The interaction between lymphoma cells and immune microenvironment cells and the impact of this functional interplay on therapeutic responses remain largely unexplored. Here, we utilized murine models with oncogenically active MYD88 and additional genetic lesions co-triggered at selected B cell stages to generate human-like lymphomas harboring the MYD88L265P mutation. Lymphomas exhibited behaviors ranging from clinically indolent small-cell tumors to aggressive diffuse large B-cell lymphoma (DLBCL). Genetically diverse lymphoma cells employ distinct immune evasion mechanisms that shape unique lymphoma microenvironment (LME) states. In this setting, clonally expanded T-cells function as a double-edged sword, either sustaining indolent lymphoma cell survival or promoting antitumor responses in DLBCL. Consequently, the efficacy of standard-of-care and novel immunotherapies was determined using individual T-cell features. Furthermore, the experimental targeting of newly identified immune mechanisms has improved therapeutic responses in vivo. Our results elucidate that genetically driven LME landscapes influence therapeutic outcomes across distinct lymphoma subtypes, providing proof-of-concept for personalized treatment based on immune LME information.

Deletion of 17p is among the most adverse cytogenetic abnormalities in multiple myeloma (MM). By integrating RNA-seq data from patient MM cells with genetic dependency data from MM cell lines, we identified the protein kinase membrane-associated tyrosine/threonine 1 (PKMYT1) kinase, a member of the Wee family, as a potential therapeutic target in MM cells harboring del(17p). Genetic suppression or pharmacological inhibition of PKMYT1 activity with the selective inhibitor RP-6306 triggered accumulation of DNA damage, micronucleus formation and mitotic catastrophe, resulting in preferential cell death in del(17p) MM cells while largely sparing del(17p)-negative MM cells and healthy cells. RP-6306 also reduced tumor burden and extended survival in vivo in both xenograft and TP53-deficient syngeneic models. Collectively, our findings nominate PKMYT1 as an actionable target and support PKMYT1 inhibition as a biomarker-driven therapeutic strategy for patients with del(17p)/TP53-deficient MM.

Abstract Multiple myeloma (MM) evolves from asymptomatic precursor conditions through progressive genetic and epigenetic remodeling, yet the regulatory mechanisms driving the development toward more active stages of the disease remain poorly understood. Here, we integrated bulk-based paired chromatin accessibility and activation, and transcriptomic profiling across disease stages to map regulatory remodeling during myeloma development. We identified a progressive increase in chromatin accessibility; furthermore, this epigenetic reconfiguration is accompanied by a stage-dependent shift from promoter-centered regulation in precursor states toward enhancer-dominated transcriptional control in active MM. Motif enrichment and regulatory network analyses identified both established and previously underappreciated transcription factors (TFs), including members of the IRF, MEF2, and FOX families, associated with disease-stage–specific transcriptional programs. Among these, MEF2D and FOXK2 emerged as candidate regulators of pathways involved in cell survival and chemotaxis. Functional perturbation demonstrated that MEF2D depletion markedly impaired MM cell viability, whereas inhibition of either MEF2D or FOXK2 reduced chemotactic migration. Together, these findings provide a stage-resolved framework of epigenetic and transcriptional remodeling across myeloma development, revealing regulatory programs established in precursor conditions and progressively reinforced during disease evolution, while identifying candidate transcriptional dependencies with potential biological and therapeutic relevance.

