Mutations In Hematopoietic Stem Cells Research Articles

Cohesin Dependency in Npm1c/+ and Flt3-ITD Mutant Hematopoietic Stem and Progenitor Cells

Acute myeloid leukemia (AML) is an aggressive myeloid malignancy with high relapse (40-60%) and low median survival rate (5-7 months post treatment in relapse setting), particularly for patients over 65 years old. Genomic investigations of AML have identified recurrent mutations, such as FMS-like tyrosine kinase 3 (FLT3, 20-25%) and Nucleophosmin (NPM1, 30%). When mutated, FLT3 acquires an internal tandem duplication (FLT3-ITD) and causes aberrant STAT5/AKT signaling. For NPM1, mutation frequently occurs at nuclear export signal, which results in cytoplasmic localization (NPM1c). In AML, these driver mutations co-occur with other genetic lesions, particularly in epigenetic regulators. Clincally, patients with FLT3-ITD mutations have a very poor prognosis. While the pathophysiology is not completely understood, direct inhibition of FLT3 by tyrosine kinase inhibitors have, to-date, been underwhelming. In contrast, the NPM1c mutation in AML patients is favorable and chemotherapy sensitive when NPM1c is the sole driver mutation. Yet, co-mutation of NPM1c with other known AML mutations, such as Flt3-ITD, negates this favorable prognosis. STAG2 (Stromal Antigen 2) is a member of cohesin complex that is recurrently mutated in >10 cancers and is essential in maintaining the integrity of the 3-dimensional genome partitioning structure known as topologically structural domains (TADs). Previously, our work has demonstrated that depletion of various cohesin factors, including Stag2, leads to increased hematopoietic stem and progenitor cell (HSPC) self-renewal and myeloid-biased differentiation. Loss of Stag2 leads to impairment of sub-TADs and affects the capability of key hematopoietic transcription factors, such as PU.1, to access and engage their target genes. In the context of AML, disruption of TADs can lead to altered transcriptional output and cellular behavior. Mutation of STAG2 frequently arises in AML (16%) and co-occurs with NPM1 and FLT3 oncogenes. To examine the consequences of co-occurring Stag2/Npm1c or Stag2/Flt3-ITD mutations, we generated two compound mutant murine models crossed to conditional alleles of Stag2, Npm1c and constitutively active Flt3-ITD in the Ubc-CreER background. At 6-8 weeks of age, tamoxifen was administered to mice harboring Stag2/Npm1c or Stag2/Flt3-ITD mutations to delete Stag2 or activate Npm1c alleles respectively, while the Flt3-ITD mutation is active since birth. Immunophenotyping analysis was performed at 4 weeks post mutation activation in the bone marrow and peripheral blood. For mice carrying double Stag2/Npm1c mutation, there was no change in the number of stem cell population (HSCLT). In contrast, comparing to Npm1c/+ mice, Stag2-/-Npm1c/+ mice had an elevated myeloid-biased multipotent progenitor population (MPP3, LSK flk2- CD150- CD48+). However, there was no change in granulocyte-monocyte progenitor (GMP) number (Figure 1A). To determine if the myeloid bias was due to lack of the entire cohesin complex, we generated the Smc3/Npm1c mutant mice, where both Stag1-cohesin and Stag2-cohesin complexes were partially depleted upon heterozygous deletion of Smc3. Interestingly, there were no changes in MPP3 population in Smc3+/-Npm1c/+ mice, pointing to a Stag2-cohesin specific phenomenon. In the Stag2/Flt3-ITD co-mutation model, loss of Stag2-cohesin resulted in no changes in MPP3 cells compared to monogenic Flt3-ITD mutation. However, we did observe a normalization of GMPs, suggesting that Stag2-cohesin loss is crucial for abnormal myeloid differentiation in Flt3-ITD background (Figure 1B). In order to evaluate whether the order of mutations impacted immunophenotypic aberrancy, we additionally generated a stepwise Stag2/Flt3-ITD mutational model by deleting Stag2 first (via Mx1-Cre) for 4 weeks followed by tamoxifen-inducible Frt activated Flt3-ITD mutation (Flt3 frt-ITD/+) for 4 weeks. Interestingly, there was no difference in the normalization effect between constitutive and conditional activation of Flt3-ITD mutation, suggesting the loss of Stag2-cohesin is not required prior to Flt3-ITD acquisition. Further characterization of these co-mutational murine models is in progress. With these models, we aim to uncover the dependency of Stag2-cohesin in leukemia maintenance. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Impact of Dnmt3a or Tet2 CH on Alzheimer's Disease in a Murine Model

