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To meet cellular bioenergetic and biosynthetic demands, cancer cells remodel their metabolism to increase glycolytic flux, a phenomenon known as the Warburg effect and believed to contribute to cancer malignancy. Among glycolytic enzymes, phosphofructokinase-1 (PFK1) has been shown to act as a rate-limiting enzyme and to facilitate the Warburg effect in cancer cells. In this study, however, we found that decreased PFK1 activity did not affect cell survival or proliferation in cancer cells. This raised a question regarding the importance of PFK1 in malignancy. To gain insights into the role of PFK1 in cancer metabolism and the possibility of adopting it as a novel anti-cancer therapeutic target, we screened for genes that caused lethality when they were knocked down in the presence of tryptolinamide (TLAM), a PFK1 inhibitor. The screen revealed a synthetic chemical-genetic interaction between genes encoding subunits of ATP synthase (complex V) and TLAM. Indeed, after TLAM treatment, the sensitivity of HeLa cells to oligomycin A (OMA), an ATP synthase inhibitor, was 13,000 times higher than that of untreated cells. Furthermore, this sensitivity potentiation by TLAM treatment was recapitulated by genetic mutations of PFK1. By contrast, TLAM did not potentiate the sensitivity of normal fibroblast cell lines to OMA, possibly due to their reduced energy demands compared to cancer cells. We also showed that the PFK1-mediated glycolytic pathway can act as an energy reservoir. Selective potentiation of the efficacy of ATP synthase inhibitors by PFK1 inhibition may serve as a foundation for novel anti-cancer therapeutic strategies.

Figure S1-13 and Table S1,3

Figure S1-S22

Table S1-S11

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

Table S2

The transcriptional repressor BCL11A is involved in hematological malignancies, B-cell development, and fetal-to-adult hemoglobin switching. However, the molecular mechanism by which it promotes the development of myeloid leukemia remains largely unknown. We find that Bcl11a cooperates with the pseudokinase Trib1 in the development of acute myeloid leukemia (AML). Bcl11a promotes the proliferation and engraftment of Trib1-expressing AML cells in vitro and in vivo. Chromatin immunoprecipitation sequencing analysis showed that, upon DNA binding, Bcl11a is significantly associated with PU.1, an inducer of myeloid differentiation, and that Bcl11a represses several PU.1 target genes, such as Asb2, Clec5a, and Fcgr3. Asb2, as a Bcl11a target gene that modulates cytoskeleton and cell-cell interaction, plays a key role in Bcl11a-induced malignant progression. The repression of PU.1 target genes by Bcl11a is achieved by sequence-specific DNA-binding activity and recruitment of corepressors by Bcl11a. Suppression of the corepressor components HDAC and LSD1 reverses the repressive activity. Moreover, treatment of AML cells with the HDAC inhibitor pracinostat and the LSD1 inhibitor GSK2879552 resulted in growth inhibition in vitro and in vivo. High BCL11A expression is associated with worse prognosis in humans with AML. Blocking of BCL11A expression upregulates the expression of PU.1 target genes and inhibits the growth of HL-60 cells and their engraftment to the bone marrow, suggesting that BCL11A is involved in human myeloid malignancies via the suppression of PU.1 transcriptional activity.

