Riggs, Daniel L.

Abstract Multiple myeloma (MM) is an incurable plasma cell neoplasm characterized by diverse and complex genetic abnormalities, including translocations of immunoglobulin (Ig) genes and a hyperdiploid (HRD) karyotype. Much of our current understanding of MM genetics, epigenetics, pathogenesis and response/resistance to myeloma directed therapies originated from studies on human MM-derived cell lines (HMCL). In this study, we conducted an integrated multi-omics analysis in five hyperdiploid cell lines established from serial samples from a single relapsed/refractory hyperdiploid MM patient, enabling detailed characterization of the genetic landscape and molecular alterations through disease progression.

Abstract Immunomodulatory drugs (IMiD) are a backbone therapy for multiple myeloma (MM). Despite their efficacy, most patients develop resistance, and the mechanisms are not fully defined. Here, we show that IMiD responses are directed by IMiD-dependent degradation of IKZF1 and IKZF3 that bind to enhancers necessary to sustain the expression of MYC and other myeloma oncogenes. IMiD treatment universally depleted chromatin-bound IKZF1, but eviction of P300 and BRD4 coactivators only occurred in IMiD-sensitive cells. IKZF1-bound enhancers overlapped other transcription factor binding motifs, including ETV4. Chromatin immunoprecipitation sequencing showed that ETV4 bound to the same enhancers as IKZF1, and ETV4 CRISPR/Cas9-mediated ablation resulted in sensitization of IMiD-resistant MM. ETV4 expression is associated with IMiD resistance in cell lines, poor prognosis in patients, and is upregulated at relapse. These data indicate that ETV4 alleviates IKZF1 and IKZF3 dependency in MM by maintaining oncogenic enhancer activity and identify transcriptional plasticity as a previously unrecognized mechanism of IMiD resistance. Significance: We show that IKZF1-bound enhancers are critical for IMiD efficacy and that the factor ETV4 can bind the same enhancers and substitute for IKZF1 and mediate IMiD resistance by maintaining MYC and other oncogenes. These data implicate transcription factor redundancy as a previously unrecognized mode of IMiD resistance in MM. See related article by Welsh, Barwick, et al., p. 34. See related commentary by Yun and Cleveland, p. 5. This article is featured in Selected Articles from This Issue, p. 4

Abstract Multiple myeloma (MM) is a malignancy that is often driven by MYC and that is sustained by IRF4, which are upregulated by super-enhancers. IKZF1 and IKZF3 bind to super-enhancers and can be degraded using immunomodulatory imide drugs (IMiD). Successful IMiD responses downregulate MYC and IRF4; however, this fails in IMiD-resistant cells. MYC and IRF4 downregulation can also be achieved in IMiD-resistant tumors using inhibitors of BET and EP300 transcriptional coactivator proteins; however, in vivo these drugs have a narrow therapeutic window. By combining IMiDs with EP300 inhibition, we demonstrate greater downregulation of MYC and IRF4, synergistic killing of myeloma in vitro and in vivo, and an increased therapeutic window. Interestingly, this potent combination failed where MYC and IRF4 expression was maintained by high levels of the AP-1 factor BATF. Our results identify an effective drug combination and a previously unrecognized mechanism of IMiD resistance. Significance: These results highlight the dependence of MM on IKZF1-bound super-enhancers, which can be effectively targeted by a potent therapeutic combination pairing IMiD-mediated degradation of IKZF1 and IKZF3 with EP300 inhibition. They also identify AP-1 factors as an unrecognized mechanism of IMiD resistance in MM. See related article by Neri, Barwick, et al., p. 56. See related commentary by Yun and Cleveland, p. 5. This article is featured in Selected Articles from This Issue, p. 4

