Oncogene In Acute Myeloid Leukemia Research Articles

Transcriptional control of leukemogenesis by the chromatin reader SGF29

AbstractAberrant expression of stem cell–associated genes is a common feature in acute myeloid leukemia (AML) and is linked to leukemic self-renewal and therapy resistance. Using AF10-rearranged leukemia as a prototypical example of the recurrently activated “stemness” network in AML, we screened for chromatin regulators that sustain its expression. We deployed a CRISPR-Cas9 screen with a bespoke domain-focused library and identified several novel chromatin-modifying complexes as regulators of the TALE domain transcription factor MEIS1, a key leukemia stem cell (LSC)–associated gene. CRISPR droplet sequencing revealed that many of these MEIS1 regulators coordinately controlled the transcription of several AML oncogenes. In particular, we identified a novel role for the Tudor-domain–containing chromatin reader protein SGF29 in the transcription of AML oncogenes. Furthermore, SGF29 deletion impaired leukemogenesis in models representative of multiple AML subtypes in multiple AML subtype models. Our studies reveal a novel role for SGF29 as a nononcogenic dependency in AML and identify the SGF29 Tudor domain as an attractive target for drug discovery.

Oncogenic drivers dictate immune control of acute myeloid leukemia

Acute myeloid leukemia (AML) is a genetically heterogeneous, aggressive hematological malignancy induced by distinct oncogenic driver mutations. The effect of specific AML oncogenes on immune activation or suppression is unclear. Here, we examine immune responses in genetically distinct models of AML and demonstrate that specific AML oncogenes dictate immunogenicity, the quality of immune response and immune escape through immunoediting. Specifically, expression of NrasG12D alone is sufficient to drive a potent anti-leukemia response through increased MHC Class II expression that can be overcome with increased expression of Myc. These data have important implications for the design and implementation of personalized immunotherapies for patients with AML.

High Expression of CD300A Predicts Poor Survival in Acute Myeloid Leukemia

Introduction: Recent studies have suggested that CD300A was an oncogene in acute myeloid leukemia (AML) development. However, the clinical relevance and biological insight into CD300A expression in AML are still not well understood. The present study aimed to examine the expression characteristics of CD300A in AML and confirmed its clinical significance for AML. Methods: Quantification of the CD300A transcript was performed in 119 AML patients by real-time quantitative PCR in bone marrow blasts. The predictive significance of CD300A expression on the clinical outcomes of AML was assessed using overall survival (OS) and relapse-free survival (RFS). The published Cancer Genome Atlas (TCGA) data were used as an external validation for survival analysis and pathway analyses. Results: In comparison with monocytes from healthy peripheral blood cells, the expression levels of CD300A in AML cells were higher. Patients in the intermediate and adverse risk categories by WHO criteria (2018) had higher CD300A expression levels than those in the favorable risk category (p < 0.001). AML patients with high expression of CD300A had a higher early death rate (p = 0.029), lower complete remission rate (p = 0.042), higher death rate (p < 0.001) and relapse rate (p = 0.002), and shorter OS (p < 0.0001) and RFS (p < 0.0001). Through multivariable analysis, high CD300A expression in AML was also an independent poor prognostic factor. The CAMP and CGMP-PKG signaling pathways may be stimulated by increased CD300A expression levels, which may be important for the development of AML. Conclusions: The expression levels of CD300A were associated with risk stratification and the clinical relevance of AML. High CD300A expression may act as an independent adverse prognostic factor for OS and RFS in AML.

A Novel Peptide that Disrupts the Lck-IP3R Protein-Protein Interaction Induces Widespread Cell Death in Leukemia and Lymphoma.

There is increasing evidence that the T-cell protein, Lck, is involved in the pathogenesis of chronic lymphocytic leukemia (CLL) as well as other leukemias and lymphomas. We previously discovered that Lck binds to domain 5 of inositol 1,4,5-trisphosphate receptors (IP3R) to regulate Ca2+ homeostasis. Using bioinformatics, we targeted a region within domain 5 of IP3R-1 predicted to facilitate protein-protein interactions (PPIs). We generated a synthetic 21 amino acid peptide, KKRMDLVLELKNNASKLLLAI, which constitutes a domain 5 sub-domain (D5SD) of IP3R-1 that specifically binds Lck via its SH2 domain. With the addition of an HIV-TAT sequence to enable cell permeability of D5SD peptide, we observed wide-spread, Ca2+-dependent, cell killing of hematological cancer cells when the Lck-IP3R PPI was disrupted by TAT-D5SD. All cell lines and primary cells were sensitive to D5SD peptide, but malignant T-cells were less sensitive compared with B-cell or myeloid malignancies. Mining of RNA-seq data showed that LCK was expressed in primary diffuse large B-cell lymphoma (DLBCL) as well as acute myeloid leukemia (AML). In fact, LCK shows a similar pattern of expression as many well-characterized AML oncogenes and is part of a protein interactome that includes FLT3-ITD, Notch-1, and Kit. Consistent with these findings, our data suggest that the Lck-IP3R PPI may protect malignant hematopoietic cells from death. Importantly, TAT-D5SD showed no cytotoxicity in three different non-hematopoietic cell lines; thus its ability to induce cell death appears specific to hematopoietic cells. Together, these data show that a peptide designed to disrupt the Lck-IP3R PPI has a wide range of pre-clinical activity in leukemia and lymphoma.

