Pathogenesis Of Myeloid Malignancies Research Articles

Insight into the mechanism of AML del(9q) progression: hnRNP K targets the myeloid master regulators CEBPA (C/EBPα) and SPI1 (PU.1)

Deletions on the long arm of chromosome 9 (del(9q)) are recurrent abnormalities in about 2 % of acute myeloid leukemia cases, which usually involve HNRNPK and are frequently associated with other known aberrations. Based on an Hnrnpk haploinsufficient mouse model, a recent study demonstrated a function of hnRNP K in pathogenesis of myeloid malignancies via the regulation of cellular proliferation and myeloid differentiation programs. Here, we provide evidence that reduced hnRNP K expression results in the dysregulated expression of C/EBPα and additional transcription factors. CyTOF analysis revealed monocytic skewing with increased levels of mature myeloid cells. To explore the role of hnRNP K during normal and pathological myeloid differentiation in humans, we characterized hnRNP K-interacting RNAs in human AML cell lines. Notably, RNA-sequencing revealed several mRNAs encoding key transcription factors involved in the regulation of myeloid differentiation as targets of hnRNP K. We showed that specific sequence motifs confer the interaction of SPI1 and CEBPA 5′ and 3′UTRs with hnRNP K. The siRNA mediated reduction of hnRNP K in human AML cells resulted in an increase of PU.1 and C/EBPα that is most pronounced for the p30 isoform. The combinatorial treatment with the inducer of myeloid differentiation valproic acid resulted in increased C/EBPα expression and myeloid differentiation. Together, our results indicate that hnRNP K post-transcriptionally regulates the expression of myeloid master transcription factors. These novel findings can inaugurate novel options for targeted treatment of AML del(9q) by modulation of hnRNP K function.

A Question of Frame: The Role of the Bone Marrow Stromal Niche in Myeloid Malignancies.

Until a few years ago, the onset of acute myeloid leukemia (AML) was entirely ascribed to genetic lesions in hematopoietic stem cells. These mutations generate leukemic stem cells, which are known to be the main ones responsible for chemoresistance and relapse. However, in the last years, increasing evidence demonstrated that dynamic interplay between leukemic cells and bone marrow (BM) niche is of paramount relevance in the pathogenesis of myeloid malignancies, including AML. Specifically, BM stromal niche components, such as mesenchymal stromal cells (MSCs) and their osteoblastic cell derivatives, play a key role not only in supporting normal hematopoiesis but also in the manifestation and progression of myeloid malignancies. Here, we reviewed recent clinical and experimental findings about how genetic and functional alterations in MSCs and osteolineage progeny can contribute to leukemogenesis and how leukemic cells in turn generate a corrupted niche able to support myeloid neoplasms. Moreover, we discussed how the newest single-cell technologies may help dissect the interactions between BM stromal cells and malignant hematopoiesis. The deep comprehension of the tangled relationship between stroma and AML blasts and their modulation during disease progression may have a valuable impact on the development of new microenvironment-directed therapeutic strategies, potentially useful for a wide cohort of patients.

Mitochondrial dynamics as a pathobiological mediator of clonal myeloid disorders.

Myeloid malignancies, including myelodysplastic syndromes and acute myeloid leukemia, are a group of clonal hematopoietic stem cell (HSC) diseases. The incidence increases with global population aging. Genome sequencing uncovered mutational profiles in patients with myeloid malignancies and healthy elderly individuals. However, the molecular and cellular basis of disease development remains unclear. Accumulating evidence shows mitochondrial involvement in the pathogenesis of myeloid malignancies, aging-related HSC phenotypes, and clonal hematopoiesis. Mitochondria are dynamic organelles that continuously undergo fission and fusion processes to maintain their function, integrity, and activity. Mitochondria could be a hub of various biological processes that underlie cellular and systemic homeostasis. Thus, mitochondrial dysfunction could directly lead to the disruption of cellular homeostasis and the development of various disorders, including cancer. Notably, emerging data have revealed that mitochondria dynamics also primarily affect not only mitochondrial function and activity but also cellular homeostasis, the aging process, and tumorigenesis. Here, by focusing on mitochondrial dynamics, we highlight the current understanding of mitochondrial roles as a pathobiological mediator of myeloid malignancies and aging-related clonal hematopoiesis.

[Role of innate immune signaling and inflammation in the pathogenesis of myeloid malignancies].

Myeloid malignancies are composed of multiple clonal hematopoietic disorders, including myelodysplastic syndrome, myeloproliferative neoplasms, and acute myeloid leukemia. Inflammation is already known to play an important role in the pathogenesis of an extensive variety of malignancies, and its significance in myeloid malignancies is becoming more widely recognized. Specifically, cell-intrinsic and -extrinsic activation of the innate immune signaling pathway, as well as elevation of proinflammatory cytokines via innate immune signaling downstream signaling, have been demonstrated. Furthermore, the inflammatory microenvironment refers to the bone marrow environment rich in inflammatory signaling molecules that surround hematopoietic malignant cells, and its role in the pathogenesis of myeloid malignancies has been extensively studied in recent years. Herein, we present the latest findings and discuss how innate immune signaling activation and the inflammatory bone marrow microenvironment contribute to the pathogenesis of myeloid malignancies.

Inflammation and myeloid malignancy: quenching the flame

AbstractChronic inflammation with aging (“inflammaging”) plays a prominent role in the pathogenesis of myeloid malignancies. Aberrant inflammatory activity affects many different cells in the marrow, including normal blood and stromal marrow elements and leukemic cells, in unique and distinct ways. Inflammation can promote selective clonal expansion through differential immune-mediated suppression of normal hematopoietic cells and malignant clones. We review these complex roles, how they can be understood by separating cell-intrinsic from extrinsic effects, and how this informs future clinical trials.

