Citations

Abstract Multiple myeloma is a blood cancer that arises in the bone marrow from malignant plasma cells. Although treatments have dramatically improved during the last decade, multiple myeloma remains an incurable disease responsible for more than 10 000 deaths per year in the United States. CD137 is a costimulatory molecule expressed on activated T cells and NK cells. We recently established that agonist monoclonal antibodies (mAbs) against CD137 (clone 3H3) significantly reduce tumor burden and improve survival in a mouse model of multiple myeloma1. We found that anti-CD137 mAb therapeutic activity requires the presence of both CD8 T cells and NK cells but is independent of Fc receptors. Moreover, we observed increased numbers of T cells in the bone marrow of anti-CD137 mAb treated mice. CD8 T cells were increased while the percentage of Tregs was reduced. Thus, anti-CD137 mAbs protect mice against multiple myeloma by inducing potent NK cell and CD8 T cell responses. In the present study, we investigate further the therapeutic interest and the potential limitations of anti-CD137 mAbs. To this aim, we used multiple myeloma cell lines derived from Vk*MYC transgenic mice. Wild-type (WT) mice intravenously injected with Vk*MYC cells develop a disease very similar to human multiple myeloma. High percentages of malignant plasma cells are observed in the bone marrow and in the spleen 5 weeks after injection and disease progression can be followed by the level of serum gamma-globulin (M protein). We first observed that, although early anti-CD137 mAb treatment efficiently protected the mice against multiple myeloma, this treatment loses efficacy when given more than 3 weeks after Vk*MYC cell injection. Interestingly, CD4 cell depletion restores the efficacy of late anti-CD137 mAb treatment, indicating that Treg-mediated suppression may limit the therapeutic efficacy of anti-CD137 mAbs. In line with previous studies, we observed that NK cell numbers in the spleen are dramatically reduced 12 days after the initiation of anti-CD137 mAb treatment. NK cell numbers are also decreased in other organs including liver, lung and blood. Importantly, we found that NK cells are still poorly represented in the spleen 3 weeks after the last treatment with anti-CD137 mAbs. Finally, we observed that anti-CD137 mAb treatment induced T cell activation and increases T cell infiltration in the liver. Consistent with the cases of severe hepatitis observed in patients treated with anti-CD137 mAbs, we noted that some of anti-CD137 mAb treated mice that had survived multiple myeloma developed severe liver damage. Such liver damage could be observed more than 100 days after the last therapy with anti-CD137 mAbs. In conclusion, we report several side effects induced by anti-CD137 mAbs treatment that last long after the treatment has been interrupted. These results encourage being particularly careful when administrating this drug to patients. Our study demonstrates the utility of mouse models to investigate the anti-tumor benefits versus toxicity of immunotherapies. We are planning to use this approach to assess the therapeutic efficacy of anti-CD137 mAbs in combination with other treatments. We speculate that the prior reduction of tumor burden by chemotherapy or stem cell transplantation will allow better efficacy of anti-CD137 mAbs at lower doses. The use of mice injected with multiple myeloma cell lines will help determining which combination allows efficient anti-tumor responses at late multiple myeloma stages with limited toxicity. 1 Guillerey C*, De Andrade LF*, Vuckovic S, Miles K, Ngiow SF, Yong MCR, Teng MWL, Colonna M, Ritchie DS, Chesi M, Bergsagel PL, Hill GR, Smyth MJ*, Martinet L* Immunosurveillance and therapy of multiple myeloma is CD226-dependent; Journal of Clinical Investigation, May 1;125(5):2077-89. Citation Format: Camille Guillerey, Lucas Ferrari de Andrade, Slavica Vuckovic, David Ritchie, Marta Chesi, Leif Bergsagel, Geoffrey R. Hill, Ludovic Martinet, Mark J. Smyth. Anti-CD137 mAb therapy of multiple myeloma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B155.