Abstract Multiple myeloma (MM) is an incurable form of plasma cell cancer in which primary and secondary chromosomal translocations routinely juxtapose oncogenes to plasma cell-specific super-enhancers. Coincidentally, drugs which target super-enhancers have had success clinically. For example, immunomodulatory imide drugs (IMiDs) degrade super-enhancer-binding pioneer factors IKAROS and AIOLOS, while glucocorticoids (Dexamethasone) and proteasome inhibitors (Bortezomib) have the ability to transrepress or block the processing of super-enhancer-forming NF-κB proteins, respectively. Currently, alternative enhancer-targeting drugs are also in clinical development, like p300 inhibitors which target the acetyl-binding bromodomains and/or histone acetyl transferase activity of the chromatin-regulating coactivator homologs CBP and EP300. Despite showing therapeutic promise, our understanding of how these drugs function, alone or together, remains incomplete. Case in point, we find that IMiD-induced degradation of its target proteins IKAROS and AIOLOS does not guarantee a therapeutic response in vitro, and patients successfully treated with IMiDs eventually relapse; meanwhile, coactivator-targeting therapies like p300 inhibitors are often too toxic in vivo, and lack a therapeutic window. To improve the outcomes of MM patients we need to understand the heterogeneous genetics and transcription-factor milieus of the myeloma enhancer landscape, as well as how to increase the precision of enhancer-disrupting drugs. To accomplish this, our lab utilizes more than 60 human myeloma cell lines that have been extensively characterized at the genetic, proteomic, and drug-therapeutic-response levels. Additionally, we have generated a highly-predictive immunocompetent mouse model (Vk*MYC hCRBN+) that develops human-like MM and is sensitive to both IMiDs and a new class of therapeutics termed "degronimids" (normal mice do not respond to IMiDs or degronimids). Our central hypothesis is that combining a broad coactivator-targeting drug (e.g., the p300 inhibitor GNE-781), with a MM-specific transcription factor-targeting drug (e.g., IMiDs) restricts toxicities to myeloma cells and thus improves the therapeutic window. Currently, we are testing a variety of coactivator-targeting compounds alongside traditional IMiD therapies and other preclinical transcription factor-targeting drugs both in vivo and in vitro. We show that Vk*MYC hCRBN+ mice are exquisitely sensitive to GNE-781, requiring one fourth of the dose needed to treat other cancers and therefore avoiding the neutropenia and thrombocytopenia seen at higher doses. Second, we show that although IMiDs and GNE-781 induce an effective but transient response in vivo as single agents, the combination of the two drugs proved curative, with a progressive deepening of the anti-tumor response occurring even after therapy is discontinued. Ongoing experiments aim to determine how this drug combination, and other coactivator + transcription factor-targeting combinations, permanently disrupt myeloma-specific super-enhancers. Disclosures Neri: BMS: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Bahlis: Sanofi: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Genentech: Consultancy. Boise: AstraZeneca: Honoraria, Research Funding; AbbVie/Genentech: Membership on an entity's Board of Directors or advisory committees. Chesi: Abcuro: Patents & Royalties: Genetically engineered mouse model of myeloma; Pi Therapeutics: Patents & Royalties: Genetically engineered mouse model of myeloma; Pfizer: Consultancy; Novartis: Consultancy, Patents & Royalties: human CRBN transgenic mouse; Palleon Pharmaceuticals: Patents & Royalties: Genetically engineered mouse model of myeloma.

Abstract BCMA/CD3-targeting bispecific antibodies (BsAb) are a recently developed immunotherapy class that shows potent tumor killing activity in multiple myeloma. Here, we investigated a murine BCMA/CD3-targeting BsAb in the immunocompetent Vk*MYC model and its immunomodulatory imide drug (IMiD)–sensitive derivative Vk*MYChCRBN model of multiple myeloma. The BCMA/CD3 BsAb was safe and efficacious in a subset of mice but failed in those with high tumor burden, consistent with clinical reports of BsAb in leukemia. The combination of BCMA/CD3 BsAb with pomalidomide expanded lytic T cells and improved activity even in IMiD-resistant high–tumor burden cases. Yet, survival was only marginally extended due to acute toxicity and T-cell exhaustion, which impaired T-cell persistence. In contrast, the combination with cyclophosphamide was safe and allowed for a tempered proinflammatory response associated with long-lasting complete remission. Concurrent cytotoxic therapy with BsAb actually improved T-cell persistence and function, offering a promising approach to patients with a large tumor burden. Significance: BCMA-targeted therapy induces deep but transient clinical responses. We developed an immunocompetent, IMiD-sensitive genetically engineered mouse model and show that IMiDs potentiate T-cell activation, increasing short-term efficacy of anti-BCMA/CD3 BsAb, but exacerbate T-cell exhaustion. Surprisingly, by reducing tumor burden and depleting regulatory T cells, cyclophosphamide prevents BsAb-induced T-cell exhaustion and promotes long-term multiple myeloma control. See related commentary by Louvet et al., p. 297.

Abstract The most common genetic abnormality in multiple myeloma is the deletion of chromosome 13, seen in almost half of newly diagnosed patients. Unlike chronic lymphocytic leukemia, where a recurrent minimally deleted region including MIR15A/MIR16-1 has been mapped, the deletions in multiple myeloma predominantly involve the entire chromosome and no specific driver gene has been identified. Additional candidate loci include RB1 and DIS3, but while biallelic deletion of RB1 is associated with disease progression, DIS3 is a common essential gene and complete inactivation is not observed. The Vk*MYC transgenic mouse model of multiple myeloma spontaneously acquires del(14), syntenic to human chromosome 13, and Rb1 complete inactivation, but not Dis3 mutations. Taking advantage of this model, we explored the role in multiple myeloma initiation and progression of two candidate loci on chromosome 13: RB1 and MIR15A/MIR16-1. Monoallelic deletion of Mir15a/Mir16-1, but not Rb1, was sufficient to accelerate the development of monoclonal gammopathy in wild-type mice and the progression of multiple myeloma in Vk*MYC mice, resulting in increased expression of Mir15a/Mir16-1 target genes and plasma cell proliferation, which was similarly observed in patients with multiple myeloma. Significance: In the absence of a defined, minimally deleted region the significance of del(13) in multiple myeloma has remained controversial. Here we show that haploinsufficiency of Mir15a/Mir16-1, but not Rb1, upregulates the cell cycle–regulatory network, inducing monoclonal gammopathy in mice and promoting multiple myeloma progression in both mice and men. See related commentary by Walker, p. 16. This article is highlighted in the In This Issue feature, p. 5