The clinical significance of one or two measurable residual disease (MRD) assessments is established in multiple myeloma (MM). However, how to stratify patients according to ≥3 MRD assessments remains unknown. The traits of MRD resistance and if treatment of persistent MRD vs relapse could improve outcomes also remains unknown. MRD dynamics were computed using next-generation flow cytometry and Connector in 539 newly-diagnosed MM patients with ≥3 assessments in the GEM2012MENOS65/GEM2014MAIN and GEM2017FIT trials. Molecular and immune profiling were performed in matched diagnostic and MRD samples. The survival impact of treating persistent MRD vs relapse was investigated with anti-BCMA CAR T cells in MIcγ1huCRBN mice. Computed MRD dynamics based on 3,610 MRD assessments identified five subgroups with different survival. Patients with late-sustained MRD response had excellent outcomes, similar to those with early-sustained MRD response. Patients with volatile results and those with primarily and resurgent MRD resistance had dismal survival. MRD dynamics outperformed transplant-eligibility and the R-ISS. These results were validated in 249 MM patients treated in routine practice. Multiomics characterization of MRD dynamics in patients and mouse models of MRD resistance revealed genomic evolution, transcriptional adaption and a pro-inflammatory tumor-immune microenvironment. Increasing clonality and exhaustion of endogenous T cells throughout disease progression urged investigating if MRD interception with anti-BCMA CAR-T cells could improve outcomes. Infusion at MRD resistance prolonged mouse survival compared to identical treatment at relapse. Altogether, MRD dynamics is the strongest predictor of progression and may help tailoring treatment to prevent additional tumor and immune alterations prior to relapse.

T cell dysfunction is an important contributor to both multiple myeloma (MM) disease progression and failure of anti-myeloma chimeric antigen receptor (CAR) T-cell and bispecific T-cell engager (TCE) therapies. Overcoming T cell dysfunction is therefore key to improving MM patient outcomes. Immunomodulatory drugs (IMiDs) and cereblon E3 ligase modulatory drugs (CELMoDs) have been observed to activate T cells, and more recently reduce T cell dysfunction, however the underlying mechanisms behind this are incompletely understood. Here, using bone marrow samples from MM patients, we demonstrate a significant reduction in dysfunctional T cell populations expressing exhaustion markers such as TIGIT, upon treatment with mezigdomide. We further demonstrate the ability of mezigdomide to improve T cell function and cytotoxicity in primary T cell models of T cell dysfunction and bispecific TCE therapy in vitro. Using concurrent ATAC-seq, ChIP-seq, HiC and RNA-seq in primary T cells treated with mezigdomide, we demonstrate the novel role of transcription factor Ikaros in regulating an important T cell exhaustion gene TIGIT. Finally, we demonstrate the ability of mezigdomide to enhance survival outcomes from anti-BCMA CAR-T therapy in vivo. Overall, our data show that mezigdomide treatment improves antimyeloma T-cell therapy efficacy and reduces T cell dysfunction by abrogating Ikaros-mediated upregulation of exhaustion genes.

Infections remain a key challenge during treatment of multiple myeloma (MM) patients with anti-BCMA and -GPRC5D bispecific antibodies (bsAbs). However, the underlying mechanism behind different rates and severity of infections induced by the two bsAbs remains poorly understood. Single-cell RNA-sequencing performed in bone marrow aspirates of 11 MM patients and 8 healthy donors revealed BCMA expression on mature B cells and, surprisingly, in small pre-B cells within B-cell precursors. By contrast, GPRC5D expression was restricted to normal and malignant plasma cells (PCs). Next-generation flow cytometry immune profiling showed that anti-BCMA bsAbs severely depleted bone marrow (BM) mature B cells (4.9%→0%; p<0.001) and normal PCs (0.17% → <0.0002%; p<0.001) during treatment of 62 relapsed MM patients. This was observed in early and late time points of therapy. Additional flow cytometry (N=31) and single-cell RNA-sequencing studies (N=8) demonstrated that, in contrast to anti-GPRC5D, anti-BCMA bsAbs also depleted immature and small pre-B cells. The MIcγ1 mouse model was used as a negative control of BCMA expression in all stages of the B-cell lineage, which confirmed no depletion of any B-cell subset after anti-BCMA treatment. In conclusion, we show that while GPRC5D bsAbs selectively target PCs, anti-BCMA bsAbs target both PCs and B cells from the small pre-B stage onwards. Our study provides mechanistic insight into the increased infection risk with anti-BCMA therapy and lays a foundation for individualized bsAb strategies in MM. Moreover, dual targeting of B cells and PCs may have therapeutic potential in other B cell malignancies or autoimmune diseases.

