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Abstract Introduction: Multiple myeloma (MM) significantly contributes to global disease burden. To facilitate tailoring of treatment strategies and improved patient (pt) management according to regional differences, there is a need for research on geographical distribution of clinical characteristics that may influence tumor expression and treatment response in RRMM. This study aimed to describe the distribution of prognostic characteristics of pts with RRMM in Europe and the US. Methods: Data were drawn from the Adelphi Real World MM III Disease Specific ProgrammeTM(DSP), a cross-sectional survey, with retrospective chart review, of physicians and their pts with RRMM. The survey was administered to hemato-oncologists and hematologists in EU4 (France, Germany, Italy, Spain) from July to Nov 2021, the UK (May to Nov 2021), and the US (Sep 2020 to Sep 2021). Physician-reported data (collected via DSP pt record forms) for consulting RRMM pts (second-line or later [2L+]) were analyzed to capture demographics, clinical characteristics, and treatment history. Descriptive analyses were performed to assess key variables. Results: In total, 236 physicians provided data on 2037 pts with 2L+ RRMM (n=259 US; n=1778 EU4+UK). Demographic characteristics in the US and EU4+UK were generally similar except there were more White pts in EU4+UK than the US (93%/59%). The mean (standard deviation [SD]) age at diagnosis was 65.8 (7.9) years in the US and 65.9 (9.2) years in EU4+UK, and at the time of data collection (i.e., at the most recent RRMM consultation) mean (SD) age was 69.1 (8.1) years in the US and 70.8 (9.1) years in EU4+UK. Most pts were male (62% in the US and 56% in EU4+UK). Most clinical characteristics were similar between the US and EU4+UK and within regions between White and non-White pts. International Staging System (ISS) stage I/II/III at the time of data collection was 10%/37%/53% in the US and 11%/26%/63% in EU4+UK. There was some variation in other clinical characteristics: ECOG performance status 0–1 at the time of data collection was 80% in the US and 68% in EU4+UK. Bone lesions were experienced by 58% in the US and 65% in EU4+UK, and high-risk cytogenetics were present in 39% in the US and 26% in EU4+UK. The most frequent comorbidities reported at the time of data collection in the US and EU4+UK were hypertension (47%/46%), anemia (24%/26%), hyperlipidemia (19% in both), diabetes (19%/24%), and mild renal disease (12%/18%). Despite some differences in populations, prescribed therapies were broadly similar between regions and between White and non-White within regions. Common 1L therapy classes prescribed in the US and EU4+UK were proteasome inhibitors (PIs) (22%/27%), immunomodulatory drugs (IMDs) (18%/15%), and PI/IMD (59%/51%). Common 2L therapies prescribed in the US and EU4+UK were PIs (15%/18%), IMDs (4%/21%), PI/IMD combination (28%/20%), and IMD/anti-CD38 combination (25%/20%). Common 3L+ therapies in the US and EU4+UK were PIs (24%/14%), IMDs (8%/20%), PI/IMD (26%/21%), and IMD/anti-CD38 (15%/16%). Minor variations in prescribed therapies included rates of stem cell transplantation (22% US/30% EU4+UK), exposure to anti-CD38 (63%/71%), exposure to lenalidomide (88%/92%), and exposure to both lenalidomide and anti-CD38 (56%/66%). The US had higher rates of pts refractory to treatments: anti-CD38 refractory (43% US/31% EU4+UK), lenalidomide refractory (32%/27%), and refractory to both lenalidomide and anti-CD38 (40%/13%). Conclusion: Our study provides critical insights into pts with RRMM across different regions, aligning closely with results from other published large population-based studies. Demographic/clinical characteristics were generally similar across the regions (except proportions of White pts) and between White and non-White pts within regions, including age of diagnosis, ISS staging, and comorbidity burden. Pts were also treated with similar combinations of classes in frontline and second line therapy during the time the study was conducted. Minor variations were seen in level of refractoriness to common therapies, including lenalidomide and anti-CD38. Similarities between the populations enable extrapolation across the regions, to inform treatment strategies.

