Citations

Abstract We have previously proposed the dysregulation of the D type cyclins by chromosome translocations to the IgH locus or by not fully characterized mechanisms in hyperdyploid patients as a unifying genetic event in the pathogenesis of Monoclonal Gammopathy of Uncertain Significance (MGUS) and multiple myeloma (MM). However, no experimental data are available to evaluate the direct consequences of cyclin Ds overexpression in plasma cells. Similarly, activating mutations of KRAS have been identified in 15% of MM patients, but never in MGUS, and have been proposed to contribute to the progression of MGUS to MM, but this hypothesis has never been experimentally validated. We evaluated the contribution of CyclinD1 and KRAS to the development and progression of monoclonal gammopathy (MG) that develops spontaneously in C57BL/6 mice. We aged a cohort of EuCCND1 mice, and lslKRASV12 mice crossed to three different CRE expressing mice: IgG1CRE, CD19CRE or Vk* CRE (KRAS/CRE). A cohort of Vk* MYC mice, Vk* MYC x EuCCND1 (MYC/CD1) and triple transgenic mice (CD1/KRAS/CRE) was also analyzed. Mice were bled every 10 weeks beginning at 30 weeks, and the presence of MG was evaluated by serum protein electrophoresis. The median Time To M-Spike (mTTS) detection was 97 weeks in C57Bl/6, 63w in EuCCND1, 54w in KRAS/CRE, 54w in Vk* MYC, 38w in CD1/KRAS/CRE and 36w in MYC/CD1 mice. This indicates that acceleration of the development of MG occurs in all these strain of mice. However, we noticed that the spontaneous MG in C57BL/6 wt, EuCCND1 or KRAS/CRE rarely progresses (as determined by detection of a M-spike >7 g/L), while in the other mouse strains it frequently progresses, with a mTTS >7 g/L around 65w. A clear progression of MGUS to MM was observed only in Vk* MYC mice, and was further accelerated in MYC/CD1 mice, which are characterized by extra-medullary disease and shorter overall survival. In conclusion, these data support our hypothesis that Cyclin Ds dysregulation in plasma cells is the initial transformation step leading to MGUS. Further oncogenic events, like activating mutations of KRAS are required to expand the monoclonal plasma cells. Among the oncogenes examined MYC provides the greatest acceleration of the MG in wildtype and EuCCND1 mice, and results in a disease with the malignant features of MM. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3015 Several preclinical models are available to assess the efficacy of novel anti-myeloma therapies, each of them with specific utilities. The most commonly utilized are: 1) Human myeloma cell lines (HMCLs) offer the advantage of being easily manipulated and well genetically characterized and therefore are best used to provide in-vitro target validation and to demonstrate specific target inhibition by a drug. On the other hand, they are highly proliferative and do not recapitulate the complexity of the human disease in an endogenous micro-environment. 2) Xenograft studies, in which HMCLs are injected into immunodeficient mice, serve the purpose of demonstrating that in-vivo target inhibition can be achieved under physiological conditions, but again fail to represent the indolence of human MM and its complex interaction with the BM stromal cells. Drug screenings performed on both these models tend inevitably to overestimate the antimyeloma activity of compounds that simply inhibit proliferation, but are ineffective in targeting the bulk of MM tumor. 3) Direct cytotoxic studies on primary patients cells, alone or in co-culture with stroma cell lines, are useful in assessing a pro-apoptotic activity in a way that is completely independent on proliferation. However, once again this in-vitro model does not capture the complete biology of human MM. The immuno-competent Vk*MYC mouse model of myeloma has already demonstrated high biological fidelity to the human disease, making it an ideal model to study the behavior of myeloma cells in the context of a native microenvironment and immune system. Furthermore, this model offers the advantage of studying both indolent, BM localized, untreated MM (primary MM), and, with the use of transplants into syngeneic hosts, advanced, more proliferative and refractory disease (secondary MM). Using the primary MM model, we have assessed the anti-myeloma activity of 30 known and novel compounds, many of which currently in clinical trials, and found that agents effective in the treatment of patients are active in this model, whereas agents ineffective in the treatment of patients are not. From this study, the most promising class of novel agents in clinical trials are the HDAC inhibitors vorinostat and panobinostat. Based on this promising single agent activity, we sought to model the effects of combination therapy in the secondary myeloma models that we specifically generated to be bortezomib refractory or multidrug resistance. Although transplanted mice treated with full dose single agent bortezomib, vorinostat or panobinostat showed no response and died within three weeks post transplant, recipient mice treated with bortezomib in combination with either vorinostat or panobinostat achieved complete response and are still alive 10 weeks post-transplant. We conclude that the bortezomib + HDAC inhibitors is an active combination that overcomes in vivo bortezomib resistance. Furthermore, the Vk*MYC model provides an excellent platform for the development of novel combinations aimed to the treatment of refractory MM disease. Disclosures: Bergsagel: Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Millennium: Speakers Bureau; Novartis: Speakers Bureau.

