The miR-106b-25 cluster mediates drug resistance in myeloid leukaemias by inactivating multiple apoptotic genes.

Drug resistance is a major obstacle to the successful treatment of cancer. The role of the miR-106b-25 cluster in drug resistance of haematologic malignancies has not yet been elucidated. Here, we show that the miR-106b-25 cluster mediates resistance to therapeutic agents with structural and mechanistic dissimilarity in vitro and in vivo. RNA sequencing data revealed that overexpression of the miR-106b-25 cluster or its individual miRNAs resulted in downregulation of multiple key regulators of apoptotic pathways. Luciferase reporter assay identified TP73 as a direct target of miR-93 and miR-106b, BAK1 as a direct target of miR-25 and CASP7 as a direct target of all three miRNAs. We also showed that inhibitors of the miR-106b-25 cluster and BCL-2 exert synergistic effects on apoptosis induction in primary myeloid leukaemic cells. Thus, the members of the miR-106b-25 cluster may jointly contribute to myeloid leukaemia drug resistance by inactivating multiple apoptotic genes. Targeting this cluster could be a promising combination strategy in patients resistant to therapeutic agents that induce apoptosis.

A novel 4-mRNA signature predicts the overall survival in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive cancer of myeloid cells with high levels of heterogeneity and great variability in prognostic behaviors. Cytogenetic abnormalities and genetic mutations have been widely used in the prognostic stratification of AML to assign patients into different risk categories. Nevertheless, nearly half of AML patients assigned to intermediate risk need more precise prognostic schemes. Here, 336 differentially expressed genes (DEGs) between AML and control samples and 206 genes representing the intratumor heterogeneity of AML were identified. By applying a LASSO Cox regression model, we generated a 4-mRNA prognostic signature comprising KLF9, ENPP4, TUBA4A and CD247. Higher risk scores were significantly associated with shorter overall survival, complex karyotype, and adverse mutations. We then validated the prognostic value of this 4-mRNA signature in two independent cohorts. We also proved that incorporation of the 4-mRNA-based signature in the 2017 European LeukemiaNet (ELN) risk classification could enhance the predictive accuracy of survival in patients with AML. Univariate and multivariate analyses showed that this signature was independent of traditional prognostic factors such as age, WBC count, and unfavorable cytogenetics. Finally, the molecular mechanisms underlying disparate outcomes in high-risk and low-risk AML patients were explored. Therefore, our findings suggest that the 4-mRNA signature refines the risk stratification and prognostic prediction of AML patients.

Reducing hyperactivated BAP1 attenuates mutant ASXL1-driven myeloid malignancies in human haematopoietic cells.

Additional sex combs-like 1 (ASXL1) is frequently mutated in a variety of myeloid malignancies, resulting in expression of a C-terminal-truncated ASXL1 protein that confers gain of function on the ASXL1-BAP1 deubiquitinase (DUB) complex. Several studies have reported that hyperactivity of BRCA-1-associated protein 1 (BAP1) in deubiquitinating mono-ubiquitinated histone H2AK119 is one of the critical molecular mechanisms in ASXL1 mutation-driven myeloid malignancies in mice. In this study, we found that human haematopoietic stem and progenitor cells (HSPCs) overexpressing truncated ASXL1 (ASXL1Y591X) developed an MDS-like phenotype similar to that induced by overexpression of BAP1. We then used shRNAs targeting BAP1 in ASXL1Y591X-overexpressing HSPCs and primary leukaemia cells with ASXL1 mutation, demonstrating that reduced BAP1 expression can partially rescue the pathological consequences. RNA sequencing and chromatin immunoprecipitation coupled with quantitative PCR analyses revealed that reduced BAP1 expression suppressed upregulation of the transcription factors AP-1 and EGR1/2, as well as myeloid dysplasia-associated genes, by retarding H2AK119Ub removal caused by ASXL1 mutation. This study indicates that targeting the hyperactive ASXL1-BAP1 DUB complex can attenuate mutant ASXL1-driven myeloid malignancies in human.

Therapeutic potential of GSK-J4, a histone demethylase KDM6B/JMJD3 inhibitor, for acute myeloid leukemia.

PURPOSE: Acute myeloid leukemia (AML) is a heterogeneous disease with poor outcomes. Despite increased evidence shows that dysregulation of histone modification contributes to AML, specific drugs targeting key histone modulators are not applied in the clinical treatment of AML. Here, we investigated whether targeting KDM6B, the demethylase of tri-methylated histone H3 lysine 27 (H3K27me3), has a therapeutic potential for AML. METHODS: A KDM6B-specific inhibitor, GSK-J4, was applied to treat the primary cells from AML patients and AML cell lines in vitro and in vivo. RNA-sequencing was performed to reveal the underlying mechanisms of inhibiting KDM6B for the treatment of AML. RESULTS: Here we observed that the mRNA expression of KDM6B was up-regulated in AML and positively correlated with poor survival. Treatment with GSK-J4 increased the global level of H3K27me3 and reduced the proliferation and colony-forming ability of primary AML cells and AML cell lines. GSK-J4 treatment significantly induced cell apoptosis and cell-cycle arrest in Kasumi-1 cells, and displayed a synergistic effect with cytosine arabinoside. Notably, injection of GSK-J4 attenuated the disease progression in a human AML xenograft mouse model in vivo. Treatment with GSK-J4 predominantly resulted in down-regulation of DNA replication and cell-cycle-related pathways, as well as abrogated the expression of critical cancer-promoting HOX genes. ChIP-qPCR validated an increased enrichment of H3K27me3 in the transcription start sites of these HOX genes. CONCLUSIONS: In summary, our findings suggest that targeting KDM6B with GSK-J4 has a therapeutic potential for the treatment of AML.

