Braggio, Esteban

Abstract Background: Monoclonal B-cell lymphocytosis (MBL), a precursor to chronic lymphocytic leukemia (CLL), is an understudied yet common premalignant condition present in ~8 million adults in the United States. Based on flow cytometry, MBL can be classified into two immunophenotypes: CLL-type (CD5, CD23, and CD20 co-expression) and non-CLL-type (CD5-negative). While the majority of the data to date pertains to CLL-type MBL, the available evidence indicates non-CLL-type MBL is also associated with an increased risk of hematologic malignancies. Clonal hematopoiesis of indeterminate potential (CHIP) is another precursor condition associated with hematologic malignancies. The relationship between non-CLL-type MBL and CHIP and their combined effect remains unknown. Herein, we report findings on these two precursor conditions in the largest screening cohort to date. Methods: Using flow cytometry, we screened for MBL in the Mayo Clinic Biobank participants who were 40 years or older with no prior history of hematologic cancers. Using whole-exome sequencing of whole-blood DNA, CHIP was screened on 291 genes related to myeloid (56 genes) or lymphoid malignancies (235 genes). Incident hematologic malignancies were detected during follow-up using International Classification of Diseases (ICD) codes in the medical record. All diagnoses were manually reviewed for confirmation. Multivariable logistic regression was used to evaluate the association between non-CLL-type MBL and CHIP. Time to incident hematologic malignancy was calculated from date of MBL screening to the earliest date of incident malignancy, withdrawal, death, last medical visit, or study end. Overall survival (OS) was calculated from date of MBL screening to date of death or last known alive. Multivariable Cox regression, adjusted for age, sex and residency, was used to investigate associations between non-CLL-type MBL and CHIP with clinical outcomes; with results reported using hazard ratios (HR) and 95% confidence intervals (CI). Results: In total, 8,802 individuals with MBL screening and CHIP data were analyzed. Non-CLL-type MBL was detected in 249 (2.8%) individuals; 121 (49%) were male, and median age was 78 years (range, 47-95). We identified 757 (8.6%) individuals with CHIP in at least one of the 291 genes tested. The most frequently mutated CHIP genes were DNMT3A, TET2, ASXL1, PPM1D, and TP53. A total of 47 (0.5%) individuals had both non-CLL-type MBL and CHIP; the four genes with the highest number of CHIP variants in these individuals were DNMT3A (n=16), TET2 (n=10), ASXL1 (n=3), and ATM (n=3). An association between non-CLL-type MBL and any CHIP (OR=1.6, 95%CI:1.2-2.3, P=0.006) was observed. When analyzing CHIP in genes related to myeloid malignancies, the association with non-CLL-type MBL was also significant (OR=1.6, 95%CI:1.1-2.3, P=0.012); however, the association was not statistically significant for genes related to lymphoid malignancies (OR=1.6, 95%CI:0.8-3.3, P=0.165). With a median follow-up of 5.4 years, 104 individuals developed incident hematologic malignancies (66 lymphoid and 38 myeloid malignancies). Non-CLL-type MBL (HR=3.5, 95%CI:1.9-6.5, P<0.001) and CHIP (HR=3.2, 95%CI:2.1-5.1, P<0.001) were each associated with increased risk of incident hematologic malignancies. Co-existence of both precursor conditions was associated with a substantially higher risk of incident hematologic malignancies (HR=10.3, 95%CI:4.0-26.2, P<0.001) compared to individuals with neither precursor condition. Among individuals with non-CLL-like MBL, any CHIP was a borderline significant predictor for incident hematologic malignancy (HR=3.6, 95%CI:1.0-12.9, P=0.049). Compared to individuals with no precursor conditions, OS was not significantly different in those with non-CLL-type MBL-only (HR=1.1, 95%CI:0.8-1.5, P=0.40), those with CHIP-only (HR=1.2, 95%CI:0.99-1.4, P=0.06), or those with both CHIP and non-CLL-type MBL (HR=1.3, 95%CI:0.8-2.1, P=0.39). Conclusions: In the largest screening cohort to date evaluating the relationship of CHIP and non-CLL-type MBL, we found that non-CLL-type MBL and CHIP are associated with each other. Individuals with non-CLL-type MBL were more likely to have CHIP variants in DNMT3A, TET2, ASXL1, and ATM. Among individuals with non-CLL-type MBL, the presence of any CHIP was associated with increased risk of developing hematologic malignancy.