Abstract LCL161 is a IAP antagonist targeting the cellular inhibitor of apoptosis proteins cIAP1 and -2 (cIAP1/2). We previously reported that loss of the ubiquitin ligase cIAP1/2 results in the stabilization of NIK and activation of the non-canonical NFkB pathway, a pathway we found frequently activated by a promiscuous array of mutations in multiple myeloma (MM), including the homozygous deletion of cIAP1/2 themselves. This suggests that LCL161 treatment will have no direct anti-tumor activity, and may even increase the already high level of NFkB in MM. However, this pathway is also a key regulator of the host immune response and LCL161 has been shown to induce some activation of the immune system that can potentiate the effects of anti-tumor vaccines. Like IMiDs, which target another ubiquitin ligase complex containing cereblon, we found that LCL161 inhibits TNFa secretion by LPS-stimulated monocytes in vitro. Also like IMiDs, LCL161 has little direct anti-tumor activity against primary MM cells and MM cell lines in vitro, or immunodeficient xenograft models in vivo, which, contrary to preclinical studies in other tumor types, is not potentiated by the addition of TNFa. At concentrations ten times higher than the ones required for target modulation (cIAP1/2 degradation, achieved at nanomolar doses), direct cytotoxicity was observed, as reported by other investigators, most likely derived from off target inhibition of the caspase inhibitor XIAP. LCL161 has been studied in a phase I study in solid tumors where it was well tolerated with a cytokine release syndrome as the dose limiting toxicity and is now being studied in a phase II study in combination with paclitaxel. Surprisingly we found that LCL161 has dramatic activity in vivo against MM that develops spontaneously in an immunocompetent genetically engineered mouse model of MM (Vk*MYC). This model has been rigorously validated and found to have a positive predictive value for clinical activity of 67%, and a negative predictive value for clinical inactivity of 88%. Among nearly 50 drugs tested as single agents, only LCL161, proteasome inhibitors and alkylating agents induce complete response in this model. We found that LCL161 has no activity in vitro against these same tumor cells, suggesting an important role for the host in mediating the anti-tumor effect. MM that develops in congenic recipient mice transplanted with unfractionated bone marrow from Vk*MYC donor mice with MM, but not from those transplanted with purified CD138+ MM cells, is sensitive to LCL161. Cell fractionation and “add back” transplantation experiments in Vk*MYC mice have identified plasmacytoid dendritic cells (pDCs) as the key mediators of LCL161 anti-MM activity, which is completely independent of adaptive immunity. pDCs infiltrate the BM of MM patients, and similarly of Vk*MYC mice, where they stimulate tumor growth through release of inflammatory cytokines and NFkB activation. It has been shown that engagement of TLR with CPG abrogates this effect. MM is a tumor exquisitely dependent on pro-inflammatory cytokines for survival, but also sensitive to the cytotoxic effects of type-I IFN. We found that the dramatic anti-MM activity of LCL161 is directly mediated by pDCs. In normal condition pDCs support MM growth by providing a cytokine rich environment. LCL161, however, turns them into killers by suppressing the MYD88 pathway responsible for IL6 and TNFa production while simultaneously activating IFNa production. Although the response rate to single agent IFNa in MM patients is approximately 20%, its use in the treatment of MM has been discontinued because of toxicity. LCL161 may represent a novel way of administering high level of IFNa directly to the tumor bed, limiting toxicity and increasing efficacy. Interestingly, IMiDs have been recently shown to augment IFNb production downstream of MYD88 in lymphoma, and their anti-lymphoma activity can be inhibited by antibodies to IFNb. We conclude that LCL161 is a novel immunomodulator with very potent anti-MM activity in vivo and well-defined molecular and cellular targets. A phase II clinical trial of LCL161 in MM is ongoing. Disclosures: No relevant conflicts of interest to declare.

