Somatic Gene Research Articles

Somatic gene mutations involved in DNA damage response/Fanconi anemia signaling are tissue- and cell-type specific in human solid tumors.

With significant advancements in the study of DNA Damage Response (DDR) and Fanconi Anemia (FA) signaling, we previously introduced the term "FA signaling" to encompass "all signaling transductions involving one or more FA proteins." This network has now evolved into the largest cellular defense network, integrating over 30 key players, including ATM, ATR, BLM, HRR6, RAD18, FANCA, FANCB, FANCC, BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, BRIP1, FANCL, FANCM, PALB2, RAD51C, SLX4, ERCC4, RAD51, BRCA1, UBE2T, XRCC2, MAD2L2, RFWD3, FAAP20, FAAP24, FAAP100, and CENPX. This system responds to both endogenous and exogenous cellular insults. However, the mutational signatures associated with this defense mechanism in non-FA human cancers have not been extensively explored. In this study, we report that different types of human cancers are characterized by distinct somatically mutated genes related to DDR/FA signaling, each accompanied by a unique spectrum of potential driver mutations. For example, in pan-cancer samples, ATM emerges as the most frequently mutated gene (5%) among the 31 genes analyzed, with the highest number of potential driver mutations (1714), followed by BRCA2 (4% with 970 putative driver mutations). However, this pattern is not universal across specific cancer types. For example, FANCT is the most frequently mutated gene in breast (14%) and liver (4%) cancers. In addition, the alteration frequency of DDR/FA signaling due to these mutations exceeds 70% in a subtype of prostate cancer, with each subtype of brain, breast, lung, and prostate cancers displaying distinct patterns of gene alteration frequency. Furthermore, these gene alteration patterns significantly impact patient survival and disease-free periods. Collectively, our findings not only enhance our understanding of cancer development and progression but also have significant implications for cancer patient care and prognosis, particularly in the development of effective therapeutic strategies.

High-risk and silent clonal hematopoietic genotypes in patients with nonhematologic cancer

AbstractClonal hematopoiesis (CH) identified by somatic gene variants with variant allele fraction (VAF) ≥ 2% is associated with an increased risk of hematologic malignancy. However, CH defined by a broader set of genotypes and lower VAFs is ubiquitous in older individuals. To improve our understanding of the relationship between CH genotype and risk of hematologic malignancy, we analyzed data from 42 714 patients who underwent blood sequencing as a normal comparator for nonhematologic tumor testing using a large cancer-related gene panel. We cataloged hematologic malignancies in this cohort using natural language processing and manual curation of medical records. We found that some CH genotypes including JAK2, RUNX1, and XPO1 variants were associated with high hematologic malignancy risk. Chronic disease was predicted better than acute disease suggesting the influence of length bias. To better understand the implications of hematopoietic clonality independent of mutational function, we evaluated a set of silent synonymous and noncoding mutations. We found that silent CH, particularly when multiple variants were present or VAF was high, was associated with increased risk of hematologic malignancy. We tracked expansion of CH mutations in 26 hematologic malignancies sequenced with the same platform. JAK2 and TP53 VAF consistently expanded at disease onset, whereas DNMT3A and silent CH VAFs mostly decreased. These data inform the clinical and biological interpretation of CH in the context of nonhematologic cancer.

Systematic analysis of Mendelian disease-associated gene variants reveals new classes of cancer-predisposing genes

BackgroundDespite the acceleration of somatic driver gene discovery facilitated by recent large-scale tumor sequencing data, the contribution of inherited variants remains largely unexplored, primarily focusing on previously known cancer predisposition genes (CPGs) due to the low statistical power associated with detecting rare pathogenic variant-phenotype associations.MethodsHere, we introduce a generalized log-regression model to measure the excess of pathogenic variants within genes in cancer patients compared to control samples. It aims to measure gene-level cancer risk enrichment by collapsing rare pathogenic variants after controlling the population differences across samples.ResultsIn this study, we investigate whether pathogenic variants in Mendelian disease-associated genes (OMIM genes) are enriched in cancer patients compared to controls. Utilizing data from PCAWG and the 1,000 Genomes Project, we identify 103 OMIM genes demonstrating significant enrichment of pathogenic variants in cancer samples (FDR 20%). Through an integrative approach considering three distinct properties, we classify these CPG-like OMIM genes into four clusters, indicating potential diverse mechanisms underlying tumor progression. Further, we explore the function of PAH (a key metabolic enzyme associated with Phenylketonuria), the gene exhibiting the highest prevalence of pathogenic variants in a pan-cancer (1.8%) compared to controls (0.6%).ConclusionsOur findings suggest a possible cancer progression mechanism through metabolic profile alterations. Overall, our data indicates that pathogenic OMIM gene variants contribute to cancer progression and introduces new CPG classifications potentially underpinning diverse tumorigenesis mechanisms.

Prediction of Metastatic Site Based On Somatic Gene Mutations in Primary Tumors in Prostate Cancer

Objective The goal of this research is to predict the most likely metastatic site(s) of a primary prostate cancer tumor that has been resected via radical prostatectomy; its genome has been sequenced to obtain a list of gene mutations; and after initial inspection of pelvic lymph nodes, there is no clinical evidence of metastasis. However, micrometastases might already be growing in distant organs and cannot be detected at the time of surgery. Background The most common metastatic targets in prostate cancer (PCa) are the pelvic lymph nodes (PLN) and bones. The PLNs are routinely dissected by a procedure called pelvic lymph node dissection (PLND) simultaneously with the surgical removal of the prostate to detect the presence of metastatic growths. Additionally, the prostate-specific antigen (PSA) level is used to assess the existence of a metastatic stage. However, micrometastases in other organs and tissues might be overlooked. Methods We downloaded publicly available prostate cancer tumor data from the website www.CbioPortal.org. After choosing the 25 most frequently mutated genes by metastatic site (MS) and finding genes that are uniquely mutated on specific metastatic sites, we found that the mutational signature of a prostate cancer tumor is associated with its MS, and thus, we developed a method to numerically predict this association. Results After executing a computational algorithm on the data set of metastatic prostate tumors, it was found that we can predict metastatic sites with the following accuracies: bone (90.9%), retroperitoneum (87.5%), liver (83.0%), kidney (80.0%), pancreas (80.0%), adrenal glands (75.0%), lung (71.1%), and brain (72.5%). Conclusions We successfully developed a method and an algorithm that predict the most likely metastatic site of a primary prostate cancer tumor based on its genetic mutations. The accuracy of the predictions for eight metastatic sites ranges from 71.1% to 90.9%, with an average of 80.5%.

