Stein, Caleb K.

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 Background: Immunoglobulin (IGH, IGL, IGK) and non-immunoglobulin (PVT1, TXNDC5, FAM46C, DUSP22, etc.) enhancers hijacking by variable genes (MYC, MAF, MAFB, CCND1/2/3, MMSET, IRF4) is a recognized oncogenic driver event in MM. However, the identity of the transcription factors (TFs) or transcriptional regulatory complexes binding and regulating the activity of these enhancers remains to be fully elucidated and may yield valuable therapeutic targets. As such the discovery of the BET family member BRD4 as the master histone acetyl mark reader at enhancers loci regulating MYC lead to promising therapeutic developments in MM and numerous other cancers. Immunomodulatory drugs (IMiDs) promote the proteasomal degradation of IKAROS (IKZF1) and AIOLOs (IKZF3) leading to the transcriptional repression of MYC and the suppression of MM cells survival and proliferation. However, acquired resistance to IMIDs and the loss of the transcriptional repression of MYC are nearly universal and occur in spite of sustained IKZF1/3 degradation suggesting that transcriptional rewiring may be sustaining hijacked enhancers activity and transcription of driver oncogenes. Methods and Results: In order to define how IMiDs repress MYC transcription, we first defined IKZF1, BRD4, the lysine acetyl transferase P300 and the mediator complex subunit MED1 mapping within the MM genome using ChIPseq. In MM cell lines (MM1S, RPMI8226, ARP1 and AMO1), IKZF1 predominantly mapped to intronic and intergenic loci which are typically enriched with enhancer and superenhancer elements. Indeed, IKZF1 mapping to the genome nearly completely (96.5%) overlapped that of P300, MED1 and BRD4 co-occupied enhancer and superenhancer loci. We also confirmed that in the MM1S sensitive cell lines IMiDs (lenalidomide 10 μM, 24h) exposure efficiently depleted IKZF1, BRD4, P300 and MED1 at enhancer loci with ensuing MYC (and MAF) downregulation. In contrast, in resistant cell lines (RPMI8226) and in spite efficient IKZF1 displacement, BRD4, P300 and MED1 were retained at the oncogenic enhancer (IGLL5) driving MYC (and MAF). These findings lead us to postulate that in IMiDs resistant cells retention of BRD4 and MED1 at oncogenic enhancers in the absence of IKZF1 likely results from rewiring of the TFs regulating MYC. To identify TFs that may co-localize with BRD4 and IKZF1, we analyzed the enrichment of DNA motifs at IKZF1and BRD4 co-occupied enhancers using the MEME suite motif-finding algorithms. This computational analysis revealed a strong enrichment at these MM enhancers of the GGAA motif recognized by the ETS family of TFs (P= 3.2 e-743) and other motifs boxes for the RUNX (P= 9.6 e-725), MYC/MYB ( P= 8.8 e-52) and interferon regulatory (IRF) (P= 3.1 e-293) TFs. We next confirmed that the ETS family TF ETV4 was indeed expressed in IMiDs resistant, but not sensitive, MM cell lines. ChiPseq occupancy profiles in IMiDs resistant RPMI8226 cell line revealed co-localization of ETV4 with IKZF1, P300 and BRD4. As predicted, lenalidomide treatment induced global depletion of IKZF1 but not ETV4 at BRD4 occupied enhancers in resistant cell lines (RPMI8226 and ARP1). Importantly, Cas9-mediated knock out of ETV4 in RPMI8226 cells sensitized them to lenalidomide with ensuing MYC downregulation and cell death. Confirming its role in MM, ETV4 transcript was indeed detectable in primary patients' samples in the CoMMpass data repository (ETV4 FPKM >1.0 in 112/724) and its expression was associated with significantly reduced survival outcomes (HR 0.64; P=0.0008). Similarly, high expression (top quartiles) of RUNX2 or MYB, TFs with enriched motifs at IKZF1 co-occupied enhancer loci, was also associated with decreased survival. Of note RNAseq analysis of paired patient samples pre- and post-IMiDs treatment (n=14 pairs) revealed significant upregulation of ETV4 at the time of acquired IMiDs resistance (7/14). Lastly transcriptome analysis of 101 patients enrolled in the RD arm (lenalidomide and dexamethasone) of the POLLUX trial (NCT02076009) confirmed the reduced survival of patients with top quartiles expression of ETV4 as well as MYB and RUNX2 (Fig.1) Conclusion: Transcriptional plasticity with expression of extra-lineage TFs such as the ETS family member ETV4 sustains oncogenic enhancers in MM overcoming IKAROS and AIOLOS dependency and promoting IMiDs resistance. Figure 1 Figure 1. Disclosures Neri: Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Soong: Jannsen: Employment. Chiu: Janssen: Employment. Bahlis: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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.

