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Clonal hematopoiesis (CH) refers to the age-related expansion of specific clones in the blood system, and manifests from somatic mutations acquired in hematopoietic stem cells (HSCs). Most CH variants occur in the gene DNMT3A, but while DNMT3A-mutant CH becomes almost ubiquitous in aging humans, a unifying molecular mechanism to illuminate how DNMT3A-mutant HSCs outcompete their counterparts is lacking. Here, we used interferon gamma (IFNγ) as a model to study the mechanisms by which Dnmt3a mutations increase HSC fitness under hematopoietic stress. We found Dnmt3a-mutant HSCs resist IFNγ-mediated depletion, and IFNγ-signaling is required for clonal expansion of Dnmt3a-mutant HSCs in vivo. Mechanistically, DNA hypomethylation–associated overexpression of Txnip in Dnmt3a-mutant HSCs leads to p53 stabilization and upregulation of p21. This preserves the functional potential of Dnmt3a-mutant HSCs through increased quiescence and resistance to IFNγ-induced apoptosis. These data identify a previously undescribed mechanism to explain increased fitness of DNMT3A-mutant clones under hematopoietic stress. Significance: DNMT3A mutations are common variants in clonal hematopoiesis, and recurrent events in blood cancers. Yet the mechanisms by which these mutations provide hematopoietic stem cells a competitive advantage as a precursor to malignant transformation remain unclear. Here, we use inflammatory stress to uncover molecular mechanisms leading to this fitness advantage. See related commentary by De Dominici and DeGregori, p. 178. This article is highlighted in the In This Issue feature, p. 171

Clonal hematopoiesis (CH) refers to the age-related expansion of specific clones in the blood system, and manifests from somatic mutations acquired in hematopoietic stem cells (HSCs). Most CH variants occur in the gene DNMT3A, but while DNMT3A-mutant CH becomes almost ubiquitous in aging humans, a unifying molecular mechanism to illuminate how DNMT3A-mutant HSCs outcompete their counterparts is lacking. Here, we used interferon gamma (IFNγ) as a model to study the mechanisms by which Dnmt3a mutations increase HSC fitness under hematopoietic stress. We found Dnmt3a-mutant HSCs resist IFNγ-mediated depletion, and IFNγ-signaling is required for clonal expansion of Dnmt3a-mutant HSCs in vivo. Mechanistically, DNA hypomethylation–associated overexpression of Txnip in Dnmt3a-mutant HSCs leads to p53 stabilization and upregulation of p21. This preserves the functional potential of Dnmt3a-mutant HSCs through increased quiescence and resistance to IFNγ-induced apoptosis. These data identify a previously undescribed mechanism to explain increased fitness of DNMT3A-mutant clones under hematopoietic stress.Significance:DNMT3A mutations are common variants in clonal hematopoiesis, and recurrent events in blood cancers. Yet the mechanisms by which these mutations provide hematopoietic stem cells a competitive advantage as a precursor to malignant transformation remain unclear. Here, we use inflammatory stress to uncover molecular mechanisms leading to this fitness advantage.See related commentary by De Dominici and DeGregori, p. 178.This article is highlighted in the In This Issue feature, p. 171

Supplementary Figure from Txnip Enhances Fitness of Dnmt3a-Mutant Hematopoietic Stem Cells via p21

Myeloproliferative neoplasms (MPN) are chronic blood diseases with significant morbidity and mortality. Although sequencing studies have elucidated the genetic mutations that drive these diseases, MPNs remain largely incurable with a significant proportion of patients progressing to rapidly fatal secondary acute myeloid leukemia (sAML). Therapeutic discovery has been hampered by the inability of genetically engineered mouse models to generate key human pathologies such as bone marrow fibrosis. To circumvent these limitations, here we present a humanized animal model of myelofibrosis (MF) patient-derived xenografts (PDX). These PDXs robustly engrafted patient cells that recapitulated the patient's genetic hierarchy and pathologies such as reticulin fibrosis and propagation of MPN-initiating stem cells. The model can select for engraftment of rare leukemic subclones to identify patients with MF at risk for sAML transformation and can be used as a platform for genetic target validation and therapeutic discovery. We present a novel but generalizable model to study human MPN biology.Significance:Although the genetic events driving MPNs are well defined, therapeutic discovery has been hampered by the inability of murine models to replicate key patient pathologies. Here, we present a PDX system to model human myelofibrosis that reproduces human pathologies and is amenable to genetic and pharmacologic manipulation.This article is highlighted in the In This Issue feature, p. 2945

