NOXA expression drives synthetic lethality to RUNX1 inhibition in pancreatic cancer.

Evasion from drug-induced apoptosis is a crucial mechanism of cancer treatment resistance. The proapoptotic protein NOXA marks an aggressive pancreatic ductal adenocarcinoma (PDAC) subtype. To identify drugs that unleash the death-inducing potential of NOXA, we performed an unbiased drug screening experiment. In NOXA-deficient isogenic cellular models, we identified an inhibitor of the transcription factor heterodimer CBFß/RUNX1. By genetic gain and loss of function experiments, we validated that the mode of action depends on RUNX1 and NOXA. Of note is that RUNX1 expression is significantly higher in PDACs compared to normal pancreas. We show that pharmacological RUNX1 inhibition significantly blocks tumor growth in vivo and in primary patient-derived PDAC organoids. Through genome-wide analysis, we detected that RUNX1-loss reshapes the epigenetic landscape, which gains H3K27ac enrichment at the NOXA promoter. Our study demonstrates a previously unknown mechanism of NOXA-dependent cell death, which can be triggered pharmaceutically. Therefore, our data show a way to target a therapy-resistant PDAC, an unmet clinical need.

RUNX1 transactivates BCR-ABL1 expression in Philadelphia chromosome positive acute lymphoblastic leukemia.

The emergence of tyrosine kinase inhibitors as part of a front-line treatment has greatly improved the clinical outcome of the patients with Ph+ acute lymphoblastic leukemia (ALL). However, a portion of them still become refractory to the therapy mainly through acquiring mutations in the BCR-ABL1 gene, necessitating a novel strategy to treat tyrosine kinase inhibitor (TKI)-resistant Ph+ ALL cases. In this report, we show evidence that RUNX1 transcription factor stringently controls the expression of BCR-ABL1, which can strategically be targeted by our novel RUNX inhibitor, Chb-M'. Through a series of in vitro experiments, we identified that RUNX1 binds to the promoter of BCR and directly transactivates BCR-ABL1 expression in Ph+ ALL cell lines. These cells showed significantly reduced expression of BCR-ABL1 with suppressed proliferation upon RUNX1 knockdown. Moreover, treatment with Chb-M' consistently downregulated the expression of BCR-ABL1 in these cells and this drug was highly effective even in an imatinib-resistant Ph+ ALL cell line. In good agreement with these findings, forced expression of BCR-ABL1 in these cells conferred relative resistance to Chb-M'. In addition, in vivo experiments with the Ph+ ALL patient-derived xenograft cells showed similar results. In summary, targeting RUNX1 therapeutically in Ph+ ALL cells may lead to overcoming TKI resistance through the transcriptional regulation of BCR-ABL1. Chb-M' could be a novel drug for patients with TKI-resistant refractory Ph+ ALL.

Runt-related transcription factor 1 (Runx1) aggravates pathological cardiac hypertrophy by promoting p53 expression.

Cardiac hypertrophy and the resultant heart failure are among the most common causes of morbidity and mortality worldwide; thus, identifying the key factor mediating pathological cardiac hypertrophy is critically important for developing the strategy to protect against heart failure. Runx1 (Runt-related transcription factor 1) acts as an essential transcription factor that functions in a variety of cellular processes including differentiation, proliferation, tissue growth and DNA damage response. However, relatively little is known about the role of Runx1 in heart, especially cardiac hypertrophy and heart failure. In the present study, we investigated the role of Runx1 in experimentally pathological cardiac hypertrophy. The in vitro model was induced by Ang II exposure to cultured neonatal rat cardiomyocytes, and the in vivo pathological cardiac hypertrophy models were induced by chronic pressure overload in mice. Runx1 expression is increased in heart tissues from mice with pressure overload-induced cardiac hypertrophy and in neonatal rat cardiomyocytes in response to Ang II stimulation. Moreover, knockdown of cardiac Runx1 alleviates the pressure overload-induced cardiac hypertrophy. Mechanistically, Runx1 activates the p53 signalling by binding to the p53 gene and promotes its transcription. Rescue experiments indicate that Runx1 promotes cardiac hypertrophy in a p53-dependent manner. Remarkably, we demonstrated that Ro5-3335 (a Runx1 inhibitor) acts as a potential therapeutic drug for treating pathological cardiac hypertrophy. In summary, we conclude that Runx1 is a novel mediator and therapeutic target for pathological cardiac hypertrophy.

