Shi, Chang-Xin

Abstract Multiple myeloma (MM) with t(11;14)(CCND1;IGH) remains the only subset sensitive to the BCL2 inhibitor venetoclax. However, not all patients with t(11;14)(CCND1;IGH) respond to treatment, and some progress early after initial response. To investigate this, we examined 44 whole-genome and whole-exome sequencing data samples from 34 patients with t(11;14) MM treated with venetoclax. The presence of mutations in the RAS pathway was strongly associated with shortened progression-free survival (PFS) and was validated in an independent cohort of 21 patients with MM. The presence of 1q gain was also associated with shorter PFS in patients without RAS mutations. In 10 patients with paired prevenetoclax and postvenetoclax treatment samples, postvenetoclax progression was recurrently driven by the selection of genomic events in the BCL2/MCL1 and RAS pathways and of high-risk features (eg, loss of TP53 and CDKN2C). Overall, our study shows that comprehensive genomic profiling can identify most mechanisms underlying resistance to BCL2 inhibition in t(11;14)(CCND1;IGH) MM.

Abstract Overview: The emergence of effective immune therapies for multiple myeloma has highlighted the importance of understanding the roles of less abundant components within the tumor microenvironment. Single-cell analyses provide snapshots of the functional relationships of individual cells at precise moments in time, as well as insight into the overall cellular composition. By integrating single-cell datasets from multiple studies, we improve our ability to identify trends within rare cell populations, such as macrophages, and explore their clinical implications. We investigate human data here because our prior research of murine models indicated that macrophages expressing C1q, particularly those co-expressing Fcgr4, tend to correlate positively with increased tumor burden. Methods: We combined 56 human bone marrow samples – primarily collected at baseline, post-induction, or post-ASCT – with three publicly available data sets: GSE210079 (post CAR-T bone marrow microenvironment); GSE223060 (single-cell analysis of the MMRF cohort); and GSE271107 (disease progression from healthy donors and MGUS to SMM and NDMM). These data sets were integrated using Seurat to generate robust cellular clusters from over 300,000 cells across 117 samples. We validated our single-cell findings with bulk Affymetrix, whole bone marrow gene expression data (collected at baseline and post-baseline) from the Total Therapy clinical trials (GSE136324). Results: A distinct cluster of C1QA/B/C-expressing macrophages was consistently identified in all single-cell cohorts. These cells co-express FCER1G, CSF1R, and CD68 (markers also expressed in murine C1Q macrophages) and typically cluster adjacent to FCGR3A-expressing monocytes. Within the disease progression cohort, C1Q macrophages were found in all phases of progression: healthy donors, MGUS, SMM, and NDMM. However, C1Q macrophages in progressive disease (SMM and NDMM) have distinct expression features including lower levels of MRC1 and higher levels of APOBEC3A and ISG15 – genes linked to inflammatory responses. Among 11 post-induction samples submitted to lenalidomide maintenance, five with shorter responses displayed elevated expression of APOBEC3A and ISG15 in C1Q-expressing macrophages compared to six samples with longer responses. In a cohort of 22 samples 100 days post-ASCT, seven poor responders showed both an increased frequency of C1Q macrophages and elevated APOBEC3A expression compared to good responders. The MMRF cohort indicated that higher baseline frequencies of C1Q macrophages often preceded rapid disease progression (e.g. MMRF_1720, MMRF_1641). Anecdotal evidence from the CAR-T therapy cohort suggests that expansion of C1Q macrophages could serve as an early warning for tumor recurrence; sample 16 showed retention and expansion of C1Q macrophages 28 days post-CAR-T, that was subsequently followed by increased tumor cell counts at 3 months post-therapy. Analysis of bulk, whole bone marrow gene expression revealed that elevated levels of C1Q genes correlated with poorer outcomes, particularly in post-baseline samples. Multivariate Cox survival analysis revealed that high C1Q expression independently predicted inferior overall survival in post-baseline samples after adjusting for age, ISS stage, high levels of tumor-associated markers (SDC1, JCHAIN, DERL3), and high-risk UAMS7 subtypes (MF, MS, PR). Moreover, post-baseline GEP70 risk scores (from matched bone marrow aspirates) positively correlated with elevated C1Q expression in whole bone marrow samples. Conclusion: Both single-cell and bulk analyses indicate that the persistence of immunosuppressive C1Q macrophages following therapy is associated with inferior clinical outcomes. Although the precise mechanisms are yet to be fully understood, the close proximity of similar macrophages to tumor cells–as observed in other cancers–likely also occurs in myeloma and reflects the crucial relationship between tumor cells and macrophages within the bone marrow. While C1Q macrophages are typically rare in individual samples, analysis of combined cohorts is building evidence of their crucial role in immune cross-talk and therapeutic resistance mechanisms.