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

Primers for q-RT-PCR

Up- and down-regulated genes

GO analysis of RUNX3-Tet2 KO MDS

RUNX3, a RUNX family transcription factor, regulates normal hematopoiesis and functions as a tumor suppressor in various tumors in humans and mice. However, emerging studies have documented increased expression of RUNX3 in hematopoietic stem/progenitor cells (HSPC) of a subset of patients with myelodysplastic syndrome (MDS) showing a worse outcome, suggesting an oncogenic function for RUNX3 in the pathogenesis of hematologic malignancies. To elucidate the oncogenic function of RUNX3 in the pathogenesis of MDS in vivo, we generated a RUNX3-expressing, Tet2-deficient mouse model with the pancytopenia and dysplastic blood cells characteristic of MDS in patients. RUNX3-expressing cells markedly suppressed the expression levels of Runx1, a critical regulator of hemaotpoiesis in normal and malignant cells, as well as its target genes, which included crucial tumor suppressors such as Cebpa and Csf1r. RUNX3 bound these genes and remodeled their Runx1-binding regions in Tet2-deficient cells. Overexpression of RUNX3 inhibited the transcriptional function of Runx1 and compromised hematopoiesis to facilitate the development of MDS in the absence of Tet2, indicating that RUNX3 is an oncogene. Furthermore, overexpression of RUNX3 activated the transcription of Myc target genes and rendered cells sensitive to inhibition of Myc-Max heterodimerization. Collectively, these results reveal the mechanism by which RUNX3 overexpression exerts oncogenic effects on the cellular function of and transcriptional program in Tet2-deficient stem cells to drive the transformation of MDS.Significance:This study defines the oncogenic effects of transcription factor RUNX3 in driving the transformation of myelodysplastic syndrome, highlighting RUNX3 as a potential target for therapeutic intervention.

Runx1 target genes

RUNX3 IHC and clinical data of MDS patients

Motif enrichment analysis

Insufficiency of polycomb repressive complex 2 (PRC2), which trimethylates histone H3 at lysine 27, is frequently found in primary myelofibrosis and promotes the development of JAK2V617F-induced myelofibrosis in mice by enhancing the production of dysplastic megakaryocytes. Polycomb group ring finger protein 1 (Pcgf1) is a component of PRC1.1, a non-canonical PRC1 that monoubiquitylates H2A at lysine 119 (H2AK119ub1). We herein investigated the impact of PRC1.1 insufficiency on myelofibrosis. The deletion of Pcgf1 in JAK2V617F mice strongly promoted the development of lethal myelofibrosis accompanied by a block in erythroid differentiation. Transcriptome and chromatin immunoprecipitation sequence analyses showed the de-repression of PRC1.1 target genes in Pcgf1-deficient JAK2V617F hematopoietic progenitors and revealed Hoxa cluster genes as direct targets. The deletion of Pcgf1 in JAK2V617F hematopoietic stem and progenitor cells (HSPCs), as well as the overexpression of Hoxa9, restored the attenuated proliferation of JAK2V617F progenitors. The overexpression of Hoxa9 also enhanced JAK2V617F-mediated myelofibrosis. The expression of PRC2 target genes identified in PRC2-insufficient JAK2V617F HSPCs was not largely altered in Pcgf1-deleted JAK2V617F HSPCs. The present results revealed a tumor suppressor function for PRC1.1 in myelofibrosis and suggest that PRC1.1 insufficiency has a different impact from that of PRC2 insufficiency on the pathogenesis of myelofibrosis.

An 80-year-old Japanese male patient presented to our hospital with complaints of fatigue. His peripheral blood tests revealed pancytopenia with predominant lymphocytes and without blasts. The bone marrow (BM) aspiration was unsuccessful due to a dry tap, and the subsequent BM biopsy revealed hypocellular marrow with fibrosis. He was diagnosed with myelodysplastic syndrome (MDS) with excess blasts (EB)-2 based on CD34-positive cells. The chromosome analysis of the BM revealed monosomy 7, and the SAMD9 W22

A 22-year-old man with a history of mediastinal germ cell tumor, which was diagnosed at age 20 and remained disease-free after chemotherapy, was diagnosed with acute myeloid leukemia (AML) M2 in January 2020. Karyotype analysis of bone marrow (BM) specimen at diagnosis detected 47,XXY, inv (16) in all cells. Following induction treatment, he achieved complete remission with a remarkable decrease in the minimal residual disease marker. Although considered related to therapy, the AML had a prognostically favorable karyotype, and the initial treatment response was very good. He had no human leukocyte antigen-matched sibling donor candidate. Thus, allogeneic hematopoietic stem cell transplantation was not scheduled at the first complete remission. After three cycles of consolidation therapy, he remained disease-free for over one year. Karyotype analysis of BM during remission revealed that all analyzed cells harbored 47,XXY, and Klinefelter syndrome (KS) was diagnosed. Although the patient experienced an adjustment disorder on KS diagnosis, he had overcome the difficulty with the assistance of psycho-oncologists, clinical psychologists, and genetic counselors. Herein, we report this rare case of KS that manifested after AML diagnosis following mediastinal germ cell tumor treatment.