Targeting Runt-Related Transcription Factor 1 Prevents Pulmonary Fibrosis and Reduces Expression of Severe Acute Respiratory Syndrome Coronavirus 2 Host Mediators.

Pulmonary fibrosis (PF) can arise from unknown causes, as in idiopathic PF, or as a consequence of infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current treatments for PF slow, but do not stop, disease progression. We report that treatment with a runt-related transcription factor 1 (RUNX1) inhibitor (Ro24-7429), previously found to be safe, although ineffective, as a Tat inhibitor in patients with HIV, robustly ameliorates lung fibrosis and inflammation in the bleomycin-induced PF mouse model. RUNX1 inhibition blunted fundamental mechanisms downstream pathologic mediators of fibrosis and inflammation, including transforming growth factor-ß1 and tumor necrosis factor-α, in cultured lung epithelial cells, fibroblasts, and vascular endothelial cells, indicating pleiotropic effects. RUNX1 inhibition also reduced the expression of angiotensin-converting enzyme 2 and FES Upstream Region (FURIN), host proteins critical for SARS-CoV-2 infection, in mice and in vitro. A subset of human lungs with SARS-CoV-2 infection overexpress RUNX1. These data suggest that RUNX1 inhibition via repurposing of Ro24-7429 may be beneficial for PF and to battle SARS-CoV-2, by reducing expression of viral mediators and by preventing respiratory complications.

Reduction of RUNX1 transcription factor activity by a CBFA2T3-mimicking peptide: application to B cell precursor acute lymphoblastic leukemia.

BACKGROUND: B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is the most common pediatric cancer. Identifying key players involved in proliferation of BCP-ALL cells is crucial to propose new therapeutic targets. Runt Related Transcription Factor 1 (RUNX1) and Core-Binding Factor Runt Domain Alpha Subunit 2 Translocated To 3 (CBFA2T3, ETO2, MTG16) are master regulators of hematopoiesis and are implicated in leukemia. METHODS: We worked with BCP-ALL mononuclear bone marrow patients' cells and BCP-ALL cell lines, and performed Chromatin Immunoprecipitations followed by Sequencing (ChIP-Seq), co-immunoprecipitations (co-IP), proximity ligation assays (PLA), luciferase reporter assays and mouse xenograft models. RESULTS: We demonstrated that CBFA2T3 transcript levels correlate with RUNX1 expression in the pediatric t(12;21) ETV6-RUNX1 BCP-ALL. By ChIP-Seq in BCP-ALL patients' cells and cell lines, we found that RUNX1 is recruited on its promoter and on an enhancer of CBFA2T3 located - 2 kb upstream CBFA2T3 promoter and that, subsequently, the transcription factor RUNX1 drives both RUNX1 and CBFA2T3 expression. We demonstrated that, mechanistically, RUNX1 and CBFA2T3 can be part of the same complex allowing CBFA2T3 to strongly potentiate the activity of the transcription factor RUNX1. Finally, we characterized a CBFA2T3-mimicking peptide that inhibits the interaction between RUNX1 and CBFA2T3, abrogating the activity of this transcription complex and reducing BCP-ALL lymphoblast proliferation. CONCLUSIONS: Altogether, our findings reveal a novel and important activation loop between the transcription regulator CBFA2T3 and the transcription factor RUNX1 that promotes BCP-ALL proliferation, supporting the development of an innovative therapeutic approach based on the NHR2 subdomain of CBFA2T3 protein.

Endothelial progenitor cells-derived exosomal microRNA-21-5p alleviates sepsis-induced acute kidney injury by inhibiting RUNX1 expression.

