Targeted therapy of multiple myeloma based upon tumor-microenvironmental interactions

Experimental Hematology 35 (2007) 155–162

Targeted therapy of multiple myeloma based upon tumor-microenvironmental interactions Kenneth C. Anderson The Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, and Harvard Medical School, Boston, MA

Multiple myeloma (MM) remains incurable, but recent advances in genomics and proteomics have allowed for advances in our understanding of disease pathogenesis, identified novel therapeutic targets, allowed for molecular classification, and provided the scientific rationale for combining targeted therapies to increase tumor cell cytotoxicity and abrogate drug resistance. Besides these advances, recognition of the role of the bone marrow (BM) milieu in conferring growth, survival, and drug resistance in MM cells, both in laboratory and animal models, has allowed for the establishment of a new treatment paradigm targeting the tumor cell and its microenvironment to overcome drug resistance and improve patient outcomes in MM. In particular, thalidomide, bortezomib, and lenalidamide all overcome conventional drug resistance, not only by directly inducing tumor cell cytotoxicity, but by inhibiting adhesion of MM cells to BM. This abrogates constitutive and MM-bindingLinduced transcription and secretion of cytokines, inhibits angiogenesis, and augments host anti-MM immunity. These three drugs have rapidly translated from bench to bedside and in treatment protocols of MM, first in patients with relapsed refractory disease, and then alone and in combination in newly diagnosed patients. Promising novel targeted agents include the novel proteasome inhibitor NPI-0052 and the heat shock protein inhibitor KOS-953. Importantly, gene-array, proteomic, and cell-signaling studies have not only helped to identify in vivo mechanisms of action and drug resistance to novel agents, but also aided in the design of promising combination-therapy protocols. Ó 2007 International Society for Experimental Hematology. Published by Elsevier Inc.

Multiple myeloma (MM) is characterized by bone marrow plasmacytosis in association with excess production of monoclonal protein. It occurs in 15,000 new patients each year and remains a uniformly fatal disease, with a median survival of 4 years, despite conventional and high-dose therapies. Recent advances in genomics and proteomics in MM have allowed for advances in our understanding of disease pathogenesis, identified novel therapeutic targets, allowed for molecular classification, and provided the scientific rationale for combining targeted therapies to increase tumor cell cytotoxicity and abrogate drug resistance. Specifically, gene-microarray profiling has shown major differences between normal plasma cells vs those from monoclonal gammopathy of unknown significance and MM, with further modulations within MM and progression

Offprint requests to: Kenneth C. Anderson, M.D., Department of Medical Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115; E-mail: [email protected]

to plasma-cell leukemia [1]. These studies identify genetic changes associated with progression of monoclonal gammopathy of unknown significance to MM, with great promise for prognostic or therapeutic application. In a recent study, correlation of expression profiling for cyclin D, five recurrent immunoglobulin-H translocations, and specific trisomies has formed the basis for a new classification system in MM, with important prognostic and therapeutic implications [2]. For example, clinical trials are ongoing with a specific tyrosine kinase inhibitor that targets fibroblast growth factor receptor 3 on the surface of tumor cells in those 15% to 20% patients with t(4:14) translocations. Moreover, known genes likely represent only the tip of the iceberg, and ongoing efforts are integrating comparative genomic hybridization, spectral karyotyping, and expression profiling data, followed by functional validation in cancer and MM models, to identify new therapeutic targets [3]. Moreover, distinct genotypic MM subtypes with differing prognoses can be defined. Ultimately, it might be necessary to profile individual patients and to use combinations of targeted therapies to overcome drug resistance [4].

0301-472X/07 $–see front matter. Copyright Ó 2007 International Society for Experimental Hematology. Published by Elsevier Inc. doi: 10.1016/j.exphem.2007.01.024

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Recognition of the role of the bone marrow (BM) milieu in conferring growth, survival, and drug resistance in MM cells, both in laboratory and animal models, has allowed for the establishment of a new treatment paradigm targeting the tumor cell and its microenvironment to overcome drug resistance and improve patient outcomes in MM [5]. Myeloma now serves as a model for target identification, drug validation, and rapid translation from the laboratory to the clinic [6].

