Supplementary Figure S3 from BRD9 Degradation Disrupts Ribosome Biogenesis in Multiple Myeloma

2025-11-25

<p>BRD9 depletion downregulates expression of ribosome biogenesis genes.</p>

Group Authors List 1 from Cancer in 2025

2025-12-02

<p>AACR Cancer Progress Report 2025 Steering Committee</p>

Spatial proteomics of the bone marrow reveals distinct Tumor–Immune niches in smoldering and relapsed myeloma and their remodeling in response to bispecific antibody and CAR-T therapy

Blood · 2025-11-03

Abstract INTRODUCTION T cell–redirecting therapies, including CAR T cells and bispecific antibodies (BsAbs), have transformed the treatment of relapsed/refractory multiple myeloma (RRMM) and are being explored in earlier stages, including high-risk smoldering myeloma (SMM). Emerging evidence suggests spatial tissue organization modulates immunotherapy outcomes. Here, we used spatial proteomics to (1) map the bone marrow (BM) microenvironment across disease stages, (2) assess associations between baseline spatial features and response, and (3) characterize longitudinal remodeling in response to immunotherapy. METHODS We analyzed 121 BM biopsies from 47 patients with high-risk SMM treated with teclistamab or cilta-cel, and 19 patients with RRMM treated with BsAbs (14 teclistamab, 2 elranatamab, 3 talquetamab) between 2022–2024. Thirty-six patients had ≥1 post-treatment biopsy. A total of 196 regions of interest (ROIs) were profiled using a 32-marker imaging mass cytometry (IMC) panel (Hyperion XTi, Standard BioTools). Cell segmentation was performed with cellpose-sam. Marker-based annotation identified 27 phenotypes across 1,116,993 single cells. Cellular neighborhoods were defined as the 10 μm microenvironment around each cell. Spatial co-localization was evaluated via permutation testing. P values were FDR-adjusted. RESULTS SMM and RRMM cohorts showed expected differences: all RRMM patients had prior BCMA therapy, 42% had extramedullary disease, and median prior lines were five (range 2–13); SMM patients were mainly treatment-naïve. High-risk cytogenetics were similarly distributed (p=0.7). PC infiltration in ROIs showed a moderate correlation with pathologist estimates based on whole-slide review (R=0.61, p<0.001). In contrast, intra-sample consistency across ROIs was high (R=0.91–1.0). Regarding overall composition, SMM samples exhibited higher PC infiltration (q=0.0008) but fewer proliferating (Ki67⁺) PCs (q<0.001). T cells were more abundant in SMM (q=0.03), whereas RRMM was enriched for activated (HLA-DR⁺) and central memory (CD45RO⁺CCR7⁺) CD4⁺ T cells (Tact, Tcm; both q<0.001), as well as exhausted (PD1⁺TIM3⁺; Tex, q<0.001) and effector memory (CD45RO⁺; Tem, q=0.005) CD8⁺ T cells—indicative of an immunosuppressed T cell milieu. Regulatory T cells (Tregs) were similar between groups (q=0.3). A cellular neighborhood clustering approach at baseline revealed two PC neighborhoods in SMM (35% and 23% PCs) versus one in RRMM (44% PCs). Most non-PC cell types were similarly distributed across SMM PC neighborhoods. However, one PC neighborhood was enriched in T cells (16%), whereas the second (8%) possessed T cell levels similar to those of the RRMM PC neighborhood (9%). The T cell–enriched SMM PC neighborhood showed significantly higher levels of Tex and Tregs (both q<0.001), suggesting localized tumor-T cell immune processes unique to the SMM microenvironment. To assess treatment-induced BM changes, we analyzed matched samples at 6 months (n=20) and 12 months (n=5). PC proportions declined rapidly, while T cells (6m q=0.12, 12m q=0.06) and Granzyme B⁺ cells (6m q=0.06, 12m q=0.04) increased over time. Within CD4⁺ T cells, Tact and Tcm trended upward, whereas Tregs declined (q=0.15). CD8⁺ T cells were more dynamic, with transient Tem and Tex increases (both q=0.005). Additionally, macrophages showed a transient rise (q=0.05). Subsequently, analyzing spatial cell-cell interactions, we observed a decline between PCs and CD4⁺ and CD8⁺ memory T cell interactions over time. PC–CD8⁺ Tex interactions initially decreased but rebounded by 12 months, while T cell–DC interactions followed the opposite pattern, increasing early and then declining (q<0.05). These temporal patterns indicate an early phase of BsAb-driven immune remodeling, which appears to subside by one year, likely due to reduced tumor burden and limited ongoing T cell engagement. Finally, to assess whether baseline spatial interaction scores predict treatment response, we stratified RRMM patients by median PC–Treg and PC–Tex interaction levels. Kaplan–Meier analysis revealed significantly shorter progression-free survival in patients with enriched PC–immune interactions (Treg: p=0.003; Tex: p=0.006).CONCLUSIONS This study provides a framework for leveraging spatial profiling to dissect the bone marrow microenvironment in myeloma across disease stages and proposes a model in which spatial PC–T cell interactions inform the efficacy of T cell–redirecting therapies.

