Mutations In Hematopoietic Stem Cells Research Articles

JAK2V617F Activates IL-10R Signalling Potentially through Transactivation of JAK1

Classical myeloproliferative neoplasms (MPNs) arise from somatic mutations in haematopoietic stem cells (HSCs) that lead to unregulated proliferation of mature myeloid cells. The most common mutation associated with MPNs is JAK2 V617F, this causes constitutive JAK-STAT signalling leading to excessive production of mature myeloid cells. In humans, for MPN to arise, a JAK2 V617F HSC must have a selective advantage over WT HSC. However, in mouse MPN models Jak2 V617F HSCs do not display an overt selective advantage. This suggests that unique selective pressures may be present among those predisposed to acquire MPN that allow for the emergence of Jak2 V617F clones. We have identified defects in the IL-10R signalling pathway among MPN patients, with evidence that this defect is an intrinsic not acquired abnormality. In mice, we found that the blockade of IL-10R extends the cycling of WT but not Jak2 V617FHSC in response to inflammatory stimuli and hastens proliferation-induced WT HSC exhaustion. We hypothesized that IL-10R blockade may create an environment that affords Jak2 V617F HSC a selective advantage, indeed we found that IL-10R blocking antibody allowed Jak2 V617F cells to outcompete WT cells in mouse transplant experiments. From these mouse data, we hypothesized that Jak2 V617Fenhances IL-10R signalling. To test this hypothesis, we created Ba/F3 cell lines co-expressing IL-10Rα and Jak2 V617F. We found that Jak2 V617F enhances growth at limiting concentrations of IL-10. We also found that cytokine-independent IL-10Rα Jak2 V617F clones (but never IL-10Rα Jak2 WTor IL-10Rα cells) emerge at day 8-10 in culture after withdrawal of cytokines. We performed a limiting dilution assay to pinpoint the proportion of IL-10R Jak2 V617F cells capable of surviving IL-3 withdrawal. One in 126 IL-10Rα Jak2 V617F cells has transforming ability, whereas 1 in 1.4 MPL Jak2 V617F has transforming ability. Together, these data support our hypothesis that Jak2 V617F enhances IL-10R signalling and can activate IL-10Rα, however with weaker transformation potential compared to Jak2V617F's activation of MPL signalling. JAK2 canonically does not participate in the IL-10R pathway. It has been previously shown that JAK1 can transactivate JAK2 when JAK2 V617F cells are under the stress of persistent JAK2 inhibitor exposure, and this is the purported mechanism of JAK inhibitor resistance among MPN patients. We hypothesized that JAK2 V617F can transactivate JAK1, leading to the activation of IL-10R. The same transfected BaF3 cells aforementioned were serum starved for 4 hours and stimulated with mIL-10 for 15 minutes to induce signalling. By way of Western Blot analysis, cytokine-independent Jak2 V617F IL-10Rα Ba/F3 cells showed constitutive activation of phosphorylated JAK1 and JAK2 as well as STAT3 and STAT5, consistent with transactivation. Studies to demonstrate direct interactions between JAK2 V617F and JAK1 are currently ongoing.

Brd4 Inhibition Abrogates Inflammation and Self-Renewal in a Murine Model of Tet2 Mutated Clonal Hematopoiesis

Clonal hematopoiesis (CH) is characterized by the somatic acquisition of malignancy associated mutations in hematopoietic stem cells (HSCs). Loss-of-function mutations in TET2 occur in one third of CH cases and are highly associated with the subsequent development of myeloid malignancies. Tet2 KO mice demonstrate increased HSC self-renewal, expansion of myeloid precursors, elevated inflammation, and progression to leukemia with long latencies, similar to CH to AML progression in humans. Additionally, leukemia development in Tet2 KO mice is dependent on increased production of pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α through hyperactive toll-like-receptor signaling (TLR). Despite extensive characterization of the adverse effects related to TET2 mutations, no effect intervention or pharmacologic therapy has been developed to slow its known progression. To investigate altered pathways in Tet2 mutant CH that might be amenable to pharmacologic targeting, we performed RNA-seq on sorted Lin -cKIT + (LK) myeloid progenitors and Lin -cKIT +Sca-1 + (LSK) stem cells from Tet2 KO vs WT mice. We found significant upregulation of hallmark JAK/STAT3 and TNF-α signaling pathways in Tet2 KO cells. The epigenetic reader protein bromo- and extra-terminal domain 4 (Brd4) interacts with both NF-κB and STAT3 to promote transcriptional activity and regulate inflammation. We hypothesized inhibition of Brd4 would reverse abnormal hematopoiesis in Tet2 KOs via reduction in inflammatory signaling. Therefore, we generated double KO mice by intercrossing Mx1-Cre mice with Tet2 conditional KO and Brd4 conditional KO mice. While deletion of Brd4 during fetal hematopoiesis is lethal, we have successfully induced deletion of Brd4 both alone and in combination with Tet2 in young adult animals without any observed lethality for 6 months. To study pharmacologic Brd4 inhibition in Tet2 KO mice, we utilized PLX51107, a structurally distinct BET inhibitor previously reported on by our laboratory. As expected, Tet2 KO marrow demonstrated increased colony formation and serial replating ability compared to WT controls. However, colony formation by Brd4 Tet2 DKO marrow was significantly decreased when compared to Tet2 KO ( p = 0.0003, n=5) or WT mice ( p = 0.0048, n=5) (Figure 1A). Similarly, PLX51107 treatment reduced Tet2 KO colony formation ( p = 0.0001, n=5) and abrogated serial replating ability ( p < 0.01, n=5). When Brd4 KO or Brd4 Tet2 DKO marrow was treated with PLX51107, which also inhibits Brd2 and Brd3, there was no significant change in colony formation compared to untreated marrow, suggesting Brd4 has a non-redundant role in colony formation. Collectively, these results suggest Brd4 plays a key role in self-renewal of Tet2 KO HSCs. To further characterize the role of Brd4 in Tet2 mutated CH, single cell RNA-seq analysis of Brd4 KO and Brd4 Tet2 DKO marrow populations is in progress. In murine models of Tet2 CH, disease progression is associated with emergence of an aberrant population of inflammatory macrophages. We therefore investigated functional differences in macrophages from WT, single, and double KO mice. LPS-stimulated bone marrow derived macrophages (BMDMs) from Brd4 Tet2 DKO mice produced significantly less TNF-α, IL-1β, IL12p40, and IL-18 when compared to single KO or WT controls ( p < 0.001, n=3) (Figure 1B). Similarly, we found mRNA expression of these cytokines to be decreased in LPS-stimulated Brd4 Tet2 DKO BMDMs when compared to single KO and WT controls ( p < 0.001, n=3). Similar results were observed in LPS-stimulated Tet2 KO and WT BMDMs treated with PLX51107 ( p < 0.005, n=3). Additionally, PLX51107 treatment reduced protein levels pSTAT3, NLRP3, and phosphorylated p65, key downstream targets of TLR signaling, in LPS-stimulated BMDMs. Together, these results suggest that Brd4 is required for the hyperinflammation characteristic of Tet2 mutated macrophages that drive progression to myeloid malignancies. In summary, we report that genetic or pharmacologic inhibition of Brd4 reduces the increased self-renewal and elevated inflammation in a murine model of Tet2 mutated CH. Our novel inducible double knockout mouse model of CH achieved simultaneous knockout of Brd4 and Tet2, which has never been investigated. Collectively, our data provides rationale for further pre-clinical investigation of BET inhibitors to prevent progression of Tet2 CH.