Abstract Progression from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) is accompanied by profound remodeling of the bone marrow microenvironment (BME), yet the contribution of its non-immune compartment remains unclear. Using single-cell RNA sequencing in genetically engineered mouse models that recapitulate disease evolution, we transcriptionally profile endothelial cells (EC) and mesenchymal stem cells (MSC). EC adopt a stress-associated program at MGUS that precedes angiogenesis in MM, while MSC undergo early and sustained loss of differentiation capacity. We identify a coordinated interferon (IFN)-driven program across EC and MSC that defines MM in the BIcγ1 model but is absent in the more aggressive MI cγ1 model. Treatment with bortezomib, lenalidomide, and dexamethasone suppresses this IFN signature, promotes endothelial adaptation, and restores osteogenic potential in MSC. Validation in patient samples reveals enrichment of this IFN-signature across disease stages. These findings define dynamic and targetable alterations in the non-immune BME during myeloma progression.

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.

Chimeric antigen receptor T-cell (CART) and T-cell engager (TCE) therapies targeting B-cell maturation antigen (BCMA) are transforming the treatment landscape for relapsed multiple myeloma (MM). However, despite impressive initial response rates, most patients eventually relapse. To investigate this unmet medical need, we applied whole-genome sequencing (WGS) to MM cells from cohorts of 102 relapsed patients treated with anti-BCMA CART and TCE therapies. Several genomic alterations were associated with clinical outcomes, particularly primary refractoriness, including high genomic complexity and mutations in genes regulating plasma cell identity, which predicted resistance to therapy. Single-cell RNA sequencing further revealed that MM cells from refractory patients exhibited high proliferation signatures and reduced expression of TNFRSF17 (encoding BCMA), while were less enriched for plasma cell-associated transcriptional programs, a phenomenon we term "plasma cell identity escape." This profile was strongly associated with immune dysregulation of CD8 T cells including increased activation and exhaustion. This evolution of MM toward a more proliferative and lineage-divergent state, refractory to the anti-BCMA T-cell redirecting therapies, was functionally validated in preclinical MM mouse models. Collectively, our results comprehensively define the cellular and molecular mechanisms underlying primary resistance to anti-BCMA therapies.

The bone marrow (BM) is a complex and compartmentalized tissue where spatial context plays a critical role in regulating cell behavior, signaling, and disease progression. To capture these dynamics, we apply spatial transcriptomics using the Visium Spatial Gene Expression platform on formalin-fixed paraffin-embedded (FFPE) BM sections from both healthy and Multiple Myeloma (MM) mouse models, as well as MM patient samples. Overcoming the technical challenges of working with mineralized long bone tissue, we develop a custom analytical framework integrating spatial and single-cell transcriptomic data to map cellular composition and interactions in situ. This approach enables the spatial characterization of transcriptionally heterogeneous malignant plasma cells (MM-PC) and their surrounding microenvironments. We identify spatially distinct gene programs linked to MM pathogenesis, including signatures of NETosis and IL-17 signalling, which are reduced in MM-PC–rich regions. Additionally, a transition gradient from effector to exhausted T cell phenotype is associated with increased remoteness from MM-PC. These spatial patterns are identified in FFPE BM biopsies from MM patients with varying tumor burdens. In summary, our study demonstrates both the capabilities and limitations of Visium technology in characterizing spatially regulated mechanisms underlying MM pathogenesis. Visium Spatial technology enables the characterization of transcriptionally heterogeneous malignant plasma cells in multiple myeloma, and identify spatially distinct gene programs linked to MM pathogenesis.