The role of microRNA-21-5p (miR-21-5p) in sepsis-induced acute kidney injury (AKI) has been seldom discussed. Therefore, the objective of this present study was to investigate the mechanism of endothelial progenitor cells-derived exosomes (EPCs-exos) in sepsis-induced AKI via miR-21-5p/runt-related transcription factor 1 (RUNX1) axis. miR-21-5p was downregulated and RUNX1 was upregulated in the kidney of cecal ligation and puncture (CLP) rats, and miR-21-5p targeted RUNX1. Elevation of miR-21-5p improved renal function and renal tissue pathological damage, attenuated serum inflammatory response, as well as reduced apoptosis and oxidative stress response in renal tissues, and regulated endothelial glycocalyx damage marker proteins syndecan-1 and heparanase-1 in CLP rats. Overexpression of RUNX1 abolished the impacts of elevated miR-21-5p in CLP rats. Also, EPCs-exos upregulated miR-21-5p expression, and functioned similar to elevation of miR-21-5p for CLP rats. Downregulating miR-21-5p partially reversed the effects of EPCs-exos on sepsis-induced AKI. Collectively, our study suggests that EPCs release miR-21-5p-containing exosomes to alleviate sepsis-induced AKI through RUNX1 silencing.

TNF-α signaling regulates RUNX1 function in endothelial cells.

Runt-related transcription factor 1 (RUNX1) acts as a mediator of aberrant retinal angiogenesis and has been implicated in the progression of proliferative diabetic retinopathy (PDR). Patients with PDR, retinopathy of prematurity (ROP), and wet age-related macular degeneration (wet AMD) have been found to have elevated levels of Tumor Necrosis Factor-alpha (TNF-α) in the eye. In fibrovascular membranes (FVMs) taken from patients with PDR RUNX1 expression was increased in the vasculature, while in human retinal microvascular endothelial cells (HRMECs), TNF-α stimulation causes increased RUNX1 expression, which can be modulated by RUNX1 inhibitors. Using TNF-α pathway inhibitors, we determined that in HRMECs, TNF-α-induced RUNX1 expression occurs via JNK activation, while NF-κB and p38/MAPK inhibition did not affect RUNX1 expression. JNK inhibitors were also effective at stopping high D-glucose-stimulated RUNX1 expression. We further linked JNK to RUNX1 through Activator Protein 1 (AP-1) and investigated the JNK-AP-1-RUNX1 regulatory feedback loop, which can be modulated by VEGF. Additionally, stimulation with TNF-α and D-glucose had an additive effect on RUNX1 expression, which was downregulated by VEGF modulation. These data suggest that the downregulation of RUNX1 in conjunction with anti-VEGF agents may be important in future treatments for the management of diseases of pathologic ocular angiogenesis.

Suppression of malignant rhabdoid tumors through Chb-M'-mediated RUNX1 inhibition.

Malignant rhabdoid tumor (MRT) is a rare and highly aggressive pediatric malignancy primarily affecting infants and young children. Intensive multimodal therapies currently given to MRT patients are not sufficiently potent to control this highly malignant tumor. Therefore, additive or alternative therapy for these patients with a poor prognosis is necessary. We herein demonstrated that the inhibition of runt-related transcription factor 1 (RUNX1) by novel alkylating conjugated pyrrole-imidazole (PI) polyamides, which specifically recognize and bind to RUNX-binding DNA sequences, was highly effective in the treatment of rhabdoid tumor cell lines in vitro as well as in an in vivo mouse model. Therefore, suppression of RUNX1 activity may be a novel strategy for MRT therapy.

Treatment of Experimental Choroidal Neovascularization via RUNX1 Inhibition.

