Mutations In Hematopoietic Stem Cells Research Articles

Increased clonal hematopoiesis mutational burden and accelerated dynamic evolution of circulating clones in patients with ischemic heart failure

Abstract Background Clonal hematopoiesis (CH) - defined as the age-related acquisition of mutations in hematopoietic stem cells giving rise to mutated circulating leukocytes - is associated with increased risk of death in patients with heart failure with reduced ejection fraction (HFrEF). While the mutational landscape of CH in peripheral blood (PB) of patients with HFrEF is well characterized, the mutational burden in bone marrow (BM) samples of patients with HFrEF as well as its dynamics over time have not been investigated. Methods/Results We analyzed matched BM and PB cells from 68 patients with ischemic HFrEF by deep error-corrected targeted sequencing of 56 CH-driver genes with a detection threshold of VAF ≥0.5%. In PB, 56/68 patients carried at least one CH mutation, whereas 63/68 patients had mutations in BM. The absolute numbers of detectable mutations were significantly higher in BM compared to PB (215 mutations vs 120 mutations; p<0.0001) (Fig. 1A). Likewise, the cumulative VAF was significantly higher in BM compared to PB (3.83 +/- 0.5% vs 3.0% +/-0.7%; mean +/- SEM, p = 0.0025) (Fig. 1B). However, there was a strong correlation between mutation load in PB and BM (r = 0.54, p<0.0001). Among the two most common mutations, DNMT3A mutations were either present in BM only or both compartments (PB only/BM only/ both compartments 2/12/10), whereas TET2 mutations were strikingly enriched in BM cells (PB only/BM only/both compartments 0/16/2). To study the dynamic evolution of CH clones in patients with HFrEF, we serially analyzed PB samples from 19 patients. At baseline, 16/19 patients carried at least one mutation in a CH driver gene and a total of 31 mutations. After a median follow-up of 12 months, 19/19 patients had at least one detectable mutation and a total of 43 mutations were identified (mean number of mutations per patient 1.63 +/- 0.033 vs 2,26 +/- 0.27; mean +/- SEM, p = 0,0410), indicating an increased mutation load over time (Fig. 2A). Moreover, cumulative VAF increased from 1.59 +/-0.36 % to 2.01 +/- 0.38 %). Conclusion Patients with ischemic HFrEF display a high burden of CH mutations compared to values reported for healthy individuals, which is further amplified in the bone marrow compartment. Clones with new mutations arise rapidly in longitudinal samples of patients with HFrEF and clonal expansion occurs with high momentum. The overrepresentation of mutations in the BM is indicative for augmented DNA damage in the hematopoietic stem cells of patients with HFrEF, while the rapid expansion of mutated circulating clones over time suggests that heart failure itself may expedite the expansion of hematopoietic cells harboring CH-driver mutations.

Identification of cell-intrinsic effects of mutated circulating cells due to clonal hematopoiesis in patients with heart failure

Abstract Clonal hematopoiesis of indeterminate potential (CHIP) is caused by somatic mutations in hematopoietic stem cells and is associated with worse prognosis in patients with heart failure with reduced ejection fraction (HFrEF). Circulating immune cells derived from patients with HFrEF harboring CHIP mutations show enhanced proinflammatory signatures. However, it is unclear, whether these signatures are derived from the relatively low number of cells harboring CHIP-driver mutations, or are indicators of a systemic pro-inflammatory activation associated with CHIP. Methods and Results We assessed the cell-intrinsic effects of the most prevalent CHIP-driver gene (DNMT3A) mutant-carrying circulating immune cells in a total of 5 patients with HFrEF. Using an improved protocol to detect mutant cells on a single cell level (MutDetect-Seq), as illustrated in figure 1A, we show that CHIP mutations can be detected in all major immune cell types (e.g. T cells, monocytes, B cells, NK cells). DNMT3A mutant monocytes demonstrated the highest number of significantly upregulated genes relative to other cell types. Specifically, DNMT3A mutant monocytes exhibit altered gene expression profiles associated with inflammation and phagolysosome function (figure 1B). In addition, DNMT3A mutant carrying CD4+, but not CD8+ T cells, showed significant changes in gene expression patterns towards effector cells and away from a naïve T cell state. Finally, increased paracrine signaling pathways involved in amplifying inflammatory circuits are predicted between mutated and non-mutated monocytes. Conclusion This is the first study to decipher the genetic signature of DNMT3A-CHIP-driver mutation carrying circulating immune cells, thereby providing first evidence for the cell intrinsic effects of DNMT3A mutations on peripheral blood cell subsets in patients with HFrEF. The altered gene expression profiles associated with inflammation and phagolysosome function of mutant monocyte as well as the skewing of mutated T cells towards effector cells provide novel insights into how CHIP might promote worse prognosis in heart failure patients.MutDetect-seq