MiR-1306-5p promotes cell proliferation and inhibits cell apoptosis in acute myeloid leukemia by downregulating PHF6 expression

BackgroundDeregulated expression of miRNAs contributes to the development of numerous malignancies, including acute myeloid leukemia (AML). The present work focused on investigating the role of miR-1306–5p in AML pathogenesis and the possible mechanisms. MethodsThe expression levels of miR-1306–5p and PHF6 were assessed in 30 healthy controls and 48 newly diagnosed AML patients. CCK-8 assay, EdU staining, quantitative real-time PCR, TUNEL assay, western blots, and flow cytometry were used to characterize the changes induced by miR-1306–5p or PHF6 overexpression or inhibition. In addition, Starbase and miRWalk databases were adopted to predict the miR-1306–5p target genes. ResultsHere we reported that upregulation of miR-1306–5p was a frequent event in both primary leukemic cells from AML patients and AML cell lines. Functional assays indicated that downregulation of miR-1306–5p leads to AML cell growth arrest, less proliferation, and elevated rates of apoptosis. Mechanistically, miR-1306–5p targets PHF6, a protein that plays a key role in gene transcription regulation. Our data further showed that PHF6 was downregulated in AML patients and cell lines, and depletion of PHF6 expression using RNA interference further enhanced the proliferation while reducing the apoptosis of those leukemic cells. ConclusionOur findings show that miR-1306–5p promotes proliferation and prevents apoptosis in AML by directly modulating PHF6 expression and consequently contributes to AML development and progression. miR-1306–5p may function as an oncogene in AML, providing a promising therapeutic target for AML patients.

Twist1 Promoter Methylation Regulates the Proliferation and Apoptosis of Acute Myeloid Leukemia Cells via PI3K/AKT Pathway.

Twist-related protein 1 (Twist1) is a widely recognized oncogene in acute myeloid leukemia (AML), and its promoter methylation is related with the progression of solid tumors. However, the association between Twist1 promoter methylation and AML has not been well studied. Twist1 mRNA expression was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of Twist1 and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signal were measured via western blotting. Methylation-specific PCR was performed to detect the methylation status of Twist1 promoter. CCK-8 assay and flow cytometry were used to reveal cellular biological effects. Twist1 expression and promoter methylation level were significantly upregulated in AML tissues and cell lines and were further downregulated in demethylating agent 5'-azacitidine (5-Aza)-treated cells. Ectopic expression of Twist1 increased AML cell viability, while reducing apoptosis, and attenuated the effects of 5-Aza on the proliferation and apoptosis. We also found that the PI3K/AKT signaling pathway was positively regulated by Twist1. Our findings revealed that Twist1 accelerates the tumorigenesis of AML cells by promoting its promoter methylation via the activation of PI3K/AKT signaling pathway.

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HnRNP K: A Driver of Myeloid Malignancy