Bioinformatics-based analysis of the relationship between disulfidptosis and prognosis and treatment response in pancreatic cancer

Tumor formation is closely associated with disulfidptosis, a new form of cell death induced by disulfide stress-induced. The exact mechanism of action of disulfidptosis in pancreatic cancer (PCa) is not clear. This study analyzed the impact of disulfidptosis-related genes (DRGs) on the prognosis of PCa and identified clusters of DRGs, and based on this, a risk score (RS) signature was developed to assess the impact of RS on the prognosis, immune and chemotherapeutic response of PCa patients. Based on transcriptomic data and clinical information from PCa tissue and normal pancreatic tissue samples obtained from the TCGA and GTEx databases, differentially expressed and differentially surviving DRGs in PCa were identified from among 15 DRGs. Two DRGs clusters were identified by consensus clustering by merging the PCa samples in the GSE183795 dataset. Analysis of DRGs clusters about the PCa tumor microenvironment and differential analysis to obtain differential genes between the two DRG clusters. Patients were then randomized into the training and testing sets, and a prognostic prediction signature associated with disulfidptosis was constructed in the training set. Then all samples were divided into high-disulfidptosis-risk (HDR) and low-disulfidptosis-risk (LDR) subgroups based on the RS calculated from the signature. The predictive efficacy of the signature was assessed by survival analysis, nomograms, correlation analysis of clinicopathological characteristics, and the receiver operating characteristic (ROC) curves. To assess differences between different risk subgroups in immune cell infiltration, expression of immune checkpoint molecules, somatic gene mutations, and effectiveness of immunotherapy and chemotherapy. The GSE57495 dataset was used as external validation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of DRGs. A total of 12 DRGs with differential expression and prognosis in PCa were identified, based on which a risk-prognosis signature containing five differentially expressed genes (DEGs) was developed. The signature was a good predictor and an independent risk factor. The nomogram and calibration curve shows the signature's excellent clinical applicability. Functional enrichment analysis showed that RS was associated with tumor and immune-related pathways. RS was strongly associated with the tumor microenvironment, and analysis of response to immunotherapy and chemotherapy suggests that the signature can be used to assess the sensitivity of treatments. External validation further demonstrated the model's efficacy in predicting the prognosis of PCa patients, with RT-qPCR and immunohistochemical maps visualizing the expression of each gene in PCa cell lines and the tissue. Our study is the first to apply the subtyping model of disulfidptosis to PCa and construct a signature based on the disulfidptosis subtype, which can provide an accurate assessment of prognosis, immunotherapy, and chemotherapy response in PCa patients, providing new targets and directions for the prognosis and treatment of PCa.

Is It Ever Wise to Edit Wild-Type Alleles? Engineered CRISPR Alleles Versus Millions of Years of Human Evolution.

The tremendous burden of lipid metabolism diseases, coupled with recent developments in human somatic gene editing, has motivated researchers to propose population-wide somatic gene editing of PCSK9 (proprotein convertase subtilisin/kexin type 9) within the livers of otherwise healthy humans. The best-characterized molecular function of PCSK9 is its ability to regulate plasma LDL (low-density lipoprotein) levels through promoting LDL receptor degradation. Individuals with loss-of-function PCSK9 variants have lower levels of plasma LDL and reduced cardiovascular disease. Gain-of-function variants of PCSK9 are strongly associated with familial hypercholesterolemia. A new therapeutic strategy delivers CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats; CRISPR-associated protein 9) specifically to liver cells to edit the wild-type alleles of PCSK9 with the goal of producing a loss-of-function allele. This direct somatic gene editing approach is being pursued despite the availability of US Food and Drug Administration-approved PCSK9 inhibitors that lower plasma LDL levels. Here, we discuss other characterized functions of PCSK9 including its role in infection and host immunity. We explore important factors that may have contributed to the evolutionary selection of PCSK9 in several vertebrates, including humans. Until such time that more fully understand the multiple biological roles of PCSK9, the ethics of permanently editing the gene locus in healthy, wild-type populations remains highly questionable.

Modifying Our Genes: Theology, Science and “Playing God”

Modifying Our Genes: Theology, Science and “Playing God”

Developing Topics.

Goal 1 of the National Plan to Address Alzheimer's Disease (AD) is to prevent and effectively treat AD and AD-related dementias (ADRD) by 2025. Toward this goal, NINDS plans 14 ADRD funding opportunities for 2024 to support training, basic mechanisms, translational, and clinical research for ADRD. Here, we describe ADRD translational therapy development initiatives, which include 1) multi-target validation, 2) early-stage therapy development, 3) genome editing and 4) animal model development and validation. Note, ADRD specific funding opportunities are not required. ADRD researchers can also take advantage of NINDS existing programs such as the NIH Blueprint for Neurotherapeutics (BPN) for Small Molecule or Biologic therapy development. These programs support optimization, manufacture, IND-enabling studies and first in human clinical trials through a combination of grants and complementary access to NIH-provided CROs and consultants. All concepts discussed here are approved, however NIH cannot guarantee future funding opportunities, award mechanisms, or funding allocation. A funding opportunity is only official when published in the NIH Guide. Multi-Target Validation for ADRD concept has the goal to support research that would validate multiple targets for future therapy development that better reflect the complexity of disease. Early-Stage Therapy Development concept would support the development of small molecule or biologic therapeutics and would be a reissue of RFA-NS-22-059, which supported assay development, therapeutic screening, optimization, and in vivo efficacy work in preparation for later-stage programs. Genome Editing for ADRD concept would support early translational research focused on somatic cell gene editing with the expectation that studies will address feasibility of the gene editing approach and would be a reissue of RFA-NS-23-017. PAR-23-154 "Development and Validation of Models for Alzheimer's Disease-Related Dementias (ADRD)" supports the development and validation of clinically- and pathophysiologically-relevant models of ADRD that can be used for future therapy development. Models should reflect multiple aspects of the human condition and include internal, face, construct, and predictive validation to the extent possible. Through these initiatives, NINDS hopes to provide a comprehensive pipeline for ADRD therapy development from target validation to the clinic.