Idiopathic CD4+ lymphocytopenia (ICL) is the depletion of CD4+ lymphocytes to 3 without human immunodeficiency virus infection or other causes of lymphocytopenia. ICL causes fatal infections; its etiology remains unclear and it lacks consensus regarding therapeutic options. We report the first patient with ICL who had a successful clinical course following a cord blood transplant (CBT). A 45-year-old woman was diagnosed with ICL and underwent partial hepatectomy for an abscess caused by the Mycobacterium avium complex. No specific gene alterations were detected through next generation sequencing-based evaluation. Following a reduced-intensity conditioning (RIC) regimen consisting of fludarabine, busulfan, and 4 Gy total body irradiation, a single-unit CBT was performed. Neutrophils were engrafted on day +14. CD4+ lymphocyte counts increased to over 300 cells/mm3 on day +436. After 75 months, she was alive without any sequelae. CBT with an RIC regimen could be a curable treatment option for ICL.

Neutrophils are generally considered as short-lived, homogenous, and terminally differentiated phagocytes that play crucial roles in conquering infection, although they occasionally cause severe collateral tissue damage or chronic inflammation. Recent reports have indicated that neutrophils also play a protective role in inflammation resolution and tissue repair. However, how terminally differentiated neutrophils have diverse functions remains unclear. Here, we show that neutrophils undergo conversion into Ly6G+SiglecF+ double-positive cells expressing neurosupportive genes in the olfactory neuroepithelium (OE) under an inflammatory state. Through comprehensive flow cytometric analysis of murine nose, we identified Ly6G+SiglecF+ double-positive cells that reside only in the OE under steady-state conditions. Double-positive cells were neutrophil-derived cells and increased by more than 10-fold during inflammation or tissue injury. We found that neutrophils infiltrate into the nose to express proinflammatory genes in the acute phase of inflammatory state, and they gradually change their surface markers and gene expression, expressing some neurogenesis-related genes in addition to inflammation related genes in the later phase. As the OE is known to have exceptionally high regeneration capacity as a nervous system, these findings suggest that neutrophils have the potential to contribute neurogenesis after conversion in peripheral nervous tissues, providing a challenge on a classic view of neutrophils as terminally differentiated leukocytes.

Gene rearrangements of MLL/KMT2A or RUNX1 are the major cause of therapy-related leukemia. Moreover, MLL rearrangements are the major cause of infant leukemia, and RUNX1 rearrangements are frequently detected in cord blood. These genes are sensitive to topoisomerase II inhibitors, and various genes have been identified as potential fusion partners. However, fetal exposure to these inhibitors is rare. Therefore, we postulated that even a proliferation signal itself might induce gene rearrangements in hematopoietic stem cells. To test this hypothesis, we detected gene rearrangements in etoposide-treated or non-treated CD34+ cells cultured with cytokines using inverse PCR. In the etoposide-treated cells, variable-sized rearrangement bands were detected in the RUNX1 and MLL genes at 3 hours of culture, which decreased after 7 days. However, more rearrangement bands were detected in the non-treated cells at 7 days of culture. Such gene rearrangements were also detected in peripheral blood stem cells mobilized by cytokines for transplantation. However, none of these rearranged genes encoded the leukemogenic oncogene, and the cells with rearrangements did not expand. These findings suggest that MLL and RUNX1 rearrangements, which occur with very low frequency in normal hematopoietic progenitor cells, may be induced under cytokine stimulation. Most of the cells with gene rearrangements are likely eliminated, except for leukemia-associated gene rearrangements, resulting in the low prevalence of leukemia development.

Platelet-derived growth factor receptor (PDGFRβ) gene is located on chromosome 5q31-33; its rearrangement leads to constitutive tyrosine kinase activity [1]. Hematological malignancies associated w...