Supplementary Figures 1-7

Hematological comparison of MF patients versus PDXs

Exome sequencing results.

Summary The DNA methylation regulators DNMT3A and TET2 are recurrently mutated in hematological disorders. Despite possessing antagonistic biochemical activities, loss-of-function murine models show overlapping phenotypes in terms of increased hematopoietic stem cell (HSC) fitness. Here, we directly compared the effects of these mutations on hematopoietic progenitor function and disease initiation. In contrast to Dnmt3a-null HSCs, which possess limitless self-renewal in vivo, Tet2-null HSCs unexpectedly exhaust at the same rate as control HSCs in serial transplantation assays despite an initial increase in self-renewal. Moreover, loss of Tet2 more acutely sensitizes hematopoietic cells to the addition of a common co-operating mutation (Flt3ITD) than loss of Dnmt3a, which is associated with a more rapid expansion of committed progenitor cells. The effect of Tet2 mutation manifests more profound myeloid lineage skewing in committed hematopoietic progenitor cells rather than long-term HSCs. Molecular characterization revealed divergent transcriptomes and chromatin accessibility underlying these functional differences., Highlights • Tet2-null HSCs exhaust at the same rate as wild-type HSCs in serial transplantation • Loss of Tet2 sensitizes cells to Flt3ITD mutation more dramatically than Dnmt3a • Loss of Dnmt3a permits epigenetic plasticity between hematopoietic progenitors • Tet2 deficiency manifests profound myeloid lineage skewing in progenitor cells, Challen and colleagues show that Dnmt3a and Tet2 loss-of-function mutations manifest distinct molecular and functional consequences in different hematopoietic stem and progenitor cell compartments. Despite producing superficially similar phenotypes in terms of stem cell function and disease initiation, we show here divergent influences on progenitor cell lineage skewing, stem cell self-renewal, and predisposition to malignant transformation.

The histone demethylase KDM6B (JMJD3) is upregulated in blood disorders, suggesting that it may have important pathogenic functions. Here we examined the function of Kdm6b in hematopoietic stem cells (HSC) to evaluate its potential as a therapeutic target. Loss of Kdm6b lead to depletion of phenotypic and functional HSCs in adult mice, and Kdm6b is necessary for HSC self-renewal in response to inflammatory and proliferative stress. Loss of Kdm6b leads to a pro-differentiation poised state in HSCs due to the increased expression of the AP-1 transcription factor complex (Fos and Jun) and immediate early response (IER) genes. These gene expression changes occurred independently of chromatin modifications. Targeting AP-1 restored function of Kdm6b-deficient HSCs, suggesting that Kdm6b regulates this complex during HSC stress response. We also show Kdm6b supports developmental context-dependent leukemogenesis for T-cell acute lymphoblastic leukemia (T-ALL) and M5 acute myeloid leukemia (AML). Kdm6b is required for effective fetal-derived T-ALL and adult-derived AML, but not vice versa. These studies identify a crucial role for Kdm6b in regulating HSC self-renewal in different contexts, and highlight the potential of KDM6B as a therapeutic target in different hematopoietic malignancies.

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.