Clonal Hematopoiesis of Indeterminate Potential (CHIP) represents a clonal expansion of mutant hematopoietic stem cells as a consequence of aging and environmental stress and is most commonly driven by mutations in DNMT3A or TET2. CHIP has been associated with a number of age-associated diseases; but controversy exists as to whether CHIP predisposes to or protects from Alzheimer's Disease (AD). Indeed, mutations in DNMT3A and TET2 have been reported to drive increased myeloid cell inflammation which would seem to increase AD; however, the effect of these mutations on microglia, a resident macrophage-like cell in the brain that plays an important role in neurodegenerative diseases including AD, remains unknown. Here, we investigated the impact of Dnmt3a and Tet2 loss-of-function CHIP in a murine model of AD. Our findings suggest opposing and mutation-specific effects by Dnmt3a versus Tet2 on AD pathogenesis. We performed total body irradiation of 5xFAD transgenic mice predisposed to familial AD and performed intravenous transplantation of either Dnmt3a Knock-out (KO), Tet2 KO or wildtype (WT) whole bone marrow. To mimic systemic chronic inflammation seen in aging, we challenged all groups weekly with LPS. We conducted behavioral assays to assess differences in recognition and cognitive memory as well as anxiety levels between the experimental and control groups. Six months following transplantation, mouse brains were harvested for cytokine analysis, measurement of beta-amyloid plaques, and characterization of myeloid and microglial populations in the brain. Mice harboring Dnmt3a -mutant bone marrow showed increased anxiety, impaired recognition, and cognitive phenotypes consistent with AD. Unexpectedly, cytokine analysis revealed that both Dnmt3a and Tet2 experimental groups showed an overall decrease in pro-inflammatory cytokines such as IFNg, IL-1 and G-CSF in the brain, suggesting lower neuroinflammatory levels compared to the WT control group. Furthermore, we found decreased CD11a expression by qRT-PCR in the brain, suggesting a decrease in infiltrating peripheral immune cells into the brain. However, quantification of immunological cells by flow cytometry revealed that 5xFAD mice transplanted with Dnmt3a -mutant marrow showed an increase in the number of CD11a- CD11b+ TMEM119+ microglial cells, but no change in CD11a+ CD11b+ TMEM119- myeloid cells. Phenotypically, the microglia from the Dnmt3a -mutant marrow mice showed lower expression of activation markers CD45, CD68 and CD11c. Interestingly, while no difference was observed in infiltrating myeloid cells, we also observed a decrease in infiltrating CD11a+ CD3+ CD4+ T cells in the brain of Dnmt3a-/- transplanted mice. These data suggest that the decreased T cell infiltration and subsequent decrease in cytokine signaling in the 5xFAD mice with Dnmt3a-/- bone marrow may contribute to decreased activation of microglia, impairing amyloid uptake and clearance. In contrast, 5xFAD mice transplanted with Tet2 mutant bone marrow showed improved recognition and cognitive memory levels compared to control, suggesting a protective effect against AD. Mice transplanted with Tet2-mutant marrow demonstrated an increase in CD11a+ cells, suggesting an increase in infiltrating peripheral immune cells into the brain. There was no significant difference in the overall frequency of microglial cells in mice transplanted with Tet2-mutant cells compared to WT. and MHC II expression was lower for both infiltrating myeloid and microglial cells in Tet2-mutant transplanted mice. Altogether, these data show that an increased myeloid cell population in the brain of Tet2 KO transplanted 5xFAD mice, despite having lower MHC II expression, may contribute to protecting against AD in our mouse model. Overall, our data suggest that CHIP-associated Dnmt3a and Tet2 mutations may confer different effects on AD pathogenesis, with DNMT3A mutations accelerating progression of AD while TET2 mutations reduce it. Further studies are ongoing to conduct immunohistochemical analysis on brain samples to measure microglia and amyloid plaques. Our work is an important first step to understanding the underlying mechanism by which CHIP mutations affect the inflammatory state and progression of AD.

Abstract 13063: Clonal Hematopoiesis Associated With TET2 and DNMT3A Gene Driver Mutations Accelerates Aortic Valve Calcification in vitro and in vivo

Calcific aortic valve disease (CAVD) is the most common age-related heart valve disease, with no medical therapy to halt progression. Age is associated with the enrichment of somatic mutations in hematopoietic stem cells leading to clonal hematopoiesis (CH). CH mutations promote inflammation, occur in more than 30% of patients with severe CAVD and CAVD patients harboring CH mutations have a worse prognosis. Single-cell RNA-sequencing of immune cells (n=127120 cells) of CAVD patients with CH-mutations showed strong proinflammatory and M1-like macrophage gene signatures (CD38, CXCL10) along with genes associated with calcification (S100A9, RUNX2, Oncostatin M=OSM) suggesting that CH might be causally involved in CAVD. Indeed, silencing of TET2 or DNMT3A in macrophages in vitro induce the prototypic osteoblastic transcription factor RUNX2 and paracrine acting genes such as OSM and S100A9, which can promote vascular calcification. Secreted factors from TET2 or DNMT3A-silenced macrophages induced osteoblastic differentiation as demonstrated by elevated calcium deposition and genes involved in the RUNX2 signaling pathway (COL1A2, ALP), which could be ablated by silencing of OSM in CH-macrophages. Atheroprone Ldlr -/- mice receiving TET2 -/- bone marrow transplants mice showed increased total calcified area along (1.49-fold) with increased numbers of calcification deposits (2.33-fold) as evidenced by von Kossa staining. Increased myeloid derived OSM and S100A9 was found in the valves of TET2 -/- BMT mice with S100A9 strongly co-occurring with OSM. This study provides insights into the development of incident CAVD for patients with CH mutations and therefore yields plausible interventions for nearly one third of CAVD cases to slow or stop disease progression.

Abstract 15374: Somatic Mutations of Dnmt3a Contribute to Pulmonary Vascular Remodeling and Right Ventricular Dysfunction in PAH