Role of the host bone microenvironment in mediating growth, survival, drug resistance, and migration of MM cells in vitro In order to identify and validate novel therapeutic targets, we have developed both in vitro systems and in vivo animal models to characterize mechanisms of MM cell interactions. These include homing to BM and identification of those factors within MM cells mediating MM cell-bone marrow stromal cell (BMSC) interactions. In addition, factors within the BM milieu (cytokines and angiogenesis) promote MM cell growth, survival, drug resistance, or migration. All such interactions can provide the potential for novel therapeutic targets [5]. In terms of factors intrinsic to MM cells, we have studied the functional significance of cell surface molecules, including CD20 [7], CD56 [8], CD59 [9], CD138 [10], Ku86 [11], caveolae [12,13], insulin-like growth factor–1 receptor [14,15], and CD40 [16–20]. Within MM cells, we have characterized the role of specific molecules, i.e., nuclear factor-k-B (NF-kB) [21,22], telomerase [23–25]; p38 mitogenactivated protein kinase (MAPK) [26–28]; and Akt [29,30] mediating growth, survival, drug resistance, and migration of MM cells within the BM milieu. In terms of MM cellhost interaction, we have examined expression and regulation of adhesion molecules mediating binding of MM cells to extracellular matrix proteins and BMSCs [11,31], as well as the resultant growth, survival, and drug-resistance advantage due to both tumor cell binding and induction of cytokines [32]. Importantly, within the BM microenvironment, we have examined the role of cytokines, including interleukin–6 [12,23,29,33,34], vascular endothelial growth factor (VEGF) [13,16,35–39], stromal-derived growth factor–1 [40], tumor necrosis factor [31,41], insulin-like growth factor1 [15,42,43], transforming growth factorb [44], and B-cell stimulating factor3 [45] in growth, survival, drug resistance, and migration of MM cells. Finally, we have examined the importance of patient and donor T and NK cells in mediating anti-MM immunity in the BM microenvironment [46–61]. These studies suggest potential utility of novel therapies targeting cytokines and signaling cascades mediating MM cell growth, survival, drug resistance, and migration in the BM milieu.

Validation of growth, survival, drug resistance, and migratory cascades triggered by MM cells in the host BM microenvironment as therapeutic targets We have delineated the signaling cascades and molecular mechanisms whereby NF-kB [21,22], p38 MAPK [26– 28], telomerase [23–25,62], Ku86/70 [11], CD40 ([16– 20]), and caveolae [12,13] mediate homing and adhesion to BM, as well as pathways mediating cytokine-induced growth, survival, and drug resistance in the BM milieu. In these studies, MM cell growth is mediated via MAPK/extracellular signalrelated kinase, survival via Janus kinase/signal transducer activator of transcription, drug resistance via phosphatidylinositol 3-kinase/Akt, and migration via phosphokinase-Cdependent signaling cascades, respectively [5]. Moreover, we have delineated apoptotic signaling and the mechanisms whereby conventional and novel therapies both trigger these cascades and inhibit growth, survival, and drug-resistance signaling in MM cells in the BM microenvironment [8,10,13,18,27,34,44,57,63–82]. To validate these signaling events as therapeutic targets, we have delineated the molecular mechanisms whereby immunomodulatory derivatives of thalidomide [32,47,52,54,57,64,74,80,83]; immunomodulatory drug FQPD [84]; proteasome inhibitor bortezomib [21,22,27,67–69,71,72,78,85–92], arsenic trioxide [93], tumor necrosis factorrelated apoptosis-inducing ligand treatment (TRAIL) [94], VEGF receptor tyrosine kinase inhibitor PTK787 [37], pan VEGF receptor inhibitor 654652 [38], transforming growth factorb receptor kinase inhibitor [44]; fibroblast growth factor receptor3 inhibitor phosphokinase-C 412 [95], 2-methoxyestradiol [68,96], CDDO [71,73], p38 MAPK inhibitor [26–28], insulin-like growth factor1 inhibitor [15,43], histone deacetylase inhibitors [70,97,98], b-lapachone [99], epothilone [77], heat shock protein (hsp) 90 inhibitor [100], telomerase inhibitors [25,62,101], Atiprimod [79], lysophosphatidic acid acyltransferase-b inhibitor [78,102], inosine monophosphate dehydrogenase inhibitor VX944 [103], the R-Enantiomer of Etodolac SDX-101 [104], seliciclib cyclindependent kinase inhibitor [105], Honokiol [106], the shingosine-1– phosphate analogue [107], a carbohydrate-based therapeutic GCS-100 [108], anti-CD40 [16–20], anti-CD56 [8], and anti-CD138 [10] can effectively target MM cells, MM cellhost BM interactions, and/or cytokines in the BM milieu and thereby potentially overcome resistance to conventional therapies. It is our hypothesis that drugs in these classes will need to be combined to achieve complete eradication of MM cells, and we are presently studying their mechanisms of action at a gene and protein level in order to provide the framework for rational combination clinical trials to overcome drug resistance and improve patient outcome. We have, in particular, used gene microarray to delineate the genetic signatures of MM vs monoclonal gammopathy of unknown significance and normal to gain insights into

