TP53 mutations are found in 10-15% of myeloid neoplasms and are associated with a dismal prognosis. Although hypomethylating agents, such as decitabine, are active in TP53-mutated myeloid neoplasms (TP53-MN), mutation clearance is rarely complete and nearly all patients relapse. Molecular determinants of response to hypomethylating agents in TP53-MN are poorly understood. Here, we show that decitabine induces replicative stress with decreased replication fork progression, induction of single-strand DNA breaks, and activation of the ATR pathway. Resolution of decitabine-induced replication stress is impaired in TP53-mutated acute myeloid leukemia (AML) cells, representing a potential therapeutic vulnerability. Indeed, the combination of decitabine and ATR inhibition (ATRi) induces synthetic lethality that is selective for TP53-AML and due, in part, to induction of mitotic catastrophe. Interestingly, this synergistic lethality was not observed with azacitidine or treatment with GSK3685032, a potent DNMT1 inhibitor, both of which produce a comparable level of global hypomethylation to decitabine. Treatment with decitabine and ATR inhibitor reduces leukemia burden and prolongs survival in in vivo mouse models of TP53-mutated AML. Collectively, these show that TP53 loss generates a selective vulnerability to decitabine-induced replication stress, with the combination of ATR inhibition and decitabine showing promise as a new therapeutic approach for TP53-MN.

BACKGROUND:Optimal consolidation therapy for patients with intermediate-risk acute myeloid leukemia (AML) in first complete remission (CR1) is controversial. Retrospective studies have suggested that the clearance of leukemia-associated mutations (LAMs) in CR1 may predict lower relapse risk and better outcomes with high-dose cytarabine (HiDAC) consolidation. We tested this hypothesis prospectively. METHODS:We performed a phase II, multicenter study of intermediate-risk, transplant-eligible, de novo AML in patients 18-60 years of age who achieved a complete remission (CR) or CR with incomplete count recovery (CRi) after induction therapy. Tumor and normal whole-exome sequencing was performed at presentation to identify somatic LAMs (median ∼30 LAMs/patient). In remission marrow samples, LAM variant allele frequencies (VAFs) were then remeasured using a VAF cutoff of less than 2.5% to define clearance. Patients who met this LAM clearance threshold received HiDAC consolidation, whereas those with persistent LAMs (VAF ≥2.5%) were recommended to undergo allogeneic hematopoietic cell transplantation. The primary endpoint compared relapse-free survival (RFS) of intermediate-risk patients with complete LAM clearance to historical cohorts with intermediate-risk AML who received HiDAC-based regimens in CR1. To account for an unplanned interim assessment, the significance threshold for the primary analysis was 0.01. RESULTS:Among 100 patients who were evaluated, intermediate-risk patients who cleared all LAMs in CR1 (n=33) had a median RFS of 33.1 months (95% confidence interval, 11.7-NA) compared to a median RFS of 11.7 months in the historical cohort (n=239; 95% confidence interval, 9.9-15.6, P=0.015). CONCLUSIONS:Among patients with intermediate-risk AML, clearance of LAMs after induction, followed by HiDAC consolidation in CR1, was associated with longer RFS compared with similarly treated historical controls. Although this result did not meet the prespecified threshold for statistical significance, the reported association sets the stage for a randomized trial to further evaluate this strategy. (ClinicalTrials.gov number, NCT02756962.).

Abstract TP53 mutations are found in 10% to 15% of myeloid neoplasms and are one of its most important prognostic factors. Emerging data show that TP53 mutational allele status is a key determinant of clinical outcomes, with multihit TP53 mutant myeloid neoplasms having a very poor prognosis. Significant differences exist among the methods used in clinical and research settings to assess TP53 mutational status, leading to variability in reported patient characteristics, response to therapy, and survival. Indeed, differences in the criteria used to define TP53 mutational states among professional societies and in landmark research studies have led to confusion, suboptimal clinical testing, and variability in therapy recommendations. We review the methods used to assess for TP53 mutational allele status and provide recommendations, based on clinically available testing, for the accurate evaluation of TP53 gene mutations in myeloid neoplasms. Hotspot mutations represent ∼35% of all TP53 missense mutations in myeloid neoplasms. There is evidence that these hotspot mutations may have dominant-negative or gain-of-function properties. Here, we review this evidence and discuss the potential impact of TP53 mutation identity on patient outcomes and clinical management.