Background: Pulmonary Arterial Hypertension (PAH) is an obliterative pulmonary vasculopathy in which structural changes are driven by inflammation, fibrosis, mitochondrial dysfunction, and pathological angiogenesis. While ~10% of cases of PAH have germline BMPR2 mutations (or one of 16 other recognized or putative PAH genes), the genetic basis for the majority of PAH patients remains unexplained. Somatic mutations in hematopoietic stem cells (HSC) are related to the disorders of clonal hematopoiesis of indeterminate potential (CHIP). Tet methylcytosine dioxygenase 2 (TET2) and DNA methyltransferase 3a (DNMT3A) are among the primary genes involved in CHIP. We found in a PAH-Biobank that 0.39% of PAH patients had somatic or germline TET2 mutations. Also, TET2-knockout mice can develop PAH spontaneously. Thus TET2 is a new PAH gene. We now hypothesize that CHIP driven by mutations of DNMT3A can also cause pulmonary vascular remodeling and right ventricular failure (RVF), contributing to PAH. Methods: Studies were performed on 9 month old DNMT3A knockout (-/-) mice and their relative controls (f/f) (N=5/group). The right ventricular (RV) function was assessed via cardiac ultrasound. Pulmonary artery medial wall thickness (PAMWT) and radial alveolar count (RAC) were assessed in lung tissue sections using light microscopy. Results: In DNMT3A -/- mice, there was a reduction in pulmonary artery acceleration time (PAAT) compared to controls ( p= 0.0268). There was also a significant reduction in percent RV free wall thickening (RVFWT %) compared to controls (57.44±9.97 DNMT3A-/-, 118.5±14.98 f/f; p= 0.017). Both Tricuspid annular plane systolic excursion (TAPSE) and cardiac output (CO) were also significantly reduced in DNMT3A -/- mice ( p < 0.0001, p = 0.0135). The medial thickness of small pulmonary arteries was significantly higher in DNMT3A -/- mice compared to controls (56.51% ±1.19 DNMT3A-/-, 39.79% ±2.07 f/f; p= 0.002). RAC was significantly reduced in the DNMT3A -/- mice when compared to controls ( p= 0.018). Conclusions: DNMT3A mutations may contribute to the development of pulmonary vascular disease and RVF in PAH.

A RUNX1-FPDMM rhesus macaque model reproduces the human phenotype and predicts challenges to curative gene therapies

AbstractGerm line loss-of-function heterozygous mutations in the RUNX1 gene cause familial platelet disorder with associated myeloid malignancies (FPDMM) characterized by thrombocytopenia and a life-long risk of hematological malignancies. Although gene therapies are being considered as promising therapeutic options, current preclinical models do not recapitulate the human phenotype and are unable to elucidate the relative fitness of mutation-corrected and RUNX1-heterozygous mutant hematopoietic stem and progenitor cells (HSPCs) in vivo long term. We generated a rhesus macaque with an FPDMM competitive repopulation model using CRISPR/Cas9 nonhomologous end joining editing in the RUNX1 gene and the AAVS1 safe-harbor control locus. We transplanted mixed populations of edited autologous HSPCs and tracked mutated allele frequencies in blood cells. In both animals, RUNX1-edited cells expanded over time compared with AAVS1-edited cells. Platelet counts remained below the normal range in the long term. Bone marrows developed megakaryocytic dysplasia similar to human FPDMM, and CD34+ HSPCs showed impaired in vitro megakaryocytic differentiation, with a striking defect in polyploidization. In conclusion, the lack of a competitive advantage for wildtype or control-edited HSPCs over RUNX1 heterozygous–mutated HSPCs long term in our preclinical model suggests that gene correction approaches for FPDMM will be challenging, particularly to reverse myelodysplastic syndrome/ acute myeloid leukemia predisposition and thrombopoietic defects.

ALL-040 The Impact of Age-Related Clonal Hematopoiesis on Outcomes of Adults With Acute Lymphoblastic Leukemia

Age-related clonal hematopoiesis (ARCH) denotes accumulation of DNA mutations in hematopoietic stem cells as a result of aging, inflammation, and other environmental factors. The role of ARCH in lymphoid leukemogenesis is unknown. We hypothesize that ARCH is a precursor lesion for acute lymphoblastic leukemia (ALL) in older adults, and ARCH-associated ALL is a unique entity with different molecular and clinical characteristics. We retrospectively studied 345 patients with ALL (83% B-ALL, 14% T-ALL, 3% ETP-ALL). The median age at diagnosis was 47 years (18-88 years), and 45% were women. Cytogenetic groups were as follows: 24% had Ph+ ALL, 13% had Ph-like ALL, and 3% had ALL with KMT2A rearrangement. The most frequent mutation was the loss of the CDKN2A gene (32%), followed by mutations in the TP53 (17%) and IKZF1 (16%) genes. Mutations involving the recurrently mutated genes in ARCH were seen in 32% of patients, with the following order of frequency: TP53 (17%), DNMT3A (5%), TET2 (4%), RUNX1 (3.5%), ASXL1 (3%), IDH1/2 (2%), and U2AF1 (1%). ARCH-associated mutations were more common in older adults (≥40 years) than in young adults (41% vs. 17%, P<0.0001). The high variant allelic frequencies for ARCH-associated mutations at diagnosis suggested that these might be founder clones. We performed single-cell multiomics (DNA+protein) analysis of ARCH-associated ALL samples to gain insights into the distribution of ARCH mutations in immunophenotypically-defined clusters. ARCH mutations were found in ALL cells as well as myeloid cells but were not found in nonmalignant mature lymphoid cells. Analysis of serial samples revealed that ARCH clones persist at the time of remission and re-emerge at relapse. The overall survival for patients with ARCH-associated ALL was shorter than for patients without ARCH (median, 32 vs. 84 months, P=0.02). In conclusion, ARCH is an ancestral event in older adults with ALL and constitutes fertile soil for acute lymphoblastic leukemogenesis leading to inferior clinical outcomes.

