning of patience

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Acknowledgments I wish to thank Patrick Bradshaw, Richard Pollenz, Darren Beaupre, Melissa Alsina, Lori Hazlehurst, Nirangan Yanamandra, Javier Cuevas and William S. Dalton for their intellectual support. I thank Paul Mackley and Yelenis Herrera for their technical suppo rt. I thank Patrick Bradshaw, Richard Pollenz, Brian Livingston, Phil Wong, Nancy Wong and Lia Perez for their kind support This research was supported in part by the following grants: American Heart Association Greater Southeastern Affiliate Grant In A id (0655291B, JC) MMRF senior research grant and a Myeloma Research Foundation Grant NIH grant: Clinical Scholars in Oncology 5K12 CA 8798902 (D.M. Beaupre). NIHClinical Scholars in Oncology grant 5K12 CA 8798902 (D.M. Beaupre and L.E. Perez) Moffit t Aging and Cancer Pilot Research grant program P20 CA103676 (D.M. Beaupre) Leukemia Research Foundation New Investigator Award (D.M. Beaupre)

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i Table of Contents L ist of Tables iv List of Figures v Abstract vii Chapter 1 Introduction and background 1 1.1 Multiple m yeloma 1 1.2 Drug resistance in multiple m yeloma 4 1.3 Farnesyl transferase inhibitors and ras proteins 6 Chapter 2 : Identification and c haracterization of a R1 15777 resistant cell l ine 8 2.1 Intr oduction 8 2.2 Materi als and m ethods 10 2.2.1 Cell lines and r eagents 10 2.2.2 Analysis of cell growth, c el l cycle arrest and cell d eath 11 2.2.3 R115777 accumulation and e fflux 12 2.2.4 Microarray analysis 14 2.2.4.1 Probe a rrays 14 2.2.4.2 Sample processing for microarray a nalysis 14 2.3 Results 15 2.3.1 8226/R5 cells are resistant to R115777induced growth arrest and cell death 15 2.3.2 Resistance to R115777 does not correlate with K Ras prenylation or farn esyl transferase activity 16 2.3.3 Resistance is not related to decreased accumulation of R115777 17 2.3.4 8226/R5 cells display a multidrugresistant ph enotype and resistance is not associated with increased expression of heat shock proteins 19 2.3.5 Transcriptional profile of 8226/R5 cells 23 2.4 Discussion 26 Chapter 3: The Akt pathway plays an important role in R115777 induced apoptosis and resist ance in multiple myeloma 30 3.1 Introduction 30 3.2 Materials and methods 33 3.2.1 Cell Culture 33 3.2.2 Drug preparation 34

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ii 3.2.3 Proliferation assay 34 3.2.4 Apoptosis assay 35 3.2.5 Immunostaining 35 3.2.6 Murine studies 37 3.2.7 Gene constructs, transfect ion and fluorescent microscopy 38 3.3 Results 38 3.3.1 Tipifarnib induces apoptosis in RPMI 8226 and U266 myeloma cell lines, but not the MM1.S cell line 38 3.3.2 Tipifar nib is cytotoxic to myeloma cell lines as exhibited in the SCID hu model of dise ase 40 3.3.3 pAkt expression levels correlate with Tipifarnib cytotoxicity and drug resistance 41 3.3.4 IHC reveals elevated levels of nuclear localized pAkt in Tipifarnib Resistant cells 42 3.3.5 Ectopic expression of constitutively active Akt promoted c ytotoxic resistance to Tipifarnib 45 3.4 Discussion 47 Chapter 4: Tipifarnib and Bortezomib are sy nergistic and overcome cell adhesionmediated drug resistance in multiple myeloma and acute myeloid leukemia 51 4.1 Introduction 54 4.2 Materials and Methods 54 4.2.1 Cell lines 54 4.2.2 Compounds 55 4.2.3 Patient samples 55 4.2.4 Combination index analysis 56 4.2.5 Fibronectin adhe sion and cell death analysis 57 4.2.6 Adhesion assays 58 4.2.7 Adhesion to bone marrow stroma and cell death analysis 58 4.2.8 Transwell analysis 59 4.2.9 Western Blotting 59 4.2.10 Proteasome assay 60 4.3 Results 60 4.3.1 Tipifarnib and Bortezomib are synergistic in multiple myeloma and AML cell lines 60 4.3.2 Tipifarnib combines wi th Bortezomib overcomes CAM DR 61 4.3.3 Reversal of CAM DR is not related to decreased tumor adherence 64 4.3.4 Activation of endoplasmic reticulum stress correlates with reversal of CAM DR. 65 4.3.5 Stroma adhered tumor cells are s ensitive to Tipifarnib and Bortezomib 66

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iii 4.3.6 HS 5 bone marrow stromal cells secrete a protective soluble factor 70 4.4 Discuss ion 71 Chapter 5 : Summary and Major C onclusions 79 5.1 Identification and characterization of t he R115777 resistant cell line 79 5.2 The role of the Akt survival pathway in R115777 induced apoptosis a nd Resistance 80 5.3 Combining R115777 and PS 341 overcomes CAM DR in multiple myeloma acute myeloid leukemia 80 Literature Cited 82

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iv List of Tables Table 2.1 Gene Expressi on Changes in 8226/R5 cells 25

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v List of Figures Figure 2.1 8226/R5 cells are highly resistant to R115777 18 Figure 2.2 Resistance in 8226/R5 cells is not linked to prenylation 20 Figure 2.3 Resistance is not related to decreased influx or increased efflux of R115777 21 Figure 2.4 8226/R5 cells harbor a multidrug resistant phenotype and R esistance does not correlate with the expression of heat shock proteins 22 Figure 2.5 8226/R5 cells are resistant to PS 341 24 Figure 3.1 Myeloma cell lines show variable sensitivity to Tipifarnib in V itro 40 Figure 3.2 Drug sensitive myeloma cell lines undergo Tipifarnib induced A poptosis as evide nced by Caspace3 cleavage 42 Figure 3.3 Tipifarnib is cytotoxic to RPMI 8226 cells in an in vivo murine M odel of multiple m yeloma 43 Figure 3.4 Tipifarnib dose response assay correlates pAkt levels 44 Figure 3.5 pAkt is nuclear localized in cells resistant to Tipifarnib 46 Figure 3.6 Ectopic expression of constitutively active Akt promotes cy totoxic r esistance to Tipifarnib 48 Figure 4.1 Tipifarnib combined with Bortezomib induces cell death in diverse multiple myeloma and AML cell lines 62 Figure 4.2 Tipifarnib and Bortezomib are syne rgistic in cytotoxicity assays 63 Figure 4.3 Tipifarnib and Bortezomib induce cell death in fibronectin adhered multiple myeloma and AML cells 67

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vi Figure 4.4 Adhesion to Fibronectin is not disrupted by Tipifarnib and Bortezomib 68 Figure 4.5 Reversal of CAM DR correlates with activation of the ER stress response 69 Figure 4.6 Bone marrow partially protects multiple myeloma an AML cell lines from Tipifarnib a nd Bortezomib induced cell death 72 Figure 4.7 Stromal cells partially protect primary isolates from the Tipifarnib and Bortezomib combination 73 Figure 4.8 HS 5 stromal cells secrete a soluble factor(s) that protects multiple m yeloma and AML cell lines 74

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vii Synergy and Resistance Mechanisms in R115777 and PS 341 Models of Myeloma and Leukemia Robert W. Buzzeo ABSTRACT The farnesyl transferase inhibitor R115777 (Zarnestra, Tipifarnib ) has been found to have clinical activity in diverse hematopoietic tumors. Clinical efficacy, however, does not correlate with Ras mutation status or inhibition of farnesyl transferase. To further elucidate the mechanisms by which R115777 induces apoptosis and to investigate drug resistance, we have identified and characterized a R115777 resistant human myeloma cell line. 8226/R5 cells were found to be at least 50 times more resistant to R11 5777 compared with the parent cell line 8226/S. 8226/R5 cells were insensitive to a diverse group of antitumor agents including PS 341 ( Bortezomib, Velcade). Comparison of gene expression profiles between resistant and sensitive cells revealed expression changes in several genes involved in myeloma survival and drug resistance. Identification and characterization of the 8226/R5 cell line helped us evaluate and confirm that the Akt tumor survival pathway plays an important role in Tipifarnib induced apoptos is and resistance in myeloma cells. Additionally, 8226/R5 cells helped to evaluate other molecules exhibiting synergistic cell death. In this study, we investigated the act ivity of R115777 combined with Bortezomib in microenvironment models of multiple my eloma and AML. The combination proved to be synergistic in multiple

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viii myeloma and AML cell lines treated in suspension culture. Even in tumor cells relatively resistant to Tipifarnib combined activity was maintained. Of importance, activation of the endopla smic reticulum stress response was enhanced and correlated with apoptosis and reversal of CAM DR. Our study provides the preclinical rationale for trials testing the Tipifarnib and Bortezomib combination in patients with multiple myeloma and AML.

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1 Chapter 1 Introduction and Background 1.1 Multiple myeloma Multiple myeloma (MM) also called Kahlers disease is a B cell malignancy in which no curative therapy exists. The disease was first characterized by Otto Kahler in the 19th century. Only 32% of individuals diagnosed with MM survive for more than 5years (Dominik et al. 2007). The high mortality rate coupled with a painful disease state makes this cancer one of the more aggressive and harmful hematologic malignancies (Kyle et al. 2004). The pathogenesis of this disease is not well understood however genes involved in cell cycle control (cyclin D) and apoptosis ( Akt ) have been implicated. DNA translocations involving chromosomes 13 and 14 have also been noted (Badros et al. 2007). Malignant B cells will over secrete t he cytokine Interleukin6 making the bone marrow microenvironment more suitable for tumorgenesis. Additionally the marrow becomes more vascular due to the tumor cells producing a protein known as VEGF (Badros et al. 2007). Diagnosis of MM is made through the use of an ELISA evaluating the levels of M Protein which is a specific protein secreted by a rapidly proliferating B cell (Dominik

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2 et al. 2004). Monoclonal Gammopathies of Undetermined Significance (MGUS) is the hypothesized precursor to multiple myeloma. In this pre disease state patients display a very low level of M protein in the serum, however this level spikes during active disease (Kyle et al. 2004). Patients with multiple myeloma typically present with b one weakness, hypercalcemia and renal damage all related to the destruction of the skeleton. Additionally, individuals may exhibit immunocompromised characteristics and anemia because of damage or eventual ablation of the bone marrow (Kyle et al 2004). Mu ltiple myeloma is a relatively uncommon disease and thus epidemiological studies have shown to be difficult. About 15,000 new cases of MM have been diagnosed each year since 2002 (Dominik et al. 2004). The disease is most common in men and the elderly (> 4 0 yrs). It accounts for 15% of all hematopoietic malignancies or 2% of all the cancers in the United States. Importantly, the disease is almost 2 times more common in African Americans and 2 times less prevalent in those of Asian descent (Dominik et al. 2004). Cohort studies have identified a notable link between obesity and multiple myeloma, however the pathophysiology of this connection remains elusive. Other linkage factors including tobacco and aclochol abuse are beginning to be evaluated (Dominik et al 2004). Current therapy for MM includes stem cell transplants and chemotherapy. Autologous stem cell transplants have been proven to increase survival time following chemotherapy. Many chemotherapeutic compounds induce a state of aplastic anemia in which the bone barrow has been ablated. To compensate for this, autologous stem cells have shown to decrease the morbidity rates of anemia (Kyle et al. 2004).

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3 Interestingly and probably as a result of the high mortality rate of MM, chemotherapeutic treatments now include drugs which target specific proteins involved in the pathogenesis of MM. Clincial trials for MM are relatively quickly approved because of the poor prognosis of those with active disease (Kyle et al. 2004) Before 2005, the drugs which were us ed for treatment included the DNA damaging drugs Melphalin and Doxorubicin (Badros et al. 2007). In 2005 the FDA approved the use of Bortezomib ; a proteosome inhibitor for the treatment of MM (Badros et al. 2007). The proteosome is a large protein complex responsible for degrading proteins in the cell. Hence, the proteosome is an essential part of protein regulation. Many of these proteins that are degraded are used by the cell to decrease proliferation and increase apoptosis two opposite hallmarks of cancer. Laboratory studies have shown that the proteosome becomes hyperactive in cancer cells most especially (Palumbo et al. 2008). Thus as predicted proteosome inhibitors have exhibited fewer side effects in patients. This form of target specific therapy is a relatively new and exciting area of research and treatment. Regardless, tumor cells eventually succumb to resistance to many of these drugs, and patients relapse (Kyle et al 2004). As such the characterization and identification of compounds that can prevent the disease altogether remains crucial. Many drugs that inhibit oxidative cellular damage and help control oncogenic proteins or prevent DNA damage have been investigated (Badros et al 2007)

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4 1.2 Drug resistance in Multiple Myeloma Myeloma patients exhibit an initial response to chemotherapy but ultimately succumb to drug resistance. U nresponsiveness to a wide spectrum of anti cancer agents is know n as multidrug resistance (MDR) (Damiano et al. 1999). Two subsets of drug resistance have been characterized. De novo drug resistance includes the mechanisms that increase survival and contribute to the intital resistance of the drugnave tumor. Aquired resistance occurs over a period of time while the tumor is expose d to the chemotherapeutic agent. Aquired resistance is frequently correlated to gene mutation(s). Many types of de novo resistance have been well characterized including cell adhesion mediated drug resistance (CAM DR) and environmental mediated drug resistance (EM DR) (Li et al. 2005) The phenomenom of CAM DR involves the interaction between the tumor cells and other cells in the bone marrow microenvironment, more specifically by contact of integrens to the substrate fibronectin (FN) EM DR has been identified as a resistance type caused by soluble factors including those produced by autocrine and paracrine loops of the tumor cells. Additionally, resistance caused by f actors released by bone marrow stromal cells is included in EM DR type resistan ce (Li et al. 2005) As such, initial survival of the tumor cells via CAM DR and/or EM DR is more significant since it is the first step for tumor cells to escape drug death and eventually obtain acquired drug resistance (Li et al. 2005). The adhesion of c ells to FN via integrins and subsequent cytotoxic protection has been well characterized as a substantial contributor to de novo drug resistance.

