Mutations In Stem Cells Research Articles

Toll-like receptor and cytokine expression throughout the bone marrow differs between patients with low- and high-risk myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders, the pathogenesis of which involves enhanced immune signaling that promotes or selects for mutant hematopoietic stem and progenitor cells (HSPCs). In particular, toll-like receptor (TLR) expression and signaling are enhanced in MDS, and their inhibition is an attractive therapeutic strategy. Although prior studies have reported increased expression of TLR2 and its binding partners TLR1 and TLR6 in the CD34+ cells of patients with MDS (especially those with low-risk disease), TLR expression in other cell types throughout the bone marrow is largely unknown. To address this, we used mass cytometry to assess the expression of TLR1, TLR2, and TLR6 and cytokines in the bone marrow hematopoietic cells of six low/intermediate-risk and six high-risk unmatched MDS bone marrow samples, as well as healthy controls, both at baseline and in response to TLR agonists. We observed several consistent differences between the groups. Most notably, TLR expression was upregulated in multiple cell populations in the low/intermediate-risk, but not high-risk, patients. In addition, many cytokines, including interleukin-6, interleukin-8, tumor necrosis factor α, transforming growth factor β, macrophage inflammatory protein 1β, and granzyme B, were highly expressed from various cell types in low/intermediate-risk patients. However, these same cytokines, with the exception of transforming growth factor β, were expressed at lower levels in high-risk MDS. Together, these findings highlight the differential role of inflammation, and specifically TLR expression, in low/intermediate- versus high-risk MDS, and suggest that elevated TLR expression and cytokine production in multiple cell types likely influences the pathogenesis of MDS in lower-risk patients.

Measures of Clonal Hematopoiesis: Are We Missing Something?

Clonal Hematopoiesis (CH) is a common, age-related phenomenon of growing scientific interest, due to its association with hematologic malignancy, cardiovascular disease and decreased overall survival. CH is commonly attributed to the preferential outgrowth of a mutant hematopoietic stem cell (HSC) with enhanced fitness, resulting in clonal imbalance. In-depth understanding of the relation between HSC clonal dynamics, CH and hematologic malignancy requires integration of fundamental lineage tracing studies with clinical data. However, this is hampered by lack of a uniform definition of CH and by inconsistency in the analytical methods used for its quantification. Here, we propose a conceptual and analytical framework for the definition and measurement of CH. First, we transformed the conceptual definition of CH into the CH index, which provides a quantitative measure of clone numbers and sizes. Next, we generated a set of synthetic data, based on the beta-distribution, to simulate clonal populations with different degrees of imbalance. Using these clonal distributions and the CH index as a reference, we tested several established indices of clonal diversity and (in-)equality for their ability to detect and quantify CH. We found that the CH index was distinct from any of the other tested indices. Nonetheless, the diversity indices (Shannon, Simpson) more closely resembled the CH index than the inequality indices (Gini, Pielou). Notably, whereas the inequality indices mainly responded to changes in clone sizes, the CH index and the tested diversity indices also responded to changes in the number of clones in a sample. Accordingly, these simulations indicate that CH can result not only by skewing clonal abundancies, but also by variation in their overall numbers. Altogether, our model-based approach illustrates how a formalized definition and quantification of CH can provide insights into its pathogenesis. In the future, use of the CH index or Shannon index to quantify clonal diversity in fundamental as well as clinical clone-tracing studies will promote cross-disciplinary discussion and progress in the field.

Disruption of the crypt niche promotes outgrowth of mutated colorectal tumor stem cells.

Recent data establish a logarithmic expansion of leucine rich repeat containing G protein coupled receptor 5–positive (Lgr5+) colonic epithelial stem cells (CESCs) in human colorectal cancer (CRC). Complementary studies using the murine 2-stage azoxymethane–dextran sulfate sodium (AOM-DSS) colitis-associated tumor model indicate early acquisition of Wnt pathway mutations drives CESC expansion during adenoma progression. Here, subdivision of the AOM-DSS model into in vivo and in vitro stages revealed DSS induced physical separation of CESCs from stem cell niche cells and basal lamina, a source of Wnt signals, within hours, disabling the stem cell program. While AOM delivery in vivo under non-adenoma-forming conditions yielded phenotypically normal mucosa and organoids derived thereof, niche injury ex vivo by progressive DSS dose escalation facilitated outgrowth of Wnt-independent dysplastic organoids. These organoids contained 10-fold increased Lgr5+ CESCs with gain-of-function Wnt mutations orthologous to human CRC driver mutations. We posit CRC originates by niche injury–induced outgrowth of normally suppressed mutated stem cells, consistent with models of adaptive oncogenesis.

