Bergsagel, P. Leif

Abstract Introduction Cila-cel is associated with a 10-15% risk of delayed neurotoxicity (DNT) with rates of cranial nerve palsies (IEC-NP) and parkinsonism (IEC-PKS) seen at a rate of 10% and 1-4% respectively and can occur as early as 3-4 weeks after infusion. We and others have shown that high post-infusion peak absolute lymphocyte count (ALCpeak) of >3x10^9/L is associated with an increased risk of delayed neurotoxicity (Lim et al., ASCO 2025) whilst the risk was negligible (~1%) when ALCpeak fell below that threshold. Since December 2024, we implemented prophylactic administration of dexamethasone (PPX DEX) when ALC was >3x10^9/L (defined as high ALCpeak). Outcome of these patients are described here. Methods Analysis of clinical outcome included 230 pts treated between Feb 2022 and Nov 2024 as a historical cohort and 102 patients treated between Dec 2024 and May 2025 after the introduction of dexamethasone prophylaxis using an oral dose of 10mg twice a day for at least three days in the high ALCpeak cohort. Results No significant differences were seen between the new and historical cohort in pre-lymphodepletion disease burden (bone marrow plasma cell %, involved light chain or M-spike level), and rates of all/high-grade CRS or ICANS, IEC-HS and rates of tocilizumub use. Median follow-up in the new cohort of 102 pts was 3.6 months (95% CI 3.3-4.2). Eleven (10.8%) developed DNT [3 (2.9%) with IEC-PKS, 8 (7.8%) with IEC-NP]. High ALCpeak of >3x10^9/L was observed in 51 (50%) pts. Among pts with high ALCpeak, DNT was observed in 10 (20%) pts [3 (5.9%) with IEC-PKS, 7 (13.7%) with IEC-NP] whereas among 51 pts with ALCpeak <3x10^9/L, DNT was observed in 1 (2%) pt [1 (2%) with IEC-NP]. In the high ALCpeak cohort, 38 (75%) received DEX [6 (12%) for toxicity management, 32 (63%) PPX DEX]. Of the 38 pts treated with DEX, DNT was observed in 6 (16%) pts (2 (5.3%) IEC-PKS, 4 (10.5%) IEC-CNP). We next compared DNT rates between this new cohort of high ALCpeak pts receiving DEX (n=38) against the historical cohort with high ALCpeak (n=98). Estimated 90-day DNT by Kaplan-Meier method was (19.2% vs 20.5%; p=0.53): IEC-PKS (5.8% vs 7.2%; p=0.60) and IEC-NP (11.7% vs 13.3%; p=0.58). In patients with high ALCpeak, the use of DEX did not appear to significantly reduce the risk of DNT compared to historical controls (HR 0.76, 0.33-1.78), IEC-PKS (HR 0.67, 0.14-3.10) or IEC-NP (0.73, 0.24-2.23). We next compared ALC expansion over the first month post CAR-T infusion (area under the curve, AUC). Among the new cohort of 102 patients, no difference was seen in the AUC of pts who received DEX but did not develop DNT vs those who developed DNT (31.0 vs 57.9, p=0.28); No difference in AUC was seen in the new high ALCpeak cohort receiving DEX vs the historical cohort with high ALCpeak (36.0 vs 43.8, p=0.15) Of the eight cases of IEC-NP, 4 received PPX DEX. No noticeable differences were observed in the severity of presentation, laterality of cranial nerve involvement and rates of multiple cranial nerve involvement between these 4 cases who received PPX DEX and the 16 cases from the historical cohort. At the time of database freeze, 3 of the 4 cases had resolved and 1 was improving. Median time to complete symptom resolution was 38 days vs 69 days in the historical cohort. Of the three cases of IEC-PKS, 2 received PPX DEX. Median time to symptom onset for IEC-PKS was 46 days (vs. 24 days in historical cohort). Both cases had severe symptoms non-responsive to first line high-dose methylprednisolone, second line intrathecal chemotherapy and third line high-dose cyclophosphamide. Both pts subsequently died from sepsis 28 and 67 days post-cyclophosphamide. Conclusion While ALCpeak >3x10^9/L predicts risk of DNT in pts treated with cilta-cel, the use of PPX DEX does not appear to significantly reduce ALC expansion and does not appear to significantly reduce the risk of DNT, IEC-PKS or IEC-NP. Other strategies to reduce ALC or alter T cell functions may need to be considered.