Myeloproliferative neoplasms (MPN) are chronic blood diseases with significant morbidity and mortality. Although sequencing studies have elucidated the genetic mutations that drive these diseases, MPNs remain largely incurable with a significant proportion of patients progressing to rapidly fatal secondary acute myeloid leukemia (sAML). Therapeutic discovery has been hampered by the inability of genetically engineered mouse models to generate key human pathologies such as bone marrow fibrosis. To circumvent these limitations, here we present a humanized animal model of myelofibrosis (MF) patient-derived xenografts (PDX). These PDXs robustly engrafted patient cells that recapitulated the patient's genetic hierarchy and pathologies such as reticulin fibrosis and propagation of MPN-initiating stem cells. The model can select for engraftment of rare leukemic subclones to identify patients with MF at risk for sAML transformation and can be used as a platform for genetic target validation and therapeutic discovery. We present a novel but generalizable model to study human MPN biology. Significance: Although the genetic events driving MPNs are well defined, therapeutic discovery has been hampered by the inability of murine models to replicate key patient pathologies. Here, we present a PDX system to model human myelofibrosis that reproduces human pathologies and is amenable to genetic and pharmacologic manipulation. This article is highlighted in the In This Issue feature, p. 2945

Clonal hematopoiesis (CH) refers to the age-related expansion of specific clones in the blood system and manifests from somatic mutations acquired in hematopoietic stem cells (HSCs). Approximately 50% of CH variants occur in the gene DNMT3A. While DNMT3A-mutant CH becomes almost ubiquitous in aging humans, 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 increase HSC fitness under recurrent hematopoietic stress. To represent the spectrum of DNMT3A variants found in humans, mouse genetic models were generated; Dnmt3a heterozygous (Vav-Cre; Dnmt3afl/+ = Dnmt3aHET) and homozygous (Vav-Cre; Dnmt3afl/fl = Dnmt3aKO) hematopoietic loss-of-function, and a knock-in model analogous to the hotspot point mutation most prevalent in AML (Vav-Cre; Dnmt3aR878H/+ = Dnmt3aR878). When Dnmt3a-mutant cells were competitively transplanted with wild-type (WT) competitor bone marrow (BM) cells and challenged with different inflammatory and proliferative stressors, Dnmt3aKO and Dnmt3aR878 HSCs were specifically resistant to the deleterious effects of IFNg on HSC self-renewal and clonal expansion. This insensitivity was also confirmed in a humanized mouse model where human CD34 + cord blood cells edited with DNMT3A-targeting gRNAs were xenografted into recipient mice and episodically exposed to human recombinant IFNg. DNMT3A mutant cells maintained their clone size, whereas AAVS1-targeted cells (control) were depleted over serial transplantation. These data suggest that Dnmt3a-mutant HSCs, mimicked DNMT3A-mutated human HSCs and are specifically resistant to IFNg-mediated depletion. One explanation for the observed resistance is that Dnmt3a-mutant HSCs have a fitness advantage under IFNg challenge. Therefore, we generated a novel mouse model to directly quantify the competition between Dnmt3a-mutant and WT HSCs. 10% donor BM cells (CD45.2; WT or Dnmt3a-mutant), 10% WT competitor BM cells (CD45.1/2; Ubc-GFP+) and 80% BM cells (CD45.1; IFNgr1KO; Rosa-M2-rtTA-IFNg) that express IFNg by doxycycline were transplanted into CD45.1 recipient mice. To normalize the effect of doxycycline, chimera made with 80% BM cells (CD45.1; IFNgr1KO; Rosa-M2-rtTA) were also transplanted into recipients. Our result from this transplantation experiment showed Dnmt3a-mutant HSCs resisted IFNg-mediated depletion due to an enhanced fitness advantage. Genetic ablation of IFNgr1 from Dnmt3a-mutant mice revealed that IFNg signaling is cell-intrinsically required by clonal expansion of Dnmt3a-mutant HSCs. In parallel, when HSCs were transplanted into IFNg-deficient recipient mice, clonal expansion of Dnmt3aKO HSCs but not WT HSCs was significantly compromised, suggesting IFNg signaling is also cell-extrinsically crucial for the clonal expansion of Dnmt3a-mutant HSCs in vivo. Mechanistically, DNA hypomethylation-associated over-expression of Thioredoxin-interacting protein (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 led to p53 stabilization and upregulation of p21 under IFNg challenge, further correlated with a retained quiescence and resistance to apoptosis 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 WT 32D murine myeloid cell line. 