Acute myeloid leukemia (AML) is increasingly defined as a heterogeneous group of malignancies divided largely by recurrent genetic and/or chromosomal aberrations. With this knowledge, development of targeted therapeutics has improved outcomes for some AML subsets. Increasing our understanding of alterations that drive disease development and progression will expand our armamentarium of therapeutic opportunities. To this end, our laboratory has identified a subset of AML that harbors increased expression of heterogeneous nuclear ribonucleoprotein K (HNRNPK; protein- hnRNP K).In a sizeable cohort of patients with AML, we have demonstrated HNRNPK gene amplification, RNA overexpression, and protein overexpression using FISH, qRT-PCR, and reverse phase protein array analyses. Importantly, the patient population with high protein expression of hnRNP K exhibited decreased median overall survival, increased WBC, and increased blast percentage in both bone marrow and peripheral blood. Together, these data suggest that hnRNP K may function as an oncogene in AML when overexpressed.To formally test this hypothesis, we generated a Vav-Cre;HnrnpkTg mouse model. Indeed, these mice overexpress hnRNP K in comparison to Vav-Cre mice or unrecombined littermates. Critically, Vav-Cre;HnrnpkTg mice develop myeloid malignancies. When transplanted, these malignancies have a decreased latency period and are rendered more aggressive. Currently, we are evaluating HSPC proliferation, differentiation potential, and using time of flight mass cytometry (CyTOF) to extensively characterize these transplanted leukemias.Functionally, hnRNP K is an RNA and ssDNA binding protein that can shuttle bidirectionally between the nucleus and cytoplasm. To further understand how hnRNP K can cause AML, we are evaluating subcellular localization of hnRNP K in the context of overexpression. As our preliminary experiments suggest a predominant cytoplasmic localization when hnRNP K is overexpressed, we are performing hnRNP K RNA-immunoprecipitation (RIP) followed by next generation sequencing in human cell lines with inducible overexpression of hnRNP K. In addition to confirming our previous observations of hnRNP K binding to the MYC transcript, we have identified several other putative targets for hnRNP K binding that may potentiate these malignant phenotypes.Additionally, we have performed RNA-Seq on HNRNPK- overexpressing human cell lines. These data suggest a significant role for hnRNP K in the regulation of innate immune response-observations that are consistent with phenotypes seen in our transplant models. Together, these studies provide insight into the pathways most critically altered in hnRNP K-overexpressing AML and will allow for appropriate utilization of therapeutic modalities. DisclosuresNo relevant conflicts of interest to declare.

Microrna-211 - a Novel Oncogene in Acute Myeloid Leukemia

MicroRNAs are a class of small, non-coding RNA molecules that regulate gene expression by post-transcriptional silencing and regulate many critical cellular processes. In cancer, microRNAs are emerging as important biomarkers of disease and a subset of microRNAs have well-established roles as drivers of cancer development. MicroRNA-211 (miR-211) is an intronic microRNA encoded on human chromosome 15, and deregulated miR-211 expression has been previously demonstrated in melanoma and a range of other cancers. A role for miR-211 in the hematopoietic system, and/or in the development of acute myeloid leukemia (AML) however remains unexplored.Using microRNA expression profiling, across two independent pediatric AML cohorts, we are the first to identify that miR-211 expression is elevated in a subset of patients, from more than 2-fold and up to 50-fold above non-leukemic remission controls. High expression of miR-211 tends to be clustered in patients with cytogenetically normal and M5 AML subtypes. Using hematopoietic reconstitution experiments in mouse models, we show for the first time that enforced expression of miR-211 alone in hematopoietic stem cells is sufficient to drive a partially-penetrant myeloproliferative disease that phenotypically resembles acute myeloid leukemia. Affected mice have an expansion of immature myeloid cells, expressing CD11b and Gr-1 cell surface markers, with monoblastic cellular morphology. This is accompanied by severe anemia and thrombocytopenia, and dissemination of immature hematopoietic cells into the spleen, liver and extramedullary spaces of the bone marrow. We have also explored the potential co-operation between miR-211 and AML oncogenes. Strikingly, preliminary data suggests miR-211 can co-operate with activating RAS mutations to immortalize cells in vitro, and drive AML in vivo .To further explore the molecular mechanisms of miR-211 in AML, we used RNA sequencing and differential expression analysis to compare transcriptional changes induced by miR-211 overexpression in disease mice relative to empty vector controls. We have identified Lin28a as the most downregulated miR-211 gene target. Lin28a is a well-established target of miR-125b in AML and its knockdown leads to an analogous expansion of myeloid cells in vivo as observed in miR-211-dependent AML.Collectively, our results demonstrate a previously unidentified role for miR-211 as an oncogenic microRNA in the development of AML and suggests that miR-211 may potentially drive the development of AML by blocking myeloid differentiation through the repression of Lin28a . DisclosuresNo relevant conflicts of interest to declare.

CircSPI1 acts as an oncogene in acute myeloid leukemia through antagonizing SPI1 and interacting with microRNAs

PU.1 (encoded by SPI1) is essential for myeloid development, and inhibition of its expression and activity can lead to acute myeloid leukemia (AML). The precise regulation of PU.1 expression is crucial for the development of AML, and the discovery of circular RNAs (circRNAs) can add a new layer of information on regulation. Here, we found that circSPI1, the circular RNA derived from the SPI1 gene, is highly expressed in AML but not in normal counterparts. Unlike SPI1, a tumor suppressor and being lowly expressed in AML, we demonstrate that circSPI1 acts as an oncogene, evidenced by the observation that circSPI1 knockdown induces myeloid differentiation and apoptosis of AML cells. We provide mechanistic evidence for multiple regulatory roles of circSPI1 in AML progression. On one hand, circSPI1 contributes to myeloid differentiation of AML cells by interacting with the translation initiation factor eIF4AIII to antagonize PU.1 expression at the translation level. On the other hand, circSPI1 contributes to proliferation and apoptosis by interacting with miR-1307-3p, miR-382-5p, and miR-767-5p; this role is uncoupled with SPI1. Finally, we illustrate the clinical significance of circSPI1 by showing that circSPI1-regulated genes are associated with the clinical outcome of AML patients. Our data provide new insight into the complex SPI1 gene regulation now involving circSPI1.