Abstract Cyclin D1, encoded by CCND1, is a cell cycle regulator, overexpressed in ~15% of multiple myeloma (MM) cases via CCND1::IGH super-enhancer juxtaposition through t(11;14). Cyclin D1 also plays a non-canonical role in transcriptional regulation, but in t(11;14) MM its function in gene regulation remains undefined. We investigated the global transcriptional and epigenomic consequences of CCND1overexpression in t(11;14) MM. To define a CCND1 signature, RNA-seq from two independent primary MM patient cohorts, CoMMpass and Mayo, was utilized to compare high CCND1 t(11;14) cases (≥300 transcripts-per-million [TPM], n=271) vs. low CCND1 hyperdiploid cases (<30 TPM, n=233). Two CCND1 knockout (KO) clones in t(11;14) MM U266B1 were also generated by CRISPR/Cas9, introducing a frameshift insertion (A) after chr11:69641478 (hg38) in exon 1. ATAC-seq and ChIP-seq (H3K4me3, H3K4me1, and H3K27ac) were performed in duplicates, and RNA-seq in triplicates in U266B1 wild-type (WT) and KO. Libraries were sequenced on Illumina NextSeq 2000 (2x51 bp for ChIP/ATAC, 2x150 bp for RNA-seq). Reads were mapped to hg38 by BWA (ChIP/ATAC) or STAR (RNA-seq), peaks called by MACS2 (FDR< 0.01), peak and gene raw counts estimated by featureCounts. Differential peaks (fold-change ≥1.5) and differentially expressed genes (fold-change ≥2) were identified by edgeR (FDR<0.05), including peaks detected in ≥2 samples and genes with TPM ≥0.1 in ≥1 sample. Interactions between promoters and distal regulatory regions were identified using publicly available chromatin interaction data in blood cell types. Pathway analysis was performed using Enrichr. Co-immunoprecipitation was performed in t(11;14) KMS12BM cells. Differential analysis comparing CCND1-high t(11;14) and CCND1-low MM cases in the two patient cohorts identified 21 upregulated and 21 downregulated genes associated with CCND1 expression which were also captured in the wild-typevs. CCND1 KO U266 cells. Enriched pathways (p<0.05) included Notch signaling, early/late estrogen response, epithelial-mesenchymal transition and reduced mitotic spindle and myogenesis. Dysregulated genes included those related to apoptosis, ubiquitin-proteosome, extracellular matrix, adhesion, and immune response, demonstrating a broader role for cyclin D1 in MM beyond cell cycle control. To explore the epigenomic basis of this transcriptional regulation, for each of the 42 differentially expressed genes we examined the changes of histone marks and chromatin accessibility in promoter and the interacting distal regulatory regions in the U266 KO model. Ten of the 21 genes upregulated in CCND1-high (downloaded in KO; e.g., argininosuccinate synthase 1 [ASS1], PMAIP1, MYEOV, CCR7, and DTX1) and 12 of the 21 genes downregulated in CCND1-high (upregulated in KO; e.g., UACA, RASAL2, PTPRZ1, PPT2, and IL23R) showed concordant changes in histone marks and/or chromatin accessibility, predominantly in distal regulatory regions, suggesting a chromatin-mediated mechanism of gene regulation by cyclin D1. The most prominent changes involved H3K27ac, a mark defining active enhancers. As H3K27ac is deposited by histone acetyltransferases CBP, we tested for the possible cyclin D1-CBP interaction. Co-IP confirmed this interaction, suggesting a role for this complex in chromatin remodeling and transcriptional control in t(11;14) MM. One gene notably upregulated in association with CCND1 is ASS1, which encodes a urea cycle enzyme responsible for endogenous arginine synthesis. Metabolomic studies demonstrated KO cells had elevated aspartate and citrulline and lowered arginine and ornithine compared to WT cells (p<0.01) demonstrating impaired urea cycle function. Further, KO cells displayed significantly reduced ASS1 protein expression, cell growth and S-phase entry compared to WT cells in arginine-replete conditions, with both WT and KO cell types further inhibited by arginine depletion (p<0.05). Together, a cyclin D1-associated gene signature was identified supporting its role in t(11;14) MM beyond the CDK4/6 mediated cell cycle control, including a role in metabolic plasticity and survival under arginine depleted conditions. These findings highlight cyclin D1 as a therapeutic vulnerability and open new avenues for personalized treatment strategies.