Mitotic Dysregulation Sensitizes Malignant Stem Cells to CHK1 Inhibition in SF3B1-Mutant Myeloid Neoplasms

Myelodysplastic syndromes (MDS) arise from the acquisition of driver mutations in hematopoietic stem cells (HSCs). Splicing factor SF3B1 mutations are recurrent initiating lesions, found in up to 30% of patients with MDS. SF3B1 mutations are associated with aberrant splicing marked by recognition of alternative 3' splice sites (a3'ss). However, the causal role of mis-spliced genes in disease pathogenesis remains largely unexplored. Although SF3B1 mutations and downstream mis-spliced genes provide attractive therapeutic targets, targeted therapies remain unavailable. Here, we developed a novel method of precise gene editing of CD34 + hematopoietic stem and progenitor cells (HSPCs) to introduce an SF3B1 K700E mutation at the endogenous locus, enabling stoichiometric expression of mutant protein. An intronic fluorescent marker was introduced upstream of the mutation for isolation of edited cells. Global splicing analysis in SF3B1 K700E knock-in HSPCs recapitulated canonical targets identified in SF3B1-mutant (SF3B1m) MDS patients. Annotation of a3'ss mis-spliced genes showed profound enrichment of gene ontology (GO) terms related to cell cycle regulation, mitotic checkpoint signaling, and chromosome segregation. Consistently, cell cycle analysis of SF3B1m HSCs revealed accumulation of cells in G2/M phase, suggesting a previously unidentified stall in G2/M progression driven by SF3B1 mutations. To identify mis-spliced genes that cause this G2/M delay, we compared a3'ss mis-spliced genes in the cell cycle GO category across multiple model systems. BUBR1 and CDC27 were consistently mis-spliced in all datasets, resulting in reduced mRNA and protein levels. BUBR1 is a mitotic kinase involved in the spindle assembly checkpoint and chromosome alignment, and CDC27 is a component of the anaphase-promoting complex. Knockdown of BUBR1 or CDC27 in normal CD34 + HSPCs recapitulated G2/M delay. BUBR1 and CDC27 are thus attractive therapeutic targets to impair cell cycle progression of SF3B1m cells. While there are no direct inhibitors of BUBR1 kinase, BUBR1 is phosphorylated by CHK1 during mitosis, and its expression is highly correlated with response to a selective CHK1 inhibitor prexasertib in a large panel of cancer cell lines (Blosser et al., Oncotarget 2020). Indeed, SF3B1m K562 cells and CD34 + HSPCs were highly sensitized to prexasertib. Knockdown of BUBR1 or CDC27 in wild-type cells induced CHK1 phosphorylation, especially upon prexasertib treatment, and sensitized normal cells to prexasertib. These data indicate that mis-splicing of BUBR1 or CDC27 delays G2/M progression leading to CHK1 activation, which sensitizes SF3B1m cells to prexasertib. To determine if CHK1 inhibition can eradicate SF3B1m HSCs, we leveraged our gene editing system to test drug responses in CD34 +CD133 +CD38 - primary immunophenotypic HSCs. Prexasertib selectively eliminated SF3B1m HSCs, including high-risk SF3B1/ RUNX1 and SF3B1/ STAG2 genotypes, while control AAVS1-edited HSCs were maintained. Furthermore, prexasertib depleted CD34 + cells in primary SF3B1m MDS patient samples. To test prexasertib efficiency in vivo, we transplanted NSGS mice with iPSC-derived HSPCs reprogrammed from a patient with high-risk MDS and SF3B1/ RUNX1 co-mutations, and treated with a standard preclinical regimen. While all the vehicle-treated mice showed consistent engraftment, none of the prexasertib-treated mice were engrafted. Together, these data indicate that prexasertib selectively targets SF3B1m HSCs in vitro and eradicates SF3B1m MDS in vivo. In conclusion, we developed a precise gene editing strategy of human HSCs to identify prexasertib as a promising therapy for SF3B1m myeloid neoplasms, and implicate the mitotic function of CHK1 as a SF3B1m sensitivity. The safety and toxicity profiles displayed in early phase clinical trials make prexasertib a suitable agent for further clinical investigation in SF3B1m MDS and its advanced stages.

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.