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BET Inhibition Enhances the Antileukemic Activity of Low-dose Venetoclax in Acute Myeloid Leukemia.

The BCL2 inhibitor, venetoclax, has transformed clinical care in acute myeloid leukemia (AML). However, subsets of patients do not respond or eventually acquire resistance. Venetoclax-based regimens can lead to considerable marrow suppression in some patients. Bromodomain and extraterminal inhibitors (BETi) are potential treatments for AML, as regulators of critical AML oncogenes. We tested the efficacy of novel BET inhibitor INCB054329, and its synergy with venetoclax to reduce AML without induction of hematopoietic toxicity. INCB054329 efficacy was assessed by changes in cell cycle and apoptosis in treated AML cell lines. In vivo efficacy was assessed by tumor reduction in MV-4-11 cell line-derived xenografts. Precision run-on and sequencing (PRO-seq) evaluated effects of INCB054329. Synergy between low-dose BETi and venetoclax was assessed in cell lines and patient samples in vitro and in vivo while efficacy and toxicity was assessed in patient-derived xenograft (PDX) models. INCB054329 induced dose-dependent apoptosis and quiescence in AML cell lines. PRO-seq analysis evaluated the effects of INCB054329 on transcription and confirmed reduced transcriptional elongation of key oncogenes, MYC and BCL2, and genes involved in the cell cycle and metabolism. Combinations of BETi and venetoclax led to reduced cell viability in cell lines and patient samples. Low-dose combinations of INCB054329 and venetoclax in cell line and PDX models reduced AML burden, regardless of the sensitivity to monotherapy without development of toxicity. Our findings suggest low dose combinations of venetoclax and BETi may be more efficacious for patients with AML than either monotherapy, potentially providing a longer, more tolerable dosing regimen.

Targeted Inhibition of the NUP98-NSD1 Fusion Oncogene in Acute Myeloid Leukemia.

Simple SummaryNUP98-NSD1-positive acute myeloid leukemia (AML) frequently shows an additional mutation in Neuroblastoma rat sarcoma (NRAS). However, the synergistic effect of NUP98-NSD1 and NRASG12D in leukemic transformation remained unclear. In addition, NUP98-NSD1 positive AML patients respond poorly to chemotherapy and lack a targeted therapeutic option. Our study aimed to identify the cooperation of NUP98-NSD1 fusion and NRASG12D mutation and to develop a novel therapeutic approach for this AML. We found that NUP98-NSD1 alone can cause leukemia with long latency, and NRASG12D contributes to the aggressiveness of this AML. Additionally, we validated a novel NUP98-NSD1-targeting siRNA/lipid nanoparticle formulation that significantly prolonged the survival of patient-derived xenograft (PDX) mice with NUP98-NSD1-positive AML.NUP98-NSD1-positive acute myeloid leukemia (AML) is a poor prognostic subgroup that is frequently diagnosed in pediatric cytogenetically normal AML. NUP98-NSD1-positive AML often carries additional mutations in genes including FLT3, NRAS, WT1, and MYC. The purpose of our study was to characterize the cooperative potential of the fusion and its associated Neuroblastoma rat sarcoma (NRAS) mutation. By constitutively expressing NUP98-NSD1 and NRASG12D in a syngeneic mouse model and using a patient-derived xenograft (PDX) model from a NUP98-NSD1-positive AML patient, we evaluated the functional role of these genes and tested a novel siRNA formulation that inhibits the oncogenic driver NUP98-NSD1. NUP98-NSD1 transformed murine bone marrow (BM) cells in vitro and induced AML in vivo. While NRASG12D expression was insufficient to transform cells alone, co-expression of NUP98-NSD1 and NRASG12D enhanced the leukemogenicity of NUP98-NSD1. We developed a NUP98-NSD1-targeting siRNA/lipid nanoparticle formulation that significantly prolonged the survival of the PDX mice. Our study demonstrates that mutated NRAS cooperates with NUP98-NSD1 and shows that direct targeting of the fusion can be exploited as a novel treatment strategy in NUP98-NSD1-positive AML patients.