A heterozygous MYBPC3 (c. 772+1G > A) mutant human induced pluripotent stem cell line (ZZUNEUi025-A) generated from a male patient with hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common heterogeneous myocardial disease. MYBPC3 variants are the leading cause of HCM. In the present study, a human induced pluripotent stem cell (iPSC) line ZZUNEUi025-A was generated from peripheral blood mononuclear cells of a male HCM patient with c. 772+1G > A in MYBPC3 gene. This cell line expressed pluripotency markers, had normal male karyotype and could differentiate into three germ layers in vitro.

Mitochondrial Fragmentation Triggers Ineffective Hematopoiesis in Myelodysplastic Syndromes.

Ineffective hematopoiesis is a fundamental process leading to the pathogenesis of myelodysplastic syndromes (MDS). However, the pathobiological mediators of ineffective hematopoiesis in MDS remain unclear. Here, we demonstrated that overwhelming mitochondrial fragmentation in mutant hematopoietic stem cells and progenitors (HSC/P) triggers ineffective hematopoiesis in MDS. Mouse modeling of CBL exon deletion with RUNX1 mutants, previously unreported comutations in patients with MDS, recapitulated not only clinically relevant MDS phenotypes but also a distinct MDS-related gene signature. Mechanistically, dynamin-related protein 1 (DRP1)-dependent excessive mitochondrial fragmentation in HSC/Ps led to excessive reactive oxygen species production, induced inflammatory signaling activation, and promoted subsequent dysplasia formation and impairment of granulopoiesis. Mitochondrial fragmentation was generally observed in patients with MDS. Pharmacologic inhibition of DRP1 attenuated mitochondrial fragmentation and rescued ineffective hematopoiesis phenotypes in mice with MDS. These findings provide mechanistic insights into ineffective hematopoiesis and indicate that dysregulated mitochondrial dynamics could be a therapeutic target for bone marrow failure in MDS. SIGNIFICANCE: We demonstrated that excessive mitochondrial fragmentation is a fundamental pathobiological phenomenon that could trigger dysplasia formation and ineffective hematopoiesis in MDS. Our findings provide mechanistic insights into ineffective hematopoiesis and suggest dysregulated mitochondrial dynamics as a therapeutic target for treating MDS.This article is highlighted in the In This Issue feature, p. 1.

A heterozygous TTN (c. 79,684C>T) mutant human induced pluripotent stem cell line (ZZUNEUi023-A) generated from a Kazakh patient with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a nonischaemic heart muscle disease with structural and functional myocardial abnormalities. TTN truncating mutations are a common cause of DCM, occurring in ∼25% of familial cases of DCM and in 18% of sporadic cases. In this study, we generated a human induced pluripotent stem cell line ZZUNEUi023-A from peripheral blood mononuclear cells of a Kazakh DCM patient with the p. Arg26562Ter (c. 79684C>T) mutation in TTN using non-integrative Sendai virus. This cell line expressed pluripotency markers, showed normal male karyotype and could differentiate into all three germ layers in vitro.

Azacitidine is a potential therapeutic drug for pyridoxine-refractory female X-linked sideroblastic anemia

AbstractX-linked sideroblastic anemia (XLSA) is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Treatment of XLSA is mainly supportive, except in patients who are pyridoxine responsive. Female XLSA often represents a late onset of severe anemia, mostly related to the acquired skewing of X chromosome inactivation. In this study, we successfully generated active wild-type and mutant ALAS2-induced pluripotent stem cell (iPSC) lines from the peripheral blood cells of an affected mother and 2 daughters in a family with pyridoxine-resistant XLSA related to a heterozygous ALAS2 missense mutation (R227C). The erythroid differentiation potential was severely impaired in active mutant iPSC lines compared with that in active wild-type iPSC lines. Most of the active mutant iPSC-derived erythroblasts revealed an immature morphological phenotype, and some showed dysplasia and perinuclear iron deposits. In addition, globin and HO-1 expression and heme biosynthesis in active mutant erythroblasts were severely impaired compared with that in active wild-type erythroblasts. Furthermore, genes associated with erythroblast maturation and karyopyknosis showed significantly reduced expression in active mutant erythroblasts, recapitulating the maturation defects. Notably, the erythroid differentiation ability and hemoglobin expression of active mutant iPSC-derived hematopoietic progenitor cells (HPCs) were improved by the administration of δ-aminolevulinic acid, verifying the suitability of the cells for drug testing. Administration of a DNA demethylating agent, azacitidine, reactivated the silent, wild-type ALAS2 allele in active mutant HPCs and ameliorated the erythroid differentiation defects, suggesting that azacitidine is a potential novel therapeutic drug for female XLSA. Our patient-specific iPSC platform provides novel biological and therapeutic insights for XLSA.

