Citations

Abstract BCMA-directed CAR T-cell and T-cell engager (TCE) therapies produce high response rates in relapsed multiple myeloma, yet most patients progress. Prior work identified TNFRSF17 alterations mediating antigen escape in up to 43% of TCE progressions. To evaluate the generalizability of these findings and examine antigen-loss-independent resistance, we performed whole-exome sequencing on 32 paired pre- and post-treatment progression bone marrow samples from 15 patients (5 CAR T, 10 TCE); two patients received both agents. TNFRSF17 alterations were infrequent and restricted to TCE recipients (one concurrent frameshift and missense mutation; two heterozygous copy-number losses); no biallelic loss or CAR T-associated events were observed. In contrast, deleterious/loss-of-function variants in NF-κB, PI3K-AKT-mTOR, and MAPK/ERK pathways were universal in pre-treatment samples and acquired or clonally enriched in 80% of patients at progression. Acquired events included loss-of-function alterations in CYLD , activating mutations in KRAS , NRAS and BRAF , and acquired TP53 mutations. In this series, antigen escape via TNFRSF17 /BCMA alteration was uncommon. Instead, pre-existing, acquired and clonally enriched pathogenic variants in NF-κB, PI3K-AKT-mTOR and MAPK cascades, together with TP53 mutation, emerged as a frequent finding. These alterations suggest that deregulation of BCMA-downstream survival signaling, enhanced proliferative fitness, and elevation of apoptotic threshold constitute antigen-loss-independent mechanisms of resistance.

BACKGROUND: It is unknown whether cardiac resynchronization therapy (CRT) improves systemic metabolomic profile by enhancing left ventricular function. The observational cohort study aimed to investigate the effect of CRT on left ventricular ejection fraction (LVEF) and circulating plasma metabolites in patients who had heart failure with reduced LVEF. METHODS: We prospectively screened patients with ischemic cardiomyopathy and nonischemic cardiomyopathy who received CRT with a defibrillator for LVEF ≤35% according to current guidelines. Clinical assessment included echocardiography and device interrogation. Blood samples for metabolomic analysis were collected before CRT and at 6-month follow-up. Plasma was subjected to gas chromatography-mass spectrometry and 1 H nuclear magnetic resonance-based metabolomic analysis. RESULTS: Totally 92 patients were enrolled, with a mean age of 67.3±11.3 years (37.0% female). LVEF was significantly improved from 28.9±7.6% at baseline to 36.0±11.3% at 6 months ( P <0.001) and to 40.1±12.6% at 12 months ( P <0.001). After CRT, 42 metabolite features were significantly decreased compared with the baseline. The ketone bodies, including 3-hydroxybutyrate ( P <0.001) and acetone ( P =0.01), were reduced. A branched-chain amino acid, isoleucine, was also decreased after CRT ( P =0.01). These metabolomic changes were mainly observed in the nonischemic cardiomyopathy group, while the metabolomic profile in the ischemic cardiomyopathy group was characterized by enhanced amino acid oxidation and elevated levels of lactate and pyruvate. The improvement in LVEF positively correlated with the ratio of changes in ketone bodies and isoleucine. CONCLUSIONS: CRT may modulate the systemic plasma metabolomic profile, which correlated with improvement in left ventricular function in patients with severe heart failure with reduced ejection fraction.

