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Abstract Background: Covalent Bruton tyrosine kinase inhibitors (cBTKi) have dramatically changed the treatment (tx) landscape of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), especially in the first-line setting. However, cBTKi resistance or intolerance eventually occurs. Pirtobrutinib is a highly selective, non-covalent BTKi that inhibits BTK with low nM potency throughout the daily dosing interval. Pirtobrutinib demonstrated safety and efficacy in the phase 1/2 BRUIN study among patients (pts) with R/R CLL/SLL, including pts previously treated with a BTKi. Pirtobrutinib is approved for treating CLL in adults in the EU after prior tx with a BTKi, and in the USA for adults with CLL/SLL who have received at least 2 prior lines of therapy, including a BTKi and a BCL-2i. Here, we report the final results from the phase 1/2 BRUIN study (NCT03740529) with more than 5 years (yrs) follow-up (f/u), focusing on efficacy and safety of pirtobrutinib in pts with CLL/SLL in the post-cBTKi setting. Methods: Pts with prior cBTKi-exposed CLL/SLL were eligible for tx with pirtobrutinib. Endpoints included overall response rate (ORR) per 2018 iwCLL criteria, ORR including partial response with lymphocytosis (PR-L), duration of response (DOR), and progression-free survival (PFS), assessed by independent review committee (IRC; presented herein) and investigator, overall survival (OS), and safety. Data cut was 27 January 2025, providing a median study f/u of 34.1 months (mos; range, 0.5-69.8). Results: Among the 282 pts with CLL/SLL who were cBTKi-exposed and treated with pirtobrutinib, of which 84.4% (238/282) received 200 mg as the starting dose, the median age was 69 yrs (range, 36-88). The median number of prior therapies was 4 (range, 1-11), with 45.4% of pts having received prior BCL2i, and 77.0% having discontinued prior cBTKi due to progressive disease (PD), 19.5% due to toxicity, and 3.5% due to other reasons/missing. High-risk features were frequent: unmutated IGHV in 85.8% (193/225), mutated TP53 in 40.4% (99/245), and del(17p) in 29.2% (59/202). The ORR was 72.3% (95% CI, 66.7-77.5). ORR including PR-L was 81.6% (95% CI, 76.5-85.9), which was consistent across subgroups including BCL2i-exposed (80.5%; 95% CI, 72.5-86.9), BCL2i-naive (82.5%; 95% CI, 75.5-88.1), unmutated IGHV (82.9%; 95% CI, 6.8-87.9), del(17p) (88.1%; 95% CI, 77.1-95.1), and prior cBTKi discontinuation due to PD (79.7%; 95% CI, 73.8, 84.9) or toxicity (87.5%; 95% CI, 76.8-94.4). The median DOR was 18.4 mos (95% CI, 14.82-20.27), with 36-mos DOR rate of 28.0% (21.6-34.7). The median PFS was 18.7 mos (95% CI, 16.6-21.8) among all pts and the 36-mos PFS rate was 25.0% (95% CI, 19.4-31.0) with 44 mos of f/u. Further, the median PFS was 22.3 mos (95% CI, 19.3-27.6) for BCL2i-naive pts, and 15.9 mos (95% CI, 13.6-17.5) for BCL2i-exposed pts; 16.6 mos (95% CI, 13.8-19.4) among those who discontinued prior cBTKi therapy due to PD, and not estimable (NE) (95% CI, 25.2-NE) in those who discontinued due to toxicity. Among all pts with CLL/SLL, with a median f/u of 46.5 mos, the median OS was NE (95% CI, 47.8-NE), with the 36-mos OS rate of 62.0% (95% CI, 55.8-67.7). Median time on pirtobrutinib was 20.0 mos (range, 0.2-69.8), and 43 (15.2%) pts were still on tx at data cutoff. The most frequent TEAEs, regardless of attribution, were fatigue (38.7%), neutropenia/neutrophil count decreased (35.8%), diarrhea (30.5%), cough (29.8%), COVID-19 (28.4%), and contusion (27.7%). The most frequent grade ≥3 TEAEs were infection (36.5%) and neutropenia/neutrophil count decreased (29.8%). Low rates of grade ≥3 TEAE of hypertension (5.3%), hemorrhage/hematoma (2.5%), and atrial fibrillation/flutter (2.1%) were observed. Grade 5 TEAEs, regardless of attribution, occurred in 35 (12.4%) pts. Overall, 11 (3.9%) pts had a TRAE leading to a dose reduction, and 9 (3.2%) pts had a TRAE leading to pirtobrutinib discontinuation. Conclusion: In heavily pretreated R/R post-cBTKi CLL/SLL pts, pirtobrutinib continues to show favorable and durable efficacy. Pirtobrutinib is well-tolerated, with low rates of hypertension, hemorrhage/hematoma, and atrial fibrillation/flutter, which can be frequently observed with cBTKi. Additionally, dose reductions/discontinuations due to pirtobrutinib TRAEs were low. No new pirtobrutinib safety signals were identified with more than 5 yrs of f/u.

