Multiple myeloma progresses from precursor states to active disease, and studying tumor microenvironment (TME) evolution across these stages is key to understanding immune dysregulation and therapeutic resistance. Here, we integrated paired single-cell RNA, T-, and B-cell receptor sequencing data from bone marrow samples of 235 patients spanning the disease spectrum. This dataset revealed dynamic changes in the abundance and functional states of diverse immune populations, including T-, natural killer, B-, and myeloid cells. Using non-negative factorization of cell-subset composition, we identified five reproducible TME subtypes, or "ecotypes," defined by coordinated cellular architectures. These ecotypes captured structured variation beyond disease stage and were associated with distinct cell-cell communication networks, cytokine signaling landscapes, transcription factor programs, and shared gene modules, reflecting coordinated immune adaptation to microenvironmental constraints. By linking tumor features with immune changes and ecotype distributions, we identified context-dependent associations influenced by both disease biology and treatment effects on the TME. We found that an ecotype enriched for bone marrow-resident populations and limited immune infiltration was associated with tumor expansion and inferior clinical outcomes, whereas ecotypes reflecting T-cell functional states showed distinct associations with immunotherapy response and survival. Our study provides a comprehensive single-cell immune atlas of multiple myeloma and its precursors, offering insights into TME organization and progression, and may guide development of stage-specific or ecotype-targeted immunotherapies.

7533 Background: In DREAMM-8 (NCT04484623), BPd demonstrated a significant improvement in the risk of progression or death vs PVd in patients (pts) with RRMM who received ≥1 prior line of therapy, including lenalidomide. Pts with high-risk cytogenetic abnormalities (HRCAs) have a poor prognosis and need more efficacious treatments. Here we present a subgroup analysis in pts with HRCAs. Methods: Pts were randomized 1:1 to BPd (28-day cycles) or PVd (21-day cycles). Pts were treated until progressive disease, unacceptable toxicity, or death. Efficacy assessments occurred every 4 weeks. Pts with HRCAs were defined as having ≥1 of the following: t(4;14), t(14;16), amp1q, and del(17p13). Descriptive statistics were used to summarize the results, along with 95% exact CIs. Hazard ratios (HRs) for progression-free survival (PFS) were estimated using the Cox model, with 95% CIs based on the Brookmeyer-Crowley method. Results: The intention-to-treat population included 302 pts: 155 in the BPd arm and 147 in the PVd arm. In the BPd arm, 68 of 155 (44%) pts had HRCAs; of them, 23 (15%) had t(4;14), 7 (5%) had t(14;16), 32 (21%) had del(17p13), and 40 (26%) had amp1q. In the PVd arm, 60 of 147 (41%) had HRCAs; of them, 20 (14%) had t(4;14), 11 (7%) had t(14;16), 26 (18%) had del(17p13), and 33 (22%) had amp1q. Median PFS in pts with ≥1 HRCA was 21.1 mo (95% CI, 13.5 mo-NR) with BPd vs 9.2 mo (95% CI, 6.5-14.8 mo) with PVd (HR, 0.58; 95% CI, 0.36-0.95); 18-mo PFS rates were 53% and 33%, respectively. PFS benefit favored BPd across HRCA subgroups (HR [95% CI]: t(14;14), 0.74 [0.31-1.76]; del(17p13), 0.45 [0.22-0.92]; and amp1q, 0.49 [0.24-1.03]). In pts with ≥1 HRCA, overall response rate (ORR) was higher with BPd (n=52; 76%; 95% CI, 64.6%-85.9%) than PVd (n=39; 65%; 95% CI, 51.6%-76.9%), and more pts achieved ≥ complete response with BPd (Table). Benefit was maintained across HRCA subgroups. Conclusions: In pts with RRMM with HRC features, BPd demonstrated clinically meaningful PFS benefit, higher ORR, and a higher rate of deep responses vs PVd. These data support the potential use of BPd as a standard-of-care regimen in this key pt population with a high unmet need. Clinical trial information: NCT04484623 . Patients achieving ≥CR in HRC groups n/N (%); 95% CI BPd PVd t(4;14) 9/23 (39); 19.7-61.5 5/20 (25); 8.7-49.1 t(14;16) 3/7 (43); 9.9-81.6 2/11 (18); 2.3-51.8 del(17p13) 11/32 (34); 18.6-53.2 0/26; 0-13.2 amp1q 17/40 (43); 27.0-59.1 3/33 (9); 1.9-24.3 ≥1 HRCA 29/68 (43); 30.7-55.2 9/60 (15); 7.1-26.6