The microbiome is a complex host factor and key determinant of the outcome of antibody-based and cellular immunotherapy. Its postbiotics are a blend of soluble commensal byproducts that are released into the host environment and have been associated with the regulation of immune homeostasis, particularly through impacts on epigenetics and cell signaling. In this study, we show that the postbiotic pentanoate is metabolized to citrate within the TCA cycle via both the acetyl- and succinyl-CoA entry points, a feature uniquely enabled by the chemical structure of the C5 aliphatic chain. We identified ATP-citrate lyase as the crucial factor that redirects pentanoate-derived citrate from the succinyl-CoA route to the nucleus, thereby linking metabolic output and histone acetylation. This epigenetic-metabolic crosstalk mitigated T cell exhaustion and promoted naive-like differentiation in pentanoate-programmed chimeric antigen receptor (CAR) T cells. The predictive and therapeutic potential of pentanoate was corroborated in two independent patient cohorts and three syngeneic models of CAR T adoptive therapy. Our data demonstrate that postbiotics are integrated into mitochondrial metabolism and subsequently incorporated as epigenetic imprints. This bridge between microbial and mammalian interspecies communication can ultimately impact T cell differentiation and efficacy.

Background: S100A8/A9 is a calcium-binding alarmin released during cellular stress and inflammation, modulating immune responses. We previously identified S100A8/A9 as a mediator of resistance to anti-BCMA CAR T-cell therapy through suppression of cytotoxic function. Here, we evaluate the role of S100A8/A9 in patients with multiple myeloma (MM) treated with bispecific antibodies (BsAb) targeting BCMA or GPRC5D. We perform in vitro and in-vivo validation to elucidate the underlying mechanisms contributing to suppression of T cell-directed therapy efficacy and test salvage strategies. Methods: Serum S100A8/A9 levels were correlated with depth of response (DOR) and progression-free survival (PFS) in patients receiving bispecific antibody therapy (n=19). We conducted extensive phenotyping of peripheral blood mononuclear cells (PBMCs) and quantified antigen-specific cytokine production and cytotoxicity by spectral flow cytometry. Post BsAb relapse EMD samples were interrogated with the Visium spatial transcriptomic platform. To study mechanistic effects, healthy donor T cells were incubated with recombinant S100A8/A9, and dynamic changes in phenotype, antigen-specific cytokine production, and cytotoxic activity were assessed. In vitro and in vivo salvage experiments were performed using blocking antibodies against S100A8/A9 in combination with bispecific therapeutics. Results: Patients with short PFS (n=11,<5 months, median 63 days) had significantly higher serum S100A8/A9 levels both at baseline and during therapy compared to those with longer PFS (n=8, median 503 days; p<0.05). S100A8/A9 levels during treatment inversely correlated with PFS (r=-0.52, p< 0.05). T cells from patients with short PFS exhibited features of exhaustion, including increased TOX+ CD3+ T cells (p=0.004) and higher CD8+ PD-1+ T cells. Upon stimulation with MM cell lines, PBMCs from these patients produced significantly lower levels of TNF-α, GM-CSF, IFN-γ, and IL-2 in both CD4+ and CD8+ subsets (p<0.05). CD4+ and CD8+ cytokine production inversely correlated with TOX expression (CD4+: r² = –0.6, p<0.05; CD8+: r² = –0.77, p<0.01). Antigen-specific cytotoxicity was significantly reduced in patients with short PFS compared to those with long PFS (p<0.001). Spatial transcriptomics from extramedullary collected samples showed colocalization of S100A8/A9 expressing CD14 monocytes with exhausted T cells in the tumor microenvironment of patients undergoing BsAb therapy. To measure the direct effect of S100A8/A9 on T cell functionality contributing to the impairment of T cell-directed therapy, we exposed healthy donor T cells to S100A8/A9. Incubation with S100A8/A9 induced T cell exhaustion in a dose-dependent manner, as evidenced by increased TOX+ CD4+ and TOX+ CD8+ populations, and impaired BsAb-mediated cytotoxicity. Importantly, blockade of anti- S100A8/A9 with monoclonal antibodies (mAbs) restored BsAb-mediated cytotoxicity (p=0.032) and prevented induction of TOX in healthy donor and patient samples, supporting a causal role of S100A8/A9 in T cell dysfunction. Direct incubation of T cells with S100A8/A9 caused a dose-dependent decrease in pERK signaling that was reversed with mAb salvage. In vivo treatment with anti-S100A8/A9 mAb activity showed a trend towards improved survival in combination with bispecific therapy as compared to bispecific therapy alone in transgenic tumor bearing mice. Conclusions: S100A8/A9 directly promotes T cell exhaustion and impairs T cell effector function, thereby reducing the efficacy of BsAb therapy in MM. Elevated serum S100A8/A9 levels are associated with inferior clinical responses and shorter PFS. Mechanistically, S100A8/A9 induces TOX expression and suppresses cytokine production and cytotoxicity in T cells. Salvage with monoclonal antibodies not only restores function but also prevents T cell exhaustion mediated by S100A8/A9 exposure. These findings position S100A8/A9 as a novel biomarker of resistance and a potential therapeutic target to enhance the efficacy of T cell-engaging immunotherapies in multiple myeloma.

