Targeting lysine demethylase 6B ameliorates ASXL1 truncation-mediated myeloid malignancies in preclinical models.

ASXL1 mutation frequently occurs in all forms of myeloid malignancies and is associated with aggressive disease and poor prognosis. ASXL1 recruits Polycomb repressive complex 2 (PRC2) to specific gene loci to repress transcription through trimethylation of histone H3 on lysine 27 (H3K27me3). ASXL1 alterations reduce H3K27me3 levels, which results in leukemogenic gene expression and the development of myeloid malignancies. Standard therapies for myeloid malignancies have limited efficacy when mutated ASXL1 is present. We discovered upregulation of lysine demethylase 6B (KDM6B), a demethylase for H3K27me3, in ASXL1-mutant leukemic cells, which further reduces H3K27me3 levels and facilitates myeloid transformation. Here, we demonstrated that heterozygous deletion of Kdm6b restored H3K27me3 levels and normalized dysregulated gene expression in Asxl1Y588XTg hematopoietic stem/progenitor cells (HSPCs). Furthermore, heterozygous deletion of Kdm6b decreased the HSPC pool, restored their self-renewal capacity, prevented biased myeloid differentiation, and abrogated progression to myeloid malignancies in Asxl1Y588XTg mice. Importantly, administration of GSK-J4, a KDM6B inhibitor, not only restored H3K27me3 levels but also reduced the disease burden in NSG mice xenografted with human ASXL1-mutant leukemic cells in vivo. This preclinical finding provides compelling evidence that targeting KDM6B may be a therapeutic strategy for myeloid malignancies with ASXL1 mutations.

ASXL1 mutations are associated with a response to alvocidib and 5-azacytidine combination in myelodysplastic neoplasms.

Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMAs) are promising therapeutic approaches in acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS). Alvocidib, a cyclin-dependent kinase 9 (CDK9) inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has previously shown clinical activity in AML. Availability of biomarkers for response to the alvocidib + 5- AZA could also extend the rationale of this treatment concept to high-risk MDS. In this study, we performed a comprehensive in vitro assessment of alvocidib and 5-AZA effects in n=45 high-risk MDS patients. Our data revealed additive cytotoxic effects of the combination treatment. Mutational profiling of MDS samples identified ASXL1 mutations as predictors of response. Further, increased response rates were associated with higher gene-expression of the pro-apoptotic factor NOXA in ASXL1 mutated samples. The higher sensitivity of ASXL1 mutant cells to the combination treatment was confirmed in vivo in ASXL1Y588X transgenic mice. Overall, our study demonstrated augmented activity for the alvocidib + 5-AZA combination in higher-risk MDS and identified ASXL1 mutations as a biomarker of response for potential stratification studies.

Loss of BRD4 induces cell senescence in HSC/HPCs by deregulating histone H3 clipping.

Bromodomain-containing protein 4 (BRD4) is overexpressed and functionally implicated in various myeloid malignancies. However, the role of BRD4 in normal hematopoiesis remains largely unknown. Here, utilizing an inducible Brd4 knockout mouse model, we find that deletion of Brd4 (Brd4Δ/Δ ) in the hematopoietic system impairs hematopoietic stem cell (HSC) self-renewal and differentiation, which associates with cell cycle arrest and senescence. ATAC-seq analysis shows increased chromatin accessibility in Brd4Δ/Δ hematopoietic stem/progenitor cells (HSC/HPCs). Genome-wide mapping with cleavage under target and release using nuclease (CUT&RUN) assays demonstrate that increased global enrichment of H3K122ac and H3K4me3 in Brd4Δ/Δ HSC/HPCs is associated with the upregulation of senescence-specific genes. Interestingly, Brd4 deletion increases clipped H3 (cH3) which correlates with the upregulation of senescence-specific genes and results in a higher frequency of senescent HSC/HPCs. Re-expression of BRD4 reduces cH3 levels and rescues the senescence rate in Brd4Δ/Δ HSC/HPCs. This study unveils an important role of BRD4 in HSC/HPC function by preventing H3 clipping and suppressing senescence gene expression.

Epigenetic regulation by ASXL1 in myeloid malignancies.