Abstract Introduction High disease burden has been associated with reduced efficacy and resistance to BCMA CAR-T therapy in relapsed/refractory multiple myeloma (van de Donk, Blood Cancer Discov, 2021; Martin, J Clin Oncol, 2023). While CAR-T cells can still induce remissions in these patients, outcomes are often worse compared to those with a lower tumor load. In addition to tumor debulking, novel strategies are needed to improve responses and durability of CAR-T cells in these patients. We previously demonstrated that immunomodulatory drugs (IMiDs) improve in vivo responses to T cell engagers (TCE) in high tumor burden setting by boosting T cell activation (Meermeier, Blood Cancer Discov, 2021; Meermeier, Blood, in press). IMiDs have also been shown to promote CAR-T cell antitumor activity in moderate to low tumor burden conditions (E:T ratios ≥1:1) (Yan, J Transl Med, 2024; Wang, Clin Cancer Res, 2018). We hypothesized that combining CAR-T with an IMiD may improve CAR-T function in high tumor burden setting. Methods We generated murine anti-BCMA.CD28 CAR-T cells by transducing Con-A stimulated T lymphocytes extracted from hCRBN+ mice, expanded with IL-7 and IL-15 to express a memory phenotype. We then tested CAR-T, or untransduced T cells, ability to kill in vitro IMiD resistant, hCRBN-, Vk*MYC myeloma cells in low (E:T 1:1) and high (E:T 1:≥5) tumor burden, with and without pomalidomide (POM) and IL-2. Tumor killing and T cell phenotype were assessed by flow cytometry at 24-72 hours. This controlled system allowed us to focus on putative CAR-T cell intrinsic impact of IMiDs. Results In low tumor burden setting, CAR-T effectively killed tumor at all timepoints, with or without POM; addition of POM increased T cell number, confirming that IMiDs support CAR-T expansion. Unexpectedly, in high tumor burden (E:T 1:≥5), adding POM diminished CAR-T mediated killing over time and hindered CAR-T expansion. Moreover, combination with POM decreased granzyme B, while increased expression of exhaustion marker TOX, ultimately leading to CAR-T apoptosis. This phenotype was further intensified by increasing concentrations of IL-2 in the co-culture, resulting in increased expression of CAR-T expressing LAG-3, TIM-3, and TIGIT, suggesting that CAR-T cells may be overactivated by POM and IL-2. Our manufacturing protocol preferentially expands memory CAR-T cells, favored for their durability, proliferative capacity, and considered to rapidly expand upon encounter with antigen. We reasoned that CAR-T are already sufficiently activated by CAR/CD3z stimulation, especially with a CD28 co-stimulatory domain (versus 4-1BB), known promote rapid, effector memory expansion upon activation (Kawalekar, Immunity, 2016). Activation is intensified by high tumor burden where antigen is highly abundant. We hypothesize under intense, sustained antigen exposure in high tumor burden, the addition of POM paradoxically triggers activation induced cell death (AICD). Conclusion Although it is well accepted that POM can boost T cell function and proliferation, we demonstrate that in high tumor burden, POM drives memory CAR-T to increase exhaustion markers and apoptotic cell death by overactivation, further exacerbated by exogenous IL-2. Interestingly, we have shown opposite effects of preclinically combining POM with TCE therapy in high tumor burden, based on dosing schedule. Concomitant administration transiently boosted T cell activation improving response rates but rapidly led to T cell exhaustion (Meermeier, Blood Cancer Discov, 2021). In contrast, IMiD pretreatment in the context of step-up dosing TCE administration improved T cell fitness and longevity (Meermeier, Blood, in press).The effect of IMiDs on T cells transcriptional program is likely to differ depending on differentiation stage and mode of T cell activation. Memory cells have more sensitive TCR signaling (Kumar, Immunity, 2011) and can be hyperactivated, leading to AICD. Thus, timing of IMiD administration, when tumor burden is low, may be a critical consideration as different T cell subsets require different levels of activation. Conversely, TCE provide only initial activation via TCR signaling, therefore POM may help in providing necessary co-stimulation in this setting. Future studies will evaluate different co-stimulatory domains and test in vivo effects of POM on CAR-T expansion and persistence, particularly as CAR-T cells diminish over time as tumor burden increases.