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

Introduction Despite continuous improvement of clinical outcome in multiple myeloma (MM), disease relapse remains a major challenge, leading to progressively shorter remissions and fewer treatment options. Strategies attempting to counteract this challenge include recent efforts resulting in an increase in the availability of novel promising anti-MM agents and targeting specific genetic profiles of the disease. In this context, we aim to develop predictive models of sensitivity and resistance to novel compounds by connecting an ex vivo high-throughput drug screen with genetic, transcriptomics, FISH, and clinical features. Methods Twenty compounds (afatinib, afuresertib, belinostat, buparlisib, cobimetinib, CPI-0610, crenolanib, dinaciclib, dovitinib, JQ1, LGH447, osimertinib, OTX015, panobinostat, romidepsin, selinexor, sunitinib, trametinib, venetoclax, and vorinostat) were selected based on overall promising anti-MM activity from an ex vivo high throughput drug screen with a panel of 79 single agents incubated for 24 hours. The area under the curve (AUC) was used to rank order the ex vivo responses for each compound and the lowest and highest quartile samples were identified for further analysis. Clinical data and FISH data, including t(11;14), t(4;14), t(14;16), del(17p), +1q, monosomy 13, and MYC rearrangement, were collected. Targeted DNA sequencing was performed using a 2.3 Mb custom capture panel covering 139 MM-relevant genes. mRNA-sequencing was performed and differential gene expression analysis in the highest and lowest quartile identified subsets of markers positively and negatively associated with the AUC response for a given compound. An additional unbiased selection of markers using lasso techniques was performed, resulting in predictive generalized linear models (GLM) for each agent. Responses from the remaining intermediate samples were estimated with the predictive models, with overall predictive ability assessed by correlating predicted AUCs with their actual counterparts. Results Our integrative analysis was performed on 50 primary patient samples (36% untreated and 64% relapsed MM). Venetoclax, dinaciclib, romidepsin, panobinostat, osimertinib, belinostat and selinexor were the most active compounds in the cohort. Interestingly, LGH447, dovitinib, selinexor, JQ1, OTX-015, cobimetinib, and trametinib showed increased activity in relapsed MM when compared to untreated samples (Wilcoxon Test; p<0.05). We generated GLMs using an average of 92 markers (range 64-107) per compound, combining mRNA-sequencing expression with FISH and mutation data. The analysis proposed in the present study was validated through the unbiased selection of BCL2 among the subset of markers included in the GLM predicting sensitivity to venetoclax, a first-in-class orally bioavailable selective BCL2 inhibitor. Expression level of critical NF-kB and cell cycle genes, such as BIRC3, CKS1B, PAX5, NFKB2, and CCND2, were included in 60% of our predictive models. Mutations of DNA repair genes (ATM,TP53) were included in the GLMs of three epigenetic therapies, one histone deacetylase inhibitor and two BET inhibitors, associated to ex vivo resistance to the drugs. The presence of monosomy 13 was also a marker for ex vivo resistance for five epigenetic therapies, four HDAC inhibitors and one BET inhibitor. The three BET inhibitors, JQ1, CPI-0610, and OTX015, were among the compounds most accurately predicted by our integrative approach, with Spearman correlation values between 0.773-0.858. Overall, our models accurately predicted the ex vivo response for 16 (80%) of the compounds (r>0.7). Five (25%) of these compounds displayed a remarkably accurate prediction model in both training (highest and lowest quartiles) and validation (intermediate quartiles) samples (r>0.8). Conclusions The GLM data integration approach enabled the establishment of effective predictive models, identifying FISH, transcriptomics, and mutations of putative driver genes important in anti-MM agent responsiveness. In addition, the resulting dataset is promising for future research focusing on the discovery of novel mechanisms of action and establishing markers of sensitivity and resistance to novel compounds. We are currently increasing our dataset and seek to create an omnibus approach that predicts responses to multiple anti-MM agents simultaneously. Disclosures Bergsagel: Celgene: Consultancy; Ionis Pharmaceuticals: Consultancy; Janssen Pharmaceuticals: Consultancy. Stewart:Amgen: Consultancy, Research Funding; Bristol Myers-Squibb: Consultancy; Celgene: Consultancy, Research Funding; Ionis: Consultancy; Janssen: Consultancy, Research Funding; Oncopeptides: Consultancy; Ono: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Takeda: Consultancy.