Myeloid malignancies are clonal hematopoietic disorders that are comprised of a spectrum of genetically heterogeneous disorders, including myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). Myeloid malignancies are characterized by excessive proliferation, abnormal self-renewal, and/or differentiation defects of hematopoietic stem cells (HSCs) and myeloid progenitor cells hematopoietic stem/progenitor cells (HSPCs). Myeloid malignancies can be caused by genetic and epigenetic alterations that provoke key cellular functions, such as self-renewal, proliferation, biased lineage commitment, and differentiation. Advances in next-generation sequencing led to the identification of multiple mutations in myeloid neoplasms, and many new gene mutations were identified as key factors in driving the pathogenesis of myeloid malignancies. The polycomb protein ASXL1 was identified to be frequently mutated in all forms of myeloid malignancies, with mutational frequencies of 20%, 43%, 10%, and 20% in MDS, CMML, MPN, and AML, respectively. Significantly, ASXL1 mutations are associated with a poor prognosis in all forms of myeloid malignancies. The fact that ASXL1 mutations are associated with poor prognosis in patients with CMML, MDS, and AML, points to the possibility that ASXL1 mutation is a key factor in the development of myeloid malignancies. This review summarizes the recent advances in understanding myeloid malignancies with a specific focus on ASXL1 mutations.

Latexin regulates sex dimorphism in hematopoiesis via gender-specific differential expression of microRNA 98-3p and thrombospondin 1.

Hematopoietic stem cells (HSCs) have the ability to self-renew and differentiate to all blood cell types. HSCs and their differentiated progeny show sex/gender differences. The fundamental mechanisms remain largely unexplored. We previously reported that latexin (Lxn) deletion increased HSC survival and repopulation capacity in female mice. Here, we find no differences in HSC function and hematopoiesis in Lxn knockout (Lxn-/-) male mice under physiologic and myelosuppressive conditions. We further find that Thbs1, a downstream target gene of Lxn in female HSCs, is repressed in male HSCs. Male-specific high expression of microRNA 98-3p (miR98-3p) contributes to Thbs1 suppression in male HSCs, thus abrogating the functional effect of Lxn in male HSCs and hematopoiesis. These findings uncover a regulatory mechanism involving a sex-chromosome-related microRNA and its differential control of Lxn-Thbs1 signaling in hematopoiesis and shed light on the process underlying sex dimorphism in both normal and malignant hematopoiesis.

The Role of PHF6 in Hematopoiesis and Hematologic Malignancies.

Epigenetic regulation of gene expression represents an important mechanism in the maintenance of stem cell function. Alterations in epigenetic regulation contribute to the pathogenesis of hematological malignancies. Plant homeodomain finger protein 6 (PHF6) is a member of the plant homeodomain (PHD)-like zinc finger family of proteins that is involved in transcriptional regulation through the modification of the chromatin state. Germline mutation of PHF6 is the causative genetic alteration of the X-linked mental retardation Borjeson-Forssman-Lehmann syndrome (BFLS). Somatic mutations in PHF6 are identified in human leukemia, such as adult T-cell acute lymphoblastic leukemia (T-ALL, ~ 38%), pediatric T-ALL (~ 16%), acute myeloid leukemia (AML, ~ 3%), chronic myeloid leukemia (CML, ~ 2.5%), mixed phenotype acute leukemia (MPAL, ~ 20%), and high-grade B-cell lymphoma (HGBCL, ~ 3%). More recent studies imply an oncogenic effect of PHF6 in B-cell acute lymphoblastic leukemia (B-ALL) and solid tumors. These data demonstrate that PHF6 could act as a double-edged sword, either a tumor suppressor or an oncogene, in a lineage-dependent manner. However, the underlying mechanisms of PHF6 in normal hematopoiesis and leukemogenesis remain largely unknown. In this review, we summarize current knowledge of PHF6, emphasizing the role of PHF6 in hematological malignancies. Epigenetic regulation of PHF6 in B-ALL. PHF6 maintains a chromatin structure that is permissive to B-cell identity genes, but not T-cell-specific genes (left). Loss of PHF6 leads to aberrant expression of B-cell- and T-cell-specific genes resulting from lineage promiscuity and binding of T-cell transcription factors (right).

ASXL1/2 mutations and myeloid malignancies.