Multiple myeloma (MM) arises from abnormal plasma cells (PCs) progressing from precursor states, including monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). Understanding this transition and progression to overt MM requires improved non-invasive strategies. We employed a liquid biopsy approach to detect and characterize circulating PCs across disease states in 68 patients (MGUS = 11, SMM = 21, NDMM = 19, RRMM = 17) using multi-channel immunofluorescence staining and machine learning-assisted rare event detection. PCs were identified by CD138 and B-cell maturation antigen (BCMA) expressions, with distinct phenotypic subpopulations stratifying disease states. The D | CD138 | BCMA-Memb phenotype was the most predictive, with incidence increasing from MGUS to SMM and overt MM (p < 0.005). Multivariate modeling distinguished precursors from overt disease with 86% accuracy. Shifts in BCMA and CD45 expression suggested immune cell profile alterations with progression and treatment. These findings underscore PB-based liquid biopsy as a promising tool for MM detection and monitoring, revealing circulating PC heterogeneity.

7544 Background: Belamaf combinations were evaluated for RRMM in the phase 3 DREAMM-7 (belamaf + bortezomib + dexamethasone [BVd]; NCT04246047) and DREAMM-8 (belamaf + pomalidomide + dexamethasone [BPd]; NCT04484623) trials, and significant progression-free survival benefits were reported over standard of care, with significant overall survival benefit reported for BVd. Ocular events (e.g., ocular adverse events [oAEs], blurred vision, dry eye) occurred with belamaf and most resolved with dose holds and modifications. We examined the baseline eye health of pts with RRMM receiving BVd or BPd, and whether baseline ocular conditions affected rates of treatment-emergent (TE) oAEs. Methods: Pts with ≥1 prior therapy were eligible for DREAMM-7/8; pts with ocular conditions were eligible except for corneal epithelial disease (mild punctate keratopathy was allowed). Mandatory ophthalmic examinations (best corrected visual acuity [BCVA], slit lamp, and funduscopic exams) were performed in both arms of the trials at baseline and routinely during treatment. oAEs were graded by Common Terminology Criteria for Adverse Events. Regardless of presence/absence of baseline ocular conditions, the same protocol-defined strategies were used for ocular event management during the studies. Results: In 392 pts treated with belamaf (n=242 DREAMM-7 and n=150 DREAMM-8), baseline ocular conditions were reported in 62% of pts (n=135 and 106); baseline conditions included cataract 50% (n=101 and 96), keratopathy 14% (n=33 and 23), dry eye 14% (n=31 and 24), visual acuity of 20/50 or worse 6% (n=18 and 7), glaucoma 6% (n=11 and 13), blepharitis 2% (n=4 and 3), age-related macular degeneration 1% (n=3 and 2), and diabetic retinopathy <1% (n=0 and 2). Any TE oAE was reported in 74% (n=100/135) and 87% (n=92/106) of pts with baseline ocular conditions in DREAMM-7 and DREAMM-8, respectively, compared with 79% (n=85/107) and 91% (n=40/44) of pts without baseline ocular conditions (Table). Conclusions: The safety profiles of belamaf combinations for oAEs were similar between patients with vs without baseline ocular conditions, suggesting that these baseline ocular conditions did not increase the risk of TE oAEs. The effect of each baseline ocular condition on TE oAEs, as well as TE corneal exam findings and visual acuity changes, will be presented. Clinical trial information: NCT04246047 , NCT04484623 . TE ocular events in patients receiving a belamaf combination in DREAMM-7/8. DREAMM-7 DREAMM-8 With any baseline ocular condition, n=135 No baseline ocular condition, n=107 With any baseline ocular condition, n=106 No baseline ocular condition, n=44 Any oAE, n (%) 100 (74) 85 (79) 92 (87) 40 (91)