STAG2 loss Induces HSC Programs By Modulating Accessibility of AP-1 Bound Enhancers

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are blood disorders characterized by clonal expansion of mutant hematopoietic stem and progenitor cells (HSPCs). Mutations in genes encoding the cohesin complex, including STAG2, RAD21, SMC3 and SMC1A, are frequent genetic drivers in MDS and AML. However, the mechanisms by which cohesin mutations lead to clonal expansion of HSPCs are not well understood. Cohesin is essential for sister chromatid cohesion, DNA damage repair, maintenance of chromatin architecture, and gene regulation. To investigate the impact of cohesin mutations on chromatin accessibility, we performed ATAC-Seq in a panel of six isogenic STAG2 wild type (WT) and knockout (KO) U937 leukemia cell lines. Among the total of 97,798 ATAC-Seq peaks, we identified significantly more STAG2 KO (8,859) versus WT (1,622) uniquely accessible chromatin sites, which were strongly enriched for intergenic regions. Integration of ChIP-Seq profiling of cohesin binding and histone modifications demonstrated cohesin binding and an increase in the active enhancer and promoter histone mark H3K27Ac in the STAG2 KO newly accessible chromatin, suggesting a direct role for cohesin in regulating the activity of such enhancers. Together, our data demonstrate that STAG2 KO-induced open chromatin may be enriched for novel STAG2 KO-specific enhancers. To further infer the regulatory function of these putative enhancers, we predicted binding of transcription factors (TFs) in ATAC-Seq-defined regions by performing TF footprinting using the Transcription factor Occupancy Prediction By Investigation of ATAC-seq Signal (TOBIAS) algorithm. We observed that binding of members of the AP-1 transcription family, including JUN FOS andATF, previously implicated in oncogenic transformation, was significantly enriched in the STAG2 KO cells (Figure 1A). Conversely, STAG2 KO cells lost CEBP TF binding in regions that lost chromatin accessibility. To identify potential regulatory targets of these TFs, we mapped TF footprints to annotated promoter regions and to enhancer-gene pairs determined by the Activity by Contact (ABC) model. We observed a strong correlation between predicted AP-1 binding and gene expression of putative regulated genes in STAG2 KO cells, further supporting the role of these newly accessible AP-1 bound sites as regulatory elements. To validate our findings using in vivo models of cohesin-mutant disease, we performed RNA-Seq and ATAC-Seq in HSPC isolated from a Tet2/Stag2 mouse model of MDS previously developed in our lab. We confirmed an increase in accessible chromatin and demonstrated a strong enrichment of predicted Ap-1 binding in intergenic regions, in agreement with our observations in the U937 model. Gene set enrichment analysis in the Tet2/Stag2-mutant versus WT HSPC revealed that putative targets of Ap-1 binding were enriched for hematopoietic stem cell gene signatures (Figure 1B) . This is consistent with our observation of clonal expansion of HSPCs in this model, and suggests a mechanism by which novel Ap-1 bound enhancers may facilitate expression of key genes involved in HSPC expansion and development of MDS/AML. To explore whether our findings were applicable to other published models of Stag2 mutant myeloid neoplasms, we performed TF footprinting using publicly available ATAC-Seq datasets generated in HSPC isolated from Stag2 and Runx1/Stag2 conditional knockout mice. We recapitulated increased chromatin accessibility and significant enrichment of cohesin-bound Ap-1 footprints in Stag2 mutant over WT conditions. Together, these data further validate conservation of STAG2 KO-induced AP-1 bound regulatory elements in human and mouse models of MDS and AML. In summary, we show that STAG2 loss leads to the establishment of a novel set of accessible intergenic regions with features of enhancer activity in vitro and in vivo. We identify multiple members of the AP-1 complex with enriched predicted binding at these putative enhancers, and demonstrate that AP-1 may drive expression of HSPC signatures in models of Stag2 mutant MDS in vivo. We illustrate here a potential role for the AP-1 complex in cohesin mutant myeloid malignancies, which we are currently functionally interrogating.

A Review of Immune Landscape of Clonal Hematopoiesis: Progression and Prospects for the Future

Context: Clonal hematopoiesis (CH), characterized by the clonal proliferation of mutant hematopoietic cells, is frequently observed in older individuals. It is widely recognized that ageing leads to somatic mutations in hematopoietic stem cells, increasing the risk of developing various diseases later in life. However, the impact of these clonal hematopoiesis-related mutations on the activation and regulation of the immune response remains uncertain. Evidence acquisition: To address this knowledge gap, a thorough review of the existing literature on clonal hematopoiesis and immune dysregulation was conducted. Relevant studies were systematically searched and their key findings were carefully analyzed to provide a comprehensive overview. Results: The review shows that mutations associated with clonal hematopoiesis have a significant impact on the modulation of immune responses in the body. These mutations are found to contribute to immune dysregulation, which has important implications for disease progression in the elderly. Conclusion: This review highlights the importance of studying immune dysregulation in the context of clonal hematopoiesis. By unravelling the relationship between CH-related mutations and immune responses, we can improve our understanding of disease progression in the elderly and identify promising therapeutic targets. This knowledge can inform the development of innovative interventions for improved disease management.