Gain-of-Function of ASXL1 Truncating Protein in the Pathogenesis of Myeloid Malignancies

Introduction: Additional Sex Combs-Like 1 (ASXL1) is mutated at a high frequency in all forms of myeloid malignancies associated with poor prognosis. We and others have reported that loss of ASXL1 impairs hematopoietic stem/progenitor cell functions (HSC/HPCs) and leads to myeloid dysplastic syndrome (MDS) in mice. It remains controversial whether truncating mutations in ASXL1 result in gain- or loss-of-function, or whether they confer dominant-negative activity in vivo . Answering this question is critical for determining how to target these mutations therapeutically.Methods: We generated a transgenic mouse model using HS321/45-vav vector that expresses the Asxl1 Y588X cDNA under the control of the Vav1 promoter. The transgene contains the entire coding region of mouse Asxl1 (full-length) with a stop codon mutation at the Y588 site (Asxl1 Y588X), leading to a production of truncating protein ASXL1aa1-587 in the hematopoietic system only. Utilizing the transgenic model, we performed a serial hematopoietic analyses, including peripheral blood (PB) counts, histological characterization on blood smear, tissue sections of femur and spleen, and flow cytometric analysis. Bone marrow(BM) transplantation was performed to identify the effects of truncating ASXL1aa1-587 on HSC/HPCs functions. RNA-seq and ATAC-seq analyses were performed to survey the differentially expressed genes in HSC/HPCs of WT and Asxl1 Y588X Tg mice. Liquid chromatography-tandem mass spectrometry (LC-MS) and co-immunoprecipitation (IP) experiments were performed to identify the specific protein binding partner of ASXL1aa1-587. An epigenetic drug screening was used to identify specific targets to block the gain-of-function protein, ASXL1aa1-587, in the pathogenesis of myeloid malignancies in vivo.Results: The Asxl1Y588X Tg mice had an enlarged hematopoietic stem cell (HSC) pool, shortened survival, and predisposition to a spectrum of myeloid malignancies, thereby recapitulating the characteristics of myeloid malignancy patientswith ASXL1 mutations. Competitive transplantation of WT and Asxl1Y588XTg BM cells revealed that expression of A SXL 1 aa587 was associated with a higher percentage of CD45.2+ cells in the PB of the recipients, indicating that A SXL11-587aa expression increases HSC self-renewal capacity in vitro and in vivo. ATAC- and RNA-Seq analyses revealed that the ASXL11-587aa truncating protein expression results in more open chromatin in cKit+ cells compared to wildtype cells, accompanied by dysregulated expression of genes critical for HSC self-renewal and differentiation. LC-MS) and co-IP experiments showed that ASXL11-587aa acquired an interaction with BRD4. An epigenetic drug screening demonstrated a hyper-sensitivity of Asxl1Y588X Tg bone marrow cells to BET bromodomain inhibitors.Conclusion: This study demonstrates that ASXL11-587aa plays a gain-of-function role in promoting myeloid malignancies and that BET bromodomain inhibitors significantly reduced the colony forming capacity of ASXL1Y588XTgHSPCs. Our model provides a powerful platform to test therapeutic approaches of targeting the ASXL1 truncation mutations in myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

TET2 and MSH6: A Novel Role for MMR in the Pathogenesis of Myeloid Malignancies

TET2 mutations (TET2MT) occur in up to 40% of myeloid neoplasms and increase linearly with age. That these lesions are founding hits can be concluded from clonal architecture and from the presence of subclinical TET2MT clones in healthy individuals at risk for subsequent MDS. Though TET2MT are heterogeneous, most disrupt TET2 catalytic domain dioxygenase activity; these widespread LOF mutations implicate TET2 as a bona fide tumor-suppressor gene (TSG). As downstream targets have not been identified as being associated with TET2MT, the precise mechanisms of TET2MT in leukemogenesis remains unclear. We studied whether TET2MT predispose cells to acquiring additional oncogenic mutations through faulty DNA repair to produce clonally acquired mutator phenotypes.In WES analyses of 435 myeloid neoplasia patient (pt) samples, we identified 95 with somatic TET2MT enriched vs controls in the ExAC database, and used 6 bioinformatics tools to predict the effect on protein function based on algorithms incorporating sequence homology, amino acid properties, sequence context, and similarity to Mendelian diseases. Most pts (63) were predicted to have damaging TET2MT in 3+ out of six of these tools.To validate TET2MT clinical relevance, we established an in-vitro 2D-UPLC-MS/MS assay to assess levels of the TET2-dependent DNA oxidation products 5hmC, 5fC, and 5CaC. While TET2 +/- knockout mouse samples had nonsignificant differences in 5hmC (1.2-fold lower levels vs TET2WT, p=.23) and 5fC (1.4-fold increase vs TET2WT, p=.07), they had significantly lower 5CaC (2.4-fold decrease vs TET2WT, p<.0001). TET2 -/- mouse samples had significantly lower 5hmC (1.6-fold reduction vs TET2WT, p<.0001), 5fC (1.3-fold reduction vs TET2WT, p=.011), and 5CaC (2.4-fold reduction vs TET2 WT, p<.0001). Human MOLM-13 TET2 -knockdown (TET2 kd) cells also had 1.6-fold lower levels of 5hmC (p<.0001). Reassuringly, eight samples from TET2MT patients also had lower 5hmC (5/8 samples, peak 2.9-fold reduction vs TET2WT, p<.0001), 5fC (6/8 samples, peak 8.2-fold reduction vs TET2WT, p<.0001), and 5CaC (5/8 samples, peak 2.6-fold reduction vs TET2 WT, p<.0001), and all 8/8 TET2MT pt samples had significant declines in at least one of the three TET2-dependent oxidation vs TET2WT samples.To test whether TET2MT predisposed cells to acquiring additional genomic mutations, we measured the total number of single nucleotide variants (SNV) in the entire exome in each pt sample, excluded pts with mutations in known DNA repair proteins, and compared the median WES SNV of TET2MT pts to TET2WT pts. Consistent with our theory, TET2MT pts had a 1.5-fold increase in median WES SNV (p<.0001), particularly at 5hmC sites. Pts with a TET 2MT variant allele frequency>50% had a 2.1-fold increase in median WES SNV (p=.03), consistent with a gene-dose effect.TET2 -/- knockout mice had 1.4-fold increased mutagenicity at TET2-dependent active demethylation sites, in a manner suggestive of defective mismatch repair (MMR). HeLa TET2 kd cells also had a 24-fold increase in spontaneous mutations, reversed with TET2 cDNA knock-in. MMR gene expression (MSH2, MSH3, MSH6, and MLH1) was equivalent among 20 TET2MT MDS pts, 71 TET2WT MDS pts, and 17 controls, indicating MMR downregulation was not responsible for the hypermutagenicity. Five pts with TET2MT were microsatellite-stable (MSS) at 5 TET2-independent poly-dA microsatellite loci, suggesting that hypermutagenicity was not driven by global MMR dysfunction and may occur only at CpG-containing microsatellites.By overexpressing Flag/V5-tagged TET2 in mouse-MEL cells and subjecting them to protein affinity purification, we identified MSH6 as a novel TET2 binding partner. TET2:MSH6 interactions were validated in co-immunofluorescence experiments in MEL and MOLM-13 cells, and TET2kd altered MSH6 nuclear co-localization. MOLM-13 TET2kd increased PARP inhibitor sensitivity by almost 3-fold, suggesting that TET2kd renders cells vulnerable to perturbations in DNA repair pathways.In sum, we uncovered novel connections among TET2, MSH6, epigenetic modifications, and genomic instability. Given that MSH6 is known to preferentially bind 5hmC, TET2 may target MSH6 to TET2-dependent DNA loci. Genomic instability due to TET2 dysfunction may allow therapeutic targeting of DNA repair proteins in the subset of hematologic malignancy pts with TET2MT. DisclosuresGreenberg:Oisin Biotechnologies: Consultancy; Acetylon Pharmaceuticals Inc.: Patents & Royalties: Treatment of Protein Degradation Disorders. Makishima:Yasuda Medical Foundation: Research Funding. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fees; Apellis Pharmaceuticals: Consultancy; Ra Pharma: Consultancy.