Elevated Functional Hematopoietic Stem and Progenitor Cells in the Peripheral Blood of G-CSF-Mobilized Dnmt3a-Mutant Mice

Clonal hematopoiesis (CH) arising from somatic mutations in hematopoietic stem cells (HSCs) is associated with increased risk of developing several non-hematologic and hematologic diseases. In the context of stem cell transplantation, donor HSCs carrying CH-associated somatic mutations can be passed from donor to recipient during stem cell transplantation. Most literature supports that there is no negative impact on recipient survival or outcomes after allogeneic stem cell transplantation (allo-HSCT) from donors with CH. However, it is less clear how allo-HSCT donors with CH respond to common mobilization methods such as Neupogen (G-CSF). As hematopoietic stem and progenitor cells (HSPCs) with CH mutations have been found to respond to external stimuli such as inflammation in a manner distinct from wild-type HSPCs, it is plausible that CH-mutant HSPCs also distinctly respond to G-CSF mobilization. It is important to understand whether the process of mobilization differentially enriches for CH-mutant HSPCs in the mobilized peripheral blood product. To address this gap in knowledge, we used a murine model of Dnmt3a-mutant CH. After five consecutive days of Neupogen (G-CSF) administration, modeling the procedure for human HSC mobilization, we observed that Dnmt3a-mutant mice had significantly increased white blood cell, neutrophil and monocyte counts compared to control mice. We observed a significant increase in cell surface marker-defined HSPCs in the peripheral blood and spleen of G-CSF-treated Dnmt3a-mutant mice compared to G-CSF-treated control mice. Using in vitro colony-forming unit assays, the peripheral blood of G-CSF-treated Dnmt3a-mutant mice was found to contain a greater number of functional hematopoietic progenitors compared to G-CSF-treated control mice. To determine if the peripheral blood of G-CSF-treated Dnmt3a-mutant mice had a greater abundance of functional HSCs, we transplanted equal volumes of mobilized blood from Dnmt3a-mutant and control mice into primary and secondary lethally irradiated wild-type recipient mice. We observed increased long-term engraftment of mobilized blood from Dnmt3a-mutant mice compared to control mice in both primary and secondary recipient animals. At a mechanistic level, HSC mobilization is facilitated by myeloid cells that respond directly to G-CSF via the G-CSF receptor ( Csf1r). In Dnmt3a-mutant mice, we observed increased Csf1r expression in myeloid progenitor and mature myeloid cells compared to control mice, providing a putative mechanism by which increased mobilization is enabled. Together, our data support differential mobilization potential of Dnmt3a-mutant compared to control HSCs in response to G-CSF, which may have implications in the context of allo-HSCT.

Mutational Changes in Solid Cancer Cells Induce DNA Damage Repair Deficiency in Hematopoietic Cells Leading to a Distinct Spectrum of Clonal Hematopoiesis Independent from Age-Related Clonal Hematopoiesis

Clonal hematopoiesis (CH) is characterized by the acquisition of defined somatic mutations in hematopoietic stem cells (HSC). It is well known that CH contributes to the development of hematological malignancies but also increases the risk of developing non-malignant conditions such as cardiovascular, pulmonary, and inflammatory liver disease. However, less is known about the association between clonal hematopoiesis and solid cancer. Reports suggest that patients with solid cancer have a higher incidence of clonal hematopoiesis at an earlier age and an inferior prognosis. To investigate whether the presence of solid cancer itself can drive the development of clonal hematopoiesis, we analyzed 392 paired tumor and blood samples from patients with advanced cancer. In our cohort, all patients had evidence of clonal hematopoiesis based on a variant allele frequency (VAF) of at least 2%. With respect to a defined set of the 20 most frequently CH-associated genes, we discovered an increased CH rate of 23% of all patients (90 cases, median age of 61 years, defined by a VAF from 2% to 30%). The frequency of mutated genes differed greatly from published data from the general population and cancer patients, with the most common mutations being PABPC1 (39%), KMT2C (28%), RUNX1 (18%), and DNMT3A (10%). In addition, we observed a strong correlation between certain mutations in tumor samples and the frequency of CH. The mutational signature of CH was not mainly associated with a typical chemotherapy-dependent mutational signature, but with an impaired DNA repair capacity. For further investigation, we performed additional experiments in mice, which showed that the presence of malignant cells impairs DNA repair capacity in HSC through inflammatory changes in the stroma. In summary, we were able to show that patients with advanced or recurrent solid tumors have a significantly increased frequency of CH, which is not only the result of prior chemotherapy exposure but the result of diminished DNA repair capacity. This suggests a possible role of solid cancer in promoting the development of CH independent from chemotherapy exposure, which is supported by our additional murine experiments.

Inflammatory Signaling Mediated By Gasdermin D Underlies Tet2-Mutant Clonal Hematopoiesis-Associated Tissue Dysfunction