Abstract Measurable residual disease (MRD)-testing is often used to evaluate therapy efficacy in chronic lymphocytic leukaemia (CLL), with undetectable MRD (uMRD) often serving as a surrogate for progression-free survival (PFS). However, its accuracy in predicting PFS with Bruton tyrosine kinase inhibitors (BTKis), with or without venetoclax, remains controversial. We searched PubMed, Web of Science, Embase, and Cochrane Library databases up to February 8, 2025 using terms chronic lymphocytic leukemiaOR small lymphocytic lymphomaAND measurable residual diseaseAND progression-free survival. Bayesian Weibull accelerated failure time models were used for individual-level analyses, while trial-level correlations used weighted Spearman rank tests and weighted least squares regression. A total of 41 trials (9,569 subjects) were included. Therapies tested included BTKis (11 trials), venetoclax (12), the combination (5), and other therapies like chemoimmunotherapy and monoclonal antibodies (23). MRD was assessed in blood (21 trials), bone marrow (7), or both (9). At the individual level, subjects with detectable MRD (dMRD) had a higher progression and/or death risk compared to uMRD (Hazard Ratio [HR] = 3.63; 95% Credibility Interval [CrI] 3.30-4.00; P < 0.01). In sensitivity analysis, MRD-testing showed reduced predictive accuracy for PFS in subjects receiving BTKi-based therapy (HR = 1.52, 1.02–2.19) compared to venetoclax-based (HR = 4.46, 3.61–5.50) or other therapies (HR = 3.86, 3.45–4.34). Across subgroups, dMRD remained consistently associated with increased risk. In subjects with del(17p)/TP53 variants, dMRD predicted greater (HR = 6.52; 3.47–12.79) versus those without (HR = 4.34; 3.24–5.81). By IGHV status, dMRD conferred a 3.54-fold risk in IGHV-mutated (95% CrI: 2.42-5.34) and 2.64-fold in unmutated subjects (95% CrI: 2.10-3.34). Among response categories, risk was higher in partial responders (HR = 2.39; 1.70–3.43) than complete responders (HR = 1.64; 1.24–2.17). dMRD also predicted increased risk in relapsed/refractory (R/R) CLL subjects (HR = 4.11, 2.54-6.84) and untreated subjects (HR = 3.59, 3.25-3.98). Bone marrow MRD testing proved a stronger predictor of PFS than blood (HR = 4.27, 3.42-5.35 versus HR = 3.52, 3.17-3.92). The highest risk was seen with MRD testing 12–32 months post-treatment (HR = 5.37, 4.56-6.40), though differences across timepoints were modest after adjusting for analytical method. At the trial level, correlations between MRD and PFS were weak (Spearman's rho [R] = -0.35; Determination Coefficients [R²] = 0.02). In sensitivity analyses, correlations remained low for BTKi-based therapy (R = -0.19 [-0.73, 0.50]; R² = 0.04) and venetoclax-based therapy (R = -0.32 [-0.85, 0.45]; R² = 0.14), but were stronger for other therapies (R = -0.80; R² = 0.58). For other therapies, MRD tests of bone marrow samples showed higher predictive value than blood (R = -0.94; R² = 0.86 versus R = -0.81; R² = 0.63). Despite blood samples and testing at 9-months of other therapies meeting the validity threshold (R² = 0.63, P = 0.21; R² = 0.63, P = 0.06), the associated P-values preclude definitive conclusions. Additional analyses examining sample source, time points and testing method within treatment classes suggest limited accuracy of predicting PFS in heterogeneous conditions (all R < -0.85, R² < 0.60). In subgroup analyses, untreated trials showed weak associations (R = -0.30 [-0.77, 0.13]; R²= 0.02), while R/R CLL trials were insufficient for analysis. MRD-testing of blood showed lower PFS predictive accuracy than bone marrow(R = -0.26 [-0.71, 0.20]; R² = 0.03 versus R = -0.37 [-1.00, 0.62]; R² = 0.03). MRD-testing at 15 months demonstrated slightly better accuracy than 9-month testing (R = -0.59 [-1.00, 0.61]; R² = 0.27 versus R = -0.26 [-0.88, 0.41]; R² = 0.04).MRD-testing by NGS outperformed MPFC (R = -0.54 [-1.00, 1.00]; R² = 0.45 versus R = -0.30 [-0.76, 0.13]; R² = 0.01), though both methods remained below the pre-specified determination coefficient threshold. Our data indicate accuracy of results of MRD-testing to predict PFS depends on whether used at the subject or trial-level, type of therapy and sample tested. Importantly MRD-testing is not an accurate predictor of PFS for BTKis with or without venetoclax. We suggest results of MRD-testing as a surrogate for PFS should not be used as a surrogate endpoint for studies of BTKis with or without venetoclax.