Disregulation of CTCF Boundary at the HOX Locus Enhances Sensitity to Multiple Drug Treatment in Myeloid Malignancies

Dysregulation of HOX genes is a dominant mechanism of leukemic transformation by altering self-renewal and differentiation properties of HS/PCs leading to leukemic transformation. The overexpression of posterior HOXA9 gene is also a poor prognostic marker in leukemia patients. Recent studies revealed that CTCF insulator constrains temporal HOX gene expression patterns within confined chromatin domains suggesting that CTCF mediated chromatin boundary activity may involve in regulating HOX gene patterns during AML prognosis.To investigate the role of CTCF in aberrant HOX gene expression and AML prognosis, we employed CRISPR-Cas9 genome editing technology to delete 47 base pairs of the core CTCF motif located between HOXA7 and HOXA9 genes (CBS7/9) in different subtype of AML cells with either MLL-rearrangement or NPM1 mutation. RNA-seq analysis revealed that transcription factors, HOXA9 , RUNX1 , and ZEB1 , as well as MAPK signaling pathway, all of them are involved in AML survival, prognosis and chemo-resistance, are significantly dowregulated in response to the CBS7/9 boundary deletion. Given that these genes and pathways are involved in AML prognosis and chemo-resistance, we then examined whether perturbation of CBS7/9 chromatin boundary resulted in alteration of drug resposes of AML cells. Both WT AML cells and the CBS7/9 defected AML cells derived from MLL-AF9 rearranged MOLM-13 and NPM mutated OCI-AML3 cells were treated with 1 mM of Ara-C or 100 nM of bromodomain (BET) inhibitors. Compared to the WT controls, the defected CBS7/9 boundary resulted in a significantly increase in AML sensitivity to both Ara-C and BET inhibitor treatment in both MOLM-13 and OCI-AML3 cells as evidence by significantly reducing percentage of proliferating cells.To further examine if treatment related drug sensitivity in AML cells is depended on the levels of HOXA9 expression, we overexpressed HOXA9 cDNA in the CBS7/9 deleted MOLM-13 and OCI-AML3 cells. Gain-of-function (GOF) expression of HOXA9 in the CBS7/9 defected AML cells rescued and restored the resistance to the Ara-C treatment of the CBS7/9 KO cells to the levels compatible to that of the WT control MOLM-13 and OCI-AML3 cells, but HOXA9 overexpression did not rescued the resistance of the CBS7/9 KO AML cells to the BET inhibitor treatment. The data suggest that HOXA9 is directly associated with cytotoxic drug Ara-C resistance, but not BET inhibitor mediated drug responses. KO of CBS7/9 specifically decreased enrichment of H3K9ac and H4K5ac, as well as recruitment of BRD4 to the posterior HOXA domain. Furthermore, overexpression of HOXA9 gene in the CBS7/9 KO AML cells indeed rescued the mRNA transcripts affected by CBS7/9 deletion to the levels compatible to that of WT control MOLM-13 or OCI-AML3 cells. Among rescued genes, ZEB1, STAT5A, and RUNX1 play essential roles in pathogenesis of myeloid malignancies and chemo-drug sensitivity.Finally, in order to further explore biological role of CTCF boundaries in AML pathogenesis, we analyze the whole genome sequencing data of 183 primary AML patients obtained from public available Cosmic Database for somatic mutations occurred in the CTCF binding sites. Several core CTCF motifs in the HOXA locus, including CBS5/6 and CBS7/9 boundaries, are highly mutated in AML patients. Thus, disruption of CTCF boundary located between HOXA7 and HOXA9 genes perturbs HOXA9 oncogene transcription and regulates chemo- or epigenetic drug sensitivity of acute myeloid leukemia. Alteration of CTCF boundaries in the oncogene loci may provide a novel therapeutic target for treatment of myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

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.