Overview: Despite advances in therapies targeting the tumor-intrinsic aspects of the disease, multiple myeloma (MM) remains functionally incurable. Given that new alternatives such as T cell redirected therapy and monoclonal antibodies engage ADCC and CDC by leveraging other elements of the immune system, we believe that deeper studies of the tumor microenvironment are warranted. In particular, we believe that certain macrophages are associated with tumor progression, serve as markers of future relapses, are immunosuppressive, and reduce the effectiveness of otherwise successful therapies. Methods: We performed 5' scRNAseq analysis of 189,025 cells collected from spleen or bone marrow of 4 wild type, 14 de novo, and 40 tumor bearing Vk*MYC mice treated with vehicle or effective anti-MM therapies. Discoveries were cross-referenced against a set of 45,626 human bone marrow cells collected from 35 samples at different stages of disease and treatment. We experimentally recreated in vitro the cross-talk between MM cells and macrophages by co-culturing bone marrow derived macrophages (BMDM) with MM cells or their conditioned medium, and correlated changes in gene expression as well as the ability of polarized BMDM to inhibit CAR T mediated killing of MM cells in vitro. Results: Gene expression analysis of murine in vivo C1q macrophages and in vitro polarized BMDM revealed a common signature identified in lipid-associated macrophages and Kupffer cell-like macrophages1 (high expression of Fabp5, ApoE, Cd63, C1qa/b/c, Cd163, Cd5l, Vcam1, Fcgr4, Folr2, Maf) which is associated with immunosuppression and disease progression. Ligand-receptor analysis via LIANA identified C1qb ligand in macrophages with C1qbp in tumor among the top interactions in both human and murine single cell analyses. C1qbp is a crucial element of cell adhesion and metastasis,2 and this link between tumor and macrophage can severely limit effective therapy: BMDM following stimulation/polarization with MM lines, but not naïve/unstimulated BMDM, strongly inhibit anti-BCMA CAR T in vitro cytotoxic activity. In total, a complement-related connection between tumor and macrophage is capable of directing an immunosuppressive, chemokine response that imparts resistance to therapy. While effective therapies reduce tumor and C1q expressing macrophage populations,3 retention of C1q expressing cells may be associated with future relapses. Presence of a specific subset of C1Q expressing cells, even in very low numbers, in human samples following ASCT was associated with early relapse (6 of 8 good responding samples had zero C1Q+ cells while 12 of 16 poor responding samples had 1 or more C1Q+ cells-Fisher test p-value < 0.05). In addition to high expression of C1QA/B/C, these cells are also highly upregulated in TYROBP (known to relate to macrophage polarization4 and found in a spatial transcriptomic analysis of cells at the periphery of MM and its environment5) as well as FCER1G (known to relate to macrophage infiltration6). Conclusion: As advanced therapies manipulate elements of the immune microenvironment to impart effective treatment, the connection between tumor and macrophage should not be overlooked since macrophages likely help create an immunosuppressive niche that assists in disease growth and eventual revival. 1. Ma, R.-Y., Black, A. & Qian, B.-Z. Macrophage diversity in cancer revisited in the era of single-cell omics. Trends Immunol. 43, 546-563 (2022). 2. Wang, Y. et al. C1QBP suppresses cell adhesion and metastasis of renal carcinoma cells. Sci. Rep. 7, 999 (2017). 3. Stein, C. K., Meermeier, E., Welsh, S. J., Chesi, M. & Bergsagel, P. L. Key Subsets of Immune Cells in Bone Marrow Myeloid Compartment Indicate Degree of Tumor Infiltration and Avenues for Successful Immunotherapy in Multiple Myeloma. Blood 140, 7082-7083 (2022). 4. Liang, T. et al. TYROBP, TLR4 and ITGAM regulated macrophages polarization and immune checkpoints expression in osteosarcoma. Sci. Rep. 11, 19315 (2021). 5. Sudupe, L. et al. Spatial Transcriptomics Unveils Novel Potential Mechanisms of Disease in a MI cγ1 Multiple Myeloma in vivo Model. Blood 142, 4076 (2023). 6. Dong, K. et al. FCER1G positively relates to macrophage infiltration in clear cell renal cell carcinoma and contributes to unfavorable prognosis by regulating tumor immunity. BMC Cancer 22, 140 (2022).