Whole-genome sequencing (WGS) is a comprehensive approach for the genomic evaluation of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). We recently described a streamlined tumor-only WGS assay (ChromoSeq) that uses Illumina short-read sequencing with targeted analysis to detect the full range of clinically relevant somatic mutations. Here we sought to determine the performance of this targeted analysis approach using long-read sequencing data from Oxford Nanopore Technologies and Pacific Biosciences. Samples from 26 patients with AML and MDS were sequenced to a mean of 52× coverage. Head-to-head comparison of reportable somatic variants to standard WGS revealed more than 96% recall and 91% precision for single nucleotide variants for both long-read platforms. Performance was lower for insertion/deletions (66% recall and 42% precision), especially in regions with few phased reads that facilitate accurate variant detection. The long-read platforms were 95% accurate for copy number calls, and they detected all recurrent structural variants with no false-positive findings. In addition, long reads properly identified intronic insertions near repetitive elements that were incorrectly identified as interchromosomal structural rearrangements by standard WGS. These results indicate that targeted, tumor-only analysis of long-read sequence data is a feasible approach for the genomic evaluation of myeloid cancers, and they show the utility of incorporating variants discovered via long-read sequencing to improve variant interpretation in short-read WGS.

Telomere biology disorders (TBDs) are genetic diseases caused by defective telomere maintenance. TBD patients often develop bone marrow failure and have an increased risk of myeloid neoplasms. To better understand the factors underlying hematopoietic outcomes in TBD, we comprehensively evaluated acquired genetic alterations in hematopoietic cells from 166 pediatric and adult TBD patients. Of these patients, 47.6% (28.8% of children, 56.1% of adults) had clonal hematopoiesis. Recurrent somatic alterations involved telomere maintenance genes (7.6%), spliceosome genes (10.4%, mainly U2AF1 p.S34), and chromosomal alterations (20.2%), including 1q gain (5.9%). Somatic variants affecting the DNA damage response (DDR) were identified in 21.5% of patients, including 20 presumed loss-of-function variants in ataxiatelangiectasia mutated (ATM). Using multimodal approaches, including single-cell sequencing, assays of ATM activation, telomere dysfunction-induced foci analysis, and cell-growth assays, we demonstrate telomere dysfunction-induced activation of the ATM-dependent DDR pathway with increased senescence and apoptosis in TBD patient cells. Pharmacologic ATM inhibition, modeling the effects of somatic ATM variants, selectively improved TBD cell fitness by allowing cells to bypass DDR-mediated senescence without detectably inducing chromosomal instability. Our results indicate that ATM-dependent DDR induced by telomere dysfunction is a key contributor to TBD pathogenesis and suggest dampening hyperactive ATM-dependent DDR as a potential therapeutic intervention.