Natural Killer Cell Subpopulations and Inhibitory Receptor Dynamics in Myelodysplastic Syndromes and Acute Myeloid Leukemia.

Natural killer (NK) cells are key innate immunity effectors that play a major role in malignant cell destruction. Based on expression patterns of CD16, CD56, CD57, and CD94, three distinct NK cell maturation stages have been described, which differ in terms of cytokine secretion, tissue migration, and the ability to kill target cells. Our study addressed NK cell maturation in bone marrow under three conditions: a normal developmental environment, during pre-leukemic state (myelodysplastic syndrome, MDS), and during leukemic transformation (acute myeloblastic leukemia, AML). In this study, we used a new tool to perform multicolor flow cytometry data analysis, based on principal component analysis, which allowed the unsupervised, accurate discrimination of immature, mature, and hypermature NK subpopulations. An impaired NK/T cell distribution was observed in the MDS bone marrow microenvironment compared with the normal and AML settings, and a phenotypic shift from the mature to the immature state was observed in NK cells under both the MDS and AML conditions. Furthermore, an impaired NK cell antitumor response, resulting in changes in NK cell receptor expression (CD159a, CD158a, CD158b, and CD158e1), was observed under MDS and AML conditions compared with the normal condition. The results of this study provide evidence for the failure of this arm of the immune response during the pathogenesis of myeloid malignancies. NK cell subpopulations display a heterogeneous and discordant dynamic on the spectrum between normal and pathological conditions. MDS does not appear to be a simple, intermediate stage but rather serves as a decisive step for the mounting of an efficient or ineffective immune response, leading to either the removal of the tumor cells or to malignancy.

PU.1-Dependent Enhancer Decommissioning Drives Transformation of Tet2 deficient Hematopoietic Stem and Progenitor Cells

Acute myeloid leukemia (AML) is the most frequent leukemia in elderly individuals with a median age at diagnosis of 67 years (Juliusson et al., Blood 2009). It arises in a step-wise process and originates from hematopoietic stem cells (HSC) (Jan et al.,Sci Transl Med. 2012). Genetic and epigenetic alterations drive the formation of pre-leukemic HSC clones with altered function, which can gain dominance and eventually give rise to AML upon the acquisition of cooperating lesions (Jan et al.,Sci Transl Med. 2012). However, it is currently impossible to predict which healthy elderly individuals with clonal hematopoiesis will eventually develop myeloid malignancies, as the pathways to leukemia are unknown. Heterozygous inactivating mutations of the epigenetic regulator Ten-Eleven Translocation-2 (TET2) are commonly found in patients with AML, yet also in a remarkable fraction of healthy elderly individuals in whom it is associated with clonal hematopoiesis (Busque, et al Nat Genet. 2012). These observations and studies in Tet2-deficient mice strongly suggest that TET2 inactivation is an early event in the pathogenesis of myeloid malignancies, but is not sufficient to fully transform HSC (Moran-Crusio et al., Cancel Cell 2011). TET2 cooperates with several transcription factors to regulate hematopoiesis (Rasmussen et al., Genome Res 2019), one of which is PU.1 (de la Rica et al., Genome Biol. 2013), an essential transcription factor governing normal hematopoiesis (Iwasaki et al., Blood 2005). In humans, PU.1 activity or expression is only moderately impaired in the majority of AML patients, and remarkably, also in aged HSC (Will et al., Nat Med. 2015), underscoring the essentiality of PU.1. Importantly, PU.1 target genes are frequently found hypermethylated in AML (Sonnet et al., Genome Med. 2014, Kaasinen et al., Nat Commun. 2019), suggesting a profound epigenetic inactivation of the PU.1 network. We hypothesized that moderate impairment of PU.1 abundance, as found in AML, can cooperate with loss-of-function mutations of Tet2 to initiate malignancy. We developed a novel tissue-specific compound mutant mouse model carrying heterozygous deletion of an upstream regulatory element (URE) of Pu.1 along with Tet2 deletion (Vav-iCre+ PU.1URΕ∆/+Tet2+/flox; Vav-iCre+ PU.1URΕ∆/+Tet2flox/flox). While none of the single mutant mice developed AML, compound mutant mice developed aggressive myeloid leukemia whose penetrance and latency exhibited Tet2 dose dependency. The disease presented with leukocytosis, anemia and splenomegaly. By cell morphology analysis of the peripheral blood, bone marrow and spleen, the leukemic mice exhibited accumulation of differentiation-blocked myeloblasts, myelocytes and/or metamyelocytes, that was confirmed using detailed myeloid differentiation markers, distinguishing the disease in immature or mature AML. Furthermore, gold standard in vitro and in vivo assays, assessing both self-renewal and differentiation capacity of double mutant mice-derived cells, revealed that the expanded differentiation-blocked stem and progenitor cells bear aberrant self-renewal and disease-initiating capacities. Comprehensive molecular profiling by next generation sequencing of disease-initiating cells uncovered a substantial overlap with human AML, such as functional GF1b loss with concomitant overexpression of CD90/Thy1 (Thivakaran et al., Haematologica 2018). Importantly, our analyses also revealed transcriptional dysregulation, hypermethylation of PU.1 regulated enhancers with concomitant loss of enhancer activity and alterations in chromatin accessibility of particularly genes co-bound by PU.1 and TET2. Current efforts focus on identifying key effectors of the dysregulated PU.1/TET2 sub-network driving malignant transformation in clonal hematopoiesis. Our collected data provide proof of concept that moderate PU.1 dose impairment can functionally cooperate with the inactivation of Tet2 in the initiation of myeloid leukemia and uncovers a likely unifying AML pathomechansim. Disclosures Will: Novartis Pharmaceuticals: Other: Service on advisory boards, Research Funding.

EZH2 in Myeloid Malignancies.