Objective Antinuclear antibody (ANA) indirect immunofluorescence (IIF) is the reference standard but depends on subjective pattern interpretation. We tested whether artificial intelligence (AI) applied to ANA IIF images could identify specific autoantibodies. Methods We assembled a cohort of ANA‐positive patients (human epithelial type 2 IIF titer ≥1:80) with anti–double‐stranded DNA (dsDNA), anti‐Smith (Sm) antibody, ribonucleoprotein (RNP), anti‐Sjögren's disease‐related antigen A (SSA), anti‐Sjögren's disease‐related antigen B (SSB), anti‐topoisomerase I (Scl‐70), or centromere antibody (CMA) testing within 90 days. The development cohort (December 12, 2016–December 31, 2022) included the first ANA test per patient. Two AI architectures were trained for each autoantibody. Performance was assessed using area under the receiver operating characteristic curve (AUROC), sensitivity, specificity, and accuracy; comparisons with IIF patterns were summarized as pattern‐antibody agreement. A prospective cohort (January 1, 2023–August 20, 2024), including a dsDNA assay transition, provided temporal validation. Results Among 512,959 patients, 47,890 met inclusion criteria (mean age 55 [SD 17.6], 78% female). Models for CMA, dsDNA, Sm, SSA, and SSB achieved AUROC ≥0.80 (CMA highest: AUROC 0.95, accuracy 94.2%, specificity 95.3%). Compared with associated IIF patterns, dsDNA and Sm models more often classified antibody‐negative cases as negative (dsDNA 88.2% vs 54.7%; Sm 82.6% vs 60.3%) while similarly classifying antibody‐positive cases. Performance for RNP (AUROC 0.68) and Scl‐70 (AUROC 0.59) was lower. In the prospective cohort (n = 21,427), performance was similar; dsDNA discrimination remained stable across assays (AUROC ~0.85). Conclusion AI applied to ANA IIF images identified specific autoantibodies and often improved classification of antibody‐negative cases versus simplified pattern‐based comparators. These findings support its potential to standardize ANA interpretation and triage or reduce confirmatory testing. image

Abstract Germinal center (GC)-derived lymphomas arise within a specialized immune ecosystem, yet whether malignant cells direct its remodeling remains unclear. Integrating genomic, transcriptomic, spatial, and functional analyses across follicular lymphoma and diffuse large B-cell lymphoma, we define a developmental framework linking malignant B-cell differentiation state to tumor microenvironment (TME) organization. Rather than segregating by histology, lymphomas align along a shared differentiation continuum in which proliferative dark zone (DZ) states associate with immune-depleted TMEs, whereas post-GC/memory B-cell (MBC) states drive inflamed but immunosuppressed TMEs. Spatial profiling reveals collapse of GC architecture and emergence of suppressive myeloid niches along this trajectory. Single-cell lineage reconstruction uncovers a DZ-to-MBC developmental axis underlying lymphoma evolution. Genetic reprogramming in vivo demonstrates that malignant differentiation state is sufficient to instruct TME remodeling. Collectively, these findings establish malignant differentiation state as a causal determinant of immune architecture across GC-derived lymphomas and identify lineage-directed immune reprogramming as a potential therapeutic strategy.

Abstract Oxidative phosphorylation (OXPHOS) is a central function and a key indicator of mitochondrial fitness, yet studies in human tissue remain limited. Inclusion body myositis (IBM) is a progressive myopathy that lies at the intersection of aging, inflammation and mitochondrial dysfunction. We aimed to perform a comprehensive profiling of mitochondrial respiration in muscle tissue from patients with IBM. A wide battery of complementary approaches from RNA level to high-resolution respirometry on permeabilized muscle fibers was employed. The relationship between mitochondrial respiration, mitochondrial content, mitochondrial DNA (mtDNA) abnormalities and mitophagy was examined, along with the correlation with various clinical parameters to determine their clinical relevance. The study included a total of 67 patients with IBM and 45 controls. On high resolution respirometry of permeabilized muscle fibers, IBM samples exhibited reduced maximal mitochondrial respiration per tissue weight in State 3 (high substrates, high ADP) and uncoupled state with decreased coupling efficiency and higher leak control ratios. When adjusting for citrate synthase reflecting mitochondrial content, male patients had decreased State 3 intrinsic respiration, whereas female patients had greater intrinsic respiration under leak states. Complex I activity was decreased mainly in female patients, in whom complex II control ratio positively correlated with disease duration and severity. IBM was further associated with decreased RNA levels of all complexes, and lower protein expression of complex I, III, IV and V, likely related to the lower mtDNA content seen in IBM samples. Regarding the production of reactive oxygen species, IBM samples exhibited lower maximal H2O2 emission, accompanied by a higher total antioxidant capacity that positively correlated with disease duration in female patients. Lastly, correlation analyses suggested that impaired mitochondrial respiration, altered mitophagy, and reduced mtDNA content are interconnected in IBM and maybe of clinical significance. IBM is characterized by multifaceted, clinically relevant impairments in mitochondrial respiration. Future studies should further explore underlying pathomechanisms and the variation of mitochondrial respiration by disease stage.