Abstract Background: Bispecific T cell engagers (BTCEs) have transformed the treatment of multiple myeloma (MM) and B cell malignancies by redirecting cytotoxic T cells toward malignant targets. While clinical responses can be deep and durable, the immunological determinants of BTCE efficacy, and mechanisms of eventual immune escape, remain poorly understood. Regulatory T cells (Tregs), enriched early in MM, are classically immunosuppressive, but whether they are modulated by BTCEs and contribute to therapeutic outcomes has not been systematically investigated. Methods We performed longitudinal single-cell RNA/TCR/ATAC-sequencing and immune profiling of 17 newly diagnosed multiple myeloma (NDMM) patients across induction therapy. 11 patients were treated with teclistamab plus daratumumab, lenalidomide, and dexamethasone (Tec-DRD, n = 11) in the ongoing MajesTEC-5 (HD10/DSMMXX) trial (NCT05695508). As controls, patients receiving standard-of-care DRD (n = 6) were included. Functional validation used newly established NDMM plasma cell-T cell co-cultures, real-time killing assays, cytokine ELISAs, intracellular flow cytometry and arrayed quantitative PCRs of key T cell signaling pathways. Mechanistic studies employed CRISPR-mediated perturbations and combination treatments with IMiDs. Generalizability was tested using three BTCEs in vitro: teclistamab (BCMA×CD3), blinatumomab (CD19×CD3), and glofitamab (CD20×CD3) to redirect primary human Tregs towards MM (MM.1S), lymphoma (Raji), and B-ALL (NALM-6) cell lines. Non-targeting BTCEs as well as anti-CD3 antibodies were used as controls. Preclinical in vivo studies used an immunocompetent, transplantable Bcma+ Vk*MYC tumor model treated with murinized BTCE surrogates and IMiDs. Results BTCE therapy induced robust transcriptional and epigenetic reprogramming of bone marrow Tregs into a FOXP3lowIL2RAlow cytotoxic phenotype (“exTregs”) expressing NKG7, GZMA/B, TBX21, and CCL5. In contrast, this Treg phenotype conversion was absent in DRD-treated controls. Although comprising only ~2% of marrow-infiltrating T cells, exTregs mediated direct plasma cell lysis in vitro, strictly dependent on BTCE-mediated antigen-specific synapse formation and not recapitulated by CD3 stimulation alone. IMiDs synergized with BTCEs by destabilizing the suppressive Treg phenotype via Helios (IKZF2) downregulation, augmenting granzyme and cytokine production. Single-cell ATAC-seq of Tregs isolated from Tec-DRD-exposed trial participants revealed increased chromatin accessibility at AP-1 motifs and we confirmed BATF/JUNB-driven AP-1 signaling as a central transcriptional axis orchestrating Treg effector conversion in vitro. This cytotoxic reprogramming was conserved across BTCEs targeting BCMA, CD19, and CD20, and validated in immunocompetent mouse models, indicating a generalizable, BTCE-driven mechanism of Treg repurposing, rather than target- or engager-specific biology. Conclusion Our findings uncover a novel mechanism by which BTCEs actively convert suppressive Tregs into cytotoxic, pro-inflammatory effectors that contribute to tumor clearance. We identified the BATF/JUNB transcriptional axis and IMiD-mediated Helios destabilization as targetable regulators of this process. These results redefine Treg function under BTCE pressure, support rational combination regimens with IMiDs, and suggest exTreg signatures as biomarkers and therapeutic targets to enhance T cell-redirecting immunotherapies across hematological malignancies.