Our understanding of the significance of epigenetic dysregulation in the pathogenesis of myeloid malignancies has greatly advanced in the past decade. Enhancer of Zeste Homolog 2 (EZH2) is the catalytic core component of the Polycomb Repressive Complex 2 (PRC2), which is responsible for gene silencing through trimethylation of H3K27. EZH2 dysregulation is highly tumorigenic and has been observed in various cancers, with EZH2 acting as an oncogene or a tumor-suppressor depending on cellular context. While loss-of-function mutations of EZH2 frequently affect patients with myelodysplastic/myeloproliferative neoplasms, myelodysplastic syndrome and myelofibrosis, cases of chronic myeloid leukemia (CML) seem to be largely characterized by EZH2 overexpression. A variety of other factors frequently aberrant in myeloid leukemia can affect PRC2 function and disease pathogenesis, including Additional Sex Combs Like 1 (ASXL1) and splicing gene mutations. As the genetic background of myeloid malignancies is largely heterogeneous, it is not surprising that EZH2 mutations act in conjunction with other aberrations. Since EZH2 mutations are considered to be early events in disease pathogenesis, they are of therapeutic interest to researchers, though targeting of EZH2 loss-of-function does present unique challenges. Preliminary research indicates that combined tyrosine kinase inhibitor (TKI) and EZH2 inhibitor therapy may provide a strategy to eliminate the residual disease burden in CML to allow patients to remain in treatment-free remission.

The Role of ASXL1/2 and Their Associated Proteins in Malignant Hematopoiesis

Advances in genomic and epigenetic research have uncovered a central role for aberrant epigenetic regulation in the pathogenesis of myeloid malignancies. In the current review, we summarize the roles of ASXL1/2 and their associated proteins in normal and malignant hematopoiesis. ASXL1/2 and their associated proteins, e.g., polycomb repressive complex 2 proteins, play key roles in regulating hematopoietic stem cell (HSC) functions. Genetic studies reveal that ASXL1/2 and their associated proteins play important roles for the establishment and maintenance of the cell fates of HSCs. Alterations of the genes coding ASXL1/2 and their associated proteins lead to the development of hematological malignancies. Epigenetic regulation is crucial for normal hematopoiesis. Alteration of multiple epigenetic modifiers contributes to myeloid malignancies. Understanding the molecular mechanisms is critical for further studying ASXL1/2 and their associated proteins in hematopoiesis and developing new therapeutic strategies to treat myeloid malignancies.

Mapping the Evolution of the Mutational Landscape of Acute Myeloid Leukemia from Diagnosis to Post-Transplantation Relapse and Its Interplay with Immune Evasion Mechanisms

Background Over the last decade, the advent of next-generation sequencing (NGS) has dramatically improved our understanding of the genomic landscape that characterize acute myeloid leukemia at time of diagnosis, leading to the identification of new prognostic subsets and therapeutic targets. Still, few studies have addressed how this landscape changes upon treatment, and in particular after allogeneic hematopoietic cell transplantation (allo-HCT). Moreover, the links between the mutational profile of AML and mechanisms of post-transplantation immune evasion remain to date largely unknown. Methods Taking advantage of the HaloPlex High Sensitivity (HS) technology, which allows a more precise definition of mutations and clonal architecture through the use of unique molecular identifiers, we designed and tested a NGS panel covering genes and miRNAs known to be involved in the pathogenesis of myeloid malignancies (n=112), in the DNA damage response (n=50) or in immune-related processes (n=30). By using an Illumina HiSeq2500n instrument,we sequenced a total of 200 AML samples collected from 131 patients, including 84 diagnoses, 45 relapses after chemotherapy (CT) and 71 relapses after allo-HCT, 31 of which characterized by genomic loss of HLA (Vago et al, N Engl J Med, 2009). Variant calling was performed using a pipeline based on the FreeBayes algorithm. Results Sequencing coverage was uniform across all target amplicons, with a 855x average depth-of-sequencing, and a mean of 122 unique barcodes for each region. Among the 84 diagnosis samples we identified 253 oncogenic mutations, with a median of 3 mutations per patient (range 0-8). Both the frequency and the prognostic value of mutations detected at diagnosis were in line with the largest published dataset on AML genomics (Papaemmanuil et al, N Engl J Med, 2016; R2= 0.88, p&amp;lt;0.0001). The overall burden of oncogenic mutations did not change significantly neither at relapses after CT nor after allo-HCT. Analyzing the profile of known oncogenic drivers of diagnoses and of the two relapse cohorts we documented high similarity between the three time-points, with significant differences limited to FLT3-ITD (present in 29% patients at diagnosis vs 49% and 48% at relapses after CT and allo-HSCT, p=0.02 and p=0.01 respectively) and WT1 mutations (5% vs 20% and 28%, p=0.006 and p&amp;lt;0.0001 respectively; Figure 1A). Also when comparing post-transplantation HLA loss relapses with their "classical" counterparts we could find no significant difference in the frequency or co-occurrence pattern of known AML driver mutations. Still, in HLA loss relapses we documented a significantly higher frequency of variants of unknown oncogenic potential befalling in genes implicated in the DNA damage response, including those belonging to the Fanconi Anemia pathway (35% of mutations in HLA loss vs 10% in classical relapses, p&amp;lt;0.0001; Figure 1B). This intriguing observation suggests that, in these cases, double strand breaks might be more frequently repaired through homologous recombination, and that this in turn might result in a higher likelihood of copy-neutral loss of heterozygosity events, the molecular mechanism at the basis of HLA loss. Finally, thanks to our panel, we were able to track possible routes of leukemic clonal evolution between diagnosis and post-transplantation relapse: interestingly, in several cases emergence of an HLA loss subclone out-competed clones carrying known oncogenic drivers, suggesting that after allo-HCT the immune privilege might outperform the proliferative advantage (Figure 1C). Discussion Taken together, our data highlight a complex interplay between the genomic landscape of AML and the environmental selective pressure represented by the graft-versus-leukemia effect. Whereas in fact some genomic alterations that carried little consequence before transplant become able in this setting to promote specific patterns of immune escape, others, that had driven AML biology before, turn out to be dispensable after allo-HCT. Understanding and tracking these evolutionary dynamics may provide new tools and targets to treat, or even prevent, AML post-transplantation relapses. Figure 1 Disclosures Sala: Gilead Company: Honoraria. Gentner:Genenta Science: Consultancy, Equity Ownership, Research Funding. Vago:GenDx: Research Funding; Moderna Therapeutics: Research Funding.