Abstract Background Pirtobrutinib is a highly selective, non-covalent Bruton tyrosine kinase inhibitor (BTKi) approved for patients (pts) with relapsed/refractory (R/R) chronic lymphocytic lymphoma (CLL) who have previously received a covalent BTKi (cBTKi) and a BCL-2 inhibitor such as venetoclax. Pirtobrutinib monotherapy has demonstrated a median duration of response of 12.2 months in the BRUIN trial. Pirtobrutinib is increasingly being used as a second-line therapy after failure of a cBTK inhibitor without prior BCL-2 inhibitor exposure. We aim to characterize short-term survival outcomes and adverse events of pirtobrutinib in venetoclax-naïve versus venetoclax-exposed pts with R/R CLL. Methods We conducted a retrospective cohort study using TriNetX Global Collaborative Network, comprising data from 152 healthcare organizations. Adults (≥18 years) with R/R CLL who received pirtobrutinib after venetoclax were classified into the “V+P” cohort. Pts who received pirtobrutinib without prior venetoclax exposure were included in the “P” cohort. 1:1 propensity score matching (PSM) was performed, and the cohorts were balanced for gender and age. The primary outcome was all-cause mortality (ACM). Secondary outcomes assessed were anemia, neutropenia, sepsis, neutropenic fever, tumor lysis syndrome (TLS), and major adverse cardiovascular and cerebrovascular events (MACCE). Kaplan-Meier analysis and risk analysis were used to assess outcomes at 6 months and 1 year. Results We identified 200 pts in V+P and 172 pts in P, with 149 matched pts in each group after PSM. The mean age of pts in V+P and P was 72.4 +/- 8.6 years and 72.7 +/- 10.5 years, respectively. 6-month ACM was 15.5% in V+P, and 12.7% in P. However, the odds ratio of ACM at 6 months, in pts in V+P when compared to P was 1.259 (95% CI 0.654 to 2.425, p = 0.490) and was not statistically significant. At 6 months, the odds ratios of the secondary outcomes in the V+P pts were higher when compared to P pts, but were not statistically significant; anemia 1.614 (95% CI 0.617 to 4.225, p = 0.327), neutropenia 1.445 (95% CI 0.599 to 3.486, p = 0.411), sepsis 1.196 (95% CI 0.477 to 2.998, p = 0.703), neutropenic fever 1.353 (95% CI 0.573 to 3.195, p = 0.490), TLS 1.180 (95% CI 0.474 to 2.939, p = 0.722), and MACCE 1.046 (95% CI 0.541 to 2.024, p = 0.893). 1 year ACM was 18.75% in V+P vs. 10.55% in P (OR 1.96; 95% CI 1.03-3.71; p=0.038) and was statistically significant. However, the secondary outcomes were clinically significant but statistically insignificant in V+P pts when compared to P pts- anemia 1.614 (95% CI 0.617 to 4.225, p = 0.327), neutropenia 1.269 (95% CI 0.568 to 2.834, p = 0.561), sepsis 1.077 (95% CI 0.438 to 2.650, p = 0.872), neutropenic fever 1.24 (95% CI 0.589 to 2.607, p = 0.571), TLS 1.180 (95% CI 0.474 to 2.939, p = 0.722), and MACCE 1.338 (95% CI 0.724 to 2.472, p = 0.352). Conclusion Pirtobrutinib use in venetoclax-exposed pts was associated with a statistically significant higher odds of all-cause mortality at 1 year, which was not present at 6 months. Although adverse event rates of anemia, neutropenia, TLS, sepsis, and neutropenic fever were numerically higher in the venetoclax-exposed cohort, these differences did not reach statistical significance. These findings suggest early survival benefit of pirtobrutinib in venetoclax-naïve patients and aligns with existing evidence supporting its role as a second-line therapy following covalent BTK inhibitor failure, including results from the BRUIN trial. Prospective studies are needed to validate these preliminary observations.

Abstract Introduction: Despite remarkable advances in treatment, patients with multiple myeloma (MM) continue to suffer relapse. Therefore, the development of novel therapeutic strategies is required. The SWI/SNF chromatin remodeling complex, especially its core ATPase subunits SMARCA2/4, is essential for super-enhancer function and MM cell survival. Furthermore, NSD2, which is overexpressed in t(4;14)-positive MM, has been reported to interact with SMARCA2/4, suggesting differential sensitivity to SMARCA2/4 inhibition. While ATPase inhibitors and PROTAC degraders targeting SMARCA2/4 have demonstrated promising preclinical efficacy in MM and other malignancies, their broader therapeutic relevance remains to be clarified. Methods: To support the clinical development of mSWI/SNF antagonists for MM, we first performed PRISM-based profiling of the anti-proliferative effects of both an mSWI/SNF ATPase PROTAC degrader and a catalytic inhibitor across a panel of 888 cancer cell lines from diverse lineages. Lineage enrichment analysis identified plasma cell myeloma as one of the most sensitive malignancies, with the PROTAC degrader demonstrating particularly potent effects. These were followed by in-depth studies in 35 human myeloma cell lines (HMCLs) using FHD-286 (SMARCA2/4 ATPase inhibitor), AU-24118 (CRBN-based degrader), and AU-15330 (VHL-based degrader). Cell viability was assessed after 5-day treatment by flow cytometry with Annexin V and Live/Dead staining. Additional assays were performed in hCRBN-engineered murine plasmacytoma cell lines and Vk*MYChCRBN MM mouse models. Chromatin profiling employed CUT&Tag with SMARCA2/4 and IKZF1/3 antibodies. Results: FHD-286 and AU-24118 showed potent anti-myeloma activity across most cell lines (median IC₅₀: 4.5 nM and 5.9 nM, respectively). HMCLs with t(4;14), as compared to those without, were more sensitive to FHD-286 (p= 0.0064), with a similar trend for AU-24118 (p=0.09). AU-24118 log2 IC₅₀ values correlated with those of FHD-286 (r=0.62, p=0.0002) and with pomalidomide (r=0.61, p=0.0002), suggesting a shared biology. Indeed, CUT&Tag analysis for SMARCA2/4 and the pomalidomide targets IKZF1/3 showed these factors co-localized at most sites throughout the genome in MM.1S cells, including at DUSP22/IRF4, POU2AF1, and IgH super-enhancers. Treatment with AU-15330, depleted SMARCA2/4 off chromatin at >80% of sites within 8 hours (FDR <0.01), and somewhat