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 re-sensitized Dnmt3aKO HSCs to IFNg-induced cell cycle activation and apoptosis. In vivo, down-regulation of p21 had no effect on WT HSCs in response to IFNg exposure, but it completely primed Dnmt3aKO and Dnmt3aR878 HSCs to IFNg-induced exhaustion in a transplantation experiment. Taken together, our data highlighted a Txnip-p53-p21 pathway that preserves the functional potential of Dnmt3a-mutant HSCs under conditions of inflammatory stress, which suggests 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. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is strictly regulated to sustain blood production throughout adult life. De novo DNA methyltransferase 3-alpha (DNMT3A) is one of the major epigenetic regulators that is essential for efficient HSC differentiation. DNMT3A mutations are prevalent in myeloid diseases that include acute myeloid leukemia (AML; ~22%) and myelodysplastic syndrome (MDS; ~10%) where they act as initiating events However, the precise molecular mechanisms of how DNMT3A regulates normal hematopoiesis and its mutations prime HSCs for leukemic formation are unclear. Although DNMT3A is described as a DNA methyltransferase enzyme, the lack of consistent correlation between changes in DNA methylation and differential gene expression in Dnmt3a-null HSCs in mouse models, and AML patients with DNMT3A mutations undermine the conventional understanding of DNMT3A's canonical role in hematopoietic cells. Hence, we hypothesized that DNMT3A may have novel functions outside of DNA methylation that regulate HSC fate decisions. To answer this question, we first ectopically expressed GFP-labeled Dnmt3a constructs (wild-type Dnmt3a, Dnmt3aE752A; complete DNA methylation dead, and Dnmt3aR832A; reduced DNA methylation target recognition) and empty vector (negative control) in Dnmt3a-null (Vav-Cre: Dnmt3afl/fl = Dnmt3a-/- in hematopoiesis) bone marrow (BM) cells. The result showed that similar to restoring wild-type Dnmt3a, ectopic expression of Dnmt3aE752A as well as Dnmt3aR832A showed a rescue effect of decreased engraftment of transduced cells in the peripheral blood as well as reduced HSC numbers in the BM. Analysis of DNA methylation by whole-genome bisulfite sequencing (WGBS) in transduced cells showed this phenotypic and functional rescue of the Dnmt3a-/- phenotype occurred in the absence of restored DNA methylation patterns. To study the importance of Dnmt3a-mediated DNA methyltransferase activity in a more physiological system, we generated knock-in mice that have one copy of either wild-type Dnmt3a, Dnmt3aE752A, or Dnmt3aR832A (CAGG-Cre-ER T2 = ER T2-Cre: Dnmt3afl/+, Dnmt3afl/E752A, and Dnmt3afl/R832A) to be compared to the Dnmt3a-null group (ER T2-Cre: Dnmt3afl/-). These mice contain one allele with loxP-flanked Dnmt3a that is deleted by tamoxifen-inducible Cre-mediated recombination and one allele of either wild-type Dnmt3a, Dnmt3aE752A, Dnmt3aR832A, or germline knockout Dnmt3anull. 5-weeks post-tamoxifen (~93% floxed allele recombination), competitive transplantation of 250 phenotypically defined test HSCs against with 2.5x10 5 congenic competitor BM cells was performed. Dnmt3a fl/R832A recipients had higher engraftment (35.6 % +/- 6.1) than Dnmt3afl/+ (28.5% +/- 7.2) and Dnmt3afl/- (10.7% +/- 2.79), while Dnmt3afl/E752A had slightly higherengraftment (12.5% +/- 3) than Dnmt3afl/-. Analysis of the BM 18 weeks post-transplant showed that Dnmt3afl/E752A and Dnmt3afl/R832A HSCs phenocopied the HSC self-renewal potential phenotype of heterozygous Dnmt3a fl/+HSCs (Fig. 1). The absolute count of donor-derived HSCs per mouse after the transplant were: ER T2-Cre control (675.7 +/- 299.3), Dnmt3afl/+ (1870 +/- 961.4), Dnmt3afl/- (3546 +/- 1019), Dnmt3afl/E752A (1130 +/- 362.7), and Dnmt3afl/R832A (1184 +/- 344.5) (mean +/- S.E.M.). While the described clonal expansion of Dnmt3a-null HSCs was observed, HSCs with one copy of full-length Dnmt3a but devoid of its methyltransferase capacity mimicked the heterozygous state rather than the homozygous loss-of-function. This is the first evidence to suggest that DNMT3A potentially regulates HSCs by non-canonical (DNA methylation independent) mechanisms. DNA methylation analysis by WGBS is ongoing to determine if Dnmt3afl/E752A and Dnmt3afl/R832A HSCs show a methylome comparable to Dnmt3a-null HSCs whilst having the functional potential of Dnmt3a-heterozygous HSCs, which will be complemented with other molecular analyses including gene expression. Our study opens new avenues for investigations into the molecular mechanisms of DNMT3A function in HSC biology, which could ultimately benefit clinical practice by identifying new therapeutic approaches for the patients with DNMT3A mutations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