Genomic Profiling of a Phase III Clinical Trial of Interferon Versus Hydroxyurea in MPN Patients Reveals Mutation-Specific and Treatment-Specific Patterns of Response

Background: To investigate the role of genomics in determining response and resistance to front line treatment in MPN, we performed somatic mutational profiling of the DALIAH trial, a randomized controlled phase III trial of interferon versus hydroxyurea in newly diagnosed MPN patients. Methods: We performed genomic analyses on 202 pre-treatment primary MPN samples obtained from patients enrolled on the DALIAH trial (NCT01387763) and 135 samples obtained after 24 months of treatment. Genomic profiling comprised targeted next generation sequencing (NGS) of 100 genes, selected on the basis of their known or suspected involvement in the pathogenesis of myeloid malignancies, and 1609 informative single nucleotide polymorphisms (SNPs) on chromosome 9p. Clinicohematological response was determined by central review using ELN 2009 (ET, PV and pre-MF) and EUMNET 2005 (PMF) criteria. We evaluated the association of somatic mutations with clinical parameters and with attainment of clinicohematological complete response (CR) at 24 months. Results: Prior to treatment, 191 of 202 (95%) patients had somatic mutations, including 93% with canonical MPN phenotypic drivers: JAK2 (74%), CALR (14%), and MPL (5%). Among those with JAK2 mutations 37% had JAK2 copy-neutral loss of heterozygosity (JAK2 CN-LOH). Patients with PV were more likely to have JAK2 CN-LOH (p = 0.0001) as compared with patients with other MPN subtypes. At baseline, patients with JAK2 CN-LOH had significantly higher hemoglobin (p = 0.0001), higher white blood cell count (WBC, p = 0.002) and lower platelet count (p=0.0001) than patients without JAK2 CN-LOH. Mutations in TET2 (24%), DNMT3A (16%), and ASXL1 (10%) were the most frequent co-occurring non-MPN phenotypic driver mutations and they occurred across all MPN subtypes. In addition, 5% of patients had spliceosome gene mutations, and 6% had mutations in genes involved in RAS/MAPK signaling. Patients with TET2, DNMT3A or ASXL1 mutations were significantly older than patients without these mutations (p= 0.0001) and there was a significant association between the presence of a TET2, DNMT3A or ASXL1 mutation and prior stroke (p = 0.004). There were no other significant associations between somatic mutation status and baseline clinical characteristics. The probability of attaining clinicohematological CR at 24 months was independent of baseline somatic mutations. Among patients with JAK2 mutations who remained on interferon treatment at 24 months, those with CR had a greater reduction in mean variant allele fraction (VAF) (28% to 8%, p&lt;0.0001) than those who did not achieve CR (34% to 23%, p=0.03). In contrast, the mutant CALR VAF did not significantly decline in either those achieving CR with interferon treatment (16% to 12%, p=0.38) or those not achieving CR (20% to 16%, p=0.35). In patients with JAK2 mutations treated with hydroxyurea, CR at 24 months was associated with a change in VAF (mean 24% to 13%, p=0.04). Among patients who remained on treatment for 2 years, 44 mutations in 35 patients were newly detected or expanded on serial sampling. DNMT3A mutations were the most frequently acquired, accounting for 41% of new mutations. ASXL1, TET2, PPM1D, TP53, IDH2, and CBL mutations were also recurrently acquired. 97% of patients who acquired new mutations were JAK2-mutant. Among those with acquired DNMT3A mutations, 85% were treated with interferon, and 23% had CR at 24 months. Among those that acquired non-DNMT3A mutations, 38% were treated with interferon and 47% had CR at 24 months. The VAF of newly detected mutations was low (median 1.5%), and half of the patients with newly acquired mutations had at least 50% reduction in JAK2V617F VAF, suggesting that new mutations could either have arisen independently or be subclonal to the dominant JAK2-mutant clone. Conclusions: Using sequential genomic analyses of a phase III clinical trial of interferon versus hydroxyurea in MPN patients, we found mutation-specific and treatment-specific patterns of response. We uncovered distinct patterns of response to interferon in JAK2-mutant MPN as compared with CALR-mutant MPN. We found that DNMT3A mutations were the most frequent acquired mutations at 24 months and that these were enriched in patients treated with interferon. In aggregate, these results provide insights into molecular response and resistance to interferon and inform the clinical use of interferon in MPN patients. Disclosures Hansen: Alexion: Research Funding. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Hasselbalch:Novartis: Research Funding; AOP Orphan Pharmaceuticals: Other: Data monitoring board. Lindsley:Jazz Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Consultancy; Medlmmune: Research Funding. Mullally:Janssen: Research Funding.