Myeloid malignancies develop through the accumulation of genetic and epigenetic alterations that dysregulate hematopoietic stem cell (HSC) self-renewal, stimulate HSC proliferation and result in differentiation defects. The polycomb group (PcG) and trithorax group (TrxG) of epigenetic regulators act antagonistically to regulate the expression of genes key to stem cell functions. The genes encoding these proteins, and the proteins that interact with them or affect their occupancy at chromatin, are frequently mutated in myeloid malignancies. PcG and TrxG proteins are regulated by Enhancers of Trithorax and Polycomb (ETP) proteins. ASXL1 and ASXL2 are ETP proteins that assemble chromatin modification complexes and transcription factors. ASXL1 mutations frequently occur in myeloid malignancies and are associated with a poor prognosis, whereas ASXL2 mutations frequently occur in AML with t(8;21)/RUNX1-RUNX1T1 and less frequently in other subtypes of myeloid malignancies. Herein, we review the role of ASXL1 and ASXL2 in normal and malignant hematopoiesis by summarizing the findings of mouse model systems and discussing their underlying molecular mechanisms.

HOTTIP-dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia.

HOTTIP lncRNA is highly expressed in acute myeloid leukemia (AML) driven by MLL rearrangements or NPM1 mutations to mediate HOXA topologically associated domain (TAD) formation and drive aberrant transcription. However, the mechanism through which HOTTIP accesses CCCTC-binding factor (CTCF) chromatin boundaries and regulates CTCF-mediated genome topology remains unknown. Here, we show that HOTTIP directly interacts with and regulates a fraction of CTCF-binding sites (CBSs) in the AML genome by recruiting CTCF/cohesin complex and R-loop-associated regulators to form R-loops. HOTTIP-mediated R-loops reinforce the CTCF boundary and facilitate formation of TADs to drive gene transcription. Either deleting CBS or targeting RNase H to eliminate R-loops in the boundary CBS of ß-catenin TAD impaired CTCF boundary activity, inhibited promoter/enhancer interactions, reduced ß-catenin target expression, and mitigated leukemogenesis in xenograft mouse models with aberrant HOTTIP expression. Thus, HOTTIP-mediated R-loop formation directly reinforces CTCF chromatin boundary activity and TAD integrity to drive oncogene transcription and leukemia development.

How fern and fern allies respond to heterogeneous habitat - a case in Yuanjiang dry-hot valley.

The Yuanjiang dry-hot valley features hot and dry climate, low vegetation and soil degradation. It had lush vegetation in the past, but has become degraded in recent decades. Understanding the interrelationship between species and the habitat is necessary to explain this change. In this study, a link between fern and fern allies - a group that is hypersensitive to environmental factors and their circumstances is constructed. Intensive transects and plots were designed to be proxies for extant fern and fern allies, and their habitats. Fifty years of meteorological records of precipitation and temperature along altitude and river running direction (latitudinal) were employed. Alpha and beta diversity are used to access diversity. Species_estimated, Singletons, Uniques, ACE, ICE, and Chao2, which associate to abundance and rarity, are subscribed to the correlation between fern and fern allies, and their ecosystem. Eight species, Selaginella pseudopaleifera, Aleuritopteris squamosa, Adiantum malesianum, Pteris vittata, Davallia trichomanoides, Sinephropteris delavayi, Selaginella jugorum, and Lygodium japonicum are used as indicators of a typical xeric and sun-drying habitat. The results indicate (1) accompanied by dramatically shrinking habitats, fern and fern allies are in very low diversity and abundance, whereas the rarity is relatively high; (2) for fern and fern allies, environmental factors are positive when altitude goes up; and (3) eight indicator species are latitudinally correlated with fern and fern allies along the river running direction.

p300 suppresses the transition of myelodysplastic syndromes to acute myeloid leukemia.

