Welsh, Seth J.

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 Bispecific T cell engagers (TCE) are novel immunotherapies for cancer that redirect a synthetic immune response against tumors. Early clinical trial data show that BCMA-targeting TCEs are highly effective immunotherapies for treating relapsed and refractory multiple myeloma (MM). Thus, it is important to define combinatorial approaches that increase the efficacy and durability of TCE. We recently investigated a murine anti-BCMA TCE in the immunocompetent IMiD-sensitive Vk*MYChCRBN model of MM. Our data show that anti-BCMA TCE was safe and efficacious in most mice, but failed in those with high-tumor burden, consistent with clinical reports of TCE in leukemia. We tested the combination of anti-BCMA TCE and IMiDs to provide an additive anti-tumor effect and stimulatory cytokines, such as IL-2 in the tumor environment. This combination expanded cytolytic T cells and improved activity even in IMiD-resistant high-tumor burden cases. Yet, survival was only marginally extended due to T cell exhaustion. We hypothesized that blockade of the inhibitory immune checkpoint PD-1 would synergize with TCE and IMiDs to potentiate the response directed by the TCE by reinvigorating exhausted T cells. We evaluated the efficacy of the triple therapy combination in Vk*MYChCRBN: the anti-BCMA TCE, pomalidomide, and anti-PD1. Checkpoint blockade alone, or in combination with TCE did not improve tumor control or survival compared to control treatments. The triple combination proved most effective at controlling tumor growth, inducing a complete response after 2 weeks of treatment, and was curative in 40% of the mice. Longitudinal analysis of the immune response showed that the addition of anti-PD1 to TCE-IMiDs rescued T cell expansion at tumor sites and sustained expression of activation markers after repeated doses. Longitudinal analysis of blood and bone marrow (BM) cytokines showed the typical profile associated with cytokine release syndrome (CRS) during T cell immunotherapy. Of note, we observed IL-2, IFN-γ, and GM-CSF production was sustained in the BM tumor environment in the triple combination. We suspect that these cytokines, especially IL-2, are key to sustaining non-exhausted effector T cell proliferation in response to TCE therapy. Expectedly, we observed toxicity with the elevated cytokine release in the triple combination. that we anticipate could be controlled with step-up dosing or CRS mitigation treatment. We conclude that combination of PD1 blockade, IMiDs and TCE provides superior efficacy in the immunocompetent preclinical model of MM, Vk*MYChCRBN, while increasing inflammatory pathways that can lead to CRS. Finally, as we found this combination to be curative in some mice, we will investigate whether this combination immunotherapy also enhanced an endogenous anti-tumor immune response leading to protective immunosurveillance. Citation Format: Erin W. Meermeier, Seth J. Welsh, Meaghen Sharik, Megan T. Du, Chang-Xin Shi, Bryant Chau, Feng Wang, Matthew Wheeler, Natalie Bezman, Pavel Strop, P. Leif Bergsagel, Marta Chesi. PD-1 blockade synergizes with IMiDs to enhance bispecific T cell engager immune responses to Vk*MYChCRBN multiple myeloma by preventing T cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5588.