T-cell acute lymphoblastic leukemia (T-ALL) arises from the accumulation of genomic abnormalities and the malignant proliferation of immature T-cells. Despite recent advancements in understanding the genetic alterations driving T cell leukemogenesis, patients still suffer from recurrent relapses and treatment-related toxicities. Genome sequencing has revealed significant heterogeneity and important insights into the genetic landscape of T-ALL. Mutations in epigenetic modifiers are frequently observed and serve as an attractive target for novel therapeutic approaches. Histone demethylase enzymes play a critical role in the regulation of gene expression programs in T-ALL. KDM6A (UTX) is known to behave as a tumor suppressor in most T-ALL subtypes. However, it's gene paralog, KDM6B (JMJD3), is never mutated and can be significantly overexpressed in patients. Here, we show that KDM6B is required for T-ALL initiation. Using genetic mouse models, Sca-1 enriched WBM from Vav-Cre: Kdm6b+/+, Vav-Cre: Kdm6bfl/+, and Vav-Cre: Kdm6bfl/fl adult mice was transduced with a retrovirus expressing Notch1 Intracellular Domain (NICD). NOTCH1 gain-of-function mutations are the most frequent driver events in adult T-ALL, and this model recapitulates many of the human pathologies. Transduced cells were transplanted into irradiated mice. While there was robust engraftment in all groups at four weeks post-transplant, T-ALL cells were not sustained in the genetic absence of Kdm6b. Mice receiving control NICD-GFP+ cells succumbed to T-ALL with median survival of 79 days, whereas the only mice receiving Kdm6b-null NICD-GFP+ cells that developed disease were found to retain one copy of the Kdm6b floxed allele (Fig 1). To investigate the translational potential, we targeted KDM6B for genetic inactivation by CRISPR/Cas9 in primary T-ALL patient cells, followed by xenograft into NSG mice. The effect of KDM6B targeting was quantified over time by monitoring the variant allele fraction (VAF) of the transplanted cells. In most patient samples, KDM6B-targeted cells were significantly outcompeted over time, thus further highlighting the requirement of KDM6B in sustaining T-ALL tumorigenesis. To examine the mechanism by which KDM6B sustain T-ALL cells, gene expression profiling was performed by RNA-seq on mouse T-ALL cells of genetic backgrounds Control, Kdm6b-Het, and Kdm6b-KO. Three gene sets were significantly downregulated in Kdm6b-deficient T-ALL cells compared to the Control group, all of which are involved in cell cycle processes. Additional validation of these findings with cell cycle functional studies is currently ongoing. Additionally, while Kdm6b has been described for its H3K27me3 histone demethylase function, recent studies have shown its involvement in regulating various gene expression programs through demethylase-independent mechanisms. To determine if the role of Kdm6b in T-ALL oncogenesis was related to it demethylase activity, we performed the same NICD retroviral transduction experiment with inclusion of a Kdm6b +/H1388Amouse genotype, a point mutation which renders Kdm6b catalytically dead. Our data shows that Kdm6b +/H1388Aphenocopies T-ALL with Kdm6b homozygous loss-of-function, showing no evidence of disease in the blood beyond 8-weeks post-transplant. We conclude from this that the leukemogenic role of Kdm6b requires it's H3K27me3 demethylase function. In summary, these data reveal Kdm6b as an oncogenic dependency in Notch1-driven T-ALL in both mouse and human systems, and present Kdm6b as a high value therapeutic target in adult T-ALL patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Genome sequencing efforts have identified virtually all of the important mutations in adult myeloid malignancies. More recently, population studies have identified cancer-associated variants in the blood of otherwise healthy individuals as they age, a phenomenon termed clonal hematopoiesis of indeterminate potential (CHIP). This suggests that these mutations may occur in hematopoietic stem cells (HSCs) long before any clinical presentation but are not necessarily harbingers of transformation because only a fraction of individuals with CHIP develop hematopoietic pathologies. Delineation between CHIP variants that predispose for disease versus those that are more benign could be used as a prognostic factor to identify individuals at greater risk for transformation. To achieve this, the biological impact of CHIP variants on HSC function must be validated. One variant that has been identified recurrently in CHIP is a gain-of-function missense mutation in the imprinted gene GNAS (Guanine Nucleotide Binding Protein, Alpha Stimulating). In this study, we examined the effect of the GNASR201C variant on HSC function. Ectopic expression of GNASR201C supported transplantable HSC activity and improved lymphoid output in secondary recipients. Because declining lymphoid output is a hallmark of aging, GNASR201C mutations may sustain lymphoid-biased HSCs over time and maintain them in a developmental state favorable for transformation.