Abstract Recent studies have highlighted the important but pleiotropic role of IL-4 in response to chimeric antigen receptor T (CART) cell therapy. IL-4 was identified as a regulator of dysfunction in CD19-targeting CART cells with a CD28 costimulatory domain (CART19-28ζ) through a genome-wide CRISPR knockout screen as well as RNA and ATAC sequencing of baseline and exhausted healthy donor CART19-28ζ cells and pre-infusion CART19-28ζ cell products from responders and non-responders in the ZUMA-1 trial (Stewart et al, Nat Commun., 2024). However, interrogation of a single-cell atlas fromactivated pre-infusion CART cell products from patients with acute lymphoblastic leukemia revealed a beneficial role for IL-4 and other type 2 cytokines in the promotion of long-term response (Bai et al, Nature, 2024). In this report, we aim to further understand how IL-4 can be detrimental to CART cell therapy. To study the regulation of CART19-28ζ cells by IL-4, we performed RNA sequencing on CART19-28ζ cells that were stimulated with CD19+target cells in the presence of either 20 ng/mL hrIL-4 or diluent for 24 hours, one week, or two weeks in an in vitro model for exhaustion as previously described (Stewart et al, Nat Commun., 2024). We observed that hrIL-4 treatment drove the development of a distinct transcriptomic profile after chronic stimulation for one week (up genes = 3253 and down genes = 3645) or two weeks (up genes = 6435 and down genes = 6224) but not following 24 hours of activation (up genes = 0 and down genes = 0). Gene set enrichment analysis on the differentially expressed genes following two weeks of chronic stimulation showed that hrIL-4 treatment pushes CART cells to a more exhausted and less naïve phenotype (FDR = 0.015) and leads to a broad downregulation in cytokine signaling (FDR = 0.012). To investigate transcription factors associated with IL-4-induced CART cell dysfunction, we used BETA analysis with publicly available transcription factor binding sites for STAT6, GATA3, and EOMES and our differential expression data. EOMES (p = 0.039), but not STAT6 (p = 0.999) nor GATA3 (p = 0.999) significantly regulated the differentially expressed genes. In support of these findings, the addition of hrIL-4 also significantly upregulated EOMES expression after one (p < 0.0001) and two (p < 0.0001) weeks of chronic stimulation and downregulated the expression of TBX21 (p < 0.0001), a binding competitor of EOMES, following two weeks of chronic stimulation. To evaluate if EOMES is critical for IL-4-induced CART cell dysfunction, we treated either control or EOMES CRISPR knockdown CART cells with 20ng/mL hrIL-4 during chronic stimulation. EOMES knockdown reduced IL-4-induced CART19-28ζ cell dysfunction as seen by an increase in antigen-specific expansion (p = 0.046) and an increase in cytokine production including IL-2 (p < 0.0001), TNF-α (p = 0.006), and IFN-γ (p = 0.020). Having identified that EOMES is at least partially responsible for IL-4-induced CART cell dysfunction, we evaluated EOMES binding sites in human CART19-28ζ cells with CUT&RUN sequencing. Our analysis revealed EOMES binding at hypersensitivity site 2 (HS2) in the IL-4 locus which, in a previous study, was more accessible in both exhausted healthy donor CART19-28ζ cells and pre-infusion CART cell products from non-responders in the ZUMA-1 clinical trial (Stewart et al, Nat Commun., 2024). To determine how HS2 can impact CART cell functions, we generated CART19-28ζ cells from either wildtype (HS2+/+) or HS2-deleted (HS2-/-) murine T-cells and tested them in a syngeneic model of lymphoma generated by engrafting C57BL6 mice with luciferase+ TBL12 cells (1x106, tail vein injection on Day -3) following preconditioning (250mg/kg cyclophosphamide, intraperitoneal injection on Day -4). Following randomization of mice via tumor burden (n = 5 per group), mice were treated with either HS2+/+ or HS2-/- CART19-28ζ cells (1x106 cells, tail vein injection). In this model, HS2-/- CART cells showed enhanced expansion (p = 0.022), increased anti-tumor activity (p = 0.018), and improved overall survival (p = 0.003). Together, our study shows that IL-4 induces a distinct cellular fate in CART19-28ζ cells in the presence of chronic antigen-specific stimulation at least partially through the upregulation of EOMES. Further, the epigenetic regulation of CART cells by the IL-4 enhancer HS2 can significantly influence CART cell activity, and deletion of HS2 can improve CART cell therapy.

