Histone demethylase PHF8 facilitates the development of chronic myeloid leukaemia by directly targeting BCR::ABL1.

Although tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myeloid leukaemia (CML), TKI resistance remains a major challenge. Here, we demonstrated that plant homeodomain finger protein 8 (PHF8), a histone demethylase was aberrantly enriched in CML samples compared to healthy controls. PHF8 inhibited CML cell differentiation and promoted CML cell proliferation. Furthermore, the proliferation-inhibited function of PHF8-knockdown have stronger effect on imatinib mesylate (IM)-resistant CML cells. Mechanistically, we identified that PHF8 as a transcriptional modulator interacted with the promoter of the BCR::ABL1 fusion gene and alters the methylation levels of H3K9me1, H3K9me2 and H3K27me1, thereby promoting BCR::ABL1 transcription. Overall, our study suggests that targeting PHF8, which directly regulates BCR::ABL1 expression, is a useful therapeutic approach for CML.

Histone chaperone ASF1A accelerates chronic myeloid leukemia blast crisis by activating Notch signaling.

The blast crisis (BC) is the final deadly phase of chronic myeloid leukemia (CML), which remains a major challenge in clinical management. However, the underlying molecular mechanism driving blastic transformation remains unclear. Here, we show that ASF1A, an essential activator, enhanced the transformation to CML-BC by mediating cell differentiation arrest. ASF1A expression was aberrantly increased in bone marrow samples from CML-BC patients compared with newly diagnosed CML-chronic phase (CP) patients. ASF1A inhibited cell differentiation and promoted CML development in vivo. Mechanistically, we identified ASF1A as a coactivator of the Notch transcriptional complex that induces H3K56ac modification in the promoter regions of Notch target genes, and subsequently enhanced RBPJ binding to these promoter regions, thereby enhancing Notch signaling activation to mediate differentiation arrest in CML cells. Thus, our work suggests that targeting ASF1A might represent a promising therapeutic approach and a biomarker to detect disease progression in CML patients.

Comprehensive Analysis of a Ferroptosis Pattern and Associated Prognostic Signature in Acute Myeloid Leukemia.

Ferroptosis is a widespread form of programmed cell death. The environment of cancer cells makes them vulnerable to ferroptosis, including AML cells, yet the specific association between ferroptosis and AML outcome is little known. In this study, we utilized ferroptosis-related genes to distinguish two subtypes in TCGA cohort, which were subsequently validated in independent AML cohorts. The subtypes were linked with tumor-related immunological abnormalities, mutation landscape and pathway dysregulation, and clinical outcome. Further, we developed a 13-gene prognostic model for AML from DEG analysis in the two subtypes. A risk score was calculated for each patient, and then the overall group was stratified into high- and low-risk groups; the higher risk score correlated with short survival. The model was validated in both independent AML cohorts and pan-cancer cohorts, which demonstrated robustness and extended the usage of the model. A nomogram was constructed that integrated risk score, FLT3-ITD, TP53, and RUNX1 mutations, and age. This model had the additional value of discriminating the sensitivity of several chemotherapeutic drugs and ferroptosis inducers in the two risk groups, which increased the translational value of this model as a potential tool in clinical management. Through integrated analysis of ferroptosis pattern and its related model, our work shed new light on the relationship between ferroptosis and AML, which may facilitate clinical application and therapeutics.

Development of a novel pyroptosis-related LncRNA signature with multiple significance in acute myeloid leukemia.

Background: Pyroptosis, a programmed cell death (PCD) with highly inflammatory form, has been recently found to be associated with the origin of hematopoietic malignancies. Long noncoding RNA (lncRNA) had emerged as an essential mediator to regulate gene expression and been involved in oncogenesis. However, the roles of pyroptosis-related lncRNA (PRlncRNA) in acute myeloid leukemia (AML) have not yet been completely clarified. Methods: We collected AML datasets from public databases to obtain PRlncRNA associated with survival and constructed a PRlncRNA signature using Lasso-Cox regression analysis. Subsequently, we employed RT-PCR to confirm its expression difference and internal training to further verify its reliability. Next, AML patients were classified into two subgroups by the median risk score. Finally, the differences between two groups in immune infiltration, enrichment analysis and drug sensitivity were further explored. Results: A PRlncRNA signature and an effective nomogram combined with clinicopathological variables to predict the prognosis of AML were constructed. The internal validations showed that the PRlncRNA risk score model was an accurate and productive indicator to predict the outcome of AML. Furthermore, this study indicated that higher inflammatory cell and immunosuppressive cells, and less sensitive to conventional chemotherapy drugs were highlighted in the high-risk group. Conclusion: Through comprehensive analysis of PRlncRNA model, our study may offer a valuable basis for future researches in targeting pyroptosis and tumor microenvironment (TME) and provide new measures for prevention and treatment in AML.