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5 Fibronectin is an extracellular matrix (ECM) glycoprotein that binds to fibrin and collagen and is predomina ntly detected at contact sites between bone marrow stromal cells ( BMSC s ) (Van der Velde Zimmermann et al. 1996) Additionally fibronectin binds to class of adhesion molecules knows as integrins. Integrins are a superfamily of adhesion molecules and are the best characterized for their role in regulating cell growth, differentiation and homing to the bone ma rrow (Hazlehurst et al. 2001). 25 of these subunits have been identified (Hazlehurst et al 2001). 1 integrins and fibronectin have been implicated in playing an important role in apoptotic suppression 51 integrins induce increased expression of the antiapoptotic protein Bcl 2. Thi s upregulation protected cells from serum starvation (Rozzo et al. 1997) Adhesion to fibronectin has been shown to protect myeloma RPMI 8226 cells from VP 16, radiation and front line chemotheurpuetic drugs such as melphalan and doxorubicin (Hazlehurst et al. 2001) Importantly, 1 integrins have been shown to activate multiple signal transduction pathways including FAK, MAPK, Src family kinases, PI3 kinase and AKT (Hanks et al. 1992, Lin et al. 1997, Meng et al. 1998, Lee et al. 2000, King et al. 1997). Many of the studies herein utilize FN adhesion so knowledge of their relevance to i ntegrin signaling is of significant importance

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6 1.3 Farnysltransferase inhibitors and Ras proteins Prenylation or isoprenylation is the addition of hydrophobic lipids to a protein. The three enzymes that prenylat e proteins in the cell include f arnesyltransfer as e (FTase) geranylgeranyltransfer as e (G G Tase) and Rab gerany lgeranyltransfeRas e (Brunner et al. 2003). FTase adds a 15 Carbon isoprenoid lipid (farne syl group) to the SH of the cysteine of proteins which have a CAAX motif on their COOH termini. Th is type of post translational modification is termed farnesylation and is considered a type of prenylation event (Maurer Stroh et al. 2003) Farnesylated proteins include the Ras superfamily of small GTP binding proteins involved in the cell cycle regulation. Farnesylation causes the proteins to associate with the plasma membrane due to the hydrophobic nature of the farnesyl group. The Ras proteins are synthesized as cytosoli c precursors that must associate with the cell membrane to transmit signals. Subsequently, membrane association can lead to heightened activation of these GTP binding proteins. Farnesyl transfe Ras e inhibitors (FTIs) represent a novel class of cancer therap eutic drugs that are designed to inhibit the farnesylation of Ras protein. The Ras superfamily of proteins is made up of 10 family proteins including Ras Rho, Rab, Rap, Arf, Ran, Rheb, Rad, Rit and Miro (Munemitsu et al. 1990). They are implicated in cell proliferation, cytoskeletal dynamic, membrane trafficking, vasicular transport, nuclear transport, mitochondrial transport and the mTOR pathway. There are over 30 members of the Ras subfamily involved in cell proliferation. Three of these (H Ras K Ras and N Ras ) are implicated in cancer when mutated and thus become

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7 constitutively active. It has been wel l established that Ras mutations are not required for tumor cell succeptibility to FTIs (Brunner et al. 2003) However Ras mutations, most especially t he N Ras protooncogene, are detectable in ~15% of those diagnosied with myelodysplasia and 25% of those with acute myelogenous leukemia (Karp et al. 2001) As such targeting the activation of Ras for the treatment of hematopoetic malignancies presents as a sound approach to treatment. Many FTIs have been synthesized. Some drugs are specific to FTase such as the drug primarily used in this study R1125777 ( Tipifarnib ). However others have exhibited high efficacy in inhibiting GGTa se as well. R115777 exhibited the highest level of specificity and lowest level of toxicity in pre clinical models. Tipifarnib was submitted to the FDA by Johnson & Johnson for the treatment of AML in patients aged 65 and over however approval was rejected in June of 2005. Howeve r, Tipifarnib exhibited similar to lower toxicity ratings in a phase III clinical trial for the treatment of solid tumors (Cunningham et al. 2002, Van cutsem et al. 2002). Besides relatively low toxicity, F TIs have a distinct spectra of activity compared w ith classical chemotherapeutic agents Additionally the growth inhibition by FTIs can supplement cytoxic effects of other drugs in an additive or synergistic way (Brunner et al. 2003). Because of these reasons, Tipifarnib should be considered a role in combination therapies for the treatment of certain tumors.

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8 Chapter 2 Identification and Characterization of a R115777 Resistant Cell Line Chapter 2 represents the following publication: Buzzeo et al. 2005 2.1 Introduction Multiple myeloma is a plasma cell malignancy with no known curative therapy. RAS mutation s occur frequ ently in myeloma (Bezieau et al. 2002, Hallek et al. 1998) and have been linked at least in some studies to a poor prognosis ( Corradina et al. 1994, Bauduer et al. 1993, Liu et al 1996). Farnesyl transfe Ras e inhibitors (FTI) inhibit Ras function by preventing its posttranslational prenylation, a modification done by the enzyme farnesyl transfe Ras e (FTase). The FTI R115777 was designed as a highly selective inhibitor of FTase (End et al. 2001) and has been clinically tested in several hematopoietic tumors. This compound has shown activity in acute myelogenous leukemia, chronic myelogenous leukemia, and myelodysplastic syndrome ( Karp et al. 2001, Cortes et al, 2003, Keating et al. 2002, Kurzrock et al. 2002) Preclinical studies have reported that FTIs have antitumor activity in myeloma cell lines and primary isolates ( Beaupre et al. 2003, Le Gouill et al. 2002, Bolick et al.

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9 2003) Based on these observations, a phase II clinical trial testing R115777 in patients with relapsed myeloma was conducted (Alsina et al. 2005). Forty three patients with a median of four prior treatment regimens entered the study. R115777 was well tolerated and 64% of patients achieved disease stabilization. Of importance, RAS mutation and inhibition of farnesyl transfe Ras e did not correlate with clinical efficacy consistent with a prior observation that R115777 can induce apoptosis via a Ras independent mechanism (Beaupre et al. 2004 ). I n myeloma cells, R115777 activates multiple int rinsic proapoptotic cascades (Beaupre et al. 2004). However, the molecules and/or signaling pathways that trigger these events remain elusive. To further elucidate the mechanisms by which R115777 induces apoptosis and to investigate drug resistance, I have established and characterized a R115777resistant human multiple myeloma cell line (8226/R5). This line is unlike a previously described R115777resistant colon cancer line (Smith et al. 2002) for resist ance is unrelated to the prenylation activity of the enzyme FTase. This finding correlates with our observation that 8226/R5 cells are insensitive to a diverse group of antitumor agents, including PS 341. In this study, I investigate and exclude several po tential mechanisms of R115777 resistance. Using comparative gene expression profiling (between sensitive and resistant cells), I have identified expression changes in several genes implicated in myeloma survival and drug resistance. Further evaluation of t hese genes may lead to the identification of novel FTI targets or resistance mechanisms that are clinically relevant.

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10 2.2 Materials and Methods 2.2.1 Cell lines and reagents The RMPI 8226 human myeloma cell line was obtained from the American Type Culture Collection (Manassas, VA). 8226/LR5 and8226/Dox40 lines were developed in our laboratory and have been previously described ( Hazlehurst et al. 2003, Dalton et al. 1986) Sensitive and resistant 8226 cells were grown in RPMI 1640 (Mediatech, Inc., He rndon, VA) supplemented with 10% fetal bovine serum (Omega Scientific, Inc., Tarzana, CA). The 8226/R5 cell line was established by continuous exposure of 8226 parental cells (8226/S) to increasing concentrations of R115777 for over 6 months. The establish ed 8 226/R5 line is maintained in 5 mol/L R115777. 8226/R5 cells were grown in R115777free supplemented medium for 2 weeks before experimentation. R115777 and [14C]R115777 (specif ic activity 1.43 GBq/mM ol, 3.68 MBq/mL) were kindly provided by David End (J ohnson and Johnson Pharmaceutical Research and development, LLC, Titusville, NJ). PS 341 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA) and FTI277 from Said M. Sebti (Moffitt Cancer Center and Research Institute, Tampa, FL). Additional dr ugs were purchased from the following vendors: doxorubicin hydrochloride, melphalan, and perillic acid (Sigma Chemical, St. Louis, MO); staurosporine and etoposide (A.G. Scientific, San Diego, CA); and tunicamycin (Calbiochem, San Diego, CA).Antibodies and Western blotting Antibodies were purchased from the following vendors: anti K Ras 2B (C 19; Santa

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11 Cruz Biotechnology, Inc., Santa Cruz, CA); anti HDJ 2 (NeoMarkers, Freemont, CA); anti hsp90, anti hsp70, anti hsp27, and anti glyceraldehyde3phosphate deh ydrogenase (Stressgen Biotechnologies, San Diego, CA); and anti a tubulin (BD Biosciences, San Diego, CA). Western bl otting was done as described (Beaupre et al. 2003). For the majority of experiments, lysates were harvested using radioimmunoprecipitation assay lysis buffer [150 M ol/L NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS, and 50 M ol/L Tris (pH 8.0)]. For evaluation of hsp27, hsp70, hsp90, and glyceraldehyde 3 phosphate dehydrogenase, lysates were harvested using Baverian lysis buffer [150 M ol/L NaCl, 1% Triton X 100, 30 M ol/L Tris (pH 7.5), and 10% glycerin]. 2.2.2 Analysis of cell growth, cell cycle arrest, and cell death Degree of resistance was determined using the tetrazolium salt 3,4,5dimethylazol 2yl 2,5diphenyl tetrazolium bromide (MTT; Sigma) reduction assay. 8226/S and 8226/R5 cells were plated into 96well microt iter plates at a density of 5x 104 cells/ mL in 200 l of supplemented media. Cells were exposed to a broad range of drug concentrations (R115777 and PS 341) in replicates of four. After a 72 hour (R115777) or 48hour ( PS341) incubation period at 37C, 50 l of 2 mg/ml MTT was added to each well and cells were incubated for an additional 4 hours. Plates were centrifuged for 5 minutes at 1,200 rpm in a Sorvall RT6000D table top centrifuge (Sorvall, Asheville, NC), supernatants were removed, and the water insoluble produc t was dissolved in 100 l of 100% DMSO (Sigma). Plates were shaken for 30 seconds and absorbance read at 540 nm

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12 on a Wallac Victor 2 1420 Multilabel Counter (PerkinElmer, Torrance, CA). The number of surviving cells was expressed as a percentage: absorban ce of the experimental sample/ a bsorbance of the control x 100. The IC50 of the drug was calculated by linear regression analysis using Excel software. Flow cytometric cell cycle analysis was done after propidium iodide stai ning as previously described (Be aupre et al. 2003). The presented histograms represent gating on live cells only. Apoptosis and cell death were evaluated by flow cytometry after Annexin V FITC and propidium iodide staining as per the recommendations of the manufacturer (BioVision Research Products, Mountain View, CA). TOPRO 3 (Molecular Probes, Eugene, OR) was substituted for propidium iodide in experiments where cells were treated with doxorubicin. Cell death was calculated as the sum of Annexin V FITC and propidium iodide or TO PRO 3positive cells. Specific cell death was determined by subtracting background death in untreated samples. Determination of P glycoprotein expression 8226/S, 8226/Dox40, and 8226/R5 cells were washed in PBS [0.8% NaCl, 0.02% KCl, 0.14% NA2HPO4, 0.02% KH2PO4 ( pH 7.4)] and resuspended in 200 l of icecold PB S containing 0.5 g/ml anti PglycoproteinFITC antibody (BD Biosciences) or 0.5 g/mL isotype control antibody anti dansyl IgG2b,nFITC (BD Biosciences). Samples were incubated on ice for 1 hour in the dark washed twice in PBS, and analyzed by flow cytometry. 2.2.3 R115777 accumulation and efflux To quantitate cellular accumulation of [14C]R115777, 8226/S and 8226/R5 cells were washed once in PBS and 1 x 106 cells were resuspended in 1 mL s upplemented

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13 me dia containing 5 mol/L R115777 (1:2.5 dilution of [14C]R115777 to cold R115777). Cells were incubated at 37 C for increasing time periods and were then washed three times in 10 mL ice cold PBS. Cell pellets were resuspended in 1 mL PBS and transferred to scintillation vials containing 10 mL Scintisafe 30% scintillation fluid (Fisher Scientific, Pittsburgh, PA). Samples were analyzed on a L56500 multipurpose scintillation counter (Beckman Coulter, Fullerton, CA). Because drug accumulation occurred rapi dly, cells were incubated in 5 mol/L R115777 at decreasing dilutions (1:10, 1:2.5, 1:1) of [14C]R115777 for 1 hour and samples were processed and analyzed as described above to confirm a dose dependent accumulation of [14C]R115777 in each cell line. For efflux experiments, 8226/S and 8226/R5 cells were washed once in PBS and 1 x 106 cells were resuspended in 1 mL supplemented media containing 5 mol/L R115777 (1:2.5 dilution of [14C]R115777 to cold R115777). Cells were incubated for 1 hour at 37C and then washed three times in 10 mL PBS and placed in R115777free supplemented media for increasing time periods. Samples were then processed and analyzed as described above. Because efflux of R115777 also occurred rapidl y, cells were incubated with 5 mol/L R115777 at increasing dilutions (1:1, 1:2.5, 1:10) of [14C]R115777 for 1 hour and then washed and placed in R115777free media for an additional hour. Samples were processed and analyzed as above to confirm a dose dependent efflux of [14C]R115777. E xperiments with 8226/S and 8226/R5 lines were done in parallel.