Abstract Background: The synergistic mechanisms of BCL-2 inhibitors and CD20-directed antibodies in the treatment of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) often lead to deep and durable responses. Although the use of venetoclax-obinutuzumab (Ven-O) in the frontline setting (1L) for CLL/SLL has led to a paradigm shift in the disease management, there are limited data on its efficacy in the relapsed/refractory (R/R) setting. In this study, we evaluated the efficacy and safety of Ven-O in both 1L and R/R CLL among patients treated outside the context of clinical trials at our institution. Methods We used the Mayo Clinic CLL Database to identify patients with CLL who were treated with Ven-O, both in 1L and R/R settings. Patients seen at Mayo Clinic Rochester between 2017 and 2025 were included. Overall survival (OS) was calculated from date of Ven-O initiation until last known alive date. Event-free survival (EFS) was calculated from date of Ven-O initiation until the next line of treatment or last known alive date. Kaplan-Meier methods estimated EFS and OS rates at 1, 3, and 5 years. Cox proportional hazards models were used to explore the association between baseline factors and EFS and OS; results are reported as hazard ratios (HR) and 95% confidence intervals (95%CI). Finally, we compared the OS to the age- and sex-matched general population of the state of Minnesota. Results A total of 154 patients (91 1L, 63 R/R) were included in the study. The median age was 68 years (69 1L, 68 R/R), and 68% were male (64% 1L, 73% R/R). Overall, TP53 disruptions (either del17p by FISH or TP53 mutation) were noted in 22 (15%) patients (9% 1L, 23% R/R). CLL FISH findings demonstrated del11q in 24 (19%) patients (19% 1L, 18% R/R), del13q in 46 (36%) patients (36% 1L, 36% R/R), and trisomy 12 in 21 (16%) patients (16% 1L, 18% R/R). IGHV genes were unmutated in 95 (68%) patients (60% 1L, 80% R/R). The CLL-IPI risk score was high in 54 (51%) patients (51% 1L, 53% R/R) and very-high in 18 (17%) patients (8% 1L, 34% R/R). A total of 121 (89%) patients completed planned treatment (1 year of venetoclax in 73 [90%] 1L and 2 years of venetoclax in 48 [87%] R/R). Median follow-up for the entire cohort was 1.8 years (2.2 1L, 1.5 R/R). Neither median EFS nor OS was reached for the overall cohort or within the 1L and R/R subgroups. The 1, 3, and 5-year EFS estimates for the overall cohort were 91% (86-96%), 85% (78-92%), and 67% (51-89%), respectively; corresponding EFS values for the 1L subgroup were 95% (91-100%), 89% (82-98%), and 67% (47-95%), respectively; and for the R/R subgroup were 85% (76-95%), 77% (65-92%), and 77% (65-92%), respectively. The 1, 3, and 5-year OS estimates were 98% (95-100%), 95% (91-99%), and 88% (76%-100%), respectively; corresponding OS values for the 1L subgroup were 99% (96-100%), 97% (94-100%), and 97% (94-100%) and for the R/R subgroup were 96% (91-100%), 91% (82-100%), and 61% (27-100%), respectively. Univariable analyses showed that TP53 disruption was associated with shorter EFS (HR 3.8, 95%CI 1.4-10.2, p=0.01), although IGHV mutation status was not associated with shorter EFS. A higher level of serum beta-2 microglobulin (HR 1.27, 95%CI 1.01-1.59, p=0.04) and R/R status (HR 4.9, 95%CI 0.9-25.8, p=0.06) were associated with a shorter OS. Interestingly, TP53 mutation status and IGHV mutation status were not associated with inferior OS. There was no difference in the observed and expected OS for both the 1L (p-0.23) and R/R (p=0.14) Ven-O treated cohorts compared to the age- and sex-matched general population of the state of Minnesota. Conclusions Our study shows that Ven-O is a well-tolerated regimen with demonstration of durable treatment responses both in the 1L and R/R groups, when used outside the context of a clinical trial. To the best of our knowledge, these data for the first time demonstrate no difference in OS of patients treated with Ven-O compared to age- and sex-matched general population, in both the 1L and R/R settings.