Aging is characterized by the progressive accumulation of somatic mutations. Acquisition of certain somatic mutations in hematopoietic stem cells (HSCs) confers on them a fitness advantage enabling them to outcompete other HSCs by clonal expansion in a phenomenon called clonal hematopoiesis (CH). CH is associated with a 40% increase in all-cause mortality linked to elevated risk for aging-associated morbidities such as cardiovascular disease (CVD), chronic kidney disease, COPD, and chronic liver disease. However, the mechanism by which CH exerts its adverse effects on disparate tissues is poorly understood. Here, we use CH-associated cardiac dysfunction as a paradigm to uncover general principles by which CH leads to the decline of tissue function. CH was modeled in mice by transplanting whole bone marrow (WBM) cells deficient for Tet2, a frequently mutated CH gene, into irradiated WT mice. We observed an elevated incidence of atrial fibrillation (AF) in Tet2 -/- WBM recipients relative to WT WBM recipients without any surgical or dietary intervention (Fig. A). The Tet2 -/- WBM recipients also had reduced left ventricular ejection fraction (LVEF), an indicator of heart failure. Mice exhibiting elevated incidence of AF and reduced LVEF are defined as having cardiac dysfunction. In addition, we observed that Tet2 -/- WBM recipients had increased systemic inflammation compared to controls, as evidenced by elevated levels of proinflammatory cytokines in the serum. Furthermore, their cardiac tissue exhibited upregulation of the inflammasome signaling pathway including the activation of Gasdermin D, the effector of pyroptotic cell death. Inflammation in the cardiac tissue was concomitantly associated with increased cardiac fibrosis in Tet2 -/- WBM recipients. Since inflammation is a known driver of CVD, we hypothesized that abrogating Gasdermin D-mediated inflammatory signaling could ameliorate CH-induced cardiac dysfunction. Indeed, Gsdmd -/- recipient mice were protected from cardiac dysfunction upon transplantation with Tet2 -/-WBM cells (Fig. A). Importantly, expansion of mutant hematopoietic cells was also mitigated in the absence of GSDMD: transplantation of Tet2 -/- WBM cells along with either WT or Gsdmd -/- WBM cells revealed that GSDMD deficiency restrained the rate of clonal expansion of Tet2 -/- cells (Fig. B). This suggests that instead of being mere bystanders, WT cells in the environment may play an active role in regulating the rate of mutant cell expansion via intercellular communication mediated by GSDMD. Identification of proinflammatory factors mediating clonal expansion and cardiac dysfunction is of significant interest for potential therapeutic intervention. Thus, we sought to determine factors that were upregulated in CH mouse models while being downregulated in Gsdmd -/- mice. Luminex multiplexed assay enabled us to identify several understudied proinflammatory cytokines present in serum satisfying the above criteria. In addition to elevated levels in the serum, some of these cytokines were upregulated in the cardiac tissue of Tet2 -/- WBM recipient mice but not in WT WBM recipients. Furthermore, some are reported to induce inflammasome signaling. CH, even in the absence of known drivers, is associated with higher CVD-associated mortality, suggesting the existence of pathways common to multiple forms of CH; systemic inflammation is one potential common mechanism. We tested whether aged WT WBM, which has low levels of mutant clone expansion but exhibits several features of CH, could cause cardiac dysfunction. We transplanted either aged or young WT WBM into young WT recipients and found that the aged WT WBM recipients exhibited elevated AF and reduced LVEF. These data suggest that aging-associated systemic inflammation may be sufficient for cardiac dysfunction. Importantly, one of the cytokines we uncovered was also upregulated in aged WT mice and has been associated with inflammation and fibrosis in multiple tissues. This cytokine is being further investigated as a top candidate for therapeutic intervention since it is a common feature of CH with and without identified drivers. Overall, our findings demonstrate the critical role of Gasdermin D-mediated inflammatory signaling in the pathogenesis of Tet2-mutant CH-induced cardiac dysfunction. We have also uncovered a cytokine that has emerged as a promising therapeutic target for CH-associated pathologies.

SF3B1 K700E Alters the Splicing of Metabolism and Stress Response Genes, Attenuating the Effect of DNMT3A Mutations in MDS

Myelodysplastic syndromes (MDS) are clonal stem cell disorders associated with low blood counts and a high risk of leukemic transformation. Genomic sequencing studies have demonstrated that MDS develops due to the acquisition of somatic mutations in hematopoietic stem cells (HSCs). Splicing factor 3b subunit 1 ( SF3B1) mutations, one of the most frequent mutations in MDS, are associated with lower-risk disease characterized by ineffective erythropoiesis. Our group and others have shown that mutations in SF3B1 cause aberrant mRNA splicing in human and murine HSCs, an expansion of long-term HSCs (LT-HSCs) in primary mice, but a competitive disadvantage in transplantation assays. DNMT3A, de novo methyltransferase 3A, is an epigenetic regulator mutated in ~10-15% of MDS. DNMT3A mutations are associated with unfavorable outcomes in MDS including a higher risk of leukemia transformation and shorter overall survival. Murine models of Dnmt3a mutations revealed that Dnmt3a-mutant HSCs have increased self-renewal over lineage-specific differentiation. SF3B1 and DNMT3A mutations co-occur more frequently than expected by chance and arise early in MDS pathogenesis, making them attractive therapeutic targets. MDS patients with SF3B1 and DNMT3A mutations have shorter progression-free survival than those with solely SF3B1 mutations. However, the mechanisms by which these mutations cooperate to cause HSC dysfunction and their possible combined role in leukemogenesis are not well understood. To investigate how Dnmt3a and Sf3b1 mutations disrupt HSC function in vivo we crossed two conditional knock-in mouse models of common point mutations in SF3B1 (K700E) and DNMT3A (R787H, murine equivalent to human DNMT3A R882H). We followed monthly blood counts and evaluated stem and progenitor cell composition in the bone marrow at 6 months. We found that Sf3b1/Dnmt3a double mutant mice display a similar phenotype to Sf3b1K700E mice, with a progressive anemia and an expansion in LT-HSCs. However, when assayed for HSC self-renewal potential, whole bone marrow and LT-HSCs isolated from Sf3b1/Dnmt3a mutant mice display increased serial colony formation capacities similar to Dnmt3a mutant hematopoietic stem and progenitor cells (HSPCs, 5 to 6 passages), but have a competitive disadvantage in secondary and tertiary transplant assays that is less severe than Sf3b1K700E HSPCs but distinct from the competitive advantage of Dnmt3a mutant HSPCs. Transcriptionally, double mutant HSCs clustered closer to Sf3b1 HSCs with shared expression of 74% of upregulated genes and 64% of downregulated genes and have decreased expression of stress response and metabolism genes. These results indicate Sf3b1 mutations may mitigate the preleukemic phenotype due to Dnmt3a mutations in MDS. To identify factors regulated by SF3B1 mutations that may mitigate the effect of DNMT3A loss on leukemic progression, we generated isogenic DNMT3A KO and SF3B1 K700E single and double mutant K562 cell lines using CRISPR/Cas9. Transcriptome analysis demonstrated that SF3B1K700E/DNMT3AKO double mutant clones clustered with and shared similar upregulated (69%) and downregulated (62%) genes as SF3B1 single mutant clones. Similar to our findings in murine HSPCs, pathway analysis of SF3B1 and SF3B1/DNMT3A mutant clones displayed enrichment of stress response and metabolic pathways. Alternative mRNA splicing analysis identified 13 events (3 alternative 3' splice site, 9 intron retention, and 1 skipped exon) in 8 genes that were upregulated in DNMT3A KO clones and downregulated in SF3B1 and SF3B1/DNMT3A mutant clones. We confirmed decreased protein expression of these mis-spliced genes in SF3B1 single and double mutant clones, but not DNMT3A KO clones. These genes include components of the integrated stress response pathway, which are also mis-spliced in SF3B1 and SF3B1/DNMT3A mutant MDS patient samples. Taken together our data suggest that aberrant splicing by mutant SF3B1 prevents metabolic reprogramming and decreases leukemic progression caused by DNMT3A mutation in MDS.