miR-181d/RBP2/NF-κB p65 Feedback Regulation Promotes Chronic Myeloid Leukemia Blast Crisis.

BACKGROUND: Chronic myeloid leukemia (CML) is a malignant clonal proliferative disease. Once it progresses into the phase of blast crisis (CML-BP), the curative effect is poor, and the fatality rate is extremely high. Therefore, it is urgent to explore the molecular mechanisms of blast crisis and identify new therapeutic targets. METHODS: The expression levels of miR-181d, RBP2 and NF-κB p65 were assessed in 42 newly diagnosed CML-CP patients and 15 CML-BP patients. Quantitative real-time PCR, Western blots, and cell proliferation assay were used to characterize the changes induced by overexpression or inhibition of miR-181d, RBP2 or p65. Luciferase reporter assay and ChIP assay was conducted to establish functional association between miR-181d, RBP2 and p65. Inhibition of miR-181d expression and its consequences in tumor growth was demonstrated in vivo models. RESULTS: We found that miR-181d was overexpressed in CML-BP, which promoted leukemia cell proliferation. Histone demethylase RBP2 was identified as a direct target of miR-181d which downregulated RBP2 expression. Moreover, RBP2 inhibited transcriptional expression of NF-κB subunit, p65 by binding to its promoter and demethylating the tri/dimethylated H3K4 region in the p65 promoter locus. In turn, p65 directly bound to miR-181d promoter and upregulated its expression. Therefore, RBP2 inhibition resulting from miR-181d overexpression led to p65 upregulation which further forwarded miR-181d expression. This miR-181d/RBP2/p65 feedback regulation caused sustained NF-κB activation, which contributed to the development of CML-BP. CONCLUSIONS: Taken together, the miR-181d/RBP2/p65 feedback regulation promoted CML-BP and miR-181d may serve as a potential therapeutic target of CML-BP.

Genome-wide identification of FHL1 as a powerful prognostic candidate and potential therapeutic target in acute myeloid leukaemia.

BACKGROUND: Acute myeloid leukaemia (AML) is a malignant haematological tumour with high heterogeneity and mortality. A reliable prognostic assessment is critical for treatment strategies. However, the current prognostic evaluation system of AML is insufficient. METHODS: Genome-wide univariate Cox regression analysis was performed on three independent AML datasets to screen for the prognostic-related genes. Kaplan-Meier survival analysis was employed to verify the efficacy of FHL1 in evaluating overall survival in 1298 de novo AML patients, 648 non-acute promyelocytic leukaemia AML patients and 407 cytogenetically normal AML patients; the data for some of these patients were also used for EFS and RFS validation. Multivariate Cox regression was performed to validate FHL1 as an independent prognostic indicator. WGCNA, GSEA, and gene correlation analysis were applied to explore the mechanism of FHL1 in AML. The synergistic cytocidal effect of FHL1 knockdown was verified in in vitro experiments. FINDINGS: Comprehensive genome-wide analyses and large-sample validation showed that FHL1 is a powerful prognostic candidate for overall survival, event-free survival, and relapse-free survival in AML and is independent of prognosis-related clinical factors and genetic abnormalities. The molecular mechanism may occur through regulation of FHL1 in leukaemia stem cells, tumour-associated signalling pathways, and transmembrane transport of chemotherapeutic drugs. FHL1-targeted intervention enhances the sensitivity of AML cells to cytarabine. INTERPRETATION: FHL1 may serve as an evaluation factor for clinical strategy selection, and its targeted intervention may be beneficial for chemotherapy in AML patients.

miR-96 acts as a tumor suppressor via targeting the BCR-ABL1 oncogene in chronic myeloid leukemia blastic transformation.