Variants of the t (8;21) (q22;q22) involving chromosome 8, 21, and other chromosomes account for about 3% of all t (8;21) (q22;q22) in patients with acute myeloid leukemia (AML). However, the prognosis of AML with variant t (8;21) remains unknown due to the scarcity of reported cases. Herein we report a case of AML with t (6;21;8) (p23;q22;q22). Fluorescence in situ hybridization confirmed a RUNX1-RUNX1T1 fusion signal on the derivative chromosome 8. This is the first report on a variant of t (8;21) involving the breakpoint 6p23. After induction chemotherapy, our patient achieved complete remission and has been stable for four years.

RUNX1 is a critical transcription factor for the emergence of definitive hematopoiesis and the precise regulation of adult hematopoiesis. Dysregulation of its regulatory network causes aberrant hematopoiesis. Recurrent genetic alterations in RUNX1, including chromosomal translocations and mutations, have been identified in both inherited and sporadic diseases. Recent genomic studies have revealed a vast mutational landscape surrounding genetic alterations in RUNX1. Accumulating pieces of evidence also indicate the leukemogenic role of wild-type RUNX1 in certain situations. Based on these efforts, part of the molecular mechanisms of disease development as a consequence of dysregulated RUNX1-regulatory networks have become increasingly evident. This review highlights the recent advances in the field of RUNX1 research and discusses the critical roles of RUNX1 in hematopoiesis and the pathobiological function of its alterations in the context of disease, particularly myeloid neoplasms, and clonal hematopoiesis.

The hypomethylating agent azacitidine (AZA) significantly extends overall survival (OS) in patients with higher risk myelodysplastic syndromes (MDS), when compared with other conventional care regimens, including supportive care and low-dose and intensive chemotherapy. However, the effects of 5- and 7-day treatment schedules of AZA (AZA-5 and AZA-7, respectively) on the OS of MDS patients had not been compared prospectively. We started a phase 3 trial comparing the effects of AZA-7 and AZA-5 on MDS patients with refractory anemia with excess blasts (RAEB) and RAEB in transformation (RAEB-T). However, this trial was prematurely terminated because of poor recruitment. Using all data, there was no significant difference in the OS of patients between AZA-7 (92 patients) and AZA-5 (95 patients), with the 2-year OS rates of AZA-7 and AZA-5 at 36.4% and 25.8%, respectively (P = 0.293). Adverse event profiles were similar between the two groups. Interestingly, data of the centrally diagnosed RAEB and RAEB-T cases showed that AZA-7 significantly prolonged the time to leukemia transformation compared with AZA-5 (P = 0.022), confirmed by multivariate analysis. Although this trial could not provide definite evidence, the results support the use of AZA-7 for RAEB and RAEB-T. (UMIN Clinical Trials Registry UMIN000009633).

Tyrosine kinase inhibitors (TKIs) are established drugs in the therapy of FLT3-ITD mutated acute myeloid leukemia (AML). However, acquired mutations, such as D835 in the tyrosine kinase domain (FLT3-ITD/D835), can induce resistance to TKIs. A cap analysis gene expression (CAGE) technology revealed that the gene expression of BCL2A1 transcription start sites was increased in primary AML cells bearing FLT3-ITD/D835 compared to FLT3-ITD. Overexpression of BCL2A1 attenuated the sensitivity to quizartinib, a type II TKI, and venetoclax, a selective BCL2 inhibitor, in AML cell lines. However, a type I TKI, gilteritinib, inhibited the expression of BCL2A1 through inactivation of STAT5 and alleviated TKI resistance of FLT3-ITD/D835. The combination of gilteritinib and venetoclax showed synergistic effects in the FLT3-ITD/D835 positive AML cells. The promoter region of BCL2A1 contains a BRD4 binding site. Thus, the blockade of BRD4 with a BET inhibitor (CPI-0610) downregulated BCL2A1 in FLT3-mutated AML cells and extended profound suppression of FLT3-ITD/D835 mutant cells. Therefore, we propose that BCL2A1 has the potential to be a novel therapeutic target in treating FLT3-ITD/D835 mutated AML.