Genetic Dissection of Dyskeratosis Congenita-Causing Mutations in TINF2 Using Human Pluripotent and Hematopoietic Stem Cell Models

The length of telomeres, which cap the ends of linear chromosomes and provide genomic stability, is tightly regulated in adult stem cells. The telomere reserve in the stem cell population sets the replicative potential of its differentiated progeny. For this reason, abnormally short telomeres in stem cells restrict the number of cell divisions that their differentiated progenies can undergo, eventually resulting in stem cell depletion and tissue failure syndromes. Telomere biology disorders (TBDs) display a broad range of clinical features, age of onset, and severity, which are all correlated with the extent of abnormal telomere shortening. One such early-onset TBD is dyskeratosis congenita (DC), which is a bone marrow predisposition syndrome characterized by a mucocutaneous triad (oral leukoplakia, nail dystrophy, and abnormal skin pigmentation) as well as other conditions driven by premature tissue aging. The leading cause of death in DC patients is bone marrow failure and hematopoietic stem cell transplantation is the only definite intervention to restore hematopoiesis.TINF2, which encodes the TIN2 protein, is mutated in 12% of patients and thereby the second most frequently altered gene in DC cases. TIN2 is a member of the shelterin protein complex bridging the double-strand binding shelterin proteins TRF1/TRF2, and the TPP1/POT1 heterodimer. Such interactions implicate a complex role of TIN2 in telomere length regulation: First, TIN2 stabilizes TRF1, which is a negative regulator of telomere length. Secondly, TPP1, which recruits telomerase, strictly requires TIN2 for telomere elongation and maintenance. TINF2-DC mutations are uniformly heterozygous and localize to a 30 amino acid coding stretch in exon 6 called the ‘DC cluster’. TINF2-DC mutations usually arise de novo and result in an earlier disease onset, shorter telomeres, and a more severe manifestation compared to other heterozygous DC-causing mutations in genes such as TR and TERT, which encode the components of the telomerase enzyme.How TINF2-DC mutations cause telomere shortening is unknown. Specifically, whether telomere shortening is caused by reduced telomerase action at telomeres or by degradation of telomeric DNA remains unresolved as studies using different model systems report contrasting results. The discrepancy could be attributed to differences in the model systems used in the studies, highlighting the need for a genetically trackable, primary preclinical human model system.Here, we report the development of two novel endogenous, isogenic model systems to study TINF2-DC mutations. First, we generated human embryonic stem cells (hESCs) engineered to express the TINF2-DC T284R mutation from the endogenous locus, which recapitulated the short telomere phenotype observed in DC patients. Using this model, we identified a gene editing strategy that elongates telomeres in the mutant stem cells and eventually restores replicative potential of the differentiated cells. Next, we used a xenotransplantation model of donor-derived human hematopoietic stem cells (hHSCs) to test the effects of target gene modifications on telomere length and proliferative capacity in vivo. We demonstrate that our models robustly complement each other and offer direct insights into the disease mechanism as well as avenues to potential therapeutic approaches. [Display omitted] DisclosuresBertuch: Elixirgen Therapeutics: Consultancy; ImmunityBio: Current equity holder in publicly-traded company; NIH/NCI,: Research Funding; DOD: Research Funding; Hyundai Hope on Wheels: Research Funding.