MicroRNA-mediated posttranscriptional regulation is an important epigenetic regulatory mechanism of gene expression, and its dysregulation is involved in the development and progression of a variety of malignancies, including chronic myeloid leukemia (CML). The BCR-ABL1 fusion gene is not only the initiating factor of CML, but it is also an important driving factor for blastic transformation. Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 tyrosine kinase activity, represented by imatinib, are currently the first-line treatment for CML. However, due to primary resistance or secondary resistance caused by mutations in the BCR-ABL1 kinase domain, TKIs cannot completely prevent the progression of CML; thus, the study of BCR-ABL1 gene expression regulation is of great significance. In this study, bioinformatics analysis and our results showed that miR-96 could directly bind to the 3'UTR region of BCR-ABL1 to regulate fusion protein expression, thereby regulating its downstream signaling pathway activity. We also found that miR-96 was downregulated during the progression from the chronic phase (CML-CP) to the blast crisis (CML-BC). Downregulation of miR-96 could promote the proliferation and participate in the cell differentiation of CML-BC cells. Additionally, we found that the novel histone deacetylase drug chidamide and the DNA methyltransferase inhibitor decitabine could restore the low expression of miR-96 in CML cells, and there were two abnormal hypermethylated sites in the promoter region of miR-96 in CML, suggesting that its low expression might be at least partially regulated by epigenetic mechanisms. In addition, re-expression of miR-96 could increase the sensitivity of CML-BC cells to imatinib. Thus, miR-96 functions as a tumor suppressor, and re-expression of this microRNA might have therapeutic benefits in CML blastic transformation.

Histone demethylase RBP2 mediates the blast crisis of chronic myeloid leukemia through an RBP2/PTEN/BCR-ABL cascade.

Epigenetic disorders play a key role in tumorigenesis and development, among which histone methylation abnormalities are common. While patients living with chronic myeloid leukemia in the chronic phase (CML-CP) have a good response to TKI, blastic phase (CML-BP) patients demonstrate poor efficacy and high fatality rates. However, while the mechanism of blast crisis of chronic myeloid leukemia remains unclear, high expression and activation of BCR-ABL are usually related to CML blast crisis transition. We found that histone H3 lysine 4 (H3K4) demethylase RBP2 expression is negatively correlated with BCR-ABL expression, which suggests a regulatory link between these two genes. We also discovered that RBP2 mediates the dephosphorylation of BCR-ABL by directly downregulating PTEN expression, depending on histone demethylase activity, while PTEN targets protein phosphatase activity of BCR-ABL, a phosphatase which directly dephosphorylates BCR-ABL. In clinical specimens, the mRNA expression of RBP2 was found to be positively correlated with that of PTEN. These data suggest that the under-expression of RBP2 promotes blast crisis transition by activating an RBP2/PTEN/BCR-ABL cascade.

The histone demethylase PHF8 promotes adult acute lymphoblastic leukemia through interaction with the MEK/ERK signaling pathway.

Adult acute lymphoblastic leukemia (ALL) is a malignant disorder of lymphoid progenitor cells that is associated with a high risk of relapse and poor prognosis. Thus, novel pathogenic mechanisms and therapeutic targets need to be explored. Histone methylation is one of the most significant chromatin post-translational modifications. Here, we show that the histone demethylase PHF8 is highly expressed in a large number of ALL clinical specimens and that PHF8 expression is associated with ALL progression. PHF8 knockdown inhibits proliferation and promotes the apoptosis of ALL cells in vitro as well as attenuates tumor growth in vivo. PHF8 transcriptionally upregulates MEK1, a key molecule in the MEK/ERK pathway, at least partially by directly binding to its promoter, thereby activating the MEK/ERK pathway. In addition, we found that an inhibitor of the MEK/ERK pathway, PD184352, subsequently suppresses PHF8 expression. Thus, PHF8 forms a positive feedback loop with the MEK/ERK pathway, and PHF8 knockdown enhances the lethality of PD184352 in ALL cells. In conclusion, this study identifies oncogenic functions of PHF8 in adult ALL and suggests a novel epigenetic strategy for disease intervention.