2001 – HUSHING LINE-1 RETROTRANSPOSITION IS AN EPIGENETIC LIABILITY OF MYELOID LEUKEMIAS

Hijacked epigenetic machinery is a hallmark of acute myeloid leukemia (AML), whereas dysregulated epigenetics also creates selective and targetable dependencies. The identification of leukemia-selective dependencies provides new knowledge in AML pathogenesis, resulting in therapeutic strategies targeting several epigenetic regulators. These strategies rely mainly on interfering with the activity of chromatin-interacting domains, highlighting the importance of understanding protein domains in cancer pathobiology. However, a comprehensive survey of epigenetic dependencies of human leukemias by a domain-based functional interrogation has been lacking. We developed a CRISPR screen system using domain-based sgRNAs targeting human chromatin regulators, and identified MPHOSPH8 (or MPP8), a methyl-H3K9-binding component of the HUSH complex, as a selective epigenetic dependency of AML cells. HUSH mediates the epigenetic silencing of LINE-1 (or L1) retrotransposons, the only autonomously mobile DNA elements in humans. While dispensable for normal hematopoiesis, MPP8 KO inhibits AML initiation and maintenance by reactivating L1 retrotransposons. Activation of endogenous or ectopic L1s phenocopied MPP8 loss, whereas blocking L1 retrotransposition abrogated MPP8-deficiency-induced phenotypes. Reactivated L1 retrotransposition increased DNA damage, activated cyclin-dependent kinase inhibitor p21, and induced AML differentiation. Furthermore, expression of AML oncogenes led to suppression of L1s in human and mouse HSPCs. Enhanced L1 suppression in human AML cells is associated with therapy resistance and poor prognosis. Hence, while retrotransposons are historically recognized as sources of genetic instability and somatic mutations to promote cancer development, we uncover an unexpected tumor-suppressive role for L1 retrotransposons as an epigenetic liability in myeloid leukemias.

An Oncogenic Enhancer Encodes Selective Selenium Dependency in AML

Deregulation of transcription is a hallmark of acute myeloid leukemia (AML) that drives oncogenic expression programs and presents opportunities for therapeutic targeting. We hypothesized that by integrating the active enhancer chromatin landscapes with pan-cancer enhancer and genetic dependency mapping, we would be able to identify targetable tumor-specific oncogenic enhancer regulation and gain mechanistic insights into the underlying basis of the regulation. Using this approach, we find that tumor-specific super enhancers statistically demarcate tumor-specific dependency (Fig. A). In addition to several well-known AML oncogenes (e.g. MYB, CDK6, FLI1…), we find SEPHS2 to have highly AML-specific enhancer regulation and genetic dependency (Fig. B). SEPHS2 is a key component of the selenoprotein production pathway and is required for the selenocysteine incorporation during protein translation (Fig. C). Across AML cell lines and primary patient samples, we observe a large AML-specific super enhancer at the SEPHS2 locus (Fig. D) that correlates with SEPHS2 genetic dependency (Fig. E) and includes a prominent binding site for AML transcription factors (Fig. D). We confirmed that the oncogenic transcription factor MYB strongly regulates SEPHS2 expression and that SEPHS2 expression correlates with poor prognosis in AML (not shown in this figure). Selenoproteins play an important role in mediating oxidative stress. We find that SEPHS2 knockout increases oxidative stress and that antioxidant treatment rescues viability effects of SEPHS2 knockout (Fig. F, G). Across murine and human in vivo AML models, genetic perturbation of selenoprotein production pathway genes strongly delays leukemogenesis (one example in Fig. H). Other cell lines (both cancerous and non-cancerous) are minimally affected by SEPHS2 knockout, confirming specificity in AML (Fig. E). As a druggable enzyme SEPHS2 merits strong consideration as a therapeutic target. In the interim, we hypothesized that selenium dietary restriction (Fig. I) may be able to phenocopy selenoprotein pathway inhibition with a lower regulatory burden. We show that mice tolerate selenium-depleted chow with no observable effects on body weight or hematopoiesis (one example in Fig. J). Confirming our hypothesis, AML transplanted into these mice exhibit a strong delay in leukemogenesis (Fig. K). Throughout the cancer biology field, there is broad interest in understanding how best to leverage pan-cancer genetic dependency data. We find that the integration of enhancer data adds another layer of specificity and helps provide mechanistic insight into the underlying basis of oncogenic deregulation and dependency. Our identification of AML-specific enhancer regulation of selenoprotein production - which has been minimally studied in this disease - validates our unbiased approach and points to selenoprotein production as a deregulated and therapeutically-actionable metabolic axis in AML. Figure Disclosures Lin: Syros Pharmaceuticals: Equity Ownership, Patents &amp; Royalties.

Rationale for a Combination Therapy Consisting of MCL1- and MEK-Inhibitors in Acute Myeloid Leukemia.