Abstract Background The prognostic relevance of fluorescence in-situ hybridization (FISH) abnormalities is well-established in multiple myeloma (MM), where high-risk (HR) abnormalities are associated with poor survival. In AL amyloidosis, however, the cytogenetic landscape and its implications might be different. For example, t(11;14), common in AL amyloidosis, has historically been linked to inferior outcomes in the pre-daratumumab era. HR FISH abnormalities are less common and less well-defined in this setting, underscoring the need to evaluate their significance in this disease. Methods We retrospectively analyzed 698 newly diagnosed AL amyloidosis patients seen at Mayo Clinic, Rochester, between 2012–2021 who had baseline FISH testing. HR FISH was defined as del(17p)/monosomy 17, t(4;14), t(14;16) or t(14;20). Baseline characteristics and treatment patterns were compared between the HR and the standard-risk (SR) groups. Staging used the Mayo 2004 system with European modification. Hematologic responses were assessed at 2 and 6 months, and cardiac/renal responses at 3, 6, 12, and 24 months per consensus criteria. Overall response rate (ORR) for hematologic and organ responses included patients who achieved partial response (PR), very good partial response (VGPR), and complete response (CR). Overall survival (OS) was estimated via Kaplan-Meier; comparisons used log-rank testing. Cox regression was used for univariate and multivariable analyses. Results Among 698 patients, 73 (10.4%) had HR FISH. The most common HR abnormality was t(14;16) (n=34, 4.9%), followed by del(17p)/monosomy 17 (n=21, 3%), and t(4;14)/t(14;20) (n=12 each, 1.7%). Across the cohort, t(11;14) was most frequent (51.3%), followed by del(13q) (36.2%), hyperdiploidy/tetraploidy (26.3%), and 1q gain/amp (23.6%). Compared to SR group, HR FISH group had fewer males (43% vs. 69%, p<0.0001), with no difference in age (62.5 vs. 65, p=0.44), cardiac (73% each, p=1) or renal involvement (52% vs. 54%, p=0.71). More HR patients had early cardiac stage (69% vs. 55.9%, p=0.04). Both groups were predominantly lambda isotype (82% vs. 74%, p=0.15), with similar baseline dFLC (21.6 vs. 28.4 mg/dL; p=0.81). Intact immunoglobulin secretion was more common in the HR group (68% vs. 45%, p=0.004). The HR group more often underwent advanced bone imaging (44% vs. 29%, p = 0.015) and had a higher incidence of lytic lesions or fractures (27% vs. 15%, p = 0.046). Use of bortezomib-based therapy, primarily CyBorD, was similar between the groups (67% each). Daratumumab-based therapy (16% vs. 11%, p=0.25) and autologous stem cell transplantation (ASCT) (33% vs. 25%, p=0.16) were numerically higher in the HR group, though not statistically significant. Post-ASCT maintenance was more frequent in the HR group (17% vs. 7%, p=0.006). At 2 months, hematologic ORR was higher in the HR group (90% vs. 72%, p=0.007), with higher ≥VGPR (67% vs. 32%, p<0.0001) and CR rates (24% vs. 8%, p=0.0004). At 6 months, the HR group maintained higher ≥VGPR (89% vs. 71%, p=0.008) and CR rates (41% vs. 26%, p=0.04), with no difference in ORR (98% vs. 92%, p=0.18). Cardiac responses at 6 months favored the HR group (VGPR or better: 29% vs. 12%, p=0.013; CR: 13% vs. 3%, p=0.007). At 12 months, cardiac ≥VGPR remained higher (50% vs. 29%, p=0.01), though CR was not significantly different (18% vs. 9%, p=0.1). Renal ≥VGPR at 6 months was more common in the HR group (47% vs. 22%, p=0.004), but not renal CR (9% vs. 7%, p=0.95). At 12 months, renal ≥VGPR remained higher in HR (61% vs. 40%, p=0.004). With a median follow-up of 87 months, median OS was not reached in HR (95% CI: 72.6–NR) vs. 88.9 months (95% CI: 72.8–110.5) in SR (p=0.03). In multivariable analysis, age ≥65 (HR 1.56, p=0.0001) and >2 organ involvement (HR 1.46, p=0.002) were associated with worse OS, while ASCT (HR 0.35, p<0.0001) and cardiac stage I/II (HR 0.48, p<0.0001) predicted better OS. HR FISH was not independently associated with OS (HR 0.74, p=0.14). Conclusions HR FISH abnormalities in AL amyloidosis are not independently associated with OS, unlike in MM. Patients with HR FISH abnormalities more often presented with earlier cardiac stage and demonstrated deeper hematologic and organ responses than patients with SR FISH abnormalities. This study underscores how clonal characteristics can potentially aid in more personalized treatment approaches for patients.