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MM pathogenesis [1], as well as the consequences of t(4;14) translocations in MM patients [76]. Importantly, we have also used microarray profiling to define mechanisms of anti-MM activity of novel therapies, including bortezomib [87,88], 2-methoxyestradiol [109], and histone deacetylase inhibitors [70,97]; as well as conventional therapies, including dexamethasone (Dex), in order to provide the rationale for optimal combination therapies that enhance cytotoxicity, overcome drug resistance, as well as provide the framework for development of the next generation of more-selective and less-toxic therapies. For example, theses studies defined the preclinical rationale for combining bortezomib with conventional DNA-damaging agents [89], stress response (hsp 90) inhibitors [87,100], and lenalidomide [64]. Moreover, our studies characterizing mechanisms of drug resistance to novel therapies have demonstrated that blockade of hsp 27 [91,110] or of CDC34 [69] can overcome resistance to bortezomib. Having shown efficacy of these novel agents in vitro against the MM cell in its microenvironment, we have then gone on to show in vivo activity of these agents against human MM cells in a severe combined immunedeficient (SCID) mouse model, evidenced by inhibition of human MM cell growth and associated angiogenesis, as well as prolonging host survival [86,111,112]. We have developed a model in which fluorochrome-labeled human MM cells injected into SCID mice migrate and grow primarily in bone, which allows for gene microarray and proteomic studies in MM cells vs BM, before and after MM cells bind to the BM milieu, in the presence or absence of novel drug treatment [113]. We have also developed a SCID-hu model of Waldenstrom’s macroglobulinemia [82], and also refined our SCID-hu model of human MM by injecting fluorochrome-labeled cytokine-dependent MM cells directly into human bone grafts within SCID mice, which allows for evaluation of cellular and gene changes triggered in tumor vs BM by MM cell binding, both before and after treatment with novel agents [34,81]. This model, therefore, allows for evaluation of the impact of binding and novel agents on human cytokines and their sequelae in the BM milieu in vivo.

Clinical trials of novel agents targeting the MM cell in its BM microenvironment The studies described here have translated rapidly to the bedside in clinical protocols treating patients with refractory relapsed MM. Thalidomide was initially used empirically in MM predicated upon increased angiogenesis in MM BM and its known antiangiogenic activity. Thirty percent of patients with relapsed refractory MM who received thalidomide decreased their M proteins [114]. Thereafter, it was combined with Dex, and 47% of patients refractory to either agent alone responded to the combination [115]. Dex/thalidomide was then explored as initial therapy