Abstract Background: Multiple myeloma (MM) is always preceded by monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). While these precursor conditions are asymptomatic, they are not entirely benign and carry a lifelong risk of progression to MM. Unlike other cancers defined by pathology, malignant transformation from MGUS or SMM to MM has so far relied on demonstration of clinical end-organ damage or high tumor cell burden (i.e. SLiM criteria, SV Rajkumar Lancet Onc 2014), as morphology and cytogenetics cannot reliably distinguish them. Methods: To identify genomic features underlying clinical behavior in MGUS and SMM, a total of 374 patients with SMM (n= 290) or MGUS (n=84) with available whole exome (whole exome sequencing, WES, n=190) or whole genome sequencing (whole genome sequencing WGS, n=184) were included in the study. Overall, none of the patients had evidence of SLiM or CRAB criteria at the time of the biopsy. Based on the center of origin and availability of WES and WGS, 227 and 97 patients were used to form our study training and validation set. The study was designed in two parts: 1) defining a new classification for MM precursor conditions; 2) integrating genomics to existing prognostic score to improve accuracy in identifying patients at imminent risk of progression. Overall, 68 patients in the training set were either lost to follow-up or enrolled in a clinical trial and they were only included in the classification part of the study. Results: By leveraging the distribution of established MM-defining genomic events (Maura et al. JCO 2024) in the training set, we developed a workflow that differentiates MGUS and SMM into two genomically distinct entities: one with evidence of neoplastic transformation (i.e., genomic MM) and one without (i.e., genomic MGUS). Overall, 39% of MGUS and 91% of SMM cases had genomic evidence of neoplastic transformation in the training set (i.e., genomic MM). When we applied the same workflow and classification to the validation set, 46% and 83% of MGUS and SMM had evidence of neoplastic transformation, respectively. Importantly, none of the patients classified as genomic-MGUS progressed to MM in either the training or validation cohorts, with a median follow-up of 40 and 61 months, respectively. Moreover, all patients with IMWG 2/20/20 high risk SMM were classified as genomic MM in both the training and validation set. Interestingly, 5 out of 62 (8%) patients enrolled in early intervention trials for high risk SMM were genomically classified as genomic-MGUS. Overall, these data demonstrated that the MM genomic background and neoplastic transformation can be acquired very early in time, even before the SMM phase, in line with what observed in solid tumors. To avoid overestimating the prognostic impact of myeloma-defining genomic events—which are, by definition, enriched in the genomic-MM group compared to genomic-MGUS—we restricted our prognostic analysis to patients in whom transformation has already occurred (genomic-MM). In fact, distinct genomic drivers may predict malignant transformation but not necessarily the risk of imminent progression to MM once transformation has occurred. After running 5-fold cross validations corrected for IMWG 2/20/20, only presence of RAS mutations, genomic events involving MYC, presence of neoplastic copy number variant (CNV) signatures and APOBEC mutagenesis were predictive of early progression in the training set. Adding the presence of high-risk myeloma-defining genomic events to the IMWG 2/20/20 model (i.e., genomic-IMWG) significantly improves the accuracy of predicting MM progression in both the training (c-index 0.69 vs. 0.74) and validation sets (c-index 0.74 vs. 0.79), compared to the IMWG 2/20/20 alone. Conclusion: By leveraging comprehensive genomic profiling integrated with the clinical IMWG 2/20/20 risk score, we propose a novel classification and prognostication system for MM precursor conditions, which will better define patients who may benefit from early therapeutic intervention versus watch-and-wait monitoring.