Abstract Introduction: Telomere biology disorders (TBDs) are caused by germline pathogenic or likely pathogenic variants in telomere maintenance genes and result in multisystem manifestations, including impaired bone health. Patients with TBDs experience reduced bone mineral density (BMD), avascular necrosis, and increased rates of fragility fractures. However, the underlying mechanisms of these bone complications remain poorly understood, and no TBD-specific treatments for bone health currently exist. Our objective was to characterize the clinical features, management, and outcomes of TBD-related bone health issues, including fractures and reduced BMD. Methods: We conducted a retrospective, multicenter, international cohort study using data from 232 patients with TBDs enrolled across 12 centers participating in the Clinical Care Consortium of Telomere-Associated Ailments. Medical records were reviewed for fracture history and BMD assessments by dual-energy X-ray absorptiometry (DXA). The primary endpoint was fracture incidence; secondary endpoints included BMD assessment and fracture management. Data were stratified by pediatric (≤18 years) and adult (>18 years) age groups. Analyses were based on data availability; specific denominators are provided where applicable. Result: Data were available for 53 (23%) pediatric and 179 (77%) adult patients. TERT mutations were the most frequently identified variants in both groups (28% pediatric, 23% adult). Fractures occurred in 25% (13/53) of pediatric patients and 35% (62/178) of adults. In contrast, fractures are estimated to occur in 10-15% of the general pediatric population. The median age at first fracture was 8.2 years (range 2.5–17) in children and 29.2 years (range 18.3–66.9) in adults. Among those with fracture data (n=20 pediatric, n=22 adult), fragility fractures were more common in adults (57%) versus predominantly traumatic fractures in children (61%). Multiple fractures occurred in 65% of affected pediatric patients and 45% of affected adults. Fracture management also differed: non-surgical treatments were used in 60% of pediatric fractures (n=12/20) versus 36% (n=8/22) of adult fractures; surgical interventions were more common in adults (36%). The inheritance pattern (autosomal dominant versus recessive or X-linked) was not associated with fracture risk but the subset of patients with CTC1 mutations were associated with increased fracture risk (n=5 of 124 patients with fracture history, 4% vs n=2 of 247 patients with no fracture history, 0.8%; p=0.04). We also assessed whether vascular manifestations were associated with fracture risk comparing patients with and without fractures. Pulmonary arteriovenous malformations (AVMs) were significantly more common in patients with fractures (17%, 7/42) versus those without (5.4%, 6/112; p=0.04). No significant differences were found in rates of retinal vasculopathy or hemorrhage (13% vs 7.2%, p=0.23) or gastrointestinal bleeding (22% vs 16%, p=0.35). DXA data were available for 171 patients (22 pediatric, 149 adult). The median age at first DXA was 11.4 years (range 4–17.7) for children and 58.2 years (range 20.8–79.4) for adults. Among pediatric patients, the median lumbar spine Z-score was –1.2 (IQR –3.0 to –0.2, n=18), with 39% (7/18) showing a Z-score < –2. Among adults, the median femoral neck T-score was –1.1 (IQR –1.7 to –0.5, n=118), with 21% (29/138) demonstrating T-scores < –2, indicating low BMD. Conclusions: Fractures and reduced BMD are common in both pediatric and adult patients with TBDs, with a substantial burden of early-onset and recurrent fractures. Our findings underscore the importance of early bone health screening and surveillance in TBDs and suggest a possible link between vascular abnormalities and skeletal fragility in this population that requires further study.