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14 2.2.4 Microarray analysis 2.2.4.1 Probe arrays. The oligonucleotide probe arrays were the Affymetrix U133A human arrays. These arrays consist of 22,215 probe sets, which target known and suspected genes as well as a number of suspected splice variants. The U133A chips detect an estimated 15,000 well characterized human genes. 2.2.4.2 Sample processing for microarray analysis. Five micrograms of total RNA derived from 8226/S, 8226/LR5, and 8226/R5 cells served as the mRNA source for microarray analysis. The polyadenylate RNA was specifically converted to cDNA and then amplified and labeled with biotin following the procedure initially de scribed by Van Gelder et al. (Va Gelder et al. 20 00). Hybridization with the biotin labeled RNA, staining, and scanning of the chips followed the proscribed procedure outlined in the Affymetrix technical manual and has been previously described (Warrington et al. 2000). Scanned output files were visuall y inspected for hybridization artifacts and then analyzed using Affymetrix Microarray Suite 5.1 software. Signal intensity was scaled to an average intensity of 500 before comparison analysis. The MAS 5.1 software uses a statistical algorithm to assess inc reases or decreases in mRNA abundance in a direct

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15 co mparison between two samples (Van Gelder et al 2000, Warrington et al. 2000) This analysis is based on the behavior of 11 different oligonucleotide probes designed to detect the same gene. The software generates a P value for the likelihood that any perceived difference was due to chance. The P values for all probe sets were exported to a text file and all pairwise comparisons were then aligned in Excel. For the comprehensive analysis, P < 0.05 was ident ified as changed (increased or decreased) for each individual comparison. Two independent samples from 8226/S, 8226/LR5, and 8226/R5 cells were collected. The samples generated from the resistant cell line were compared with the sensitive lines in all poss ible combinations. Genes were ultimately selected if they were identified as increased in all eight comparisons or decreased in all eight comparisons 2.3 Results 2.3.1 8226/R5 cells are resistant to R115777induced growth arrest and cell death. Continuous culture of 8226/S cells with increasing concentrations of R115777 established the 8226/R5 line. In cytotoxicity assays, 8226/R5 cells were at least 50 times more resistant to R115777 compared with parental 8226/S cells (Fig. 2.1A). The IC50 for the 8226/S line was 0.1 mol/L and tha t for the 8226/R5 line was 5.4 mol/L. Cell cycle analysis revealed that R115777 induced G1 growth arrest in 8226/S cells and this effect was largely abolis hed in the 8226/ R5 line (Fig. 2.1B). 8226/R5 cells were also protected

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16 from apoptosis at concentra tions of R115777 as high as 20 mol/L (Fig. 1C; data not shown). The observed resistance was stable because cells cultured in R11577free conditioned media for several months continued to display the resistant phenotype. Relative to doxorubicin(8226/Dox 40) and melphalan resistant (8226/LR5) isogenic lines, 8226/R5 cells were the m ost resistant to R115777 (Fig. 2.1C). 2.3.2 Resistance to R115777 does not correlate with K Ras prenylation or farnesyl transfe Ras e activit y. To determine whether 8226/R5 cells were cross resistant to other compounds that inhibit prenylation, cells were exposed to inhibitors of FTase and geranylgeranyl transfe Ras e I (GGTase I). Similar to our results with R115777, 8226/R5 cells were resista nt to the specific FTase inhibitor FTI 277 (22) when compared with parental 8226/S cells (Fig. 2.1). In addition, resistance was maintained in the presence of perillic acid, a compound that inhibits both FTase and GGTase I (Fig. 2.1B; ref. 23). 8226 cells express K Ras and harbor a codon 12 K RAS mutation. Similar to our prior observation in U266 cells (Beaupre et al. 2004), K Ras remained prenylated in both sensitive and resistant cells after R115777 treatment (Fig. 2.2C). Moreover, farnesylation of HDJ 2 (a protein that can be farnesylated but not geranylgeranylated) was inhibited in both lines (Fig. 2.2D). These results indicate that R115777 resistance does not correlate with the prenylation status of K Ras or HDJ 2. Furthermore, the fact that HDJ 2 farne sylation can

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17 be inhibited in 8226/R5 cells implies that mutation of FTase (the drug target) is not responsible for the development of resistance. 2.3.3 Resistance is not related to decreased accumulation of R115777. Many 8226 lines that acquire drug re sistance have elevated expression of P glycoprotein. An example of this is the 8226/Dox 40 cell line that expresses high levels of membrane P glycoprotein that participates in the doxorubicinresistant phenotype (Dalton et al. 1986). As expected, 8226/Dox 40 cells were found to have a marked increase in surface P glycoprotein expression when compared with parent 8226/S cells (Fig. 2.3A). 8226/R5 cells, however, had membrane levels that were similar to that noted for the 8226/S line (Fig. 2.3A). Because incr eased expression or activity of other membrane pumps may also produce a resistant phenotype, we investigated the influx and efflux of R115777 in both 8226/S and 8226/R5 cells. Influx of R115777 occurred rapidly in both lines and reached steady state within 15 minutes of incubation with radiolabeled R115777 (Fig. 2.3B). A dose dependent increase in R115777 uptake was also observed (Fig. 2.3C) and both time and concentration dependent experiments revealed that R115777 uptake was increased in 8226/R5 cells compared with the parental line. With regard to R115777 efflux, intracellular R115777 decreased rapidly (within 15 minutes) in both sensitive and resistant cells in time course experiments (Fig. 2.3D). Furthermore, a dose dependent efflux of R115777 was note d with resistant cells retaining more R115777

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18 compared with sensitive 8226/S cells (Fig. 2.3E). These results indicate that 8226/R5 resistance is not related to decreased influx or increased efflux of R115777. Fig. 2.1 8226/R5 cells are highly resistant to R115777. A degree of resistance was determined using the MTT reduction assay. 8226/S and 8226/R5 cells were treated with increasing concentrations of R115777 for 72 hours. Percentage surviving cells was calculated relative to cells treated with control media only (see Materials and Methods). IC50 determinations were done by linear regression analysis. B cell cycle analysis after R115777 treatment. Sensitive and resistant 8226 lines were treated with increasing concen trations of R115777 for 24 hours. Cell cycle arrest was determined by flow cytometry after propidium iodide staining. Gating was on live cells only. C evaluation of cell death after R115777 treatment. 8226/S, 8226/Dox40, 8226/LR5, and 8226/R5 cells were t reated with increasing concentrations of R115777 for 72 hours. Cell death was determined by flow cytometry after Annexin V FITC and propidium iodide staining. Specific cell death was calculated by subtracting background death in untreated samples. The pres ented data is representative of three independent experiments.

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19 2.3.4 8226/R5 cells display a multidrugresistant phenotype and resistance is not associated with increased expression of heat shock proteins. In an attempt to further categorize resistance, 8226/R5 cells were exposed to agents that induce apoptosis with diverse mechanisms of action. Compounds that have been reported to promote mitochondrial dysfunction, endoplasmic reticulum stress, and nuclear stress were tested (Fig 2.4 A E) Sur prisingly, 8226/R5 cells were resistant to all evaluated agents when compared with the parent 8226/S line. The proteosome inhibitor PS341 has been shown to have cytotoxic activity in several chemoresistant myeloma lines (Ma et al. 2003). In cytotoxicity a ssays, 8226/R5 cells were three times more resistant to PS 341 when compared with 8226/S cells with an IC50 of 41.8 and 13.2 nmol/L, respectively (Fig. 2.5A) Unlike our findings with R115777, PS 341 induced G2M arrest in both sensitive and resistant cell s with 8226/R5 cells being relatively protected (Fig. 2.5B). 8226/R5 cells were also protected from PS341induced apoptosis at concentrations as high as 20 nmol/L (Fig. 2.5C: data not shown). 8226/R5 cells maintained a PS 341resistant phenotype when cult ured in the absence of R11577 for several months, indicating stable resistance. Heat shock proteins have antiapoptotic properties that can protect cells from stressful conditions. It has been previously reported that upregulation of hsp70 can protect ovarian cancer cells from FTI induced apoptosis (Hu et al. 2003). In addition, blockade of hsp27 expression has been shown to overcome PS 341 resistance in

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20 Fig. 2.2. Resistance in 8226/R5 cells is not linked to prenylation. A, 8226/R5 cells are r esistant to the FTase specific inhibitor FTI 277. 8226/S and8226 /R5 cellswere treated with increasing concentrations of FTI 277 for 72 hours. Cell death was determined by flow cytometry after AnnexinV FITC and propidiumiodide staining. Specific cell death was calculated by subtracting background death in untreated samples. B, 8226/R5 cells are resistant to perillic acid, an inhibitor of both FTase and GGTase I. Sensitive and resistant cells were treated with increasing concentrations of perillic acid for 7 2 hours. Samples were analyzed as in (A). C, K Ras remains prenylated in sensitive and resistant cells after R115777 treatment. 8226/S and8226 /R5 cells were t reated with control media or 5 mol/LR115777 for 72 hours. Cell lysates were harvested and evaluated by Western blotting using the indicated antibodies. D, HDJ 2 farnesylation is inhibited in both 8226/S and 8226/R5 cell lines. Sensitive and resistant cells were treated and analyzed as in (C) using the indicated antibodies. u, unprocessed form; p, processed form. (A) and (B) are representative of three independent experiments; (C) and (D) are representative of two independent experiments.

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21 Fig. 2.3. Resistance is not related to decreased influx or increased efflux of R115777. A, P glycoprotein surface expression is not increased in 8226/R5 cells. 8226/S, 8226/Dox40, and 8226/R5 cells were stained with anti P glycoprotein (dashed line histograms) or isotype control antibody (solid line histograms) as described in Materials and Methods. Samples were analyzed by flow cytometry. Decreased accumulation and/or increased efflux of R115777 is not responsible for resistance in the 8226/R5 line (B E). B, 8226 /S and8226 /R5 cells were exposed to 5 mol/LR115777 (1:2.5 dilution of [14C]R115777 to coldR115777) in supplemented media for increasing time periods. Cells were washed and accumulation of [14C]R115777 was quantitated by scintillation counting (see Materials and Methods). C, 8226/S and8226 /R5 cells were incubated in supplemented m edia containing 5 mol/LR115777 at decreasing dilutions (1:10,1:2.5,1:1) of [14C]R115777 for1hour. Samples were processed and a nalyzed as in (B) to confirm a dose dependent accumulation of [14C]R115777 in each line. D, sensitive and resis tant lines were treated with 5 mol/L R115777 (1:2.5 dilution of [14C]R115777 to cold R115777) for 1 hour, then washed and placed in R115777free supplemented media for increasing time period s. Samples were processed and analyzed as in (B). E, 8226/S and8226/R5 cells were treated with 5 mol/LR115777 at increasing dilutions (1:1,1:2.5,1:10) of [14C]R115777 for 1 hour and then washed and placed in R115777 free media for an additional hour. Samp les were processed and analyzed as in (B) to confirm a dosedependent efflux of [14C]R115777. (A), (B), and( D) are representative of two independent experiments; (C) and (E) are representative of three independent experiments.

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22 Fig. 2.4. 8226/R5 cells h arbor a multidrug resistant phenotype and resistance does not correlate with the expression of heat shock proteins. 8226/R5 cells display multidrug resistance (A E). 8226/S and8226 /R5 lines were treated with the indicated compounds for 48 hours (A and B) or 24 hours (C E). Cell death was determined by flow cytometry after Annexin V FITC and propidiumiodide staining. Specific cell death was calculated by subtracting background death in untreated samples. The presented d ata is representative of three independent experiments. F, resistance in 8226/R5 cells is not associated with increased expression of heat shock proteins. 8226/S and8226 /R5 cells were treated with control media or 5 mol/L R115777 for 72 hours. Cell lysat es were harvested and analyzed by Western blotting using the indicated antibodies (see Materials and Methods). The presented data is representative of two independent experiments. lymphoma cell lines ( Chauhan et al. 2003, Chauhan et al. 2004) To determ ine whether multidrug resistance was related to overexpression of heat shock proteins, sensitive and resistant 8226 lines were evaluated for heat shock protein expression (Fig. 2.4F).

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23 Expression levels of hsp70 and hsp90 were similar in sensitive and resis tant lines and treatment with R115777 (druginduced stress) had no appreciable effect. Hsp27 expression was also not effected in 8226/S cells after R115777 treatment but was undetectable in the 8226/R5 line. These results indicate that resistance does not correlate with the expression of heat shock proteins hsp27, hsp70, and hsp90. 2.3.5 Transcriptional profile of 8226/R5 cells. To identify potential targets of R115777 and/or molecules associated with R115777 resistance, we compared gene expression profiles between sensitive and resistant variants of 8226 cells. Direct comparisons were made between the 8226/S and 8226/R5 cell lines. In an attempt to reduce the number of nonspecific gene expression changes identified, comparisons were also made between 8226/LR5 (which possessed an intermediate degree of resistance to R115777) (Fig. 2.1C) and 8226/R5 cells. This approach produced 2,064 probe sets (representing 1,666 genes) with changes unique to the 8226/R5 line. A selected list of genes involved in cel l survival and growth, maintenance of cell cytostructure, cell microenvironment contact, cholesterol biosynthesis, and protein degradation are presented in (Table 2.1)

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24 Fig. 2.5 8226/R5 cells are resistant to PS 341. A degree of resistance was determi ned by the MTT reduction assay. 8226/S and 8226/R5 cells were treated with increasing concentrations of PS 341 for 48 hours. Percentage surviving cells and IC50 determinations were done as in Fig 2.1. B analysis of cell cycle after PS 341 treatment. Sensi tive and resistant 8226 lines were treated with increasing concentrations of PS 341 for 24 hours. Cell cycle arrest was determined by flow cytometry after propidium iodide staining. Gating was on live cells only. C evaluation of cell death after PS 341 tr eatment. 8226/S and 8226/R5 cells were treated with increasing concentrations of PS 341 for 48 hours. Cell death was determined by flow cytometry after Annexin V FITC and propidium iodide staining. Specific cell death was calculated by subtracting backgrou nd death in untreated samples. The presented data is representative of three independent experiments.

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25 Expression Increased Expression Decreased Janus Kinase 2 Interleukin 6 receptor Signal transducer and a ctivator of transcription 1 Insulin like growth factor 2 receptor Bcl XL Mcl 1 Phosphoinositide 3 kinase HSP27 FGFR activating protein 1 Retinoblastoma 1 Fibroblast growth factor 20 Transforming growth factor Mavalonate kinase Tumor protein p53 Rap 2A Farnesyl diphosphate synthetase Rab GGase, B subunit Lamin A/C Rab 4A Ras protein activator like 1 Ras and Rab interactor 3 Ras and Rab interactor 2 Mitogen activated protein Kinase 1 Rab 36 Mitogen activated protein kinase 9 Rho GTPase activating protein 6 Mitogen activated protein kinase 14 Mitogen activated protein kinase phosphatase Cyclin D3 Cyclin D1 Fas apoptotic inhibitory molecule Syndecan 1 Calmodulin 3 X box binding protein 1 Calpain 2 Beta Actin S100 calcium binding protein A13 and A14 Fibronectin 1 Collagan type XXI Integrin B5 Paxillin Integrin B7 Integrin 4 Ubiquitin conjugating enzymes Proteosome inhibitor subunit 1 A100 calcium binding protein (A11 calgizzarin) Table 2.1 Gene expression changes in 8226/R5 cells

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26 2.4 Discussion FTIs were designed as specific inhibitors of Ras intended to interfere with a crucial posttranslational processing step. Ras requires the addition of a 15carbon farnesyl group to its carboxyl terminal cysteine, which permits its localization to the plasma membrane (a requirement for function). This modification is done by the enzyme FTase, the purported target of R115777. It has been well established that alternate prenylation of Ras may occur in the presence of FTIs (Sebti et al. 2000) This has been most notably described for K Ras but also applies to the N Ras protein. These reports are consistent with our prior observation in U266 cells (N Ras expressing line; ref. Beaupre et al. 2004) and our present finding that K Ras remains prenylated in 8226/S cells after R115777 treatment. These results suggest a Ras independent mechanism of cell death. This is further supported by the clinical observation that responses to R115777 do not correlate with Ras mutation status or inhibition of farnesyl transfe Ras e measured ex vivo (Karp et al. 2001)Karp et al. 2001, Kurzrock et al. 2003, Alsina et al. 2004). We hypothesized that the isolation of a R115777resistant human myeloma cell line (8226/R5) might provide insight concerning the potential targets of R115777 or identify novel mechanisms of FTI resistance. R115777 resistance was not associated with an increase in the surface expression of P glycoprotein nor was it associated with decreased influx or increased efflux of R115777. In a ddition, the expression of heat shock proteins (hsp27, hsp70, hsp90) did not correlate with the drug resistant phenotype. We therefore undertook molecular profiling of sensitive and resistant 8226 lines and

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27 identified expression changes in several genes im plicated in cell survival and drug resistance. They included genes involved in cell signaling, cholesterol biosynthesis, and protein degradation. It remains possible that molecules associated with one of these genes is a major target of R115777 and/or dire ctly participates in FTI resistance. Our microarray data identified increased expression of several proteins associated with Jak Stat signaling, including Jak2. In primary isolates, constitutive Stat3 activation has been observed in the majority of patients with multiple myeloma (Catlette Falcone et al. 1999, Bharti et al. 2004. Stat3 homodimers and Stat1:Stat3 heterodimers seem to be the predominant DNA binding forms (Catlette Falcone et al. 1999) It has been reported that inhibition of Stat3 can sensitize resistant myeloma cells to chemotherapy mediated apoptosis (Alas et al. 2003). These results imply that Jak Stat signaling may contribute to the drug resistant phenotype. Stat3 activity has been linked to upregulation of Bcl XL (Catlette Falcone et al. 1999) and consistent with this, Bcl XL expression was increased in 8226/R5 cells. Elevated expression of Bcl XL has been observed in several hematopoietic tumors, including blast crisis chronic myelogenous leukemia and nonHodgkin's lymphoma (Grad et al. 2000) and one study suggested that it was an indicator of chemoresistance in multiple myeloma (Tu et al. 1998) We have previously reported that R115777 can partially overcome drug resistance in U266 cells that maintain high levels of stable Bcl XL expression (Beaupre et al. 2004). These findings imply that R115777 resistance may only in part be regulated by increased Jak Stat signaling or BclXL expression in 8226/R5 cells.