Amplification and overexpression of the myeloid cell leukemia differentiation protein MCL1 and the murine double minute protein MDM2 have been reported in various human tumors as well as hematological malignancies including acute myeloid leukemia (AML). While MCL1 is an anti-apoptotic member of the BCL-2 family proteins, MDM2 is an important cellular inhibitor of the p53 tumor suppressor. The key oncogene in AML is the FLT3 growth factor receptor gene. FLT3 signaling pathways including the MAPK cascade (RAS-RAF-MEK-ERK) are highly active in AML cells, leading to induced protein translation and cell proliferation as well as reduced apoptosis. Consequently, combined administration of MCL1-, MDM2-, and MEK-inhibitors may present a promising anti-leukemic treatment strategy. Here, we assessed the MCL1-antagonist S63845, the MDM2-inhibitor HDM201, and the MEK1/2-inhibitor trametinib as single agents and in combination in a variety of AML cell lines and mononuclear cells isolated from patients with hematological malignancies centered on myeloid leukemia, some lymphatic leukemia, as well as some lymphomas, for their ability to induce apoptosis and cell death. We observed a considerably varying anti-leukemic efficacy of the MCL1-inhibitor S63845 and the MEK1/2-inhibitor trametinib. Hematological cells with susceptibility to the single compounds as well as to the combined treatment were defined by elevated MCL1- and MEK-protein levels, independent of the mutational status of FLT3 and TP53. Our data indicate that hematological cells with elevated MCL1- and MEK-protein levels are most sensitive to the combined treatment with S63845 and trametinib. MCL1- and MEK1/2-protein expression may be valid biomarkers for treatment response to S63845 and trametinib, respectively.

MicroRNA-21 promotes proliferation in acute myeloid leukemia by targeting Krüppel-like factor 5.

Abnormal expression of microRNA (miR)-21 has been reported in various types of cancers. However, the role and mechanism of miR-21 remain to be elucidated in acute myeloid leukemia (AML). In the present study, it was observed that miR-21 was upregulated and Krüppel-like factor 5 (KLF5) was downregulated in AML cells compared with normal bone marrow cells. Dual luciferase reporter assays revealed that KLF5 was a direct target of miR-21. Indeed, miR-21 overexpression resulted in a downregulation of KLF5 expression, while miR-21 inhibition had the opposite effect in AML cells. In addition, miR-21 overexpression promoted the proliferation of AML cells in vitro. Notably, using a mouse xenograft model, miR-21 overexpression was demonstrated to result in enhanced tumor growth and suppressed KLF5 expression in the xenograft tumors in vivo. In conclusion, the present results indicated that miR-21 promoted proliferation through directly regulating KLF5 expression in AML cells. miR-21 may thus serve as an oncogene in AML, providing a potential target for AML therapy.

Nucleosome Binding Protein HMGN1 Promotes HSPC Competitive Advantage with Decreased Quiescence and Cooperates with AML Oncogenes to Block Differentiation and Increase Clonogenicity