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[116,117], and 63% of patients achieved responses, compared to 41% patients treated with Dex alone [118]. Moreover, peripheral blood stem cells could be harvested readily after this induction therapy. Subsequently, this regimen has become the most common initial therapy for stem cell transplantation candidates. Thalidomide has also been added to melphalan and prednisone in the elderly nontransplantation candidates and has markedly increased overall response rates, extent of responses, and has prolonged survival [119]. As such, it represents the first agent to improve upon outcomes from melphalan and prednisone alone. To date, studies show that thalidomide not only abrogates angiogenesis, but also directly induces apoptosis of drugresistant MM cells, abrogates MM cell adhesion to BMSCs, inhibits cytokine secretion in BMSCs, and stimulates patient T- and natural killercell responses against MM [83]. The proteasome inhibitor bortezomib was utilized originally based on stabilizing inhibitor of kB kinase, thereby inactivating NF-B. We have shown that it maintains its cytotoxicity against MM cells even in the BM milieu by directly inducing apoptosis of drug-resistant MM cells; decreasing their adhesion to BMSCs and extracellular matrix proteins; downregulating transcription and secretion of cytokines in the BM milieu that mediate tumor cell growth, survival, and migration; and inhibiting angiogenesis [85]. It also downregulates gp 130 interleukin-6 receptor on MM cells [92]; activates c-Jun NH2-terminal kinase, increase reactive oxygen species, and caspase 9O8 [65,66]; induces cytotoxicity independent of p53 status [88]; induces cleavage of Mcl-1 [39]; inhibits DNA repair [89]; and induces apoptosis of endothelial cells [120]. Which of these activities is most important for cytotoxicity is unknown. It was first shown to have antitumor activity in a Phase II trial in relapsed and refractory MM, both alone and when combined with Dex, and was approved by the U.S. Food and Drug Administration in 2003 [121]. Approval was extended to relapsed MM as it significantly prolonged time to progression and survival compared to Dex [122]. Clinical trials then demonstrated its utility alone and in combination in newly diagnosed patients. Both the frequency and extent of response was increased markedly. For example, bortezomib combined with Dex can achieve responses in the majority of patients with newly diagnosed MM, including one third complete or near-complete responses [123]. Moreover, bortezomib has been combined with melphalan and prednisone as initial therapy for the nontransplantation candidates and achieved high overall as well as complete and near-complete response rates [124]. Most recently, lenalidomide has translated from the bench to the bedside in MM. We have shown that it induces caspase-8mediated apoptosis even of MM cells bound to BM, inhibits angiogenesis, as well as induces T- and natural killercell responses to MM cells via activation of T-cell costimulation via CD28 and IL-2 secretion [32,47,52,54,57,64,74,80,83]. We completed a Phase I trial

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of the immunomodulatory drug lenalidomide, which demonstrated benefit in 20 of 24 patients with refractory MM [125]. This was moved rapidly to a Phase II trial that confirms responses, including some complete responses, and further showed the addition of Dex-enhanced responses to lenalidomide responses [121]. Two Phase III trials, therefore, compared lenalidomide/Dex vs Dex/placebo and showed increased overall and complete response rates, as well as prolonged progression-free and overall survival, with combination therapy [126]. These studies provided the basis for the U.S. Food and Drug Administration approval of lenalidomide/Dex to treat relapsed myeloma after one prior therapy. Just as with thalidomidide and bortezomib, lenalidomide has been combined with Dex or with melphalan and prednisone as initial therapy for transplantation candidates and elderly MM patients, respectively, and in each case achieved remarkable frequency and extent of responses [127,128].

Ongoing and future clinical trials Clinical PhaseI/II protocols are also evaluating hsp-90 inhibitors (17 AAG) [100], VEGF tyrosine kinase inhibitor (PTK 787) [37] , histone deacetylase inhibitor (SAHA) [70], Akt inhibitor perifosine [30], arsenic trioxide [93], TRAIL [129], Atiprimod [79], anti-CD40 [19], and antiCD56 [8] based upon our preclinical leads. In particular, a first human trial of the novel proteasome inhibitor NPI0052 [130] is ongoing, and we have developed assays to measure the extent of inhibition of all three proteasome activities [131,132] in tumor cells of such treated patients. NPI-0052 induces apoptosis in MM cells resistant to conventional and bortezomib therapies and is distinct from bortezomib in its chemical structure, effects on proteasome activities, mechanisms of action, and toxicity profile against normal cells. Moreover, NPI-0052 is orally bioactive. In animal tumor model studies, NPI-0052 is well tolerated and prolongs survival, with significantly reduced tumor recurrence. Combining NPI-0052 and bortezomib induces synergistic anti-MM activity. Another example of rapid bench to bedside translation to clinical trials is the hsp-90 inhibitor KOS-953. Heat shock protein 90 is overexpressed in MM cells and functions as a chaperone to shuttle proteins in the proper conformation to mediate growth, survival, and drug resistance signaling on the one hand; as well as to shuttle and unfold ubiquitin-labeled proteins prior to their degradation either via proteasomes or aggresomes [100]. A clinical trial of single-agent KOS-953 hsp-90 inhibitor in relapsed refractory MM has already demonstrated promising activity. Importantly, gene array, proteomic, and cell signaling studies have helped to identify in vivo mechanisms of action and drug resistance, as well as aiding in the clinical application of combination therapies. For example,