Clonal hematopoiesis of indeterminate potential, DNA methylation, and risk for coronary artery disease

Age-related changes to the genome-wide DNA methylation (DNAm) pattern observed in blood are well-documented. Clonal hematopoiesis of indeterminate potential (CHIP), characterized by the age-related acquisition and expansion of leukemogenic mutations in hematopoietic stem cells (HSCs), is associated with blood cancer and coronary artery disease (CAD). Epigenetic regulators DNMT3A and TET2 are the two most frequently mutated CHIP genes. Here, we present results from an epigenome-wide association study for CHIP in 582 Cardiovascular Health Study (CHS) participants, with replication in 2655 Atherosclerosis Risk in Communities (ARIC) Study participants. We show that DNMT3A and TET2 CHIP have distinct and directionally opposing genome-wide DNAm association patterns consistent with their regulatory roles, albeit both promoting self-renewal of HSCs. Mendelian randomization analyses indicate that a subset of DNAm alterations associated with these two leading CHIP genes may promote the risk for CAD.

TERT and TET2 Genetic Variants Affect Leukocyte Telomere Length and Clinical Outcome in Coronary Artery Disease Patients-A Possible Link to Clonal Hematopoiesis.

Inherited and acquired mutations in hematopoietic stem cells can cause clonal expansion with increased risk of cardiovascular disease (CVD), a condition known for the clonal hematopoiesis of indeterminate potential (CHIP). Inherited genetic variants in two CHIP-associated genome loci, the telomerase gene telomerase enzyme reverse transcriptase (TERT) (rs7705526) and the epigenetic regulator ten–eleven translocation 2 (TET2) (rs2454206), were investigated in 1001 patients with stable coronary artery disease (CAD) (mean age 62 years, 22% women), with regards to cardiovascular outcome, comorbidities, and leukocyte telomere length. Over 2 years, mutated TERT increased the risk two-fold for major clinical events (MACEs) in all patients (p = 0.004), acute myocardial infarction (AMI) in male patients (p = 0.011), and stroke in female patients (p < 0.001). Mutated TET2 correlated with type 2 diabetes (p < 0.001), the metabolic syndrome (p = 0.002), as well as fasting glucose, HbA1c, and shorter telomeres (p = 0.032, p = 0.003, and p = 0.016, respectively). In conclusion, our results from stable CAD patients highlight TERTs’ role in CVD, and underline TET2s’ role in the epigenetic regulation of lifestyle-related diseases.

Discovering the drivers of clonal hematopoiesis

Mutations in genes that confer a selective advantage to hematopoietic stem cells (HSCs) drive clonal hematopoiesis (CH). While some CH drivers have been identified, the compendium of all genes able to drive CH upon mutations in HSCs remains incomplete. Exploiting signals of positive selection in blood somatic mutations may be an effective way to identify CH driver genes, analogously to cancer. Using the tumor sample in blood/tumor pairs as reference, we identify blood somatic mutations across more than 12,000 donors from two large cancer genomics cohorts. The application of IntOGen, a driver discovery pipeline, to both cohorts, and more than 24,000 targeted sequenced samples yields a list of close to 70 genes with signals of positive selection in CH, available at http://www.intogen.org/ch. This approach recovers known CH genes, and discovers other candidates.

Clonal haematopoiesis is associated with higher mortality in patients with cardiogenic shock.

Cardiogenic shock (CS) with variable systemic inflammation may be responsible for patient heterogeneity and the exceedingly high mortality rate. Cardiovascular events have been associated with clonal haematopoiesis (CH) where specific gene mutations in haematopoietic stem cells lead to clonal expansion and the development of inflammation. This study aims to assess the prevalence of CH and its association with survival in a population of CS patients in a quaternary centre. We compared the frequency of CH mutations among 341 CS patients and 345 ambulatory heart failure (HF) patients matched for age, sex, ejection fraction, and HF aetiology. The association of CH with survival and levels of circulating inflammatory cytokines was analysed. We detected 266 CH mutations in 149 of 686 (22%) patients. CS patients had a higher prevalence of CH-related mutations than HF patients (odds ratio 1.5; 95% confidence interval [CI] 1.0-2.1, p = 0.02) and was associated with decreased survival (30 days: hazard ratio [HR] 2.7; 95% CI 1.3-5.7, p = 0.006; 90 days: HR 2.2; 95% CI 1.3-3.9, p = 0.003; and 3 years: HR 1.7; 95% CI 1.1-2.8, p = 0.01). TET2 or ASXL1 mutations were associated with lower survival in CS patients at all time-points (p ≤ 0.03). CS patients with TET2 mutations had higher circulating levels of SCD40L, interferon-γ, interleukin-4, and tumour necrosis factor-α (p ≤ 0.04), while those with ASXL1 mutations had decreased levels of CCL7 (p = 0.03). Cardiogenic shock patients have high frequency of CH, notably mutations in TET2 and ASXL1. This was associated with reduced survival and dysregulation of circulating inflammatory cytokines in those CS patients with CH.

P748: SINGLE CELL GENOTYPING OF MATCHED BONE MARROW AND PERIPHERAL BLOOD CELLS IN TREATMENT NAIVE AND AZA-TREATED MDS AND CMML