e15017 Background: Poly (ADP-ribose) polymerase inhibitors (PARPi) have become a critical part of treatment for multiple solid tumors, especially among patients with germline homologous recombination deficiency (HRD). With their significant impact on survival has come a concern for therapy-related myeloid neoplasms (tMN). DNA damage response (DDR) mutations causing clonal hematopoiesis (CH) are the driving force behind the tMNs. We hypothesized that platinum and PARPi therapy have significant variation in their impact on CH, further altered by the presence of HRD. Methods: Serial blood samples from 250 patients with a variety of cancers, primarily prostate, ovarian, and lung cancer treated with PARPi (n = 100) or carboplatin (n = 150) were compared to 176 age-matched patients who were not treated for cancer. Samples were sequenced at an average depth of 19,964x using a sequencing assay that captured the exonic regions of nine common CH genes ( DNMT3A, TET2, ASXL1, TP53, CHEK2 , JAK2, SRSF2, SF3B1, PPM1D ). Mixed bone marrow chimeric mice were created by competitive transplant of Trp53 R172H/+ cells—the murine equivalent of the R175H TP53 hotspot mutation seen in myeloid neoplasms— against wild-type cells. Mice were treated with vehicle (n = 19), cisplatin (n = 16), olaparib (n = 9), or talazoparib (n = 14) for 4 weeks before sequential bleeding and bone marrow collection. Results: The average length of carboplatin treatment was 5.6 months (range 1.15-40.8 months) and 8.76 months (range 1.78-44.4 months) for PARPi treatment. Among patients treated with either carboplatin or PARPi, 51% had on average two DDR CH mutations (range 0-19) versus 23% of untreated patients. Carboplatin induced significantly greater growth of DDR CH compared to PARPi (p = 2.6e-2). Expansion of DDR CH mutations during treatment was significantly reduced in patients with a germline HRD pathogenic variant (N = 44) as compared to those without (N = 276, p < 0.001). No significant expansion of DDR CH mutations was observed after either PARPi (p = 3.9e-2) or carboplatin exposure (p = 1.4e-2) in patients with a germline HRD pathogenic variant. Similarly, within our murine model we observed that cisplatin (p = 5.5e-11) and to a lesser extent talazoparib (p = 0.15) increased Trp53-mutant blood leukocytes. However, the competitive advantage of Trp53-mutant cells during treatment with both cisplatin and talazoparib was lost among mice with hematopoietic-specific Brca1 heterozygote mutations. Conclusions: We observed a high frequency of DDR CH expansion after carboplatin and to a lesser extent PARPi treatment. This expansion was largely abrogated in patients with germline HRD. We validated these findings in a mouse model of Trp53-mutated CH. Our findings suggest that the increased risk of tMN following PARPi is likely influenced by prior exposure to other oncologic therapies, including platinum therapy, and may be modified by HRD status.

Hematologic malignancies, such as multiple myeloma, exhibit a markedly increased incidence with age and are profoundly influenced by the bone marrow microenvironment. The aged microenvironment can lead to the accumulation of senescent cells, characterized by permanent cell cycle arrest and a pro-inflammatory secretory phenotype. Despite its significance in age-related diseases, the role of senescent cells in the bone marrow microenvironment and their impact on aging and hematologic malignancies remain poorly understood.As part of the Cellular Senescence Network (SenNet), we created a multimodal atlas to characterize senescent cells in the human bone marrow using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics. Analysis of 55 scRNA-seq samples from healthy donors (aged 25-84) revealed that mesenchymal cells exhibited the strongest senescence signatures, which were elevated in aged individuals. While monocytes also displayed senescence signatures, they did not show an age-associated increase. Hematopoietic stem cell (HSC) subpopulations showed senescent profiles, suggesting that senescence spans multiple cell types in the bone marrow.Using the Xenium spatial transcriptomics platform, we profiled the bone marrow microenvironment in 17 samples from 15 healthy donors across a similar age range. We identified 32 distinct cell types and uncovered putative senescent subpopulations in plasma cells and vascular smooth muscle cells. Our analysis revealed 9 unique cellular neighborhoods, some anchored around established niches like the endosteum and arteriole, while others suggested novel niche candidates such as adipocytes, early myeloid progenitors, and megakaryocytes. Aging was associated with significant microenvironment remodeling, including increased mature myeloid cell types and lymphoid aggregates, along with a decline in lymphoid and progenitor populations.In 22 multiple myeloma samples from 15 untreated and 4 relapsed patients, we found significant microenvironment changes, such as the formation of plasma cell-rich pockets. These neighborhoods were enriched with monocytes and CD8 T cells, the latter showing elevated expression of exhaustion markers. In agreement with our neighborhood analysis, monocytes and CD8 T cells were found in closer proximity to plasma cells in samples with high tumor burden. Mesenchymal stem cells near plasma cells also showed increased senescence signatures compared to age-matched controls.Overall, our findings illuminate the senescent landscape of the bone marrow and provide a resource for understanding its role in aging and multiple myeloma. Wen-hung Chou, Julia T. Wang, Xiang Li, Kapur B. Dhami, Daniel Rapp, André Luiz N. Targino da Costa, Andrew Houston, Xiyi Wei, Yuting Zhao, Maede Shahin, Yizhe Song, Xiangwei Fang, Julie Fortier, Yuwei Zhang, Matthew Wyczalkowski, Reyka G. Jayasinghe, Kelsey Gallant, Michael Slade, Karolyn A. Oetjen, Kiran Vij, Daniel C. Link, Ryan Nunley, Ravi Vij, Feng Chen, Ryan C. Fields, Li Ding. Multimodal atlas of cellular senescence in human bone marrow reveals insights into aging and multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2555.