Drug Repurposing in a Zebrafish AML Model

Acute myeloid leukemia (AML) is a genetically heterogeneous hematological malignancy. AML arises from the stepwise accumulation of mutations in hematopoietic stem cells (HSCs). The t(9;11)(p22;q23) translocation is a translocation detected in about 5 to 7% of AML patients, which gives rise to the KMT2A/MLLT3 or MLL/AF9 ( MA9) fusion gene. Zebrafish (ZF) are well-established model organisms for investigating human hematopoiesis. Approximately 70% of human genes have corresponding orthologues in zebrafish, and over 80% of disease-related genes are conserved between the two species. Previously, we established a transgenic MA9 zebrafish AML model (Runx1+23:MA9), in which the AML was serially transplantable. Our investigations revealed that MA9 expression alters hematopoiesis in transgenic embryos before the onset of AML, leading to myeloid expansion. Whole-mount in situ hybridization (WISH) of embryos showed increased expression of granulocyte markers such as lyz 24 and 48 hours after fertilization (hpf). Based on these findings, we developed a Runx1+23:MA9/lyz-EGFP zebrafish leukemia line, in which lyz-expressing myeloid cells are EGFP positive. This model provides a phenotype-based platform to evaluate the therapeutic potential of any drug that can affect the myeloid cell population. We used this ZF model to investigate the therapeutic potential of nine drugs in MA9-driven AML. MA9 transgenic fish (Runx1+23:MA9) were crossed with lyz-EGFP fish to generate a MA9/lyz-EFGP line. In this line, myeloid cells can be visualized with a fluorescent microscope. The expression of lyz in the MA9/lyz-EFGP transgenic embryos was compared to that in the lyz-EGFP line. In our previous study on MA9 transgenic leukemic fish, RNA-seq analysis had revealed higher expression of genes acting in the EGFR, MEK, RAF, KRAS pathways and downregulation of genes in TP53. Based on these results, we selected nine drugs that are known to affect some of these pathways as well as drugs that are used to treat leukemia for screening in our model. Control and MA9/lyz-EFGP ZF 48 hpf embryos were treated with 10 µM of each drug. 24 hrs after drug treatment, the number of lyz expressing cells was analyzed by visualizing the embryos under a fluorescent microscope, and lyz transcript levels were measured by qPCR. Treatment with erlotinib, lapatinib, cytarabine, and gefitinib led to a visible reduction in the number of lyz-expressing cells in the MA9 transgenic embryos but not in the controls (Figure 1). In comparison, treatment with decitabine resulted in a decrease in the number of lyz expressing cells in both the MA9/lyz-EGFP and the control embryos. Treatment with doxorubicin, zoledronic and arsenic trioxide did not have an effect on the number of lyz-expressing population in the transgenic nor in the control embryos. These results were confirmed by qPCR, which showed that expression of lyz was reduced by 33% to 51% in the erlotinib, lapatinib, cytarabine, and gefitinib treated MA9 transgenic embryos (Figure 2). Treatment with these drugs did not significantly change the lyz expression in lyz-EFGP control embryos. Our results provide strong evidence for the successful establishment of a zebrafish model of KMT2A-rearranged AML that can be used for low to medium-throughput drug screening by employing the expression level of an endogenous gene (lyz) as a read-out. This model is a valuable tool to assess the drug's specificity in targeting oncogene-expressing cells and normal hematopoietic cells and, therefore, to identify novel therapeutic agents that can target the cancer cells with fewer side effects on normal hematopoietic cells.

Stag2-Cohesin Loss Attenuates Flt3 ITD Myeloid Blast Expansion Yet Preserves Mutant HSC

FLT3 is mutated through an internal tandem duplication (ITD) in 20-25% of acute myeloid leukemia (AML), driving aberrant STAT5/AKT signaling and leukemogenesis. Murine modelling of ITD showed marked myeloid progenitor expansion and hematopoietic stem cell (HSC) exhaustion in various co-mutant settings. Here we present a Stag2 Δ Flt3 ITD model with stark reversal of these two aforementioned features. STAG2, a cohesin complex member, maintains the integrity of the 3D genome partitioning structure known as topologically structural domains. Loss of Stag2 impairs the access and engagement of key hematopoietic transcription factors such as PU.1 to their target genes. sAML is diagnosed when patients present leukemia with known history of hematological malignancies or chemotherapy/radiotherapy treatment. Comparing to de novo AML, sAML often arises in older patients and harbors a poor prognosis with a 5-year overall survival rate of <30%. Advances in genomic studies found various epigenetic mutations, such as STAG2, are often associated with sAML and AML-myelodysplastic related changes (MRC) subtype. STAG2 mutations are found in 14-20% of sAML cases and is suggested to reside within a dominant clone during the pre-leukemia phase, MDS to sAML transformation, such as with FLT3 ITD acquisition and persists during remission. To understand the mechanistic contribution of STAG2-cohesin loss with FLT3 ITD, we generated sequential mutagenesis murine models where the order of Stag2 and Flt3 ITD mutation is set as either ITD 1st Stag2 2nd ( de novo like) or Stag2 1stITD 2nd (sAML like) using tamoxifen-inducible Cre/Flpo recombinase or pIpC-inducible Mx1Cre. In the de novo like model, ITD is constitutively active then Stag2 is deleted when mice reach 6-8 weeks of age. Surprisingly, loss of Stag2 attenuates LSK to MP transition at 4 weeks post deletion, while MPP3 remains elevated, suggesting aberrant remodeling of myeloid differentiation. In the sAML like model, Stag2 is deleted via Mx1Cre and waited for 4 months to mimic the MDS phase, which is then followed by activation of ITD mutation via Flpo, which represents the MDS to sAML transformation. After activating both mutations, mice were followed for another 4 months before analyzing the hematopoietic stem and progenitor compartment. In contrast to the de novo like model, sequential Stag2 1stITD 2nd preserves the HSC population defined by either immunophenotyping or transcriptome via scRNAseq ( Figure 1). The mutant HSC is more quiescent but retains the capacity to reconstitute lethally irradiated recipients in the short term. Similar to the de novo like model, sequential Stag2 1stITD 2nd mice also exhibits a blocked myeloid differentiation. Comparing to ITD mutant, Stag2 1stITD 2nd LSK cells have decreased expression of Socs2 and Cish. While functionally determining the role of mutant HSC, we are performing RNA-seq during at early timepoints post ITD activation to determine how preceding Stag2 mutation could have altered the stem cell fate decision. Targeted therapy with inhibitors of FLT3 have had an overall survival benefit in FLT3-mutant AML, though the magnitude of effect has been modest. STAG2 mutations are more likely to be identified in poor responders to FLT3 inhibition as both reported by us with Pexidartinib treatment, as well as in the setting of Crenolanib treatment where expansion of the STAG2-mutant clone was observed during treatment. Thus, remodeling of the chromatin landscape though altered CTCF binding or cohesin function might impact leukemia identity in FLT3-mutant AML. Our data highlights an important regulatory role of Stag2-cohesin in Flt3 ITD mediated leukemogenesis, while generating a model that mimics the genetic evolution of sAML. This model will not only shed light on the sAML pathogenesis but also with creates a pre-clinical testing platform with potential therapeutic relevance.