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28 Our comparative gene expression profiling also identified other genes pote ntially involved in R115777 resistance. Phosphatidylinositol 3kinase is a heterodimer consisting of p85 and p110 subunits (Otsu et al. 1991, Hiles et al. 1992) The p110 isoform is preferentially expressed in cells of hematopoietic origin (Chantry et al. 1997, Vanhaesebroeck et al. 1997) and elevated expression of the catalytic polypeptide was observed in the 8226/R5 line. An important downstream target of phosphatidylinositol 3kinase is Akt /protein kinase B, a protein that is known to play a role in myeloma survival and drug resistance ( Hideshima et al. 2001, Tu et al. 2000, Hsu et al. 2002) Interestingly, it has been reported that FTI induced apoptosis can be prevented by a constitutively activated form of Akt 2 ( Jiang et al. 2000) Therefore, it r emains possible that phosphatidylinositol 3kinase/ Akt signaling also contributes to R115777 resistance and the multidrug resistant phenotype. Because the p110 subunit is mainly expressed in hematopoietic cells, it potentially represents a novel therapeut ic target particularly for resistant tumors of hematopoietic origin. Our analysis also identified increased expression of mevalonate kinase, an enzyme associated with cholesterol biosynthesis. Influx and efflux experiments suggest that R115777 is retained in 8226/R5 cells compared with the parent 8266/S line. This is relevant because an increase in cholesterol rich microdomains, such as lipid rafts, have been associated with the development of multidrug resistance (Lavie et al. 2000) R115777 is a lipophil ic molecule that could potentially be sequestered and compartmentalized in such microdomains; however, in 8226/R5 cells, R115777 efficiently inhibits FTase. Nevertheless, it remains possible that subcompartmentalization

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29 of R115777 influences its interaction with unforeseen targets. Also of importance was the observation that 8226/R5 cells were resistant to the proteosome inhibitor PS 341. It has been previously reported that increased expression of hsp27 produces a PS 341resistant phenotype in multiple myeloma cells ( Chauhan et al. 2003) In 8226/R5 cells, however, hsp27 protein expression was undetectable. Our microarray analysis revealed decreased expression of the 26S proteosome subunit (the target of PS 341) in 8226/R5 cells when compared with the parental 8226/S line. This perhaps represents a novel mechanism of proteosome inhibitor resistance. In conclusion, I have identified a myeloma cell line with resistance to both R115777 and PS 341. Further characterization of this line may lead to ide ntification of novel drug targets or resistance mechanisms that are clinically relevant.

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30 Chapter 3 The Akt Pathway plays an Important Role in R115777 Induced Apoptosis and Resistance in Multiple Myeloma 3.1 Introduction Multiple myeloma (MM) is a malignant B Cell neoplasm characterized by the proliferation of clonal plasma cells preferentially in the bone marrow (Hallek et al. 1998, Ghafoor et al 2002) While chemotherapy exhibits an initial response, patients eventually succumb to acquired drug resistance (Bergsagel et al. 1995, Dalton et al. 1997) Current research is focusing on identification of targets in the myeloma cell implicated in tumor cell growth, survival and drug resistance. This research has led to the development an d study of innovative compounds such as thalidomide, immunomodulatory drugs (IMIDs) and protea some inhibitors for the treatment of MM (Weber et al. 2003, Richardson et al. 2004, Pei et al 2004, Weber et al. 2002, Santucci et al 2003) Ras mutation s occur f requently in myeloma as then are found in 50% of patients with advanced disease (Neri et al. 1989). Furthermore, patients with mutated Ras are less likely to respond to chemotherapy and have a shorter median survival (Liu et al. 1996). Therefore, the dev elopment of compounds to inhibit Ras activity may exhibit antitumor activity in MM as well as other cancers. One such family of Ras inhibiting agents is the

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31 FarnesyltransfeRas e inhibitors (FTIs) (Datlon et al. 1997) Ras prenylation is necessary for onco genic activi ty Prenylation is a post translational addition of an isoprene lipid moiety at the cysteine residue in a consensus sequence (CAAX box) of the COOH terminus (Kato et al 1992, Willumsen et al. 1984, Hancock et al 1989, Sebti et al. 2000) Ras i s preferentially prenylated by FarnesyltransfeRas e (FTase), however GeranylgeranyltransfeRas e (GGTase) will alternatively prenylate the k Ras isoforms (Sebti et al. 1997, Sebti et al 2000, Sun et al. 1998) While early generation FTIs were designed as pep tidomimetics of the CAAX box, current molecules are mostly non peptide, nonthiol containing compounds with nanomolar potency including farnesyl pyrophosphate analogues and nonpeptide compounds derive d from high throughput screening (Cox et al, 2001) Ti pifarnib also known as FTI R115777 or Zarnestra is a nonpeptidomimmetic substituted quinol i ne inhibitor of the CAAX binding site of FTase (Hahn et al. 2001, Tamoni et al. 2001) In vitro, Tipifarnib has shown to inhibit the prenylation of lamin A in MCF 7 breast cancer cells (Kellard et al. 2001) and the prenylation of lamin B, H Ras and N Ras in colon, pancreatic, and melanoma cell lines (End et al. 2001). Tipifarnib treatment of patients with adv anced myeloma in a phase II clinical trial exhibited disease stabilization in 64% of patients (Alsina et al. 2004) Of importance, RAS mutation status and inhibition of FTase did not correlate with clinical efficacy. This is consisten t with prior observations that Tipifarnib can induce apoptosis via Ras independent mechanisms (Beaupre et al. 2004). Interestingly, treatment with

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32 Tipifarnib decreased levels of phosphorylated Akt ( pAkt ) and STAT3 in bone marrow from patients where these tumor survival pathways were constitutively active. The serine/threonine kinase Akt is an important downstream target of phosphatidylinositol 3kinase (PI3K) (Cantly et al. 2002) The PI3K/ Akt signaling pathway is a key mediator of cell survival while interacting with pro apoptotic proteins including Bad, and Caspase 9, as well as the anti apoptotic protein IK K(Nrunet et al. 1999, Madrid et al. 2000) The phase II clinical trial demonstrated disease stabilization in 2 out of 2 patients where Tipifarnib inhibited Akt phosphorylation. This suggests that either pAkt or an upstream kinase or modulator of pAkt may be targeted by Tipifarnib These findings are consistent with those of other groups which indicate that the efficacy of FTIs anti proliferative and apoptotic responses on tumor cells may be due to the drug affecting other proteins than Ras (Takada et al. 2004). It has been reported that the PI3 kinase/ Akt pathway is a critical target for FTI induced apoptosis in ovarian and pancreatic cancer cell lines and in Ras transformed rodent fibroblasts, respectively (Du et al. 1999). In MM primary isolates, Akt has been found to be predominately activated and localized to the nucleus (Alkan et al. 200). pAkt has show to be primarily localized to the nucleus following rec eptor activation in B lymphocytes (Tu et al. 2000). Additionally, activated Akt in the nucleus has been implicated in the modulation and activity of regulatory proteins including the Forkhead/FOXO family of transcription factors (Brunet et al. 1996). No direct correlation between Akt localization and drug resistance has yet been characterized. It has however been well established that Akt activation inhibits

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33 transcription of RhoB (Jiang et al. 2000) and that RhoB controls Akt nuclear/cytoplasmic traffick ing in retinal cells (Adini et al. 2003) As such, we have examined the mechanisms of the cytotoxicity of Tipifarnib in vitro and in vivo and its correlation with the status of Akt activation. We have demonstrated that Tipifarnib induces apoptosis in a dose dependent manner in two out of three MM cell lines. Using a SCID hu model of myeloma we show that Tipifarnib was cytotoxic to myeloma cells in vivo Additionally, we have correlated levels of pAkt expression to Tipifarnib resis tance with cell lines harboring higher levels of pAkt such as the Tipifarnib resistant RPMI 8226 cell line (8226/R5) (Buzzeo et al. 2005). Ectopic expression of constitutively active Akt in B cells induces Tipifarnib resistance and we identify a correlat ion between nuclear localization of pAkt and drug resistance in MM cell lines. To this end, we can confirm that the Akt tumor survival pathway plays an important role in Tipifarnib induced apoptosis and resistance in myeloma cell lines. 3.2 Material and M ethods 3.2.1 Cell culture Myeloma cell lines RPMI 8226, and U266 were obtained from ATCC (Manassas, VA). The murine proB cell, Ba/F3 was obtained from DSMZ (Braunschweig, Germany). MM1.S cells were generously donated by Steven T. Rosen (Northwestern Uni versity, Chicago, Illinois). 8226/S and 8226/R5 cells have been previously characterized (Buzzeo

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34 et al. 2005) and were developed in this lab. All cell lines were maintained in RPMI medium (CellGro, Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin/streptomycin, and 100 M L glutamine (Gemini Bio Products, Calabasas, CA). Medium for the IL 3 dependant Ba/F3 cells was supplemented with 10% WEHI conditioned medium. WEHI cells were obtained from ATCC and grown in RPMI supplemented with 10% FBS. Cells were grown to high con fluency, and conditioned medium filtered through a 0.4 filter. 8226/R5 cells were cultured in the presence of 5M Tipifarnib however drug was removed two weeks prior to experimentation. 3.2.2 Drug preparation Tipifarnib (FTI R115777, Janssen Research Institute, Beerse, Belgium) was diluted in DMSO (Fisher Chemical, Pittsburg, PA) to a concentration of 5mM, sonicated 10 minutes in a water bath sonicator (Branson 3510) and stored at 20C. 3.2.3 Proliferation assay Half log serial dilutions of Tipifarnib from 5x1010 to 5x106 M were plated in quadruplicate in 96 well plates. RPMI 8226, U266, and MM1.S cells in log phase growth were added at 10,000 cells per well (Ba/F3 cells at 5,000 cells/well) and incubated for 72 hours at 37oC. 50 L of 2mg/ml 3 [4,5Dimethyl 2yl] 2,5 diphenyl tetrazolium bromide

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35 (MTT dye) (Sigma, St. Louis, MO) was added to each well. Plates were incubated an additional 4 hours and centrifuged at 1,200 rpm. The supernatant was aspirated, the precip itate solubilized in 100 L DMSO and the plates were shaken for 30 seconds with sample absorbances read at 450nM. IC50 values were calculated by linear regression analysis of the dose response curve. Data shown represents an average of 3 independent experi ments and includes standard error. 3.2.4 Apoptosis assay Myeloma cells were grown 48 hours in the presence of vehicle control (DMSO), or varying concentrations of Tipifarnib Cells were collected, washed in PBS, and stained with 5l Annexin V FITC (Clo netech, Palo Alto, CA) and 5l Propidium Iodide (Clonetech, Palo Alto, CA) in 0.5 ml annexin binding buffer. Apoptosis was determined by Flow Cytometry and analyzed using CellQuest software (Becton Dickinson, Carpinteria, CA). Data shown represents an av erage of 3 independent experiments and includes standard error. 3.2.5 Immunostaining For western blotting, cells were washed in PBS and lysed in Baverian lysis buffer (30 mM HEPES, pH 7.5, 10 mM NaCl, 1% Triton X 100, 10% glycerol, 5mM MgCl2, 1 mM EGTA) with freshly added protease and phosphatase inhibitors (25 mM NaF, 25

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36 cytoplasmic fr actions were separated using NE PER (Pierce, Rockford, IL.) per the manufacturers instructions. 50 g protein was run on 12.0% acrylamide gels and transferred to PVDF membranes (Millipore, Billerica, MA). Blots were blocked in 5% milk in 0.1% PBS Tween for 2 hrs, incubated overnight in primary antibody in 5% BSA, washed for 45 min in 0.1% Tween and incubated for 1 hr in secondary antibody. The wash was repeated and bands were visualized using SuperSignal West Dura Extended Duration Substrate (Thermo Sci entific). Antibodies were purchased from the following vendors: Caspase 3, Cleaved Caspase 3 (Asp175), Akt pAkt (Ser473), pAkt (Ser473) IHC Specific, HRP anti mouse and HRP anti rabbit (Cell Signaling Technologies, tubulin, GAPDH a nd anti HA (Sigma Chemical, St. Louis, MO). Data shown is representative of three independent experiments. For immunohistochemistry (IHC), 0.5 x 106 8226/S, 8226/R5 and MM1.S cells were cytospun and fixed in 4 % paraformaldehyde for 1 min, air dried and incubated in methanol for 5 min at 20C. Slides were inc ubated in a 1:00 dilution in Tris Bufferred Saline (TBS) (3%BSA) with 1:100 diluted pAkt (Ser473) IHC Specific antibody for 1 hr, and washed three times in TBS (10 min/wash). Slides were incubated w ith secondary at a dilution of 1:80 in TBS(1%BSA) for 1 hr, washed three times in TBS and incubated for 1 hr in avidinbiotin peroxidase complex (ABC reagent). Slides were then washed three times in TBS and incubated with DAB reagent (15 min) (Zymed Labora tories) then stained with or without Hematoxylin (2 min) (Zymed Laboratories). Cells were