Clonal Hematopoiesis of Indeterminate Potential (CHIP) and Incident Type 2 Diabetes Risk.

Clonal hematopoiesis of indeterminate potential (CHIP) is an aging-related accumulation of somatic mutations in hematopoietic stem cells, leading to clonal expansion. CHIP presence has been implicated in atherosclerotic coronary heart disease (CHD) and all-cause mortality, but its association with incident type 2 diabetes (T2D) is unknown. We hypothesized that CHIP is associated with elevated risk of T2D. CHIP was derived from whole-genome sequencing of blood DNA in the National Heart, Lung, and Blood Institute Trans-Omics for Precision Medicine (TOPMed) prospective cohorts. We performed analysis for 17,637 participants from six cohorts, without prior T2D, cardiovascular disease, or cancer. We evaluated baseline CHIP versus no CHIP prevalence with incident T2D, including associations with DNMT3A, TET2, ASXL1, JAK2, and TP53 variants. We estimated multivariable-adjusted hazard ratios (HRs) and 95% CIs with adjustment for age, sex, BMI, smoking, alcohol, education, self-reported race/ethnicity, and combined cohorts' estimates via fixed-effects meta-analysis. Mean (SD) age was 63.4 (11.5) years, 76% were female, and CHIP prevalence was 6.0% (n = 1,055) at baseline. T2D was diagnosed in n = 2,467 over mean follow-up of 9.8 years. Participants with CHIP had 23% (CI 1.04, 1.45) higher risk of T2D than those with no CHIP. Specifically, higher risk was for TET2 (HR 1.48; CI 1.05, 2.08) and ASXL1 (HR 1.76; CI 1.03, 2.99) mutations; DNMT3A was nonsignificant (HR 1.15; CI 0.93, 1.43). Statistical power was limited for JAK2 and TP53 analyses. CHIP was associated with higher incidence of T2D. CHIP mutations located on genes implicated in CHD and mortality were also related to T2D, suggesting shared aging-related pathology.

Synergistic regulation of Notch signaling by different O-glycans promotes hematopoiesis.

Glycosylation of Notch receptors by O-fucose glycans regulates Notch ligand binding and Notch signaling during hematopoiesis. However, roles in hematopoiesis for other O-glycans that modify Notch receptors have not been determined. Here we show that the EGF domain specific GlcNAc transferase EOGT is required in mice for the optimal production of lymphoid and myeloid cells. The phenotype of Eogt null mice was largely cell-autonomous, and Notch target gene expression was reduced in T cell progenitors. Moreover, EOGT supported residual Notch signaling following conditional deletion of Pofut1 in hematopoietic stem cells (HSC). Eogt : Pofut1 double mutant HSC had more severe defects in bone marrow and in T and B cell development in thymus and spleen, compared to deletion of Pofut1 alone. The combined results show that EOGT and O-GlcNAc glycans are required for optimal hematopoiesis and T and B cell development, and that they act synergistically with POFUT1 and O-fucose glycans to promote Notch signaling in lymphoid and myeloid differentiation.

Clonal hematopoiesis and acute myeloid leukemia

During aging phenotypic changes in the hematopoietic system occur, and possible reason of these changes can be accumulation of gene mutations in hematopoietic stem cells or early blood progenitors. Although these mutations are mostly neutral, some may give hematopoietic stem cells and progenitor cells a proliferative advantage. In this case clonal hematopoiesis will arise, which is characterized by the formation of a genetically distinct subpopulation of blood cells. Clonal hematopoiesis may become a basis for the development of hematologic malignancies, such as acute myeloid leukemia. Clonal hematopoiesis associated genes which are most commonly mutated in acute myeloid leukemia patients are DNMT3A, TET2 and ASXL1. The prognostic significance of these gene mutations currently remains a subject of study.

Cell-intrinsic effects of clonal hematopoiesis in heart failure.