Two complementary studies used whole-genome sequencing of single cell-derived hematopoietic colonies to show that chemotherapy results in a marked decrease in hematopoietic stem and progenitor cell (HSPC) diversity, with the parallel evolution of multiple independent HSPCs harboring mutations in DNA damage response genes.

AbstractPurpose: Clonal hematopoiesis (CH) is thought to be the origin of myeloid neoplasms (MN). Yet, our understanding of the mechanisms driving CH progression to MN and clinical risk prediction of MN remains limited. The human proteome reflects complex interactions between genetic and epigenetic regulation of biological systems. We hypothesized that the plasma proteome might predict MN risk and inform our understanding of the mechanisms promoting MN development. Experimental Design: We jointly characterized CH and plasma proteomic profiles of 46,237 individuals in the UK Biobank at baseline study entry. During 500,036 person-years of follow-up, 115 individuals developed MN. Cox proportional hazard regression was used to test for an association between plasma protein levels and MN risk. Results: We identified 115 proteins associated with MN risk, of which 30% (N = 34) were also associated with CH. These were enriched for known regulators of the innate and adaptive immune system. Plasma proteomics improved the prediction of MN risk (AUC = 0.85; P = 5×10–9) beyond clinical factors and CH (AUC = 0.80). In an independent group (N = 381,485), we used inherited polygenic risk scores (PRS) for plasma protein levels to validate the relevance of these proteins toMNdevelopment. PRS analyses suggest that most MN-associated proteins we identified are not directly causally linked toMN risk, but rather represent downstream markers of pathways regulating the progression of CH to MN. Conclusions: These data highlight the role of immune cell regulation in the progression of CH to MN and the promise of leveraging multi-omic characterization of CH to improveMN risk stratification. See related commentary by Bhalgat and Taylor, p. 3095

Association between plasma protein levels and individual clonal hematopoiesis (CH) genes with frequency greater than 20 (N=13 genes).

Association between plasma protein levels and biologically informed categories of clonal hematopoiesis (CH) genes.