Abstract In this study, we investigated tumor intrinsic mechanisms of resistance to cevostamab, a bispecific T-cell engager targeting Fc receptor-like protein 5 (FCRL5) and CD3 that has shown promising clinical activity and durable responses in advanced MM. Longitudinal bone marrow aspirates from nine MM patients treated with cevostamab were analyzed. All patients had previously been treated with both immunomodulatory drugs (IMiDs) and proteasome inhibitors (PI) and eight patients had received daratumumab prior to cevostamab, which was, on average, the 6th line of treatment (mean treatment duration 220±273 days). Five of nine patients responded with ≥PR. Whole genome and transcriptome sequencing was performed on sorted CD138+ MM cells at baseline, and whole exome and transcriptome sequencing was performed on five and three samples, respectively, following cevostamab discontinuation. Baseline FCRL5 expression levels did not correlate with clinical response. However, in one non-responder patient, FCRL5 expression increased four-fold after 28 days of treatment. Given the location of the FCRL5 gene on chromosome 1q—commonly amplified in advanced MM—we evaluated the presence of Gain(1q) in our study cohort. All patients whose best response was ≤PR (n=6) harbored a Gain(1q), whereas it was absent in those who achieved VGPR and sCR (n=3). Crucially, one of the patients that achieved sCR was initially treated with 60 mg of cevostamab for one year and then escalated to a higher dose cohort (90 mg) in the second year. At progression, a clonal FCRL5E115A missense mutation (allelic frequency = 56%) and a subclonal FCRL5G455Efs*19(allelic frequency = 5%) frameshift deletion mutation were detected, both absent at baseline. The missense mutation was located on the extracellular domain of FCRL5 targeted by cevostamab. To validate the functional impact of FCRL5E115Aand FCRL5G455Efs*19 on the sensitivity to cevostamab in vitro, we generated a FCRL5 knockout (KO) model utilizing CRISPR-Cas9 technology in OPM2 MM cells. In vitro cevostamab cytotoxicity assay with PBMCs and pan T cells from healthy donors at various effector to target (E:T) ratios confirmed a complete loss of cevostamab sensitivity in our FCRL5KO models. cDNA encoding patient-derived FCRL5 alterations (FCRL5E115Aand FCRL5G455Efs*19) as well as FCRL5wtwere cloned into mammalian expression vector and reintroduced into FCRL5KO cells. Cevostamab sensitivity was reinstated by FCRL5wt expression in KO cells. In contrast, FCRL5E115A conferred complete resistance to cevostamab (p<0,001) and FCRL5G455Efs*19 depicted a significant decline in cevostamab sensitivity relative to FCRL5wt expressing models. Additional experiment utilizing direct stochastic optical reconstruction microscopy (dSTORM) are ongoing to monitor the membrane localization of FCRL5 mutants. By integrating genomic, transcriptomic and functional data with clinical outcomes, this work is the first to report on acquired FCRL5 mutations causing resistance to cevostamab. We hypothesize that the prolonged treatment duration may have contributed to the acquisition of the target mutations and note a subsequent durable sCR response to BCMA-targeted immunotherapy in the same patient, suggesting immune-related factors such as T cell exhaustion, were not drivers of cevostamab resistance. Due to the limited sample size, these findings require validation in larger cohorts. Nonetheless, our findings begin to elucidate tumor intrinsic mechanisms associated with cevostamab resistance.

