Therapeutic Target In Acute Myeloid Leukemia Research Articles

IL2RA Promotes Aggressiveness and Stem Cell–Related Properties of Acute Myeloid Leukemia

Overexpression of IL2RA, which encodes the alpha chain of the IL2 receptor, is associated with chemotherapy resistance and poor outcome in acute myeloid leukemia (AML). The clinical potential of anti-IL2RA therapy is, therefore, being explored in early-stage clinical trials. Notwithstanding, only very limited information regarding the biological function of IL2RA in AML is available. Using genetic manipulation of IL2RA expression as well as antibody-mediated inhibition of IL2RA in human cell lines, mouse models, and primary patient samples, we investigated the effects of IL2RA on AML cell proliferation and apoptosis, and on pertinent signaling pathways. The impact of IL2RA on the properties of leukemic stem cells (LSC) and on leukemogenesis were queried. IL2RA promoted proliferation and cell-cycle activity and inhibited apoptosis in human AML cell lines and primary cells. These phenotypes were accompanied by corresponding alterations in cell-cycle machinery and in pathways associated with cell survival and apoptosis. The biological roles of IL2RA were confirmed in two genetically distinct AML mouse models, revealing that IL2RA inhibits differentiation, promotes stem cell-related properties, and is required for leukemogenesis. IL2RA antibodies inhibited leukemic, but not normal, hematopoietic cells and synergized with other antileukemic agents in this regard. Collectively, these data show for the first time that IL2RA plays key biological roles in AML and underscore its value as a potential therapeutic target in this disease. SIGNIFICANCE: This study identifies IL2RA as a potential therapeutic target in AML, where it is shown to regulate proliferation, differentiation, apoptosis, stem cell-related properties, and leukemogenesis.

CircRAD18 Accelerates the Progression of Acute Myeloid Leukemia by Modulation of miR-206/PRKACB Axis.

BackgroundCircular RNAs (circRNAs) play a crucial role in tumorigenesis. However, the effects of circRNAs on acute myeloid leukemia (AML) remain largely unexplored. We explored the function of circRAD18 in AML development.MethodsQRT-PCR was performed for the levels of circRAD18, RAD18, microRNA-206 (miR-206) and protein kinase CAMP-activated catalytic subunit beta (PRKACB). Cell Counting Kit-8 (CCK-8) assay and colony formation assay were utilized for cell proliferation. Flow cytometry analysis was carried out to analyze cell apoptosis and cell cycle process. Transwell assay was manipulated for cell migration and invasion. Western blot assay was conducted for protein levels. Dual-luciferase reporter assay was adopted to verify the interaction between miR-206 and circRAD18 or PRKACB.ResultsCircRAD18 level was increased in AML patients’ blood specimens and AML cell lines compared to normal controls. CircRAD18 knockdown impeded the proliferation, migration and invasion and facilitated the apoptosis and cell cycle arrest in AML cells. Moreover, circRAD18 was identified as a sponge for miR-206, and circRAD18 knockdown-mediated effect on AML cell progression was reversed by miR-206 suppression. Additionally, PRKACB was the target gene of miR-206. MiR-206 overexpression suppressed the malignant behaviors of AML cells, while PRKACB elevation restored the effects.ConclusionCircRAD18 aggravated the malignancy of AML cells through reducing miR-206 expression and elevating PRKACB expression, indicating circRAD18 might be a therapeutic target for AML.

Clinical significance of dysregulation of miR-381 in pediatric acute myeloid leukemia

BackgroundmicroRNA-381 is dysregulated in a variety of cancers. However, its clinical significance in pediatric acute myeloid leukemia (AML) is still unclear. The purpose of this study was to detect the expression level of miR-381 in pediatric AML patients and to explore its potential clinical significance.MethodsThe levels of miR-381 in bone marrow and serum of 102 pediatric AML patients were measured by quantitative real-time polymorperase chain reaction (qRT-PCR). The diagnostic value of serum miR-381 in pediatric AML patients was evaluated by the receiver operating characteristic (ROC) curve. A Chi square test was used to analyze the relationship between serum miR-381 and the clinical characteristics of patients. Cox regression analysis and Kaplan–Meier evaluated the prognostic value of serum miR-381 in patients. Finally, the proliferation of the cells was analyzed by the CCK-8 assay.ResultsCompared with healthy controls, the levels of miR-381 in serum and bone marrow of pediatric AML patients were significantly decreased (P < 0.001). ROC curve showed that miR-381 could distinguish pediatric AML cases from normal controls. At the same time, the downregulation of miR-381 was associated with M7 in the French–American–British (FAB) classifications and unfavorable cytogenetic risks (P < 0.05). Low serum miR-381 levels were associated with poor overall survival of pediatric AML (log-rank test, P = 0.011) and poor relapse-free survival (log-rank test, P = 0.004). Cox regression analysis confirmed that reduced serum miR-381 was an independent predictor of poor prognosis in AML (HR = 3.794, 95% CI 1.3633–10.559, P = 0.011). In addition, low expression of miR-381 significantly reduced the proliferation of cells (P < 0.05).ConclusionAll experimental results confirm that miR-381 has reduced bone marrow and serum expression in pediatric AML, and low levels of serum miR-381 have certain diagnostic and prognostic value in pediatric AML and may be a potential therapeutic target for AML.