Context Previous studies have linked poly(ADP-ribose) polymerase inhibitors (PARPi) and platinum-based agents to therapy-related myeloid neoplasia. However, interpretation of these findings is limited by confounding variables, including germline status and prior therapy. We hypothesized that exposure to PARPi confers a competitive advantage to hematopoietic stem/progenitor cells (HSPCs) containing mutations in DNA damage response (DDR) genes. Moreover, in patients with germline mutations in HRD genes, genotoxic stress induced by PARPi may be increased, further enhancing the fitness of HSPCs carrying DDR gene mutations. Methods We performed longitudinal studies on peripheral blood samples collected from 418 patients before and after treatment with PARPi or carboplatin. We also developed a chimeric mouse model of Trp53-mutated CH. The percentage of Trp53-mutant cells was measured by flow cytometry before and after treatment with vehicle, talazoparib, olaparib, or cisplatin. Results In patients, both carboplatin and PARPi treatment significantly increased the growth of CH mutations, driven almost entirely by mutations in DDR genes. The strongest PARP-trapping member, talazoparib, had the largest effect on the growth rate of CH mutations in DDR genes but was lower than carboplatin. Surprisingly, DDR-CH expansion was largely abrogated in patients harboring a germline HRD mutation, for both carboplatin and PARPi. In the mouse model, treatment with cisplatin resulted in a significant expansion of Trp53-mutated leukocytes and HSPCs. A smaller but significant increase was also observed after talazoparib but not olaparib. We next modeled the effect of germline HRD mutations in mice containing Brca1+/- bone marrow competed with Brca1+/-/Trp53-mutant bone marrow. Consistent with the human CH data, no expansion of Trp53-mutated leukocytes or HSPCs was observed after treatment with cisplatin or talazoparib. Conclusion Collectively, these data support the hypothesis that mutations in DDR genes provide a fitness advantage to HSPCs following PARPi and platinum therapy. However, PARPi shows less selective pressure on DDR CH than platinum. Importantly, these data strongly argue that germline HRD variants do not enhance this fitness advantage. PARPi therapy may have less of an impact on leukemia risk compared to carboplatin and may synergize with HRD in blocking the competitive advantage of DDR CH during genotoxic stress.

Significantly enriched Reactome gene sets after multiple hypothesis testing correction (pFDR <= 0.05).

MOTIVATION:The acquisition of somatic mutations in hematopoietic stem and progenitor stem cells with resultant clonal expansion, termed clonal hematopoiesis (CH), is associated with increased risk of hematologic malignancies and other adverse outcomes. CH is generally present at low allelic fractions, but clonal expansion and acquisition of additional mutations leads to hematologic cancers in a small proportion of individuals. With high depth and high sensitivity sequencing, CH can be detected in most adults and its clonal trajectory mapped over time. However, accurate CH variant calling is challenging due to the difficulty in distinguishing low frequency CH mutations from sequencing artifacts. The lack of well-validated bioinformatic pipelines for CH calling may contribute to lack of reproducibility in studies of CH. RESULTS:Here, we developed ArCH, an Artifact filtering Clonal Hematopoiesis variant calling pipeline for detecting single nucleotide variants and short insertions/deletions by combining the output of four variant calling tools and filtering based on variant characteristics and sequencing error rate estimation. ArCH is an end-to-end cloud-based pipeline optimized to accept a variety of inputs with customizable parameters adaptable to multiple sequencing technologies, research questions, and datasets. Using deep targeted sequencing data generated from six acute myeloid leukemia patient tumor: normal dilutions, 31 blood samples with orthogonal validation, and 26 blood samples with technical replicates, we show that ArCH improves the sensitivity and positive predictive value of CH variant detection at low allele frequencies compared to standard application of commonly used variant calling approaches. AVAILABILITY AND IMPLEMENTATION:The code for this workflow is available at: https://github.com/kbolton-lab/ArCH.

Association between plasma proteins and common CH genes.

Somatic mutations arising in hematopoietic stem cells (HSCs) may provide the latter with a fitness advantage, allowing the mutant HSC to clonally expand. Such mutations have been recurrently identified in the chromatin modifier, SRCAP, in both non-malignant and leukemic clones, suggesting that this gene plays a significant role in hematopoiesis. We generated a conditional Srcap loss of function murine model and determined the consequences of hematopoietic-specific loss of this gene. We show that Srcap is essential for normal fetal liver erythropoiesis and monocytopoiesis. In Srcap deficient fetal livers, the number of phenotypic HSCs is similar to that of controls, but these HSCs exhibit a profound repopulating defect. Likewise, conditional deletion of Srcap during adult hematopoiesis results in a rapid loss of HSCs. Loss of Srcap is associated with evidence of increased DNA damage in HSCs and lineage-restricted progenitors as assessed by y-H2AX expression. Consistent with this finding, we observed strong transcriptional upregulation of the p53 pathway in Srcap deficient erythroid precursors. Collectively our data highlight the importance of Srcap in maintaining HSC function and supporting hematopoietic differentiation and suggests that it plays an essential role in maintaining genomic integrity.