Choroidal neovascularization (CNV) is a prevalent cause of vision loss in patients with age-related macular degeneration. Runt-related transcription factor 1 (RUNX1) has been identified as an important mediator of aberrant retinal angiogenesis in proliferative diabetic retinopathy and its modulation has proven to be effective in curbing pathologic angiogenesis in experimental oxygen-induced retinopathy. However, its role in CNV remains to be elucidated. This study demonstrates RUNX1 expression in critical cell types involved in a laser-induced model of CNV in mice. Furthermore, the preclinical efficacy of Ro5-3335, a small molecule inhibitor of RUNX1, in experimental CNV is reported. RUNX1 inhibitor Ro5-3335, aflibercept-an FDA-approved vascular endothelial growth factor (VEGF) inhibitor, or a combination of both, were administered by intravitreal injection immediately after laser injury. The CNV area of choroidal flatmounts was evaluated by immunostaining with isolectin B4, and vascular permeability was analyzed by fluorescein angiography. A single intravitreal injection of Ro5-3335 significantly decreased the CNV area 7 days after laser injury, and when combined with aflibercept, reduced vascular leakage more effectively than aflibercept alone. These data suggest that RUNX1 inhibition alone or in combination with anti-VEGF drugs may be a new therapy upon further clinical validation for patients with neovascular age-related macular degeneration.

Topical delivery of a small molecule RUNX1 transcription factor inhibitor for the treatment of proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is the leading cause of retinal detachment surgery failure. Despite significant advances in vitreoretinal surgery, it still remains without an effective prophylactic or therapeutic medical treatment. After ocular injury or retinal detachment, misplaced retinal cells undergo epithelial to mesenchymal transition (EMT) to form contractile membranes within the eye. We identified Runt-related transcription factor 1 (RUNX1) as a gene highly expressed in surgically-removed human PVR specimens. RUNX1 upregulation was a hallmark of EMT in primary cultures derived from human PVR membranes (C-PVR). The inhibition of RUNX1 reduced proliferation of human C-PVR cells in vitro, and curbed growth of freshly isolated human PVR membranes in an explant assay. We formulated Ro5-3335, a lipophilic small molecule RUNX1 inhibitor, into a nanoemulsion that when administered topically curbed the progression of disease in a novel rabbit model of mild PVR developed using C-PVR cells. Mass spectrometry analysis detected 2.67 ng/mL of Ro5-3335 within the vitreous cavity after treatment. This work shows a critical role for RUNX1 in PVR and supports the feasibility of targeting RUNX1 within the eye for the treatment of an EMT-mediated condition using a topical ophthalmic agent.

Inhibition of RUNX1 promotes cisplatin-induced apoptosis in ovarian cancer cells.

Runt-related transcription factor 1 (RUNX1), one subunit of core-binding factors in hematopoiesis and leukemia, was highly expressed in ovarian cancer (OC), but the role of RUNX1 in OC is largely unknown. Since we found that high expression of RUNX1 is correlated with poor survival in patients with OC through bioinformatic analysis of TCGA database, we developed RUNX1-knockout clones by CRISPR/Cas9 technique and discovered that RUNX1 depletion could promote cisplatin-induced apoptosis in OC cells, which was further confirmed by RUNX1 knockdown and overexpression. We also proved that RUNX1 could elevate the expression of BCL2. We then examined a total of 32 candidate miRNAs that might mediate the regulation between RUNX1 and BCL2, of which three miRNAs from the miR-17~92 cluster were found to be negatively regulated by RUNX1. Consistently, our analysis of data from TCGA database revealed the negative correlation between RUNX1 and the cluster. We further confirmed that miR-17~92 cluster could enhance cisplatin-induced apoptosis by directly targeting BCL2 3'UTR. Since rescue experiments proved that RUNX1 could repress cisplatin-induced apoptosis by up-regulating BCL2 via miR-17~92 cluster, combining RUNX1 inhibitor Ro5-3335 and cisplatin showed synergic effect in triggering OC cell apoptosis. Collectively, these findings show for the first time that combinational treatment of cisplatin and RUNX1 inhibitor could be used to potentiate apoptosis of ovarian cancer cells, and reveal the potential of targeting RUNX1 in ovarian cancer chemotherapy.

Acute Myeloid Leukemia iPSCs Reveal a Role for RUNX1 in the Maintenance of Human Leukemia Stem Cells.