Background: Somatic mutations in hematopoietic stem cells (HSCs) are a central pathogenic event in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) where they induce proliferative advantages and impaired differentiation with subsequent cytopenias in peripheral blood (PB). Patients with high-risk disease who are ineligible for allogenic stem cell transplantation are treated with hypomethylating agents, including 5-azacytidine (AZA). AZA treatment can improve PB counts and delay progression to Akute Myeloid Leukemia. However, AZA response does not require eradication of mutated HSCs and may be related to improved differentiation capacity of mutated hematopoietic stem and progenitor cells (HSPCs). However, the contribution of mutated HSPCs to steadystate hematopoiesis in MDS and CMML is unclear. Aims: In this project, we aimed to determine the distribution of individual subclones and even wild-type cells within the HSPC-compartment and the contribution of these subclones and wild-type cells to effective hematopoiesis in patients with MDS or CMML. Methods: We used a combination of index sorting and single cell genotyping to characterize the somatic mutations of individual stem- and progenitor cells and matched high-turnover circulating cells in three patients, one treatment naïve and two AZA-treated. Using this approach, myeloid driver mutations previously detected in a traditional approach were tracked in HSC/multipotent progenitors (MPP), MDS stem cells MDS-SC, progenitor cells (common myeloid progenitor (CMP), granulocyte monocyte progenitor (GMP), megakaryocyte erythroid progenitor (MEP)), as well as in monocytes, neutrophils and naïve B cells (nBC). Results: Patient H198302 and Patient H198303 both had CMML and had been treated with AZA for ̴10 years with complete response. Patient H198304 had MDS-EB1 and has never been treated with hypomethylating agents. For each patient, the figure shows the proportions of cells across the haematopoietic hierarchy carrying zero, one, two, three, or four mutations in the specified alleles. Only very small numbers of HSPCs carrying no mutation could be detected. In all three patients, most stem and progenitor cells carried a majority of tracked mutations, with no major differences between stem cells, progenitor cells and MDS-stem cells. In one patient, H198304, MDS-stem cells had a higher proportion of cells with 3 or 4 mutations detected. Overall, in all three patients, a similar frequency of mutations was observed in differentiated monocytes and neutrophils with a substantial proportion of the circulating cells derived from highly mutated progenitors. One notable exception were nBCs in Patient H198304, which were mostly wild-type, suggesting that the small wildtype HSC population is the predominant origin of nBCs in this individual. Analysis of additional lymphoid populations in PB from this patient revealed that naïve T, but not NK cells were also predominantly wild-type, suggesting specific impairment of B- and T-lineage maturation in the mutated cells. Image:Summary/Conclusion: In conclusion, attrition of highly mutated cells during myeloid maturation was not observed in any of the three patients, irrespective of HMA therapy. This suggests that in vivo, highly mutated stem and progenitor cells retain the capacity to differentiate to mature myeloid and in some patients mature lymphoid cells and contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment.

P980: JAK2V617F-DEPENDENT DOWN REGULATION OF SHP1 EXPRESSION PARTICIPATES IN THE SELECTION OF MPN CELLS IN THE PRESENCE OF TGFΒ

Background: Myeloproliferative neoplasms (MPNs) are characterized by an increased production of blood cells, due to acquisition in HSC of mutations in the JAK2, CALR or MPL genes. The JAK2V617F mutation, leading to a constitutively active form of JAK2, is the most frequent mutation. During disease evolution the clonal selection of mutated cells is dependent on cell-intrinsic and extrinsic factors present in the niche microenvironment such as cytokines. TGFβ, whose local secretion is increased in MPNs, and more particularly in myelofibrosis is known to negatively regulate normal hematopoietic stem cells (HSC) proliferation. However, its mode of action is not clearly established. We hypothesized that JAK2V617F mutant HSC are resistant to TGFβ antiproliferative effect explaining the pathological stem cell invasion in myelofibrosis. Using multiomics approaches to study the intracellular signaling pathways in MPNs we recently identified the down regulation of SHP-1 expression in JAK2V617F cells compared to wild type cells. Of note, the phosphatase SHP-1 has recently been shown to be required for the TGFβ-induced quiescence of HSCs. Aims: We hypothesized a relationship between SHP-1 down regulation and the resistance of JAK2V617F mutated cells to the antiproliferative effect of TGFβ. The work presented herein aimed at verifying this hypothesis. Methods: We used the HEL erythroleukemia cell line which carries several copies of the JAK2V617F mutated gene. We also used an isogenic model obtained by transducing either the JAK2 wild type (JAK2wt) of JAK2V617F allele in the UT-7 megakaryoblastic cell line. Proliferation was measured using the CCK8. Protein expression was measured using western blot while gene expression was measured using RT-qPCR. Results: The expression of SHP-1 was reduced by 30% at the RNA level and 50% at the protein level in UT-7 cells transduced with JAK2V617F compared to JAK2wt -transduced cells. In bone marrow cells taken from JAK2V617F knock-in mice the protein level of SHP1 was reduced by 70% compared to wild-type mice. EPO treatment of UT-7 cells (which express the EPO-R) showed a down regulation of SHP-1 expression which was abrogated in the presence of the JAK2 inhibitor ruxolitinib. These results suggest that SHP-1 expression is modulated by JAK2 signaling leading to its down regulation in JAK2V617F cells. Although TGFβ receptor and SMAD2/3 expression was similar in both cell type, JAK2V617F UT-7 cells were less responsive to TGFβ signaling compared to JAK2wt cells (reduced phosphorylation of SMAD2/3). The proliferation of JAK2wt UT-7 cells was significantly reduced in the presence of TGFβ 10ng/mL while JAK2V617F UT-7 cells were not affected. To confirm the involvement of SHP-1 we transduced HEL cells with the cDNA coding for SHP-1 in order to restore higher levels of the protein in this JAK2V617F mutated cell line and observed a significant reduction in the proliferation when treated with TGFβ compared to cells transduced with the empty vector. Finally, we performed an in vitro competitive assay where JAK2V617F UT-7 cells transduced by an m-cherry fluorescent vector were mixed 50/50 with JAK2wt UT-7 and cultured with TGFβ. After 14 days of culture with TGFβ 10ng/mL we observed a significant positive selection of the JAK2 mutant cells over JAK2wt cells. Summary/Conclusion: In conclusion, we found a JAK2-dependent down-regulation of SHP1 expression which is related to the resistance of JAK2V617F cells to the antiproliferative effect of TGFβ explaining clonal selection of mutant stem cells in myelofibrosis

Hematopoiesis of Indeterminate Potential and Atherothrombotic Risk.