Myeloproliferative neoplasms (MPNs) are characterized by recurrent driver mutations in JAK2, CALR and MPL, with JAK2 being the most frequently enriched in patients with polycythemia vera. There is an expansion of often dysmorphic megakaryocytes, which occurs with disease progression to myelofibrosis. This is of potential biological and clinical significance, since megakaryocytes are considered an important component of the stem cell niche producing key niche factors, such as TGF-β and CXCL4. However, the mechanisms mediating megakaryocyte expansion and the impact of mutant JAK2 on megakaryocyte gene expression are not fully understood. To address these issues, we established bone marrow chimeras by transplanting congenic wildtype and inducible Jak2V617F cells into irradiated recipients; the wildtype cells included a GFP transgene to distinguish wildtype from Jak2V617F cells. Specifically, a 5:1 ratio of wildtype to Jak2V617F cells was used and tamoxifen was given 4 weeks after transplantation to induce UBC-ERT2-Cre, and thereby activate the Jak2V617F allele, and mice were analyzed 12 weeks later. Whereas the contribution of Jak2V617F cells to hematopoietic stem cells and myeloid progenitors increased modestly (~2-fold) from input, a marked (~5-fold) expansion of Jak2V617F megakaryocytic-erythroid progenitors (MEPs) was observed, suggesting that Jak2V617F induces a selective cell-intrinsic expansion of MEPs. Single cell sequencing of sorted Kit+ lineage- Sca+ cells from these mice was performed (n = 4) using the 10X Genomics platform: this experimental approach allowed us to compare MEP transcriptomes under a shared altered (bone marrow microenvironment. We identified 148 differentially expressed genes that were enriched for cell cycling genes. Enriched Hallmark gene expression signatures included Myc and E2F targets and MTORC1 signaling. To address the impact of Jak2V617F on megakaryocyte gene expression, we performed scRNA sequencing of enriched megakaryocyte populations from wildtype or Jak2V617F mice (n = 3, each). In brief, wildtype or Jak2V617F mice (8-10 weeks old) were treated with polyinosinic:polycytidylic acid to induce Jak2V617F expression. Six weeks later, bone marrow was harvested and megakaryocytes enriched by bovine serum albumin gradient followed by CD61-positive magnetic bead selection. Single cell RNA sequencing was then performed using the 10X Genomics platform. Megakaryocytes were identified by common protein identifiers (Pf4, Vwf, Cd34, Gp1ba and Mpl) as well as unbiased cell identifiers from the literature. In total, 1,228 differentially expressed genes were identified in Jak2V617F megakaryocytes. The top most enriched gene expression signature was Myc targets (FDR = 0). Consistent with this, we identified increased Myc mRNA expression in Jak2V617F megakaryocytes (mean 0.67 vs 0.28, adjusted p-value = 3.77E-16.). We also observed significant enrichment for TNF-a/NF-kB, p53 and JAK/STAT signaling pathways. Megakaryocytes are a major source of important stem cell niche factors, including TGF-β, CXCL4, IL6, IL12, CCL3 and others). We observed increase in the number TGF- β transcripts (adjusted p-value = 2.14E-7) in megakaryocytes, which colocalized with mature megakaryocytes (expressing high Gp1ba/Gp1bb/Vwf) and was in contrast to higher Myc expression in the megakaryocyte precursors (expressing high Kit/Cd34.) Altogether, these data show that Jak2V617F expression results in a marked cell-intrinsic expansion of MEPs and the production of atypical megakaryocytes marked by increased Myc expression and expression of selected stem cell niche genes.