Background Phosphatidylserine (PS) exposed on apoptotic cells promotes immune clearance of dead cells without inducing inflammation. Conversely, PS exposure on live tumor cells promotes an immunosuppressive tumor microenvironment that hinders antitumor immune responses. After confirming elevated PS levels in various tumor cell lines and cancer tissues, we aimed to investigate its potential as a target antigen for chimeric antigen receptor T cell (CAR-T) therapy.Methods We used two different approaches to target PS. First, we employed the adaptor proteins, EDAnnexin or BCMAnnexin comprising annexin V and EDA (extra domain A of fibronectin) or B-cell maturation antigen (BCMA) antigens, to redirect the lytic activity of EDA CAR-T or BCMA CAR-T cells toward PS-expressing tumor cells. In a second approach, we developed an annexin V-based CAR (Anxa CAR-T) to directly recognize PS-positive tumor cells.Results The adaptors proteins EDAnnexin and BCMAnnexin successfully redirected EDA CAR-T or BCMA CAR-T cell activity, leading to an efficient recognition of PS+ tumor cells in vitro. However, the established immunological synapse differs significantly from that observed when CAR-T cells recognize the tumor cells directly. In vivo administration of the adaptor proteins, combined with the corresponding CAR-T cells, displayed antitumor activity in mice bearing PS+ tumors. Regarding the second approach, Anxa CAR-T cells effectively recognized and killed PS+ tumor cells in vitro. Nonetheless, PS exposure on T-cell membranes during T-cell activation impeded efficient Anxa CAR-T cell manufacturing due to fratricide. By optimizing retroviral dose to reduce Anxa CAR expression on the cell membrane, or by using the multikinase inhibitor dasatinib, the fratricide effect was mitigated, enabling successful Anxa CARLow-T cell production. Remarkably, Anxa CARLow-T cells demonstrated antitumor activity in in vivo murine models of PS+ hepatocarcinoma and teratocarcinoma. No signs of toxicity were observed after Anxa CAR-T cell administration.Conclusions PS holds promise as a target antigen for CAR-T cell therapy, underscoring the need to address fratricide as a key challenge in the development of PS-targeting CAR-T cells.