Since the first identification of hypoxic cells in sections of carcinomas in the 1950s, hypoxia has been known as a central hallmark of cancer cells and their microenvironment. Indeed, hypoxia benefits cancer cells in their growth, survival, and metastasis. The historical discovery of hypoxia-inducible factor-1α (HIF1A) in the early 1990s had a great influence on the field as many phenomena in hypoxia could be explained by HIF1A. However, not all regions or types of tumors are necessarily hypoxic. Thus, it is difficult to explain whole cancer pathobiology by hypoxia, especially in the early stage of cancer. Upregulation of glucose metabolism in cancer cells has been well known. Oxygen-independent glycolysis is activated in cancer cells even in the normoxia condition, which is known as the Warburg effect. Accumulating evidence and recent advances in cancer metabolism research suggest that hypoxia-independent mechanisms for HIF signaling activation is a hallmark for cancer. There are various mechanisms that generate pseudohypoxic conditions, even in normoxia. Given the importance of HIF1A for cancer pathobiology, the pseudohypoxia concept could shed light on the longstanding mystery of the Warburg effect and accelerate better understanding of the diverse phenomena seen in a variety of cancers.

Although several causal genes of familial myelodysplastic syndromes (MDS) have been identified, the genetic landscape and the molecular pathogenesis are not totally understood. To explore novel driver genes and their pathogenetic significance, we performed whole-exome sequence analysis of four individuals from a familial MDS pedigree and 10 candidate single-nucleotide variants (C9orf43, CYP7B1, EFHB, ENTPD7, FAM160B2, HELZ2, HLTF, INPP5J, ITPKB, and RYK) were identified. Knockdown screening revealed that Hltf downregulation enhanced colony-forming capacity of primary murine bone marrow (BM) stem/progenitor cells. γH2AX immunofluorescent staining assay revealed increased DNA damage in a human acute myeloid leukemia (AML) cell line ectopically expressing HLTF E259K, which was not observed in cells expressing wild-type HLTF. Silencing of HLTF in human AML cells also led to DNA damage, indicating that HLTF E259K is a loss-of-function mutation. Molecularly, we found that an E259K mutation reduced the binding capacity of HLTF with ubiquitin-conjugating enzymes, methanesulfonate sensitive 2 and ubiquitin-conjugating enzyme E2N, resulting in impaired polyubiquitination of proliferating cell nuclear antigen (PCNA) in HLTF E259K-transduced cells. In summary, our results indicate that a familial MDS-associated HLTF E259K germline mutation induces accumulation of DNA double-strand breaks, possibly through impaired PCNA polyubiquitination.

Insufficiency of polycomb repressive complex 2 (PRC2), which trimethylates histone H3 at lysine 27, is frequently found in primary myelofibrosis and promotes the development of JAK2V617F-induced myelofibrosis in mice by enhancing the production of dysplastic megakaryocytes. Polycomb group ring finger protein 1 (Pcgf1) is a component of PRC1.1, a non-canonical PRC1 that monoubiquitylates H2A at lysine 119 (H2AK119ub1). We herein investigated the impact of PRC1.1 insufficiency on myelofibrosis. The deletion of Pcgf1 in JAK2V617F mice strongly promoted the development of lethal myelofibrosis accompanied by a block in erythroid differentiation. Transcriptome and chromatin immunoprecipitation sequence analyses showed the de-repression of PRC1.1 target genes in Pcgf1-deficient JAK2V617F hematopoietic progenitors and revealed Hoxa cluster genes as direct targets. The deletion of Pcgf1 in JAK2V617F hematopoietic stem and progenitor cells (HSPCs), as well as the overexpression of Hoxa9, restored the attenuated proliferation of JAK2V617F progenitors. The overexpression of Hoxa9 also enhanced JAK2V617F-mediated myelofibrosis. The expression of PRC2 target genes identified in PRC2-insufficient JAK2V617F HSPCs was not largely altered in Pcgf1-deleted JAK2V617F HSPCs. The present results revealed a tumor suppressor function for PRC1.1 in myelofibrosis and suggest that PRC1.1 insufficiency has a different impact from that of PRC2 insufficiency on the pathogenesis of myelofibrosis.