Genomic Landscape of Juvenile Myelomonocytic Leukemia: A Real World Context

Introduction: Juvenile myelomonocytic leukaemia (JMML) is a clonal haematopoietic disorder occurring in pediatric age group usually characterized by uncontrolled granulocytic and monocytic lineage proliferation and a poor outcome. JMML is characterised by aberrant signal transduction of the RAS signalling pathways, usually manifesting in molecular alteration of one of the five cardinal genes: NF1, NRAS, KRAS, PTPN11 and CBL. Recent high-throughput studies have started to push the horizon of JMML associated mutations wider and have proposed molecular-based risk algorithms. We comprehensively evaluated the genomic profile of JMML that were referred to our hospital for diagnosis and treatment. Methods: We developed a 51 gene (103 kB) low-cost targeted sequencing myeloid panel based on single molecule molecular inversion probes. This focussed panel interrogated sets of genes implicated in pathogenesis of myeloid malignancies. A total of 50 children with clinical and pathological features of JMML were sequenced at high coverage using this assay on an Illumina MiSeq. Results:The median age of our cohort was 2 years, with a male preponderance. Among the 50 patients, 43 (86%) harboured mutations in one of the RAS/MAPK pathway genes. The most frequently mutated gene in our cohort was PTPN11 (14, 28%), and NRAS (14, 28%), followed by NF1 mutation in 22% (11) cases (Figure 1). Interestingly, 20% (10) of children had more than one mutation, with 5 cases harbouring two RAS pathway mutations. Of these 5 patients, one patient harboured two coexistent mutations in the NF1 gene, three patients had coexistent PTPN11 and NF1 mutations and one patient had a coexistent NF1 and NRAS mutations. Additionally, two patients also had an ASXL1 mutation, coexistent with NF1 and NRAS mutations respectively. Other non-RAS pathway mutations involved ABL1, ATRX, SETBP1, SH2B3 and ZRSR2 genes. The median variant allele frequency of RAS pathway mutations detected was 40.75%. Monosomy 7 was detected in 32% (16) patients, and five of these did not harbour any RAS pathway mutations. The follow up data revealed that 37 (74%) of these patients have succumbed to the disease. In our cohort, only five patients received an allogenic stem cell transplant, owing to economic constraints; two of these patients have succumbed to the disease. The median time period from diagnosis to death was 7.2 months in the cohort. On survival analysis, patients with PTPN11 mutations showed a trend to shorter overall survival, compared to their wildtype counterparts (p=0.7, median OS 10.6 months vs 38.1 months). Additionally, patients harbouring more than one mutation also showed a trend to shorter OS (p=0.64, median OS 10.3 months vs 38.1 months). Among the thirteen patients who are still on regular follow-up, two patients have relapsed with disease progression to acute myeloid leukemia and T/myeloid mixed phenotypic acute leukemia respectively. Conclusion: Our study represents the largest cohort of JMML from a single centre in the Indian subcontinent. This study depicted that almost 90% cases of JMML harbor at least one mutation with 86% harbouring at least one RAS pathway mutation. Presence of PTPN11 mutations and co-existence of more than one mutation may be the major determinants of outcome in JMML. Figure 1 Disclosures No relevant conflicts of interest to declare.

A Novel Machine Learning Derived Genomics-Based Scoring System Is Highly Predictive of Outcome in Core Binding Factor Acute Myeloid Leukemia

Introduction: Core binding factor acute myeloid leukemia (CBF-AML) is one of the commonest subtypes of AML characterized presence of t(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22). It is characterised by a high frequency of somatic mutations especially in RAS and tyrosine kinase signalling pathways. Here we investigated the feasibility of improving risk prediction of CBF-AML using machine learning algorithms. Methods: We developed a next generation sequencing panel that targeted 50 genes implicated in the pathogenesis of myeloid malignancies using single molecule molecular inversion probes. This panel was used to sequence 106 patients of CBF-AML accrued over a six year period (March 2012 - December 2018) treated with conventional "3 + 7" chemotherapy. Post data analysis, we devised a supervised machine learning (ML) approach for identification of mutations most likely to predict for favorable outcome in CBF-AML. We included somatic mutations in genes occurring in CBF-AML at a frequency of &gt;5%. A total of 11 variables were included for feature selection to predict for favorable outcome (including mutations in ASXL2, CSF3R,FLT3, KIT, NF1, NRAS, RAD21, TET2 and WT1 genes as well as mutation burden). Approaches for supervised ML were naïve bayes, generalized linear model, logistic regression, deep learning and random forest methods. Based on the ML results top 6 selected variables were allotted an individual score. A final score for that case was devised as a sum total of the individual scores. These sum were used to generate a genetic risk for a patient. Overall survival (OS) was calculated from date of diagnosis to time of last follow up or death. Relapse free survival (RFS) was calculated from date of CR till time to relapse or death or last follow up if in CR. Results of the genetic risk were analyzed for their impact on OS and RFS using log rank test. Multivariate analysis was performed using cox proportional hazards regression model. Results: The median follow up of the cohort was 27.6 months. A total of 181 somatic mutations were identified in this subset of AML with 86.7% harbouring at least one somatic mutation (median = 2). Based on ML data, a genetic score was formulated that incorporated mutations in RAD21, FLT3, KIT D816, ASXL2, NRAS genes as well as high mutation burden (≥2) into two genetic risk classes (favorable risk and poor ML derived genetic genetic risk). Patients classified as poor genetic risk had a significantly lower OS [median OS: 34.8 months; 95% confidence interval (CI) (14.2-34.8); p=0.0086] and RFS [median RFS: 17.9 months; 95%CI (12.7-33.6); p=0.0043] as compared to patients with favorable genetic risk (median OS and RFS not reached). These results can be seen in Figure 1. On multivariate analysis poor genetic risk was the most important independent risk factor that predicted for inferior OS [hazard ratio(HR), 2.7; 95% CI 1.3 to 5.7] and RFS (HR, 2.6; 95% CI:1.3 to 5.1). Conclusions In a proof of concept, we describe a novel ML derived genomics scoring model that provides a mechanism to risk stratify CBF-AML, a seemingly homogeneous disease entity. This study, to the best of our knowledge represents a novel application of ML to CBF mutated AML. Our data indicates that this scoring system will be useful in identifying CBF mutated AML patients who are at higher risk of relapse and distinguishes them from patients who are truly good risk. Figure 1 Disclosures No relevant conflicts of interest to declare.

Quantitative and qualitative impairments in GATA2 and myeloid neoplasms.