Abstract A048: Novel murine models of ARID1A and PBRM1-deficient cholangiocarcinoma for preclinical discovery and development

Abstract Genes encoding SWI/SNF chromatin remodeling complex subunits are recurrently mutated across human cancers. In cholangiocarcinoma (CCA), AT-rich interaction domain-1A (ARID1A) and polybromo-1 (PBRM1) loss of function mutations occur in approximately 18 and 10% of cases respectively. Yet our mechanistic understanding and ability to therapeutically exploit ARID1A and PBRM1 deficiency are immature. Genetically accurate preclinical models are needed to define the role of ARID1A and PBRM1 and test therapeutic approaches in CCA. Analysis of human CCA tumors showed that loss of function mutations in ARID1A and PBRM1 frequently co-occur with each other and with PI3K/AKT pathway activation. Therefore, we developed novel murine models of cholangiocarcinoma initiated by Arid1a knockout or dual Arid1a/Pbrm1 knockout in the context of AKT activation, employing a surgical biliary transfection method for somatic gene editing with CRISPR/Cas9. Mice with cholangiocyte Arid1a knockout and AKT activation (AAC) or dual Arid1a/Pbrm1 knockout and AKT activation (APAC) formed numerous liver tumors within 8 months. Histologically, tumors were predominantly CCA and mixed HCC/CCA, with a similar spectrum of tumors across AAC and APAC mice. Gene editing with CRISPR/Cas9 was confirmed at the DNA level, and loss of protein expression was confirmed in tumor cells by immunohistochemistry. Similar to human CCA, many tumors were densely infiltrated with cancer-associated fibroblasts and a variable degree of CD45+ immune cells. To generate scalable models for preclinical testing, tumors from AAC and APAC mice were dissociated to create cell lines for in vitro studies and propagated by orthotopic transplantation for in vivo studies. Cell lines and orthotopic tumors were validated and retained the genetic features and histologic characteristics of the parental tumors. Collectively these studies have produced a suite of novel, well-characterized, genetically relevant mouse models of epigenetic dysregulation in CCA due to ARID1A and PBRM1 deficiency, which can be used broadly for discovery research and preclinical testing of novel treatment approaches. Citation Format: Caitlin B. Conboy, Jennifer L. Tomlinson, Ryan D. Watkins, Jayla T. Millender, Danielle M. Carlson, Rory L. Smoot, Keith D. Robertson, Greg J. Gores. Novel murine models of ARID1A and PBRM1-deficient cholangiocarcinoma for preclinical discovery and development [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A048.

CD7 Chimeric Antigen Receptor T-Cell Therapy Bridging to Allogeneic Hematopoietic Stem Cell Transplantation Remarkably Improved Long-Term Disease-Free Survival in Refractory/Relapsed T-Cell Acute Lymphoblastic Leukemia/Lymphoma

Introduction: The prognosis of refractory/relapsed T-cell acute lymphoblastic leukemia/lymphoma (r/r T-ALL/LBL) is poor, and salvaged allogeneic hematopoietic stem cell transplantation (allo-HSCT) can only result in 20%-30% disease-free survival (DFS). Our previous clinical trials have shown that the patients with r/r T-ALL have achieved 90% complete remission (CR) with CD7 chimeric antigen receptor T cell (CART) therapy (Pan J. et al. JCO 2021), then quick received allo-HSCT to obtain 57% of 1-year DFS in 12 patients (Li ZH. et al. Transplantation and Cellular Therapy 2022). Objectives : In current study, the long-term outcomes of allo-HSCT in r/r T-ALL/LBL after CD7 CART therapy in larger cohort are investigated, and compared with that chemotherapy only before allo-HSCT simultaneously. The risk factors for prognosis after allo-HSCT in this setting are also analyzed. Methods: Between February 2018 and January 2023, total 90 patients with r/r T-ALL/LBL who underwent allo-HSCT in our hospital were included. The median age was 14 (2-65) years old. Somatic and germline gene mutations were detected by sequencing pre-transplant. Thirty-two (35.6%) patients were sensitive to chemotherapy and achieved CR before transplant (group A), and 58 (64.4%) cases were resistant to chemotherapy and in non-remission (NR) pre-HSCT. Forty-one of 58 patients in NR received CD7 CART before allo-HSCT (group B) and the rest 17 patients in NR underwent salvaged transplant (group C). Donor types included haploidential (60, 66.7%), unrelated (16, 17.8%) and identical sibling (14, 15.6%). Myeloablative conditioning regimens with either total body irradiation (TBI)/fludarabine (51, 56.7%) based or busulfan/fludarabine (39, 43.3%) based were applied. Antithymocyte globulin was used for haploidentical and unrelated transplants. Cyclosporine, mycophenolate mofetil and short-term methotrexate were employed for graft-versus-host disease (GVHD) prophylaxis. Results: All patients achieved durable engraftment. No significant difference was found in the incidences of acute GVHD (aGVHD), chronic GVHD (cGVHD) and infections in 3 groups (p=0.612, p=0.091, p=0.649). With a median follow-up of 25.5 (19.6-32) months, 2-year overall survival (OS) in group B was similar to that in group A (54.4% vs. 69.9%, p=0.33) and much higher than that in group C (35.3%, p=0.0065), and 2-year DFS in group B was similar to that in group A (51.0% vs. 61.1%, p=0.24) and much higher than that in group C (17.6%, p=0.0029). No statistical difference was seen on transplant-related mortality (TRM) (17.3% vs. 9.8% vs. 11.8%, p=0.65) among group B, A, C. Two-year cumulative incidence of relapse in group B (31.7%) was similar to group A (29.0%), and much lower than that in group C (70.5%). The risk for relapse and death decreased remarkably in group B (p=0.0047) and group A (p=0.0003) compared with that in group C. NR before transplant (p=0.0062), aGVHD (p=0.013) and viral infections (p=0.047) were risk factors for relapse-related death, but TBI-based conditioning (p=0.01) was positive impact factor on OS. The most common somatic gene mutations were NOTCH 1(55.7%), JAK 3 (18.0%), TP53 (16.4%), JAK1 (14.8%) and N R AS(14.8%). The most common germline gene mutations were EP300 (10.3%), ATM (8.6%), F7 (8.6%), KIT (8.6%) and N CF2 (8%). The patients with somatic TP53 or MED12 mutation had significantly higher risk for relapse and death (p=0.0018, p=0.0068). Germline gene mutations had no significant influence on prognosis post-transplant. Multivariate analysis had shown that CD7 CART (p=0.037), TBI-based conditioning (p=0.002) and cGVHD (p=0.004) were independent positive impact factors on OS, but aGVHD (p=0.034) and TP53 mutation (p=0.002) were independent negative impact factors for OS. Meanwhile, CD7 CART (p=0.005) and TBI-based conditioning (p=0.024) had positive impact on DFS, but TP53 mutation (p=0.02) also had negative impact on DFS. Conclusion s : Our study has demonstrated that CD7 CART followed by allo-HSCT has remarkably improved long-term DFS for chemotherapy-resistant T-ALL/LBL, and achieved comparable results in safety and efficacy with chemotherapy-sensitive disease post-transplant. Our study has also shown the profiles of somatic and germline gene mutations in r/r T-ALL/LBL, and identified some positive and negative impact factors for prognosis after transplant in this setting.