surprisingly also depleted IKZF1/3 off chromatin at 70% of bound regions, indicating IKZF1/3 function is dependent upon SWI/SNF. Resultantly, combination studies of SWI/SNF targeting agents and pomalidomide showed a lack of synergy and in some cases antagonism, highlighting the importance of careful evaluation when considering combination with pomalidomide. We and others have demonstrated that murine cells are IMiDs resistant due to species-specific differences in CRBN. Using three distinct mouse plasmacytoma cell lines (+/-hCRBN), hCRBN-expressing cells showed a 100-fold greater sensitivity to AU-24118 compared hCRBN- cell lines. In vivo safety testing of AU-24118 using hCRBN transgenic mice exhibited early mortality within 7 days at doses ≥1.875 mpk and within 3 days at doses ≥7.5 mpk, accompanied by rapid body weight loss. In contrast, a preliminary efficacy study using de novo Vk*MYChCRBN mice, AU-24118 administered at 1 mpk three times per week was well tolerated and led to approximately 40% reduction in serum M-protein within 2 weeks (p<0.05), with no notable weight loss, demonstrating a therapeutic window. Conclusions: Broad PRISM profiling confirms lineage-specific vulnerability of MM to SWI/SNF inhibition, with in vitro and in vivo validation supporting selective efficacy in high-risk t(4;14)-positive subtypes. FHD-286, AU-15330, and AU-24118 demonstrated strong in vitro activity, particularly in t(4;14)-positive HMCLs. This activity was due, in part, to chromatin compaction and decommissioning of lineage and oncogenic enhancers as well as eviction of IKZF1/3 off chromatin. AU-24118 showed high selectivity, with picomolar range IC₅₀ in sensitive HMCLs. Due to species-specific CRBN differences, accurate toxicity evaluation of AU-24118 requires hCRBN-expressing models, and preliminary data indicate in vivo efficacy in the highly predictive Vk*MYChCRBN model. These findings support the potential of SMARCA2/4 targeting agents, especially AU-24118, as promising therapies for MM, particularly in high-risk t(4;14)-positive cases.

Abstract Background: Proteasome inhibitors (PIs) are a cornerstone of multiple myeloma (MM) therapy, exploiting the high rate of immunoglobulin production and protein turnover in malignant plasma cells. However, intrinsic and acquired resistance to PIs remains a significant barrier to durable clinical responses. Hypoalbuminemia is a frequent and clinically relevant symptom in MM, often associated with poor prognosis, but its underlying causes are not fully understood. To date, it has not been explored whether MM plasma cells actively consume albumin and whether this contributes to hypoalbuminemia or interferes with current therapeutics.Although albumin catabolism has been described in endothelial cells and some solid tumors, its role in hematologic malignancies has not been investigated. Here, we report for the first time that MM cells internalize and degrade extracellular albumin, predominantly via lysosomal pathways, and that this mechanism contributes directly to resistance against proteasome inhibition. Methods: Human MM cell lines (RPMI-8226, JJN3, MM1.S, H929, and KMS11), representing a range of PI sensitivities, were cultured in media with defined amino acid composition and varying concentrations of human serum albumin. Cell viability in response to PIs (bortezomib and carfilzomib) was measured using MTT and Cell Titer-Glo assays. To determine whether albumin affected drug sensitivity, PI dose-response assays were conducted under low and high albumin conditions, with or without lysosomal inhibitors (ammonium chloride and leupeptin). Intracellular albumin accumulation and degradation pathway activation were assessed via Western blotting for proteasomal and lysosomal markers. Results: MM cells exhibited active uptake of extracellular albumin through endocytosis. Once internalized, albumin was metabolized to provide both essential and nonessential amino acids, supporting cell proliferation and enhancing antioxidant defenses. A strong correlation was observed between the extent of albumin consumption and resistance to PIs. Notably, under high-albumin conditions, MM cells became significantly more resistant to PI-induced cytotoxicity. Even in the absence of other metabolic stressors, high concentrations of albumin alone were sufficient to trigger PI resistance. Mechanistically, although small quantities of albumin could be degraded by the proteasome, elevated albumin levels induced robust lysosomal activation, diverting protein degradation away from the proteasome and effectively bypassing PI-induced stress. Under low-albumin conditions, PI treatment led to intracellular albumin accumulation in sensitive cells. In contrast, high-albumin conditions enhanced lysosomal degradation, mitigating the effects of proteasome blockade. Protein profiling revealed the upregulation of lysosomal enzymes and reduced proteasome dependence in albumin-treated cells. Importantly, inhibition of lysosomal function with ammonium chloride (partial) or leupeptin (complete) restored PI sensitivity, even under high-albumin conditions. Conclusion: This study identifies albumin-driven lysosomal activation as a novel and clinically relevant mechanism of PI resistance in MM. For the first time, we demonstrate that MM cells consume albumin as a nutrient source, offering a plausible explanation for the hypoalbuminemia commonly observed in patients. Our findings suggest that lysosomal degradation serves as a compensatory protein catabolism pathway during elevated extracellular albumin uptake. This has important therapeutic implications: reliance on lysosomal degradation renders MM cells less susceptible to standard PIs, and dual inhibition of lysosomal and proteasomal pathways may overcome resistance. These findings lay the groundwork for designing next-generation therapeutics that simultaneously target proteasomal and lysosomal degradation pathways to overcome drug resistance in multiple myeloma and related hematological malignancies.