ABSTRACT:Plasma cell dyscrasias encompass a spectrum from the precursors monoclonal gammopathy of undetermined significance and smoldering myeloma to symptomatic myeloma, but the genes that enable progression and confer poor prognosis are incompletely understood. Using single-cell transcriptomics, we identified the caseinolytic protease proteolytic subunit (CLPP), a key component of the mitochondrial caseinolytic protease (CLP) serine endopeptidase, as being overexpressed in CD138+ neoplastic vs normal and in symptomatic vs precursor plasma cells. Its high expression was associated with an adverse prognosis across multiple molecularly defined subgroups in the newly diagnosed and relapsed/refractory settings and with extramedullary disease. Pharmacologic CLPP inhibition and genetic suppression reduced organoid growth, cell viability, and cell cycle progression, and triggering an unfolded protein response and apoptosis. This occurred in association with mitochondrial transmembrane potential loss and caspase and proteasome activation in a reactive oxygen species-dependent manner. Downstream consequences included autophagy and mitophagy induction and reductions in oxidative phosphorylation and glycolysis with consequent compromise of mitochondrial and cytoplasmic adenosine triphosphate (ATP) production. CLP endopeptidase inhibition overcame conventional and novel drug resistance, induced apoptosis in primary samples, showed efficacy in vivo, and could be achieved with the clinically relevant agent inobrodib. Finally, regimens combining a CLPP and proteasome inhibitor showed enhanced efficacy, as did combinations with inhibitors of intermediary metabolism and autophagy. Taken together, our data indicate that CLPP is a key contributor to transformed plasma cells, a novel mediator of high-risk behavior, and a legitimate target for myeloma therapy whose inhibitors could be rationally combined with current therapeutics to improve outcomes.

Multiple myeloma (MM) is an aggressive hematologic malignancy arising from plasma cell (PC) proliferation in the bone marrow, progressing from its precursor states MGUS and SMM. Despite therapeutic advances, MM remains incurable, underscoring the need for better risk stratification and early detection. Tumor heterogeneity and dynamic immune microenvironment changes drive progression, yet bulk analyses overlook rare subpopulations critical to disease evolution and resistance. This study employed multiplexed targeted proteomics to characterize bone marrow aspirates (BMA) from 22 patients to observe the change in the distribution of PCs and tumor immune microenvironment (TiME) cells across MM disease states and controls. Bone marrow samples were processed, stained with a 29-metal-labeled antibody panel, and analyzed using computational clustering approaches. Clustering of PCs revealed changes in subtypes with disease progression, marked by shifts from CD45-positive/CD138-low subpopulations in precursor states to CD45-negative/CD138-high populations in advanced MM. Analysis of the TiME identified distinct immune phenotypes, with significant reductions in monocyte/macrophage and lymphoid clusters across MM states compared to controls. Notably, a distinct PC cluster enriched in NDMM and RRMM exhibited high BCMA and CD138 expression, suggesting a potential role in disease progression. These findings provide critical insights into MM evolution and immune landscape alterations, with implications for targeted therapeutic strategies.