Abstract Background Pediatric patients undergoing hematopoietic cell transplantation (HCT) may develop life-threatening complications and need for ICU admission (ICU). They may display more severe and/or unique manifestations of organ dysfunction syndromes, including acute respiratory failure, sepsis and delirium, compared to the general pediatric In this interim analysis of the POEM study, the utility of a p-MODS screening tool as a predictor of endotheliopathies and/or ICU is reported. Methods: NCT05090345 is a prospective multi-center registry/biorepository trial of children and adolescents/young adults (AYA) undergoing HCT to assess the impact of endotheliopathies on morbidity and mortality. Patients undergoing all HCT types are prospectively monitored for 100 days post-HCT for sinusoidal obstructive syndrome (SOS), thrombotic microangiopathy (TMA), capillary leak syndrome (CLS), diffuse alveolar hemorrhage (DAH) and ICU. The risk of p-MODS was assessed within 7 days of HCT admission and weekly for 4 weeks, to dynamically evaluate organ function across 8 systems: respiratory, cardiovascular, renal, hepatic, hematologic, neurologic, immune, and gastrointestinal. Each system was graded on a 0–5 scale based on clinical and laboratory parameters, with total pMODS calculated using the worst score per organ. Descriptive statistics were used to compare p-MODS score between groups with “events” (defined as endotheliopathy or ICU) versus “no events”. The “no event” comparison group was matched 1:1 by diagnosis, age, gender and HCT type. Results: Among all subjects (n=151), the median age was 11.3 (range: 0.2–26.9) years and the majority (64%) male and Caucasian ( 60%) and 20% Black. Most patients (86.8%) underwent allogeneic HCT. Common indications were hematologic malignancy (45%), solid tumors (19%) and hemoglobinopathies (8.6%).Events (n=50) were observed in 35 of 151 patients, including ICU (n=26), SOS (n=19), TMA (n=3) CLS (n=1) and DAH (n=1). Events occurred at a median of 16 (range: –7 to 85) days post-HCT. Median time to ICU was 15 (range: –7 to 82) days post-HCT. The median pMODS score at the time of ICU was 10 (range: 1–22). All three patients with TMA had preceding ICU admissions and 4 of 19 patients with SOS required ICU management. The median time to onset of endotheliopathy was 17 (range: 6–33) days post-HCT. The median pMODS score at the time of event was 11 (range: 7–18), compared to a median of 8 (range: 5–21) the week prior to onset. Patients who developed events exhibited progressively higher scores compared to matched controls starting at week 1 (p < 0.05). This difference became more pronounced over time, reaching statistical significance at week 2 (p < 0.001). Since physiologic decline may precede overt events, we next analyzed pMODS score change between Day 0 and week 1. A significant difference was observed with median increase of 2 (range: –2 to 16) in the event group versus 1 (range: –3 to 7) in the control group (p = 0.01). Analysis of individual organ system scores revealed notable differences between the event and control groups by Week 1, prior to median event onset. The event group had higher rates of indicators of organ dysfunction in 6 of 8 organ system. The event group had more subjects with acute kidney injury, elevated delirium scores, need for respiratory support, increased bilirubin, moderate to significant weight gain and infections. However, rates of neutropenia and lymphopenia were comparable between groups. Conclusion In this interim analysis, a significant increase in pMODS between Day 0 and week 1 post-HCT may reflect early physiologic decline, with notable changes in respiratory status, acute kidney injury, elevated CAPD scores, and infectious burden. These findings highlight the potential utility of pMODS as an early screening tool to identify pediatric HCT recipients at risk for clinical deterioration. Prospective and dynamic monitoring of pMODS may support timely intervention and improve outcomes in this vulnerable population.

Abstract Background: Sickle cell disease (SCD) is marked by wide variability in clinical phenotypes and severity due to environmental, clinical, and genetic factors. Variants in iron-regulatory genes are well described modifiers of clinical presentation in different diseases, but their role in modulating SCD phenotype has been sparsely studied in African populations. In this cross-sectional study, we investigated the role of single nucleotide polymorphisms (SNPs) in iron-regulatory genes in SCD severity and lifetime transfusion(Tx) requirement. Methods: The study was approved by the institutional review board of Lagos University Teaching Hospital, Lagos, Nigeria. Clinical data were abstracted from medical records and patient interviews using a structured questionnaire. Participants were