Abstract Altered Bone Marrow (BM) Plasma Amino Acid (AA) Composition Reflects Microenvironmental Remodeling in Patients with Multiple Myeloma (MM) compared to Monoclonal Gammopathy of Unknown Significance (MGUS) Michael Egan, Yogesh Chawla, Puja Dey, Dragan Jevremovic, Taro Hitosugi, Shaji Kumar and Wilson Gonsalves BACKGROUND: MYC overexpression, a hallmark of progression from MGUS to MM, drives metabolic reprogramming, including enhanced AA uptake and utilization. However, the contribution of AAs to disease progression from MGUS to MM remains poorly defined. In this study, we comprehensively profiled AA levels in the BM plasma of MGUS and MM patients to determine whether shifts in the availability of specific AAs reflect microenvironmental remodeling and evolving metabolic demands of malignant plasma cells (PCs). METHODS: AA profiles of BM plasma samples—reflective of the BM microenvironment—from two different cohorts of patients with either MGUS or MM (Cohort #1: 49 MGUS and 50 newly diagnosed MM (NDMM) and Cohort #2: 50 MGUS and 64 NDMM) were acquired using liquid chromatography (LC) coupled with tandem mass spectrometry (MS). Individual AA were quantified by MS using single-point quantification with isotopically labeled internal standards of AAs. Individual relative and quantitative AA data were analyzed for differences between the groups using the parametric t-test, and significance was defined as p < 0.05. We utilized a Pearson correlation coefficient method to determine the association of the percentage of clonal PCs in the BM to the levels of the AAs. The areas under the curve (AUC) of receiver operating characteristic (ROC) analyses were created on the web-based tool MetaboAnalyst 6.0 to determine the ability of quantitative AA levels to be utilized as a model to distinguish MGUS vs. MM. RESULTS: In BM plasma samples from cohort #1, using a global untargeted metabolite profiling, we identified 28 AAs and their metabolites out of the 1,089 known metabolites detected. A targeted quantification of these AAs was performed in the same samples to validate any observed differences between the groups. Both aspartate and glycine were found to be more abundant in NDMM compared to MGUS in both untargeted and targeted assays. In contrast,12 AAs (branched chain AAs: valine, leucine and isoleucine, aromatic AAs: tryptophan, kynurenine, tyrosine and phenylalanine; essential AAs: threonine and lysine; non-essential AAs: ornithine, citrulline and asparagine) were found to be less abundant in NDMM compared to MGUS. We then validated the presence of these differences using targeted quantification of AAs in the BM plasma samples from cohort #2. Aspartate was the only AA consistently found to be present at higher levels in NDMM vs. MGUS. Whereas, 8 AAs—threonine, valine, citrulline, tyrosine, tryptophan, isoleucine, phenylalanine, and kynurenine—were consistently lower in NDMM vs. MGUS. We observed a statistically significant positive correlation between the concentration of aspartate and the percentage of clonal PCs present in the BM (r = 0.56). In contrast, there were statistically significant negative correlations between the concentrations of all 8 AAs consistently lower in NDMM vs. MGUS and the percentage of clonal PCs in the BM: threonine (r = -0.41), valine (r = -0.35), citrulline (r = -0.37), tyrosine (r = -0.28), tryptophan (r = -0.31), isoleucine (r = -0.21), phenylalanine (r = -0.19), and kynurenine (r = -0.19). Using only the quantitative concentrations of the 9 AAs that were significantly different between NDMM and MGUS across both patient cohorts, we performed ROC analyses to develop predictive models distinguishing NDMM from MGUS. In cohort #1, the models yielded AUC values ranging from 0.738 to 0.785, while in cohort #2, AUC values ranged from 0.798 to 0.835, depending on the number of AAs included in the model. CONCLUSIONS: Several AAs are present at significantly different levels in the BM plasma of patients with NDMM compared to MGUS. These findings support the emerging role of AA availability, across a diverse set of AAs, in the pathogenesis and progression of MGUS to MM. Future studies are warranted to validate these observations and to determine whether alterations in the BM AA landscape can be leveraged for diagnostic or therapeutic strategies.