KPT-8602 Induced Hematopoietic Differentiation in U2AF1 S34F Mutant Cells and Myelodysplastic Syndromes Bone Marrow Stem and Progenitor Cells

Myelodysplastic syndromes (MDS) are a group of clonal bone marrow (BM) neoplasms characterized by ineffective hematopoiesis, cytopenia's, and a high risk of progression to acute myeloid leukemia. Current standard therapies include erythropoiesis stimulating agents for lower risk disease and DNMTi for higher risk MDS; however, less than half of patients respond and even the best responses are transient and non-curative. The majority of MDS patients harbor at least one of ~40 commonly found somatic gene mutations, most of which occur in splicing factor genes such as U2AF1, which also have non-canonical roles in nuclear export. KPT-8602 (Eltanexor) is a novel selective inhibitor of nuclear export (SINE) that acts by disrupting nuclear export of cargo relevant in MDS. We hypothesized that U2AF1 S34 F mutant cells would be selectively sensitive to treatment with KPT-8602. We created immortalized murine hematopoietic progenitor cells by retroviral transduction of stimulated bone marrow (BM) cells isolated from U2AF1 WT or U2AF1 S34F mice with a HOXB8_ER vector. Estrogen drives the hematopoietic transcription factor immortalizing the cells in a progenitor state, however, upon estrogen withdrawal the cells will differentiate and eventually die. This differentiation can be driven by treatment with specific growth factors such as erythropoietin (EPO) for erythroid differentiation or granulocyte macrophage colony stimulating factor (GM-CSF) for myeloid differentiation. KPT-8602 exposure induced differentiation and increased hematopoiesis in murine cell models and primary MDS samples. U2AF1 WT and U2AF1 S34F cells were plated in medium lacking estrogen and treated with GM-CSF (10nM) or EPO (1nM) with or without KPT-8602 (10nM) for 72h, then stained with Ly6C, CD11b, Ter119, and CD71 for flow cytometry analysis. In addition, we performed colony forming capacity assays in both the murine cells and primary MDS BM stem and progenitor cells and age-comparable healthy donors, with 14 day KPT-8602 exposure. Treatment with GM-CSF significantly increased the expression of Ly6C in both U2AF1 WT and U2AF1 S34F cells (WT fold change untreated vs GMCSF =1.9, p=0.03; S34F fold change untreated vs GMCSF =2.3, p<0.01). However, addition of KPT-8602 caused significantly more Ly6C expression than GM-CSF alone in the mutant cells (WT fold change GMCSF vs combination =3.1, p=0.06; S34F fold change GMCSF vs combination =4.0, p<0.01). There were no significant changes in CD11b expression with KPT-8602 treatment. We did not find a statistically significant increase in erythroid markers assessed by Ter-119 and CD71 after EPO treatment, however, KPT-8602 treatment alone showed a trend of increased expression of Ter-119 and CD71 in the mutant cells that fell just below statistical significance (Ter119 p=0.08 for WT, p=0.09 for S34F; CD71 p=0.21 for WT, p=0.06 for S34F). Additionally, we looked at selective toxicity of U2AF1 S34Fby WST-1 reagent and found no differences in toxicity between the WT or mutant cells with KPT-8602. However, U2AF1 WT and U2AF1 S34F progenitor cells showed an increase in colony forming capacity (n=1 with technical duplicates) when plated in the presence of KPT-8602 showing an average fold change in colony growth of 1.4 for U2AF1 WT and 1.3 for U2AF1 S34F underscoring the ability of KPT-8602 to induce differentiation. To confirm these results were not cell line artifacts, we next treated primary BM stem and progenitor cells from MDS patients (n=2) and healthy donors (n=4). MDS patient 1 (age 73y) was diagnosed with MDS-MLD-EB1 and harbored mutations in TP53, ASXL1, BCOR, TET2 and PPM1D. MDS patient 2 (age 79y) was diagnosed with MDS/MPN and harbored an SF3B1 mutation. Healthy donors (n=4) were all male and had a median age of 65y. Treatment with KPT-8602 (10nM) significantly increased burst and colony forming unit-erythroid (BFU-E/CFU-E) in MDS primary cells (p=0.02) with no significant increase in the healthy donors (p=0.57) (BFU-E/CFU-E fold increase was 1.1 for healthy donors and 3.5 for MDS). Collectively, these findings suggest that KPT-8602 promotes myeloid and erythroid differentiation and further validates the use of KPT-8602 as a therapeutic strategy for MDS. Future studies will fully elucidate the specific mechanisms by which KPT-8602 acts in the context of splicing factor mutations, and other commonly found MDS mutation types.

Combining Gene Mutation with Transcriptomic Data Improves Outcome Prediction in Myelodysplastic Syndromes

Background and Aim. Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by peripheral blood cytopenias and risk of progression to acute myeloid leukemia (AML). Disease management is challenged by heterogeneity in clinical courses and survival probability. Recently, the genomic screening integration (by Molecular International Prognostic Scoring System, IPSS-M) into patient's assessment has resulted into a significant improvement in predicting clinical outcomes compared to the conventional prognostic score (Revised IPSS, IPSS-R). Many of the consequences of genetic and cytogenetic alterations will affect gene expression by means of transcriptional and epigenetic instability and altered microenviromental signaling. The aim of this project conducted by GenoMed4All and Synthema EU consortia is to link genomic information with transcriptomic data for possibly improving the prediction of clinical outcomes in MDS patients. Patients and Methods.Clinical, cytogenetic, genomic (somatic mutations screening of 31 target genes) and transcriptomic (bulk RNA-seq of CD34 + bone marrow cells) data were collected at diagnosis in 389 MDS patients. Transcriptomic and genomic profiles were processed and the former were normalized before Principal Component Analysis (PCA) dimensionality reduction to mine the interdependency of expression-wide perturbation and recurrent genomic alterations. The prognostic impacts of genetic, cytogenetic, transcriptomic, clinical and demographic features were assessed with a penalized Cox's proportional hazards model [Gerstung M et al, Nat Commun. 2015. 6, 5901] considering the Overall Survival (OS) as primary end point. A 5-fold cross-validating (CV) scheme was exploited to control bias in risk estimation. Model accuracy was assessed using Harrell's concordance index (C-index). An independent validation of the results on 202 patients was planned. Results.We first processed each data layer assessing data robustness, removed not informative variables and scaled quantitative ones. We considered recurrent genomic and cytogenetic lesions (present in ≥5 patients), platelets, hemoglobin and bone marrow blasts (%), age and sex as covariates. To explore the main patterns of expression changes, PCA was performed to reduce multidimensional correlated expression features (20 PCs was selected, explaining 42% of the total transcriptomic variability). To evaluate the prognostic power of each data layer we grouped all available features into five groups: gene mutations (n=15), cytogenetic alterations (n=7), expression data (n=20), blood counts (n=3) and demographic variables (n=2). Within a 5-fold CV we combined these variables in our integrative model to calculate MDS patients risk. The obtained predictive accuracy (C-index) for OS was 0.83, underlying that transcriptomic data significantly improved the current standard prognostic scoring systems. Accordingly, in our patient population, the C-index of the conventional IPSS-R score and the new IPSS-M were 0.68 and 0.76, respectively. A similar improvement by adding transcriptomic data was observed in prediction of the risk of AML evolution. Moreover, by analyzing the contribution of each feature category to the OS probability ( Figure 1), in term of explained variance, the relative impact of transcriptomic is 40%, with the remaining prognostic information distributed among genomic features (somatic gene mutations and cytogenetics lesions, 24%), demographics (20%) and clinical features (15%). An independent validation of these results on 202 patients is currently ongoing. Figure 2 shows an example of personalized survival prediction using patients from the study population. In two subjects with same clinical phenotype and mutations leading to a similar IPSS-M prognosis, the integrative model captures additional prognostic information and efficiently predicts clinical outcome. Given the complexity of our model, specific technological support is needed to combine data at individual patient level and to translate it into a personalized outcome prediction. To this aim, we created a prototype web portal based on our dataset for user-defined genomic/transcriptomic and clinical features. Conclusion. In predicting survival of MDS patients, genomic, transcriptomic and diagnostic clinical variables all have utility, with a significant contribution from the transcriptome.