Single Cell Transcriptomics Reveal That HMGA1 Chromatin Regulators Drive Transcriptional Networks Involved in Megakaryocyte Expansion, Platelet Activation, and Interferon Signaling in JAK2 V617F Myeloproliferative Neoplasms

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by hyperactive JAK/STAT signaling, thromboses, abnormal bleeding, and increased risk of transformation to myelofibrosis (MF) or acute myeloid leukemia (AML). Importantly, clinical outcomes are poor after transformation and actionable mechanisms that drive progression remain elusive. We discovered that High Mobility Group A1 ( HMGA1) chromatin regulators are required for transformation to AML in preclinical models of MPN by up-regulating transcriptional networks involved in GATA2 and proliferation (Li et al, Blood, 2022). Unexpectedly, loss of just a single Hmga1 allele within HSC prevents progression to MF in JAK2 V617F mice. Intriguingly, Hmga1 also drives fibrosis and tumor progression in pancreatic tumor models (Chia et al, JCI 2023). We therefore sought to elucidate mechanisms underlying HMGA1 early in progression that could be targeted in therapy to prevent MF. Methods : To elucidate Hmga1 function early in MPN, we performed single cell RNA sequencing (scRNAseq) of bone marrow (BM)-derived Lin -, Sca +, c-kit + (LSK) cells from JAK2 V617F mice at 16 weeks old before the development of MF with both Hmga1 alleles intact ( Hmga1+/+) compared to those with haploinsufficiency ( Hmga1+/-). We used gene set enrichment analysis (GSEA) to identify Hmga1-dependent transcriptional networks, CellChat to define cell interactions, and trajectory analysis to assess differentiation. To identify relevant networks in patients, we assessed HMGA1 pathways in transcriptomes from platelets, peripheral blood mononuclear cells, or CD34 + stem and progenitor cells from MPN patients and healthy, age-matched controls via bulk or scRNAseq. Results: We discovered that loss of just a single Hmga1 allele in HSPC is sufficient to dampen expansion of long-term, quiescent HSC (qHSC), megakaryocyte-biased HSC (Mk-HSC), and megakaryocyte-erythroid-biased HSC (MEP-HSC) while expanding HSC with a lymphoid bias (Ly-HSC) in JAK2 V617Fmice by scRNAseq. Pathway analysis in the most quiescent HSC clusters reveal that Hmga1 induces pathways involved in interferon α (IFNα) and interferon γ (IFNγ) signaling. Trajectory analysis show that JAK2 V617F qHSC with intact Hmga1 have diminished differentiation capacity, leading to decreased Ly-HSC. Because Hmga1 haploinsufficiency dramatically decreased megakaryocyte hyperplasia and thrombocytosis in JAK2 V617Fmice, we focused on HMGA1 transcriptional networks in Mk-HSC and MEP-HSC. Similar to qHSC, Hmga1 up-regulates networks involved in IFNα and IFNγ signaling in Mk- and MEP-biased HSC. In contrast to the qHSC, Hmga1 activates transcriptional networks involved in cell cycle progression in HSC poised to differentiate into Mk and MEP, including: 1) MYC Target pathways, 2) E2F Targets, and, 3) Mitotic Spindle genes. In addition, gene networks involved in oxidative phosphorylation are activated, which associate with proliferative states. Unexpectedly, we also discovered that Hmga1 up-regulates genes involved in coagulation and platelet activation, including genes encoding Platelet factor 4, von Willebrand factor, glycoprotein 2b, glycoprotein 3a, and P-selectin. Because megakaryocytes are associated with inflammatory signals in MPN, we queried inflammatory gene networks, which revealed that Hmga1 induces genes encoding IL6, IL1-β, and gene networks involved in signaling through TNFα and NF-κB. CellChat shows that Hmga1 increases MIF receptor-ligand signaling. Moreover, HMGA1 and similar networks are up-regulated in transcriptomes of platelets, PBMC, and CD34 + cells from patients with JAK2 V617F MPN. Conclusions: We uncovered a previously unknown epigenetic program in JAK2 V617FMPN whereby Hmga1 induces transcriptional networks that drive expansion in mutant HSC with greatest impact on HSC biased to differentiate into megakaryocytes (Mk-HSC, MEP-HSC). Mechanistically, Hmga1 up-regulates genes involved in cell cycle progression, platelet activation, and inflammatory signaling in Mk-biased HSC. HMGA1 also induces genes involved in IFNα and IFNγ signaling. Together, our studies reveal a new paradigm whereby HMGA1 drives progression early in MPN by activating transcriptional networks required for megakaryocyte expansion, inflammatory signaling, and IFN networks, the latter of which may sensitize JAK2 mutant HSC to IFN therapy.

UBE2N Regulates Oncoprotein Networks in Myeloid Malignancies

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) originate from clonal mutant hematopoietic stem and progenitor cells, referred to as leukemic stem and progenitor cells (LSPCs). Ubiquitination is an essential process for various cellular maintenance functions, including protein degradation and scaffolding, which is often perturbed in a variety of cancers. Ubiquitination of protein substrates involves three related enzymes: the E1 activating enzyme, the E2 conjugating enzyme, and the E3 ligase. In humans, there are up to 40 E2 conjugating enzymes, among which Ubiquitin conjugating enzyme E2 N (UBE2N) stands out as one of the most crucial and overexpressed E2 enzymes in AML cells. Our recent study reported that UBE2N is a druggable target in MDS and AML (Barreyro et al., Science Translational Medicine, 2022), and we found that its catalytic active site cysteine 87 is required for various AML mouse models, including MLL-AF9, FLT3-ITD, AML1-ETO9a, and MN1 (unpublished). These observations indicate that the catalytic function of UBE2N is critical for AML cells but dispensable for normal hematopoiesis. To examine the active state of UBE2N, we investigated the “charged” version of UBE2N, which contains a ubiquitin bound to cysteine 87, in normal hematopoietic cells and in a AML cell line panels. Our findings confirmed that the activated form of UBE2N is highly expressed in human AML cells compared to normal hematopoietic cells. UBE2N exclusively synthesizes Lysine-63 (K63)-linked polyubiquitin chains on target proteins in conjunction with selected E3 ligases, leading to the stabilization and/or activation of protein substrates. To identify the relevant targets of UBE2N in AML, we conducted a ubiquitin-enrichment followed by a proteomic analysis in isogenic UBE2N-deficient AML cells compared to WT AML cells. We identified ~300 proteins that are ubiquitinated by UBE2N. Importantly, UBE2N regulates a network of leukemia-associated oncoproteins, including SYNCRIP, HUWE1, NPM1, TYK2, IKBKG, VAV1, BAX, IGF2BP2, IRF4, IRAK4, RBMX, STAT3, and BTK. UBE2N can interact with various E3 ligases in a cell- and context-dependent manner. To gain insight into the major E3 ligase that cooperates with activated UBE2N in AML, we performed a proximity-based labeling assay followed by mass spectrometry. This interactome screen identified Tripartite motif containing 21 (TRIM21) as the top E3 ligase that partners with UBE2N in AML cells. TRIM21 is overexpressed in AML patients compared to healthy controls. Moreover, deletion of TRIM21 in AML cells impaired cell viability and proliferation in vitro and prolonged survival in a xenograft model in vivo, which phenocopies the loss of UBE2N. Importantly, restoration of TRIM21 rescued the functional defect of UBE2N-deficient AML cells, indicating that the UBE2N-TRIM21 axis is operational in AML. To prioritize the UBE2N-dependent oncoprotein network, we focused on UBE2N-TRIM21 regulated signaling in AML. For this, a gene expression analysis was performed in UBE2N-deficient AML cells that were restored with TRIM21. The top UBE2N-TRIM21 oncoprotein network that emerged was related to interferon-STAT signaling. Additionally, both UBE2N and TRIM21 were found to regulate STAT3 ubiquitination. Deletion or inhibition of UBE2N catalytic activity significantly reduced total and activated STAT3 in AML cells. Finally, the expression of constitutively active STAT3 partially restored the functional defect of AML cells treated with a UBE2N inhibitor, indicating that STAT3 function, a known driver of LSPCs, is regulated via the UBE2N-TRIM21 axis in AML. In summary, our data reveal that UBE2N regulates oncoprotein networks in AML and that inhibiting UBE2N catalytic function, such as with small molecule inhibitors, effectively suppresses LSPCs.