Abstract B cell maturation antigen (BCMA)-targeted chimeric antigen receptor T (CART) cell therapies have emerged as a potentially curative treatment for multiple myeloma (MM). However, 50-70% of patients relapse within 3 years. The MM tumor microenvironment (TME) is highly immunosuppressive with an abundance of bone marrow (BM) cancer-associated fibroblasts (CAFs), which contribute to BCMA-CART cell failure. Therapeutic targeting of BM-CAFs has been beset by 1) an incomplete understanding of CAF interactions with CART cells and other BM cells and 2) toxicity risks due to prolonged depletion of healthy fibroblasts and inefficient drug delivery to the BM. To elucidate how BM-CAFs drive BCMA-CART resistance, we performed single cell RNA sequencing (scRNAseq) of baseline BM aspirates from responders (R, n = 6) and non-responders (NR, n = 6) to BCMA-CART cell therapy. NR BMs were enriched for inhibitory CAFs (iCAFs) that highly express fibroblast activation protein (FAP). Spatial analyses of BM cells revealed greater iCAF-iCAF and iCAF-inhibitory macrophage interactions in NR. These findings suggest that FAP+ iCAFs in NR are abundant in the BM and exhibit significant interactions with immunosuppressive macrophages. Next, we sought to validate our scRNAseq data in preclinical models. Xenograft mice were intravenously (IV) engrafted with luciferase+ (luc+) BCMA+ OPM2 cells and/or patient-derived (pd) BM-CAFs (1x106 each). Following engraftment, mice received 1x106 BCMA-CART cells (IV). BCMA-CART cells eradicated tumors in mice engrafted with OPM2 cells, but not in mice engrafted with pdBM-CAFs and OPM2 cells (p<0.01). In NSG and humanized mice (NSG mice engrafted with human CD34+ cells in the first 48 hours of life), tumor growth was significantly accelerated in the presence of pdBM-CAFs (NSG: p<0.01; humanized: p<0.001). BCMA-CART, OPM2, and pdBM-CAF co-cultures also showed significant increases in immunosuppressive cytokines including IL-1Rα, IL-4 and IL-10 and inhibited CART cell proliferation compared to co-cultures without pdBM-CAFs (IL-1Rα, IL-10: p<0.01; IL-4: p<0.001; proliferation: p<0.05). To overcome CAF-mediated CART dysfunction, we engineered BCMA-CART cells to secrete molecules to target BM FAP+ iCAFs and remodel the MM-TME (STriKEs). We designed two BCMA-CART-STriKE strategies: 1) a tri-specific composed of IL-15, anti-FAP, and anti-CD16 molecules to activate innate immune cells to kill CAFs and 2) bi-specific anti-FAP, anti-SIRPα molecules linked to an antibody Fc region (Fc fusion) targeting the CD47/SIRPα axis to induce phagocytosis of FAP+ iCAFs. We hypothesized that BCMA-CART-STriKEs would surmount CAF-mediated immunosuppression without toxicity. We generated BCMA-CART-STriKEs from healthy human donor T cells. CAR expression, STriKE secretion, and binding were confirmed via flow cytometry. Co-cultures of BCMA-CART-STriKEs, donor-matched macrophages, OPM2 cells or BCMA+ MM1S cells, and pdBM-CAFs showed significantly higher OPM2 or MM1S killing (p<0.001), immune cell activation (p<0.01), and phagocytosis (p<0.05), compared to control BCMA-CART cells secreting IL-15 or Fc fusions targeting only FAP or only SIRPα. Next, we assessed BCMA-CART-STriKE efficacy in vivo. NOD-SCID-γ-/- (NSG) mice received 1x106 pdBM-CAFs and 1x106 luc+ OPM2 or luc+ MM1S cells via IV or subcutaneous (SQ) injection. Mice were randomized after confirming tumor engraftment (via bioluminescence imaging for IV or tumor caliper for SQ) and received IV treatment with 1x106 BCMA-CART-STriKEs (or controls) and 5x105 donor-matched macrophages or CD16+ immune cells. BCMA-CART-STriKE-treated mice achieved total tumor clearance. Treatment with BCMA-CART cells secreting IL-15 led to lethal weight loss despite tumor clearance while BCMA-CART cells secreting Fc fusions targeting only FAP or SIRPα failed to eradicate tumors. Lastly, we assessed the potential toxicities of BCMA-CART-STriKEs in humanized mice engrafted with OPM2 cells and pdBM-CAFs (1x106 each). CART cells expressing BCMA and FAP CARs (BCMA-FAP CART) served as a control. BCMA-FAP CART treatment resulted in significant toxicity and weight loss while BCMA-CART-STriKE treatment did not (p<0.01). In summary, we have identified iCAF interactions with immunosuppressive immune cells as key contributors to BCMA-CART cell failure and designed novel BCMA-CART-STriKEs that overcome this failure in MM preclinical models. These findings support the translation of BCMA-CART-STriKEs to a phase I clinical trial.