Abstract Introduction: Multiple myeloma (MM) is an incurable plasma cell (PC) malignancy, in which nearly all patients are expected to relapse and undergo multiple lines of treatment over the course of their disease. By the time patients receive CAR-T cell therapy, they have typically been exposed to numerous prior therapies, including high-dose melphalan (HDM). Chemotherapeutic agents that cause genotoxic stress, such as HDM, can induce cellular senescence in normal cells. More recently, it has been demonstrated that tumor cells can also exhibit features of therapy-induced senescence (TIS). TIS in tumor cells is associated with distinct gene expression patterns that have been implicated in resistance to therapy and relapse. To investigate whether TIS is activated in MM cells following exposure to chemotherapies, particularly in the context of late-stage disease, we performed an integrated analysis of single-cell RNA sequencing (scRNA-seq) datasets. Methods: We integrated scRNA-seq data from bone marrow plasma cells and MM cells using the following three sources: 1) Boiarsky, et al. (GSE193531, dbGaP: phs001323.v3.p1), which includes healthy donors, MGUS, SMM, and newly diagnosed MM (NDMM) patients; 2) Dhodapkar, et al. (GSE210079), which includes late-stage MM patients pre- and post-CAR-T cell therapy; and 3) newly generated scRNA-seq data from healthy donor (N=3) and late-stage MM patients pre- (N=10) and post- (N=2) CAR-T cell therapy. Non-plasma cells were excluded based on canonical plasma cell markers. We used Seurat v4.3 for data integration, employing canonical correlation analysis (CCA) to define anchors using the FindIntegrationAnchors and IntegrateData functions. Integration quality across datasets was validated by the consistent clustering of healthy plasma cells and expected expression patterns of chromosomal translocation markers CCND1 in t(11;14) cases and FGFR3 in t(4;14) cases, as previously described (PMID: 36396631). To evaluate senescence, we applied single-cell gene set enrichment analysis (scGSEA) using previously published gene sets (PMID: 40164720): SenUp (Senescence Upregulated), SenGA (Senescence Growth Arrest), and SCAPs (Senescent Cell Anti-apoptosis Pathways). We also curated a Plasma Cell Senescence (PCSen) gene set, composed of senescence-related genes that were differentially upregulated in pre-malignant plasma cells across two independent datasets (GSE5900, GSE47552).A normal plasma cell gene set was also generated, comprising genes differentially expressed between healthy and abnormal plasma cells in GSE193531. Results:In the integrated dataset, scGSEA revealed significantly higher enrichment of PCSen, SenUp, SenGA, and SCAPs in late-stage MM cells (both pre- and post-CAR-T cell therapy) compared to NDMM and healthy donors (p<0.05, Kruskal-Wallis with Dunn's posttest). Conversely, the Normal gene set was significantly enriched in healthy plasma cells relative to MM (p<0.05). Expression of CDKN2A, which encodes the senescence marker p16, was also increased in late-stage MM relative to NDMM or healthy plasma cells. Notably, in our newly generated scRNA-seq samples collected within 6 months before CAR-T therapy, we observed a significant inverse correlation between senescence marker expression and progression free survival (PFS) post-CAR-T, suggesting that MM cells exhibiting TIS may be more resistant to T cell mediated cytotoxicity. Supporting this, preliminary in vitro experiments showed that MM cells treated with HDM displayed reduced sensitivity to T cell-mediated cytotoxicity, consistent with a senescence-associated resistant phenotype. Conclusion: Our findings demonstrate that late-stage MM cells exhibit transcriptional signatures consistent with therapy-induced senescence, potentially driven by prior exposure to chemotherapy. This senescent phenotype may contribute to resistance to immunotherapies, including CAR-T cells. These data highlight senescence as a mechanism of resistance and a potential biomarker for predicting CAR-T outcomes. Further studies are needed to explore senescence-targeted strategies to enhance immunotherapeutic efficacy in MM.