Mitigating Oxidative Stress Promotes Quiescence of Hematopoietic Stem Cells from Concurrent TLR4 Activation and IL-10R Blockade Mediated Inflammation

Inflammatory stress provides a clonogenic advantage towards CHIP mutant hematopoietic stem and progenitor cells (HSPCs) via different mechanisms including resistance to apoptosis, enhanced self-renewal and limited generation of reactive oxygen species. Reactive oxygen species (ROS) play a causative role in hematopoietic stem cell (HSC) dysfunction due to exit from quiescence and replication stress-induced stem cell exhaustion. Oxidative stress mediated decline in the fitness of WT hematopoietic stem cells (HSCs) may allow mutant HSCs to gain a selective advantage. Therefore, protection of WT HSCs from ROS may maintain their fitness, and prevent the selective expansion of mutant cells. To test whether the anti-oxidant N-Acetylcysteine (N-AC) protects HSCs from chronic inflammatory stress, we mice were challenged daily with lipopolysaccharide (LPS) (5μg, intraperitoneal) or saline control for 30 days while their drinking water was supplemented with N-AC at 2mg/ml or untreated regular water. At day 30, bone marrow was harvested for competitive repopulation assays using equal numbers of whole bone marrow cells from each competitor (Figure 1A). As expected, bone marrow from mice exposed to 30 days of chronic LPS on regular water showed inferior reconstitution compared to saline exposed mice. However, bone marrow from mice exposed to 30 days of LPS and maintained on N-AC supplemented water performed equivalently in competitive transplants as saline exposed mice. To demonstrate that this functional defect of HSCs stemming from chronic LPS treatment is dependent on increased HSC divisions, we challenged TetOP-H2B-GFP mice with LPS for 30 days and assessed H2B-GFP label retention in immunophenotypically defined long-term HSCs. Progenitor populations in the bone marrow demonstrated low levels of H2B-GFP expression while LT-HSCs showed high GFP signal indicating low turnover. Meanwhile, LPS treatment led to increased cell divisions in LT-HSCs compared to saline treated mice. We hypothesized that N-AC protects HSCs via reducing entry into cell cycle in response to inflammatory stimuli, which is ROS dependent. We assessed the impact of N-AC treatment on ROS in HSCs and its correlation with cell cycling. To induce acute inflammation, wildtype (WT) mice were injected with LPS (5μg) and IL-10R blocking antibody (0.1mg). IL-10R blocking antibody was used in conjunction with LPS because we have previously found that IL-10R blockade extends LPS induced HSC proliferation. Intracellular ROS was determined using the ROS sensitive dye, CM-H 2DCFDA (DCF). The DCF signal was increased in HSCs (Lin -, c-Kit +, Sca1 +, CD150 +, CD48 -) in response to TLR4 activation by LPS and the addition of αIL-10R further augmented this increase in ROS (Figure 1B). Pre-treatment with N-AC significantly reduced the increase in ROS mediated by LPS alone. Cell cycle status was determined by intracellular Ki67 staining of HSCs. LPS significantly increased HSC proliferation as determined by increased percentage of cells in the G1 phase of the cell cycle while IL-10R blockade further augmented HSC cycling. Mice pre-treated with N-AC did not show significant increase in HSC proliferation indicating protection from ROS mediated exit from quiescence. In conclusion, incorporation of N-AC into drinking water protected WT HSCs from inflammation induced exhaustion and promoted persistence of quiescence in HSCs. N-AC could potentially be utilized as a preventative method to preserve fitness of HSCs, particularly in those with chronic inflammatory stress. Moreover, preservation of HSC fitness should be evaluated for its ability to prevent emergence of clonal hematopoiesis.

The Zymogen Form of Caspase-1 Is Required to Fine Tune Excessive Cell-Intrinsic Inflammation in Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is characterized by the expansion of clonally-derived mutant hematopoietic stem and progenitor cells. Many patients do not respond to standard chemotherapy because AML cells acquire mutations that exploit inflammatory and cell death pathways, allowing them to evade cell death. Targeted therapies are urgently needed to block AML cell pro-survival and inflammatory signaling for more effective outcomes. One promising area of interest is Caspase-1 (CASP1), a cysteine-aspartic acid protease responsible for cleaving the precursors of interleukin-1β (IL-1β) and Gasdermin D, which induces pyroptosis, into their active mature peptides. In myeloid malignancies, IL-1β levels are significantly elevated, and pyroptosis has been linked to ineffective hematopoiesis as observed in MDS, thereby implicating CASP1. CASP1 is typically expressed as a zymogen (Pro-CASP1) that can be cleaved into an active enzyme. In this report, we present a novel and unexpected role for Pro-CASP1, which appears to be independent of its proteolytic function, in MDS/AML leukemic cells. Pyroptosis, an inflammatory cell death mechanism, is driven by CASP1-mediated osmotic lysis and IL-1b secretion. Notably, CASP1 expression is significantly higher in AML patients compared to healthy hematopoietic cells, and elevated CASP1 expression in AML patients correlates with shorter overall survival. In leukemic cells, CASP1 primarily exists in its zymogen form, lacking the ability to induce pyroptosis. It is thus unexpected that elevated CASP1 expression, which typically leads to heightened susceptibility to pyroptotic cell death, does not result in pyroptosis in leukemic cells, and the underlying mechanisms for this evasion remain unknown. To investigate Pro-CASP1's significance in AML cells, we used CRISPR-Cas9 to create Pro-CASP1 knockout (Pro-CASP1 KO) human isogenic MDS and AML cell lines (THP1 and MDSL). Pro-CASP1 KO MDS/AML cells showed reduced cell growth and leukemic colony forming potential in methylcellulose compared to isogenic WT control cells. When Pro-CASP1 KO THP1 AML cells were transplanted into immunocompromised mice, the loss of Pro-CASP1 significantly reduced leukemic cell burden and extended overall survival. We next preformed experiments to probe the mechanism of action of Pro-CASP1 in AML cells. Previous studies suggest that Pro-CASP1 can function as a scaffold to elicit downstream signaling without cleaving into its activated form. The functional defect in Pro-CASP1 KO THP1 AML cells was rescued with full-length Pro-CASP1, but not with a scaffolding mutant of CASP1, indicating a specific dependency of leukemic cells on the zymogen form of CASP1. To precisely delineate which domains of Pro-CASP1 are essential for AML, we performed a tiled CRISPR dropout screen in MDSL and THP1 cells. The results showed similar CRISPR-Knockout High Sensitivity (CKHS) domain footprints in both cell lines mapping to the N-terminal CARD and the Inter-Domain Linker (IDL) domains. These results indicate that Pro-CASP1's proteolytic function was found to be dispensable, whereas the CARD domain, a scaffolding region, was crucial for leukemogenesis. We next investigated how loss of Pro-CASP1 affects inflammatory signaling in AML cells. Deletion of Pro-CASP1 in THP1 AML cells led to excessive and constitutive activation of NF-kB as indicated by nuclear translocation and phosphorylation of p65/RelA. Restoring NF-kB regulation using the inhibitor of kappa B (IkB) super-repressor (IkB-SR) was sufficient to reverse the leukemic progenitor defect and cell viability in Pro-CASP1 KO AML cells. These findings suggest that Pro-CASP1 is required for AML cells to finetune canonical NF-kB activation. Conversely, loss of Pro-CASP1 results in excessive NF-kB activation that is incompatible with AML, consistent with previous observations (Ellegast et al., Cancer Discovery 2022). In summary, Pro-CASP1 serves as a critical signaling hub, fine-tuning NF-kB pathways independently of its proteolytic function in leukemic cells. Its loss exposes leukemic cells to functional and therapeutic vulnerabilities.