The Proliferative History Index, Epicmit, Derived from Genome-Wide Epigenomic Profiling, Is a Key Driver of Clinical Survival in Splenic Marginal Zone Lymphoma

The epiCMIT (epigenetically-determined Cumulative MIToses) mitotic clock (Durran-Ferrer, 2020) tracks DNA hypo- and hyper-methylation changes in heterochromatin and H3K27me3-containing chromatin, respectively, providing a comprehensive record of B cell mitotic history (pre- and post-malignant transformation). Higher epiCMIT levels correlate with poor survival in CLL and MCL, suggesting that a greater mitotic history predicts future proliferative capacity. However, no studies have investigated epiCMIT in splenic marginal zone lymphoma (SMZL). We studied 142 SMZL cases, a rare lymphoid malignancy with specific genetic characteristics [del(7q), KLF2and NOTCH2 mutations, biased use of IGHV1-2*04]. After calculating epiCMIT scores from 450/850K Illumina arrays we compared these data with gene mutations from targeted resequencing and WGS, copy number variations, telomere length, RNA-Seq, miRNA-Seq data, and clinical outcome data. By defining epiCMIT-hyper [mean: 0.6, range: 0.29-0.91] and -hypo [mean: 0.71, range: 0.39-0.90] values in our cohort, we demonstrate a strong positive correlation between hyper- and hypo- scores, as previously observed in MCL and CLL (Durran-Ferrer, 2020). The highest score obtained for epiCMIT-hyper or -hypo per sample was then selected as the epiCMIT score per patient [mean: 0.72, range: 0.39-0.91]. We identified significant correlations between epiCMIT and key clinico-biological characteristics, including a significant negative correlation between epiCMIT and telomere length (R 2=0.67 p<0.001) supporting cell proliferation and concomitant telomere attrition. High epiCMIT scores were associated with female gender (p=0.02), IGHV1-2*04 alleles (p<0.001) with intermediate levels of IGHV somatic hypermutation (SHM) load (97-99.9%, p=0.04), driver somatic gene mutations, including those in KLF2 (p<0.001), NOTCH2 (p<0.01), TP53 (p=0.01) and KMT2D (p<0.001) and deletion of 7q (p=0.01) ( Fig1). Using previously defined classification systems (Arribas, 2015 and Bonfiglio, 2022), we show enrichment of high-risk subgroups [Arribas- high global methylation subgroup (High-M, p<0.001) and Bonfiglio- NFKB, NOTCH and KLF2 genetic subgroup (NNK, p=0.01)] in cases with high epiCMIT. In a sub-cohort of 42 SMZL cases, with a spectrum of epiCMIT values and WGS/ (mi)RNA-Seq data available, we demonstrated 1) a significantly higher genome-wide somatic mutational burden in patients with high epiCMIT-total scores (25 v 17mut/Mb, p=0.001), and 2), correlations between epiCMIT and specific gene/miRNA expression associated with cellular survival and proliferation ( CARD11 [R 2=0.6, p<0.01], MAP2K1 [R 2=0.61, p<0.01], miRNA-155 [R 2=0.58, p=0.018]), apoptosis ( BCOR [R 2=0.61, p<0.01]), and epigenetic regulation ( EZH2 [R 2=0.58, p<0.01]). We then conducted univariate Cox Proportional Hazards analysis to investigate the prognostic significance of clinical and molecular features on time to first treatment (TTFT; most prevalent treatments: splenectomy, n=51; rituximab, n=20) and overall survival (OS). The epiCMIT score and epiCMIT-hyper values showed the highest hazard ratios (HR) for TTFT (HR=26.6, p=0.001) and OS (HR=9.45, p=0.043) respectively ( Fig2). Other factors associated with shorter TTFT included TP53 mutation, 3q gain, IGHV1-2*04 usage, High-M, short telomeres and female gender. Shorter OS was significantly (p<0.05) linked to mutations in key genes (including TP53 and NOTCH2), genomic complexity and del(7q). To avoid redundancy and collinearity, only the most significant epiCMIT metric was included in a multivariate Cox Proportional Hazards model (backwards selection, 94 patients/63 events), and epiCMIT-total emerged as an independent marker for shorter TTFT (HR=44.4, p=0.004), together with gain of chromosome 3q (HR=2.6, p=0.002). Kaplan-Meier analysis further confirmed the importance of epiCMIT-total, with higher scores (>median: 0.73) associated with significantly shorter TTFT (median 23 vs 3.5 months) and higher mortality (p=0.004). To conclude, we demonstrate the potential clinical utility of epiCMIT score in SMZL patients, identifying that SMZL patients with high epiCMIT harbour specific genetic characteristics, and exhibit reduced treatment-free survival. EpiCMIT could be valuable in clinical practice, helping to identify those patients destined to progress and require closer monitoring.

Del(20q) As a Somatic Gene Rescue in Adult Patients: Can Germline Mutations Other Than SBDS be Found in Affected Patients?