Chromosome 1q21 amplification is closely associated with disease progression and adverse prognosis in multiple myeloma (MM), rising from approximately 20% incidence in MGUS to 70–80% in relapsed or refractory MM. The CKS1B gene, located in the 1q21 region, acts as an important prognostic biomarker; patients with the highest CKS1B mRNA levels in the COMPASS study exhibited the poorest survival. As a core partner of SKP2 in the SCF E3 ubiquitin ligase complex, CKS1B promotes degradation of the cell cycle inhibitor p27 and contributes to MM cell proliferation. Using CRISPR-Cas9 gene editing of 17 genes located in 1q21 individually knocked out in two MM cell lines with genomic 1q21 amplification, CKS1B was identified as key vulnerability. To validate and extend these findings, CKS1B, as well as SKP2, were knocked out in 19 MM cell lines of varying 1q21 copy number. Strong proliferation inhibition was observed in 13 of 19 cell lines, characterized by increased G2/M cell cycle arrest, and induction of senescence. While no clear association was found between 1q21 copy number and response, 13q loss was present in 4 of 6 robustly inhibited lines but only one of three lines with limited response. To explore the mechanism of action, systematic analysis of 22 published SKP2/CKS1B substrates in the MM1R cell line after SKP2/CKS1B knockout demonstrated increased protein levels of p27, Cyclin E, ORC1, and FOXO3a. The levels of p27 were further explored across all 19 MM cell lines, resulting to be upregulated in a large majority (17/19) including two insensitive lines. Strikingly, knocking out p27 in SKP2- or CKS1B-dependent cell lines rescued the proliferation defect, confirming the central role of p27 in mediating the effect. To further investigate the SKP2/CKS1B dependency in MM, we analyzed Cks1b, Skp2, and p27 expression along with whole-exome sequencing in high-risk P53-BIcγ1-derived mouse MM cell lines (Larrayoz et al., Nat Med 2023). Based on molecular profiling, the MM5080 cell line exhibited amplification of the chromosome 3q region syntenic to the human 1q21, leading to high expression of Cks1b. MM5080 cells were then used to generate stable, doxycycline-inducible shSkp2 clones, which progressively led to a significant proliferation reduction and cell cycle arrest ex vivo, together with upregulation of p27 levels. Notably, these effects were reversed upon doxycycline withdrawal. Finally, in vivo Skp2 inhibition in MM5080 cells engrafted into in Rag2−/−γc−/− mice delayed disease onset and extended mouse survival (median OS: 26 vs 22 days, p<0.001). These results validate SKP2 and CKS1B as critical dependencies in MM, support the therapeutic potential of targeting this pathway—especially in high-risk, 1q21-amplified or 13q-deleted disease—and provide a strong rationale for further preclinical development and patient stratification with SKP2/CKS1B inhibitors.