Loss of nr4a1 abrogates Fitness of asxl1-mutant Hematopoietic Clones

Clonal fitness of mutant hematopoietic stem and progenitor cells (HSPCs) underlies clonal hematopoiesis (CH), a state of clonal expansion associated with increased risk of blood malignancies and cardiovascular disease. Mechanisms by which acquired mutations lead to clonal fitness are not known. We used a zebrafish model to study the effect of acquired asxl1 mutations on HSPC clonality with TWISTR (Tissue editing With Inducible Stem cell Tagging via Recombination) that combined mosaic CRISPR-Cas9 mutagenesis with color labeling of HSPC clones. TWISTR asxl1 mutants showed clonal dominant states with the expansion of single-colored clones contributing to over 30% of myelopoiesis. These zebrafish had normal hematopoietic output and no major lineage skewing. Single-cell RNA sequencing analysis of TWISTR mutant marrow cells harboring asxl1 mutations showed >10-fold upregulation of inflammatory cytokines in mutant mature myeloid cells and >30-fold upregulation of anti-inflammatory modulators in immature progenitors. Increased inflammation has been widely documented in persons with CH with acquired mutations in DNMT3A, TET2, ASXL1 and other genes. Moreover, chronic inflammation due to infection was shown to promote relative clonal fitness in Dnmt3a mutant mice. Based on our results, we proposed the hypothesis that upregulation of the anti-inflammatory genes, including nr4a1, served as a mechanism of resistance to chronic inflammation created by the mutant HSPC progeny, resulting in a self-perpetuating cycle of clonal fitness in that environment. To test this, we used TWISTR to generate mosaic mutants of asxl1 and nr4a1 by injecting zebrafish with guide RNAs targeting exon 12 of asxl1 and exon 3 of nr4a1 together. Our model would predict that abrogation of nr4a1 expression in asxl1-mutant clones would weaken their fitness relative to clones that maintained nr4a1 expression. We sorted over 300 clones of various sizes from this cohort of zebrafish and sequenced the two targeted genes. We found that clones with frameshift mutations in asxl1 with either no nr4a1 mutations or heterozygous nr4a1 mutations had a clone size of 20%±14% or 19.7%±15% in myeloid cells, respectively. Asxl1-mutant clones with biallelic frameshift mutations in nr4a1 were significantly smaller with a clone size of 13.8±11.5% (p < 0.015). Clones with intact asxl1 did not differ in their clone size independent of nr4a1 genetic status (11.9% wildtype nr4a1 vs 11.5% homozygous nr4a1 mutant). Chemical inhibition of nr4a1 over 3 months resulted in reduced change of edited clones in asxl1-mutant zebrafish compared to vehicle-treated zebrafish, with median change in allelic fraction of 3%±4.8% vs 5.3%±7.5%, respectively. This suggested that upregulation of nr4a1 in asxl1-mutant clones maintains their fitness in inflammatory conditions, potentially by limiting HSPC exhaustion. We successfully used TWISTR to study asxl1 induced CH in zebrafish and identified nr4a1 upregulation as a critical pathway engaged for establishing clonal fitness. DisclosuresZon: Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; CAMP4 Therapeutics: Current holder of individual stocks in a privately-held company, Other: Founder; Amagma Therapeutics: Current holder of individual stocks in a privately-held company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Branch Biosciences: Current holder of individual stocks in a privately-held company, Other: Founder; Celularity: Consultancy; Cellarity: Consultancy.

DNMT3A and TET2 mutant Clonal Hematopoiesis May Drive a Proinflammatory State and Predict Enhanced Response to Immune Checkpoint Inhibitors

DNMT3A and TET2 mutant Clonal Hematopoiesis May Drive a Proinflammatory State and Predict Enhanced Response to Immune Checkpoint Inhibitors

A Murine Model With JAK2V617F Expression in Both Hematopoietic Cells and Vascular Endothelial Cells Recapitulates the Key Features of Human Myeloproliferative Neoplasm.

The myeloproliferative neoplasms (MPNs) are characterized by an expansion of the neoplastic hematopoietic stem/progenitor cells (HSPC) and an increased risk of cardiovascular complications. The acquired kinase mutation JAK2V617F is present in hematopoietic cells in a majority of patients with MPNs. Vascular endothelial cells (ECs) carrying the JAK2V617F mutation can also be detected in patients with MPNs. In this study, we show that a murine model with both JAK2V617F-bearing hematopoietic cells and JAK2V617F-bearing vascular ECs recapitulated all the key features of the human MPN disease, which include disease transformation from essential thrombocythemia to myelofibrosis, extramedullary splenic hematopoiesis, and spontaneous cardiovascular complications. We also found that, during aging and MPN disease progression, there was a loss of both HSPC number and HSPC function in the marrow while the neoplastic hematopoiesis was relatively maintained in the spleen, mimicking the advanced phases of human MPN disease. Different vascular niche of the marrow and spleen could contribute to the different JAK2V617F mutant stem cell functions we have observed in this JAK2V617F-positive murine model. These results indicate that the spleen is functionally important for the JAK2V617F mutant neoplastic hematopoiesis during aging and MPN disease progression. Compared to other MPN murine models reported so far, our studies demonstrate that JAK2V617F-bearing vascular ECs play an important role in both the hematologic and cardiovascular abnormalities of MPN.

Nature or Nurture? Role of the Bone Marrow Microenvironment in the Genesis and Maintenance of Myelodysplastic Syndromes.