Myelodysplastic syndromes (MDS) are hematopoietic stem and progenitor cell (HSPC) malignancies characterized by ineffective hematopoiesis and an increased risk of leukemia transformation. Epigenetic regulators are recurrently mutated in MDS, directly implicating epigenetic dysregulation in MDS pathogenesis. Here, we identified a tumor suppressor role of the acetyltransferase p300 in clinically relevant MDS models driven by mutations in the epigenetic regulators TET2, ASXL1, and SRSF2. The loss of p300 enhanced the proliferation and self-renewal capacity of Tet2-deficient HSPCs, resulting in an increased HSPC pool and leukemogenicity in primary and transplantation mouse models. Mechanistically, the loss of p300 in Tet2-deficient HSPCs altered enhancer accessibility and the expression of genes associated with differentiation, proliferation, and leukemia development. Particularly, p300 loss led to an increased expression of Myb, and the depletion of Myb attenuated the proliferation of HSPCs and improved the survival of leukemia-bearing mice. Additionally, we show that chemical inhibition of p300 acetyltransferase activity phenocopied Ep300 deletion in Tet2-deficient HSPCs, whereas activation of p300 activity with a small molecule impaired the self-renewal and leukemogenicity of Tet2-deficient cells. This suggests a potential therapeutic application of p300 activators in the treatment of MDS with TET2 inactivating mutations.

INTS11 regulates hematopoiesis by promoting PRC2 function.

INTS11, the catalytic subunit of the Integrator (INT) complex, is crucial for the biogenesis of small nuclear RNAs and enhancer RNAs. However, the role of INTS11 in hematopoietic stem and progenitor cell (HSPC) biology is unknown. Here, we report that INTS11 is required for normal hematopoiesis and hematopoietic-specific genetic deletion of Ints11 leads to cell cycle arrest and impairment of fetal and adult HSPCs. We identified a novel INTS11-interacting protein complex, Polycomb repressive complex 2 (PRC2), that maintains HSPC functions. Loss of INTS11 destabilizes the PRC2 complex, decreases the level of histone H3 lysine 27 trimethylation (H3K27me3), and derepresses PRC2 target genes. Reexpression of INTS11 or PRC2 proteins in Ints11-deficient HSPCs restores the levels of PRC2 and H3K27me3 as well as HSPC functions. Collectively, our data demonstrate that INTS11 is an essential regulator of HSPC homeostasis through the INTS11-PRC2 axis.

HOXBLINC long non-coding RNA activation promotes leukemogenesis in NPM1-mutant acute myeloid leukemia.

Nucleophosmin (NPM1) is the most commonly mutated gene in acute myeloid leukemia (AML) resulting in aberrant cytoplasmic translocation of the encoded nucleolar protein (NPM1c+). NPM1c+ maintains a unique leukemic gene expression program, characterized by activation of HOXA/B clusters and MEIS1 oncogene to facilitate leukemogenesis. However, the mechanisms by which NPM1c+ controls such gene expression patterns to promote leukemogenesis remain largely unknown. Here, we show that the activation of HOXBLINC, a HOXB locus-associated long non-coding RNA (lncRNA), is a critical downstream mediator of NPM1c+-associated leukemic transcription program and leukemogenesis. HOXBLINC loss attenuates NPM1c+-driven leukemogenesis by rectifying the signature of NPM1c+ leukemic transcription programs. Furthermore, overexpression of HoxBlinc (HoxBlincTg) in mice enhances HSC self-renewal and expands myelopoiesis, leading to the development of AML-like disease, reminiscent of the phenotypes seen in the Npm1 mutant knock-in (Npm1c/+) mice. HoxBlincTg and Npm1c/+ HSPCs share significantly overlapped transcriptome and chromatin structure. Mechanistically, HoxBlinc binds to the promoter regions of NPM1c+ signature genes to control their activation in HoxBlincTg HSPCs, via MLL1 recruitment and promoter H3K4me3 modification. Our study reveals that HOXBLINC lncRNA activation plays an essential oncogenic role in NPM1c+ leukemia. HOXBLINC and its partner MLL1 are potential therapeutic targets for NPM1c+ AML.

Early administration of imatinib mesylate reduces plexiform neurofibroma tumor burden with durable results after drug discontinuation in a mouse model of neurofibromatosis type 1.