Deletions involving chromosome 20 (20q-) are present in up to 5-8% of myeloid neoplasms (MN) and have been associated with either myelodysplastic (MDS) or myeloproliferative (MPN) features, chiefly myelofibrosis. Theoretically, any chromosomal deletion may result in haploinsufficient expression of tumor suppressor genes (TSGs) or in loss of a protective allele in heterozygous carriers of germline (GL) variants or heterozygous somatic (SM) mutations. However, deletions have also been implicated as a maladaptive somatic gene rescue (SGR) event in carriers of GL bone marrow failure (BMF) genes such as SAMD9 and SBDS. The latter is found in Shwachman-Diamond syndrome whereby the upregulated EIF6 gene, responsible for hematopoietic dysfunction, is clonally removed by SM 20q deletion event. We identified an interesting 35-years-old patient with mild cytopenia and 20q- in which a cryptic homozygous SBDS c.A148T GL mutation was found. This prompted us to search in other adult patients with 20q deletion for: i) other cases with SBDS gene alteration (both hetero and homozygous), ii) other GL variants which could be clonally relieved by compensatory 20q- or, iii) disease-prone GL variants on 20q, whereby either disease-prone suppressive allele is included in the deleted region. Within a cohort of 5,308 cases with various MN, 215 patients with 20q- were identified. WES results were available for 350 patients. We applied a stringent bioanalytic algorithm to identify unique GL alterations (not deposited in SM databases) in genes expressed in hematopoietic tissues and involved in BMF and cancer, and a population allelic frequency of <1% which were classified as pathogenic (P), likely pathogenic (LP), and VUS with particular level of clinical suspicion (sVUS). In 76 of 215 patients with 20q-, 346 GL mutations in genes of potential interest were identified and further analyzed. No P/LP variants were detected on genes within commonly deleted region on chr.20q, and no additional carriers of SBDS variants were found. However, we identified 9 pathogenic GL variants in potentially pertinent genes including GL alterations in DDX41, STAG2 and TP53 which have been described in the context of MN. However, these gene variants were also present in patients with diploid 20q. The remaining 6 variants included heterozygous P/LP GL alterations in 3 different Fanconi anemia genes ( FANCA, FANCM, FANCD2), SUZ12, and TINF2. The latter, TINF2, a telosome gene, known to be mutated in dyskeratosis congenita (DC), carried an LOF c.605-1G>A in a patient with MDS/MPN with a family history of cancer who later developed 20q-. The patient did not show any symptoms of DC-like features, which usually present at an early age. Since FANCA LOF with SGR via overepression of EIF6 has previously been described 1, we investigated and did not identify any cases in our cohort of EIF6 SGR resultant overexpression. While occasional patients with no previously diagnosed SBDS can be found among MDS with 20q-, screening for SBDS genes demonstrates that such a genetic configuration among adult patients with 20q is very rare. In addition, only 9 out of 215 patients with 20q- carried other pathogenic GL variants in non-recurrent genes which appear to be likely coincidental. This suggests that del20q is not a SGR in response to a GL mutation, but rather a SGR due to other aforementioned mechanisms, which are currently being explored in our cohort of patients.

Looking ahead: ethical and social challenges of somatic gene therapy for sickle cell disease in Africa.

Somatic gene therapy will be one of the most exciting practices of genetic medicine in Africa and is primed to offer a "new life" for persons living with sickle cell disease (SCD). Recently, successful gene therapy trials for SCD in the USA have sparked a ray of hope within the SCD community in Africa. However, the high cost, estimated to exceed 1.5 million USD, continues to be a major concern for many stakeholders. While affordability is a key global health equity consideration, it is equally important to reflect on other ethical, legal and social issues (ELSIs) that may impact the responsible implementation of gene therapy for SCD in Africa. These include informed consent comprehension, risk of therapeutic misestimation and optimistic bias; priorities for SCD therapy trials; dearth of ethical and regulatory oversight for gene therapy in many African countries; identifying a favourable risk-benefit ratio; criteria for the selection of trial participants; decisional conflict in consent; standards of care; bounded justice; and genetic tourism. Given these ELSIs, we suggest that researchers, pharma, funders, global health agencies, ethics committees, science councils and SCD patient support/advocacy groups shouldwork together to co-develop: (1) patient-centric governance for gene therapy in Africa, (2) public engagement and education materials, and (3) decision making toolkits for trial participants. It is also critical to establish harmonised ethicaland regulatory frameworks for gene therapy in Africa, and for global health agencies to accelerate access to basic care for SCD in Africa, while simultaneously strengthening capacity for gene therapy.

Energetic costs of mounting an immune response in a coral reef damselfish (Pomacentrus amboinensis)

While immune responses can be energetically costly, quantifying these costs is challenging. We tested the metabolic costs of immune activation in damselfish ( Pomacentrus amboinensis Bleeker, 1868) following a mass-adjusted injection of lipopolysaccharide (LPS) endotoxin. Fish were divided into eight treatments: two controls (handling and saline injection) and six LPS groups with concentrations ranging from 3 to 100 mg kg−1. We used intermittent flow respirometry to measure differences in oxygen uptake (delta ṀO2) 20 h before versus 20 h after LPS injection and changes in metabolic traits (lowest, routine, and peak metabolic rates) as proxies of the aerobic costs of metabolism. Spleen somatic index (SSI) and gene expression in spleens were measured to assess immune activation. We found no difference in metabolic traits or SSI but observed different nonlinear patterns of delta ṀO2 in fish exposed to 50 and 100 mg kg−1 LPS compared to lower doses and controls. Fish exposed to high doses of LPS also had lower residual aerobic scope compared to controls and lower LPS doses. Fish exposed to doses of 3, 50, and 100 mg kg−1 showed altered gene expression compared to the handling control. Overall, our results suggest that immune activation has measurable effects on metabolic traits that are both dose- and time-dependent.

Abstract 15180: Inhibition of Tgfβ Signaling Enables Durable Ventricular Pacing by TBX18 Gene Transfer

Introduction: Somatic gene transfer of an embryonic transcription factor, TBX18, could directly reprogram ventricular myocytes to induced pacemaker myocytes. We and others have previously reported that direct myocardial gene delivery of TBX18 created ventricular pacing in heart block animals in vivo. However, the de novo biological pacing was short-lived, diminishing its translational value. Hypothesis: Our preliminary RNAseq and mass spec data indicate pronounced Tgfβ signaling in TBX18-transduced cardiomyocytes. Tgfβ signaling induces fibrosis in multiple organs including the heart. We hypothesized that TBX18 triggers Tgfβ signaling and results in fibrosis, leading to loss of durable ventricular pacing. Methods: Adenoviral vectors expressing TBX18 or GFP was delivered to neonatal rat ventricular myocytes (NRVMs) in vitro or to the left ventricular apex of adult rats with complete atrioventricular block (CAVB). Results: In vitro experiments showed increased population of vimentin+ fibroblasts and alpha smooth muscle actin+ myofibroblasts in TBX18-NRVMs compared to GFP-NRVMs at 2 weeks after gene transfer. Single cell transcriptome data indicated that TBX18 triggered substantial changes in extracellular matrix-related gene expression and Tgfβ signaling. Quantitative RT-PCR and ELISA validated scRNA-seq data, indicating fibroblast activation. TBX18-NRVMs showed faster automaticity than that control did, but their automaticity began wavering after day 7. Treatment of TBX18-NRVMs with a Tgfβ signaling inhibitor, A83-01, mitigated fibroblast activation by TBX18 and preserved their automaticity. Direct delivery of TBX18 into ventricular myocardium resulted in focal fibrosis as well as expansion of vimentin+ fibroblasts at day 7. TBX18 gene transfer initiated ventricular pacing in a rat model of CAVB, but ventricular pacing diminished after one week. In contrast, treating TBX18-injected animals with A83-01 enabled sustained biological pacing beyond three weeks of after gene delivery, accompanied by reduced fibrosis in situ. Conclusions: Inhibition of Tgfβ signaling is sufficient to achieve durable cardiac pacing by TBX18-induced biological pacemakers.