Simple SummaryMyelodysplastic syndromes (MDS) like many other blood cancers is a disease of the bone marrow, in which the spongy part of the bone is not able to produce enough healthy blood cells. MDS is primarily a disease of the elderly, but it can affect people at a younger age as well. The disease arises as a result of a combination of complex processes that is thought to be primarily driven by accumulation of genetic mutations in the stem cells. However, there is an increasing evidence implicating the bone marrow environment as a fertile milieu where these mutated stem cells thrive and give rise to the disease. In this review, we have discussed the role of blood stem cells and how other cell types in the bone marrow environment interact with each other, therefore contributing to MDS. In addition, we discuss the therapeutic strategies that can be exploited to treat MDS. Myelodysplastic syndrome (MDS) are clonal haematopoietic stem cell (HSC) disorders driven by a complex combination(s) of changes within the genome that result in heterogeneity in both clinical phenotype and disease outcomes. MDS is among the most common of the haematological cancers and its incidence markedly increases with age. Currently available treatments have limited success, with <5% of patients undergoing allogeneic HSC transplantation, a procedure that offers the only possible cure. Critical contributions of the bone marrow microenvironment to the MDS have recently been investigated. Although the better understanding of the underlying biology, particularly genetics of haematopoietic stem cells, has led to better disease and risk classification; however, the role that the bone marrow microenvironment plays in the development of MDS remains largely unclear. This review provides a comprehensive overview of the latest developments in understanding the aetiology of MDS, particularly focussing on understanding how HSCs and the surrounding immune/non-immune bone marrow niche interacts together.

Generation of a COL4A5 heterozygous mutation human embryonic stem cell line (WAe009-A-58) using an episomal vector-based CRISPR/Cas9 system

X-linked Alport syndrome (XLAS) is the second most common inherited kidney disease which pathogenic variants related to a mutation in the COL4A5 gene encoding the type IV collagen α5 chain. Here, we have generated a COL4A5 heterozygous mutant human embryonic stem cell (hESC) line (H9-COL4A5+/−) by an episomal vector-based CRISPR/Cas9 system. The generated H9-COL4A5+/− maintained a normal stem cell morphology, stably expressed pluripotent markers, and could differentiate into all three germ layers in vivo. This cell line offers an in vitro efficient platform to explore pathogenic mechanisms in XLAS and provides a cell-based disease model for drug testing.

The ability of bone marrow progenitor cells to form colonies in ex vivo culture of MDS patients

The results of in vitro hematopoiesis studies have provided most of the knowledge about the organization, regulation, and development of the human hematopoietic system over the past three to four decades. However, due to the impossibility of an appropriate assessment of hematopoietic stem cells (HSC) in humans and because of the shortcomings of methodological approaches to determining the role of hematopoietic progenitor cells in the pathogenesis of MDS and to predicting the course of the pathological process, semiliquid agar cultures of bone marrow from patients with myelodysplastic syndrome were used. Myelodysplastic syndrome (MDS) refers to a clinically, morphologically, and genetically heterogeneous group of diseases characterized by clonalism and arising from mutations at the level of hematopoietic progenitor cells. Proliferation of such a mutated stem cell progenitors leads to ineffective maturation of myeloid lineage cells and dysplastic changes in the bone marrow (BM). The aim of the study was to establish the relationship between the functional activity of hematopoietic progenitor cells in the ex vivo culture and the activity of the pathological process in the myelodysplastic syndrome. We studied bone marrow samples from patients with the myelodysplastic syndrome, namely refractory anemia with excess blasts I (MDS RAEB I) and refractory anemia with excess blasts II (MDS RAEB II) and AML under conditions in vitro, as well as their clinical laboratory data. It was found that the percentage of blasts and myeloblasts in the samples of patients with AML and MDS RAEB II increased, compared to the samples of patients with MDS RAEB I (63.5±3.9 %, 18.05±1.01 % and 9.49±1.53 % respectively). An increase in the number of erythrocytes and hemoglobin content was noted in the group of patients with MDS RAEB I compared with MDS RAEB II (2.9±1.4×1012 / l and 105.04±3.6 g / l versus 9±0.8×1012 / l and 84.5±4.8 g / l, respectively). The analysis of the results of BM studies of patients with MDS in in vitro culture indicated a significant lag in the formation of cell aggregates during cultivation and a pronounced inhibition of the colony-forming ability of progenitor cells, compared to the control. A noticeable decrease in the colony-forming ability was observed in patients with MDS RAEB I, MDS RAEB II and AML in this sequence – 4.1±1.2 per 1×105 explanted cells, 3.2±0.9 per 1×105 explanted cells and 2.0±0.6 per 1×105 explanted cells, respectively. The analysis of hematological parameters and the results of BM cells cultivation at different stages of MDS indicates that the colony-forming ability of progenitor cells correlates with the depth of the pathological process.