Abstract Introduction: Chimeric Antigen Receptor T-cell (CAR-T) therapy has revolutionized the treatment landscape for many types of non-Hodgkin's lymphoma (NHL), offering the potential for prolonged remission and even cure in relapsed/refractory disease. Despite being a one-time treatment, patients spend a significant number of days in contact with the healthcare system in the preparation, monitoring and follow-up required for safe administration. Time toxicity describes the burden on patients' time required to receive all care associated with treatment. This study seeks to characterize the number of healthcare contact days (the so-called time toxicity) associated with CAR-T cell therapy for NHL. Methods: We conducted a claims database analysis of healthcare contact days using the OptumLabs platform. Patients who received CAR-T were identified using a combination of ICD-10-Procedure Coding System (PCS) codes, Current Procedural Terminology (CPT)/revenue codes and Healthcare Common Procedure Coding System (HCPCS) codes. NHL diagnosis was confirmed using ICD-10 codes (C82.X, C83.X and C85.X) to include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL) or other NHL subtype. All claims indicating a healthcare contact day were captured in the time between apheresis and administration and at 1 month, 3 months and 1 year following CAR-T administration. Patients were excluded from analysis if they did not have continuous coverage with available claims data within the periods of interest. Negative binomial regression was used to estimate incident rate ratios (IRR), adjusting for demographic and clinical factors. Results: A total of 813 patients received CAR-T cell therapy for NHL between October 2017 and December 2024. The mean age of patients was 64.4 (range 18-87 years); 64.7% of patients were male, and 79.3% of patients received CAR-T inpatient. Median length of follow-up from CAR-T administration to coverage end or death was 340 days (IQR 164-639 days). Eleven patients (1.4%), 54 patients (6.6%), and 205 patients (25.2%) died in the 1 month, 3 months, and 1 year following CAR-T, respectively. DLBCL was the most common NHL subtype (86.0%) followed by MCL (6.8%), other NHL subtype (4.1%) and FL (3.2%). Patients received axi-cel (32.1%), liso-cel (21.2%), brexu-cel (9.7%), tisa-cel (4.7%) or unspecified CAR-T product (32.4%). Between apheresis and CAR-T administration, patients spent a median of 7 days (IQR 5-11 days) or 23.7% of days with healthcare contact (n=470). At 1 month after CAR-T, patients spent a median of 20 days (IQR 16-25 days) or 67.2% of days with healthcare contact (n=806). At 3 months, patients spent a median of 28 days (IQR 22-38 days) or 31.5% of days with healthcare contact (n=773). At 1 year, patients spent a median of 52 days (IQR 37-72 days) or 18.9% of days with healthcare contact (n=591). Outpatient CAR-T administration was associated with lower time toxicity at all timepoints compared to inpatient administration (p<0.05). FL and MCL had lower time toxicity only at the 1-year mark compared to DLBCL (FL IRR 0.72, 95% CI 0.57-0.90, p<0.01, MCL IRR 0.74, 95% CI 0.57-0.97, p=0.03). Brexu-cel was associated with higher time toxicity at all timepoints compared to liso-cel (p<0.05). Axi-cel was associated with higher time toxicity at 1 month (IRR 1.08, 95% CI 1.01-1.15, p=0.03) and 3 months (IRR 1.20, 95% CI 1.08-1.34, p<0.01), but not at 1 year when compared to liso-cel (IRR 1.09, 95% CI 0.91-1.30, p=0.35). Conclusions: Patients receiving CAR-T cell therapy for NHL spent a significant number of days in contact with the healthcare system, particularly in the first month following administration. Time toxicity lessened over time but patients still spent nearly 20% of their days with healthcare contact in the year following CAR-T. Outpatient administration was associated with significantly lower time toxicity. FL and MCL had lower time toxicity at 1 year compared to DLBCL, likely due to selection for surviving patients. Axi-cel and brexu-cel were associated with higher time toxicity when compared to liso-cel, reflecting the increased toxicity profiles of these products. These findings help clinicians and patients understand their true time commitment beyond the first month and can potentially inform future innovations for patient-centered delivery of cellular therapy that decrease time toxicity and improve patient quality of life.

Abstract INTRODUCTION: Bruton tyrosine kinase inhibitors (BTKis) are becoming the preferred treatment for patients with chronic lymphocytic leukemia (CLL), taking the place of novel chemoimmunotherapies. While these medications significantly improve both progression-free and overall survival, they are also linked to various adverse effects, including cardiovascular toxicity. In contrast, glucagon-like peptide-1 (GLP-1) agonists have shown benefits for patients with diabetes mellitus who are at high risk for cardiovascular disease. Studies indicate that these drugs not only improve glycemic control but also lead to a reduction in the incidence of major cardiovascular events. This study aimed to assess the incidence of cardiovascular events and overall survival among CLL patients receiving BTK inhibitors who were also taking GLP-1 agonists for diabetes mellitus and/or obesity, compared to diabetic or obese CLL patients on BTK inhibitors not receiving GLP-1 agonists. METHODS: We utilized the TriNetX Global Collaborative Network to identify patients with CLL who are being treated with BTKis such as ibrutinib, acalabrutinib, or zanubrutinib, and who also had diabetes mellitus and/or obesity. Cohorts were formed based on the use of GLP-1 agonists. Propensity score matching was performed to adjust for potential confounders, including age, gender, ethnicity, alcohol consumption, tobacco use, diabetes mellitus, obesity, hypertension, and end-stage renal disease. Outcomes such as all causes of mortality, atrial fibrillation/flutter, acute heart failure, and acute ischemic heart disease were evaluated within one year of initiating GLP-1 agonist therapy. Risk differences (RD), risk ratios (RR), and 95% confidence intervals (CIs), were calculated. Kaplan-Meier survival analysis was performed, and the log-rank test was used to compare survival probability between groups. A p-value < 0.05 was considered statistically significant. RESULTS: A total of 844 patients with CLL were included in the study, with 422 patients in each propensity-matched cohort. The incidence of atrial fibrillation/atrial flutter was significantly lower among patients receiving concurrent BTKis and GLP-1 agonists compared to those on BTKis alone (12.08% vs. 17.29%; RD –5.21%; p= 0.03; RR 0.699, 95% CI: 0.502% to 0.973%). Kaplan–Meier analysis demonstrated a significantly lower cumulative incidence of atrial fibrillation/atrial flutter at 365 days in the GLP-1 agonist cohort compared to non-users (log-rank test, p = 0.0245), indicating a higher atrial fibrillation–free survival probability among GLP-1 users. There were no statistically significant differences between groups in the incidence of ischemic heart disease (27.48% vs. 23.46%; RD 4.08%; p= 0.17; RR 1.172, 95% CI: 0.929% to 1.477%) or heart failure (13.27% vs. 13.27%; RD 0%; p= 1; RR 1, 95% CI: 0.708% to 1.412%). Although not statistically significant, a trend toward lower all-cause mortality was observed in patients receiving concurrent BTK inhibitors and GLP-1 agonists compared to those on BTK inhibitors alone (4.976% vs. 8.294%; risk difference –3.31%; p = 0.052; relative risk 0.6, 95% CI: 0.355 to 1.013; Kaplan–Meier analysis, log-rank test p = 0.055). A large cohort is needed to confirm this last finding. CONCLUSION: The use of GLP-1 agonists was associated with a significantly reduced incidence of atrial fibrillation/atrial flutter and a trend toward improved overall survival in diabetic or obese CLL patients receiving BTK inhibitors. These findings suggest a potential cardioprotective effect of GLP-1 agonists in this high-risk population. However, further prospective cohort studies or randomized controlled clinical trials are needed to confirm these observations.