Loss of ASXL1 in the bone marrow niche dysregulates hematopoietic stem and progenitor cell fates.

Somatic or de novo mutations of Additional sex combs-like 1 (ASXL1) frequently occur in patients with myeloid malignancies or Bohring-Opitz syndrome, respectively. We have reported that global loss of Asxl1 leads to the development of myeloid malignancies and impairs bone marrow stromal cell (BMSC) fates in mice. However, the impact of Asxl1 deletion in the BM niche on hematopoiesis remains unclear. Here, we showed that BMSCs derived from chronic myelomonocytic leukemia patients had reduced expression of ASXL1, which impaired the maintaining cord blood CD34+ cell colony-forming capacity with a myeloid differentiation bias. Furthermore, Asxl1 deletion in the mouse BMSCs altered hematopoietic stem and progenitor cell (HSC/HPC) pool and a preferential myeloid lineage increment. Immunoprecipitation and ChIP-seq analyses demonstrated a novel interaction of ASXL1 with the core subunits of RNA polymerase II (RNAPII) complex. Convergent analyses of RNA-seq and ChIP-seq data revealed that loss of Asxl1 deregulated RNAPII transcriptional function and altered the expression of genes critical for HSC/HPC maintenance, such as Vcam1. Altogether, our study provides a mechanistic insight into the function of ASXL1 in the niche to maintain normal hematopoiesis; and ASXL1 alteration in, at least, a subset of the niche cells induces myeloid differentiation bias, thus, contributes the progression of myeloid malignancies.

TET2 Loss Dysregulates the Behavior of Bone Marrow Mesenchymal Stromal Cells and Accelerates Tet2-/--Driven Myeloid Malignancy Progression.

TET2 is a methylcytosine dioxygenase that regulates cytosine hydroxymethylation. Although there are extensive data implicating a pivotal role of TET2 in hematopoietic stem/progenitor cells (HSPCs), the importance of TET2 in bone marrow mesenchymal stromal cells (BMSCs) remains unknown. In this study, we show that loss of TET2 in BMSCs increases cell proliferation and self-renewal and enhances osteoblast differentiation potential of BMSCs, which may in turn alter their behavior in supporting HSPC proliferation and differentiation. In addition, Tet2 loss alters BMSCs in promoting Tet2-deficiency-mediated myeloid malignancy progression. Tet2 loss in BMSCs also dysregulates hydroxylation of 5-methylcytosine (5mC) and the expression of genes that are key for BMSC proliferation and osteoblast differentiation, leading to alteration of biological characteristics in vivo. These results highlight the critical role of TET2 in the maintenance of BMSC functions and osteoblast differentiation and provide evidence that dysregulation of epigenetic modifiers in BMSCs contributes to the progression of myeloid malignancies.

Gain of function of ASXL1 truncating protein in the pathogenesis of myeloid malignancies.

Additional Sex Combs-Like 1 (ASXL1) is mutated at a high frequency in all forms of myeloid malignancies associated with poor prognosis. We generated a Vav1 promoter-driven Flag-Asxl1Y588X transgenic mouse model, Asxl1Y588X Tg, to express a truncated FLAG-ASXL1aa1-587 protein in the hematopoietic system. 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 patients with ASXL1 mutations. ATAC- and RNA-sequencing analyses revealed that the ASXL1aa1-587 truncating protein expression results in more open chromatin in cKit+ cells compared with wild-type cells, accompanied by dysregulated expression of genes critical for HSC self-renewal and differentiation. Liquid chromatography-tandem mass spectrometry and coimmunoprecipitation experiments showed that ASXL1aa1-587 acquired an interaction with BRD4. An epigenetic drug screening demonstrated a hypersensitivity of Asxl1Y588X Tg bone marrow cells to BET bromodomain inhibitors. This study demonstrates that ASXL1aa1-587 plays a gain-of-function role in promoting myeloid malignancies. Our model provides a powerful platform to test therapeutic approaches of targeting the ASXL1 truncation mutations in myeloid malignancies.

Upregulation of miR-99a is associated with poor prognosis of acute myeloid leukemia and promotes myeloid leukemia cell expansion.

Leukemia stem cells (LSCs) can resist available treatments that results in disease progression and/or relapse. To dissect the microRNA (miRNA) expression signature of relapse in acute myeloid leukemia (AML), miRNA array analysis was performed using enriched LSCs from paired bone marrow samples of an AML patient at different disease stages. We identified that miR-99a was significantly upregulated in the LSCs obtained at relapse compared to the LSCs collected at the time of initial diagnosis. We also found that miR-99a was upregulated in LSCs compared to non-LSCs in a larger cohort of AML patients, and higher expression levels of miR-99a were significantly correlated with worse overall survival and event-free survival in these AML patients. Ectopic expression of miR-99a led to increased colony forming ability and expansion in myeloid leukemia cells after exposure to chemotherapeutic drugs in vitro and in vivo, partially due to overcoming of chemotherapeutic agent-mediated cell cycle arrest. Gene profiling and bioinformatic analyses indicated that ectopic expression of miR-99a significantly upregulated genes that are critical for LSC maintenance, cell cycle, and downstream targets of E2F and MYC. This study suggests that miR-99a has a novel role and potential use as a biomarker in myeloid leukemia progression.