Primary resistance to BCMA-directed bispecific antibodies (BCMA-BsAb) is a major clinical hurdle despite their outstanding efficacy for relapsed and refractory multiple myeloma (MM). Primary resistance has been linked clinically to patient characteristics including tumor burden. Our previous work in the immunocompetent Vk*MYC model of MM credentialed that tumor burden also correlates with resistance to a murine BCMA-BsAb, which we addressed through drug combinations with MM backbone agents. Pairing the BsAb with cyclophosphamide (Cy) was well-tolerated, reversed primary resistance, reshaped an immunosuppressive tumor microenvironment (TME) and led to durable responses with immunosurveillance in all but the most aggressive settings. Here, we aimed to improve the BCMA-BsAb and Cy combination strategy with a goal of curing models representing clinically relevant Cy-refractory aggressive settings, by adding T cell checkpoint blockade. Despite a lack of single agent activity of checkpoint inhibitors in MM (and in our models), PD1 and TIGIT are relevant targets as their expression on T cells correlates with both primary and acquired resistance to BCMA-directed cell therapies. Our team hypothesized that blockade of either PD1 or TIGIT with concurrent BCMA-BsAb and Cy would deepen anti-MM responses and provide a more durable immunotherapy regimen by sustaining activation of the T cell repertoire. Methods: We preclinically tested the ability of two weeks of concurrent dosing of BCMA-BsAb with Cy, checkpoint inhibitors targeting either PD1 or TIGIT, or the triple combination to overcome primary resistance and improve survival in a Cy-resistant transplantable Vk*MYC model of MM. We followed the response longitudinally in the blood, and in the bone marrow and spleen after complete responses were achieved at five weeks. Results: The double combination of a checkpoint inhibitor to the BCMA-BsAb was well-tolerated and abolished primary resistance, yet, failed to provide curative responses. Whereas the triple combination with either PD1 or TIGIT blockade remained safe, provided robust initial responses that overcame primary resistance and resulted in durable complete and curative responses in the entire cohort. These survival benefits represent significant improvements over control combinations tested here but also over all historical preclinical anti-MM therapy tested in our models to date. Both PD1 and TIGIT blockade with Cy/BCMA-BsAb triple combinations delivered a similar multiphasic potent anti-tumor response. This response was first characterized by highly effective T cell-mediated lysis of the tumor, as evidenced by deep reduction of M-spike after two weeks of treatment. As we previously published, the addition of Cy to BCMA-BsAb rapidly reshapes the TME by depleting Tregs and exhausted T cells and brings naive T cells to the tumor. However, we observed elevated T cell checkpoint expression at times of BsAb-relapse in aggressive models, suggesting that blockade of these pathways may help sustain T cell activation. Indeed, the ensuing T cell response in the triple combination gradually builds and encourages a pool of less terminally differentiated T cells to proliferate to allow for a more sustained response with repeat dosing. For the first time, we observe that the triple combination significantly engages the CD4 T cell compartment, which can be essential for a complete anti-tumor response. At the time of complete response, single cell analysis displays that T cells retained in the BM were less exhausted and defined by elevated CCR2 expression, a known marker of activated memory T cells, suggestive of ongoing immunosurveillance. Conclusions: Here we demonstrated through controlled in vivo experimentation how the addition of Cy reverts primary resistance to BCMA-BsAb by reshaping the TME in our Cy-insensitive preclinical model. Adding checkpoint inhibitors targeting TIGIT or PD1 further improves the durability of BsAb and Cy combination to cure all mice in these cohorts, by critically lysing the tumor, resetting the suppressive TME, and instigating a gradual, multiphasic, and sustained anti-MM T cell response. The concurrently delivered triple combination is well tolerated and provides optimal temporal control of MM especially in a preclinical model reflecting primary resistance mediated by high tumor burden and may have translational benefit to MM patients.

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.