Identification of persister cells and evolution of tumor clones during immunotherapy and at minimal residual disease timepoints decoded by single-cell whole-genome sequencing in multiple myeloma

Blood · 2025-11-03

Abstract Background: Mutations in cancer driver genes and target antigens are associated with poor prognosis in multiple myeloma (MM), but their role in minimal residual disease (MRD) and in driving progression under immunotherapy remains unclear. To detect the cells responsible for relapse, we used longitudinal single-cell whole-genome sequencing (scWGS) to isolate and characterize tumor cells - including rare MRD cells - from patients treated with quadruplet therapy (Daratumumab, Lenalidomide, Bortezomib, Dexamethasone (DRVd)), anti-BCMA-directed T-cell-redirecting therapy (Teclistamab), or anti-BCMA CAR-T cell therapy (Cilta-cel). In addition, we present a method to assess MRD cells using peripheral blood (PB) in conjunction with bone marrow (BM) biopsies. Methods Tumor plasma cells from 11 patients with high-risk smoldering MM (HR-SMM, n=9) or relapsed/refractory MM (RRMM, n=2) were isolated using 9-color fluorescence-activated cell sorting (FACS) after magnetic bead enrichment for CD138 on BM and PB samples. For scWGS, ResolveDNA (Bioskryb Genomics) was used to amplify DNA prior to library preparation. All DNA libraries were prepared using Ultra II FS (New England Biolabs), and sequencing was performed at a depth of 60X (WGS) or 10X per single cell (scWGS). Subclonal dynamics were analyzed using the PhylogicNDT suite, and phylogenetic trees were inferred with the Sequoia method. Results We performed longitudinal WGS at baseline, MRD timepoints, and/or relapse in patients receiving DRVd (n=6), Teclistamab (n=4), or Cilta-cel (n=1). In patients treated with Daratumumab, CD38 expression was low in tumor samples collected during treatment (n=3) but high in those collected after the end of treatment (n=3). In one of the 3 on-treatment samples, a heterozygous CD38 gene deletion was identified in 3 out of 3 single tumor cells, from 2 independent clonal branches. No genomic alterations at the CD38 locus were detected in the remaining samples, suggesting that they could be rechallenged with CD38-targeted therapies irrespective of clinical response and clonal selection. Notably, the CD38 deletion was detected at an MRD timepoint and was later confirmed by bulk sequencing at disease progression. In contrast, scWGS of another hyperdiploid patient who remained MRD+ revealed unexpected subclonal chromosomal heterogeneity (including within trisomies 9 and 15) across all 22 single cells analyzed at the baseline timepoint - findings that were not detectable by bulk sequencing. After 12 cycles of therapy, the patient achieved a very good partial response (VGPR), and we profiled 12 tumor cells as part of the planned MRD assessment, which were CD38 low/negative. The tumor cells branched out of the clone found at baseline and harbored additional copy-number abnormalities (del(16q) and del(6q)). Across the five patients treated with Teclistamab (n=4) or Cilta-cel (n=1), BCMA expression remained high throughout treatment despite 4 of them not responding or relapsing after treatment. The fifth patient received Teclistamab, we profiled 4 single cells collected after 6 months of therapy (MRD negative 10⁻⁵, positive 10⁻⁶). We identified a new heterozygous loss of chromosome 16 (encompassing BCMA) in the single BCMA+ tumor plasma cell, while 3 other candidate cells were found to be normal plasma cells (false positives by FACS). The patient continued therapy and achieved CR and MRD negativity (<10⁻⁶), suggesting that even cells with a single copy of BCMA remaining can still be eliminated post-therapy. Lastly, a patient receiving Cilta-cel achieved CR with MRD positivity (>10⁻⁶). We performed WGS of 7 and 30 BCMA+ single circulating tumor cells at 10 and 18 months post-infusion, respectively, prior to clinical progression. Our analysis revealed genomically mature, persistent tumor cells lacking BCMA mutations, branching from the baseline tumor and acquiring additional mutations. Relapsed disease at 18 months originated from several of the clones already present at 10 months. Conclusion Using scWGS, we profiled persistent tumor cells during immunotherapy with minimal disease burden available. Subclonal analysis suggested that these persister cells primarily branched from a genomically mature, late clone - some acquiring additional mutations targeting the antigen as early as 6 months into therapy - rather than from an ancestral population.