Background: Evidence suggests that CTCs are responsible for MM spreading and therefore prognostic. However, some pts unexpectedly have undetectable CTCs and we hypothesized that this feature defines a distinct MM subtype. Aim: Investigate the clinical and biological features of MM pts with undetectable CTCs. Methods: This study included 3,146 pts. Disease characteristics and clinical outcomes associated with undetectable CTCs defined by next-generation flow (NGF) with a limit of detection (LOD) of 2x10-6 were investigated in 1,093 transplant-eligible and ineligible newly diagnosed MM (NDMM) pts enrolled in the GEM2012MENOS65, CLARIDEX and GEM2017FIT clinical trials. Validation was performed in a European pooled-analysis of 1,601 NDMM pts having CTC assessments by flow cytometry with LOD of ≤1x10-5. We further investigated the prognostic value of undetectable CTCs after 2 cycles of induction in the GEM2017FIT trial (n=251), and prior salvage therapy in 242 relapsed MM (RRMM) pts enrolled in the GEM KyCyDex and SeliBorDara trials. Risk of transformation in the absence of CTCs was investigated in 1,487 MGUS and 324 smoldering MM (SMM) pts. Molecular alterations associated with CTC egress were evaluated by exome and RNA sequencing. Mechanistic validation of selected genes was performed in MM5080 cells derived from Trp53-BIcγ1 mice, a model that recapitulates tumor dissemination from early to late stages of MM progression. Results: Among NDMM pts, 10% showed undetectable CTCs before treatment. Compared to those with detectable CTCs, they displayed significantly less anemia and tumor burden, only one had R-ISS 3 and MGUS-like profiles were more frequent. In contrast, the incidence of plasmacytomas was higher (33% vs 15%, p<.001). Altogether, pts with undetectable CTCs showed clinical features linked to macrofocal disease. When compared to NDMM pts with detectable CTCs, those with undetectable CTCs showed significantly higher 5y rates of PFS (80% vs 50%) and OS (92% vs 72%). In multivariate analyses of PFS and OS including transplant-eligibility and the R-ISS, undetectable CTCs showed independent prognostic value for PFS (HR: 0.5; p=.005) and OS (HR: 0.4, p=.02). Pts with undetectable CTCs achieving MRD negative CR displayed unprecedented 5y rates of PFS and OS (92% and 98%). In the European pooled analysis, undetectable CTCs was confirmed as an independent prognostic factor of PFS (HR: 0.5; p<.001) and OS (HR: 0.4, p<.001). Of note, a 2x10-6 LOD was required to define this group of pts with unprecedented prognosis since detection of CTCs in between ≥0.0002% and <0.001% was associated with inferior survival in the Spanish and European cohorts. Absence of CTCs was also associated with longer PFS after cycle 2 of induction in transplant-ineligible NDMM (HR: 0.47, p=.003) and before a new line of therapy in RRMM (HR: 0.5, p=.01). Although the absence of CTCs was associated with lower risk of progression in MGUS (HR: 0.1, p<.001) and SMM (HR:0.4, p<.001), 1% of MGUS and 10% of SMM pts with sustained undetectable CTCs progressed to active MM. These results suggest the presence of a subgroup of pts with undetectable CTCs throughout the disease course, which displays less aggressive tumors and favorable clinical outcomes by the time they progress to active MM. We next investigated the biology of bone marrow tumor cells in this subgroup. While the median number of coding mutations was similar, copy number alterations were less frequent in pts with undetectable vs detectable CTCs (29 vs 61, p=.03). The former displayed lower frequency of del(17p) (5% vs 11%, p=.03) and +1q21 (23% vs 48%, p<.001). RNAseq uncovered 66 differentially expressed genes, 9 of which were located on 1q, in line with the genetic findings. To gain more insights into the functional role of candidate genes we selected LGALS1, which was among the genes showing the highest correlation between mRNA expression and CTC levels, for CRISPR/Cas9 knockout experiments in MM5080 cells inoculated in Trp53-BIcγ1 mice. Compared to Lgals1 wild-type, biallelic loss of Lgals1 resulted in longer OS (median not reached vs 46 days). Conclusions: NDMMpts with undetectable CTCs have less genetic alterations and lower expression of genes linked to CTC egress, which results in less aggressive and macrofocal disease. Thus, this study recognizes a new myeloma subtype that accounts for 10% of NDMM pts, who show unprecedented survival and can be defined by undetectable CTCs with NGF.