Acquired factor V deficiency in a patient with a urinary tract infection presenting with haematuria followed by multiple haemorrhages with an extremely low level of factor V inhibitor: a case report and review of the literature.

: Acquired factor V deficiency (AFVD) is a rare haemostatic disorder that is primarily because of the development of factor V inhibitors. Approximately, 200 cases have been reported and the greatest portion of these cases was because of bovine thrombin exposure. We report a case of a man who presented with haematuria followed by multiple haemorrhages associated with an elevated prothrombin time and an activated partial thromboplastin time. A workup revealed reduced factor V activity and a factor V inhibitor level of 1.9 BU, which were likely secondary to a urinary tract infection. Using corticosteroids, we successfully eliminated the inhibitor and controlled the bleeding. We review the published literature to identify the conditions that are associated with nonbovine thrombin AFVD. We assume that AFVD should be kept in mind for patients who present with multiple haemorrhages.

RBP2 Promotes Adult Acute Lymphoblastic Leukemia by Upregulating BCL2.

Despite recent increases in the cure rate of acute lymphoblastic leukemia (ALL), adult ALL remains a high-risk disease that exhibits a high relapse rate. In this study, we found that the histone demethylase retinoblastoma binding protein-2 (RBP2) was overexpressed in both on-going and relapse cases of adult ALL, which revealed that RBP2 overexpression was not only involved in the pathogenesis of ALL but that its overexpression might also be related to relapse of the disease. RBP2 knockdown induced apoptosis and attenuated leukemic cell viability. Our results demonstrated that BCL2 is a novel target of RBP2 and supported the notion of RBP2 being a regulator of BCL2 expression via directly binding to its promoter. As the role of RBP2 in regulating apoptosis was confirmed, RBP2 overexpression and activation of BCL2 might play important roles in ALL development and progression.

Histone demethylase RBP2 decreases miR-21 in blast crisis of chronic myeloid leukemia.

Chronic myeloid leukemia in the blastic phase (CML-BP) responds poorly to clinical treatments and is usually fatal. In this study, we found that the histone H3 lysine 4 (H3K4) demethylase RBP2 (also called JARID1A and KDM5A) is underexpressed in CML-BP. The RBP2 histone demethylase stimulates leukemia cell differentiation and inhibits cell proliferation. We identified miR-21 was directly downregulated by RBP2 and found that miR-21 downregulated PDCD4 expression in leukemia cells. By binding to miR-21 promoter and by demethylating of trimethylated H3K4 at the miR-21 locus, RBP2 downregulated miR-21 expression. This in turn activated PDCD4. In conclusion, RBP2 epigenetically downregulated miR-21 in blast transformation of CML.

Critical role of histone demethylase RBP2 in human gastric cancer angiogenesis.

BACKGROUND: The molecular mechanisms responsible for angiogenesis and abnormal expression of angiogenic factors in gastric cancer, including vascular endothelial growth factor (VEGF), remain unclear. The histone demethylase retinoblastoma binding protein 2 (RBP2) is involved in gastric tumorgenesis by inhibiting the expression of cyclin-dependent kinase inhibitors (CDKIs). METHODS: The expression of RBP2, VEGF, CD31, CD34 and Ki67 was assessed in 30 human gastric cancer samples and normal control samples. We used quantitative RT-PCR, western blot analysis, ELISA, tube-formation assay and colony-formation assay to characterize the change in VEGF expression and associated biological activities induced by RBP2 silencing or overexpression. Luciferase assay and ChIP were used to explore the direct regulation of RBP2 on the promoter activity of VEGF. Nude mice and RBP2-targeted mutant mice were used to detect the role of RBP2 in VEGF expression and angiogenesis in vivo. RESULTS: RBP2 and VEGF were both overexpressed in human gastric cancer tissue, with greater microvessel density (MVD) and cell proliferation as compared with normal tissue. In gastric epithelial cell lines, RBP2 overexpression significantly promoted the expression of VEGF and the growth and angiogenesis of the cells, while RBP2 knockdown had the reverse effect. RBP2 directly bound to the promoter of VEGF to regulate its expression by histone H3K4 demethylation. The subcutis of nude mice transfected with BGC-823 cells with RBP2 knockdown showed reduced VEGF expression and MVD, with reduced carcinogenesis and cell proliferation. In addition, the gastric epithelia of RBP2 mutant mice with increased H3K4 trimethylation showed reduced VEGF expression and MVD. CONCLUSIONS: The promotion of gastric tumorigenesis by RBP2 was significantly associated with transactivation of VEGF expression and elevated angiogenesis. Overexpression of RBP2 and activation of VEGF might play important roles in human gastric cancer development and progression.