PSMD3 orchestrates metabolic reprogramming and immunogenic cell death in multiple myeloma

Blood · 2025-11-03

Abstract Background and Rationale Multiple myeloma (MM) is a cancer of the plasma cells characterized by excessive production of immunoglobulins and dependence on the protein degradation system, which makes proteasome inhibitors (PIs) an important treatment for MM patients. However, PI resistance remains an unsolved problem in MM. PIs directly target the 20S core particle (CP) of the proteasome, which is responsible for proteolysis. The 19S regulatory particle (RP) is responsible for recognizing and delivering ubiquitinated proteins to the 20S CP. We identified PSMD3/Rpn3, a scaffold subunit of the 19S RP essential for proteasome assembly, as a potential target in MM. Using preclinical in vitro and in vivo models, we demonstrated that targeting PSMD3 induces MM cell apoptosis and overcomes PI resistance. Mechanically, targeting PSMD3 disrupts protein homeostasis, impairs metabolic adaptation, and relieves immune suppression in MM. Results Bioinformatic analyses revealed that PSMD3 is highly expressed in MM patient samples and correlates with poor overall survival. IHC and immunoblotting confirmed elevated PSMD3 levels in MM patient plasma cells compared to healthy controls. siRNA-mediated PSMD3 knockdown significantly reduced the viability of multiple MM cell lines, including those resistant to bortezomib (ANBL6-BR), carfilzomib (AMO1-CFZR), and pomalidomide (H929-PomR), suggesting its role in overcoming drug resistance. Inducible PSMD3 knockout (PSMD3-iKO) in AMO1 and KMS11 cells suppressed proliferation, while re-expression of wild-type PSMD3 restored growth, confirming target specificity. PSMD3 silencing induced apoptosis and cell cycle arrest, validated by flow cytometry and immunoblotting. Given PSMD3's role in proteasome assembly, we evaluated proteasome function and found marked accumulation of K48-linked polyubiquitinated proteins and activation of ER stress pathways in PSMD3-depleted MM cells. A degron-linked reporter assay further confirmed impaired proteasome degradation. In-gel proteasome assays demonstrated that PSMD3 knockout disrupted 26S proteasome assembly without affecting 20S core activity, indicating a deficiency in 19S regulatory particle incorporation. Finally, in a xenograft mouse model, PSMD3 depletion significantly suppressed tumor growth and prolonged survival, validating its therapeutic relevance in vivo. To elucidate the mechanism underlying PSMD3 depletion–induced cell death, we performed quantitative proteomic analyses. Pathway enrichment analysis revealed significant alterations in mitochondrial-related processes including glycolysis and oxidative phosphorylation, indicating that PSMD3 knockdown disrupts mitochondrial homeostasis. Functional metabolic profiling using the Seahorse XF Analyzer demonstrated that PSMD3 silencing led to profound mitochondrial dysfunction, evidenced by elevated mitochondrial superoxide levels and a marked reduction in maximal respiratory capacity, spare respiratory capacity, and ATP-linked respiration. This mitochondrial impairment resulted in the release of mitochondrial double-stranded DNA (mtDNA) into the cytosol, which in turn activated the cGAS-STING pathway. Activation of this innate immune sensor triggered a robust type I interferon response, as evidenced by upregulation of interferon-stimulated genes (ISGs). Moreover, PSMD3 depletion induced hallmark features of immunogenic cell death (ICD), including surface exposure of calreticulin and release of high mobility group box 1 (HMGB1), both of which enhance the immunogenicity of MM cells and promote anti-tumor immune recognition. Conclusion Our in vitro and in vivo data highlight the therapeutic potential of targeting PSMD3, a central regulator of protein homeostasis which links mitochondrial metabolism to immune surveillance in multiple myeloma. Targeting PSMD3 not only abrogates protein degradation but also disrupts energy metabolism and induces immunogenic cell death via cGAS-STING activation. These findings provide a compelling rationale for developing PSMD3-targeted strategies to overcome drug resistance and promote immune-mediated clearance of MM cells.