Abstract Multiple myeloma tumors universally target one of the three human cyclin D genes (CCND1, CCND2 or CCND3) for dysregulation (Bergsagel et al., 2005, Blood, 106:296). Using a lentivirus expressing CCND2 RNAi we first tested the effects of selective cyclin D2 knock down on My5 and H929 myeloma cell lines and found G0/G1 phase arrest, increased apoptosis and significant selective disadvantage in transfected cells. By comparison, knockout mouse models indicate that most somatic tissues can develop in the total absence of cyclin D1, D2 and D3 (Kozar et al., 2004, Cell,118:477). Targeted inhibition of specific cyclin D expression is therefore a rational therapeutic strategy in myeloma. To identify novel pharmaceutical inhibitors of CCND2 transactivation we developed an assay employing NIH 3T3 cells stably co-expressing the CCND2 transactivator c-Maf and the cyclin D2 promoter driving firefly luciferase (luc) and screened the Lopac (n=1280), Prestwick (n=1120) and Spectrum (n=2000) libraries of drugs and natural compounds. In a parallel MTS assay, the effect of each compound on 3T3 viability was determined, allowing exclusion of compounds that caused secondary suppression of CCND2 due to non-specific cytotoxicity. From the screen we identified 10 c-Maf independent putative CCND2 inhibitors. These included monensin, patulin, β-lapachone, camptothecin, dihydrogambogic acid, gentian violet, thapsigargin, brefeldin A, pristimerin and kinetin riboside. Three of the 10 compounds (gentian violet, thapsigargin and patulin) were not studied further due to toxicity cited in the literature. Subsequent validation studies using selected compounds in human myeloma cell lines (HMCL) confirmed successful suppression of both cyclin D2 and D1 proteins. Each of these compounds was then shown to be cytotoxic to a genetically diverse and standardized panel of 14 HMCL in MTT assays: monensin (10–760 nM), camptothecin (5–700nM), dihydrogambogic acid (250–800 nM), pristimerin (150–500 nM) and kinetin riboside (2.5–20μM). Cell cycle analysis confirmed induction of G0/G1 phase arrest for most compounds, consistent with cyclin D inhibition. However, camptothecin and b-lapachone induced S-phase arrest, suggesting secondary suppression of cyclin D by virtue of S-phase activity. Unsorted myeloma patient bone marrow samples demonstrated selective activity for pristimerin, dihydrogambogic acid and kinetin riboside against CD138+ myeloma cells compared with non malignant hematopoietic cells; by contrast monensin showed almost equal toxicity for normal cells. The triterpenoid, pristimerin, showed potent anti-myeloma activity and was examined in greater detail. Studies confirm that pristimerin rapidly inhibits cyclin D1, D2 and D3 expression (<6 hours) at nanomolar concentrations and induces apoptosis of primary myeloma cells characterized by caspase 9 cleavage and Annexin V binding. While pristimerin is cytotoxic to HMCL and patient myeloma cells at 0.1–0.15 mg/L, toxicity studies in vivo indicate that the drug is tolerated in mice at 2.5 mg/kg i.p. daily. In vivo activity against a xenograft model is currently being determined. Overall this targeted chemical biology screen has identified several compounds, including the triterpenoid, pristimerin, that are being further characterized for promising preclinical anti-myeloma activity.