Abstract Introduction Chimeric antigen receptor (CAR) T-cell therapy and bispecific antibodies have emerged as transformative options for patients with relapsed or refractory multiple myeloma (MM). Both modalities commonly target B-cell maturation antigen (BCMA) and have demonstrated high overall response rates in pretreated populations ranging from 65-83%. However, the optimal sequencing and comparative effectiveness of these two novel therapies remain unclear. Understanding their treatment outcomes is critical to inform treatment decisions in this rapidly evolving therapeutic landscape. Method A retrospective cohort study was conducted using TriNetX, a global federated health research network that provides access to electronic medical records from approximately 132 million patients, primarily in the United States. Adult patients (≥18 years) diagnosed with relapsed or refractory MM between January 1, 2022, and January 1, 2024, were identified using ICD-10-CM codes. Patients were categorized into two cohorts: those who received CAR T-cell therapy including idecabtagene vicleucel and ciltacabtagene autoleucel and those who received bispecific antibodies including teclistamab and elranatamab. Propensity score matching (1:1) using nearest neighbor matching with a 0.1 pooled standard deviation caliper was performed for demographics, comorbidities, prior stem cell transplantation, medications, and performance status. Study outcomes included 1-year rates of all-cause mortality, all-cause hospitalization, cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), anemia, neutropenia, thrombocytopenia, sepsis, and pneumonia. Statistical analyses were conducted on the TriNetX platform, with significance set at P<0.05 (two-sided). TriNetX calculates hazard ratios (HRs) and cumulative incidences (CIs) using R survival package version 3.2-3 with the proportional hazard assumption tested using Schoenfeld residuals. This study does not require Institutional Review Board review or informed consent due to the use of de-identified data. Results Our study identified 2,023 patients, including 911 patients who received CAR T-cell therapy and 1,112 who received bispecific antibiotics. After propensity score matching (PSM), each cohort included 649 patients. The CAR T-cell therapy group had a mean age of 66.8 ± 8.4 years, with 41.4% female, 68.0% White, 20.6% African American, and 2.3% Asian. The bispecific antibody group had a mean age of 66.3 ± 9.3 years, with 41.1% female, 67.3% White, 21.1% African American, and 2.2% Asian. CAR T-cell therapy group experienced a 64% lower risk of all-cause mortality (HR 0.36; 95% CI, 0.26–0.48) and a 44% lower risk of all-cause ICU admission (HR 0.56; 95% CI, 0.42–0.75) compared with bispecific antibodies group. However, CAR T-cell therapy group was associated with higher risk of CRS (HR: 1.91; 95% CI: 1.57–2.31), ICANS (HR: 1.50; 95% CI: 1.12–2.01), neutropenia (HR: 2.32; 95% CI: 1.96–2.76), and thrombocytopenia (HR: 1.80; 95% CI: 1.43–2.28) compared to bispecific antibodies group. There was no significant difference observed in anemia (HR: 0.96; 95% CI: 0.82–1.13) between these two treatment groups. Additionally, CAR T-cell therapy group was associated with 46% lower risk of sepsis (HR: 0.54; 95% CI: 0.39–0.75) and 68% lower risk of pneumonia (HR: 0.32; 95% CI: 0.17–0.61) compared to bispecific antibodies group. ConclusionIn this real-world analysis, CAR T-cell therapy demonstrated a substantial lower mortality and reduced ICU utilization compared with bispecific antibodies in patients with relapsed or refractory MM, despite its higher risks of CRS, ICANS, and hematologic toxicities. An important limitation of our study is the lack of data on the number of prior lines of therapy, which likely differed between groups, as bispecific antibodies are typically used in the fourth line or beyond, while CAR T-cell therapies are approved to be used as the second line. This discrepancy may have influenced both mortality and infection rates. Despite the limitation, our findings underscore the need for prospective clinical trials directly comparing CAR T-cell therapy and bispecific antibodies to validate these observations and define the optimal sequencing of these modalities. Furthermore, research into predictive biomarkers and personalized selection algorithms will be crucial to optimize treatment decisions in this rapidly evolving therapeutic landscape.