Stein, Caleb K.

Abstract Purpose: Naturally occurring tumor-specific T cells derived from peripheral blood (PB) offer a clinically accessible source for adoptive immunotherapy. However, the expansion of these T cells from patients with cancer remains a challenge. We hypothesized that mimicking innate immune activation could optimally stimulate antigen-driven T-cell expansion in vitro, unlocking the therapeutic potential of PB-derived T cells. Patients and Methods: We previously developed an ex vivo culture system that selectively expands tumor antigen–activated T cells from PB mononuclear cells (PBMC), generating multiclonal effector and central memory T cells. In this hypothesis-generating study, we evaluated the therapeutic potential of MUC1-activated T cells in patients with relapsed and/or refractory multiple myeloma (r/rMM) in a phase I clinical trial (NCT05411497). MUC1 is an oncoprotein overexpressed in r/rMM. We translated our small-scale culture into GMP-compliant, large-scale manufacturing, which achieved expansion of T cells from heavily pretreated patients’ PBMCs without exhaustion. Five patients were treated with escalating doses of up to 1 × 1010 T cells. Results: Although the cell infusions were well tolerated, no objective responses occurred. One patient, who received the highest dose, has had stable disease for 2 years after infusion. This patient exhibited transient dermatitis with localized MUC1 and CD3 staining, as potential evidence of on-target, off-tumor T-cell reactivity, perhaps contributing to disease stabilization. T-cell receptor sequencing revealed the T-cell product in four of the patients’ blood, which correlated with the product’s degree of polyfunctionality and MUC1 reactivity. Conclusions: These findings demonstrate the feasibility, safety, and biological activity of PB-derived, MUC1-specific T cells as adoptive immunotherapy. Significance: Expanding tumor antigen–specific T cells for clinical use from the blood of heavily pretreated patients with cancer remains a significant hurdle. We scale up a novel culture method that integrates innate immune signals to stimulate T-cell expansion and demonstrate safety, feasibility, and a case of long-term disease stabilization upon treatment of patients with multiple myeloma in a phase I clinical trial.

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 Background: B-cell maturation antigen (BCMA)-targeted T cell engagers (TCEs) have emerged as a highly potent immunotherapy for relapsed/refractory multiple myeloma (MM). However, both primary and acquired resistance significantly limit the durability of responses. Antigen escape and development of dysfunctional T cell responses are a significant driver of relapse post-therapy. Here, we preclinically interrogate if continual versus intermittent/response-adapted dosing differently affects development of resistance, the translatability to clinical resistance mechanisms, and how tumor-intrinsic or microenvironmental factors shape the development of resistance to BCMA-TCEs. Methods: We utilized a syngeneic transplantable Vk*MYC MM model which permits clonal tracking through unique immunoglobulin rearrangements. Two cohorts of tumor-bearing mice were treated with a murine surrogate BCMA-TCE using distinct strategies: (1) continuous weekly dosing until disease progression, and (2) an “ intermittent/response-adapted” strategy, where treatment was paused after initial response and resumed upon detection of M-spike. At time of relapse on therapy, RNA-seq was performed on isolated tumor cells to investigate mutations in- or loss of Tnfrsf17/BCMA. Single-cell RNA sequencing (scRNA-seq) was also performed on 189,025 cells isolated from bone marrow and extramedullary sites. Results: Both continuous and intermittent treatment regimens resulted in similar overall survival. BCMA-TCE treatment induced a distinctive pattern of disease progression, with para- and extra-medullary dissemination observed in 31% (17/54) of treated mice—regardless of treatment schedule. Given this treatment-induced dissemination hasn't been historically observed in our model, we further profiled BCMA expression and infiltrating immune cells to understand relapse mechanisms at these sites. Furthermore, we noted lower BCMA expression in tumors from continuously treated mice compared to untreated or stop-and-go cohorts. Importantly, reduced BCMA expression occurred without loss of plasma cell identity indicating that lineage switching may not be occurring as observed in other hematological malignancies treated similarly (Ruella, 2023, Nat Rev Drug Discov); malignant cells retained high expression of canonical plasma cell markers Irf4, Prdm1, Xbp1, Sdc1. We identified three mechanisms of acquired resistance:Genomic BCMA loss: Complete and irreversible loss of BCMA due to retrotransposition of IAP particles into the first intron of Tnfrsf17, disrupting transcription of downstream exons. This represented a genomic mechanism of antigen escape occurring with continual and prolonged TCE dosing, disrupting transcription and eliminating surface BCMA.Transient BCMA downregulation: Transient downregulation of BCMA expression associated with rapid disease relapse under continuous treatment. Tumors exhibited low BCMA levels and widespread dissemination in paramedullary sites. However, re-transplantation into naïve hosts led to restoration of BCMA expression and re-sensitization to TCE, indicating antigen loss was not permanent.Resistance without antigen loss: In the intermittent dosing regimen, several cases achieved complete remission after two doses, relapsed after treatment pause, and were not controlled after subsequent retreatment. Despite preserved BCMA expression and absence of BCMA mutations or deletions, tumors were refractory to retreatment, suggesting a tumor extrinsic mechanism of resistance. scRNA-seq analysis of the tumor microenvironment is ongoing and the role of uniquely identified immune infiltrates, like NK-like lymphocytes and distinct macrophages, in shaping the cell responses is under consideration. Conclusion: Resistance to BCMA-TCE therapy in the Vk*MYC model of MM arises through multiple mechanisms including genomic antigen loss and transient antigen downregulation. Continuous therapy drives selective pressure leading to target loss via genomic or epigenetic routes, while intermittent dosing preserves antigen but permits resistance to TCE, potentially through immune escape mediated by microenvironmental changes. These insights underscore the translatability of the Vk*MYC model for evaluating rational combination therapies to mitigate these resistance mechanisms. All procedures performed on animals were in accordance with regulations and established guidelines and were reviewed and approved by an IACUC or through an ethical review process.

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).

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