Abstract Introduction: Although therapies targeting Bruton Tyrosine Kinase (BTK), either as monotherapy or in combination have revolutionized the treatment in chronic lymphocytic leukemia (CLL), the disease remains incurable. Continuous treatment with these agents frequently leads to the development of resistance and disease progression, mostly associated with mutations in BTK or PLCG2. However, a significant fraction of BTKi-resistant patients does not harbor mutations in these genes, revealing that multiple distinct mechanisms of resistance can independently develop in these patient cohorts. Identifying novel molecular pathways associated with drug resistance in the relapsed/refractory (RR) setting of CLL requires more exploration. Materials and Methods: Using the Mayo Clinic CLL Database and tissue bank, we identified 30 RR patients, who had had disease progressionon a BTKi. The median lines of prior therapies were four. The median age at start of treatment was 57.9 (range, 36-78), 77% were male. Rai stage at diagnosis was 0 in 10% of patients, stage I in 37%, stage II in 20%, stage III in 6%, and stage IV in 27%. IGHV unmutated was observed in 87% of cases. Five patients subsequently evolved to Richter's Transformation (RT). A total of 64 blood samples were collected at different timepoints: baseline (before BTKi therapy), during treatment and at disease progression from BTKi (Ibrutinib/Acalabrutinib/Pirtobrutinib/Nemtabrutinib). The CD5+/CD19+ tumor population was enriched by flow sorting. We performed whole genome sequencing (WGS) in tumor/germline pairs with an average sequencing depth of 60X. Copy number variants (CNVpytor), structural variants – SV (Manta and DELLY2), single nucleotide variants (SNV) and indels (MuTect2, Strelka) were calculated. Mutational signature analysis was conducted independently on each disease timepoint with sigProfiler. De novo signatures were extracted with non-negative matrix factorization, then decomposed and mapped to known signatures from the COSMIC Human Cancer Signatures database. Results: Prior to start of BTKi treatment, the most common abnormality was del13q (65%), followed by abnormalities in TP53 (mutation and/or del17p) in 59% of patients, del8p, del11q (23% each), and mutations in SF3B1 (35%), NFKIE, NOTCH1 and MAPK signaling pathway (BRAF, KRAS, NRAS, PTPN11) in 23% of cases, each. Complex karyotype, defined as >3 copy number abnormalities was found in 59% of cases. Disease progression was observed 4.4 years (median; range 1.9-10.8 yrs) after starting BTKi therapy. At disease progression, there was an overall increase in genomic complexity. Overall, 17 cases (56%) showed mutations in BTK (p.C481S, p.C481Y, p.L528W, p.A428D) and/or PLCG2 (p.P844H). Four cases showed mutations in both genes or two or more independent mutations in BTK. Del17p and/or TP53 mutations were found in 20 (67%) cases, 9 of them with biallelic abnormalities. A third of patients (N=10) were found to have MAPK mutations, and 9 (30%) cases had mutations in the epigenetic regulators CREBBP, SETD2, SETD1A andKMT2D. Loss of heterozygosity, including 11q and 17p was found in 13% of cases. Chromothripsis was found in 23% of cases. In patients without BTK or PLCG2 mutations (N=13), TP53 abnormalities were present in 92% of cases and mutations in the MAPK pathway in 46% of these patients. Finally, we identified several SVs involving driver genes at disease progression, including inversions in RB1 (N=1), NOTCH1 (N=1) and translocations in MYC (N=1) and IGH (N=2). Analyzing the mutational signatures, baseline timepoints were dominated heavily by aging signatures (SBS5, SBS1). However, in cases that progressed after therapy, we found enrichment of mutational signatures associated with defective DNA mismatch repair (SBS44, 6%), chemotherapy treatment (SBS25, 14%), homologous and nucleotide excision repair deficiency (SBS8, 48%), and indirect effects of AID-induced somatic mutagenesis (SBS85, 7%). Additionally, the Melphalan signature (SBS99, 10%) was found in patients treated alkylating agents. Conclusions: Disease progression of CLL on BTKi confirmed the existence of multiple driver genes beyond BTK, PLCG2 andTP53, including the high prevalence of mutations in MAPK pathway and epigenetic regulators, and increased genomic complexity. Ongoing efforts are focused on integrating these findings with clinical demographics, epigenetic modifications, and preclinical drug sensitivity profiles.