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37 visualized by light microscopy and captures are representative of three independent experiments. 3.2.6 Murine studies To develop myeloma SCID hu mice, C.B 17 mice homozygous for both the severe combined immunodeficiency ( scid ) and beige ( bg) mutations (C.B 1 7/GbmsTac Prkdcscid Lyst bg, herein referred to as SCID; Taconic, Germantown, New York) were used. Human fetal bones of 1823 weeks of gestation w ere obtained from Advance Bioscience Resource (Alameda, California) and from the General Hospital of Vienna (Vienna, Austria) in compliance with the regulations issued by the state and federal governments. SCID mice 68 weeks old were transplanted subcutan eously with fetal human bones (humerus, femur or tibia) and fetal thymus, as previously described (U Ras hima et al. 1997) Six weeks post implant 5x104 RPMI 8226 cells were injected directly into the implanted bones. Beginning at 10 weeks post implant, and each week thereafter, urine was collected from 12 mice light chain immunoglobulin levels by Radial Immunodiffusion (BIND A RID human kappa and lambda free, The Binding Site Limited, Birmingham, UK). At 12 weeks postimplant and afte r the presence of disease was established, Tipifarnib was administered by gastric lavage at a dose of 50 mg/kg/d, once per day for two weeks. At the conclusion of the drug course 10 mice were euthanized via decapitation and implanted bones were removed for

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38 histology. Wright Gimsa staining on all 10 bone sets were performed and evaluated by light microscopy. 3.2.7 Gene constructs, transfection and fluorescent microscopy Constitutively active HA Myr Akt 1 (Myr Akt ) was previously described (Frank et al. 1995), pDsRed2 (Clontech), pDsRed2HAMyr Akt 1 and pcDNA3 (Invitrogen) were generous gifts from Jin Cheng (University of South Florida, Tampa, Fl.). Ba/F3 cells were transfected using Nucleofect ion SolutionV (Amaxa Biosystems) according to the manufacturers instructions. Cells were selected for 2 weeks in 300g/ml G418 (Gibco Scientific). 5,000 cells were stained with DAPI + mounting medium (Vector Labs, Burlingame, CA) and viewed with a fully automated, upright Zeiss AxioImagerZ.1 microscope with a 63x /1.40NA oil immersion objective, and DAPI and Rhodamine filter cubes. Images were produced using the AxioCam MRm CCD camera and Axiovision (v.4.5). 3.3 Results 3.3.1 Tipifarnib induces apoptosis in RPMI 8226, and U266 myeloma cell lines, but not the MM1.S cell line. MTT and AnnexinV binding assays were used to determine Tipifarnib sensitivity in three myeloma cell lines. MTT binds active mitochondrial reductase enzymes and its

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39 resulting formazin product is associated with cellular proliferation and viability. Of the three cell lines examined RPMI 8226, and U266 cell lines had IC50 manufacturers recommended maximum dose for specific inhibition (Fig. 3.1A ). RPMI 8226 cells were most sensitive (IC50=64 nM) while U266 (IC50 (IC50 an IC50 value 190 times greater than that of the RPMI 8226 cells. AnnexinV binding indicated that the decrease in proliferation observed in MTT data was due to apoptosis, not growth arrest (Fig. 3.1B). Consistent with the MTT results the apoptosis assay showed the RPMI 8226 cells to be most sensitive, followed by U266 cells, with the M M1.S cells most resistant to Tipifarnib. Expression of effector caspase (Caspase 3) cleavage was analyzed by western blotting (Fig 3.2A B). A 48hour time course with 1 M Tipifarnib treating RPMI 8226 and MM1.S cells showed induction of caspase 3 activat ion in RPMI 8226 cells beginning at 24 and continuing through 48 hours. However, the same dose failed to do so in MM1.S cells (Fig 3.2A). After the appropriate time time of caspase cleavage was found, a dose dependent increase in Caspase3 cleavage in RPMI 8226, and U266 cell lines following seventy similar response was not observed in MM1.S cells. This data further substantiates the previous annexin data (Fig 3.1) that Tipifar nib is inducing apoptosis in cells sensitive to it, while MM1.S cells remain resistant.

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40 Figure 3.1. Myeloma cell lines show variable sensitivity to Tipifarnib in vitro (A ) Three myeloma cell lines were treated with log dilutions of Tipifarnib for 72 hours and viability measured by the MTT proliferation assay. Tipifarnib sensitivity was greatest in RPM I 8226 > U266 > MM1.S cells. (B ) These Tipifarnib for 48 hours. Cells were assayed for apoptosis using Annexin V FITC/Propidium Iodide staining as determined by flow cytometry. To include early and late apoptotic events, values were derived as follows 100% [(% viable of treated / % viable of DMSO ctrl /)100]. The same pattern of sensitivity as seen in the MTT assay can observed here. Data is presented as the avg of three independent experiments +/ SE. 3.3.2 Tipifarnib is cytotoxic to myeloma cell lines as exhibited in the SCID hu model of disease To invest igate the effect of Tipifarnib on MM cell lines i n vivo we used a SCID hu model of human myeloma previously described (U Ras hima et al. 1997) SCID hu mice had human fetal bone fragments implanted into their flanks and RPMI 8226 cells injected into the implant site. Urine immunoglobulin levels (M Spike) were used to detect the establishment of disease in the mice. Mice were treated for 2 weeks with

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41 Tipifarnib and sacrificed. The im planted bone tissue was removed and lymphocytes identified by Wright Gim sa staining. light chain levels in the untreated mice nearly doubled from 38.7 to 73.6 mg/L during the four we ek Tipifarnib treatment (Fig 3.3A ). In cont Ras light chain levels in Tipifarnib treated mice were reduced from 28.6 mg/L to undetectable levels. The samples from mice receiving Tipifarnib treatment had significantly fewer myeloma cells compared to the control samples. Femur cross sections from a control animal display a bone with extensive disease (Fig 3.3B ). In cont Ras t, the sections from the Tipifarnib treated mice exhibited myloma cells to be located on the periphery of the sections in close proximity to the bone marrow stroma 3.3.3 pAkt expression levels correlate with Tipifarnib cytotoxicity and drug resistance To determine if there was a correlation between Tipifarnib sensitivity and pAkt levels we examined pAkt expression on Tipifarnib treated cell lines. The pAkt levels in the highly sensitive RPMI 8226 cell line were negligible while a dose dependent decrease in Akt phosphorylation was observed in U266 cells (Fig. 3.4A ). In cont Ras t, the highly resistant MM1.S cells consistently express high levels of pAkt and exhibit no dose dependent decrease in its expression. To examine the relationship between Akt phos phorylation and Tipifarnib sensitivity in an isogenic model I utilized the Tipifarnib resistant cell line, 8226/R5. Western blot analysis showed a dose dependent increase in Caspase3 cleavage in the

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42 Tipifarnib sensitive parental line, but no Caspase 3 activation was observed in 8226/R5 cells (Fig 3.4B). Furthermore, much like MM1.S cells, 8226/R5 cells express significantly high levels of pAkt both at baseline levels and under the treatment of increasing concentrations of Tipifarnib. 3.3.4 IHC reveals e levated levels of nuclear localized pAkt in Tipifarnib resistant cells. Immunohistochemical staining can complement western blotting data when attempting to evaluate the cellular localization of proteins. It has been implied that the signal mediating AKT localization is conformation dependant or is dependent upon the Fig 3.2 Drug sensitive Myeloma cell lines undergo Tipifarnib induced apoptosis as evidenced by Caspase 3 cleavage. (A) Time course experiment: 2 x 106 RPMI 8226 (8226) and MM1.S cell lin es were Tipifarnib for the time points indicated. Vehicle control (vc) samples were cells treated with DMSO for 48 hrs. Cell lysates were then immunoblotted for Procaspase and cleaved Caspase3. The RPMI 8226 line showed evidence of Caspase 3 activation at 24 hours. While no Caspase activity was observed in MM1.S cells at the time points indicated. (b) Tipifarnib dose response assay: 2 x 106 RPMI 8226 (8226), U266 and MM1.S cells were treated with DMSO (vc), 10 Tipifarnib for 48 hours. Cell lysates were then immunoblotted for Procaspase and cleaved Caspase 3. A dose dependent increase in Caspase 3 cleavage can be observed in 8226 and U266 cells however not in the highly Tipifarnib resistant MM1.S cell line.

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43 Figure 3 .3 Tipifarnib is cytotoxic to RPMI 8226 cells in an in vivo murine model of multiple myeloma. Six to eight week old SCID mice were transplanted bilaterally, in the flanks with fetal human bone. Six weeks post implant, human MM cells were injected into the implanted bones. Treatment commenced when the presence of disease was confirmed by ELISA dete light chain protein in the urine. (A ) At 12 weeks post implantation (week 12), before Tipifarnib treatment, both mice populations (control and Tipifarnib light chain protein, 38.7 and 28.6 mg/L respectively in the ir urine. After 2 weeks of treatment with 50 mg/kg/d Tipifarnib light chain levels in control group mice nearly doubled to 73.6 mg/L while those in the Tipifarnib treated group were undetectable. (B ) Following post mortem removal and subseque nt Wright Gimsa staining of the implanted bones, histology exhibits a marked difference between the control and drug treated groups. Cross sections of bones from control mice show cavities nearly full of myeloma cells in the ctrl population. In cont Rast th e Tipifarnib treated group show far less disease integration with most myeloma confined to the periphery of the marrow.

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44 Figure 3.4 Tipifarnib dose response assay: 2 x 106 MM cells were treated with DMSO (vc), 10 nM, 100 Tipifarnib for 48 hours. Cell lysates were then immunoblotted for Akt pAkt (Ser473) or cleaved Caspase3. (A) While endogenous Akt is expressed steadily across all three cell lines, the more resistant cell lines exhibit increased expression of activated Akt pAkt expression in RPMI 8226 cells is not detectable by this method, whereas it is relatively highly expressed in the MM1.S cell line. Furthermore, we observed a dose dependent decrease in pAkt expression in U266 cells treated with Tipifarnib (B ) The RPMI 8226 Tipifarnib resistant cell line (8226/R5) exhibits constitutive activation of Akt and increased endogenous levels of Akt as compared to the parental cells (8226/S). 8226/R5 cells, like MM1.S do not exhibit Caspase 3 activatio n with Tipifarn ib treatment. association with other proteins (Brattain et al. 2006). Because we and others believe Tipifarnib may be utilizing multiple pathways to induce apoptosis, we determined it possible that drug resistance to Tipifarnib may be not only associated with the hyperexpression of AKT but also its cellular localization. In this study, MM1.S and 8226/R5 cells expressing high levels of pAkt also exhibit a majority of the pAkt localized to the nucleus. This is exhibited by IHC utilizing the nuclear stain h ematoxylin (Fig 3.5 A). Activated AKT is still not readily detectable in 8226/S cells using this method. For further verification, nuclear and cytoplasmic extracts were prepared from 8226/S and 8226/R5 cells and probed with anti pAkt and anti Akt antibodi es using anti Poly ADP Ribose Polyme r as e (PARP) antibody as a control for the purity of the extr acts

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45 (Fig 3.5B). Even though 50 g of protein was evaluated in both nuclear and cytoplasmic extracts, the percentages of the target protein may be heavily increased. Thus, when evaluating AKT levels using a concentrated lysate such as obtained from a nuclear extract, pAkt is detectable in 8226/S cells however pAkt and basal levels of AKT remain higher in the resistant cell lin e as made evident by consistent levels of the predominantly nuclear protein PARP between 8226/S and 8226/R5 nuclear extracts. 3.3.5 Ectopic expression of constitutively active AKT promotes cytotoxic resistance to Tipifarnib Because of grave limitations w ith maintaining stable expression of exogenous proteins in 8226/S cells, the IL 3 dependant murine proB cells (Ba/F3) were used to ectopically express constitutively active AKT via the N term addition of a myrostilation sequence which tethers Akt to the plasma membrane. Stable cell lines were created via selection in G418. This tethering increases its association with PI3K and phosphatidylinositol biphosphate rendering AKT constitutively active. Upon activation, AKT will release itself from the membrane an d activate proteins such as MDM2, mTOR and NF kB while inhibiting proteins such as BAD and FKHR. Membrane association exposes two crucial amino acids that are phosphorylated and necessary for activation. Expression was confirmed by immunoblotting with AKT, pAkt and the cloned HA tag. The 30 kD shift noted between in dsRED Myr Akt and is accounted for by the

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46 Figure 3.5 pAkt is nuclear localized in cells resistant to Tipifarnib To perform IHC: 0.5 x 106 8226/S, 8226/R5 and MM1.S cells were cytospun fixed in 4 % paraformaldehyde and methanol methanol then incubated in saline plus pAkt (Ser473) IHC Specific antibody. Slides were then washed in TBS, incubated with DAB reagent and stained with or without Hema toxylin. Cells were visualized by light microscopy and captures are representative of three independent experiments (A). Nuclear and cytoplasmic extracts were prepared from 8226/S and 8226/R5 cells. 50g of cytoplasmic or nuclear protein was run on a 12% a crylamide gel and proteins were probed with antibodies for basal and activated AKT Additionally uncleaved PARP expression was evaluated to asses the purity of the extracts. fusion of the ~30 kD dsRED fluorescent protein ( Fig 3.6 A ). Importantly however Ba/F3 cells exhibit a very similar sensitivity to Tipifarnib as RPMI 8226 cells ( Fig 3.6 C ) where 8226 (IC50=114.348 nM) are only ~2.5fold resistant to Tipifarnib compared with Ba/F3 cells (IC50=40.562 nM). Induction of Myr Akt rendered Ba/F3 cells IL 3 independent as exhibited by Ba/F3(Myr Akt ) cells proliferating in the absence of IL 3. This transformed phenotype

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47 has been seen with other well characterized tumor associated proteins including BCR ABL, JAK1, JAK2 and STAT5 (Huang et al. 2005, Onishi et al. 1998, Daley et al. 1988) Addition of Ba/F3(Myr Akt ) conditioned medium to Ba/F3 cells did not support proliferation of the nontransformed cells. This suggests that Ba/F3(Myr Akt ) cells did not develop an autocrine growth factor loop capable of sustaining the cells in culture in the absence of IL 3 (d ata not shown). Expression of pAkt in Ba/F3 cells rendered them to be ~57 fold resistant to Tipifarnib as compared to cells transfected with empty vector ( Fig 3.6 C ). Importantly, this fold resistance is similar to the phenotype held by 8226/S and 8226/R5 (~48 fold resistance) Because of the presence of the Discosoma sp. red fluorescent protein in this construct, the localization of AKT could be evaluated and was found to be predominately nuclear in when stably expressed in this murine proB cell (Fig 3.6B). 3.4 Discussion Understanding Tipifarnibs mechanism(s) of action is of significant importance. The drug has been shown to effectively induce apoptosis in both myeloma cell lines and primary isolates (Beaupre et al. 2003, Ochiai et al. 2003). Alsina et al. showed that 64% of patients experienced disease stabilization in a phase II clinical trial of patients with advanced multiple myeloma (Alsina et al. 2004). Tipifarnib did not receive Federal Drug Administration approval for the treatment of acute myeloid leukemia in 2005. Most

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48 notably however the drug has exhibited extremely low toxicity in patients (Alsina et al. 2004) while we and others have postulated that Tipifarnib like other RAS inhibitors can be highly synergistic with a broad range of compounds including paclita xel, tamoxifen Fig 3.6 Ectopic expression of constitutively active AKT promotes cytotoxic resistance to Tipifarnib Ba/F3 cells stably expressing pDsRed2 and pDsRed2HA Myr Akt 1 were lysed and probed anti HA, anti pAkt and antiAkt antibodies to verify expression. pAkt is only detectable in those cells transfected with the myrostilated engineered version of AKT (A ). dsRED fluorescence was evaluated using DAPI staining to visualize the nucleus. Ba/F3 dsRED2 Myr Akt cells exhibited dsRED fluorescence in th e nucleus of cells (B). The MTT cytotoxity assay was utilized to determine the IC50 of Tipifarnib against Ba/F3 cells overexpressing active AKT Ba/F3 cells exhibit a very similar sensitivity to Tipifarnib as RPMI 8226 cells (IC50=114.348 nM) where 8226 (IC50=114.348 nM) are only ~2.5 fold resistant to Tipifarnib compared with Ba/F3 cells (IC50=40.562 nM). pAkt in Ba/F3 cells rendered them to be ~57 fold resistant to Tipifarnib as compared to cells transfected with empty vector (C). Data shown is representative of three independent experiments (A), 6 images taken on three separate days (B) or the average of 3 independent experiments +/ Standard Error (SE).