recruited in steady state and were stratified according to the severity score previously described by Shah et al. (Clinicoecon Outcomes Res. 2020;12:625). Biochemical analyses included serum transferrin and serum iron; transferrin saturation (TS) was calculated. Genotyping was performed using the Axiom Precision Medicine Research Array (PMRA; Thermo Fisher Scientific). Seventy-one single nucleotide polymorphisms (SNPs) in genes implicated in iron metabolism were analyzed, including SLC40A1 (ferroportin), HFE (homeostatic iron regulator), HJV (hemojuvelin), HAMP (hepcidin), and TFR2 (transferrin receptor 2). SNPs whose variants were not represented in this cohort, including HFE C282Y and H63D, were excluded. Ensembl and NCBI databases were used to describe the wild-type (WT)/variant alleles and gene consequences. Univariate and multivariate logistic regression were used to analyze binary outcomes, and age-adjusted linear regression for Tx burden. Odds ratios (OR) and regression coefficients (β) were estimated using the WT as the reference. Multiple-testing correction was performed as necessary. Statistical analyses were conducted in R version 4.4.1, with p<0.05 considered significant. Results: We included 60 genotyped, confirmed HbSS patients; median age was 22 years (range: 18–43), 22 (37.3%) were female, 42 (70.0%) were classified as mild severity (Class I), 5 (8.0%) as Class II, and 13 (22.0%) as severe (Class III). Twenty-seven (45.0%) had never received a blood Tx, while 33 (55.0%) had received Tx, median of 2units (range: 1-10). On univariate analysis (UVA), five SNPs (rs10218795, rs10188230, rs114250626, rs4287798, and rs634349) were significantly associated with severe phenotype; three of these remained significant on multivariable analysis (MVA). Two were associated with higher odds of severe phenotype: rs10177654 (OR = 28.9, 95% CI: 3.0–100.0, p = 0.03) and rs10218795 (OR = 15.0, 95% CI: 2.4–327.0, p<0.01), while rs10188230 was associated with lower odds (OR = 0.03, 95% CI: 0.008–0.28, p<0.01). Results remained consistent after adjusting for age. For lifetime transfusion burden, both UVA and MVA identified two significant SNPs: rs4667289, associated with higher transfusion burden (β = 2.6, 95% CI: 0.04–5.2, p = 0.047) and rs11568344, associated with lower transfusion burden (β = –1.8, 95% CI: –3.5 to –0.36, p = 0.02). Genotype distributions for the five significant SNPs were as follows: rs10177654 (SLC40A1, transcription factor binding site), TT(WT) 12 (20.3%), TC 30 (50.8%), CC 17 (28.8%); rs10188230 (SLC40A1, intron variant), TT (WT) 32 (54.2%), TC 22 (37.3%), CC 5 (8.5%); rs10218795 (HJV, intron variant), CC (WT) 22 (37.3%), TC 29 (49.2%), TT 8 (13.6%); rs4667289 (SLC40A1 proximal intergenic), GG (WT) 46 (78.0%), AG 10 (16.9%), AA 3 (5.1%); rs11568344 (SLC40A1 splice region variant), GG (WT) 47 (79.7%), AG 9 (15.3%), AA 3 (5.1%). Compared with the homozygous WT, patients with rs10188230 had a lower median TS (21.65% vs. 43.3%, p = 0.04), while those with rs4667289 had a higher median TS (83% vs. 31%, p<0.01). When adjusted for age and lifetime Tx, rs4667289 remained significant (β = 47.8, 95% CI 3.6-82.8, p = 0.03). No other SNPs showed association with TS. Conclusion: We present novel genetic data on iron-regulatory variants in African patients with sickle cell disease, identifying SNPs in SLC40A1 and HJV associated with disease severity. Most of these are yet to be fully described in the literature, and their intronic locations suggest possible regulatory roles. We hope to further validate these findings in larger cohorts and through functional studies to clarify their impact on iron metabolism and clinical significance.

Abstract Introduction Detecting genetic abnormalities is mandatory during diagnostic workup and for potential individualization of therapy selection in multiple myeloma (MM) and its precursor conditions. At present, this requires invasive bone marrow (BM) biopsies, severely limiting early detection, patient monitoring, and the precise selection of therapy. The current standard for detecting genetic alterations in MM is fluorescence in situ hybridization (FISH), which cannot detect point mutations and other clinically relevant alterations. Consequently, the IMS-IMWG guidelines were recently updated to require next-generation sequencing for the classification of high-risk MM. Moreover, the importance of identifying alterations in therapeutic targets (e.g., BCMA, GPRC5D) for guiding immunotherapies is becoming increasingly recognized. To address this need, we recently launched GenoPredicta, a CLIA-approved Laboratory Developed Test that enables routine monitoring, informing diagnosis, and treatment selection by comprehensively and robustly characterizing MM genomes with whole genome sequencing (WGS) from a minimum of ~50 