Myelofibrosis (MF) is the deadliest subtype of myeloproliferative neoplasm (MPN) with a median survival of approximately 5 years. Ruxolitinib, a front line therapy for JAK2V617F mutant MPN, can alleviate symptoms of the disease, but does not eliminate the malignant clone and has minimal impact on BM fibrosis and overall survival. Current mouse models do not recapitulate the clinical heterogeneity, clonal genetic composition, or morphological features of MF. Most notably, these models do not generate robust reticulin fibrosis in the bone marrow, the most significant MF pathology. This lack of clinically relevant MF models presents a major barrier to deciphering the complex genetic drivers of the disease and developing effective therapies against it. We evaluated the ability of CD34+ hematopoietic stem and progenitor cells (HSPCs) from MF patients (that contain the MPN-disease initiating population) to give rise to MF in xenotransplanted NSGS mice. >5x104 FACS-sorted CD34+ HSPCs from the peripheral blood of MF patients with JAK2V617F (n=12), CALRindels (n=7) and MPLW515L (n=2) were transplanted into sublethally irradiated (200rads) NSGS mice via X-ray guided intra-tibial injection. We observed robust engraftment of patient-derived cells at 12 weeks post-transplant regardless of their genetic background or donor patient disease severity (Fig 1A). Post-transplant, BM analysis revealed robust expansion of phenotypically defined MF HSCs relative to cord blood CD34+ control recipients, suggesting a permissive niche for MF HSCs to undergo self-renewal. Remarkably, transplantation of CD34+ cells produced other hallmarks of MF in recipient animals such as splenomegaly, thrombocytosis and most importantly BM reticulin fibrosis in all recipients (Fig 1B). We assessed the clonal architecture of engrafted human cells compared to the primary disease in the donor patients through exome sequencing of CD34+ cells prior to transplantation and hCD45+ cells from MF xenografts. We found that the clonal and subclonal mutational landscape observed in CD34+ cells prior to transplantation was maintained in recipient mice (Fig 1C), suggesting that the PDX model accurately reflects the cellular composition of the primary disease. Intriguingly, in two of the xenografted patient samples, we identified additional mutations that were not detected in the primary patient samples using standard sequencing - namely TP53R248Q and EZH2Y663H respectively. Two years after we detected these mutations in PDXs, these MF patients transformed into sAML with acquisition of TP53R248Q and EZH2Y663H mutations. We performed droplet digital PCR and demonstrated that indeed rare pre-leukemic subclones containing these mutations were present at low levels (< 0.01% VAF) in chronic stage MF patients at least two years prior to sAML progression. These data also suggested that these rare subclones responsible for leukemic transformation expand significantly (>300 fold) under the selective pressure of transplantation in NSGS mice. Additional validation of these findings in a further six pre-sAML MF patient samples is currently ongoing. If successful, this model could be used to prospectively identify rising leukemic clones in chronic stage MF patients, which are below the level of detect of standard sequencing as a mechanism to stratify such patients for more aggressive treatments. While sequencing can identify ultra-rare variants, it cannot discern their functional potential for sAML transformation, which is the advantage of this approach. Finally, we harnessed this system for pre-clinical studies, initially focusing on inhibiting the JAK/STAT signaling pathway. Ruxolitinib treatment in MF PDXs produced remarkably similar phenotypes as observed in patients. We observed a small, but significant reduction in engraftment of MF cells in the BM and a sharp reduction in spleen size in Ruxolitinib-treated group compared to vehicle control. Ruxoltinib treatment however did not reduce the frequency of MF HSCs, the disease initiating population or lessen the degree of reticulin fibrosis. These data suggest that this system can be used as a reliable, clinically-relevant drug screening platform. Taken together, we offer the field a critical, previously missing biologically relevant screening system for validation of MPN drug targets identified in cell lines or genetic mouse models prior to moving forward into clinical trials. Disclosures Oh: Blueprint Medicines: Consultancy; Celgene/BMS: Consultancy; Constellation: Consultancy; CTI Biopharma: Consultancy; Disc Medicine: Consultancy; Gilead Sciences: Consultancy; Incyte Corporation: Consultancy; Kartos Therapeutics: Consultancy; Novartis: Consultancy; PharmaEssentia: Consultancy.

