Garbitt, Victoria M.

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 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 A genetic rearrangement of the MYC locus, resulting in dysregulated expression of MYC, is the most common mutation in human multiple myeloma (MM). The genetically engineered Vk*MYC mouse model is based on dysregulation of MYC, and has been extensively validated as a clinically and biologically faithful model of untreated MM. We previously reported 9 drugs or classes of drugs (DNA alkylators, glucocorticoids, proteasome inhibitors, IMiDs, nab-paclitaxel, histone deacetylase inhibitors, TACI-Ig, perifosine and SNS-032, a CDK2,7,9 inhibitor) with more than 20% partial response (PR) rate in Vk*MYC MM. Among those, the first 5 also have more than 20% PR in patients with MM for a positive predictive value of 56%. Although the HDACi did not show single agent activity in relapsed/refractory MM patients, the PANORAMA phase 3 study of panobinostat in combination with bor+dex has shown superior PFS compared to bor+dex, suggesting the possibility that HDACi may have shown single agent activity if tested in a less advanced clinical setting. In contrast, 11/12 drugs that have less than 20% PR in Vk*MYC MM also have less than 20% PR in patients with MM for a NPV of 92%. Confident that drugs with activity in Vk*MYC mice will likely be effective in the treatment of MM, we have used this model to study novel drugs for prioritization in clinical trials. Pim kinases are constitutively active serine/threonine kinases identified using retroviral mutagenesis as potent suppressors of MYC induced apoptosis and can be inhibited by the pan-PIM kinase inhibitor LGH447. KPT-276 is a selective inhibitor of nuclear export CRM1/XPO1 inhibitor. Both drugs are active in Vk*MYC MM and are showing early evidence of promising clinical activity, increasing the PPV of the Vk*MYC model of MM to 64% (or 73% if HDACi are considered clinically active). Also active in the Vk*MYC model were the bromodomain inhibitors GSK I-BET151, CPI-203, CPI-456, OTX-015, which, like JQ1, compete with acetylated histones for the binding to BRD4, inhibiting super-enhancer activity and MYC transcription. The histone methyltransferase enhancer of Zeste homolog 2 (EZH2) induces transcriptional repression through histone H3 lysine 27 trimethylation (H3K27me3) at the promoter of silenced genes. Activating mutations in the SET domain of EZH2 are prevalent in germinal center DLBCL and follicular lymphoma but have not been identified in MM, where the frequent dysregulation of MMSET by t(4;14) translocation or biallelic deletion of UTX are thought to play a tumorigenic role equivalent to EZH2 activation. EZH2 inhibitors have demonstrated activity against lymphoma with EZH2 activating mutations. Interestingly, we found the EZH2 inhibitor CPI-169 active against Vk*MYC MM, identifying EZH2 as a promising new epigenetic target in MM. EDO-S101 is a novel drug resulting from the fusion of a molecule of bendamustine with a molecule of vorinostat, with the aim of increasing the efficacy of the alkylator through the HDACi-mediated chromatin relaxation that would make DNA more accessible to the damaging effect of bendamustine. It induced a high rate of response in Vk*MYC MM that was sustained for more than 3 months in mice receiving only 2 doses, one week apart. Remarkably EDO-S101 is the only drug we have identified with single agent activity in the very aggressive, multi-drug resistant Vk12653 transplant model of relapsed/refractory MM. Finally, 2 agents (anti-murine PD-L1 and LCL161) that primarily modulate the immune microenvironment, with little to no direct anti-MM activity, also induced responses. Anti-PD-L1 blocks immunosuppressive signaling from MM cells to PD1 on T cells. LCL161 is an IAP antagonist that leads to constitutive activation of the alternative NFkB pathway with direct stimulation of the innate and adaptive immune systems. Although antibodies to CD38 are among the most exciting new agents for the treatment of MM, they cannot be evaluated in Vk*MYC MM since murine normal or malignant plasma cells do not express CD38. In contrast while SLAMF7 is expressed in Vk*MYC MM, there is no murine equivalent of elotuzumab. In summary, we report 7 new drugs with single agent activity in Vk*MYC MM, nearly doubling the number of drugs or classes of drugs with promising anti-MM activity. Two (LGH447 and KPT-330) are already beginning to demonstrate clinical efficacy in MM. All of these agents should be prioritized for rapid evaluation in clinical trials of patients with MM. Disclosures Bergsagel: Novartis: Research Funding; Constellation Pharmaceutical: Research Funding; OncoEthix: Research Funding; MundiPharma: Research Funding.