Purpose The aim of the International Myeloma Working Group was to develop practical recommendations for the diagnosis and management of multiple myeloma–related renal impairment (RI). Methods Recommendations were based on published data through December 2015, and were developed using the system developed by the Grading of Recommendation, Assessment, Development, and Evaluation Working Group. Recommendations All patients with myeloma at diagnosis and at disease assessment should have serum creatinine, estimated glomerular filtration rate, and electrolytes measurements as well as free light chain, if available, and urine electrophoresis of a sample from a 24-hour urine collection (grade A). The Chronic Kidney Disease Epidemiology Collaboration, preferably, or the Modification of Diet in Renal Disease formula should be used for the evaluation of estimated glomerular filtration rate in patients with stabilized serum creatinine (grade A). International Myeloma Working Group criteria for renal reversibility should be used (grade B). For the management of RI in patients with multiple myeloma, high fluid intake is indicated along with antimyeloma therapy (grade B). The use of high-cutoff hemodialysis membranes in combination with antimyeloma therapy can be considered (grade B). Bortezomib-based regimens remain the cornerstone of the management of myeloma-related RI (grade A). High-dose dexamethasone should be administered at least for the first month of therapy (grade B). Thalidomide is effective in patients with myeloma with RI, and no dose modifications are needed (grade B). Lenalidomide is effective and safe, mainly in patients with mild to moderate RI (grade B); for patients with severe RI or on dialysis, lenalidomide should be given with close monitoring for hematologic toxicity (grade B) with dose reduction as needed. High-dose therapy with autologous stem cell transplantation (with melphalan 100 mg/m 2 to 140 mg/m 2 ) is feasible in patients with RI (grade C). Carfilzomib can be safely administered to patients with creatinine clearance > 15 mL/min, whereas ixazomib in combination with lenalidomide and dexamethasone can be safely administered to patients with creatinine clearance > 30 mL/min (grade A).

Abstract Angiogenesis has been postulated to be critical for the pathogenesis of multiple myeloma, a neoplastic disease characterized by abnormal proliferation of malignant plasma cells in the bone marrow (BM). Cleavage of the N- and C-terminal regions of circulating chromogranin A (CgA, CHGA), classically an antiangiogenic protein, can activate latent antiangiogenic and proangiogenic sites, respectively. In this study, we investigated the distribution of CgA-derived polypeptides in multiple myeloma patients and the subsequent implications for disease progression. We show that the ratio of pro/antiangiogenic forms of CgA is altered in multiple myeloma patients compared with healthy subjects and that this ratio is higher in BM plasma compared with peripheral plasma, suggesting enhanced local cleavage of the CgA C-terminal region. Enhanced cleavage correlated with increased VEGF and FGF2 BM plasma levels and BM microvascular density. Using the Vk*MYC mouse model of multiple myeloma, we further demonstrate that exogenously administered CgA was cleaved in favor of the proangiogenic form and was associated with increased microvessel density. Mechanistic studies revealed that multiple myeloma and proliferating endothelial cells can promote CgA C-terminal cleavage by activating the plasminogen activator/plasmin system. Moreover, cleaved and full-length forms could also counter balance the pro/antiangiogenic activity of each other in in vitro angiogenesis assays. These findings suggest that the CgA-angiogenic switch is activated in the BM of multiple myeloma patients and prompt further investigation of this CgA imbalance as a prognostic or therapeutic target. Cancer Res; 76(7); 1781–91. ©2016 AACR.

Abstract Multiple myeloma (MM) cells are the malignant counterpart of post-germinal center (GC) long-lived PCs, characterized by strong BM dependence, somatic hypermutation (SHM) of immunoglobulin (Ig) genes, and isotype class switch recombination (CSR) resulting in absence of IgM expression in all but 1% of tumors. Virtually every case of MM is preceded by a pre-malignant PC tumor called monoclonal gammopathy of undetermined significance (MGUS) that, like MM, produces a typical M-spike by SPEP or free light chain in the urine. Translocations involving the immunoglobulin heavy chain (IgH) locus are present in at least half of MM cases and are thought to result from errors during the physiological process of CSR or SHM. These translocations result in dysregulated expression of an oncogene that is juxtaposed to the strong Ig enhancers. There are three recurrent primary IgH translocation groups in MM: CYCLIN D (11q13-CYCLIN D1; 12p13-CYCLIN D2; 6p25-CYCLIN D3) MAF (16q23-MAF; 20q12-MAFB; 8q24.3-MAFA; MMSET/(FGFR3)-4p16-(MMSET in all but also FGFR3 in 80% of these tumors. Multiple trisomies represent an alternative pathogenetic pathway. There is a consensus that chromosome content reflects at least two pathways of pathogenesis. Nearly half of MGUS and MM tumors are hyperdiploid (HRD), usually with extra copies of three or more specific chromosomes (3,5,7,9,11,15,19, 21). Strikingly, HRD tumors rarely (~10%) have a primary IgH translocation, whereas Non-hyperdiploid (NHRD) tumors usually (~70%) have an IgH translocation. Almost all cases of plasma cell neoplasm starting from the MGUS stage