TNFα-Driven Interplay between Hematopoietic Stem Cells and Antigen-Specific Cytotoxic T Cells

Clonal hematopoiesis (CH) is governed by aberrant hematopoietic stem cell (HSC) clones with leukemia-associated somatic mutations that arise spontaneously during organismal aging or after exposure to genotoxic insults. Cells with a somatic mutation can be eliminated through presentation of neoantigens on MHC class I (MHC-I) to antigen-specific cytotoxic T cells. Indeed, many of the CH-related mutations, such as JAK2V617F, are reported or predicted to elicit antigen-specific cytotoxic T cell response. On the other hand, accumulating evidence suggests that inflammatory environment facilitates clonal expansion of abnormal HSCs. However, much remains to be elucidated on whether HSCs expressing such non-self peptides can be eliminated by antigen-specific cytotoxic T cells and how inflammation affects the T cell-mediated HSC regulation. Our RNA-seq analyses revealed that genes involved in MHC-I-dependent antigen presentations, such as Nlrc5, H2-kb and B2m, are most abundantly expressed in HSCs (Lin -/c-Kit +/Sca-1 +/Flk2 -/CD48 -/CD150 +) among murine bone marrow immature hematopoietic cells. Using transgenic mice that ubiquitously express a model antigen ovalbumin (OVA) and a monoclonal antibody that specifically recognizes MHC-I-OVA complex, we found that while MHC-I protein is abundantly expressed on the surface of hematopoietic cells except granulocytes, HSCs and multipotent progenitor cells (MPPs) possess greater capacity to present intracellular antigens through MHC-I compared to myeloid progenitor and mature cells. Remarkably, in vitro co-culture experiments revealed that whereas OVA-expressing (OVA +) HSCs, MPPs and myeloid progenitors could directly activate OVA-specific OT-I CD8 + T cells through MHC-I-dependent antigen presentation, HSCs are the most susceptible population to the killing effect by cytotoxic T cells. Indeed, co-infused OT-I CD8 + T cells totally abolished the long-term repopulation capacity of OVA + HSCs in a serial transplantation setting, and adoptive transfer of OT-I CD8 + T cells to mixed chimeric mice that harbor both wild-type and OVA + hematopoietic cells completely and specifically eliminated hematopoiesis governed by OVA + HSCs. TNFα is not only a prototypical inflammatory cytokine but also one of the major cytokines that are secreted by CD8 + cytotoxic T cells. Notably, genes directly involved in T cell regulation including Cxcl9, Cd274, and Pdcd1lg2 were induced in HSCs, but not myeloid progenitor cells, upon exposure to TNFα (Yamashita and Passegué, Cell Stem Cell 2019). Indeed, upon co-culturing OT-I CD8 + T cells produced TNFα as well as IFNγ and Granzyme B and OVA + HSCs but not myeloid progenitor cells produced CXCL9 largely in a TNFα receptor-dependent manner. Interestingly, such HSC response to T cell-derived TNFα was critical for naïve CD8 + T cells to rapidly differentiate to effector cytotoxic T cells, as TNFα receptor-deficient ( Tnfr1 -/-Tnfr2 -/-) OVA + HSCs stimulated proliferation and cytokine production of OT-I CD8 + T cells but failed to stimulate Granzyme B production, whereas IFNγ receptor deficiency ( Ifngr -/-) did not have such effect. As a result, Tnfr1 -/-Tnfr2 -/- OVA + HSCs, but not Ifngr -/- OVA + HSCs, could escape from the killing by OT-I CD8 +T cells. In sharp contrast, prior exposure to environmental TNFα, but not to IL-1β or IL-6, before interaction with OT-I CD8 + T cells rendered OVA + HSCs resistant to the killing by OT-I CD8 + T cells largely through upregulation of PD-L1 and PD-L2. Of note, upon 24-month aging or acquisition of Jak2V617F mutation, OVA + HSCs still maintained robust MHC-I-dependent antigen presentation activity and high susceptibility to the killing by OT-I CD8 + T cells, though prior exposure to TNFα allowed for their immune escape. Consequently, adoptive transfer of OT-I CD8 + T cells completely and specifically eliminated Jak2V617F-mutant OVA + HSCs and reversed myeloproliferative neoplasms in the mixed chimeric mice. Taken together, our results reveal the robustness of HSC quality control via MHC-I-dependent antigen presentation to antigen-specific cytotoxic T cells. They also highlight a critical but complex role of TNFα in active interplay between HSCs and antigen-specific cytotoxic T cells as well as immune evasion by HSCs during inflammation. Specifically targeting abnormal HSCs by antigen-specific cytotoxic T cells may pave the way to eradicate mutant HSC clones and prevent CH-associated disease.