Gene abnormalities, including mutations and fusions, are important determinants in the molecular diagnosis of myeloid neoplasms. The use of bone marrow (BM) smears as a source of DNA and RNA for next-generation sequencing (NGS) enables molecular diagnosis to be done with small amounts of bone marrow and is especially useful for patients without stocked cells, DNA or RNA. The present study aimed to analyze the quality of DNA and RNA derived from smear samples and the utility of NGS for diagnosing myeloid neoplasms. Targeted DNA sequencing using paired BM cells and smears yielded sequencing data of adequate quality for variant calling. The detected variants were analyzed using the bioinformatics approach to detect mutations reliably and increase sensitivity. Noise deriving from variants with extremely low variant allele frequency (VAF) was detected in smear sample data and removed by filtering. Consequently, various driver gene mutations were detected across a wide range of allele frequencies in patients with myeloid neoplasms. Moreover, targeted RNA sequencing successfully detected fusion genes using smear-derived, very low-quality RNA, even in a patient with a normal karyotype. These findings demonstrated that smear samples can be used for clinical molecular diagnosis with adequate noise-reduction methods even if the DNA and RNA quality is inferior.

DNA methyltransferase inhibitors have improved the prognosis of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, because these agents are easily degraded by cytidine deaminase (CDA), they must be administered intravenously or subcutaneously. Recently, two orally bioavailable DNA methyltransferase inhibitors, CC-486 and ASTX727, were approved. In previous work, we developed 5-O-trialkylsilylated decitabines that resist degradation by CDA. However, the effects of silylation of a deoxynucleotide analog and enzymatic cleavage of silylation have not been fully elucidated. Enteric administration of OR21 in a cynomolgus monkey model led to high plasma concentrations and hypomethylation, and in a mouse model, oral administration of enteric-coated OR21 led to high plasma concentrations. The drug became biologically active after release of decitabine (DAC) from OR21 following removal of the 5′-O-trisilylate substituent. Toxicities were tolerable and lower than those of DAC. Transcriptome and methylome analysis of MDS and AML cell lines revealed that OR21 increased expression of genes associated with tumor suppression, cell differentiation, and immune system processes by altering regional promoter methylation, indicating that these pathways play pivotal roles in the action of hypomethylating agents. OR21 induced cell differentiation via upregulation of the late cell differentiation drivers CEBPE and GATA-1. Thus, silylation of a deoxynucleotide analog can confer oral bioavailability without new toxicities. Both in vivo and in vitro, OR21 exerted antileukemia effects, and had a better safety profile than DAC. Together, our findings indicate that OR21 is a promising candidate drug for phase I study as an alternative to azacitidine or decitabine.

High Mobility Group AT-hook 2 (HMGA2) is a chromatin modifier and its overexpression has been found in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Level of Hmga2 expression is fine-tuned by Lin28b-Let-7 axis and Polycomb Repressive Complex 2, in which deletion of Ezh2 leads to activation of Hmga2 expression in hematopoietic stem cells. To elucidate the mechanisms by which the overexpression of HMGA2 helps transformation of stem cells harboring a driver mutation of TET2, we generated an Hmga2-expressing Tet2-deficient mouse model showing the progressive phenotypes of MDS and AML. The overexpression of Hmga2 remodeled the transcriptional program of Tet2-deficient stem and progenitor cells, leading to the impaired differentiation of myeloid cells. Furthermore, Hmga2 was bound to a proximal region of Igf2bp2 oncogene, and activated its transcription, leading to enhancing self-renewal of Tet2-deficient stem cells that was suppressed by inhibition of the DNA binding of Hmga2. These combinatory effects on the transcriptional program and cellular function were not redundant to those in Tet2-deficient cells. The present results elucidate that Hmga2 targets key oncogenic pathways during the transformation and highlight the Hmga2-Igf2bp2 axis as a potential target for therapeutic intervention.