The B2M–LILRB1 axis as a novel myeloid immune checkpoint and therapeutic target in multiple myeloma

Blood · 2025-11-03

Abstract Cancer immunotherapies have shown significant promise in multiple myeloma (MM). However, their long-term efficacy is limited by immune evasion, and MM remains incurable. A key evasion strategy involves upregulating inhibitory phagocytosis checkpoints (IPCs), which prevent antigen-presenting cells (APCs) from phagocytosing cancer cells. This pathway is critical for the immune effects of standard anti-MM agents, including bortezomib (BTZ), an inducer of immunogenic cell death (ICD), and the monoclonal antibody daratumumab (DARA). Yet, the mechanisms regulating IPCs in MM remain poorly understood. To address this gap, we screened IPC axes in MM cells and APCs using bone marrow (BM) aspirates from MM patients (n=39) and healthy donors (n=5) via multiparametric flow cytometry. Immunophenotyping included 9 parameters for MM cells and 16 for APCs, covering IPC and lineage markers. We identified the B2 microglobulin (B2M) / leukocyte immunoglobulin-like receptor B1 (LILRB1) axis as the dominantly expressed IPC in MM. B2M, expressed on MM cells, binds LILRB1 on APCs. Notably, B2M is a known adverse prognostic factor in MM. Computational analysis of flow-cytometry data using a semi-automated pipeline allowed us to define 8 distinct myeloid cell clusters, with consistently high LILRB1 abundance, especially in conventional type 1 dendritic cells (DCs). Single-cell RNA-seq data from 90 MM BM samples confirmed LILRB1 expression in DCs, monocytes (Mo) and macrophages (MΦ). qRT-PCR of BM Mo-derived DCs found that LILRB1 mRNA levels increased from healthy donor to smoldering MM to overt MM, suggesting a link to disease progression. To functionally assess the B2M/LILRB1 axis, we knocked out B2M in MM cell lines (n=3) and re-expressed a chimeric B2M mutant (B2MMUT) lacking functional LILRB1-binding site by introducing 16 missense mutations. B2M functions as an MHC-I subunit, so this mutant allowed us to isolate its checkpoint role without the confounding effects from complete B2M loss. Yeast 2-hybrid assays confirmed a direct interaction between B2M and LILRB1, but not for B2MMUT. Phagocytosis assays found that B2MKO and B2MMUT cells were more susceptible (more than 2-fold) than WT to phagocytosis by DCs and MΦ after BTZ or DARA treatment (p<0.01). LILRB1 knockdown (KD) in APCs, using an antisense oligonucleotide, similarly enhanced the phagocytic potential after BTZ treatment (p<0.05). Since MM cells actively secrete B2M (s-B2M), we analyzed the media from MM cell lines with different B2M statuses: B2M WT, B2MKO, and B2MMUT. We found s-B2M in the media from B2M WT cells, but no B2M in the KO cells and s-B2MMUT in the B2MMUT. Exposure of DCs to the conditioned media of WT s-B2M, but not s-B2MMUT, reduced phagocytosis after BTZ or DARA (p<0.05) of B2MKO cells, suggesting that s-B2M impairs APC function in a LILRB1-dependent manner. Treatment of APCs with His-tagged recombinant B2M (rB2M) confirmed activation of downstream LILRB1 signaling, assessed by modulation of p-SHP1. Interestingly, APCs internalized rB2M in a LILRB1-dependent manner, as both LILRB1 KD and treatment with rB2MMUT resulted in reduced internalization. Correlative light and electron microscopy (CLEM) and TEM revealed that internalized rB2M localized to perinuclear vesicles near the microtubule-organizing center (MTOC). Confocal imaging found that rB2M uptake increased microtubule stability, as assessed by a higher acetylated-to-tyrosinated tubulin ratio. Functionally, this correlated with impaired APC maturation following lipopolysaccharide stimulation (assessed by phalloidin staining) and a reduced ability to activate T cells after ICD, as measured shown by reduced T cell-mediated MM cell lysis. To evaluate the in vivo relevance, we generated a murine MM cell line (5TGM1) expressing a chimeric B2M (B2Mmut), which cannot bind the murine LILRB1 ortholog. Mice injected with B2Mmut cells exhibited reduced tumor engraftment and growth compared to those injected with WT cells, accompanied by increased infiltration of CD3⁺ and CD8⁺ T cells.Ongoing studies are assessing the effect of the B2M/LILRB1 axis on BTZ or DARA efficacy in vivo and will be presented at the meeting. This work provides fundamental insight into the immune role of B2M in MM, revealing the B2M/LILRB1 axis as a novel immune checkpoint in MM driven by impaired APC function. Targeting this axis represents a new therapeutic vulnerability to restore immune competence and improve MM outcomes.