Clonal hematopoiesis of indeterminate potential (CHIP) is caused by somatic mutations in hematopoietic stem cells and associates with worse prognosis in patients with heart failure. Patients harboring CHIP mutations show enhanced inflammation. However, whether these signatures are derived from the relatively low number of cells harboring mutations or are indicators of systemic pro-inflammatory activation that is associated with CHIP is unclear. Here we assess the cell-intrinsic effects of CHIP mutant cells in patients with heart failure. Using an improved single-cell sequencing pipeline (MutDetect-Seq), we show that DNMT3A mutant monocytes, CD4+ T cells and NK cells exhibit altered gene expression profiles. While monocytes showed increased genes associated with inflammation and phagocytosis, T cells and NK cells present increased activation signatures and effector functions. Increased paracrine signaling pathways are predicted and validated between mutant and wild-type monocytes and T cells, which amplify inflammatory circuits. Altogether, these data provide novel insights into how CHIP might promote a worse prognosis in patients with heart failure.

Hematopoietic-specific heterozygous loss of Dnmt3a exacerbates colitis-associated colon cancer.

Clonal hematopoiesis (CH) is defined as clonal expansion of mutant hematopoietic stem cells absent diagnosis of a hematologic malignancy. Presence of CH in solid tumor patients, including colon cancer, correlates with shorter survival. We hypothesized that bone marrow-derived cells with heterozygous loss-of-function mutations of DNMT3A, the most common genetic alteration in CH, contribute to the pathogenesis of colon cancer. In a mouse model that combines colitis-associated colon cancer (CAC) with experimental CH driven by Dnmt3a+/Δ, we found higher tumor penetrance and increased tumor burden compared with controls. Histopathological analysis revealed accentuated colonic epithelium injury, dysplasia, and adenocarcinoma formation. Transcriptome profiling of colon tumors identified enrichment of gene signatures associated with carcinogenesis, including angiogenesis. Treatment with the angiogenesis inhibitor axitinib eliminated the colon tumor-promoting effect of experimental CH driven by Dnmt3a haploinsufficiency and rebalanced hematopoiesis. This study provides conceptually novel insights into non-tumor-cell-autonomous effects of hematopoietic alterations on colon carcinogenesis and identifies potential therapeutic strategies.

Gene editing of hematopoietic stem cells restores T-cell response in familial hemophagocytic lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by a life-threatening cytokine storm and immunopathology. Familial HLH type 3 (FHL3) accounts for approximately 30% of all inborn HLH cases worldwide. It is caused by mutations in the UNC13D gene that result in impaired degranulation of cytotoxic vesicles and hence compromised T-cell- and natural killer-cell-mediated killing. Current treatment protocols, including allogeneic hematopoietic stem cell (HSC) transplantation, still show high mortality. We sought to develop and evaluate a curative genome editing strategy in the preclinical FHL3 Jinx mouse model. Jinx mice harbor a cryptic splice donor site in Unc13d intron 26 and develop clinical symptoms of human FHL3 upon infection with lymphocytic choriomeningitis virus (LCMV). We employed clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology to delete the disease-causing mutation in HSCs and transplanted Unc13d-edited stem cells into busulfan-conditioned Jinx recipient mice. Safety studies included extensive genotyping and chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-Seq)-based off-target analyses. Cure from HLH predisposition was assessed by LCMV infection. Hematopoietic cells isolated from transplanted mice revealed efficient gene editing (>95%), polyclonality of the T-cell receptor repertoire, and neither signs of off-target effects nor leukemogenesis. Unc13d transcription levels of edited and wild-type cells were comparable. While LCMV challenge resulted in acute HLH in Jinx mice transplanted with mock-edited HSCs, Jinx mice grafted with Unc13d-edited cells showed rapid virus clearance and protection from HLH. Our study demonstrates that transplantation of CRISPR-Cas edited HSCs supports the development of a functional polyclonal T-cell response in the absence of genotoxicity-associated clonal outgrowth.

Sézary syndrome originates from heavily mutated hematopoietic progenitors

AbstractThe pathogenesis of cutaneous T-cell lymphoma (CTCL) remains unclear. Using single-cell RNA or T-cell receptor (TCR) sequencing of 32 619 CD3+CD4+ and CD26+/CD7+ and 29 932 CD3+CD4+ and CD26−/CD7− lymphocytes from the peripheral blood of 7 patients with CTCL, coupled to single-cell ATAC-sequencing of 26,411 CD3+CD4+ and CD26+/CD7+ and 33 841 CD3+CD4+ and CD26−/CD7− lymphocytes, we show that tumor cells in Sézary syndrome and mycosis fungoides (MF) exhibit different phenotypes and trajectories of differentiation. When compared to MF, Sézary cells exhibit narrower repertoires of TCRs and exhibit clonal enrichment. Surprisingly, we identified ≥200 mutations in hematopoietic stem cells from multiple patients with Sézary syndrome. Mutations in key oncogenes were also present in peripheral Sézary cells, which also showed the hallmarks of recent thymic egression. Together our data suggest that CTCL arises from mutated lymphocyte progenitors that acquire TCRs in the thymus, which complete their malignant transformation in the periphery.