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 Multiple myeloma (MM) is a predominantly incurable form of plasma cell cancer. Although immunomodulatory imide drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide constitute the backbone of most MM therapies, little is understood about how these drugs function to alter the tumor/microenvironment in vivo. Recently, it was demonstrated that IMiDs bind to the substrate receptor protein cereblon (CRBN) leading to the ubiquitination and degradation of multiple neoantigen protein targets, often DNA-binding zinc finger regulatory proteins such as ZFP91, IKZF1 and IKZF3. Interestingly, CRBN differs in amino acid sequence between mice and humans rendering mice non-responsive to IMiDs’ effects. This difference has made it difficult to study the complex pleotropic actions of IMiDs in vivo. Consequently, our lab has generated an immunocompetent transgenic mouse that expresses full-length human CRBN under the control of its endogenous regulatory elements (hCRBN+). We crossed this mouse with our well-established Vk*MYC mouse to generate a novel humanized Vk*MYChCRBN+ strain that develops human-like multiple myeloma disease, is sensitive to IMiD therapy, and maintains a fully competent immune system. The purpose of generating our Vk*MYChCRBN+mouse is to determine the mechanism of action of IMiDs on the tumor only, the host only, and to understand the cross-talk between tumor and microenvironment in a clinically relevant in vivo model of MM. We find that hCRBN+ mice are sensitive to IMiD treatment in vivo by displaying degradation of known target proteins such as Ikzf1/Ikzf3 in splenocytes. We also show that non-tumor immune cells such as T and NK cells respond to IMiD treatment by increased proliferation and expression of effector molecules such as IL-2 and IFN-γ. Furthermore, by engrafting IMiD-sensitive Vk*MYChCRBN+ MM cells into an IMiD-insensitive WT host we establish that IMiDs have tumor-intrinsic effects in vivo that are independent of the host’s immune system. These tumor-intrinsic IMiD effects synergize not only with dexamethasone, which has been shown clinically, but also with enhancer-targeting BET or p300/CBP inhibitors. Conversely, when engrafting IMiD-insensitive Vk*MYC MM cells into an IMiD-sensitive hCRBN+ host we were unable to demonstrate significant IMiD-induced host immune activation or tumor suppression. We conclude, based on our preliminary data, that IMiD activity is predominantly tumor-intrinsic. Moving forward, we are currently utilizing single-cell RNA sequencing to map the cellular and transcriptional changes across time in both tumor cells and the immune microenvironment during IMiD treatment. Ultimately, our goal is to gain mechanistic understanding in order to optimize clinical practice. Citation Format: Seth J. Welsh, Meaghen E. Sharik, Victoria M. Garbitt, Link R. Taylor, Daniel L. Riggs, Caleb Stein, Grady Day, Courtney J. Hillukka, Zach J. Hammond, P Leif Bergsagel, Marta Chesi. A clinically relevant mouse model to understand how IMiDs modulate the host-tumor immunolandscape in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4628.

Abstract MYC dysregulation, the most common genetic aberration in multiple myeloma, is frequently due to the translocation of super-enhancers to the MYC locus. Several drugs target proteins that are enriched at many of these enhancers including BRD4 (BET inhibitors, BETi) and Ikaros (IMiDs), although their mechanism of action remains poorly understood. Here we present a characterization of the responses to these drugs in a collection of over sixty myeloma cell lines having a diversity of MYC rearrangements. We found that the anti-proliferative effects of these drugs significantly correlated with changes in MYC protein levels, consistent with both drugs targeting MYC expression. Despite this common target, there was no statistically significant correlation between the individual BETi and IMiD responses, suggesting that they act through different mechanisms. Of those lines having extremes of sensitivity or resistance, there were two major groups (BETiS/IMiDS and BETiS/IMiDR), a smaller group of four lines resistant to both drugs individually (BETiR/IMiDR) and only one line was BETiR/IMiDS. In the BETiR/IMiDR group, resistance to BETi was mediated by a BRD4-independent mechanism as BRD4 was efficiently released from the MYC-associated enhancers. In all three of BETiR/IMiDR cell lines that we examined, treatment with BETi and IMiD together abolished proliferation and down-regulated MYC, consistent with parallel BRD4- and Ikaros-dependent pathways driving MYC expression. These resistant lines all expressed high levels of the transcription factor ETV4 and knocking out its gene sensitized a line to each drug individually. Thus ETV4 appears to be necessary for the parallel pathways driving MYC expression. There were nine lines in the BETiS/IMiDR group. Sensitivity to BETi in these lines could be explained by either low ETV4 expression or by BETi repressing Ikaros levels (which was only observed in BETiS lines, suggesting that BRD4 drives IKZF1 expression in these lines). Thus, in ETV4-containing lines, BETi sensitivity is due to the simultaneously targeting of the BRD4- and Ikaros-dependent pathways. IMiD resistance likely was due to several reasons. In one cell line, OCIMY5, IMiD had little effect on Ikaros levels, likely due to the previously reported low levels of Cereblon. Seven of the eight remaining lines expressed high levels of either ETV4, or the other potential super-enhancer binding factors IRF4 or RUNX1. In the eight BETiS/IMiDS cell lines, IMiD strongly reduced both Ikaros and Aiolos protein levels, which likely caused IMiD sensitivity. As with the BETiS lines described above, the lines in this group either lacked ETV4 or BETi repressed Ikaros levels. In conclusion, by examining drug response in a collection of genetically annotated myeloma cell lines we have been able to identify factors that contribute the broad range of responses to BETi and IMiDs in myeloma cells. Citation Format: Daniel L. Riggs, Camille Herzog, Victoria M. Garbitt, Niamh Keane, Courtney J. Hillukka, Zachary J. Hammond, Julia E. Wiedmeier, Seth J. Welsh, Shulan Tian, Huihuang Yan, Ranjan Maity, Nizar Bahlis, Paola Neri, W Michael Kuehl, Marta Chesi, P Leif Bergsagel. IMiDs and BET inhibitors target distinct pathways of MYC dysregulation by super-enhancers in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3015.

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.

Abstract The attrition rate for anticancer drugs entering clinical trials is unacceptably high. For multiple myeloma (MM), we postulate that this is because of preclinical models that overemphasize the antiproliferative activity of drugs, and clinical trials performed in refractory end-stage patients. We validate the Vk*MYC transgenic mouse as a faithful model to predict single-agent drug activity in MM with a positive predictive value of 67% (4 of 6) for clinical activity, and a negative predictive value of 86% (6 of 7) for clinical inactivity. We identify 4 novel agents that should be prioritized for evaluation in clinical trials. Transplantation of Vk*MYC tumor cells into congenic mice selected for a more aggressive disease that models end-stage drug-resistant MM and responds only to combinations of drugs with single-agent activity in untreated Vk*MYC MM. We predict that combinations of standard agents, histone deacetylase inhibitors, bromodomain inhibitors, and hypoxia-activated prodrugs will demonstrate efficacy in the treatment of relapsed MM.