Abstract Background Calcium release channel deficiency syndrome (CRCDS) arises from loss-of-function variants in the RYR2-encoded type 2 ryanodine receptor (RyR2), predisposing patients to sudden cardiac arrest/death (SCA/SCD) despite normal exercise testing. Previously, a novel homozygous RYR2 duplication causing ~ 80% RyR2 reduction was associated with exertion-related SCD. However, how Ca2+ homeostasis is maintained despite RyR2-loss remains unknown, as RYR2-/- animals are non-viable. Purpose To generate a RYR2-knockout (RYR2-KO-/-) induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model to dissect compensatory mechanisms sustaining Ca2+ homeostasis and identify arrhythmogenic pathways predisposing to SCD. Methods CRISPR/Cas9 editing was used to introduce a homozygous c.163delT variant into a wild-type iPSC line (control), generating a RYR2-KO-/- model. A patient-derived RYR2-duplication (RYR2-DUP+/+) iPSC line was used for comparison. RyR2 expression was assessed by western blot (WB) and immunofluorescence (IF). Proteome and phospho-proteome were assessed using omics approaches. Intracellular Ca2+ handling was evaluated by Fluo-4 AM imaging (0.5 Hz and long-burst–long-pause [LBLP] protocol). Electrophysiological properties (action potential [AP], and sarcolemmal currents [ICaL, INa and INCX]) were assessed by patch-clamp. Results Despite complete RyR2-loss in RYR2-KO-/- iPSC-CMs as confirmed by WB and IF, contractility and contractile machinery expression was preserved. Ca2+ transient amplitude (ΔF/F0) was reduced markedly in RYR2-KO (0.14±0.01, p&lt;0.01) and RYR2-DUP (0.27±0.2, p&lt;0.01) compared to control (0.52±0.02). Proteomics revealed a significant NCX1 up-regulation and IP3R-interacting protein (ITPRIP) down-regulation in RYR2-KO compared to control, with pathway-analyses showing significant up-regulation of IP3-related pathways. Pharmacological IP3Rs block (2-APB, 10μM) abolished Ca2+ transient generation in RYR2-KO iPSC-CMs (Amplitude: baseline: 0.15±0.01, 2-APB: 0.06±0.01, p&lt;0.01). RyR2-ablation induced electrical remodeling (pA/pF), including ICaL reduction, INCX up-regulation and hyperpolarization of the INa window current in RYR2-KO (ICaL [at 0mV]: -8.5±1.6; INCX [at -110mV]: -4.1±1.1) as compared to control (ICaL:-7.5±3.7, p&lt;0.01; INCX:-1.5±0.5, p=0.02). RYR2-KO CMs demonstrated increased delayed after-depolarizations and beat-to-beat variability as compared to control. Despite Ca2+ sparks abolishment, RYR2-KO CMs displayed higher erratic beating (at 0.5Hz) and arrhythmogenic propensity than control, showing ventricular tachycardia-like events during LBLP stimulation. Conclusions Genetic RYR2-ablation disrupts physiological Ca2+ cycling and triggers electrical remodeling, including INCX up-regulation, overall promoting arrhythmogenesis. Compensatory IP3Rs activation sustains Ca2+ transient generation, revealing a mechanism preserving excitation-contraction coupling that may paradoxically promote arrhythmogenesis in CRCDS.RYR2-KO: Generation and CharacterizationElectrophysiological Assessment