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 BCMA is an ideal target antigen for myeloma immunotherapy as it is specifically expressed by plasma cells and T cell-engaging, BCMA-bispecific antibodies show potent tumor killing activity in human multiple myeloma (MM). However, a lack of longevity, potential immunotoxicity, and efficacy in high tumor burden situations are limitations to effective clinical utility. In order to optimize this therapeutic approach, we performed in vitro and in vivo assessments of a BCMA bispecific tool using the Vk*MYC immunocompetent mouse model of MM. Analysis at gene and protein level revealed variable levels of BCMA in Vk*MYC tumors, mirroring the heterogeneity of BCMA observed in human MM. In addition, we found that treatment with a gamma secretase inhibitor enhanced BCMA expression in Vk*MYC MM cells, similar to observations from patients. Having found the Vk*MYC model as a suitable model for BCMA targeted therapy, we treated Vk*MYC de novo and transplant mice with the BCMA targeting tool and established that the treatment was safe and efficacious (significant reduction in M-spike levels compared to control antibody). However, the response was transient and modest activity was noted in high tumor burden setting, likely because of poor T cell infiltration. Therefore, we hypothesized that combining BCMA therapy with agents capable of debulking the tumors would prolong and deepen therapeutic benefits and thus selected IMiDs for their anti-tumor and immunomodulatory properties. Because mice are resistant to therapy with IMiDs, we generated a humanized transgenic mouse strain with robust human CRBN expression and crossed it with the Vk*MYC mouse to obtain a novel immunocompetent, IMiD-responsive model, Vk*MYChCRBN. When combined with the IMiD pomalidomide (POM), bispecific-directed T cell control of MM was enhanced relative to bispecific alone. M-spike levels in two different transplantable models were reduced regardless of tumor burden. In addition, POM increased T cell effector activation, proliferation, and cytokine production. We also monitored transient weight loss unique to the bispecific-POM combination. Following this promising combinatorial result in vivo, we studied the relative roles of POM on target vs. effector cells by an in vitro killing assay using Vk*MYC tumor and splenocytes with or without expression of CRBN. Separately from the direct tumoricidal effects of POM on the tumor, we observed T cell-intrinsic effects of POM on the enhancement of BCMA-bispecific directed tumor lysis that correlated with T cell activation and proliferation. In summary, POM potentiated BCMA-CD3 bispecific directed T cell cytolysis of MM in the Vk*MYChCRBN animal model by enhancing effector cell activation, proliferation, cytokine production and directly reducing tumor burden. These results support ongoing preclinical studies of the benefits of combining standard of care therapy with potent T cell immunotherapy for MM. Citation Format: Erin W. Meermeier, Meaghen E. Sharik, Seth J. Welsh, Caleb K. Stein, Victoria M. Garbitt, Sochilt Brown, Kennedi T. Todd, Marco Bergsagel, P. Leif Bergsagel, Marta Chesi. Anti-BCMA bispecific tool therapy against Vk*MYC multiple myeloma is enhanced by IMiDs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5630.

Abstract Previously, the IAP antagonist LCL161 was shown to have robust anti-myeloma activity in a transgenic mouse model where an acute inflammatory response accompanied a reduction in tumor burden. In order to fully examine alterations to the immune microenvironment, murine splenocytes from twenty mice transplanted with Vk12598 myeloma and administered single-agent treatment regimens of LCL161, cyclophosphamide, or bortezomib were harvested after 24 hours and subjected to single cell RNA-seq sequencing. After initial removal of erythrocytic and mitochondrial expressing cells, count matrices of over 50,000 total cells span twenty murine whole spleen samples. In an examination of the myeloid component, we identified 13 clusters of immune cells with unique compositions across samples due to differing treatment regimens and initial tumor burden. While minimally present in all seven non-transplanted mice, macrophages with elevated expression of complement activation system genes (C1q) were the most abundant myeloid cell population in each of our untreated, Vk12598 transplanted mice. Alternatively, populations of cDC2, pDC, moDC, and monocytes lacking expression of MHC class II markers were fixtures for healthy controls while markedly less abundant in tumor-bearing mice. Tumor burdens in three samples subjected to LCL161 therapy were significantly reduced 24-hours post treatment and accompanied by an expansion of late-stage neutrophils and migratory DCs with expression features mirroring those of DCs from mesenteric lymph nodes. This composition of immune cells was distinct from all other sample including those treated with cyclophosphamide or bortezomib. In addition, relative proportions of B, T, and NK cells were nearly fully reconstituted, matching levels observed in healthy controls, in two of these samples. In a direct functional assessment, expression levels of Fscn1 and Ccr7 (key over-expressed genes in these migratory DCs) markedly increased following treatment of bone marrow derived DCs with LCL161. Overall, the transcriptional profile of these migratory DCs indicates their pivotal role in maturation of DCs (Fscn1, Tmem123) as well as the recruitment of T and NKT cells (Cxcl16, Ccr7) and increase in antigen presentation (Cd63)–all of which may serve to ‘warm' tumors, increasing T cell infiltration necessary for immunotherapy approaches. With the increased resolution of scRNA-seq, we observe unique alterations in the composition of the immune microenvironment of MM induced by treatment with LCL161 that further detail its unique mechanism of anti-MM activity. Citation Format: Caleb K. Stein, Erin Meermeier, Seth Welsh, Victoria Garbitt, Meaghen Sharik, P. Leif Bergsagel, Nicholas Banovich, Jonathan Keats, Marta Chesi. scRNA-seq reveals restructuring of the immune microenvironment of multiple myeloma following treatment with an IAP antagonist [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5622.