The Global State of Blood Cancers: An Ongoing Challenge

Blood Cancer Discovery · 2025-11-13

The state of the blood cancer field and its toll on patient mortality and morbidity, adapted from the 15th edition of the annual AACR Cancer Progress Report (https://cancerprogressreport.aacr.org/progress/), is presented to the US Congress and the public.

Supplementary Figure S1 from BRD9 Degradation Disrupts Ribosome Biogenesis in Multiple Myeloma

2025-11-25

<p>Clinical impact and biological significance of BRD9 in multiple myeloma (MM) in vitro and in vivo.</p>

Supplementary Data S2 from BRD9 Degradation Disrupts Ribosome Biogenesis in Multiple Myeloma

2025-11-25

<p>DifferentialExpression_RNAseq</p>

The impact of Duffy genotype on progression-free survival (PFS) with lenalidomide, Bortezomib, and dexamethasone (RVd) alone or RVd plus autologous stem cell transplantation (ASCT) and continuous R maintenance in patients (pts) with newly diagnosed multiple myeloma (NDMM): Updated subgroup analysis of the phase 3 DETERMINATION trial

Blood · 2025-11-03 · 1 citations

Abstract Background: A single nucleotide polymorphism (SNP) in ACKR1/DARC results in erythrocyte Duffy null phenotype in ~66% of African American (AA) and <1% of White pts. This is associated with lower absolute neutrophil count (ANC) and has a key role in cytokine homeostasis, which may influence MM pathobiology, response to inflammatory stressors, and treatment (tx) outcomes. DETERMINATION found a PFS benefit with RVd+ASCT (RVd-alone vs RVd+ASCT: hazard ratio [HR] 1.53; 95% confidence interval (CI) 1.23–1.91), but subgroup analysis showed differential PFS effect by race (AA pts: HR 1.07, 95% CI 0.61–1.89; White pts: HR 1.67, 95% CI 1.29–2.15). As race is a social construct, and with equitable access to care and cost-free therapy provision in DETERMINATION, we sought a pathobiological explanation. Given the prevalence of Duffy null and its known impact on the inflammasome, we hypothesized that Duffy status may affect magnitude of PFS benefit. We report updated PFS analyses in all pts in DETERMINATION with Duffy status available. Methods: Pts received RVd-alone or RVd+ASCT followed by R maintenance until progression in both groups. Peripheral blood samples underwent genomics analysis for the SNP rs2814778 and were classified as C/C (Duffy null) or non-C/C (Duffy non-null). Impact of Duffy status on PFS was evaluated with Cox proportional hazards regression in univariate models. Heterogeneity of tx effect was assessed by a test for interaction. Results: Overall, 592 enrolled pts were evaluable for Duffy status; consistent with US population data, 63.3% (n=62/98) of AA pts and 1.1% (n=5/465) of White/other pts were Duffy null (n=4/29 pts with missing race). 