Summary How specific genetic lesions contribute to transformation of non-malignant myeloproliferative neoplasms (MPNs) and myelodysplastic syndromes (MDSs) to secondary acute myeloid leukemia (sAML) are poorly understood. JARID2 is lost by chromosomal deletions in a proportion of MPN/MDS cases that progress to sAML. In this study, genetic mouse models and patient-derived xenografts demonstrated that JARID2 acts as a tumor suppressor in chronic myeloid disorders. Genetic deletion of Jarid2 either reduced overall survival of animals with MPNs or drove transformation to sAML, depending on the timing and context of co-operating mutations. Mechanistically, JARID2 recruits PRC2 to epigenetically repress self-renewal pathways in hematopoietic progenitor cells. These studies establish JARID2 as a bona fide hematopoietic tumor suppressor and highlight potential therapeutic targets.

The DNA end resection process dictates the cellular response to DNA double strand break damage and is essential for genome maintenance. Although insufficient DNA resection hinders homology-directed repair and ATR (ataxia telangiectasia and Rad3 related)-dependent checkpoint activation, overresection produces excessive single-stranded DNA that could lead to genomic instability. However, the mechanisms controlling DNA end resection are poorly understood. Here we show that the major resection nuclease Exo1 is regulated both positively and negatively by protein-protein interactions to ensure a proper level of DNA resection. We have shown previously that the sliding DNA clamp proliferating cell nuclear antigen (PCNA) associates with the C-terminal domain of Exo1 and promotes Exo1 damage association and DNA resection. In this report, we show that 14-3-3 proteins interact with a central region of Exo1 and negatively regulate Exo1 damage recruitment and subsequent resection. 14-3-3s limit Exo1 damage association, at least in part, by suppressing its association with PCNA. Disruption of the Exo1 interaction with 14-3-3 proteins results in elevated sensitivity of cells to DNA damage. Unlike Exo1, the Dna2 resection pathway is apparently not regulated by PCNA and 14-3-3s. Our results provide critical insights into the mechanism and regulation of the DNA end resection process and may have implications for cancer treatment.