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 Background Mosaic chromosomal alterations (mCAs) are large-scale somatic gains, losses, or copy-neutral loss of heterozygosity (CNLOH) detectable by genotyping of peripheral blood DNA. They represent a form of clonal hematopoiesis and are observed in 0.5-5% of cancer-free individuals. The presence of any autosomal mCA has been associated with >3-fold risk of hematological malignancies. In particular, mCAs occurring in regions related to lymphoid malignancies have an 11-fold increased risk of lymphoid malignancies and a 68.6-fold risk for chronic lymphocytic leukemia (CLL). Monoclonal B-cell lymphocytosis (MBL), the precursor state to CLL, is defined by <5×10⁹/L clonal B cells in otherwise healthy individuals and is classified as low-count (<0.5x109/L) or high-count (0.5-5x10⁹/L) based on clonal B-cell count. MBL is also associated with increased risk of lymphoid malignancy (beyond CLL), with 4.3-fold and 74-fold risk in individuals with low- and high-count MBL, respectively. We previously reported a significant enrichment of autosomal mCAs in MBL, demonstrating a 1.7-fold association in those with low-count MBL and a 50.5-fold association in those with high-count MBL. Whether mCAs independently predict lymphoid malignancy risk after accounting for MBL status is unknown. Here, we evaluated the impact of mCAs on risk of incident lymphoid malignancies in individuals with MBL and in individuals screened negative for MBL. Methods CLL-type MBL (CD5 positive) was identified using flow cytometry assay on peripheral blood. Participants were age ≥40, had no prior hematologic malignancy, and were enrolled through the Mayo Clinic Biobank or the Division of Hematology at Mayo Clinic, Rochester. Participants were followed for incident lymphoid malignancy, including CLL, with diagnoses confirmed by medical record review. mCAs with cell fraction >1% were detected from array-based genotyping of whole blood DNA. Cox proportional hazards models, adjusted for age and sex, estimated hazard ratios (HRs) and 95% confidence intervals (CIs) for risk of developing an incident lymphoid malignancy in individuals with autosomal mCA versus those without an autosomal mCA. Results In 4,331 participants with available MBL and mCA status, the median age was 67 years, and 45% were male. Flow cytometry identified 3,231 individuals who screened negative for MBL, 754 individuals with low-count MBL, and 346 individuals with high-count MBL. Autosomal mCAs were present in 6.5% of individuals without MBL, 7.6% with low-count MBL, and 71.1% with high-count MBL. Over a median follow-up of 6.9 years, 237 individuals developed an incident lymphoid malignancy. The most common types were CLL, multiple myeloma, and diffuse large B-cell lymphoma. Ignoring MBL status, the presence of an autosomal mCA was associated with increased risk of lymphoid malignancy (HR = 2.65; 95% CI: 1.45–4.86; P = 0.002), replicating previous results. However, after excluding individuals with MBL, this association attenuated; those with mCA alone did not have a significantly elevated risk (HR = 1.60; 95% CI: 0.57-4.52; P = 0.38) compared to individuals without MBL or mCA. We next examined the effect of mCAs among individuals with MBL. In those with low-count MBL, the most frequent mCA events were 13q loss (2.1%), 12 gain (1.9%), and 4q CNLOH (1.2%), and individuals with an mCA had a borderline significant risk of developing a lymphoid malignancy (HR = 2.78; 95% CI: 1.01–7.67; P = 0.049) compared to those without an mCA. Similarly, in those with high-count MBL, the most frequent mCA events were 13q loss/CNLOH (31.2%), 12 gain (13.0%), and 11q loss (4.9%), and the presence of an mCA conferred an increased risk compared to those without mCA (HR = 1.51; 95% CI: 1.09-2.08; P = 0.012). Conclusions Autosomal mCAs were more frequent in individuals with high-count MBL compared to those with low-count or no MBL. mCAs showed limited predictive value for lymphoid malignancy among individuals without MBL. In contrast, in individuals with low-count MBL, autosomal mCAs emerged as a potential biomarker for identifying those at risk of developing a lymphoid malignancy. In high-count MBL, mCAs were also a biomarker, but were driven by known chromosomal abnormalities identified through clinical testing. These findings suggest that the utility of mCAs as a predictive marker for lymphoid malignancy is contingent on the presence of MBL and may inform risk stratification and surveillance strategies in this population.