MiR-370 sensitizes chronic myeloid leukemia K562 cells to homoharringtonine by targeting Forkhead box M1.

BACKGROUND: Homoharringtonine (HHT) is a kind of cephalotaxus alkaloid used in traditional Chinese medicine. Although HHT has been successfully used as a therapeutic agent for leukemia, the drug resistance and toxicity are major concerns. MicroRNAs (miRNAs) have been identified to modulate cellular sensitivity to anticancer drugs. We examined the synergistic action between miR-370 and HHT in vitro and in vivo. METHODS: The synergistic action between miR-370 and HHT was examined by flow cytometry. The effect of HHT on miR-370 expression was determined by quantitative RT-PCR (qRT-PCR). The expression of miR-370 and Forkhead box M1 (FoxM1) in 23 patients with newly diagnosed chronic-phase chronic myeloid leukemia (CML-CP) and 10 patients with blast-crisis CML (CML-BP) as well as miR-370-targeted FoxM1 was determined by qRT-PCR and western blot analysis. RESULTS: Ectopic expression of miR-370 sensitized the CML K562 cell line to HHT by targeting FoxM1, the major regulator in cell proliferation and apoptosis. miR-370 significantly promoted HHT-mediated cell apoptosis and miR-370 and HHT cooperated in affecting FoxM1 expression. As well, miR-370 was moderately upregulated after HHT treatment in K562 cells. In addition, the expression of miR-370 was significantly reduced in CML patients as compared with healthy controls. Furthermore, the expression of miR-370 was lower in CML-BP than CML-CP patients. CONCLUSIONS: MiR-370 sensitized K562 cells to HHT by inducing apoptosis in part by downregulation of FoxM1 expression. These findings may provide further information for CML treatment with HHT.

The tumor suppressive role of miRNA-370 by targeting FoxM1 in acute myeloid leukemia.

BACKGROUND: Recent evidence has accumulated that MicroRNA (miRNA) dysregulation occurs in the majority of human malignancies including acute myeloid leukemia (AML) and may contribute to onco-/leukemo-genesis. METHODS: The expression levels of miR-370 and FoxM1 were assessed in 48 newly diagnosed AML patients, 40 AML patients in 1st complete remission (CR) and 21 healthy controls. Quantitative real-time PCR, western blots, colony formation assay, and ß-Galactosidase ( SA-ß-Gal) staining were used to characterize the changes induced by overexpression or inhibition of miR-370 or FoxM1. RESULTS: We found that the down-regulation of miR-370 expression was a frequent event in both leukemia cell lines and primary leukemic cells from patients with de novo AML. Lower levels of miR-370 expression were found in 37 of 48 leukemic samples from AML patients compared to those in bone marrow cells derived from healthy adult individuals. Ectopic expression of miR-370 in HL60 and K562 cells led to cell growth arrest and senescence. In contrast, depletion of miR-370 expression using RNA interference enhanced the proliferation of those leukemic cells. Mechanistically, miR-370 targets the transcription factor FoxM1, a well established oncogenic factor promoting cell cycle progression. Moreover, when HL60 and K562 cells were treated with 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, miR-370 expression was up-regulated, which indicates epigenetic silencing of miR-370 in leukemic cells. CONCLUSIONS: Taken together, miR-370 may function as a tumor suppressor by targeting FoxM1, and the epigenetic silence of miR-370 thus leads to derepression of FoxM1 expression and consequently contributes to AML development and progression.