Multi-Dimensional Molecular and Tumor-Microenvironment Analysis of Classic Hodgkin Lymphoma

Introduction Classic Hodgkin Lymphoma (CHL) is currently classified into histological subtypes based on morphology and antigen marker expression. However, additional biological features might help guide treatment strategies and inform prognosis. Here, we aimed to uncover disease heterogeneity and biologic subtypes of CHL based on multi-dimensional molecular profiling ( Fig A) capturing somatic gene mutations, malignant cell expression phenotypes and altered tumor microenvironment (TME) architecture. METHODS We performed exome/targeted sequencing on flow-sorted or micro-dissected HRS cells from fresh-frozen CHL biopsies (n=116) of patients treated at BC Cancer. In addition, we constructed tissue microarrays (TMA) from this cohort on which we performed GeoMx® Whole Transcriptome Assay (WTA) to obtain gene expression profiles from CD30+ HRS cells and multiplexed imaging analyses using multi-colour immunofluorescence (mIF) to delineate the spatial TME ecosystem. Cox regression analysis was performed to identify outcome differences according to molecular features in CHL patients treated with ABVD-like chemotherapy (n=104). Results Mutational and copy number analyses identified known recurrent driver events including mutations and copy number changes in SOCS1, STAT6, TNFAIP3, B2M, REL and the PDL1/PDL2 locus. These mutational findings provided a statistically powered framework to investigate the association between genomic alterations with pathological and clinical disease parameters including outcomes. Mutations affecting BCL7A, TNFAIP3, and 2p15 (REL) amplifications were strongly enriched in EBV- CHL compared to EBV+ CHL. In line with “thymic mutations” reported in grey zone lymphoma, STAT6, GNA13, ITPKB, and TBL1XR1 mutations were significantly enriched in CHL with involvement of the anterior mediastinum. ZNF217, SPEN and CD58 mutations were significantly associated with progression-free survival (PFS) (all P < .01). When investigating outcome correlation according to age group, genomic alteration in STAT6 including both single nucleotide variants and STAT6 amplification, was the most significant feature associated with unfavorable PFS in patients <45 years of age ( P = .013). Alterations in the antigen recognition system, including loss of major histocompatibility complex (MHC) is one of the most studied mechanisms of immune escape in CHL. Mutations in CSF2RB, GNA13, TNFAIP3, and STAT6 were significantly associated with MHC-I loss. Strikingly, all cases harboring CSF2RB mutations (n=23) also showed loss of MHC-I. GeoMx analysis revealed that IFN-a and IFN-g pathways were significantly upregulated in MHC-I+ HRS cells. Mutations in HIST1H1E and BCL7A were significantly enriched in cases with loss of both MHC-I and MHC-II expression. To define mutational subtypes of CHL, we next applied non-negative matrix factorization (NMF) consensus clustering and discovered four robust subsets of tumors (clusters) using recurrent genomic events. Cluster1 (C1) is characterized by mutations in TNFAIP3, CSF2RB and GNA13, younger age, loss of MHC-I, and up-regulation of GCB and fatty acid metabolism signatures. C2 was associated with old age at initial diagnosis, EBV positivity and significant upregulation of the IFN-g pathway. C3 is characterized by REL and STAT6 amplification andexpression of a DNA repair signature. C4 includes samples from mostly younger patients with STAT6 and B2M mutations and high expression of CCL17 by HRS cells. MIF analyses further identified correlations between each mutational NMF cluster and TME composition ( Fig B), and in particular specific CD4+ T cell subsets. The TME of tumors was significantly enriched by C1: FOXP3+Tregs, C2: LAG3+CD4+ cells, and C3: PD1+CD4+ cells. C4 tumors showed no positive correlation with any of these immune cell compartments. C2 tumors were also significantly associated with the abundance of CD68+ macrophages. Conclusion Our multi-dimensional profiling approach enabled us to delineate molecular profiles of HRS cells that are linked to distinct TME patterns. These linkages have implications for current pathogenesis models, molecular subtyping of CHL, and identification of cellular vulnerabilities that might be therapeutically exploitable via targeting of HRS cell phenotypes and/or immune escape mechanisms.

Somatic Genetic Rescue Involving CSF3R and Other Novel Phosphothyrosine Kinase Receptor Mutations Occurring in Myeloid Malignancies