Abstract Deletions of chromosome 13 occur in about 50% of multiple myeloma (MM) patients, with monosomy being the most common (85%), and small interstitial deletions, often involving the Rb gene, representing only 15% of cases. In contrast to CLL, where a minimal deleted region (MDR) has been mapped to the DLEU2/miR-15a/16-1 locus, no MDR has been identified in MM. The prognostic significance of monosomy 13 has been overshadowed by its strong association with high-risk MM, mostly t(4;14) and t(14;16) translocations. Genomic analysis of 28 MM tumors from Vk*MYC mice identified recurrent (27%) monosomy of the syntenic chromosome 14, with focal bi-allelic deletions of Rb1 in three independent tumors. One tumor presented a focal monoallelic interstitial deletion of the mirR15a-16.1 cluster. Reasoning that the biological significance of monosomy 13 would be conserved between human and murine MM, we carried out genetic crosses between Vk*MYC and mice heterozygous for Rb1 (MYC/Rb1), for the DLEU2/miR-15a/16-1 locus (MYC/MDR) or for the miR-15a/16-1 cluster only (MYC/MIR). All of the MYC/Rb1 mice died around one year of age of pituitary tumors without a significant plasma cell expansion compared to the Vk*MYC mice alone. In contrast, both the MYC/MDR+/- and MYC/MIR+/- mice exhibited a progressive monoclonal plasmacytosis affecting secondary lymphoid organs, with associated shorter overall survival. The development of extra-medullary MM was further accelerated in MYC/MDR -/- and MYC/MIR -/- mice, and was reminiscent of that observed in Vk*MYC crossed with Emu-BCL2 mice. As previously shown for the CLL-like disease that develops in the MIR and MDR mice, the MM phenotype is more pronounced in the MYC/MDR than the MYC/MIR, and in the homozygous than the heterozygous mice. Although BCL2 has been proposed as a target of the miR-15a/16-1, it has not been identified among genes whose expression is altered in MIR null B lymphocytes, where instead miR-15a/16-1 deletion promotes proliferation. In contrast, we found significant BCL2 upregulation in some MM tumors from MYC/MIR and MYC/MDR, suggesting that miR-15a/16-1 deletion in MM provides a survival signal that renders plasma cells less dependent on the BM microenvironment. We propose that selection for monosomy 13 in MM is driven in at least in part by loss of one copy of the miR cluster. Disclosures: No relevant conflicts of interest to declare.

Abstract The search for novel and clinically effective anti-myeloma therapies has been hampered by a paucity of good pre-clinical animal models. Most established animal models of multiple myeloma (MM) fail in one or more crucial features to resemble human MM, frequently exhibiting extramedullary tumors or lacking a competent immune system. In addition, competing models are either too expensive or time consuming to generate and maintain. Finally, no model has been shown to reliably predict both drug response and drug inactivity. We have previously described a C57Bl6/J transgenic mouse model in which the expression of the human c-myc oncogene is activated in post-germinal B cells by somatic hypermutation. These mice (Vk*myc) spontaneously develop monoclonal gammopathies and plasma cell expansion beginning at 20 weeks of age. However, in contrast to other models, their plasma cells cannot be found in secondary lymphoid tissues. Now, we report that this model closely reproduces the clinical behavior of human MM. Serum protein electrophoresis (SPEP) as well as ELISA for serum IgG demonstrate that the incidence and quantity of monoclonal paraproteinemia are greatly elevated in the Vk*myc mice as compared to age matched wild type C57Bl6/J controls (50 weeks mean IgG 1.92g/dL Vk*myc vs 0.2g/dL controls; peak IgG levels up to 7.5g/dL). As these mice age, their paraproteins continue rising and remain far higher than that of controls at every time point. Mice with significant paraproteinemia demonstrate marrow with up to 50% plasma cells with evidence of a low proliferative index, similar to what is observed in human MM and unlike what is seen in other models of MM. Anemia is observed, with a mean hemoglobin of 8.9g/dL in the Vk*myc mice vs 13.4g/dL in the wild type. Vk*myc mice analyzed show marked bone thinning with a 20% reduction in total bone volume and in the number of trabeculae per unit area as shown by microCT. In addition a 15% reduction in bone mineral density was demonstrated in affected mice. We next demonstrated the efficacy of 3 drugs, used commonly in clinical practice to treat myeloma (melphalan, dexamethasone and bortezomib). These were given as single courses of daily IP injections for 5 days, for the first two drugs, and bi-weekly IP for 4 weeks for the third. As early as two weeks post treatment, a statistically significant reduction in serum paraprotein levels was observed in the treated mice versus vehicle treated controls. The reductions were maximal at 21d post treatment in the melphalan (−77.2%±39 p<0.02) and dexamethasone (−67.4%±29 p<0.02) groups and at 28d in the bortezomib group (−68.9%±9 p<.001). No reduction was seen in the vehicle treated controls at any time point. As with human myeloma, the effect of one course of treatment was transient in nature. In contrast, treatment of VK*myc mice with 3 drugs that have not shown clinical activity against myeloma as single agents (hydroxyurea, vincristine and fludarabine) did not reduce serum monoclonal proteins. We conclude that our Vk*myc model closely reproduces the clinical characteristics of human MM. We have also shown that this model responds to drugs known to be clinically active, while showing no response to drugs that have little or no effect against human MM. To our knowledge, this is the first time that this level of fidelity to human MM has been demonstrated in a pre-clinical animal model. Our model therefore represents a unique and powerful tool for pre-clinical anti-myeloma drug development.