Plasma Proteomic Signature Predicts Risk of Myeloid Neoplasm

Introduction The presence of clonally expanded mutant hematopoietic stem cells, clonal hematopoiesis (CH) is known to predispose to hematologic malignancy, particularly myeloid neoplasms (MN). However, the overall risk of developing MNs among individuals with CH is low (<1% per year). Limitations in our ability to predict who with CH will progress to MN is a barrier to prevention strategies. The human proteome reflects the downstream impact of complex interactions between genetic and epigenetic regulation of biologic systems. We hypothesized that plasma proteomic markers might be associated with MN risk and if so could inform our understanding of the mechanisms promoting the progression of CH to MN. Methods Among 46,237 individuals in the UK Biobank (UKB) with whole exome sequencing data and single nucleotide polymorphism (SNP) array profiling of the blood and plasma proteomic profiling using the Olink Explore platform (1,463 unique proteins), we jointly characterized CH and plasma proteomic profile. Cox proportional hazard models were used to assess the association between the level of each plasma protein and risk of MN; while controlling for age, sex, genetic ancestry, smoking status, blood counts, and sample collection year. The relative contribution of clinical, CH, and proteomics features in prediction of incident MN was assessed using lasso regression with 100-fold cross validation. We performed protein quantitative trait loci (pQTL) mapping to identify independent germline autosomal genetic loci that significantly regulate plasma proteomic levels (p <5x10 -8). We calculated genetically predicted risk scores for plasma protein levels as the as the sum of the effect estimates from linear regression analysis with additive genetic effect, multiplied by the number of risk alleles for each locus. As a validation cohort, we used an independent set of 427,614 individuals in the UKB to test for an association between genetically predicted levels of proteomic markers and MN risk. Results A total of 115 participants developed MN at least 3 months after study entry (median 83 months). As expected, participants who developed MN (cases) had a higher frequency of CH at the time of study entry compared to those free of hematologic malignancy (controls); 42 versus 6% for SNV/Indels and 23 versus 3% for mosaic copy number events in cases compared to controls respectively. We identified 115 proteins significantly associated (qFDR <0.05) with incident MN (Figure 1). These include known regulators of the adaptive immune system, innate immune system, leukocyte trafficking, hematopoietic stem cell self-renewal, and platelet activation. Given that CH is a genetic precursor to MN, we assessed the relationship between plasma proteomic markers of MN and CH. Among the 115 plasma proteins that were significantly associated with MN, 35 were significantly (qFDR <0.05) and 59 nominally (p <0.05) associated with CH. After adjusting for CH, the strength of the association between plasma protein markers and MN risk was generally reduced (mean 7%), but remained significant for many (N=44). We identified a total of 79 plasma proteins that were associated with the presence of CH at the time of study entry, 34 of which overlapped with MN risk proteins. Inclusion of proteomics features in a lasso regression model (AUC = 0.85) improved prediction of incident MN beyond clinical factors and CH (AUC = 0.80; Figure 2). There were 1,282 independent genetic loci that were significantly associated with 115 proteomic markers of MN risk. On average, genetic risk scores captured 12% of the total variance of plasma protein levels (range 0.1-64%). In an independent cohort of 427,614 UKB participants (1,099 of whom developed MN), we found that of the 110 plasma protein markers with a genetic risk score R 2 >1%, 12 were significantly associated with MN (p <0.05). Of the remaining, 63 were directionally consistent. Conclusions We show that plasma proteomic markers predict risk of MN. Some, but not all of the impact of plasma proteomic profile on MN risk is mediated by baseline CH. Inclusion of proteomic markers improves MN risk prediction beyond clinical and CH features. Genetic risk score analysis provides orthogonal validation of the plasma proteomic signature for MN risk. These data highlight the promise of protein and genetic biomarker integration for early diagnosis of MN and further support the role of immune cell regulation in early leukemogenesis.

Leukemic Driver Clones Can Arise Independently and Outcompete MPN Cells in Post-MPN Secondary AML

The JAK2V617F variant is an acquired somatic mutation recurrently found in myeloproliferative neoplasm (MPN) patients and is used as a diagnostic marker. While considered a strong MPN driver mutation, this variant is sometimes absent in secondary acute myeloid leukemia (sAML) samples which transform from antecedent JAK2-mutant MPNs. The transformative evolution of MPN to sAML is thought to be driven by acquisition of additional genetic mutations affecting epigenetic modifiers (e.g. TET2), tumor suppressor genes (e.g. TP53), and splicing regulation, but the contribution and sequence of onset for these specific genetic events is not well understood. It is currently unknown why or how patients with a JAK2-mutant MPN may develop a JAK2 wild-type sAML. This transformation sequence is coupled with very limited treatment options and a survival of less than 6-months. Elucidating the evolutionary trajectory of secondary leukemic mutation occurrence is paramount for disease monitoring and early detection. To understand this evolutionary trajectory, we employed single cell proteogenomic analysis for three separate paired MPN patient samples (2 MF; 1 PV) who progressed to sAML stage. Clinical sequencing data indicated presence of the JAK2 V617F mutation in all three samples at the MPN stage whereas this mutation was not detected at the sAML stage. We employed the MissionBio Tapestri platform to delineate the clonal trajectory of transformation to determine if sAML and MPN driver clones share a common founder clone or if they arise through independent, non-clonally related founders. Utilization of this single-cell platform enabled us to investigate two potential models of transformation. First, two distinct mutation phases of MPN to sAML occur wherein the JAK2 -mutant clone and sAML driver mutations originate form a shared founder clone. In this model, an MPN phenotype driven by JAK2-mutant clones arises in patients first, followed by the sAML disease stage driven by leukemic mutations. An alternative tested model is that MPN and sAML originate as separate clones wherein the JAK2 mutant hematopoietic stem cells (HSCs) give rise to an MPN phenotype first, while the independent sAML clone(s) grow slowly yet separately in the background of the existing MPN. Both models provide a plausible explanation which results in leukemic blast stage clones harboring leukemic mutations but lacking the MPN driving mutation JAK2V617F. Single-cell analysis detected the JAK2V617F mutation in both the MPN (MF) as well as the sAML stage in one of the paired patient samples in which JAK2 was previously reported as wild-type at the sAML stage. This sample also contained a RUNX1 mutation at the sAML stage as well as a clonally unrelated ATM mutation (Figure 1A). The single-cell data for the remaining two paired patient samples (1MF; 1PV) demonstrated that pre-leukemic driver mutations were not detected within the JAK2-mutant clones at the MPN stage or during a middle time-point stage before AML diagnosis. Single-cell sequencing from the sAML time point for both samples demonstrated loss of the JAK2-mutant clone (verified by ddPCR analysis), amidst presence of leukemic driver mutant clones (Figure 1B). This clonal hierarchy suggests that the leukemic mutations were not present within the JAK2-mutant clones during the MPN stage and arose in the background of the MPN where they outcompeted the MPN driver clone during sAML transformation. Further, this hierarchy suggests that the AML driver mutations do not share a clonal intermediate ancestor with the JAK2 mutant cells which drove the MPN. These data demonstrate that one clonal trajectory of JAK2-mutant MPN to JAK2 wild-type sAML is through independent leukemic clones arising in the background of MPN and outcompeting the JAK2-mutant clones. In these instances, it is more appropriate to view these patients as de novo AML cases arising in a background of pre-existing MPN, which could influence treatment decisions and disease surveillance.

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

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.