Gain of Function Mutant p53 Drives Clonal Hematopoiesis and the Pathogenesis of Myelodysplastic Syndromes

Clonal hematopoiesis increases with age, where a single mutant hematopoietic stem or progenitor cell (HSPC) contributes to a significant, measurable clonal proportion of mature blood lineages. Evolution of mutant clonal hematopoiesis with age predisposes the elderly to myelodysplastic syndromes (MDS) and other aging-associated diseases, suggesting that mutations identified in HSPCs contribute to disease development. Previous studies using whole exome sequencing of blood DNAs identified a high frequency of TP53 mutations in aged healthy individuals . TP53 mutations are also present in 10% of MDS cases and correlate with worse clinical outcomes. However, the role of TP53 mutations in clonal hematopoiesis and the pathogenesis of MDS is largely unknown. Ample data indicate that mutant p53 proteins not only lose their tumor suppressive functions, but also gain new abilities that promote tumorigenesis. Here, we report that specific gain-of-function (GOF) mutant p53 proteins drive the development of pre-leukemic hematopoietic stem cells (HSCs), predisposing aged mutant p53 mice to MDS development.Pre-leukemic HSCs have been shown to contribute to normal blood development and harbor a selective growth advantage compared to normal HSCs. We found that GOF mutant p53 (p53R248W) enhances the competitive advantage of HSCs without affecting terminal differentiation in steady state. Further, we observed that p53R248W mutant promotes clonal expansion of HSCs following genotoxic stresses. RNA-seq analysis revealed that HSC and leukemia stem cell signatures were significantly enriched in mutantHSCs compared to wild-type HSCs. Thus, HSCs expressing GOF mutant p53 recapitulate several key features of pre-leukemic HSCs. GOF p53 proteins are capable of dramatically altering tumor cell behavior, primarily through their interactions with other cellular proteins and regulation of cancer cell transcriptional programs. We discovered that GOF mutant p53 proteins, including p53R248W, p53R273H, and p53R175H, but not wild-type p53, interact with histone methyltransferase EZH2 and enhance H3K27me3 in HSCs. EZH2, a catalytic component of the Polycomb repressive complex 2 (PRC2), trimethylates histone H3 at lysine 27 (H3K27me3) to repress the transcription of target genes. Indeed, we found that the expression of HSC regulators, including Elf4 and Gadd45g, was significantly downregulated in mutant HSCs compared to wild-type HSCs. ChIP-seq analysis show enrichment of H3K27me3 in the promoter regions of Elf4 and Gadd45g in mutant HSCs. Importantly, we discovered that EZH2 inhibitor treatment decreased the replating potential of mutantbone marrow cells to wild-type cell level. Thus, we demonstrated that GOF mutant p53 drives the development of pre-leukemic HSCs through enhancing EZH2 activity.Given that most homozygous p53-/- and p53R248W/R248Wmice develop spontaneous tumors, including lymphoma, thymoma, and sarcoma, and die within 3 to 6 months after birth, it was not clear whether heterozygous mutant p53 mice (p53R248W/+) would develop myeloid malignancies during aging. To evaluate the role of GOF mutant p53 in the pathogenesis of myeloid malignancies, we maintained p53+/+ and p53R248W/+ mice for more than a year and monitored their survival and tumor development. While p53R248W/+ mice show extended life span compared to p53-/- and p53R248W/R248W mice, most of these mice died within 16-18 months after birth. During the observation period, approximately 30 % of heterozygous mutant p53mice developed MDS based upon pathological analysis of BM, spleen, and peripheral blood smear. Histological assessment revealed hypercellular BM in some aged p53R248W/+ mice. BM of some aged p53R248W/+ mice show neutrophil, erythroid precursor and megakaryocyte dysplasia. We further demonstrated that mutant p53 driven-MDS is transplantable. Thus, we provided evidence that GOF mutant p53 drives MDS development during aging.In summary, we have identified a critical role for GOF mutant p53 in clonal hematopoiesis and the pathogenesis of MDS. DisclosuresNo relevant conflicts of interest to declare.