Background: Chimeric Antigen Receptor (CAR) T cell therapy is a promising cancer immunotherapy that is growing exponentially. The doubling time of medical knowledge in 2010 was 3.5 years, and the projection for 2020 is just 73 days. In the last five years, the number of PubMed publications on cancer applications of CAR T cells has tripled. Therefore, to remain updated in the field represents a challenge for patients, care providers and researchers. In this review we provide a focused summary of the currently ongoing clinical trials, with a comprehensive overview of advances in CAR T cell therapy, beyond CD19, emphasizing on antigenic targets, development phases, and leading sponsor pharmaceutical companies. Methods: We retrieved the available data from the national registry of clinical trials (clinicaltrials.gov) using the following keywords: "CAR T cell", "CAR T cell and cancer", "chimeric antigen receptor", "CAR T AND tumor antigen", 'CAR T cell antigens", "Tumor antigens targeted by CAR T cells", "engineered T cells", "modified T cell", "CAR T cells in Cancer", "CAR T cell therapy", "CAR T cell therapy AND Cancer" until December 31, 2018 and manually excluded the trials unrelated to CAR T-cell therapies on cancer, by reviewing the detailed information provided on the website as well as preliminary data published. Results: The analysis included 271 clinical trials posted on the clinicaltrials.gov website from the United States by the cut-off date. For efficacy analysis, we retrieved information from 52 trials, by NCT number on a PubMed search. The majority of CAR T clinical research is focused on hematological cancer (57%), followed by CNS 8%, GI 6%, Skin 5%, Genitourinary 4%, Breast 4%, Gynecologic 4%, Respiratory 3%, Sarcoma 2%, Mesothelioma 2% and others 5%. The most used target in CAR T cell therapy and the leaders in phase 3 trials are CD19 (42%) and BCMA (12%), followed by CD20, NY-ESO-1, Mesothelin, HER2, GD2, MAGE-A3 and CD30. An essential step in CAR T cell therapy development is the selection of the right antigen/target. Here, we provide an overview of the clinically relevant targets that are actively being using by clinical trials in the United States. For example, CD19 appears to be a leading target regarding CAR T cell therapy on cancer with 116 trials (42% of total CAR T cells trials) on going just in the United States with a significant increment in the previous years. Similarly, with BCMA is one of the targets with more phase 3 trials (Figure 1) with promising results on patients with Multiple Myeloma with and the objective response of 85%, CR 45%, and PFS of 11.8 months. Second-generation CARs with either CD28 or 4-1BB as costimulatory signaling domain are preferred, with 4-1BB being the most commonly chosen. Conclusions: Our findings show growing trends in the development of CAR T cell-based therapies, combination and possible retargeting therapies in the future for solid tumor and hematologic malignances; taking into account the amount of important information and the complexity of the database, we have developed this analysis to understand how to generate in the future a friendly platform for researchers and patients to have an detailed overview of the clinical trials in cellular therapies Disclosures No relevant conflicts of interest to declare.

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.