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.

Abstract Mutations in non-coding regulatory elements are increasingly recognized as critical drivers of cancer development and progression. Among these, structural alterations such as duplications of super-enhancers (SEs), large clusters of enhancers that regulate key oncogenes, have emerged as significant contributors to oncogenesis. In T-cell acute lymphoblastic leukemia (T-ALL), a particularly aggressive hematologic malignancy, SE duplications have been identified in approximately 5% of human cases. These duplications frequently involve a non-coding region located approximately 1.3 megabases downstream of the Myc oncogene, a region known as N-Me. Despite the growing recognition of the role of SE duplications in T-ALL, functional studies to determine their molecular origins and contributions to leukemogenesis have been limited, largely due to the absence of suitable and genetically tractable preclinical models. This gap in knowledge hinders both our understanding of disease pathogenesis and our ability to design targeted therapeutic interventions. Here, we report the generation and characterization of a novel genetically engineered mouse model of T-ALL that spontaneously develops oncogenic SE duplications at the Myc locus. This model offers the first spontaneous in vivo system to investigate the origin, regulation, and oncogenic function of SE duplications. This model was created by conditionally stabilizing β-catenin in CD4⁺CD8⁺ double-positive (DP) thymocytes in combination with targeted deletion of the transcription factor HEB. This combination is sufficient to drive the development of aggressive T-cell leukemias that consistently harbor duplications of the N-Me super-enhancer. This finding recapitulates the genomic alteration observed in a subset of human T-ALL and provides a unique platform for mechanistic studies. We further demonstrate that the molecular events leading to SE duplication and oncogenic Myc activation unfold in a stepwise manner. At the preleukemic stage, stabilization of β-catenin alone is sufficient to enhance recruitment of HEB to the N-Me region. The simultaneous loss of HEB greatly increased chromatin accessibility at the N-Me enhancer. This altered chromatin landscape permits aberrant binding of transcription factors including TCF-1, which plays a critical role in T-cell development and leads to N-Me SE activation associated with significant three-dimensional chromatin reorganization. Chromosome conformation analyses in the leukemic cells reveal the formation of a novel chromatin loop that brings the activated N-Me SE into close spatial proximity with the Myc promoter, enabling oncogenic Myc transcription. These findings uncover a novel mechanistic pathway by which β-catenin and HEB interact to reshape the enhancer landscape and three-dimensional chromatin architecture, ultimately promoting SE duplication and aberrant Myc expression in T-ALL. β-catenin appears to play a dual role, first facilitating HEB recruitment under physiological conditions and then, upon HEB loss, permitting the reprogramming of enhancer activity through chromatin opening and ectopic transcription factor binding. Our data suggest that the cooperative interplay between signaling pathways and transcriptional regulators at enhancer elements can initiate structural genomic changes that drive malignancy. This work provides a long-sought preclinical model that faithfully mirrors key features of human T-ALL. Our findings open new avenues for understanding how non-coding regulatory mutations arise and exert oncogenic effects and have the potential to provide insights that may extend beyond T-ALL to other cancers driven by enhancer dysregulation. Moreover, by revealing molecular intermediates such as β-catenin, HEB, and TCF-1 in the regulation of enhancer activity, our findings identify potential therapeutic targets to disrupt the oncogenic enhancer-promoter interactions that underlie Myc-driven leukemogenesis.