gene-microarray profiling of bortezomib-treated MM cells has shown induction of hsp-90 stress response [87,88,100], providing the rationale for the combined clinical use of bortezomib and 17 AAG to block this stress response and enhance anti-MM activity of bortezomib; clinical trials of 17 AAG alone and combined with bortezomib are ongoing and show promising results in overcoming Bortezomib resistance. Proteomics also form the basis for clinical application of novel agents. For example, protein profiling of bortezomib-treated MM cells revealed cleavage of DNA repair enzymes [89], providing the rationale for combining bortezomib with DNA-damaging agents to enhance sensitivity or overcome resistance to these conventional therapies. Cell-signaling studies suggested that combining lenalidomide with bortezomib would trigger dual apoptotic signaling [64]; an ongoing clinical trial has shown responses to the combination in 68% patients who were refractory to either agent alone. Interestingly, preclinical signaling studies show that bortezomib blocks MAPK/ extracellular signalrelated kinase growth signaling, Janus kinase/signal transducer activator of transcription survival signaling, and phosphokinase-C migration signaling, but activates phosphatidylinositol 3-kinase/Akt signaling; trials of the Akt inhibitor perifosine are underway, and combined protocols with bortezomib are planned [30]. Most important, we have shown recently that the histone deacetylase6 inhibitor tubacin can inhibit protein degradation in the aggresome autophagy pathway; blockade of the proteasome with bortezomib upregulates aggresome activity, whereas blockade of the aggresome with a histone deacetylase6 inhibitor upregulates proteasome activity, providing the preclinical rationale for protocols combining these agents to enhance tumor cytotoxicity [133]. Lenalidomide can augment anti-CD40induced antibody-dependent cell-mediated cytotoxicity [20], providing the rationale for combination clinical trials. Correlative science studies on blood and BM samples from patients on clinical protocols can both define targets of drug sensitivity and resistance as well as guide the design of protocols. For example, gene-array studies showed that hsp 27 was overexpressed in patients with intrinsic or acquired resistance to bortezomib. Our studies then showed that overexpressing hsp 27 in sensitive MM cells conferred bortezomib resistance and, conversely, that inhibition of hsp 27 in resistant MM cells via antisense oligonucleotide or small interfering RNA restored sensitivity to bortezomib [68,110]. Subsequently, we showed that p38 MAPK inhibitor downregulates hsp27 in bortezomib-resistant MM cell lines and patient cells, thereby restoring sensitivity to bortezomib [27,28] and providing the rationale for a completed clinical trial combining p38 MAPK inhibitor and bortezomib. Our studies have demonstrated the critical role of host BMtumor cell interactions both in MM pathogenesis and as targets for novel therapies [5]. They have provided the framework for a new treatment paradigm targeting

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MM cellhost BMSC interactions in the BM milieu, as well as their sequelae, including induction of cytokines, in order to overcome drug resistance and improve patient outcomes in MM. They serve as a model of the power of collaborations between academia, pharmaceutical companies, U.S. Food and Drug Administration, National Cancer Institute, and advocacy groups to rapidly identify therapeutic targets in the MM cell and its BM microenvironment, use laboratory and animal models of human MM to validate novel agents directed at these targets, and then design clinical trials evaluating these agents, which ultimately lead to their rapid U.S. Food and Drug Administration approval [6].

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Acknowledgments This study was supported by National Institutes of Health grants CA 50947, CA 78373, and CA10070.

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