and independently on the chromosome content aberrantly express one or more of the CYCLIN D genes and it has been proposed that dysregulation of a CYCLIN D gene provides a unifying, early oncogenic event in MGUS and MM. Consistently, the dysregulation of CYCLIN D1 in mouse B cells is sufficient to induce a monoclonal gammopathy that does not progress to MM. Secondary oncogenic events drive MGUS and MM progression MYC dysregulation. There is increased expression of c-MYC in most newly diagnosed MM tumors compared to MGUS tumor. Recently, it was shown that sporadic activation of a MYC transgene (Vk*MYC) in GC B cells in an MGUS prone mouse strain led to the universal development of MM tumor. Hence, increased MYC expression may be responsible for progression from MGUS to MM. Complex translocations involving MYC appear to be secondary progression events that often do not involve Ig loci, but rather other super enhancers that are very active in plasma cells. They are rare or absent in MGUS, but recently identified in over 40% of untreated MM, and 90% of MM cell lines. Activating mutations of RAS and BRAF. The prevalence of activating NRAS or KRAS mutations is about 15-18% each in newly diagnosed and relapsed MM tumors, and recently, BRAF mutations were described in 4% of MM tumors. Expression of an activated of KRAS in mouse B cells is sufficient to induce a monoclonal gammopathy but it does not progress to MM. Not surprisingly, dysregulating pairs of CYCLIN D1, KRAS and/or MYC in mouse B cells accelerated the progression of MM. Activating mutations of NFkappaB pathway. Extrinsic ligands (APRIL and BAFF) produced by BM stromal cells provide critical survival signals to long-lived PCs by stimulating TACI, BCMA, and BAFF receptors to activate the NFKB pathways. Most MGUS and MM tumors highly express NFKB target genes, suggesting a continued role of extrinsic signaling in PC tumors and at least in part explaining the constant dependency of MM cells on the BM microenvironment. Activating mutations in positive regulators and inactivating mutations in negative regulators of the NFKB pathway have been identified in at least 20% of untreated MM tumors and ~50% of HMCLs, rendering the cells less dependent on ligand-mediated NFKB activation and most likely contributing to extra-medullary spread of the disease. Additional changes include monosomy 13 (DIS3, mir15/16, RB1), del17p (TP53), add1q (MCL1) and del1p (CDKN2C, FAM46C). While the role of these putative tumor suppressor/oncogene remain to be defined, we found that loss of one copy of miR15/16 cooperated with MYC to cause the progression of MM in mice, while loss of one copy of RB1 did not, implicating loss of miR15/16 as one driving force behind selection for monosomy 13 in human MM. High-risk MM is associated with intra-clonal tumor heterogeneity Recent evidence indicates that tumor heterogeneity is prevalent in MM, as in many other cancers, and that different subclones are present within the tumor population, characterized by distinct genetic mutations that contributed independently to the tumor progression. The degree of clonal heterogeneity is presumably related to the degree of genomic instability, and appears to correlate with the presence of high-risk genetics. Three patterns of relapse are seen following successful therapy. A third of the time the dominant clone at diagnosis predominates, unchanged, at relapse. A third of the time the diagnostic clone has acquired additional genetic changes at relapse. And a third of the time an ancestral clone to the one dominant at diagnosis emerges, lacking some of the genetic changes present in the diagnostic clone, and having acquired additional genetic changes. Activating the immune system to overcome tumor heterogeneity MM cells develop a malignant relationship with components of the innate immune system in the BM. Initially inflammatory macrophages support MM tumor development, but over time convert to an immunosuppressive M2 phenotype. This relationship can be disrupted by activating signaling downstream of TLR and TNFR receptors in macrophages through the use of IAP antagonists which target for destruction negative regulators of these pathways (cIAP1/2). In MM bearing mice with a mature (but not those with an immature) immune system this results in a complete response, with no evidence of a direct anti-tumor effect of IAP antagonists on the tumor cells at clinically relevant doses. This has led to a phase II clinical trial of IAP antagonists to activate the innate immune system in patients with MM. The treatment is associated with dramatic activation and mobilization of macrophages, with cytokine release and early evidence of anti-tumor effects. Conclusion Significant progress has been made is understanding the molecular pathogenesis and biology of MM. Oncogenic pathways can be activated through cell intrinsic or extrinsic mechanisms. Similar to other cancers, MM is characterized by multi-stage accumulation of genetic abnormalities deregulating different pathways. Much of this knowledge is already being utilized for diagnosis, prognosis and risk-stratification of patients. Importantly, from a clinical standpoint, this knowledge has led to development of novel therapeutic strategies, some of which are already in clinical use, and many others showing promise in pre-clinical and early clinical studies. Citation Format: Marta Chesi, P. Leif Bergsagel. Targeting genetic heterogeneity in multiple myeloma through immune activation. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA25.