Abstract Introduction: Large B-cell lymphoma (LBCL) harboring chromosomal rearrangements (double-hit, DH) or coexpression of MYC and BCL2 (double-expressor, DE) represents biologically aggressive subgroups with inferior clinical outcomes. Oncogene-driven DNA replication stress (ODRS) is a hallmark of MYC and BCL2 dysregulation; however, the DNA damage response (DDR) pathways that sustain replication and cell survival under ODRS remain incompletely defined in DLBCL. Identifying DDR dependencies induced by ODRS may reveal therapeutically actionable vulnerabilities in DH/DE-LBCL. Methods: We studied DH/DE-LBCL cell lines (OCILY1, SUDHL2, DOHH2), non-DH/DE-LBCL cell lines (HT, KARPAS422, HBL1), and benign retinal pigment epithelial (RPE) cells engineered to overexpress MYC and/or BCL2. ODRS and DNA damage were assessed by phosphorylation of RPA (S4/S8, S33) and H2AX histone (γH2AX), respectively, using immunofluorescence, immunohistochemistry, and western blot (WB). Activity of homologous recombination (HR), nonhomologous end-joining (NHEJ), and microhomology-mediated end joining (MMEJ) pathways were quantified with established reporter assays. POLΘ (gene: POLQ) expression, an MMEJ marker, was evaluated by qPCR and WB. DDR activity was assessed using genomic signatures from whole-exome sequencing data of 404 LBCL patients. In vitro sensitivity of LBCL cells to POLΘ and PARP inhibitors was assessed with publicly available small-molecule inhibitors using CellTiterGlo and clonogenic assays, and therapeutic efficacy was validated in a DH-LBCL xenograft model in NRG mice. Results: DH/DE-LBCL cells and primary tumors exhibited increased ODRS and persistent DNA damage compared with non-DH/DE-LBCL models, including accumulation of γH2AX in mitosis. Functional profiling revealed selective upregulation of MMEJ in MYC/BCL2-overexpressing cells, with minimal changes in HR or NHEJ activity. POLQ expression was significantly increased in DH/DE-LBCL cell lines and primary samples, and the ID6 signature (associated with MMEJ activity) was significantly enriched in DH-LBCL tumors compared to non-DH/DE-LBCL tumors. POLΘ inhibition demonstrated greater toxicity in DH/DE-LBCL and in MYC-overexpressing RPE cells compared to non-DH/DE-LBCL and wild-type RPEs. Combined inhibition of POLΘ and PARP resulted in marked synergy in vitro. The therapeutic efficacy of POLΘ inhibition, with the synthetic lethality of concurrent PARP inhibition, was validated in the DH-LBCL xenograft model. Concurrent POLΘ and PARP inhibition with small-molecule inhibitors induced an antitumor effect in vivo, with increased overall survival in mice compared with vehicle- or olaparib-single-agent-treated groups. Conclusions: ODRS in DH/DE-LBCL creates a selective dependence on MMEJ mediated by POLΘ. Therapeutic targeting of POLΘ, alone or in combination with PARP inhibition, exploits this vulnerability and represents a promising strategy for this high-risk LBCL subset. These findings provide a biological rationale for clinical translation of POLΘ-directed therapies in DH/DE-LBCL. Citation Format: Cecilia Ayala-Zambrano, Dongni Yi, Michelle Manske, Kimberly Gwin, Richar Kandasamy, Surendra Dasari, Anne Novak, Shuhei Asada, Xiaosheng Wu, Zhenkun Lou, Thomas Witzig, Jithma Abeykoon. c-MYC/BCL2 induced replication stress drives dependence on POLQ-mediated microhomology-mediated end joining in aggressive large B-cell lymphoma [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr A029.

Abstract Therapeutic options for Waldenström macroglobulinemia (WM) have expanded but improvements in overall-survival (OS) are difficult to interpret due to competing non–WM mortality. We conducted a large single-center retrospective study of 1,020 patients with WM to evaluate survival trends. Patients were stratified by era of active WM diagnosis using a two-era model (1996–2005 and post-2005) and a four-era model (pre-2001, 2001–2007, 2008–2013, and post-2013). OS did not differ significantly across eras in the overall cohort (p = 0.64 and p = 0.86 for the two- and four-era models). However, among patients aged < 65 years at the time of active WM diagnosis in the 2-era model, OS improved in the post-2006 era (median 18.8 vs 14.1 years, p = 0.018). Cause-specific-survival (CSS) improved across all eras, increasing from 15.6 years in 1996–2005 to 18.9 years post-2006 (p < 0.0001), and from 13.7 years pre-2001 to not reached in later periods in the four-era model (p < 0.0001). Competing-risk-analysis (CRA) demonstrated a reduction in WM-related mortality over time (p < 0.001). However, excess mortality persisted, with a standardized mortality ratio (SMR) of 2.79 (p < 0.001) among patients < 65 years compared with a matched U.S. population. Our findings highlight improvements in WM-specific survival, while gains in OS are observed in younger patients.