Multiple myeloma (MM) is consistently preceded by monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). While these precursor conditions are asymptomatic, they are not entirely benign and carry a lifelong risk of progression to MM. Unlike other cancers defined by pathology, malignant transformation from MGUS or SMM to MM has so far relied on demonstration of clinical end-organ damage as morphology and cytogenetics cannot reliably distinguish them. In this study, using genomic data from 374 patients with MGUS or SMM (277 training, 97 validation), to our knowledge, we demonstrate for the first time the ability to identify malignant transformation in MGUS and SMM. We introduce the concept of genomic MM and genomic MGUS to differentiate the subsets of MGUS and SMM that are biologically malignant with genomic features indistinguishable from MM from the subset that is premalignant and unlikely to progress to malignancy. Importantly, we find that most SMM has biological features of malignant transformation indistinguishable from MM. As expected, this subset that we consider having genomic MM is associated with a high risk of progression to MM although some patients remained progression-free beyond 5 years. Conversely, 60% of MGUS and 10% of SMM have no evidence of malignant transformation (genomic MGUS), with no progression during follow-up. Integration of genomic features with the 2/20/20 International Myeloma Working Group model significantly improved the prediction of progression among genomic MM. These findings support the use of genomic criteria to refine the classification and the risk stratification in myeloma precursor conditions.

SummaryIn individuals with high‐count monoclonal B‐cell lymphocytosis (MBL), we investigated if lymphadenopathy or splenomegaly found by imaging was associated with shorter time to first chronic lymphocytic leukaemia (CLL) therapy (TTFT) and overall survival (OS). Individuals with MBL seen at Mayo Clinic (2002–2019) were retrospectively divided into three cohorts based on imaging studies within 1 year of diagnosis: no imaging studies (Cohort A); imaging with no evidence of lymphadenopathy/splenomegaly (Cohort B); imaging with evidence of lymphadenopathy/splenomegaly (Cohort C). We compared baseline characteristics, TTFT and OS across the MBL cohorts to a cohort of individuals with small lymphocytic lymphoma (SLL). A total of 1078 patients were included: 640 with MBL and 438 with SLL. Compared to Cohort B, individuals in Cohort C were more likely to have unmutated immunoglobulin heavy chain variable region (IGHV) (43% vs. 25% p = 0.016), high‐risk fluorescence in situ hybridization (FISH) (del17p and del11q in 15% vs. 4%, p = 0.038) and higher expression of CD38 (31% vs. 16%; p = 0.011). After adjusting for sex and CLL‐International Prognostic Index (IPI), lymphadenopathy/splenomegaly was associated with a shorter TTFT (hazard ratio [HR] = 2.04, 95% confidence interval [CI]: 1.02–4.04, p = 0.042) but not OS (HR = 1.09, 95% CI: 0.62–1.92, p = 0.775). Lymphadenopathy/splenomegaly on imaging in individuals with high‐count MBL is associated with a more unfavourable risk profile and shorter time to first CLL‐directed therapy.