SummaryMultiple myeloma (MM) is a tumor of indolent, bone marrow (BM) localized, isotype‐switched plasma cells. Recently, the diagnostic criteria have been amended to include some patients who would previously have been diagnosed with ultra‐high‐risk smoldering MM and benefit from immediate treatment. Genetically it can be divided into tumors with different recurrent immunoglobulin heavy chain gene translocations (4p16, 11q13, 6p21, 16q23, 20q11) and tumors characterized by hyperdiploidy with multiple trisomies. Recent genomic studies have shown that almost half of untreated patients have a genetic rearrangements of the MYC locus that result in juxtaposition of ectopic super‐enhancers adjacent to MYC, as well as somatic mutations that activate the RAS/MAPK pathway (NRAS, KRAS, BRAF, FGFR3). Mutations that result in constitutive activation of the NFkB pathway and that inactivate TP53, CDKN2C, KDM6A, FAM46C, and DIS3 are also recurrent. A major insight from these studies has been the recognition of the high degree of subclonal heterogeneity in MM, which is more frequent in patients with high‐risk genetics. The subclones may alternate in dominance under alternating therapeutic pressure, a phenomenon known as ‘clonal tides’. The identification of marked subclonal heterogeneity argues in those patients for the use of therapeutic strategies to maximize response, and long‐term suppressive therapies to prevent tumor regrowth and development of additional subclones.

Abstract Myeloma remains a virtually incurable malignancy. The inevitable evolution of multidrug-resistant clones and widespread clonal heterogeneity limit the potential of traditional and novel therapies to eliminate minimal residual disease (MRD), a reliable harbinger of relapse. Here, we show potent anti-myeloma activity of macrophage-activating immunotherapy (αCD40+CpG) that resulted in prolongation of progression-free survival (PFS) and overall survival (OS) in an immunocompetent, preclinically validated, transplant-based model of multidrug-resistant, relapsed/refractory myeloma (t-Vκ*MYC). αCD40+CpG was effective in vivo in the absence of cytolytic natural killer, T, or B cells and resulted in expansion of M1-polarized (cytolytic/tumoricidal) macrophages in the bone marrow. Moreover, we show that concurrent loss/inhibition of Tpl2 kinase (Cot, Map3k8), a MAP3K that is recruited to activated CD40 complex and regulates macrophage activation/cytokine production, potentiated direct, ex vivo anti-myeloma tumoricidal activity of αCD40+CpG–activated macrophages, promoted production of antitumor cytokine IL12 in vitro and in vivo, and synergized with αCD40+CpG to further prolong PFS and OS in vivo. Our results support the combination of αCD40-based macrophage activation and TPL2 inhibition for myeloma immunotherapy. We propose that αCD40-mediated activation of innate antitumor immunity may be a promising approach to control/eradicate MRD following cytoreduction with traditional or novel anti-myeloma therapies. Cancer Immunol Res; 3(8); 881–90. ©2015 AACR.