circulating tumor cells (CTCs) isolated from peripheral blood (PB) or tumor cells from BM. Here, we describe the characterization of clinical and research samples with GenoPredicta, highlighting resistance-conferring genetic alterations that can only be detected by sequencing and that are consistent with patients' clinical histories, while further demonstrating the complete concordance of GenoPredicta with prior FISH results. Methods Tumor cells were isolated from BM and/or PB samples using fluorescence-activated cell sorting (FACS) (after CD138+ enrichment for PB) and subjected to whole genome sequencing (WGS), from which we identified copy number alterations, structural variants, and single nucleotide variants and indels. For a subset of samples, the BM was also characterized by FISH, using a standard clinical panel for characterizing MM; for other samples prior FISH results were available from patients' clinical records. Results The analytical validation of GenoPredicta, conducted on a retrospective cohort of >60 viably frozen samples from >30 patients, showed complete concordance between BM and PB for detected genomic abnormalities, with all clinically relevant events (11 translocations and 125 CNVs) observed in BM recapitulated in PB (100% recall). All events identified by FISH in 14 samples (8 translocations and 30 CNVs) were also identified in the BM (100% recall), with 50 translocations and 30 CNVs not detected by FISH also absent in the WGS data (100% precision). The abnormalities detected in these samples also included multiple high-risk events not assayed by FISH, including biallelic TP53 loss (Del 17p coupled with a deleterious TP53 mutation, observed in 4 patients) and MYC::IGL translocations (2 patients). Moreover, for all fresh peripheral blood clinical samples with FISH results available from patients' histories, GenoPredicta recalled 100% of reported events. Among these clinical samples, we notably observed a case with both a biallelic GPRC5D deletion and a TNFRSF17 (BCMA) deletion combined with a truncating mutation on the remaining allele. This was consistent with the patient's clinical history showing progression after therapy with talquetamab (anti-GPRC5D bispecific), and previous nonresponse to teclistamab (anti-BCMA bispecific) after prior therapy with Cilta-cel (BCMA-directed CAR T). GenoPredicta also detected other mutations likely to confer resistance to immunotherapies, including loss-of-function mutations in IKZF3 and deletions of FCRL5 in frozen peripheral blood research samples. Conclusion We demonstrate that WGS-based characterization of MM from BM or CTCs is a viable replacement for FISH for clinical diagnosis and monitoring, with blood-based measurements enabling comprehensive profiling of the myeloma cancer genome. Importantly, this includes genetic alterations that confer resistance to therapy, allowing for both early detection of such alterations and more precise selection of therapy.

Abstract Cyclin D1, encoded by CCND1, is a cell cycle regulator, overexpressed in ~15% of multiple myeloma (MM) cases via CCND1::IGH super-enhancer juxtaposition through t(11;14). Cyclin D1 also plays a non-canonical role in transcriptional regulation, but in t(11;14) MM its function in gene regulation remains undefined. We investigated the global transcriptional and epigenomic consequences of CCND1overexpression in t(11;14) MM. To define a CCND1 signature, RNA-seq from two independent primary MM patient cohorts, CoMMpass and Mayo, was utilized to compare high CCND1 t(11;14) cases (≥300 transcripts-per-million [TPM], n=271) vs. low CCND1 hyperdiploid cases (<30 TPM, n=233). Two CCND1 knockout (KO) clones in t(11;14) MM U266B1 were also generated by CRISPR/Cas9, introducing a frameshift insertion (A) after chr11:69641478 (hg38) in exon 1. ATAC-seq and ChIP-seq (H3K4me3, H3K4me1, and H3K27ac) were performed in duplicates, and RNA-seq in triplicates in U266B1 wild-type (WT) and KO. Libraries were sequenced on Illumina NextSeq 2000 (2x51 bp for ChIP/ATAC, 2x150 bp for RNA-seq). Reads were mapped to hg38 by BWA (ChIP/ATAC) or STAR (RNA-seq), peaks called by MACS2 (FDR< 0.01), peak and gene raw counts estimated by featureCounts. Differential peaks (fold-change ≥1.5) and differentially expressed genes (fold-change ≥2) were identified by edgeR (FDR<0.05), including peaks detected in ≥2 samples and genes with TPM ≥0.1 in ≥1 sample. Interactions between promoters and distal regulatory regions were identified using publicly available chromatin interaction data in blood cell types. Pathway analysis was performed using Enrichr. Co-immunoprecipitation was performed in t(11;14) KMS12BM cells. Differential analysis comparing CCND1-high t(11;14) and CCND1-low MM cases in the two patient cohorts identified 21 upregulated and 21 downregulated genes associated