Leukemia stem cells (LSCs) are believed to have more distinct vulnerabilities than the bulk acute myeloid leukemia (AML) cells, but their rarity and the lack of universal markers for their prospective isolation hamper their study. We report that genetically clonal induced pluripotent stem cells (iPSCs) derived from an AML patient and characterized by exceptionally high engraftment potential give rise, upon hematopoietic differentiation, to a phenotypic hierarchy. Through fate-tracking experiments, xenotransplantation, and single-cell transcriptomics, we identify a cell fraction (iLSC) that can be isolated prospectively by means of adherent in vitro growth that resides on the apex of this hierarchy and fulfills the hallmark features of LSCs. Through integrative genomic studies of the iLSC transcriptome and chromatin landscape, we derive an LSC gene signature that predicts patient survival and uncovers a dependency of LSCs, across AML genotypes, on the RUNX1 transcription factor. These findings can empower efforts to therapeutically target AML LSCs.

RUNX1 and RUNX2 transcription factors function in opposing roles to regulate breast cancer stem cells.

Breast cancer stem cells (BCSCs) are competent to initiate tumor formation and growth and refractory to conventional therapies. Consequently BCSCs are implicated in tumor recurrence. Many signaling cascades associated with BCSCs are critical for epithelial-to-mesenchymal transition (EMT). We developed a model system to mechanistically examine BCSCs in basal-like breast cancer using MCF10AT1 FACS sorted for CD24 (negative/low in BCSCs) and CD44 (positive/high in BCSCs). Ingenuity Pathway Analysis comparing RNA-seq on the CD24-/low versus CD24+/high MCF10AT1 indicates that the top activated upstream regulators include TWIST1, TGFß1, OCT4, and other factors known to be increased in BCSCs and during EMT. The top inhibited upstream regulators include ESR1, TP63, and FAS. Consistent with our results, many genes previously demonstrated to be regulated by RUNX factors are altered in BCSCs. The RUNX2 interaction network is the top significant pathway altered between CD24-/low and CD24+/high MCF10AT1. RUNX1 is higher in expression at the RNA level than RUNX2. RUNX3 is not expressed. While, human-specific quantitative polymerase chain reaction primers demonstrate that RUNX1 and CDH1 decrease in human MCF10CA1a cells that have grown tumors within the murine mammary fat pad microenvironment, RUNX2 and VIM increase. Treatment with an inhibitor of RUNX binding to CBFß for 5 days followed by a 7-day recovery period results in EMT suggesting that loss of RUNX1, rather than increase in RUNX2, is a driver of EMT in early stage breast cancer. Increased understanding of RUNX regulation on BCSCs and EMT will provide novel insight into therapeutic strategies to prevent recurrence.

Benzodiazepines Drive Alteration of Chromatin at the Integrated HIV-1 LTR.

Antiretroviral therapy (ART) lowers human immunodeficiency virus type 1 (HIV-1) viral load to undetectable levels, but does not eliminate the latent reservoir. One of the factors controlling the latent reservoir is transcriptional silencing of the integrated HIV-1 long terminal repeat (LTR). The molecular mechanisms that control HIV-1 transcription are not completely understood. We have previously shown that RUNX1, a host transcription factor, may play a role in the establishment and maintenance of HIV-1 latency. Prior work has demonstrated that inhibition of RUNX1 by the benzodiazepine (BDZ) Ro5-3335 synergizes with suberanilohydroxamic acid (SAHA) to activate HIV-1 transcription. In this current work, we examine the effect of RUNX1 inhibition on the chromatin state of the integrated HIV-1 LTR. Using chromatin immunoprecipitation (ChIP), we found that Ro5-3335 significantly increased the occupancy of STAT5 at the HIV-1 LTR. We also screened other BDZs for their ability to regulate HIV-1 transcription and demonstrate their ability to increase transcription and alter chromatin at the LTR without negatively affecting Tat activity. These findings shed further light on the mechanism by which RUNX proteins control HIV-1 transcription and suggest that BDZ compounds might be useful in activating HIV-1 transcription through STAT5 recruitment to the HIV-1 LTR.

miR-215-5p is an anticancer gene in multiple myeloma by targeting RUNX1 and deactivating the PI3K/AKT/mTOR pathway.