GATA2 is a key transcription factor critical for hematopoietic cell development. During the past decade, it became clear that heterozygous germline mutations in the GATA2 gene cause bone marrow failure and primary immunodeficiency syndrome, conditions that lead to a predisposition toward myeloid neoplasms, such as myelodysplastic syndrome, acute myeloid leukemia, and chronic myelomonocytic leukemia. Somatic mutations of the GATA2 gene are also involved in the pathogenesis of myeloid malignancies. Cases with GATA2 gene mutations are divided into two groups, resulting in either a quantitative deficiency or a qualitative defect in the GATA2 protein depending on the mutation position and type. In the former case, GATA2 mRNA expression from the mutant allele is markedly reduced or completely abrogated, and reduced GATA2 protein expression is involved in the pathogenesis. In the latter case, almost equal amounts of structurally abnormal and wildtype GATA2 proteins are predicted to be present and contribute to the pathogenesis. The development of mouse models of these human GATA2-related diseases has been undertaken, which naturally develop myeloid neoplasms.

RUNX1 mutations promote leukemogenesis of myeloid malignancies in ASXL1-mutated leukemia

BackgroundAdditional sex combs-like 1 (ASXL1) mutations have been described in all forms of myeloid neoplasms including chronic myelomonocytic leukemia (CMML) and associated with inferior outcomes, yet the molecular pathogenesis of ASXL1 mutations (ASXL1-MT) remains poorly understood. Transformation of CMML to secondary AML (sAML) is one of the leading causes of death in CMML patients. Previously, we observed that transcription factor RUNX1 mutations (RUNX1-MT) coexisted with ASXL1-MT in CMML and at myeloid blast phase of chronic myeloid leukemia. The contribution of RUNX1 mutations in the pathogenesis of myeloid transformation in ASXL1-mutated leukemia, however, remains unclear.MethodsTo evaluate the leukemogenic role of RUNX1-MT in ASXL1-mutated cells, we co-expressed RUNX1-MT (R135T) and ASXL1-MT (R693X) in different cell lines and performed immunoblot, co-immunoprecipitation, gene expression microarray, quantitative RT-PCR, cell proliferation, differentiation, and clonogenic assays for in vitro functional analyses. The in vivo effect was investigated using the C57BL/6 mouse bone marrow transplantation (BMT) model.ResultsCo-expression of two mutant genes increased myeloid stem cells in animal model, suggesting that cooperation of RUNX1 and ASXL1 mutations played a critical role in leukemia transformation. The expression of RUNX1 mutant in ASXL1-mutated myeloid cells augmented proliferation, blocked differentiation, and increased self-renewal activity. At 9 months post-BMT, mice harboring combined RUNX1 and ASXL1 mutations developed disease characterized by marked splenomegaly, hepatomegaly, and leukocytosis with a shorter latency. Mice transduced with both ASXL1 and RUNX1 mutations enhanced inhibitor of DNA binding 1 (ID1) expression in the spleen, liver, and bone marrow cells. Bone marrow samples from CMML showed that ID1 overexpressed in coexisted mutations of RUNX1 and ASXL1 compared to normal control and either RUNX1-MT or ASXL1-MT samples. Moreover, the RUNX1 mutant protein was more stable than WT and increased HIF1-α and its target ID1 gene expression in ASXL1 mutant cells.ConclusionThe present study demonstrated the biological and functional evidence for the critical role of RUNX1-MT in ASXL1-mutated leukemia in the pathogenesis of myeloid malignancies.

PF542 MITOCHONDRIAL GENOMIC ANALYSIS IN PATIENTS WITH MYELOID MALIGNANCIES

Background: Mitochondrial DNA (mtDNA) somatic mutation frequently occur in neoplasms. Because of heteroplasmy of mDNA, it is difficult to determine whether genetic variations in mtDNA occur as germline variants or truly somatic variant. Genetic abnormalities in nuclear DNA in myelodysplastic syndrome (MDS) and other myeloid malignancies are massively investigated. However, the molecular abnormalities in mtDNA in MDS and other myeloid malignancies have not been poorly revealed. In this study, we analyzed full sequence of mtDNA using next generation sequencing (NGS) technique in patients with MDS or other myeloid malignancies to investigate possible variants in mtDNA in these patients. Aims: The aim of this study is to find mtDNA variants and to select possible somatic mtDNA variants and investigate possible association with disease characteristics. Methods: We collected total DNA from bone marrow or peripheral blood samples with malignant cells in 35 patients with various myeloid malignancies [15 MDS, 7 acute myeloid leukemia (AML) without recurrent genetic abnormalities, 6 primrary myelofibrosis (PMF), 4 myelomomocytic leukemia (CMML), and 2 atypical chronic myeloid leukemia (aCML)]. In addition, 49 patients with non-hematologic diseases are included as controls. Whole mitochondrial genomic sequencing was amplified, and NGS was performed by HiSeq 2000 (illumine). In addition, targeted sequencing using genomic DNA for 138 genes including well-known genes in MDS was also performed. Results: We selected candidate mtDNA single nucleotide variants (mt-SNVs) according to the heteroplasmy levels of variants and filtering from reference database and control patients’ results. We discarded small insertion or deletions from our analysis. When same selection criteria were applied in control samples, no candidate mt-SNVs were found except 1 known mutation in MELAS patients. We finally selected 53 candidate mt-SNVs in 26 patients (74.3%). The 40/53 (75.5%) of mt-SNVs were found in protein coding genes, 10/53 (18.9%) were found in rRNA genes, and 3/53 (5.7%) were found in tRNA genes. The mt-SNVs were most frequently found in CYTB and CO3, and ND3, ND4, ND4, RNR1, and RNR2 in descending order. mt-SNVs in CO3 were most frequently found in MPN patients, and variants in ND3 and ND4 were most frequently found in AML patients. Most of patients with ND3 variants had ASXL1 mutations. Summary/Conclusion: Our results suggested that somatic mtDNA may present different distributions among disease subtypes of myeloid malignancies, which may give new insight on the pathogenesis of myeloid malignancies.