Somatic gene rescue (SGR), via acquisition and selection of somatic (SM) genetic hits, neutralizes functional defects resulting from hypomorphic germline (GL) mutations. However, this process may be maladaptive and lead to the development of leukemia. Acquisition of SM gain-of-function CSF3R mutations in the context of severe congenital neutropenia (SCN) illustrates such a scenario. Here, we further explored this and similar mechanisms in a cohort of 7121 adults with myeloid neoplasia and bone marrow failure (AML=1473, MDS=2248, other=3400). CSF3R mutations were found in 97 patients. In total, there were 37 and 56 patients with SM and GL mutations, respectively; biallelic GL and SM were 4. Among SM alterations, 18 were pathogenic or likely pathogenic including 8 nonsense truncating variants in class III isoform specific region of the cytoplasmic domain and 10 classical missense mutations in the juxtamembrane region, previously defined in the context of post-SCN AML and CNL. We thus hypothesized that these CSF3R SM mutations may also represent SGR in adult patients harboring hypomorphic GL variants and investigated the presence of SCN-associated alterations ( ELANE, WAS, GFI1) and in other phosphothyrosine kinase receptors (PTKR) such as CSF1R, CSF2RA, CSF2RB, FLT3 and KIT. Indeed, we found biallelic WAS p.D264H GL and CSF3R p.A119T SM in a patient with aplastic anemia (AA). Biallelic CSF3R were present in 4 patients: a) p.Q749X GL withp.M696T SM, b) E835K GL with p.T618I SM, c) p.Y752X GL withp.Q754E SM and d) biallelic p.Y752X GL with p.Q754E GL configuration with T615A SM. Compound heterozygous configuration of CSF3R SM included CSF1R SM variants( CSF3R p.M696T SM & CSF1R p.R294Q GL in a 64 year old. patient with MPN, CSF3R p.M696T SM & CSF1R c.1626+3G>A GL in a 61 year old patient with MDS) and CSF2RB ( CSF3R p.T618I SM & CSF2RB p.V890I GL in a 69 year old female patient with AML with leukopenia). When we analyzed the remaining 66 GL CSF3R variants in 56 carriers (in addition to the aforementioned biallelic combinations), we identified 9 compound heterozygous mutants involving CSF1R SM (n=1), CSF2RA (n=1), CSF2RB (n=3), FLT3(TKD/ITD, n=4/1). Specifically, we found the following configurations and disease associations: a) CSF3R p.V372E GLCSF2RB p.P842L SM in a 49 yo. AA patient who subsequently developed AML with FLT3 TKD, b) MDS patients with CSF3R p.E149 GLCSF1R p.G413S SM, c) CSF3R p.R440Q GL& CSF2RA p.R164Q SM and d) CSF3R p.E405K GL& CSF2RB c.1152+6G>A SM and e) AML patients with CSF3R p.E405K GL& CSF2RB p.R432C SM; f) CSF3R p.L619S GLandg,h)2 AML cases with CSF3R p.E808K GL of which all 3 acquired compound heterozygosity with FLT3 TKD, i) CSF3R p.E808K GLCSF3R p.E835K GL in a 71 year old patient with MDS with FLT3ITD AML. In addition to FLT3 GOF mutations, the most common SM hits observed in CSF3RGLcarriers were ASXL1 (n=12), TET2 (n=12), and SF3B1 (n=12). Identification of somatic and GL alterations in PTKR genes other than CSF3R, led us to systematically analyze these genes as targets of GL and SM CSF3R alterations. In total, we identified 72 GL and 73 SM CSF1R variants, 7 SM and 19 GL CSF2RA and 47 GL and 69 SM CSFR2B. Among these, we found 3 patients with compound heterozygous CSF2RBSMCSF3RGL, one with CSF2RBGLCSF3RGLas described above. We also identified one patient with CSF2RASMCSF3RGL. Finally, we identified two patients with CSF1RGL and CSF3RSM, and one with CSF1RSMCSF3RGLmutations. In summary, our study provides evidence for a tremendous complexity of interaction between less penetrant GL alterations of receptor genes and somatic GOF mutations, which may in some instances, correspond to the result of SGR. It is likely that the degree of LOF corresponds to the selection pressure for somatic rescue lesions.

Clonal Hematopoiesis-Related Mutations Are Associated with Favorable Clinical Benefit Following Luspatercept Treatment in Patients with Lower-Risk Myelodysplastic Syndromes: A Subgroup Analysis from the Phase 3 COMMANDS Trial

Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) is a common age-related condition arising from somatic mutations in hematopoietic stem cells that leads to mutant cell clonal progeny in the blood (variant allele frequency [VAF] > 2%) and is found in 10%-30% of people > 70 years of age. CHIP-related mutations (eg, DNA methyltransferase 3 alpha [ DNMT3A], tet methylcytosine dioxygenase 2 [ TET2], ASXL transcriptional regulator 1 [ ASXL1], splicing factor 3b subunit 1 [ SF3B1]) are associated with increased inflammation and elevated risk of developing hematologic malignancies, including myelodysplastic syndromes (MDS)/acute myeloid leukemia, and are an independent risk factor for cardiovascular complications. Recently, patients with MDS were reported to have high rates of cardiovascular co-morbidities including elevated levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), a marker of cardiovascular and all-cause mortality. Currently, no treatments targeting CHIP mutations or associated complications are available. Luspatercept is currently approved for anemia in adult patients with lower-risk MDS (LR-MDS) with ring sideroblasts who require red blood cell (RBC) transfusions (≥ 2 RBC units/8 weeks) after erythropoiesis-stimulating agent (ESA) treatment failure. Recently, the phase 3 COMMANDS trial (NCT03682536) reported superiority of luspatercept over ESAs in reducing transfusion burden in ESA-naive patients with LR-MDS. Aims: We investigated the association between the most frequent CHIP-related mutations ( DNMT3A, TET2, ASXL1, SF3B1) and clinical outcomes in patients treated with luspatercept from the COMMANDS trial. Methods: Genomic DNA was isolated from bone marrow (BM) mononuclear cells, and 36 myeloid-specific somatic gene mutations were identified by targeted next-generation sequencing (400X; sensitivity 3%; Munich Leukemia Laboratory information system) at baseline. Hematologic parameters (eg, hemoglobin [Hb], complete blood counts), transcriptome data (bulk BM RNA sequencing [RNA-Seq] data) and cytokines/chemokines (peripheral blood) were assessed at baseline and week 24. Results: Overall, 145 patients in the luspatercept arm of the COMMANDS trial were included in the current analyses, of whom 85% (123/145) (Figure 1A) had the most frequent CHIP-related mutations (eg, DNMT3A, TET2, ASXL1, and SF3B1) with a VAF of 3%-50%. Modest positive correlations were observed between DNMT3A, TET2 and SF3B1 baseline VAF and monocyte percentage ( P = 0.024, P = 0.015, and P = 0.005, respectively). Baseline Hb levels were lower in patients with frequent CHIP-related mutations than in those with less frequent/other somatic mutations. Luspatercept treatment significantly reduced anemia from baseline in patients with CHIP-related mutations (primary endpoint response 62%; n/N = 76/123). Concomitant with the Hb increase (∆ = +1.0 mg/dL; P < 0.001), we observed improvements in other cell counts, including white blood cell counts (∆ = +0.7 × 10 9 cells/L; P = 0.01). In addition, luspatercept treatment in patients with CHIP-related mutations was associated with downregulation of inflammatory gene signatures (Figure 1B) and proinflammatory cytokines/chemokines, including hepcidin (∆ = −43.9 ng/mL; P < 0.001). Furthermore, we observed upregulation of an anti-inflammatory regulator (growth/differentiation factor 15) following treatment. Most importantly, significant downregulation of NT-proBNP levels (∆ = −330 pg/mL; P < 0.05) was observed in patients with LR-MDS who received luspatercept. Conclusions: This retrospective subgroup analysis of patients in the COMMANDS trial with the most frequent CHIP-related mutations ( DNMT3A, TET2, ASXL1 and SF3B1) demonstrated novel effects of luspatercept in clonal hematopoiesis-related consequences including improvements in anemia, cytopenias, and reduced inflammation. Most importantly, NT-proBNP and elevated hepcidin levels were significantly downregulated in responders following luspatercept treatment. These results warrant evaluation of luspatercept in patients with high-risk CHIP mutations including clonal cytopenia of undetermined significance and patients with anemia of inflammation.