Hematopoiesis is the process of blood production, essential for the continued supply of immune cells and red blood cells. However, the proliferative nature of hematopoietic stem cells (HSCs) renders them susceptible to developing somatic mutations. HSCs carrying a mutation can gain a selective advantage over normal HSCs and result in hematological disorders. One such disorder is termed clonal hematopoiesis of indeterminate potential (CHIP), a premalignant state associated with aging, where the mutant HSCs are responsible for producing a small portion of mature immune cells in the circulation and subsequently in tissues. People with CHIP have been shown to have an increased risk of mortality due to cardiovascular disease (CVD). Why this occurs is under rigorous investigation, but the majority of the studies to date have suggested that increased atherosclerosis is due to heightened inflammatory cytokine release from mutant lesional macrophages. However, given CHIP is driven by several mutations, other hematopoietic lineages can be altered to promote CVD. In this review we explore the relationship between mutations in genes causing CHIP and atherothrombotic disorders, along with potential mechanisms of enhanced clonal outgrowth and potential therapies and strategies to slow CHIP progression.

Abstract LB555: Augmented Txnip-p53-p21 axis preserves Dnmt3a mutant hematopoietic stem cells during inflammatory stress

Abstract Clonal hematopoiesis (CH), the age-related expansion of specific clones in the blood system has been considered as a pre-malignant condition of blood cancers. Approximately 50% of CH manifests from DNMT3A mutations acquired in the hematopoietic stem cells (HSCs), suggesting these mutations may convey a fitness advantage to HSCs during hematopoietic stress. However, a unifying molecular mechanism to illuminate how DNMT3A-mutant HSCs outcompete their counterparts is still lacking. Here, we used interferon-gamma (IFNg) as a model to study the mechanisms by which Dnmt3a mutations enhances HSC fitness under recurrent inflammatory stress. To represent the spectrum of DNMT3A variants found in humans, mouse genetic models were generated by specifically deleting Dnmt3a heterozygously (Vav-Cre;Dnmt3afl/+ = Dnmt3aHET) or homozygously (Vav-Cre;Dnmt3afl/fl = Dnmt3aKO) from the hematopoietic system. A hematopoietic system-specific knockin model analogous to the hotspot point mutation most prevalent in AML (Vav-Cre;Dnmt3aR878H/+ = Dnmt3aR878) was also included in the analysis. Competitive transplantation assays coupled with inflammatory and proliferative challenges suggested that Dnmt3aKO and Dnmt3aR878 HSCs were specifically tolerant of the deleterious effects of IFNg on HSC self-renewal and clonal expansion. When the competition between Dnmt3a-mutant and control HSCs under IFNg exposure was directly quantified in a novel mouse model, we found Dnmt3a-mutant HSCs resisted IFNg-mediated depletion due to an enhanced fitness advantage. Mechanistically, DNA hypomethylation-associated over-expression of Txnip in Dnmt3a-mutant HSCs was identified by coupling single-cell RNA-sequencing and Whole-Genome Bisulfite sequencing. The sustained Txnip levels in Dnmt3aKO HSCs lead to p53 stabilization and upregulation of p21 under IFNg challenge, further correlated with a retained quiescence due to a prolonged G0 exit in response to IFNg exposure. Implementing biochemical studies, we observed Txnip mediated an enrichment of p53 at p21 promoter under IFNg exposure in Dnmt3aKO but not control 32D cells. Knocking down Txnip by shRNA normalized p53 occupancy at p21 promoter and rescued IFNg-associated p21 upregulation in Dnmt3aKO 32D cells. Functionally, knocking down Txnip and p21 sensitized Dnmt3aKO HSCs to IFNg-induced cell cycle activation ex vivo. In vivo, down-regulation of p21 had no effect on control HSCs in response to IFNg exposure, but it completely primed Dnmt3aKO and Dnmt3aR878 HSCs to IFNg-associated exhaustion in a transplantation experiment. In summary, our data suggest an augmented Txnip-p53-p21 axis preserves the functional potential of Dnmt3a-mutant HSCs under inflammatory stress, which highlights a novel mechanism to explain the increased fitness of Dnmt3a-mutant HSCs and supports rationale for developing interventions to mitigate expansion of pre-malignant clones as a method of blood cancer prevention. Citation Format: Christine R. Zhang, Elizabeth Ostrander, Won Kyun Koh, Ostap kukhar, Hamza Celik, Jeffrey Magee, Grant Challen. Augmented Txnip-p53-p21 axis preserves Dnmt3a mutant hematopoietic stem cells during inflammatory stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB555.

In utero origin of myelofibrosis presenting in adult monozygotic twins

The latency between acquisition of an initiating somatic driver mutation by a single-cell and clinical presentation with cancer is largely unknown. We describe a remarkable case of monozygotic twins presenting with CALR mutation-positive myeloproliferative neoplasms (MPNs) (aged 37 and 38 years), with a clinical phenotype of primary myelofibrosis. The CALR mutation was absent in T cells and dermal fibroblasts, confirming somatic acquisition. Whole-genome sequencing lineage tracing revealed a common clonal origin of the CALR-mutant MPN clone, which occurred in utero followed by twin-to-twin transplacental transmission and subsequent similar disease latency. Index sorting and single-colony genotyping revealed phenotypic hematopoietic stem cells (HSCs) as the likely MPN-propagating cell. Furthermore, neonatal blood spot analysis confirmed in utero origin of the JAK2V617F mutation in a patient presenting with polycythemia vera (aged 34 years). These findings provide a unique window into the prolonged evolutionary dynamics of MPNs and fitness advantage exerted by MPN-associated driver mutations in HSCs.