Abstract Background: The AAA-ATPase p97/VCP facilitates the extraction and degradation of ubiquitinated proteins by converting chemical energy into mechanical force. p97 is closely involved in several facets of protein homeostasis, including ubiquitin-dependent protein degradation, endoplasmic reticulum-associated degradation (ERAD) and autophagy. p97 has been increasingly linked to cancer: it showed elevated protein expression in tumors, it can mediate the degradation of proteins in cancer-relevant pathways and is required for orchestrating the ubiquitin-governed DNA-damage response. In this context, p97 inhibitors may have an advantage versus other protein homeostasis inhibitors and may be active in solid tumors where 26S proteasome inhibitors, bortezomib and carfilzomib, have shown poor efficacy. We report here p97 inhibition as a novel approach to exploit cancer cell addiction to protein homeostatic mechanisms. Results: We have discovered novel small molecule inhibitors of p97 ATPase activity with nanomolar enzymatic and cellular potency. In cellular models, treatment of cancer cells with our lead compound CB-5083 causes disruptions in specific p97 functions, including ubiquitin-dependent protein degradation, ERAD, endocytosis and autophagy. In mouse models, CB-5083 is orally bio-available and causes rapid and sustained accumulation of poly-ubiquitin in tumor xenografts after a single administration. Concurrent with increases in polyubiquitin levels, activation of ER stress response pathways and induction of apoptosis markers are also observed. Sub-chronic oral administration of CB-5083 is generally well-tolerated with <10% body weight loss and results in potent tumor growth inhibition in several solid tumor xenograft models. This result is in marked contrast to proteasome inhibitors that are inactive in the same solid tumor models. In the Vk*MYC transgenic mouse model of multiple myeloma, CB-5083 treatment resulted in >50% reduction in M-spike. Additional efforts are focused on the development of translational assays to monitor p97 target engagement and antitumor efficacy in upcoming clinical trials of CB-5083. Conclusion: These data demonstrate that CB-5083 is a potent inhibitor of p97 that translates to tumor growth inhibition in multiple rodent models of human cancer. Furthermore, CB-5083 appears to exhibit greater potency over current proteasome inhibitors that further validate targeting p97 and protein homeostasis in the treatment of cancer. Citation Format: Ronan Le Moigne, Steve Wong, Ferdie Soriano, Eduardo Valle, Daniel J. Anderson, Stevan Djakovic, Mary-Kamala Menon, Bing Yao, Julie Rice, Jinhai Wang, Szerenke Kiss Von Soly, Brajesh Kumar, Marta Chesi, P. Leif Bergsagel, Han-Jie Zhou, David Wustrow, Mark Rolfe, F. Michael Yakes. CB-5083 is a novel first in class p97 inhibitor that disrupts cellular protein homeostasis and demonstrates anti-tumor activity in solid and hematological models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 951. doi:10.1158/1538-7445.AM2014-951

Abstract The numerous and diverse genetic lesions in multiple myeloma (MM) make it difficult to discern those genetic abnormalities that may be suitable therapeutic targets. We have observed that the MYC locus is frequently rearranged in MM (about half of both patients and cell lines), suggesting that MYC dysregulation is a common feature of this disease. To evaluate MYC suppression as a therapeutic strategy in MM, we treated human myeloma cell lines with CPI203, a potent and selective inhibitor of bromodomain and extra-terminal (BET) proteins. The bromodomain inhibitor CPI203 has potency and selectivity similar to that of JQ1, but has improved pharmacokinetics for in vivo studies. From our collection of MM cell lines we have identified sensitive and resistant cell lines and characterized their response to CPI203 in detail. A number of conclusions have emerged from this work. First, response correlates with MYC protein levels after 3 days of treatment. MYC levels are significantly suppressed in sensitive lines and much less affected in resistant lines, supporting the hypothesis that MYC suppression alone determines cell fate in MM cell lines. In sensitive cell lines treatment with CPI203 caused a rapid (within 90 min.) decrease in MYC transcription that is consistent with the response being an intrinsic property of the cell lines. Inhibition of MYC transcription was followed by eventual cell cycle arrest and limited apoptosis in these sensitive cell lines. Both sensitive and resistant cell lines exhibited induction of Histone2H2BE gene in response to CPI203 treatment, indicating that drug efflux pumps alone do not mediate resistance. Surprisingly, several of the resistant cell lines exhibited an initial phase of sensitivity to BET inhibition. Upon drug addition, MYC transcription and MYC protein levels are repressed 2- to 5-fold, followed by a gradual recovery of MYC message and protein levels to the initial levels. In this second phase the cells are truly insensitive to BET inhibition: the drug remains active during this incubation and addition of fresh medium with drug does not affect MYC transcription. Furthermore, many aspects of the BET inhibition gene expression profile remain constant throughout this transition between sensitivity and resistance. These results suggest that the recovery phase is not a simple reversal of BET inhibition but rather the emergence of an alternative-signaling pathway that activates MYC expression in the presence of BET inhibition. Presently we are analyzing gene expression data to identify genes and signaling pathways responsible for this “induced resistance”. The identification of such pathways will help us better understand the molecular basis of the response to BET inhibition, which in turn might enable us to identify predictive biomarkers of the therapeutic response. Citation Format: Daniel L. Riggs, Marta Chesi, P Leif Bergsagel. Targeting MYC in multiple myeloma by BET protein inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5489. doi:10.1158/1538-7445.AM2014-5489