with CCND1 expression which were also captured in the wild-typevs. CCND1 KO U266 cells. Enriched pathways (p<0.05) included Notch signaling, early/late estrogen response, epithelial-mesenchymal transition and reduced mitotic spindle and myogenesis. Dysregulated genes included those related to apoptosis, ubiquitin-proteosome, extracellular matrix, adhesion, and immune response, demonstrating a broader role for cyclin D1 in MM beyond cell cycle control. To explore the epigenomic basis of this transcriptional regulation, for each of the 42 differentially expressed genes we examined the changes of histone marks and chromatin accessibility in promoter and the interacting distal regulatory regions in the U266 KO model. Ten of the 21 genes upregulated in CCND1-high (downloaded in KO; e.g., argininosuccinate synthase 1 [ASS1], PMAIP1, MYEOV, CCR7, and DTX1) and 12 of the 21 genes downregulated in CCND1-high (upregulated in KO; e.g., UACA, RASAL2, PTPRZ1, PPT2, and IL23R) showed concordant changes in histone marks and/or chromatin accessibility, predominantly in distal regulatory regions, suggesting a chromatin-mediated mechanism of gene regulation by cyclin D1. The most prominent changes involved H3K27ac, a mark defining active enhancers. As H3K27ac is deposited by histone acetyltransferases CBP, we tested for the possible cyclin D1-CBP interaction. Co-IP confirmed this interaction, suggesting a role for this complex in chromatin remodeling and transcriptional control in t(11;14) MM. One gene notably upregulated in association with CCND1 is ASS1, which encodes a urea cycle enzyme responsible for endogenous arginine synthesis. Metabolomic studies demonstrated KO cells had elevated aspartate and citrulline and lowered arginine and ornithine compared to WT cells (p<0.01) demonstrating impaired urea cycle function. Further, KO cells displayed significantly reduced ASS1 protein expression, cell growth and S-phase entry compared to WT cells in arginine-replete conditions, with both WT and KO cell types further inhibited by arginine depletion (p<0.05). Together, a cyclin D1-associated gene signature was identified supporting its role in t(11;14) MM beyond the CDK4/6 mediated cell cycle control, including a role in metabolic plasticity and survival under arginine depleted conditions. These findings highlight cyclin D1 as a therapeutic vulnerability and open new avenues for personalized treatment strategies.

Abstract Multiple myeloma (MM) is the second-most common hematological malignancy in the United States. Myeloma tumors have significant genetic heterogeneity that impacts disease progression and promotes resistance to treatment. RAS is a key oncogenic driver that activates the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K)/AKT pathways..Mutations in genes that code for RAS isozymes, KRAS, HRAS, or NRAS, lead to increased levels of activated GTP-RAS and are associated with the most aggressive forms of cancer. Mutations in the RAS family of genes are most common (40-50%) in myeloma. We and others have reported that RAS mutations may not be disease-initiating but rather arise during disease progression and relapse or as subclonal events that impart selective growth advantage and emerging drug resistance. In addition, non-mutant RAS can also be hyperactivated from upstream signaling (like receptor tyrosine kinases) or loss-of-function mutations in GTPase-activating proteins (GAPs). Given the importance of RAS mutations, allele-specific RAS inhibitors have long been considered an attractive strategy for myeloma treatment. However, multiple studies have reported various mechanisms of resistance to allele-specific RAS inhibitors as well as isoform-specific pan-RAS inhibitors, including newly acquired activating RAS mutations, RAS amplification, unchecked activation of co-expressed wild-type RAS (WT-RAS) isozymes, or acquired bypass mechanisms like new oncogenic fusions. Since MM remains incurable, we sought to improve upon currently available treatments by developing small-molecule inhibitors that preferentially inhibit myeloma cells with activated RAS isozymes. We have created NRasQ61X and KRasG12X mutations by CRISPR-Cas directed edits in a single endogenous RAS allele in human myeloma cell lines (HMCLs) that initially had two wild-type alleles. We found that these mutant-NRAS and mutant-KRAS edited cells have increased proliferation compared to cells with WT-RAS, as well as acquired resistance to several commonly used therapeutics. In this study, we used a triangulation method that combines our drug combination candidate prediction algorithm called ‘secDrug’ and the ‘Direct to Drug’ screening platform that evaluated 84 FDA-approved oncology drugs and emerging therapeutics in these RAS mutant myeloma lines. We identified BIRC5 as a potential target for RAS-mutant