Acute myeloid leukemia (AML) is characterized by rapid growth and differentiation arrest of immature myeloblasts. The AML blast is defined by having “open” chromatin, thus, we hypothesized that alterations of chromatin compaction may promote AML. The nucleosome binding protein HMGN1 is known to decompact chromatin and is located on chr21q22 in one of the most recurrently amplified regions in AML. We previously presented in vitro data showing that HMGN1 overexpression delayed myeloid differentiation in association with an increase in global levels of Histone H3K27 acetylation downstream of the histone acetyltransferases (HATs) CBP and p300. The goal of this study was to define the effect of HMGN1 overexpression on hematopoiesis in vivo and to identify leukemia oncogenes that cooperate with HMGN1 in primary hematopoietic cells.We analyzed native WT and OE-HMGN1 (transgenic overexpressing HMGN1) hematopoiesis at steady state in young and older mice. OE-HMGN1 transgenic mice had a significant increase in lineage-negative cells with age (Fig A, *p=0.02). We next performed competitive bone marrow transplantation with CD45.1 (WT) vs CD45.2 (WT or OE-HMGN1) donors measuring the relative contribution of CD45.1 and 45.2 cells to hematopoiesis over time after 3 consecutive serial transplants, each at 16 weeks. OE-HMGN1 stem and progenitor cells (HSPCs) had a progressive clonal advantage during serial transplantation (Fig B, *p<0.0002). This clonal advantage was observed across multiple HSPC populations and in all mature lineages. Lin-Sca1+Kit+ (LSK) and Lin-Kit+ (LK) HSPCs were sorted 4 months after transplantation for cell cycle analysis, ex vivo differentiation assays, and RNA-sequencing. OE-HMGN1 HSPCs were less quiescent in vivo, with fewer cells in G0 (Fig C, LK p= 0.0007, LSK p=0.01), and had a relative defect in differentiation capacity in vitro compared to WT. Gene set enrichment analysis was consistent with these phenotypes, showing enrichment of senescence and cell cycle regulatory gene expression programs in WT compared to OE-HMGN1 HSPCs.HMGN1 overexpression alone did not cause leukemia in mice. To screen for synergy between HMGN1 and AML oncogenes, we immortalized myeloid progenitors from WT or OE-HMGN1 mice with a HoxB8 transgene and measured growth properties after transduction with retroviruses expressing one of several AML-associated fusion proteins (BCR-ABL, MLL-AF9, PML-RAR, AML-ETO9a, MOZ-TIF2) or mutated oncogenes (FLT3, NPM1, NRAS, GATA2) with GFP. AML-ETO9a (AE9a) and MOZ-TIF2 had the most significant cooperation with HMGN1, most notably by enhancing their ability to block myeloid differentiation in vitro. This is of interest because, like we found for HMGN1, AE9a and MT2 are known promote leukemia via chromatin effects dependent on CBP/p300 and/or acetyltransferase activity. In confirmatory experiments, combination of AE9a and HMGN1 exacerbated the delay in myeloid differentiation induced by either gene alone (p<0.05), and HMGN1 enhanced serial colony formation capacity of AE9a in semi-solid media (p<0.05).In vivo transplantation of WT or OE-HMGN1 c-Kit+ HSPCs transduced with AE9a led to development of AML ~110 days after injection in both groups. However, subpopulation analysis of AE9a-expressing leukemias showed that LSK and MPP-like populations were expanded in the context of OE-HMGN1 compared to WT (p<0.05). Remarkably, global H3K27ac levels were significantly higher in LT-HSC-like HMGN1-OE AE9a leukemia cells using single cell subpopulation analysis by intracellular flow cytometry compared to WT (Fig D p=0.008), similar to what we observed in HSPCs after competitive transplant. Ex vivo clonogenic capacity was increased in AE9a leukemias that overexpressed HMGN1 in an LTC-IC assay (Fig E *p<0.05). Limiting dilution secondary transplants are ongoing to test if these differences correlate with increased leukemia-initiating capacity per cell in vivo in leukemias that overexpress HMGN1.Our study suggests that overexpression of HMGN1 provides a clonal advantage to hematopoietic populations including long-term stem cells. HMGN1 also cooperates with specific AML oncogenes, possibly those associated with H3K27 acetylation/HAT activity, to enhance leukemic phenotypes. Further study is needed to determine if HMGN1 overexpression confers specific therapeutic vulnerabilities, such as to HAT inhibitors or other chromatin-targeting agents. [Display omitted] DisclosuresLane:Stemline Therapeutics: Research Funding; N-of-one: Consultancy.

The HACE1-NRF2 Axis a Novel Target in Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is a genetically heterogenous disease characterized by clonal expansion of immature myeloid progenitors cells in the bone marrow (BM). Despite this genetic heterogeneity, AML patients share Leukemia associated oncogenes such as NF-E2-related factor 2 (Nrf2) (Rushworth SA et al.). NRF2 is a transcription factor that activates genes with antioxidant response elements (ARE)-containing promoters and protects cancer cells from apoptosis. Inhibition of NRF2 or antioxidant defense increases the level of Radical Oxygen Species (ROS), leading to tumor supression (Chio IIC et al.). Recently, the E3 Ubiquitin-Protein Ligase HACE1, a tumor suppressor in solid tumors, was demonstrated to promote the expression of NRF2 in Huntigton disease (Rotblat B et al.). Thus, we hypothesized a role for HACE1 as an oncogenic factor acting through NRF2 activation in myeloid malignancies and provide first data supporting the HACE1-NRF2 axis to be a novel target in acute myeloid leukemias.Material and methodsThe mRNA expression data from AML patients (296 samples) vs normal Hematopoietic Stem Cells (HSC) (6 samples) were exported from the bloodSpot database. HACE1 mRNA and protein expression was measured by q-RT-PCR and western blot in 12 commercially available Myeloid Malignancies cell lines. The HACE1 inducible knock down (KD) was carried out by Sleeping Beauty Transposon system in U937 and NOMO-1 cell lines. The cell viability was analyzed by Cell Titer Glo Luminescent assay. Apoptosis was measured by Annexin V (AV)/Propidium Iodide (PI) assay.Results and discussionHACE1 mRNA is downregulated in AML patients compared to HSC (***p<0.001, Bloodspot database). However mRNA and HACE1 protein expression do not correlate in AML cell lines, suggesting post translational modifications. High HACE1 protein expression was observed in most AML cell lines. HACE1 KD reduced drastically the cell viability of U937 cells through caspase activation and NRF2 degradation. However, no effect on cell viability was observed in NOMO-1 cells. Recently, non-programmed cell death necroptosis induction has been described by TNFR1 activation in HACE1 knock out Mouse Embrionic Fibroblast cells (Tortola L et al.). In line with this study, we observed that TNF induces strong cell death in HACE1 KD NOMO-1 cells within 48 hours.In addition HACE1 KD promotes autophagy through p62 degradation (late autophagy marker) in U937 cells. Autophagy has recently been described to contribute to the differentiation and death of AML cells, and to the promotion of immunostimulatory signals activating immune responses against cancer cells (Chen L et al.; Pietrocola F et al.). Thus HACE1 might be a potential target to induce autophagy, providing a novel therapeutical target in the treatment of myeloid malignancies.Finally, HACE1 KD in our hands promoted sensitization of U937 and NOMO-1 cells to cytatarabine, the backbone therapy in AML patients. This treatment promotes HACE1 protein expression at 24 and 48 hours in NOMO-1 cells, which may explain the better response rates of HACE1 KD cells to cytarabine.Taken together, we provide first evidence of HACE1 being a novel oncogene in AML and that the HACE1-NRF2 axis is a promising target in the treatment of Acute Myeloid Leukemias. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