Genome sequencing efforts have identified virtually all of the important mutations in adult myeloid malignancies. More recently, population studies have identified cancer-associated variants in the blood of otherwise healthy individuals as they age, a phenomenon termed clonal hematopoiesis of indeterminate potential (CHIP). This suggests these mutations may occur in HSCs long before any clinical presentation, but are not necessarily harbingers of transformation as only a fraction of individuals with CHIP develop hematopoietic pathologies. Delineation between CHIP variants that predispose for disease versus those which are more benign could be used as a prognostic factor to identify individuals more at-risk for transformation. To achieve this, the biological impact of CHIP variants on hematopoietic stem cell (HSC) function must be validated. One variant that has been recurrently identified in CHIP is a gain-of-function missense mutation in the imprinted gene GNAS. In this study, we examined the effect of the GNASR201C variant on HSC function. Ecoptic expression of GNASR201C supported transplantable HSC activity, and improved lymphoid output in secondary recipients. As declining lymphoid output is a hallmark of aging, GNASR201C mutations may sustain lymphoid-biased HSCs over time and maintain them in a developmental state favorable for transformation.

Background Mutations in the Cu/Zn superoxide dismutase gene (SOD1) are responsible for 20% of familial forms of amyotrophic lateral sclerosis (ALS), and mutant SOD1 has been shown to have increased surface hydrophobicity in vitro. Mutant SOD1 may adopt a complex array of conformations with varying toxicity in vivo. We have used a novel florescence-based proteomic assay using 4,4’-bis-1-anilinonaphthalene-8-sulfonate (bisANS) to assess the surface hydrophobicity, and thereby distinguish between different conformations, of SOD1and other proteins in situ. Results Covalent bisANS labeling of spinal cord extracts revealed that alterations in surface hydrophobicity of H46R/H48Q mutations in SOD1 provoke formation of high molecular weight SOD1 species with lowered solubility, likely due to increased exposure of hydrophobic surfaces. BisANS was docked on the H46R/H48Q SOD1 structure at the disordered copper binding and electrostatic loops of mutant SOD1, but not non-mutant WT SOD1. 16 non-SOD1 proteins were also identified that exhibited altered surface hydrophobicity in the H46R/H48Q mutant mouse model of ALS, including proteins involved in energy metabolism, cytoskeleton, signaling, and protein quality control. Heat shock proteins (HSPs) were also enriched in the detergent-insoluble fractions with SOD1. Given that chaperones recognize proteins with exposed hydrophobic surfaces as substrates and the importance of protein homeostasis in ALS, we crossed SOD1 H46R/H48Q mutant mice with mice over-expressing the heat shock factor 1 (HSF1) transcription factor. Here we showed that HSF1 over-expression in H46R/H48Q ALS mice enhanced proteostasis as evidenced by increased expression of HSPs in motor neurons and astrocytes and increased solubility of mutant SOD1. HSF1 over-expression significantly reduced body weight loss, delayed ALS disease onset, decreases cases of early disease, and increased survival for the 25th percentile in an H46R/H48Q SOD1 background. HSF1 overexpression did not affect macroautophagy in the ALS background, but was associated with maintenance of carboxyl terminus of Hsp70 interacting protein (CHIP) expression which declined in H46R/H48Q mice. Conclusion Our results uncover the potential importance of changes in protein surface hydrophobicity of SOD1 and other non-SOD1 proteins in ALS, and how strategies that activate HSF1 are valid therapies for ALS and other age-associated proteinopathies.