Abstract MYC expression is frequently dysregulated in multiple myeloma (MM). In one comprehensive study, MYC structural variations (SV) were found in nearly half of MM cases (Affer et al. Leukemia 2014). The prevalence was higher in hyperdiploid (HRD) tumors (65%) compared to non-hyperdiploid (NHRD) tumors (36%). The large amount of tumor DNA required for all of the genomic studies performed may have biased the samples analyzed (e.g., to those with higher tumor burdens). To validate the findings of recurrent MYC SV in another dataset, we analyzed the CoMMpass data. We analyzed long-insert whole genome sequencing (WGS) data from diagnostic samples in 420 patients from the IA7 release (dbGAP phs000748) for SV. The results of clinical data, processed WES (SNV), RNASeq (gene expression) and WGS (copy number) data from IA8 (http://research.themmrf.org) were used to calculate survival, RAS and NFKB pathway mutation (WES), TC class (RNA), NFKB index (RNA) and hyperdiploid index (WGS). MYC SVs were identified in 38% of tumors. They were present in 53% HRD and 28% NHRD, and by TC in 55% D1, 43% D2, 36% MMSET, 26% MAF, 13% CCND. Juxtaposition of an Ig enhancer (IgH, IgK, IgL) close to MYC was the most common MYC SV, representing ~40% of the MYC SV. Other enhancers identified have mostly been reported previously, with the most frequent being NSMCE2 (12% of SV) and TXNDC5 (5% of SV). Intrachromosomal SV (deletions, inversions, duplications) not associated with any known enhancer were also frequent (18% of SV). As expected, MYC expression was higher in tumors with MYC SV compared to those without (~2.4 fold, p-value<0.0001). We used Gene Set Enrichment Analysis (GSEA) to identify activated pathways that might substitute for MYC in HRD patients without MYC SV and observed significant activation of the NFkB pathway. Interestingly, examining patients with RAS/MAPK pathway (NRAS, KRAS, BRAF, FGFR3 - abbreviated RAS) mutations (identified in 50% of all patients) the same pattern was observed: in the absence of RAS mutation, there was a significantly higher NFkB index. In general, the HRD tumors seem to have either activation of RAS and/or MYC, or activation of NFkB (Figure). There was no difference in overall survival in patients with versus those without MYC SV. We developed a clinically applicable sequencing platform to identify MYC SV, which cannot be reliably identified by FISH. We sought to validate this targeted capture approach, where in addition to the coding exons of 81 interesting genes described previously (M3P), we also pulled down the region surrounding MYC (2 Mb), IgH (0.5 Mb), IgK (50 kb) and IgL (100 kb) allowing us to additionally identify SV in MYC and Ig loci. Using this approach we identified IgH translocations in 29/30 samples with translocations previously identified by FISH (97%). Moreover, we identified MYC SV in 19/22 patients with SV previously identified by mate-pair WGS (86%). Importantly, sequencing identified the precise translocation breakpoint, and identity of the enhancer dysregulating MYC, which may be important variables. In one informative patient two different MYC SV were present at diagnosis, only one of which was still present following a partial response to four cycles of lenalidomide and dexamethasone. This suggests that the two MYC SV are in different subclones, one of which was much more sensitive to the treatment. Interestingly, the enhancer dysregulating MYC in the sensitive subclone harbors 5 strong Ikaros binding sites identified by ChIPseq, suggesting one intriguing mechanism for sensitivity to lenalidomide. To summarize, we verified in a large dataset that MYC expression is frequently dysregulated by SV in MM (38%), and the RAS/MAPK (50%) and NFKB (23%) pathways are frequently activated by mutations. Surprisingly, given their generally good prognosis, nearly half of HRD tumors seem to be MYC driven, while this is true for only a quarter of NHRD tumors. HRD tumors not driven by MYC or RAS appear to be driven by NFKB. Remarkably, the pathways most commonly activated by mutation in MM: CCND, MYC, RAS, NFKB are common to many cancers and have been studied extensively individually. To understand their clinical impact in MM we have developed a comprehensive custom capture sequencing panel that identified 97% of IgH translocations, 86% of MYC SV, and as well as SNV and CNV of 81 recurrently mutated genes. It will be important to include such a comprehensive genetic analysis to complement clinical trials in the future. Figure. Figure. Disclosures Stewart: Bristol Myers Squibb: Consultancy; Celgene: Consultancy; Takeda Oncology: Consultancy; Janssen Pharmaceuticals: 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.