Longitudinal profiling of clonal hematopoiesis provides insight into clonal dynamics

BackgroundClonal hematopoiesis of indeterminate potential (CHIP), the age-related expansion of mutant hematopoietic stem cells, confers risk for multiple diseases of aging including hematologic cancer and cardiovascular disease. Whole-exome or genome sequencing can detect CHIP, but due to those assays’ high cost, most population studies have been cross-sectional, sequencing only a single timepoint per individual.ResultsWe developed and validated a cost-effective single molecule molecular inversion probe sequencing (smMIPS) assay for detecting CHIP, targeting the 11 most frequently mutated genes in CHIP along with 4 recurrent mutational hotspots. We sequenced 548 multi-timepoint samples collected from 182 participants in the Women’s Health Initiative cohort, across a median span of 16 years. We detected 178 driver mutations reaching variant allele frequency ≥ 2% in at least one timepoint, many of which were detectable well below this threshold at earlier timepoints. The majority of clonal mutations (52.1%) expanded over time (with a median doubling period of 7.43 years), with the others remaining static or decreasing in size in the absence of any cytotoxic therapy.ConclusionsTargeted smMIPS sequencing can sensitively measure clonal dynamics in CHIP. Mutations that reached the conventional threshold for CHIP (2% frequency) tended to continue growing, indicating that after CHIP is acquired, it is generally not lost. The ability to cost-effectively profile CHIP longitudinally will enable future studies to investigate why some CHIP clones expand, and how their dynamics relate to health outcomes at a biobank scale.

In This Issue

Patients with B-lymphoid malignancies are at increased risk of severe COVID-19, but the impact of antineoplastic therapies on COVID-19 outcomes in this population is uncertain. Here Rubinstein et al. compare the outcomes of COVID-19 infection among 995 patients with B-lymphoid malignancies and multiple control populations of patients with cancer. The cohort size enables distinguishing contributions of lymphoid malignancy and of antineoplastic therapy, with adjustment for important prognostic factors. The results indicate that patients recently treated for B-lymphoid malignancies are at increased risk of severe COVID-19.See article, p. 181.Genomic characterization of pediatric acute myeloid leukemia (AML) at relapse identified recurrent somatic tandem duplications of exon 13 of UBTF (upstream binding transcription factor) in nearly 10% of cases. These UBTF tandem duplication (TD) AMLs are associated with adolescence, FLT3 and WT1 cooperating mutations, yet do not occur with other known driver alterations. Extension into additional datasets demonstrates that UBTF-TDs are rare in adults and present in approximately 4% of children with AML at diagnosis. These UBTF-TD AMLs are associated with poor outcome and high rates of minimal residual disease positivity. Collectively, these findings suggest that UBTF-TD represents a new subtype of pediatric AML.See article, p. 194.Bulk DNA sequencing can identify a pool of mutations within a sample. With serial sampling of the same tumor, one can infer clonal evolution: which mutations likely cooccur in the same subclones and how the subclonal frequencies change through time, space, or lineage differentiation. The complex and noisy nature of bulk genomic sequencing data requires considerable computational resources to reconstruct clonal evolution, limiting applications to high-throughput and high-resolution datasets. Wintersinger et al. developed Pairtree, a computational method for building clonal evolution trees from deeply sampled cancer to construct much more detailed and accurate trees than previously possible. In B-cell leukemias, Pairtree used up to 90 samples per cancer to reveal up to 26 distinct cell populations. Increasing data complexity impairs performance of the existing algorithms, whereas Pairtree performs stably and with increased accuracy.See article, p. 208.DNMT3A mutations are recurrent in hematopoietic malignancies and in clonal hematopoiesis (CH), a preneoplastic state characterized by asymptomatic expansion of mutant hematopoietic stem and progenitor cells (HSPC). The expansion is explained by increased competitive fitness of mutant HSPC under specific selection pressure, such as Dnmt3a-mutant HSPC in IFNγ-driven emergency hematopoiesis. However, molecular and functional underpinnings of competitive fitness in CH remain uncharacterized. Zhang et al. identify the Dnmt3a–Txnip–p53–p21 axis as a molecular mechanism of Dnmt3a-mutant clonal advantage. Specifically, activation of this axis boosts quiescence and survival of Dnmt3a-mutant HSPC relative to their wild-type counterparts during IFNγ challenge, enabling the mutant HSPC to prevail.See article, p. 220.ZNF384 fusion oncoproteins define a subtype of lineage ambiguous leukemia. In this article, Dickerson et al. utilize mouse and human models of ZNF384-rearranged leukemia to investigate the mechanisms of leukemogenesis. They show that expression of ZNF384 fusions in mouse and human progenitors skews hematopoietic development and drives lineage aberrancy, and cooccurring Ras mutations promote development of leukemia. This is accompanied by histone acetylation and deregulation of core hematopoietic transcription factors. In addition, ZNF384-fusion leukemia exhibits overexpression of FLT3 with sensitivity to FLT3 inhibition in xenograft models, providing a therapeutic approach in this high-risk subtype of leukemia.See article, p. 240.

Association of Clonal Hematopoiesis of Indeterminate Potential with Worse Kidney Function and Anemia in Two Cohorts of Patients with Advanced Chronic Kidney Disease.