493 randomized pts had Duffy status evaluated (68.3% of intent-to-treat [ITT] population, N=722), with 238 assigned to RVd-alone and 255 to RVd+ASCT. Pts were broadly representative of the ITT population. PFS with RVd-alone vs RVd+ASCT in the analysis cohort, overall and by race, was consistent with the ITT population. Of the 493 pts, 59 (12.0%) pts were Duffy null (53 [89.8%] AA, 5 [8.5%] white/other race, 1 missing), and 434 (88.0%) were Duffy non-null (403 [92.9%] white/other race, 28 [6.5%] AA, 3 missing). Median (interquartile range) baseline ANC was 2.8 (2.1–4.3) x 109/L in Duffy null pts vs 3.4 (2.6–4.6) x 109/L in Duffy non-null pts. In Duffy null vs Duffy non-null pts, median duration of tx from randomization (35.1 vs 33.8 months [mos]) and of R maintenance (42.1 vs 36.4 mos) were similar in the RVd-alone arm but numerically shorter (30.3 vs 40.4 mos; 32.5 vs 42.4 mos) in the RVd+ASCT arm. Rates of grade ≥3 neutropenia in the RVd-alone vs RVd+ASCT arms (for all tx) were 48.3% vs 80.0% in Duffy null pts and 40.7% vs 88.9% in Duffy non-null pts; rates of grade ≥3 febrile neutropenia (FN) were 0% vs 3.3% in Duffy null pts and 7.2% vs 9.3% in Duffy non-null pts. Overall pooled PFS was similar in Duffy null vs Duffy non-null pts (median 62.5 vs 56.7 mos; HR 0.94, 95% CI 0.62–1.42). Duffy non-null pts had PFS findings consistent with the ITT analysis (RVd-alone vs RVd+ASCT: 120/209 vs 85/225 events/pts; median 46.7 vs 67.5 mos; HR 1.76, 95% CI 1.33–2.34). In contrast, Duffy null pts had longer PFS with RVd-alone vs RVd+ASCT (9/29 vs 16/30 events/pts; median NR vs 44.0 mos; HR 0.64, 95% CI 0.27–1.50) (interaction p-value 0.005). When these analyses were restricted to AA pts, the same PFS pattern was seen with RVd-alone vs RVd+ASCT among Duffy null (8/25 vs 15/28 events/pts; median NR vs 45.4 mos; HR 0.66, 95% CI 0.27–1.60) compared to Duffy non-null pts (6/13 vs 3/15 events/pts; median 64.4 mos vs NR; HR 5.29, 95% CI 1.20–23.4). On univariate analysis by Duffy status (null vs non-null), PFS HR was 0.51 (95% CI 0.26–1.00) with RVd-alone and 1.63 (95% CI 0.95–2.78) with RVd+ASCT. Conclusions: These exploratory analyses of DETERMINATION suggest that Duffy status drives a difference in tx effect that is more pronounced than for PFS by race. With RVd-alone vs RVd+ASCT, PFS appeared better in Duffy null pts and poorer in Duffy non-null pts. In Duffy null vs non-null pts, PFS appeared better with RVd-alone and poorer with RVd+ASCT. Similar trends were seen in analyses restricted to AA pts, indicating Duffy status may provide a biological rationale for observed differential tx effects, rather than race. Further studies are warranted to assess impact of Duffy status on clinical outcomes such as treatment response, overall survival, and effects on the inflammasome, as well as ANC and FN.