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49 and Bortezomib among others (Lebowitz et al. 2005, Yanamandra et al. 2006, Zhu et al.) This suggests a promising future for FTIs in combination therapy. Although FTIs were designed to obstruct Ras prenylation, mounting evidence indicated that FTI induced cytotoxicity involves other pathways. FTI SCH66336, FTI 277, and Perillic acid have al l been suggested to function through non Ras pathways Akt 2, and RhoA and Rac pathways ( Takada et al. 2004, Jiang et al. 2000, Beaupre et al. 2003) Studies with Tipifarnib have likewise indicated that Ras independent mechanisms may be involved. It has been r eported that Tipifarnib cytotoxicity in neoplastically transformed mouse cells is RhoB dependent (DuHadaway et al. 2003). In human myeloma cell lines Tipifarnib has been suggested to induce apoptosis via other Ras independent mechanisms, including the JAK/STAT pathway and/or through Bax activation of an ER stress response (Beaupre et al. 2004, Le Gouill et al. 2002) Here we report a correlation between pAkt levels and sensitivity to Tipifarnib where the cell lines expressing low or minimal levels of pAkt show to be relatively more sensitive than those cells with high levels of pAkt These results are consistent with observations made b y one group i n a phase II clinical trial (Alsina et al. 2004). A variance in p Akt express ion was observed between all three myeloma lines tested. Furthermore, the variation in pAkt expression was even more pronounced between standard RPMI 8226/S cells and the Tipifarnib resistant R PMI 8226/R5 cell line. Additionally, it has been reported that overexpressed constitutively active myr Akt can

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50 overcome the proapoptotic effects of FTI (Du et al. 1999). These findings indicate Akt may be an excellent target for combination drug therapy.

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51 Chapter 4 Tipifarnib and Bortezomib Are Synergistic and Overcome Cell Adhesion Mediated Drug Resistance in Multiple Myeloma and Acute Myeloid Leukemia Chapter 4 represents the following publication: Yanamandra et al. 2006 4.1 Introduction It has been established in preclinical models of multiple myeloma and acute myeloid leukemia (AML) that the bone marrow microenvironment provides protection from chemotherapy and death receptor mediated apoptosis. This form of resistance, termed de novo drug resistance, occurs independent of chronic exposure to cancer related therapies and likely promotes the development of multidrug resistance. Consequently, it is of major interest to identify compounds or drug combinations that can overcome environment mediated resistance. In this study, we investigate d the activity of Tipifarnib (Zarnestra, formerly R115777) combined with Bortezomib (Velcade, formerly PS 341) in microenvironment models of multiple myeloma and AML. The combination proved to be synergistic in multiple myeloma and AML cell lines treated i n suspension culture. Even in tumor cells relatively resistant to Tipifarnib combined activity was maintained. Tipifarnib and Bortezomib were also effective when multiple myeloma and AML cells

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52 were adhered to fibronectin, providing evidence that the combination overcomes cell adhesionmediated drug resistance (CAM DR). Of importance, activation of the endoplasmic reticulum stress response was enhanced and correlated with apoptosis and reversal of CAM DR. Multiple myeloma and AML cells cocultured with bone marrow stromal cells also remained sensitive, although stromaladhered tumor cells were partially protected (relative to cells in suspension or fibronectin adhered). Evaluation of the combination using a transwell apparatus revealed that stromal cells produce a protective soluble factor. Investigations are under way to identify the cytokines and/or growth factors involved. In summary, our study provides the preclinical rationale for trials testing the Tipifarnib and Bortezomib combination in patients w ith multiple myeloma and AML. Multiple myeloma and acute myeloid leukemia (AML) are cancers with high mortality rates, where novel strategies are required to improve on current treatment standards. It has been well established in multiple myeloma and othe r malignancies that the interaction between tumor cells and elements of their microenvironment results in resistance to chemotherapy and death receptor mediated apoptosis (Hazlehurst et al. 2003) This form of resistance, termed de novo drug resistance, o ccurs independent of chronic exposure to chemotherapy and likely promotes the development of multidrug resistance (Shain et al. 2001) Two basic components comprise environment mediated drug resistance: physical contact between tumor cells and microenvironment components (cell adhesion mediated drug resistance, CAM DR) and the local production of soluble factors. More specifically, in multiple myeloma and AML, it has been found that the adhesion of tumor cells (via

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53 integrin receptors) to fibronectin results in a drug resistant phenotype (Hazlehurst et al. 2003, Matsunaga et al. 2003) Of importance, in a small series of AML patients, it was noted that those whose leukemic cells expressed VLA 4 (41 integrin) had a high rate of relapse compared with those with low VLA 4 expression (Matsunaga et al. 2003) These results imply that the physical interaction between tumor cells and bone marrow constituents provides a refuge for minimal residual disease. Tumor microenvironment contact also results in the pr oduction of soluble factors that can further accentuate the drug resistant phenotype (Nefedova et al. 2003) In multiple myeloma and AML, cytokines, such as interleukin 6 (IL 6), IL 1, and vascular endothelial growth factor, have been implicated as import ant growth and survival factors, and these molecules may also contribute to environment mediated resistance. Based on the knowledge that the mechanisms of de novo drug resistance are unique and genetically distinct from those associated with acquired resis tance (Hazlehurst et al. 2003) it is of major interest to identify compounds or drug combinations that are specifically active on tumor cells protected by the microenvironment compartment. The proteasome inhibitor Bortezomib (Velcade, formerly PS 341) has been found to have clinical activity in patients with relapsed multiple myeloma (Kane et al. 2003). Bortezomib is a reversible inhibitor of the 26S proteasome, a complex that plays a major role in protein degradation. Inhibition of this complex ultimately leads to inactivation of the transcription factor nuclear factor B, a survival protein that is thought to be one of the drugs main targets. A previous study has reported that Bortezomib can reverse the CAM DR phenotype in a multiple myeloma cell line (Mitsiades et al. Blood 2003)

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54 Interestingly, we made similar observations testing the compound Tipifarnib (Zarnestra, formerly R115777) in multiple myeloma and AML cells Tipifarnib is a farnesyl transfe Ras e inhibitor (FTI) that inhibits the membrane loc alization of Ras resulting in a loss of function (End et al. 2001) Tipifarnib has been clinically tested in patients with multiple myeloma and AML and was found to be active in both diseases (Alsina et al. 2004, Karp et al. 2001) In this study, we invest igate the combination of Tipifarnib and Bortezomib in microenvironment models of multiple myeloma and AML. Our data provide preclinical evidence of activity for this novel drug combination. 4.2 Materials and Methods 4.2.1 Cell Lines RPMI 8226/S, H929, U266, KG 1, and U937 lines were obtained from the American Type Culture Collection (Manassas, VA). MM1s cells were kindly provided by Steven Rosen (Northwestern University, Chicago, IL). 8226/S, U266, U937, and MM1s cells were maintained in RPMI 1640 supplemented with 100 mmol/L l glutamine (Mediatech, Inc., Herndon, VA) and 10% fetal bovine serum (FBS; Omega Scientific, Inc., Tarzana, CA). H929 cells were maintained in RPMI 1640 supplemented with 10% FBS and 0.05 mol/L 2mercaptoethanol (Sigma Chemical, St. Louis, MO). The KG 1 line was maintained in Iscove's modification of DMEM (Mediatech) with 4 mmol/L L glutamine, 25 mmol/L HEPES, and 20% FBS. HS 5 bone marrow stromal cells were

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55 obtained from the American Type Culture Collection and maintained in RPMI 1640 supplemented with 100 mmol/L L glutamine, 10% FBS, and 1% penicillin/streptomycin. HS 5 green fluorescent protein (GFP) stromal cells were developed by stably expressing enhanced green fluorescent protein under hygromycin (Invitrogen, Carlsbad, CA) selection (50 g/mL). 4.2.2.Compounds Tipifarnib was kindly provided by David End (Johnson & Johnson Pharmaceutical Research and Development, LLC, Titusville, NJ). Tipifarnib was dissolved in 100% DMSO (Sigma Chemical) and sonicated for 10 minutes a t room temperature. Bortezomib (Millennium Pharmaceuticals, Cambridge, MA) was also dissolved in 100% DMSO, and both compounds were stored at 20C before use. 4.2.3 Patient samples. Multiple myeloma and AML patient samples were collected under two Ins titutional Review Board approved protocols (MCC# 13715 and MCC# 13947/13355). After obtaining informed consent for bone marrow aspiration, mononuclear cells were isolated by FicollHypaque gradient purification as per the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ). Primary isolates were exposed to Tipifarnib and Bortezomib and analyzed as described below.

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56 To establish patient bone marrow stromal cells, multiple myeloma patient specimens with <20% myeloma cells were cultured co ntinuously in MEM medium (Invitrogen) supplemented with 15% FBS and 1% penicillin with streptomycin until an adherent layer of stromal cells predominated. AML patient specimens were processed by CD33+ selection using CD33 microbeads and the AutoMacs magn etic cell sorter (Miltenyi Biotec, Inc., Auburn, CA). CD33 populations were cultured continuously as above. For coculture experiments, stromal cells were seeded to near confluence and incubated overnight at 37C. Cell lines were adhered, exposed to Tipifa rnib and Bortezomib and analyzed as described below. 4.2.4 Combination index analysis. The dose effect relationship between Tipifarnib and Bortezomib was analyzed using CalcuSyn software (Biosoft, Ferguson, MO). The combination index equation is based on the following multiple drug effect equation of ChouTalalay (Chou et al. 1984): combination index = ( D )1 / ( Dx)1 + ( D )2 / ( Dx)2 + ( D )1( D )2 / ( Dx)1 / ( Dx)2. Combination index = 1, >1, or <1 is considered additive, antagonistic, or synergistic, respectively. Drug combination studies were based on the fraction of cells affected relative to untreated controls. The mean and SD of the combination index were calculated using the Monte Carlo algorithm. To evaluate the relative contribution of each agent, 8226 and U937 cells were seeded at 1 x 103 per well and exposed to five concentrations of Tipifarnib Bortezomib and the combination (constant molar ratio, 100:1). After 72 hours at 37C,

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57 cytotoxicity was measured by 3 (4,5dimethylthiazol 2yl) 2,5di phenyltetrazolium bromide assay as previously described (Damiano et al. 1999). 4.2.5 Fibronectin adhesion and cell death analysis. Adhesion of primary isolates and tumor cell lines to fibronectin was done as previously described (Damiano et al. 1999) For cell lines, adhered tumor cells were incubated overnight at 37C, and then control supplemented media or Bortezomib Tipifarnib was added for an additional 24 hours. In primary isolates, control supplemented media or Bortezomib Tipifarnib was added for 24 hours after 2 hours of adhesion. In parallel, primary isolates or tumor cell lines were cultured in 0.1% bovine serum albumin coated plates (Boehringer Mannheim, Indianapolis, IN; multiple myeloma) or 0.1% poly hema (Sigma, St. Louis, MO) coated plates (AML) and exposed to control media or Bortezomib Tipifarnib as above. Cell death was determined by flow cytometry after Annexin V/FITC (Biovision, Mountain View, CA) and propidium iodide (Biovision) or 7amino actinomycin D (BD PharMingen, San Jose, CA) staining as described previously (Beaupre et al. 2003) In primary isolates, samples were also stained with anti CD138 (BD PharMingen, San Jose, CA; myeloma cells) or anti CD33 (BD PharMingen; leukemic cells) antibodies to identify tumor cell populatio ns.