Clonal hematopoiesis of indeterminate potential (CHIP) is an inflammatory premalignant disorder resulting from acquired genetic mutations in hematopoietic stem cells. This condition is common in aging populations and associated with cardiovascular morbidity and overall mortality, but its role in CKD is unknown. We performed targeted sequencing to detect CHIP mutations in two independent cohorts of 87 and 85 adults with an eGFR<60 ml/min per 1.73m2. We also assessed kidney function, hematologic, and mineral bone disease parameters cross-sectionally at baseline, and collected creatinine measurements over the following 5-year period. At baseline, CHIP was detected in 18 of 87 (21%) and 25 of 85 (29%) cohort participants. Participants with CHIP were at higher risk of kidney failure, as predicted by the Kidney Failure Risk Equation (KFRE), compared with those without CHIP. Individuals with CHIP manifested a 2.2-fold increased risk of a 50% decline in eGFR or ESKD over 5 years of follow-up (hazard ratio 2.2; 95% confidence interval, 1.2 to 3.8) in a Cox proportional hazard model adjusted for age, sex, and baseline eGFR. The addition of CHIP to 2-year and 5-year calibrated KFRE risk models improved ESKD predictions. Those with CHIP also had lower hemoglobin, higher ferritin, and higher red blood cell mean corpuscular volume versus those without CHIP. In this exploratory analysis of individuals with preexisting CKD, CHIP was associated with higher baseline KFRE scores, greater progression of CKD, and anemia. Further research is needed to define the nature of the relationship between CHIP and kidney disease progression.

High Throughput Multi‐omic Characterization of Clonal Hematopoietic Stem Cells During Progression of Myeloid Malignancies

Hematopoietic stem cells (HSCs) are responsible for giving rise to all other lineages of blood cells in the body. Over time, mutations in HSCs can promote the outgrowth of clonal populations that outcompete other HSCs, resulting in a phenomenon called Clonal Hematopoiesis of Indeterminate Potential (CHIP). Though not cancerous in and of itself, CHIP can progress to more serious hematologic disorders, such as the Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). The mechanisms of clonal expansion, by which certain mutant HSCs acquire a competitive advantage over other HSCs, currently remain largely undeciphered, as are the mechanisms by which clonal HSCs drive the initiation of MDS and contribute to the development of AML. Moreover, previous studies have shown that such mutant HSCs are resistant to conventional therapies and may act as reservoirs for disease relapse and progression. In the past, researchers were hindered by bulk cell analyses and the relative rarity of HSCs, but advances in single‐cell omics have now enabled us to explore the molecular heterogeneity of clonal HSCs and identify distinct clonal populations based on genotype and cell surface phenotype. We used single cell RNA sequencing to examine the transcriptional dynamics of purified HSCs from MDS and AML patients before and after treatment, as well as from age matched elderly controls. Interestingly, dimensionality reduction methods such as UMAP and tSNE revealed a reservoir of control HSCs that clustered with MDS HSCs. Upon comparison with other normal control HSCs, we found that genes associated with aging, mitochondrial function, and particular ion channels were strongly upregulated in these “MDS‐like” control HSCs, while genes involved in ribosomal and translation activity, along with certain surface markers, were substantially downregulated. Additionally, ribosomal transcripts were depleted in MDS HSCs from patients who did not respond to treatment. These results support the notion that the most immature HSCs, which impose the strictest constraints on translation, might clonally expand and initiate CHIP and/or MDS upon acquiring driver mutations, also serving as treatment resistant populations that underlie disease relapse. We also subjected purified MDS HSCs to simultaneous single cell targeted DNA sequencing and cell surface phenotyping, which allowed us to correlate the cell surface phenotype and genotype for specific clonal populations and identify cell surface markers that can be used to isolate HSCs with enhanced engraftment ability. Taken together, these results indicate that combined single cell genotyping and phenotyping can be used to track clonal populations across different stages of pathogenesis, providing further insight about the development of CHIP and its progression to MDS and AML.

Profound abnormalities in thymic epithelial cells in Rag1 hypomorphic mice: implications for immune reconstitution after stem cell transplantation

Abstract Thymic epithelial cells (TEC) are a heterogeneous population of stromal cells which orchestrate T cell development and selection. Although recent reports highlighted TEC complexity, their developmental origin, hierarchy and differentiation still remain ill-defined. We compared TEC distribution and gene expression in wild-type (WT) mice and in mice carrying Rag1 hypomorphic mutations (Rag1mut) observed in patients with immunedeficiency and immune dysregulation. Single cell RNA-seq analysis of TECs isolated from Rag1mut mice revealed an excess of cortical TECs (cTECs). Medullary TECs (mTECs), albeit reduced in number, showed a similar distribution in previously described mature subsets. To address whether TEC abnormalities in Rag1mut mice might result from a developmental block, we compared them to TECs from neonate WT mice. Indeed, we observed a similar TEC distribution in adult Rag1mut mice and newborn WT mice, although we identified peculiar features in cTEC subsets of Rag1mut mice. Transplantation of WT hematopoietic stem cells (HSC) into Rag1mut mice was able not only to correct thymocyte development, but also to revert TEC phenotype to normalcy. However, in a competitive transplantation setting where 10% of WT and 90% mutant HSCs were used, only partial restoration of thymocyte subsets was observed, associated with incomplete rescue of TEC phenotype. In summary, we show that abnormalities of TEC composition in Rag1 mutant mice correlate with impaired development of thymocytes, highlighting the role of lymphostromal cross-talk in TEC maturation. Moreover, these data point to the need to achieve full donor chimerism after HSC transplantation in order to rescue the thymic compartment and prevent defects of immune tolerance. This research was supported by the Intramural Research Program of the NIAID, NIH