Abstract Consumption of a Western diet and high body mass index (BMI) are risk factors for progression from premalignant phenotypes to multiple myeloma, a hematologic cancer. In the NUTRIVENTION trial (NCT04920084), we administered a high-fiber, plant-based diet (HFPBD; meals for 12 weeks, coaching for 24 weeks) to 23 participants with myeloma precursor states and elevated BMI. The intervention was feasible and improved quality of life and modifiable risk factors: metabolic (BMI, insulin resistance), microbiome (diversity, composition), and immune (inflammation, monocyte subsets). Disease progression trajectory improved (n = 2) or was stable. Findings were translated to Vk*MYC mice modeling the myeloma precursor state, in which a high-fiber diet (HFD) delayed disease progression through improved metabolism and microbiome composition, leading to increased short-chain fatty acid production that reinvigorated antitumor immunity and inhibited tumor growth. These effects from fiber consumption were independent of calorie restriction and weight loss. A HFD is a low-risk intervention that may delay progression to myeloma. Significance: A HFPBD in participants with precursor plasma cell disorders and an elevated BMI improved metabolic, microbiome, and immune biomarkers of disease. In a subset, it may delay progression to myeloma. In mouse models, a HFD delayed progression to myeloma independent of calorie restriction. See related commentary by Fairfield and Reagan, p. 623

Multiple myeloma (MM) is consistently preceded by monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). While these precursor conditions are asymptomatic, they are not entirely benign and carry a lifelong risk of progression to MM. Unlike other cancers defined by pathology, malignant transformation from MGUS or SMM to MM has so far relied on demonstration of clinical end-organ damage as morphology and cytogenetics cannot reliably distinguish them. In this study, using genomic data from 374 patients with MGUS or SMM (277 training, 97 validation), to our knowledge, we demonstrate for the first time the ability to identify malignant transformation in MGUS and SMM. We introduce the concept of genomic MM and genomic MGUS to differentiate the subsets of MGUS and SMM that are biologically malignant with genomic features indistinguishable from MM from the subset that is premalignant and unlikely to progress to malignancy. Importantly, we find that most SMM has biological features of malignant transformation indistinguishable from MM. As expected, this subset that we consider having genomic MM is associated with a high risk of progression to MM although some patients remained progression-free beyond 5 years. Conversely, 60% of MGUS and 10% of SMM have no evidence of malignant transformation (genomic MGUS), with no progression during follow-up. Integration of genomic features with the 2/20/20 International Myeloma Working Group model significantly improved the prediction of progression among genomic MM. These findings support the use of genomic criteria to refine the classification and the risk stratification in myeloma precursor conditions.

Abstract Glutamine dependence of cancer cells reduces local glutamine availability, which hinders antitumor T-cell functionality and facilitates immune evasion. We thus speculated that glutamine deprivation might be limiting efficacy of chimeric antigen receptor (CAR) T-cell therapies in patients with cancer. We have seen that antigen-specific T cells are unable to proliferate or produce interferon gamma (IFN-γ) in response to antigen stimulation when glutamine concentration is limited. Using multiple myeloma (MM) as a glutamine-dependent disease model, we found that murine CAR T cells selectively targeting B-cell maturation antigen (Bcma) in MM cells were sensitive to glutamine deprivation. However, CAR T cells engineered to increase glutamine uptake by expression of the glutamine transporter Asct2 exhibited enhanced proliferation and responsiveness to antigen stimulation, increased production of IFN-γ, and heightened cytotoxic activity, even under conditions of low glutamine concentration. Mechanistically, Asct2 overexpression reprogrammed the metabolic fitness of CAR T cells by upregulating the mechanistic target of rapamycin complex 1 gene signature, modifying the solute carrier transporter repertoire, and improving both basal oxygen consumption rate and glycolytic function, thereby enhancing CAR T-cell persistence in vivo. Accordingly, expression of Asct2 increased the efficacy of Bcma-CAR T cells in syngeneic and genetically engineered mouse models of MM, which prolonged mouse survival. In patients, higher-level expression of ASCT2 by MM cells predicted poor outcome to combined immunotherapy and BCMA-CAR T-cell therapy. Our results indicate that reprogramming glutamine metabolism may enhance antitumor CAR T-cell functionality in MM. This approach may also be effective for other cancers that depend on glutamine as a key energy source and metabolic hallmark.