FAM46C/Tent5c, a non-canonical polyA-polymerase expressed by plasma cells, is recurrently mutated, or genetically deleted, in 20% of multiple myeloma (MM) patients but rarely in other cancers. While the role of FAM46C as a tumor suppressor has been defined in MM cell lines, a comprehensive mechanistic investigation of its physiological and pathological function in immunocompetent in vivo models is lacking. Here, we generated isogenic mouse MM cell lines with Tent5c loss by CRISPR-Cas9 editing, which promoted tumor cell proliferation upon in vivo transplantation that shortened survival of C57BL/6 immunocompetent mice. Reintroduction of full-length wild-type Tent5c, but not of several mutant Tent5c isoforms, decreased MM cell proliferation and clinical aggressiveness, concordant with the loss-of-function nature of common FAM46C mutations. To further explore the role of FAM46C, we engineered mice using Cre-LoxP recombination to conditionally delete Tent5c in mature B lymphocytes and plasma cells by a cγ1-cre allele upon T cell driven-immunization with SRBCs. Tent5c-KO mice exhibited a higher number of germinal center B cells that shifted towards activated B-cell differentiation at the expense of memory B lymphocytes, promoting formation of aberrant plasma cells in spleen and bone marrow (BM) with increased proliferation and expression of B-cell markers. To determine FAM46C/Tent5c oncogenic function in MM, Tent5c-KO mice were crossed with BIcγ1 mice, which progressively develop human-like MM preceded by asymptomatic conditions (Larrayoz et al, Nat. Med. 2023). Tent5c-BIcγ1 mice showed marked acceleration of MM development from precursor stages with respect to BIcγ1 mice (median OS, 187 vs 296 days; p<0,0001), exhibiting an augmented number of MM cells infiltrating the BM and circulating in peripheral blood. Transcriptional analyses of MM cells at single-cell resolution revealed enrichment in cell proliferation signatures and high expression of MYC and its target genes, leading to MYC protein stabilization. To validate mouse data in patients, a series of 658 newly diagnosed MM cases from the CoMMPass study with available genomic and transcriptomic data was characterized, with 41 (6%) with FAM46C somatic mutation, 128 (20%) with chromosome 1(p12) deletion, and 24 (4%) with both mutation and deletion. MM cells with FAM46C mutation and/or deletion showed enrichment in proliferation signatures and increased MYC expression, as well as an association with elevated circulating tumor cells (p=0.05). In a second series of 87 untreated smoldering MM patients, cases with FAM46C genetic loss and/or mutation exhibited accelerated progression into clinically active MM (progression rate at 2 years, 50% vs 25%; p=0.00072). Notably, increased degradation of immunoglobulin mRNAs caused by reduced polyadenylation was found in MM cells from Tent5c-BIcγ1 mice, which accordingly exhibited marked reduction of Ig secretion. To evaluate whether Ig production/secretion defects could be MYC-dependent, MYC expression was genetically induced in MM cells from genetically engineered mice with Tent5c loss, called the Tent5c-MYC-Icγ1 model. While MM with an aggressive, proliferative phenotype was found in Tent5c-MYC-Icγ1 mice, which was similar to that of Tent5c-BIcγ1 mice, full restoration of Ig production and secretion was observed. Further transcriptional investigations revealed that MM cells with Tent5c loss showed reduced expression of antigen presentation machinery and MHC-I/II genes, and increased expression of IFN pathway genes, suggesting aberrant tumor immunogenicity. Accordingly, major changes in the BM immune microenvironment were found, with prominent infiltration of CD4 and CD8 T cells with effector-memory phenotypes and expression of co-inhibitory molecules PD1, TIGIT and LAG3, and abundant mature LAG3+TIGIT- NK cells. Overall, our study uncovered a new role of FAM46C/Tent5c in regulating B-cell differentiation from the germinal centers. In addition, these results highlight that FAM46C/Tent5c inactivation promotes a MYC-driven proliferative and non-secretory MM phenotype with profound remodeling of the immune microenvironment, which collectively accelerate disease progression from early stages.

Triplet regimens that include an immunomodulatory agent, proteasome inhibitor, and dexamethasone are widely used in newly diagnosed and relapsed/refractory (R/R) multiple myeloma (MM). Mezigdomide (MEZI; CC-92480) is a cereblon E3 ubiquitin ligase modulator that is being clinically investigated in combination with bortezomib (BTZ) and low-dose dexamethasone (DEX) for safety and efficacy in pretreated R/RMM. The single-agent mechanism of action (MOA) of MEZI has been defined by the recruitment and degradation of essential MM transcription factors Ikaros and Aiolos, leading to cell autonomous antitumor effects and immune modulation. These effects were confirmed in patients based on pharmacodynamic measurements of Ikaros/Aiolos degradation in biomarker evaluations of immune subsets. However, the MOA of triplet regimens, including that of MEZI/BTZ/DEX remain poorly defined. To better understand the MOA of this triplet combination, we compared the mechanistic contributions of MEZI, BTZ, or DEX alone, or in combination, in preclinical MM models in vitro and in vivo. Additionally, we have compared these results with similar combinations with the immunomodulatory agent pomalidomide (POM). Our studies indicate that the MEZI/BTZ/DEX triplet is superior to all single agents and POM/BTZ/DEX in terms of potency of antiproliferative and proapoptotic activities, substrate degradation depth and kinetics in the presence of BTZ, and in vivo efficacy. We show that the combination of MEZI with BTZ increases cell death through disruption of multiple phases of the cell cycle and this thereby enhances the direct cytotoxic effects of the combination treatment.