This study examined the underlying mechanism of miR-215-5p in multiple myeloma (MM) at the molecular level. miR-215-5p was upregulated in bone marrow specimens of patients with MM and MM cell lines through real-time polymerase chain reaction, and downregulation of miR-215-5p was related to poor survival of patients with MM. The results of Western blot assay showed that the PI3K/AKT/mTOR signaling pathway was activated in bone marrow specimens of patients with MM. miR-215-5p was found to negatively correlate with runt-related transcription factor 1 (RUNX1) expression in MM clinical bone marrow samples. Therefore, to test the probable mechanisms of the regulation of MM cell proliferation and apoptosis, a miR-215-5p mimics/inhibitors/negative control was transfected into MM cells. The targets of miR-215-5p were estimated using four bioinformatics databases (including miRDB, miRTarBase, Starbase, and Targetscan). Furthermore, enrichment analyses of gene ontology and Kyoto Encyclopedia of Genomes pathway were carried out at the Enrichr website. Cell viability was detected using the MTT method, while apoptosis and cell cycle were measured using flow cytometry. RUNX1, CDK2, CDC25B, cleaved-caspase-3, cleaved-PARP, p-PI3K, PI3K, p-AKT, AKT, p-mTOR as well as mTOR protein were also investigated using Western blot analysis. According to our findings, miR-215-5p overexpression could induce apoptosis while rendering cell cycle arrest at G1 phase and reducing the proliferation of cells. Meanwhile, miRNA-215-5p could negatively regulate messengerRNA and protein levels of RUNX1 in MM cells. In addition, there was a similar effect in cell proliferation and apoptosis after miR-215-5p mimics or siRNA-RUNX1 transfection. miR-215-5p inhibition inhibited apoptosis while increased the phosphorylation levels of PI3K, AKT, and mTOR proteins, whereas, miR-215-5p overexpression increased cell proliferation. Therefore, miR-215-5p inhibited MM cell apoptosis via targeting RUNX1 and suppressing the activation of the PI3K/AKT/mTOR pathway.

Use of polymeric CXCR4 inhibitors as siRNA delivery vehicles for the treatment of acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults and is associated with poor long-term survival often owing to relapse. Current treatments for AML are associated with considerable toxicity and are frequently not effective after relapse. Thus, it is important to develop novel therapeutic strategies. Short interfering RNA (siRNA)-based therapeutics targeting key oncogenes have been proposed as treatments for AML. We recently developed novel siRNA delivery polycations (PCX) based on AMD3100 (plerixafor), an FDA-approved inhibitor of the CXC chemokine receptor 4 (CXCR4). Inhibitors of CXCR4 have been shown to sensitize leukemia cells to chemotherapy. Therefore, PCX has the potential to target leukemia cells via two mechanisms: inhibition of CXCR4 and delivery of siRNAs against critical genes. In this report, we show that PCX exerts a cytotoxic effect on leukemia cells more effectively than other CXCR4 inhibitors, including AMD3100. In addition, we show that PCX can deliver siRNAs against the transcription factor RUNX1 to mouse and human leukemia cells. Overall, our study provides the first evidence that dual-function PCX/siRNA nanoparticles can simultaneously inhibit CXCR4 and deliver siRNAs, targeting key oncogenes in leukemia cells and that PCX/siRNA has clinical potential for the treatment of AML.

Myeloid translocation gene CBFA2T3 directs a relapse gene program and determines patient-specific outcomes in AML.