Abstract P3055: Identification Of Cell-intrinsic Effects Of Clonal Hematopoiesis In Heart Failure

Background: Clonal hematopoiesis of indeterminate potential (CHIP) is caused by somatic mutations in hematopoietic stem cells and associates with worse prognosis in patients with heart failure (HF). While, circulating immune cells derived from patients with HF harboring CHIP mutations show heightened proinflammatory signatures, it is unclear whether these signatures are derived from the relatively low number of cells harboring CHIP-driver mutations, or are indicators of a systemic pro-inflammatory activation associated with CHIP. Methods and Results: We assessed the cell-intrinsic effects of the most prevalent CHIP-driver gene (DNMT3A) mutant-carrying circulating immune cells in a total of 5 HF patients. Using a novel protocol to detect DNMT3A mutant cells on a single cell level (MutDetect-Seq), we show that CHIP mutations can be detected in all major immune cell types (e.g. T cells, monocytes, B cells, NK cells), with detected mutation similar to that predicted by traditional targeted DNA sequencing. DNMT3A mutant monocytes demonstrated the highest number of significantly upregulated genes relative to other cell types. DNMT3A mutant monocytes exhibit altered gene expression signatures associated with inflammation and phagolysosome function. Additionally, DNMT3A mutant carrying CD4+, but not CD8+ T cells, showed significant changes in gene expression patterns towards an activated and cytotoxic profile, which was validated in vitro. Finally, increased paracrine signaling pathways involved in amplifying inflammatory circuits are predicted between mutated and non-mutated monocytes, this was validated in vitro with further implications for cardiac fibroblast activation via aSMA and COL1A1 expression. Conclusion: This is the first study to decipher the genetic signature of DNMT3A-CHIP-driver mutation carrying circulating immune cells, thereby providing first evidence for the cell intrinsic effects of DNMT3A mutations on peripheral blood cell subsets in patients with HF. The altered gene expression profiles associated with inflammation and phagolysosome function of mutant monocyte, as well as skewing of mutated T cells towards effector cells provide novel insights into how CHIP may promote worse prognosis in heart failure patients.

Common in atherogenesis and carcinogenesis: clonal hematopoiesis

Clonal hematopoiesis is a common age-dependent state accompanied by the expansion of mutant hematopoietic stem cells as a result of somatic mutations and is associated with a high risk of hematopoietic neoplasms and cardiovascular diseases. Clonal hematopoiesis in human ontogenesis occurs asymptomatically, and the fraction of mutant clones can exceed more than 2% of the total pool of circulating nucleated blood cells by age 70. Due to the variability of the accumulation rate of mutant clones, signs of clonal hematopoiesis can be observed at a younger age. Clonal hematopoiesis may act as a benign, precancerous condition and a strong factor for acute cardiovascular events such as myocardial infarction and stroke. Current evidence indicates that somatic mutations in driver genes of clonal hematopoiesis significantly increase the risk of acute conditions such as acute myeloid leukemia and acute myocardial infarction. The high mortality and morbidity of cardiovascular and cancer diseases, and their strong association with clonal hematopoiesis, make it of indeterminate potential worthy of close attention.

Clinical relevance of clonal hematopoiesis and its interference in cell-free DNA profiling of patients with gastric cancer.

Clonal hematopoiesis (CH) is a condition in which healthy individuals have somatic mutations in hematopoietic stem cells. It has been reported with increased risk of hematologic malignancy and cardiovascular disease in the general population, but studies of Korean populations with comorbid disease entities are scarce. White blood cells (WBCs) from patients with gastric cancer (GC) (n=121) were analyzed using a DNA-based targeted (531 genes) panel with customized pipeline designed to detect single nucleotide variants and small indels with low-allele-frequency of≥0.2 %. We defined significant CH variants as having variant allele frequency (VAF)≥2 % among variants found in WBCs. Matched cell-free DNA (cfDNA) samples were also analyzed with the same pipeline to investigate the false-positive results caused by WBC variants in cfDNA profiling. Significant CH variants were detected in 29.8 % ofpatients and were associated with age and male sex. Thenumber of CH variants was associated with a history of anti-cancer therapy and age. DNMT3A and TET2 were recurrently mutated. Overall survival rate of treatment-naïve patients with stage IV GC was higher in those with CH, but Cox regression showed no significant association after adjustment for age, sex, anti-cancer therapy, and smoking history. In addition, we analyzed the potential interference of WBC variants in plasma cell-free DNA testing, which has attracted interest as a complementary method for tissue biopsy. Results showed that 37.0 % (47/127) of plasma specimens harbored at least one WBC variant. VAFs of interfering WBC variants in the plasma and WBC were correlated, and WBC variants with VAF≥4 % in WBC were frequently detected in plasma with the same VAF. This study revealed the clinical impact of CH in Korean patients and suggests the potential for its interference in cfDNA tests.