Abstract Abstract 573 Using high throughput RNA interference screening on 6,722 druggable genes we previously identified XPO1/CRM1 as one of the 50 most vulnerable targets in Multiple Myeloma (MM)1. XPO1 knockdown proved lethal in MM cell lines, but had no effect on human embryonic kidney (293) cells or lung cancer (A549) cells, showing that XPO1 is a specific myeloma vulnerability, and that myeloma cell survival is dependent upon XPO1 expression. XPO1 encodes the protein exportin 1, a nuclear transport protein that exports tumor suppressor proteins from the nucleus, where they are active, to the cytoplasm, where they become inactive. We next analyzed XPO1 in MM via gene expression profiling (GEP). XPO1 expression is up-regulated as the disease progresses: patients with active MM have a higher level of XPO1 compared to normal plasma cells (p<0.04) and to patients with monoclonal gammopathy of undetermined significance or smoldering MM (p<0.0001). The highest levels were in human MM cell lines. TC classification revealed highest levels in t(11;14) and lowest levels in t(4;14) disease. Selective inhibitors of nuclear export (SINE) compounds have recently been developed that irreversibly inhibit XPO1/CRM1 and its nuclear export function. One such inhibitor, KPT-276, decreased the viability of all 12 MM cell lines tested in vitro, as shown by MTT assay. After 72 hours of drug treatment, a median IC50 value of approximately 175 nM (range 30–1000 nM) was observed. No synergy with other commonly used anti-MM therapeutics was observed in vitro. In contrast, the drug had little effect in 8 solid tumor cell lines with the exception of the B cell lymphoma line Ramos. KPT-276 was also consistently active in inducing apoptosis against MM primary patient samples. Using an IC80 dose of KPT-276, drug-treated samples had a reduced population of cells in S phase (8%) compared to cells treated with DMSO (21%). Using the vkappa*myc transgenic MM model, KPT-276 reduced monoclonal spikes (by a mean of 56%) in all mice treated orally with 150 mg/kg dose three times per week for 4 weeks. Furthermore, KPT-276 significantly reduced tumor growth in a xenograft MM1.S mouse model. GEP was performed in the presence or absence of drug in two different MM cell lines. Two genes of probable relevance, cell division cycle 25 homolog A (CDC25A) and Bromodomain-containing protein 4 (BRD4), were dysregulated by SINE treatment. Both are involved in cell cycle control and have been linked to MYC. RT-PCR and western blotting confirm that MYC, CDC25A and BRD4 are down-regulated, as soon as six hours, after treatment with KPT-276. KPT-276 has shown marked anticancer activities against B cell malignancies in vitro and is active and tolerated in Phase I canine studies. KPT-330, a close analog of KPT-276, is currently in Phase 1 studies in human with advanced hematological and solid tumors. Disclosures: Schmidt: Karyopharm: Research Funding. McCauley:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy.

Abstract Emerging evidence indicates that tumors can follow several evolutionary paths over a patient's disease course. With the use of serial genomic analysis of samples collected at different points during the disease course of 28 patients with multiple myeloma, we found that the genomes of standard-risk patients show few changes over time, whereas those of cytogenetically high-risk patients show significantly more changes over time. The results indicate the existence of 3 temporal tumor types, which can either be genetically stable, linearly evolving, or heterogeneous clonal mixtures with shifting predominant clones. A detailed analysis of one high-risk patient sampled at 7 time points over the entire disease course identified 2 competing subclones that alternate in a back and forth manner for dominance with therapy until one clone underwent a dramatic linear evolution. With the use of the Vk*MYC genetically engineered mouse model of myeloma we modeled this competition between subclones for predominance occurring spontaneously and with therapeutic selection.