CBFA2T3 is a master transcriptional coregulator in hematopoiesis. In this study, we report novel functions of CBFA2T3 in acute myeloid leukemia (AML) relapse. CBFA2T3 regulates cell-fate genes to establish gene expression signatures associated with leukemia stem cell (LSC) transformation and relapse. Gene set enrichment analysis showed that CBFA2T3 expression marks LSC signatures in primary AML samples. Analysis of paired primary and relapsed samples showed that acquisition of LSC gene signatures involves cell type-specific activation of CBFA2T3 transcription via the NM_005187 promoter by GCN5. Short hairpin RNA-mediated downregulation of CBFA2T3 arrests G1/S cell cycle progression, diminishes LSC gene signatures, and attenuates in vitro and in vivo proliferation of AML cells. We also found that the RUNX1-RUNX1T1 fusion protein transcriptionally represses NM_005187 to confer t(8;21) AML patients a natural resistance to relapse, whereas lacking a similar repression mechanism renders non-core-binding factor AML patients highly susceptible to relapse. These studies show that 2 related primary AML-associated factors, the expression level of CBFA2T3 and the ability of leukemia cells to repress cell type-specific CBFA2T3 gene transcription, play important roles in patient prognosis, providing a paradigm that differential abilities to repress hematopoietic coregulator gene transcription are correlated with patient-specific outcomes in AML.

Targeting miR-193a-AML1-ETO-ß-catenin axis by melatonin suppresses the self-renewal of leukaemia stem cells in leukaemia with t (8;21) translocation.

AML1-ETO, the most common fusion oncoprotein by t (8;21) in acute myeloid leukaemia (AML), enhances hematopoietic self-renewal and leukemogenesis. However, currently no specific therapies have been reported for t (8;21) AML patients as AML1-ETO is still intractable as a pharmacological target. For this purpose, leukaemia cells and AML1-ETO-induced murine leukaemia model were used to investigate the degradation of AML1-ETO by melatonin (MLT), synthesized and secreted by the pineal gland. MLT remarkedly decreased AML1-ETO protein in leukemic cells. Meanwhile, MLT induced apoptosis, decreased proliferation and reduced colony formation. Furthermore, MLT reduced the expansion of human leukemic cells and extended the overall survival in U937T-AML1-ETO-xenografted NSG mice. Most importantly, MLT reduced the infiltration of leukaemia blasts, decreased the frequency of leukaemia stem cells (LSCs) and prolonged the overall survival in AML1-ETO-induced murine leukaemia. Mechanistically, MLT increased the expression of miR-193a, which inhibited AML1-ETO expression via targeting its putative binding sites. Furthermore, MLT decreased the expression of ß-catenin, which is required for the self-renewal of LSC and is the downstream of AML1-ETO. Thus, MLT presents anti-self-renewal of LSC through miR-193a-AML1-ETO-ß-catenin axis. In conclusion, MLT might be a potential treatment for t (8;21) leukaemia by targeting AML1-ETO oncoprotein.

RUNX1-targeted therapy for AML expressing somatic or germline mutation in RUNX1.

RUNX1 transcription factor regulates normal and malignant hematopoiesis. Somatic or germline mutant RUNX1 (mtRUNX1) is associated with poorer outcome in acute myeloid leukemia (AML). Knockdown or inhibition of RUNX1 induced more apoptosis of AML expressing mtRUNX1 versus wild-type RUNX1 and improved survival of mice engrafted with mtRUNX1-expressing AML. CRISPR/Cas9-mediated editing-out of RUNX1 enhancer (eR1) within its intragenic super-enhancer, or BET protein BRD4 depletion by short hairpin RNA, repressed RUNX1, inhibited cell growth, and induced cell lethality in AML cells expressing mtRUNX1. Moreover, treatment with BET protein inhibitor or degrader (BET-proteolysis targeting chimera) repressed RUNX1 and its targets, inducing apoptosis and improving survival of mice engrafted with AML expressing mtRUNX1. Library of Integrated Network-based Cellular Signatures 1000-connectivity mapping data sets queried with messenger RNA signature of RUNX1 knockdown identified novel expression-mimickers (EMs), which repressed RUNX1 and exerted in vitro and in vivo efficacy against AML cells expressing mtRUNX1. In addition, the EMs cinobufagin, anisomycin, and narciclasine induced more lethality in hematopoietic progenitor cells (HPCs) expressing germline mtRUNX1 from patients with AML compared with HPCs from patients with familial platelet disorder (FPD), or normal untransformed HPCs. These findings highlight novel therapeutic agents for AML expressing somatic or germline mtRUNX1.