Targeting the Epigenetic and Metabolic Vulnerabilities of Ezh2-Deficiency-Induced Myeloid Neoplasms

Myeloproliferative neoplasms (MPNs) are clonal, progressive hematopoietic malignancies characterized by alterations of multiple signaling and epigenetic pathways in hematopoietic stem cells (HSCs). The identification of driver oncogenic mutations in JAK2, CALR or MPL has transformed our knowledge of MPNs; however, patients with non-mutated JAK2, CALR and MPL (so-called ‘triple-negative‘) have the highest incidence of leukemic transformation and the lowest overall survival (Rumi et al., 2014 Blood 124:1062-1069), indicating that other factors may contribute to MPNs. The molecular processes controlling MPN progression from chronic to acute leukemic transformation remain unknown. This is a major impediment for developing target-based therapeutics to selectively eliminate the mutant stem cells to prevent disease progression and/or relapse. Therefore, the challenge is twofold: 1) to identify and characterize the genetic complexity of MPN pathophysiology, and 2) to understand and control the progression of MPNs from chronic to life-threatening stages.We have developed a new mouse model of MPNs by hematopoietic-selective (Mx1-cre) activation of oncogenic Ras (NRasG12D+/-) and inactivation of Ezh2, the enzymatic subunit of the Polycomb Repressive Complex 2 (PRC2), which mimics concurrent mutations in human MPNs. While NRasG12D+/- alone led to an indolent myeloproliferative disorder, Ezh2 loss markedly accelerated disease progression to life-threatening stages, resulting in thrombocytosis, neutrophilia, splenomegaly, primary myelofibrosis, leukemic transformation and mortality. Strikingly, while loss of Ezh1, a homolog of Ezh2, had no effect on normal hematopoiesis, concurrent inactivation of Ezh1 and Ezh2 with NRasG12D+/- completely abolished MPN development and myelofibrosis in vivo . These results establish distinct functions of Ezh1 and Ezh2 in myeloid neoplasms, and identify a selective epigenetic vulnerability for MPNs induced by Ezh2-deficiency. Follow-up studies of Ezh1/2-mediated chromatin (by ChIP-seq) and transcriptomic (by RNA-seq) changes in normal HSCs and leukemia-initiating cells (LICs) revealed non-overlapping gene targets and cellular pathways required for the distinct cellular phenotypes.Reprogrammed cellular metabolism underlies the progression of various human cancers including leukemia. We noted by RNA-seq transcriptomic profiling that Bcat1, the first and rate-limiting enzyme catalyzing the transamination of branched-chain amino acids (BCAAs), was aberrantly activated in Ezh2-deficient leukemia-initiating cells (LICs). By ChIP-seq analysis, we observed that the Bcat1 promoter is highly enriched with tri-methylated H3-Lys27 (H3K27me3), suggesting that it is epigenetically silenced by Ezh2-PRC2 in normal HSCs. Furthermore, genetic or pharmacologic inhibition of BCAT1 activity selectively abrogated the clonogenic activity of Ezh2-deficient LICs in vitro and the leukemogenic potential in transplanted recipients in vivo, highlighting a metabolic dependency for Ezh2-mutant myeloid neoplasms. Follow-up targeted metabolomic analysis of Ezh2-deficiency-induced metabolic changes established the critical role of Bcat1 and the associated BCAA metabolism in the progression of myeloid neoplasms and leukemia.Collectively, our results not only elucidate the mechanisms by which Ezh1 is specifically required for Ezh2-deficient LICs, but also validate an important epigenetic vulnerability for MPN patients carrying loss-of-function Ezh2 mutations. Moreover, in-depth analysis of BCAA metabolism in myeloid neoplasms identifies a new metabolic dependency for Ezh2-deficient LICs. Because normal hematopoietic stem cells are unaffected by loss of Ezh1 or Bcat1, the selective vulnerabilities raise the possibility of leveraging Ezh1 or BCAA metabolism as targeted therapies to specifically eradicate Ezh2-mutant LICs. Hence, our study promises to provide critical insights into developing new and generalizable therapeutic strategies to selectively eliminate leukemia-initiating cells harboring alterations of epigenetic pathways. DisclosuresDeberardinis:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Cell competition between wild-type and JAK2V617F mutant cells in a murine model of a myeloproliferative neoplasm