Optimizing IFN alpha therapy against Myeloproliferative Neoplasms.

Myeloproliferative Neoplasms (MPNs) are hematological malignancies that result from acquired driver mutations in hematopoietic stem cells (HSCs), causing overproduction of blood cells and an increased risk of thrombo-hemorrhagic events. The most common MPN driver mutation affects the JAK2 gene (JAK2V617F ). Interferon alpha (IFNα) is a promising treatment against MPNs by inducing a hematological response and molecular remission for some patients. Mathematical models have been proposed to describe how IFNα targets mutated HSCs, indicating that a minimal dose is necessary for long-term remission. This study aims to determine a personalized treatment strategy. First, we show the capacity of an existing model to predict cell dynamics for new patients from data that can be easily obtained in clinic. Then, we study different treatment scenarios in silico for three patients, considering potential IFNα dose-toxicity relations. We assess when the treatment should be interrupted, depending on the response, the patient's age, and the inferred development of the malignant clone without IFNα. We find that an optimal strategy would be to treat the patients with a constant dose so that the treatment could be interrupted as fast as possible. Higher doses result in earlier discontinuation but also higher toxicity. Without knowledge of the dose-toxicity relationship, trade-off strategies can be found for each patient. A compromise strategy is to treat patients with medium doses (60-120 μg/week) for 10-15 years. Altogether, this work demonstrates how a mathematical model calibrated from real data can help build a clinical decision-support tool to optimize long-term IFNα therapy for MPN patients. Significance Statement Myeloproliferative Neoplasms (MPNs) are chronic blood cancers. Interferon alpha (IFNα) is a promising treatment with the potential to induce a molecular response by targeting mutated hematopoietic stem cells. MPN patients are treated over several years, and there is a lack of knowledge concerning the posology strategy and the best timing for interrupting therapy. The study opens avenues for rationalizing how to treat MPN patients with IFNα over several years, promoting a more personalized approach to treatment.

Inflammatory signals from fatty bone marrow support DNMT3A driven clonal hematopoiesis

Both fatty bone marrow (FBM) and somatic mutations in hematopoietic stem cells (HSCs), also termed clonal hematopoiesis (CH) accumulate with human aging. However it remains unclear whether FBM can modify the evolution of CH. To address this question, we herein present the interaction between CH and FBM in two preclinical male mouse models: after sub-lethal irradiation or after castration. An adipogenesis inhibitor (PPARγ inhibitor) is used in both models as a control. A significant increase in self-renewal can be detected in both human and rodent DNMT3AMut-HSCs when exposed to FBM. DNMT3AMut-HSCs derived from older mice interacting with FBM have even higher self-renewal in comparison to DNMT3AMut-HSCs derived from younger mice. Single cell RNA-sequencing on rodent HSCs after exposing them to FBM reveal a 6-10 fold increase in DNMT3AMut-HSCs and an activated inflammatory signaling. Cytokine analysis of BM fluid and BM derived adipocytes grown in vitro demonstrates an increased IL-6 levels under FBM conditions. Anti-IL-6 neutralizing antibodies significantly reduce the selective advantage of DNMT3AMut-HSCs exposed to FBM. Overall, paracrine FBM inflammatory signals promote DNMT3A-driven clonal hematopoiesis, which can be inhibited by blocking the IL-6 pathway.

Aberrant activation of TCL1A promotes stem cell expansion in clonal haematopoiesis.