7515 Background: In DREAMM-8 (NCT04484623), BPd demonstrated a statistically significant, clinically meaningful benefit to progression-free survival (PFS) vs PVd in pts with RRMM who received ≥1 prior line of treatment including lenalidomide. MRD neg has been shown to be a predictor of PFS and overall survival (OS) in MM. We assessed efficacy outcomes by MRD status. Methods: Ptswere randomized (1:1) to BPd or PVd. The primary endpoint was independent review committee (IRC)–assessed PFS; OS, duration of response, and MRD status determined by next-generation sequencing with 10 −5 sensitivity threshold was assessed in pts with complete response or better (≥CR) every 6 mo until progressive disease. Post hoc subgroup analyses of PFS and OS were conducted based on IRC-assessed response (≥CR or ≥VGPR) and MRD-neg status using Kaplan-Meier method; CIs were estimated using Brookmeyer-Crowley method. Results: 302 pts were randomized to BPd (n=155) or PVd (n=147). As previously reported (median follow-up, 21.8 mo), more pts with BPd had CR-based MRD neg vs PVd (37/155 [24%] vs 7/147 [5%]). A similar trend was seen in pts with ≥VGPR; 50/155 pts (32%) had VGPR-based MRD neg with BPd vs 8/147 (5%) with PVd. In the DREAMM-8 trial, MRD neg was associated with improved efficacy outcomes (Table). Pts with CR-based MRD neg had a lower risk of disease progression or death compared with pts without MRD neg (PFS HR, 0.14; 95% CI, 0.06-0.32; Table); median was NR overall and in each treatment arm (HR [BPd vs PVd], 0.90; 95% CI, 0.10-7.76). MRD neg pts had a lower risk of death (OS HR, 0.18; 95% CI, 0.07-0.49). In all pts who did not have CR-based MRD neg, pooled median PFS was 14.0 mo (95% CI, 11.1-18.6 mo; BPd, 19.6 mo; PVd, 10.2 mo; HR [BPd vs PVd], 0.67; 95% CI, 0.47-0.94), and the pooled 18-mo PFS rate was 46% (95% CI, 39%-52%; BPd, 52%; PVd, 39%). Median OS was NR overall, 33.0 mo (95% CI, 23.7-NR) with BPd and NR with PVd; 18-mo OS rate was 69% (95% CI, 63%-75%; BPd, 72%; PVd, 67%). Conclusions: Consistent with previous reports, MRD neg was associated with a robust benefit in PFS and OS, highlighting the significance of a greater response depth. Pts with BPd achieved a 5-fold improvement in CR-based MRD neg vs PVd (24% vs 5%). Pts who did not achieve CR-based MRD neg had a clinically meaningful benefit in PFS with BPd vs PVd. Clinical trial information: NCT04484623 . MRD neg Non-MRD neg MRD status (≥CR), n Pooled (44) BPd (37) PVd (7) Pooled (258) BPd (118) PVd (140) Median PFS (95% CI), mo NR (NR-NR) NR (NR-NR) NR (NR-NR) 14.0 (11.1-18.6) 19.6 (13.5-NR) 10.2 (8.4-17.1) 18-mo PFS rate (95% CI), % 93 (79-98) 91 (75-97) 100 (100-100) 46 (39-52) 52 (42-62) 39 (30-48) 18-mo OS rate (95% CI), % 93 (80-98) 92 (76-97) 100 (100-100) 69 (63-75) 72 (62-79) 67 (59-74)

Plasma cell dyscrasias encompass a spectrum from the precursors monoclonal gammopathy of undetermined significance and smoldering myeloma to symptomatic myeloma, but the genes that enable progression and confer poor prognosis are incompletely understood. Using single cell transcriptomics, we identified the Caseinolytic protease proteolytic subunit (CLPP), a key component of the mitochondrial CLP serine endopeptidase, as being over-expressed in CD138+ neoplastic versus normal, and in symptomatic versus precursor plasma cells. Its high expression was associated with an adverse prognosis across multiple molecularly defined subgroups in the newly diagnosed and relapsed/refractory settings, and with extramedullary disease. Pharmacologic CLPP inhibition and genetic suppression reduced organoid growth, cell viability, and cell cycle progression, while triggering an unfolded protein response and apoptosis. This occurred in association with mitochondrial transmembrane potential loss, and caspase and proteasome activation in a reactive oxygen species-dependent manner. Downstream consequences included autophagy and mitophagy induction, and reductions in oxidative phosphorylation and glycolysis with consequent compromise of mitochondrial and cytoplasmic ATP production. CLP endopeptidase inhibition overcame conventional and novel drug resistance, induced apoptosis in primary samples, showed efficacy in vivo, and could be achieved with the clinically relevant agent inobrodib. Finally, regimens combining a CLPP and proteasome inhibitor showed enhanced efficacy, as did combinations with inhibitors of intermediary metabolism and autophagy. Taken together, our data indicate that CLPP is a key contributor to transformed plasma cells, a novel mediator of high-risk behavior, and a legitimate target for myeloma therapy whose inhibitors could be rationally combined with current therapeutics to improve outcomes.