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58 4.2.6 Adhesion assays. Adhesion assays were done similar to previously described (Damiano et al. 1999) using the cell tracker 5 chloromethylfluorescein diacetate (Molecular Probes, Eugene, OR). For pre adhesion drug treatment, tumor cells were incubated with Tipifarnib (5 mol/L), Bortezomib (5 nmol/L), or the combination for 2 hours before adhesion to fibronectin. After 2 hours of adhesion, wells were washed and fluorescence was measured at 490 nm on a Wallac Victor 2 1420 Multilabel Counter. For post adhesion drug treatment, tumor cells were stained with 5 chloromethylfluorescein diacetate (as above) and then adhered to fibronectin for 2 hours followed by exposure to Tipifarnib (5 mol/L), Bortezomib (5 nmol/L), or t he combination for an additional 2 hours. Wells were then washed, and absorbance was read as described. 4.2.7 Adhesion to bone marrow stroma and cell death analysis In coculture experiments, HS 5 GFP stromal cells were seeded to near confluence and incubated overnight at 37C. The next morning, stromal cells were washed once with serum free medium, and primary isolates or tumor cell lines were allowed to adhere for 2 hours in either serum free MEM (Alpha MEM, Invitrogen) or RPMI 1640, respectively. N onadhered cells were removed, and samples were then exposed to control supplemented media or Bortezomib Tipifarnib for 24 to 36 hours (see figure legends). Percent cell death was determined by flow cytometry as described above. For coculture experiments

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59 involving cell lines, gating on GFP populations identified tumor cells. In primary isolates, additional staining with antiCD138 (myeloma cells) or anti CD33 (leukemic cells) antibodies distinguished tumor cells from background mononuclear cells. For our experiments, we also determined the number of live cells per sample using flow cytometry as previously described (Beaupre et al. 2003) 4.2.8 Transwell analysis 8226 myeloma and U937 leukemia cells were either adhered (as described above) or separated f rom HS 5 GFP stromal cells by a Transwell insert (costar, 0.4 m mesh, 12mm diameter; Corning, Corning, NY). Cells were treated with either control supplemented media or the Tipifarnib (5 mol/L) and Bortezomib (5 nmol/L) combination for 36 hours. Cell death was determined by flow cytometry after staining with Annexin V/FITC and 7 amino actinomycin D. Cell death of multiple myeloma and AML cells was determined in coculture by gating on GFP populations. 4.2.9 Western blotting Western blotting was done as described previously (Beaupre et al. 2003) Antibodies were purchased from the following vendors: GADD153 and anti K Ras 2B (Santa Cruz Biotechnology, Santa Cruz, CA), actin (Sigma Chemical), antiHDJ 2 (Ne oMarkers, Freemont, CA). Densitometry was done by scanning radiographic images,

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60 and bands were quantitated using Alpha Ease image analysis software (Alpha Innotech Corp., San Leandro, CA) 4.2.10 Proteasome assay 8226/S myeloma cells (4 x 106) were expo sed to control media, Tipifarnib Bortezomib or the combination for 2 hours at 37C. Cells were harvested, washed twice with ice cold PBS, and then lysed in 50 L of TE buffer [10 mmol/L Tris, 1 mmol/L EDTA (pH 7.9)]. Total protein was quantitated using a Bio Rad protein assay kit (Hercules, CA), and 10 g of protein were analyzed for proteasome activity using a 20S proteasome activity assay as per the manufacturer's instructions (Chemicon International, Temecula, CA) 4.3 Results 4.3.1 Tipifarnib and Bo rtezomib are synergistic in multiple myeloma and AML cell lines. It has previously been reported that FTIs and proteasome inhibitors induce apoptosis in multiple myeloma (Mitsiades et al. 2003, Beaupre et al. 2003, Le Gouill et al. 2002, Bolick et al. 2003, Hideshima et al. 2001) and leukemic (Beaupre et al. 1999, Dai et al. 2003) (cell lines. By analyzing the activity of Tipifarnib and Bortezomib as single agents in several representative lines, we defined concentrations with low toxicity

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61 for combination studies (data not shown). We first tested Tipifarnib and Bortezomib on a diverse group of myeloma lines maintained in suspension culture (Fig 4.1 A C) Evidence for g reater than additive cell death was observed and was most pronounced in H929 and MM1s cells (Fig 4.1 A and C) Interestingly, the MM1s line was relatively resistant to single agent Tipifarnib yet the combination maintained its activity. We also tested KG1 leukemia cells and found that they were sensitive although to a lesser degree when compared with our myeloma lines (Fig 4.1D) To confirm that Tipifarnib and Bortezomib were synergistic in vitro 8226/S myeloma cells and U937 leukemia cells were treated with single agent Tipifarnib Bortezomib and the combination in cell suspension. Combination index analysis revealed synergistic activity between the two compounds at several dose combinations tested (Fig 4.2 A and B). These results indicate that Tipifar nib combined with Bortezomib is an active regimen in diverse multiple myeloma and AML cell lines 4.3.2 Tipifarnib combined with Bortezomib overcomes CAM DR. It has previously been established that adherence of multiple myeloma and AML cells to the extracellular matrix component fibronectin results in resistance to chemotherapeutic agents, including melphalan, doxorubicin, and cytosine arabinoside (Matsunaga et al. 2003). To determine whether Tipifarnib and Bortezomib overcome CAM DR, 8226/S myeloma (Fig 4.3A and B) and U937 leukemia cells (Fig 4.3 C and D) were adhered

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62 Fig. 4.1 Tipifarnib combined with Bortezomib induces cell death in diverse multiple myeloma and AML cell lines. H929 ( A ), U266 ( B ), MM1s ( C ), or KG 1 ( D ) cell lines were treated with the indicated concentrations of Tipifarnib Bortezomib or the combination for 24 hours in cell suspension. Cell death was determined by flow cytometry after Annexin V/FITC and propidium iodide staining. Specific cell death w as calculated relative to untreated controls. Three independent experiments. To identify evidence for greater than additive effect, a simple linear regression (no intercept model) was used that included drug effect for each individual drug and the interact ion effect of the combination. SAS software was used in the calculations using Proc REG ( n = 9 observations for each cell line). P s are provided. Columns, mean; bars, SE.

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63 Fig. 4.2 Tipifarnib and Bortezomib are synergistic in cytotoxicity assays. 8226/S ( A ) and U937 ( B ) cells were treated with Tipifarnib Bortezomib or a constant molar ratio (100:1) of the combination for 72 hours. Cytotoxicity was determined using 3 (4,5 dimethylthiazol 2 yl) 2,5 diphenylt etrazolium bromide assays (as described in Materials and Methods). The resulting data was used to generate combination index plots ( CI ) for various dose combinations. The dashed line indicates additive affect (CI = 1). Antagonism ( above dashed line ) and sy nergism ( below dashed line ). Tables are included to provide the drug concentrations tested. Points, average of quadruplicate values of three independent experiments; bars, SD. to fibronectin and evaluated for sensitivity to single agent Tipifarnib and Bortezomib In both lines and with both compounds, a dose dependent increase in cell death was observed in suspension and fibronectinadhered tumor cells. Importantly, fibronectin adherence did not protect tumor cells from Tipifarnib or Bortezomib induced apoptosis. Furthermore, the Tipifarnib and Bortezomib combination consistently induced cell death more efficiently in adhered 8226/S and U937 cells when compared with cells treated in

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64 suspension (Fig 4.3 E) Evaluation of three multiple myeloma and five A ML patient samples also revealed that fibronectin adhered primary tumor isolates were not protected (Fig 4.3E). These results indicate that Tipifarnib combined with Bortezomib overcomes the CAM DR phenotype. 4.3.3 Reversal of CAM DR is not related to dec reased tumor adherence. Because it has been reported that Bortezomib can alter the expression of adhesion molecules (Read et al. 1995) we addressed whether the activity of Tipifarnib and Bortezomib was related to decreased adherence of tumor cells to fibronectin. 8226/S myeloma cells (Fig 4.4 A) and U937 leukemia cells (Fig 4.4 C) were exposed to Tipifarnib Bortezomib, or the combination for 2 hours before adhesion to fibronectin. Tumor cell attachment was not prevented in either line, and pre adhesio n drug exposure for up to 8 hours revealed no significant decrease in attachment relative to controls (data not shown). To verify that Tipifarnib and Bortezomib did not reverse cell adhesion in attached tumor cells, 8226/S (Fig 4.4 B) and U937 (Fig 4.4 D) cells were exposed to Tipifarnib Bortezomib, or the combination for 2 hours after being adhered to fibronectin. Once again, we detected no reduction in tumor cell adhesion that could explain sensitivity to the Tipifarnib and Bortezomib combination.

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65 4.3.4 Activation of endoplasmic reticulum stress correlates with reversal of CAM DR. It has been reported that the antitumor activity of isoprenoid inhibitors (such as FTIs and geranylgeranyl transfeRas e inhibitors) may in part be related to inhibition of the proteasome proteolytic pathway. We therefore evaluated proteasome proteolysis in tumor cells after Tipifarnib and Bortezomib treatment. We found that Tipifarnib had little to no effect on endogenous proteasome activity, and the combination was no more active than Bortezomib alone (Fig 4.5 A) Similarly, inhibition of FTase (using HDJ 2 prenylation as a surrogate marker) was also not enhanced (Fig 4.5 B) Inhibition of K Ras farnesylation was not observed after treatment with Tipifarnib and/or Bortezomib, consistent with our prior report of a Ras independent mechanism of cell death (Beaupre et al. 2004) These results indicate that enhanced activity at the purported targets of Tipifarnib and Bortezomib is not responsible for drug synergy; rather, events downstream of the 26S proteosome and FTase likely cooperate to activate intrinsic proapoptotic cascades. It has been shown that proteasome inhibitors induce apoptosis in myeloma cells via triggering of endoplasmic reticulum (ER) stress with subsequent disruption of the unfolded protein response (Lee et al. 2003) It has also been observed that proteasome inhibitors combined with other compounds that activate ER stress (such as tunicamycin) induce cell death in synergy (Lee et al. 2003) Of relevance, we found that Tipifarnib activates the ER stress response in multiple myeloma cell lines (Beaupre et al. 2004) Based on these observations, we surmised that apoptosis induced by Tipifarnib and Bortezomib may be related to activation of the ER stress respons e. Therefore, 8226/S

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66 myeloma cells were exposed to Tipifarnib Bortezomib or the combination for 24 hours either in suspension culture or after being adhered to fibronectin (Fig 4.5 C) Activation of ER stress was determined by monitoring the expression of GADD153, a well established ER stress marker (Wang et al. 1996). We found that both Tipifarnib and Bortezomib increased GADD153 expression under suspension and adhered conditions consistent with their ability to activate ER stress related cascades. Importantly, the combination enhanced the ER stress response particularly under fibronectinadhered conditions. These results imply a link between reversal of CAM DR and activation of the ER stress response. 4.3.5 Stroma adhered tumor cells are sensitive to Tipifarnib and Bortezomib. Due to the fact that soluble factors may also participate in environment mediated drug resistance, we tested Tipifarnib and Bortezomib in coculture models of the bone marrow microenvironment. 8226/S myeloma cells were adher ed to HS 5 bone marrow stromal cells and then exposed to increasing concentrations of Bortezomib (Fig 4.6 A) or Tipifarnib (Fig 4.6 B) for 24 hours. The percentage of live cells decreased in a dosedependent manner after treatment with both compounds, and importantly, stroma adhered 8226/S cells were nearly as sensitive as suspension cells. Also of significance was the fact that high concentrations of Bortezomib and Tipifarnib were not cytotoxic to cocultured HS 5 stromal cells (Fig 4.6 A and B). Combined a ctivity was maintained in both stroma

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67 Fig. 4.3. Tipifarnib and Bortezomib induce cell death in fibronectin adhered multiple myeloma ( MM) and AML cells. 8226/S myeloma cells ( A and B ) and U937 leukemic cells ( C and D ) were treated with the indicated concentrations of Tipifarnib or Bortezomib for 24 hours either in cell suspension or after adhesion to fibronectin. Cell death was determined by flow cytometry after Annexin V/FITC and propidium iodide staining. Combined f rom three independent experiments ( A D ). E, 8226/S and U937 cell lines along with mononuclear cells from three multiple myeloma and five AML patients were exposed to 5 mol/L Tipifarnib and 5 nmol/L Bortezomib for 24 hours either in cell suspension or afte r adhesion to fibronectin. Cell death was determined as in ( A D ). In primary isolates, tumor cells were identified by staining with anti CD138 (myeloma cells) or anti CD33 (leukemic cells) antibodies. For cell lines, five (8226/S) and seven (U937) independent experiments were compared using a paired t test with P s. Columns/points, mean; bars, SE.

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68 Fig. 4.4 Adhesion to fibronectin is not disrupted by Tipifarnib and Bortezomib 8226/S myeloma cells ( A and B ) and U937 leukemic cells ( C and D ) were stained for 30 minutes with the cell tracker 5 chloromethylfluorescein diacetate (see Materials and Methods). A and C, cells were washed and then treated with the indicated concentrations of Tipifarnib Bortezomib or the combination for 2 hours (pr e adhesion exposure). After drug treatment, cells were adhered to fibronectin for an additional 2 hours and washed, and fluorescence was analyzed on a fluorescence plate reader. B and D, cells were adhered to fibronectin for 2 hours and then exposed to the indicated concentrations of Tipifarnib Bortezomib or the combination for an additional 2 hours (post adhesion exposure). Samples were washed, and fluorescence was measured as in ( C and D ). Combined from three independent experiments. Percent adhesion to fibronectin is relative to cells treated with control media only. Columns, mean; bars, SE.

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69 Fig. 4.5 Reversal of CAM DR correlates with activation of the ER stress response. A, 8226 myeloma cells were treated with the indicated concentrations of Tipifarnib and Bortezomib in cell suspension for 2 hours. Proteasome activity was measured as described in Materials and Methods. Percent proteasome ( PS ) activity is relative to cells treated with control media. B, 8226/S myeloma cells were treated with co ntrol media ( lane 1), 5 mol/L Tipifarnib ( lane 2), 5 nmol/L Bortezomib ( lane 3), or the combination ( lane 4) for 24 hours in cell suspension. Cell lysates were harvested and analyzed by Western blotting using the indicated antibodies. u, unprocessed HDJ 2 ; p, processed HDJ 2 or K Ras C, 8226 myeloma cells were treated for 12 hours in cell suspension or after adherence to fibronectin as follows: suspension, control media ( lane 1), 5 mol/L Tipifarnib ( lane 2), 5 nmol/L Bortezomib ( lane 3), 10 nmol/L Bortez omib ( lane 4), 5 mol/L Tipifarnib and 5 nmol/L Bortezomib ( lane 5), 5 mol/L Tipifarnib and 10 nmol/L Bortezomib ( lane 6); fibronectin, control media ( lane 7), 5 mol/L Tipifarnib ( lane 8 ), 5 nmol/L Bortezomib ( lane 9), 10 nmol/L Bortezomib ( lane 10), 5 mol/L Tipifarnib and 5 nmol/L Bortezomib ( lane 11), 5 mol/L Tipifarnib and 10 nmol/L Bortezomib ( lane 12). As a control for ER stress, U266 cells were either treated with control media ( lane 13) or 25 mol/L tunicamycin ( lane 14) for 12 hours. Cell lysates were harvested and analyzed by Western blotting using the indicated antibodies. Densitometric evaluation of bands. A, combined from three independent experiments. B and C, representative of two independent experiments. Columns, mean; bars, SE.