Mutations in a diverse set of driver genes increase the fitness of haematopoietic stem cells (HSCs), leading to clonal haematopoiesis1. These lesions are precursors for blood cancers2-6, but the basis of their fitness advantage remains largely unknown, partly owing to a paucity of large cohorts in which the clonal expansion rate has been assessed by longitudinal sampling. Here, to circumvent this limitation, we developed a method to infer the expansion rate from data from a single time point. We applied this method to 5,071 people with clonal haematopoiesis. A genome-wide association study revealed that a common inherited polymorphism in the TCL1A promoter was associated with a slower expansion rate in clonal haematopoiesis overall, but the effect varied by driver gene. Those carrying this protective allele exhibited markedly reduced growth rates or prevalence of clones with driver mutations in TET2, ASXL1, SF3B1 and SRSF2, butthis effect was not seen inclones withdriver mutations in DNMT3A. TCL1A was not expressed in normal or DNMT3A-mutated HSCs, but the introduction of mutations in TET2 or ASXL1 led to the expression of TCL1A protein and the expansion of HSCs in vitro. The protective allele restricted TCL1A expression and expansion of mutant HSCs, as did experimentalknockdown of TCL1A expression. Forced expression of TCL1A promoted the expansion of human HSCs in vitro and mouse HSCs in vivo. Our results indicate that the fitness advantage of several commonly mutated driver genes in clonal haematopoiesis may be mediated by TCL1A activation.

Abstract 5928: Prevalence of clonal hematopoiesis in neuroendocrine tumor patients prior to lutetium 177 dotatate therapy (Lu177): A prospective trial of 36 patients

Abstract Introduction: Clonal hematopoiesis (CH) is defined by acquisition of somatic mutations in hematopoietic stem cells with potential for expansion over time. CH is influenced by exposures to chemotherapy/radiation & has been associated with therapy related myeloid neoplasms (tMN). Peptide receptor radionuclide therapy (PRRT) is a radionuclide therapy and is FDA approved for neuroendocrine tumors (NET). PRRT has been associated with a 2-10% risk of tMN, especially with prior exposure to alkylating agents. A prior study of 13 NET patients found CH prevalence of 64%. We carried out a prospective assessment of CH in NET patients prior to PRRT. Methods: We evaluated pre-PRRT blood samples in 37 NET patients. Genomic DNA from mononuclear cells was analyzed for CH using a custom panel targeting 229 genes to a targeted depth of >1000X. Results: After approval by Mayo Clinic IRB, 45 patients with Stage IV NET were enrolled & CH data was available for 37. Fifty-one percent of patients were female, with a median age of 68yrs (range, 34-84yrs). Types of NET included small bowel (51%), pancreatic (29%), carcinoid (8%), paraganglioma (5%), & other (5%). The median number of therapies prior to PRRT was 1 (range, 0-4) & included somatostatin analogues (SSA)-81%, alkylating agents-30%, & prior radiation-13%. Thirty-six of 37 patients had variants identified, 5 of which were presumed germline (DDX41, CDKN2B, CHEK2, POT1, ERBB2), & 17 (45.9%) had pathogenic variants meeting the operational definition of CH (DNMT3A, TET2, PPM1D, TP53, SF3B1, ASXL1). The median number of pathogenic variants per patient was 1 (range, 0-2), & 6 patients had >1. In patients >60yrs old, 46% had pathogenic variants & 3 patients with pathogenic variants (TET2, TP53, DNMT3A) were <60 years old. The most common pathogenic variants were in epigenetic regulators, DNMT3A (37.5%) & TET2 (16.7%) with a median VAF of 2.8% (range, 0.70-50%). Eight patients (47%) with pathogenic CH had grade 1 cytopenias at baseline, compared to 10 patients (50%) without pathogenic CH. One patient had grade 3/4 cytopenia at baseline & had a missense variant in TP53 (VAF 50%) & history of exposure to anthracyclines, alkylating agents, platinum & radiation. The patient developed MDS before receiving PRRT. Pathogenic CH variants were more common in patients with previous exposure to radiation (p=0.008) or SSA (p=0.005). Two patients had PPM1D variants, both with a history of prior radiation, one with a history of prior alkylating treatment. Conclusion: To our knowledge this is the largest prospective evaluation of CH in NET. We identified CH to be present in 46% of patients & DNMT3A mutations were most common. This establishes the clonal landscape with mutations involving epigenetic regulators, DNA damage repair, transcription & splicing factors. Prospective longitudinal evaluation will evaluate the association between CH, cytopenias and tMN. Citation Format: Yael Kusne, Terra Lasho, Zaid Elsabbagh, Abhishek Mangaonkar, Rachel Eiring, Timothy Hobday, Jason Starr, Tanios Bekaii-Saab, Thorvardur Halfdanarson, Mrinal Patnaik, Mohamad Sonbol. Prevalence of clonal hematopoiesis in neuroendocrine tumor patients prior to lutetium 177 dotatate therapy (Lu177): A prospective trial of 36 patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5928.