BACKGROUND: Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by plexiform neurofibromas (pNF), which are thought to be congenital tumors that arise in utero and enlarge throughout life. Genetic studies in murine models delineated an indispensable role for the stem cell factor (SCF)/c-kit pathway in pNF initiation and progression. A subsequent phase 2 clinical trial using imatinib mesylate to inhibit SCF/c-kit demonstrated tumor shrinkage in a subset of preexisting pNF; however, imatinib's role on preventing pNF development has yet to be explored. PROCEDURE: We evaluated the effect of imatinib dosed at 10-100 mg/kg/day for 12 weeks to one-month-old Nf1flox/flox ;PostnCre(+) mice, prior to onset of pNF formation. To determine durability of response, we then monitored for pNF growth at later time points, comparing imatinib- with vehicle-treated mice. We assessed gross and histopathological analysis of tumor burden. RESULTS: Imatinib administered preventatively led to a significant decrease in pNF number, even at doses as low as 10 mg/kg/day. Tumor development continued to be significantly inhibited after cessation of imatinib dosed at 50 and 100 mg/kg/day. In the cohort of treated mice that underwent prolonged follow-up, the size of residual tumors was significantly reduced as compared with age-matched littermates that received vehicle control. CONCLUSIONS: Early administration of imatinib inhibits pNF genesis in vivo, and effects are sustained after discontinuation of therapy. These findings may guide clinical use of imatinib in young NF1 patients prior to the substantial development of pNF.

Novel combinations to improve hematopoiesis in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) represents a heterogeneous group of clonal hematopoietic disorders, which is characterized by cytopenias in the peripheral blood and bone marrow dysplasia due to ineffective hematopoiesis. Patients with MDS have an increased risk of transformation to acute myeloid leukemia (AML). Although the molecular basis of MDS is heterogeneous, several studies demonstrated the significant contribution of the dysregulated immune system in accelerating MDS progression. The immunosuppressive tumor microenvironment is shown to induce tolerance of MDS blasts, which may result in a further accumulation of genetic aberrations and lead to the disease progression. Increasing evidence shows an expansion of myeloid-derived suppressor cells (MDSCs), a population of inflammation-associated immature cells, in patients with MDS. Interestingly, the increased MDSC populations are shown to be correlated with a risk of disease progression in MDS. In addition, MDS is highly prevalent in aged individuals with non-hematology co-morbidities who are fragile for chemotherapy. Increasing research effort is devoting to identify novel agents to specific targeting of the MDSC population for MDS treatment.

HOTTIP lncRNA Promotes Hematopoietic Stem Cell Self-Renewal Leading to AML-like Disease in Mice.

Long non-coding RNAs (lncRNAs) are critical for regulating HOX genes, aberration of which is a dominant mechanism for leukemic transformation. How HOX gene-associated lncRNAs regulate hematopoietic stem cell (HSC) function and contribute to leukemogenesis remains elusive. We found that HOTTIP is aberrantly activated in acute myeloid leukemia (AML) to alter HOXA-driven topologically associated domain (TAD) and gene expression. HOTTIP loss attenuates leukemogenesis of transplanted mice, while reactivation of HOTTIP restores leukemic TADs, transcription, and leukemogenesis in the CTCF-boundary-attenuated AML cells. Hottip aberration in mice abnormally promotes HSC self-renewal leading to AML-like disease by altering the homeotic/hematopoietic gene-associated chromatin signature and transcription program. Hottip aberration acts as an oncogenic event to perturb HSC function by reprogramming leukemic-associated chromatin and gene transcription.

Cardiac Sca-1+ Cells Are Not Intrinsic Stem Cells for Myocardial Development, Renewal, and Repair.

BACKGROUND: For more than a decade, Sca-1+ cells within the mouse heart have been widely recognized as a stem cell population with multipotency that can give rise to cardiomyocytes, endothelial cells, and smooth muscle cells in vitro and after cardiac grafting. However, the developmental origin and authentic nature of these cells remain elusive. METHODS: Here, we used a series of high-fidelity genetic mouse models to characterize the identity and regenerative potential of cardiac resident Sca-1+ cells. RESULTS: With these novel genetic tools, we found that Sca-1 does not label cardiac precursor cells during early embryonic heart formation. Postnatal cardiac resident Sca-1+ cells are in fact a pure endothelial cell population. They retain endothelial properties and exhibit minimal cardiomyogenic potential during development, normal aging and upon ischemic injury. CONCLUSIONS: Our study provides definitive insights into the nature of cardiac resident Sca-1+ cells. The observations challenge the current dogma that cardiac resident Sca-1+ cells are intrinsic stem cells for myocardial development, renewal, and repair, and suggest that the mechanisms of transplanted Sca-1+ cells in heart repair need to be reassessed.