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70 adhered and suspension 8226/S cells after treatment with the Tipifarnib and Bortezomib combination (Fig 4.6. C). Once again, however, stroma adhered tumor cells were partially protected. A similar trend was observed in U937 leukemia cells (Fig 4.6 D). To determine whether HS 5 stromal cells could also protect primary tumor isolates, primary multiple myeloma and AML cells were adhered to HS 5 stroma and treated with the Tipifarnib and Bortezomib combination (Fig 4.7 A and B) Similar to our observations in tumor cell lines, stroma adhered primary isolates were partially protected relative to suspension cells, whereas fibronectin adhered tumor cells seemed more sensitive. Stromal cells derived from a multiple myeloma and two AML patients were also able to prevent apoptosis of adhered 8226/S and U937 cells, respectively (Fig 4.7 C E) 4.3.6 HS 5 bone marrow stromal cells secrete a protective soluble factor. Partial resistance provided by bone marrow stromal cells could be explained by eithe r CAM DR or by the participation of protective soluble factors. To address these two possibilities, 8226/S myeloma cells (Fig 4.8 A) and U937 leukemia cells (Fig 4.8 B) were treated with the combination of Tipifarnib and Bortezomib either in suspension cul ture (suspension), after adhesion to HS 5 stromal cells (coculture), or with separation between the two populations using a transwell insert (transwell). As described previously, stroma adhered tumor cells were less sensitive to the drug combination when compared with cells treated in suspension (36 hour incubation). Interestingly, tumor cells separated from

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71 stromal cells using a transwell insert remained protected. These results imply that HS 5 cells produce a soluble factor(s) that can attenuate the toxi city of Tipifarnib and Bortezomib To determine whether tumor stroma contact enhanced the production of this factor(s), tumor cells were adhered to HS 5 stromal cells and additional tumor cells were separated from coculture by a transwell insert (coculture /transwell). The nonadhered tumor cells were protected similarly to tumor cells separated by the transwell insert (no coculture), suggesting that HS 5 cells constitutively secrete a protective soluble factor(s), and its production is not enhanced by tumor stroma contact. 4.4 Discussion The emergence of drug resistance continues to be a major obstacle to the successful treatment of patients with multiple myeloma and AML. Acquired drug resistance is contingent upon the survival of tumor cells during their initial exposure to cancer chemotherapy ( de novo drug resistance). The interaction between tumor cells and components of their microenvironment remains critical during this early phase of treatment. Environment mediated resistance is a result of both physi cal contact between tumor cells and environmental components as well as the exposure of tumor cells to soluble factors. Both of these interactions participate in prosurvival processes that allow tumor cells to resist chemotherapy and acquire multidrug resistance (Hazlehurst et al. 2003, Nefedova et al. 2003).

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72 Fig. 4.6 Bone marrow stroma partially protects multiple myeloma and AML cell lines from Tipifarnib and Bortezomib induced cell death. 8226/S myeloma cells were treated with the indicated concentrations of Bortezomib ( A ), Tipifarnib ( B ), or the combination ( C ) for 24 hours. Cells were treated either in suspension culture (suspension) or after adhesion to HS 5 stromal cells (adhered). The HS 5 stroma line (stroma) expres ses GFP in a stable fashion. Myeloma cells were distinguished from stromal cells in coculture by using flow cytometry with gating on GFP+ and GFP populations. D, U937 leukemia cells were treated and analyzed as in ( C ). Cell death was determined by staining with Annexin V/FITC and 7 amino actinomycin D as described in Materials and Methods. Percent live cells are relative to tumor cells treated with control media only. Combined from three independent experiments ( A D ). Columns/points, mean; bars, SE.

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73 Fig. 4.7 Stromal cells partially protect primary isolates from the Tipifarnib and Bortezomib combination. After obtaining informed consent for bone marrow aspiration, mononuclear cells from a multiple myeloma ( A ) and an AML ( B ) patient were isolated by FicollHypaque gradient purification. Cells were treated with the combination of 5 mol/L Tipifarnib and 5 nmol/L Bortezomib for 36 hours either in suspension or after adhesion to fibronectin or HS 5 bone marrow stromal cells. Cell death was determined by flow cytometry after Annexin V/FITC and 7 amino actinomycinD staining. Tumor cells were identified in coculture by staining with antiCD138 (myeloma cells) or anti CD33 (leukemic cells) antibodies. 8226/S myeloma cells ( C ) and U937 leukemic cells ( D an d E ) were adhered to HS 5 stroma and bone marrow stromal cells derived from a patient with multiple myeloma ( C ) and AML ( D and E ), respectively. Tumor cells were exposed to 5 mol/L Tipifarnib and 5 nmol/L Bortezomib for 36 hours, and cell death was determ ined as described above. Cell death in adhered samples was compared with tumor cell lines treated in suspension culture. Columns, mean; bars, SE.

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74 Fig. 4.8 HS 5 stromal cells secrete a soluble factor(s) that protects multiple myeloma and AML cell lines. 8226/S myeloma ( A ) and U937 leukemia cells ( B ) were treated with 5 mol/L Tipifarnib and 5 nmol/L Bortezomib for 36 hours. Tumor cells were maintained as fol lows: in suspension culture (suspension), adhered to HS 5 GFP stromal cells (coculture), separated from HS 5 GFP stromal cells by a transwell insert (transwell), or adhered to HS 5 GFP stromal cells with additional tumor cells separated by a transwell inse rt (coculture/transwell). In the latter sample, nonadhered tumor cells were harvested for evaluation. Cell death was determined by flow cytometry after staining with Annexin V/FITC and 7 amino actinomycinD. In coculture, death of myeloma and leukemia cell s was determined by gating on GFP cells. Combined from three independent experiments ( A and B ). Individual conditions were compared using a paired t test with P s. Columns, mean; bars, SE.

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75 Novel agents or drug combinations that overcome de novo drug resistance are eagerly sought. Tipifarnib is a FTI that has clinical activity in multiple myeloma and AML (Alsina et al. 2004, Karp et al. 2001). Interestingly, Tipifarnib accumulates in bone marrow (Karp et al. 2001), a desirable property in hematopoietic malignancies that are dependent on the bone marrow microenvironment. We observed that as a single agent Tipifarnib can overcome the CAM DR phenotype in multiple myeloma and AML cell lines (Fig 4.3 A and C) and primary isolates (data not shown). It h ad previously been reported that Bortezomib shares similar activity in fibronectin adhered (Mitsiades et al. 2006) and stroma adhered (Hideshima et al. 2001) MM1s myeloma cells, and consistent with this, we found that Bortezomib efficiently induced cell de ath in stroma adhered 8226/S myeloma cells (Fig 4.6 A) Because Bortezomib has been shown to sensitize fibronectinadhered myeloma cells to chemotherapy mediated apoptosis (Mitsiades et al. 2003) we set out to discern whether low doses of Bortezomib (5 nm ol/L) could enhance the activity of Tipifarnib in microenvironment models of multiple myeloma and AML. Our data reveal that this combination has activity not only in multiple myeloma and AML cells maintained in suspension culture (Fig 4.1) but also in tum or cells adhered to the extracellular matrix component fibronectin (Fig 4.3) indicating that Tipifarnib combined with Bortezomib effectively overcomes CAM DR. It has been previously reported that Bortezomib decreases the adhesion of myeloma cells to bone marrow stromal cells (Hideshima et al. 2001) Conversely, Tipifarnib did not reduce the adhesion of AML blasts to either primary bone marrow stroma or human umbilical endothelial cells (Liesveld et al. 2003) In our models, Tipifarnib Bortezomib nor the combination

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76 decreased tumor cell adhesion to fibronectin implying that cell death was not associated with the loss of tumor microenvironment contact (Fig 4.4) Stroma adhered tumor cells were also sensitive to the combination, although they were partially protected relative to cells maintained in suspension culture (Fig 4.6 and 4.7) The fact that Tipifarnib and Bortezomib were particularly active in fibronectin adhered tumor cells leads us to consider soluble factors as a possible source of stroma mediated resistance. In experiments where tumor cells were physically separated from stromal cells using a transwell insert, protection persisted confirming that a soluble factor(s) could partially suppress the activity of the drug combination (Fig 4.8) Several key cytokines serve as autocrine and paracrine growth factors for multiple myeloma and AML cells. IL 6 is known to be a major growth and survival factor in multiple myeloma (Hallek et al. 1998) IL 6 is secreted by HS 5 bone marrow stromal cells (Roecklein et al. 1995) and protects multiple myeloma cells from dexamethasone mediated apoptosis (Chauhan et al. 2000) Of importance, it has been reported that IL 6 is incapable of protecting myeloma cells from Bortezomibinduced cell death (Hideshima et al. 2001) It therefore remains possible that other cytokines and/or growth factors are involved. HS 5 stromal cells also secrete IL 1 that can contribute to autocrine and paracrine growth loops in multiple myeloma (Kawano et al. 1989, Nagata et al. 1991) and AML (Sakai et al. 1987, Cozzolino et al. 1989) However, we previously reported that an FTI inhibits the expression of IL 1 in leukemic cell lines (Beupre et al. 1999) implying that IL 1 is not the protective cytokine in our models. Vascular endothelial gro wth factor has also been shown to promote the growth of multiple myeloma (Podar et al. 2001) and

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77 AML cells (List et al. 2004) and its mRNA is expressed in HS 5 stromal cells (Roecklein et al. 1995) It remains to be seen whether any of the abovementioned cytokines are relevant to the observed stromal mediated resistance, but experiments are under way to define the cytokine(s) and/or growth factor(s) involved. Our findings will have clinical relevance for antagonists to all three of these factors, as well as other soluble proteins are being clinically tested. With respect to the mechanism of action of this drug combination, as single agents, both Tipifarnib and Bortezomib have been shown to induce ER stress related apoptosis (Beaupre et al. 2004, Lee et al. 2003, Landowski et al. 2005, Fribley et al. 2004) We found that the combination enhanced activation of the ER stress marker GADD153 (Fig 4.5 C) and this correlated with apoptosis and reversal of CAM DR. We have previously observed that Tipifarnib increases the expression and activity of Bax (Beaupre et al. 2004) and Bim in myeloma cell lines. Interestingly, it has been reported that the expression of Bax and Bim can be regulated via a proteasome mediated pathway (Luciano et al. 2003, Li et al. 2000) It therefore remains possible that Tipifarnib and Bortezomib cooperate to enhance the activity of these proapoptotic proteins. Bax and Bim are known to target the ER, leading to ER calcium release (Scorrano et al. 2003) activation of caspase12 (Morishima et al. 2003, and apoptosis in a mitochondria independent fashion (Morishima et al. 2002) Mitochondrial dysfunction also occurs after Tipifarnib treatment (Beaupre et al. 2004) and is likely the result of localization of Bax and Bim to mitochondria. It remains possible, however, that intracytoplasmic calcium participates in mitochondrial membrane depolarization by opening of the mitochondrial

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78 permeability transition pore (Landowshi et al. 2005, Rizzuot et al. 2003) Importantly, Tipifarnib and B ortezomib are active when tumor cells are adhered to fibronectin. Bim expression is known to decrease in adhered myeloma cells (Hazlehurst et al. 2003) and it remains possible that the combination reverses this effect. Current investigations are determini ng the role of Bax and Bim in reversal of the CAM DR phenotype. Delineation of the ER stress related mechanisms responsible for Tipifarnib and Bortezomib combined activity may ultimately lead to treatment strategies that specifically target environment med iated drug resistance. In conclusion, in this study, we provide the preclinical rationale for clinical trials testing Tipifarnib and Bortezomib in patients with multiple myeloma and AML. Future trials may also include a cytokine and/or growth factor neutr alization strategy once protective soluble factors are identified. In theory, such a regimen would eradicate tumor cells protected by the microenvironment compartment, leaving sensitive tumor cell populations for standard or highdose chemotherapy

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79 Chapter 5 Summary and Major Conclusions 5.1 Identification and characterization of the R115777 resistant cell line The farnesyl transfer as e inhibitor R115777 has been found to have clinical activity in diverse hematopoietic tumors. Clinical efficacy, however, does not correlate with Ras mutation status or inhibition of farnesyl transfe r as e. To further elucidate the mechanisms by which R115777 induces apoptosis and to investigate drug resistance, we identified and characterized a R115777resistant human myeloma cell line. 8226/R5 cells were found to be at least 50 times more resistant to R115777 compared with the parent cell line 8226/S. K Ras remained prenylated in both resistant and sensitive cells after R115777 treatment; howeve r, HDJ 2 farnesylation was inhibited in both lines, implying that farnesyl transfeRas e (the drug target) has not been mutated. Whereas many 8226 lines that acquire drug resistance have elevated expression of P glycoprotein, we found that P glycoprotein exp ression is not increased in the 8226/R5 line and intracellular accumulation of R115777 was not reduced. In fact, 8226/R5 cells were insensitive to a diverse group of antitumor agents including PS 341, and multidrug resistance did not correlate with the exp ression of heat shock proteins. Comparison of gene expression

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80 profiles between resistant and sensitive cells revealed expression changes in several genes involved in myeloma survival and drug resistance. Identification of molecules associated with R115777 and PS 341 resistance is clinically relevant because both compounds are being tested in solid tumors and hematopoietic malignancies. 5.2 The role of the Akt survival pathway in R115777 induced apoptosis and resistance It has been well described that R11 5777 induces apoptosis in a RAS independent manner. In this study I have implicated Akt as a possible culprit in the cytotxic resistance to R115777. I have demonstrated that Tipifarnib induces apoptosis in myeloma cell lines when AKT in not endogenously active. As such, we were able to correlate levels of pAkt expression to drug resistance in multiple myeloma in cell lines. In MM primary isolates, AKT has been found to be predominately activated and localized to the nucleus. However, no direct correlati on between Akt localization and drug resistance has yet been characterized until now. Here we have demonstrated a correlation between nuclear localization of pAkt and drug resistance in MM cells. Utalizing a SCID hu model of myeloma we were successful in exhibiting the in vivo cytoxicity of myeloma cells from R115777. 5.3 Combination treatment using R15777 and PS 341 It has been established in preclinical models of multiple myeloma and acute myeloid leukemia (AML) that the bone marrow microenvironment provides protection

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81 from chemotherapy and death receptor mediated apoptosis. This form of resistance, termed de novo drug resistance, occurs independent of chronic exposure to cancer related therapies and likely promotes the development of multidrug resistance. Consequently, it is of major interest to identify compounds or drug combinations that can overcome environment mediated resistance. In this study, we investi gated the activity of Tipifarnib (Zarnestra, formerly R115777) combined with Bortezomib (Velcade, formerly PS 341) in microenvironment models of multiple myeloma and AML. The combination proved to be synergistic in multiple myeloma and AML cell lines treat ed in suspension culture. Even in tumor cells relatively resistant to Tipifarnib combined activity was maintained. Tipifarnib and Bortezomib were also effective when multiple myeloma and AML cells were adhered to fibronectin, providing evidence that the c ombination overcomes cell adhesionmediated drug resistance (CAM DR). Of importance, activation of the endoplasmic reticulum stress response was enhanced and correlated with apoptosis and reversal of CAM DR. Multiple myeloma and AML cells cocultured with b one marrow stromal cells also remained sensitive, although stromaladhered tumor cells were partially protected (relative to cells in suspension or fibronectin adhered). Evaluation of the combination using a transwell apparatus revealed that stromal cells produce a protective soluble factor. Investigations are under way to identify the cytokines and/or growth factors involved. In summary, our study provides the preclinical rationale for trials testing the Tipifarnib and Bortezomib combination in patients wi th multiple myeloma and AML.

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