The myeloproliferative neoplasms (MPNs) are characterized by overproduction of mature blood cells and increased risk of transformation to frank leukemia. The acquired kinase mutation JAK2V617F plays a central role in a majority of patients with these diseases. As MPNs are clonal stem cell disorders (i.e. arise from a single stem cell which eventually expands), the hematopoietic stem/progenitor cell (HSPC) compartment in MPNs is heterogeneous with the presence of both JAK2 wild-type and JAK2V617F mutant cells. Mechanisms responsible for the mutant stem cell expansion in MPNs are not fully understood. Utilizing in vitro co-culture assays and in vivo competitive transplantation assays, we show that the presence of wild-type cells alters both the gene expression profile and cellular function of JAK2V617F mutant HSPCs and inhibits the expansion of co-existing JAK2V617F mutant cells in a normal microenvironment. In contrast, we found that a microenvironment bearing the mutant kinase promotes JAK2V617F mutant HSPC expansion over wild-type cells due in part to altered CXCL12/CXCR4 signaling. Further understanding of the molecular mechanisms controlling the competitive interactions between normal and JAK2V617F mutant cells, and how these mechanisms break down during MPN disease progression hold great potential for advances in treating patients with these diseases.

JAK2V617F mutant endothelial cells promote neoplastic hematopoiesis in a mixed vascular microenvironment

The chronic myeloproliferative neoplasms (MPNs) are clonal stem cell disorders. The hematopoietic stem/progenitor cell (HSPC) compartment in patients with MPNs is heterogeneous with the presence of both wild-type and JAK2V617F mutant cells. Mechanisms responsible for mutant stem cell expansion in MPNs are not fully understood. Vascular endothelial cells (ECs) are an essential component of the hematopoietic microenvironment. ECs carrying the JAK2V617F mutation can be detected in patients with MPNs. Utilizing an ex vivo EC-HSPC co-culture system with mixed wild-type and JAK2V617F mutant ECs, we show that even small numbers of JAK2V617F mutant ECs can promote the expansion of JAK2V617F mutant HSPCs in preference to wild-type HSPCs during irradiation or cytotoxic chemotherapy, the two treatments commonly used in the conditioning regimen for stem cell transplantation, the only curative treatment for patients with MPNs. Mechanistically, we found that both cell-cell interactions and secreted factors are important for JAK2V617F mutant EC-mediated neoplastic hematopoiesis. Further understanding of how the JAK2V617F mutation alters vascular niche function will help identify new strategies to not only control neoplastic cell expansion but also prevent disease relapse in patients with MPNs.

Novel fragile X syndrome 2D and 3D brain models based on human isogenic FMRP-KO iPSCs

Fragile X syndrome (FXS) is a neurodevelopmental disorder, characterized by intellectual disability and sensory deficits, caused by epigenetic silencing of the FMR1 gene and subsequent loss of its protein product, fragile X mental retardation protein (FMRP). Delays in synaptic and neuronal development in the cortex have been reported in FXS mouse models; however, the main goal of translating lab research into pharmacological treatments in clinical trials has been so far largely unsuccessful, leaving FXS a still incurable disease. Here, we generated 2D and 3D in vitro human FXS model systems based on isogenic FMR1 knock-out mutant and wild-type human induced pluripotent stem cell (hiPSC) lines. Phenotypical and functional characterization of cortical neurons derived from FMRP-deficient hiPSCs display altered gene expression and impaired differentiation when compared with the healthy counterpart. FXS cortical cultures show an increased number of GFAP positive cells, likely astrocytes, increased spontaneous network activity, and depolarizing GABAergic transmission. Cortical brain organoid models show an increased number of glial cells, and bigger organoid size. Our findings demonstrate that FMRP is required to correctly support neuronal and glial cell proliferation, and to set the correct excitation/inhibition ratio in human brain development.

Chronic infection drives Dnmt3a-loss-of-function clonal hematopoiesis via IFNγ signaling.

Age-related clonal hematopoiesis (CH) is a risk factor for malignancy, cardiovascular disease, and all-cause mortality. Somatic mutations in DNMT3A are drivers of CH, but decades may elapse between the acquisition of a mutation and CH, suggesting that environmental factors contribute to clonal expansion. We tested whether infection provides selective pressure favoring the expansion of Dnmt3a mutant hematopoietic stem cells (HSCs) in mouse chimeras. We created Dnmt3a-mosaic mice by transplanting Dnmt3a-/- and WT HSCs into WT mice and observed the substantial expansion of Dnmt3a-/- HSCs during chronic mycobacterial infection. Injection of recombinant IFNγ alone was sufficient to phenocopy CH by Dnmt3a-/- HSCs upon infection. Transcriptional and epigenetic profiling and functional studies indicate reduced differentiation associated with widespread methylation alterations, and reduced secondary stress-induced apoptosis accounts for Dnmt3a-/- clonal expansion during infection. DNMT3A mutant human HSCs similarly exhibit defective IFNγ-induced differentiation. We thus demonstrate that IFNγ signaling induced during chronic infection can drive DNMT3A-loss-of-function CH.