7509 Background: Randomized controlled trials oflenalidomide (LEN) maintenance after AuSCT have shown a progression free survival (PFS) benefit in pts with MM. We report final results of a phase 2 trial evaluating the efficacy and safety of adding elotuzumab (ELO) to LEN as maintenance therapy post- AuSCT. Methods: On 28-day cycles, patients received ELO 10mg/kg iv weekly for cycles 1-2.Between 4/15/2015-1/27/2016, 27 pts received ELO 10mg/kg q2 weeks for cycles 3-6 and 20mg/kg monthly for cycles 7+. From 1/28/2016 forward, 74 pts received ELO 20 mg/kg monthly from cycle 3+. LEN was dosed at 10 mg/day for cycles 1-3 and increased to 15 mg/day at physician discretion starting with cycle 4 in the absence of non-hematologic (HEME) toxicity > grade 1, ANC < 1000/mL and platelet count < 100K/ml. For the 1st 8 weeks, pts <75 yrs received 28 mg of dexamethasone (DEX) 3-24 hours pre-infusion and pts ≥75yrs received 8 mg; pts received 4-10 mg iv DEX pre-infusion for all cycles. Zoster and thromboembolic prophylaxis were prescribed as per standard recommendations. The primary endpoint was PFS, defined as time from AuSCT to progressive disease (PD) or death (whichever occurred first), or censored at last contact date. Secondary objectives were best response, overall survival (OS), incidence of second primary malignancies (SPMs) and adverse event (AE) profile. Enrollment was completed on 6/5/2019. Eligible pts received ≤2 induction lines of therapy (LOT) and were 60-210 days post-AuSCT. Results: Pts (n=100) were treated for a median of 40 cycles (2-109). With a median follow up of 74.3 mos, 78% of pts (n=79) remain alive. Median PFS was 75.4 months. Median OS has not been reached; 5-year OS rate was 86.7%. At study entry, rate of ≥VGPR was 77.3% and ≥CR was 37.7%. On study, this improved to 91.1% ≥VGPR and 69.3% ≥CR. Of pts in ≥VGPR and tested for minimal residual disease (10-5 sensitivity by flow cytometry), 37/44 were negative. High-risk cytogenetics (n=44; p<0.0001), International Staging System (ISS) Stage III (n=10/95, p=0.049) and Revised-ISS Stage III (n = 3/89; p= 0.026) predicted shorter median PFS (39.2, 53.2 and 32.2 mos. respectively). Median PFS on next LOT was 21.8 mos. Grade 3-4 HEME AEs (n=102) were: neutropenia 35%, thrombocytopenia 7%, and anemia 10% . Grade 3-4 non-HEME AEs in >3 pts were: respiratory infections 23%, hypophosphatemia 19%, diarrhea 13% , fatigue 11% , peripheral neuropathy 7%, other infections 6%, myalgias 4%, and high ALT 4%. Incidence of HEME, non-invasive and solid tumor SPMs was 8%, 8% and 7%. Median time to HEME SPM diagnosis was 45.5 mos. Conclusions: ELO-LEN is a well-tolerated maintenance therapy on which 53% of patients had improved quality of response. Median PFS and 5-year OS compare favorably with results from CALGB 100104 and IFM 2005-02 trials of lenalidomide alone. SPM incidence is similar to rates reported in these trials. Clinical trial information: NCT02420860 .