myeloma. BIRC5 expression is a key mechanism for the evasion of apoptosis. Notably, oncogenic RAS has been shown to promote MYC stabilization via upregulation of BIRC5. Earlier studies suggested that BIRC5 inhibitors are potent in targeting drug-resistant subclones in hematological malignancies. We performed in vitro cytotoxicity studies and cell-based assays using an in-house panel of HMCLs (n>50) representing WT- and mutant-RAS, as well as innate and acquired response/resistance to the standard of care drugs, proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs), to confirm the potency of novel selective BIRC5 inhibitors, as single agent (IC50 range 0.22-15.30 nM; Mean 4.431±1.041 nM) and in combination with PIs and IMiDs (Chou-Talalay's Combination Index or CI <0.3, indicating strong synergism). Further, we used bulk and single-cell whole-transcriptomics, siRNA-mediated gene knockdown studies, and functional validation to characterize the treatment-induced genes and molecular pathways underpinning the mechanism of action and drug synergy at the subclone level. Finally, we compared the efficacy of these BIRC5 inhibitors against several RAS inhibitors, including cyclorasin B4-27, a bicyclic peptidyl pan-RAS inhibitor, RMC-6236, a multi-selective noncovalent RAS(on) inhibitor, and ADT-007, a novel, highly potent, and selective pan-RAS inhibitor that we have synthesized, and observed comparable effects on cell death and apoptosis. Ex vivo analysis in bone marrow-derived CD138+ cells from myeloma patients using high-throughput Mass Cytometry (CyTOF or Cytometry by Time-Of-Flight) confirmed the dose-dependent reduction of MCL1, Ki-67, CCND1, c-KIT, and MYC. We will present these results that demonstrate the clinical potential of BIRC5 inhibitors as novel candidates in curbing progression and drug resistance in RAS-mutant myeloma.

Background Contact aspiration remains a frontline approach for mechanical thrombectomy (MT) in acute ischemic stroke (AIS). Early evidence correlates catheter size with procedural efficiency and clinical outcomes. Larger‐bore (LB) aspiration catheters may enhance clot ingestion, increase aspiration force, and facilitate faster and more complete recanalization. However, the transition from standard LB catheters (0.068‐0.072″) to super‐large‐bore (SLB, ≥0.080″) devices has raised questions regarding both safety and clinical benefit. Although the use of SLB catheters is increasing, comparative data on their performance versus LB catheters remain limited. This study aims to evaluate the safety and efficacy of SLB compared with LB catheters including data from a large multicenter international registry. Methods We retrospectively analyzed patients with AIS due to LVO treated at 46 international stroke centers between January 2013 and June 2025. Among eligible patients, propensity score matching (PSM) at a 5:1 ratio was used to balance baseline characteristics between those treated with standard LB catheters and those treated with SLB catheters. Safety endpoints included intraprocedural complications, symptomatic intracranial hemorrhage (sICH), distal embolization, and 90‐day mortality. Efficacy endpoints included functional independence at 90 days (modified Rankin Scale [mRS] 0‐2), successful recanalization (TICI ≥2b), first‐pass effect (TICI ≥2b on the first aspiration attempt), and procedural timing to TICI2b. Results A total of 3,100 patients were included (LB n=3,024; SLB n=67). After PSM (LB n=335; SLB n=67), safety outcomes were comparable between groups in terms of intraprocedural complications occurred (p=0.5), sICH (p>0.9), embolization to new territory (p=0.2), and 90‐day mortality (p>0.9). Efficacy outcomes were similarly consistent for functional independence (mRS 0‐2) (p>0.9), successful recanalization (p>0.9), first‐pass effect (p=0.5), and time to TICI ≥2b (27.6 vs. 24.5 minutes; p=0.9). These findings remained consistent in the subgroup with ICA and M1 occlusions, with no significant differences in safety (intraprocedural complications [p=0.4], sICH [p>0.9], embolization to new territory [p=0.6], or 90‐day mortality [p>0.9], nor in efficacy outcomes (functional independence at 90 days [p>0.9], successful recanalization [p=0.9], first‐pass effect [p=0.6], and time to TICI ≥2b [p=0.9]). Conclusion In this large multicenter analysis, the use of SLB aspiration catheters demonstrated comparable safety and efficacy to standard LB catheters for MT in AIS. Despite theoretical advantages in aspiration dynamics, SLB devices did not confer significant procedural or clinical benefit over LB catheters. These findings suggest that while SLB catheters are a feasible alternative, technical and clinical outcomes remain comparable to LB catheters. Further prospective studies are warranted to clarify potential clinical advantages. image