ALKBH5 Functions As an Oncogene in Acute Myeloid Leukemia

N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic messenger RNAs (mRNAs) has been shown to play important roles in diverse cellular and pathological processes (Deng X, et al. Cell Res. 2018;28:507-517). ALKBH5, recently identified as a m6A demethylase, was reported to promote tumorigenesis and proliferation in glioblastoma stem-like cells (GSCs) (Zhang, S. et al. Cancer Cell. 2017;31:591-606) and breast cancer stem cells (BCSCs) (Zhang, C et al. PNAS. 2016;113: E2047-E2056). While ALKBH5 is well-recognized to function as an oncogene in solid tumors, it was reported that shallow/deep deletion of ALKBH5 is associated poor prognosis in patients with acute myeloid leukemia (AML), and is frequently co-existing with TP53 mutation (Kwok, C. T et al. J Hematol Oncol. 2017; 10(1): 39), implying that ALKBH5 may function as a tumor suppressor in AML. Thus, a systematic investigation of the definitive role of ALKBH5 in AML is warranted.To this end, we performed series of in vitro and in vivo experiments to determine the function of ALKBH5 in AML. For the in vitro experiments, we used three lentiviral shRNAs (shALKBH5-A, shALKBH5-D and shALKBH5-E) to deplete ALKBH5 expression in three human AML cell lines with different TP53 mutation status: NOMO-1 (TP53-mutant), MV4;11 (TP53-WT) and MA9.3 cells (TP53-WT). Somewhat surprisingly, ALKBH5 depletion significantly (p<0.05) inhibited AML cell proliferation/growth in all three AML cells lines, regardless of the status of TP53 mutation. We next conducted colony forming assays and found that ALKBH5 knockdown significantly (p<0.01) impaired the colony forming ability to 18% ~45% of the control group level in all three AML cell lines. We further showed that ALKBH5 depletion caused a significant increase in apoptosis (with a 1.5 ~ 4 fold increase; p<0.001) in all three AML cell lines, which is consistent with the previous report that knockout of ALKBH5 caused severe apoptosis of mouse testis cells (Zheng G et al. Mol Cell. 2013; 49:18-29). In contrast, ALKBH5 knockdown did not significantly affect cell cycles.To further confirm ALKBH5's role in AML development in vivo. We utilized Xenografted AML model as well as mouse bone marrow transplantation (BMT) model. Consistent with the in vitro results, we found that NSGS mice xeno-transplanted with MV4;11-ALKBH5-knockdown cells survived significantly longer than those with MV4;11 control cells (p<0.001). Moreover, we have also conducted mouse bone marrow transplantation (BMT) assays with MLL-AF9-transduced mouse bone marrow lineage negative (Lin-) progenitor cells collected from mice carrying Alkbh5 wild-type (Alkbh5+/+), or heterozygous (Alkbh5+/-) or homozygous (Alkbh5-/-) deletion. Consistent with the xeno-transplanted mouse model results, our BMT assays also showed that Alkbh5 depletion significantly inhibited leukemogenesis and prolonged survival in BMT recipient mice (median survival of ALKBH5wt/wt +MA9 vs. ALKBH5+/- +MA9 or ALKBH5-/- +MA9: 32 days vs. 64 days or 68 days; p<0.005).Taken together, our in vitro and in vivo functional studies data indicate ALKBH5 also functions as an oncogene in AML regardless of TP53 mutation status, similar to its role in solid tumors. We are currently conducting as series of studies to reveal the molecular mechanism(s) underlying the oncogenic role of ALKBH5 in AML. DisclosuresNo relevant conflicts of interest to declare.