Abstract 3652: EED or BRD4 inhibition as a novel therapeutic strategy in acute myeloid leukemia with monosomy 7

Abstract Backgrounds: Monosomy of chromosome 7 (monosomy 7) is observed in about 9% of acute myeloid leukemia (AML) patients and related to poor prognosis. It is required to establish an effective treatment strategy based on the specific molecular pathogenesis associated with the chromosomal abnormality. Methods: Chromosome 7 encodes multiple genes regulating histone methylation. We hypothesized that haploinsufficiency of the epigenetic modifiers in the chromosome 7 is compensated by other epigenetic molecules in different chromosomes, and the AML cells with monosomy 7 are vulnerable to inhibition of those molecules. Based on this notion, we performed an RNA-interference (RNAi)-based screening by targeting 53 genes encoding epigenetic enzymes regulating histone methylation by using AML cell lines with monosomy 7 and non-monosomy 7. Results: We found that the siRNA-mediated knockdown of EED, or BRD4 significantly decreased cell viability in AML cells with monosomy 7 compared to the control leukemia cell lines. Stable knockdown of EED markedly attenuated proliferation as well as increased cell apoptosis specifically in leukemia cells with monosomy 7. Interestingly, PRC1 rather than PRC2 activity of EED had a predominant role in the survival of monosomy 7 leukemia cells. We then explored whether the deletion of specific genes within the chromosome 7 was responsible for increased sensitivity to EED inhibition. We found that knockdown of GTF2I increased the sensitivity to EED inhibition via increased cell apoptosis. Because both GTF2I and EED suppressed expression levels of BCL2, it is possible that EED inhibition additively abrogate BCL2 activity in AML with monosomy 7. In addition to EED, we identified BRD4 as another essential survival factor in AML with monosomy 7. Although inhibition of BET bromodomain proteins have already shown to be effective for a variety of AML, we showed that blockade of bromodomain proteins preferentially affected cellular proliferation of AML cells with monosomy 7. Mechanistically, we found that knockout of MLL3 or MLL5, both encoded in the chromosome 7, resulted in increased vulnerability to JQ-1 treatment. In monosomy 7, functional loss of MLL3 and MLL5 showed reduced expression levels of c-MYC and CCND1. JQ-1 treatment additively impaired the functions of these genes, and strongly inhibited cell proliferation of AML cells with monosomy 7. Conclusion: Our synthetic-lethality based approach identified EED and BRD4 as novel promising therapeutic targets in AML with monosomy 7. Citation Format: Kensuke Matsuda, Yuki Kagoya, Hideaki Mizuno, Sho Yamazaki, Masashi Miyauchi, Mineo Kurokawa. EED or BRD4 inhibition as a novel therapeutic strategy in acute myeloid leukemia with monosomy 7 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3652.

The role of mitochondrial proteases in leukemic cells and leukemic stem cells.

The biological function of most mitochondrial proteases has not been well characterized. Moreover, most of the available information on the normal function of these proteases has been derived from studies in model organisms. Recently, the mitochondrial proteases caseinolytic protease P (CLPP) and neurolysin (NLN) have been identified as therapeutic targets in acute myeloid leukemia (AML). Both proteases are overexpressed in approximately 40% of AML patients. Mechanistically, CLPP and NLN maintain the integrity of the mitochondrial respiratory chain: CLPP cleaves defective respiratory chain proteins, while NLN promotes the formation of respiratory chain supercomplexes. In this review, we highlight the functional consequences of inhibiting and activating mitochondrial proteases and discuss their potential as therapeutic targets in AML.

SRC-Family Kinases in Acute Myeloid Leukaemia and Mastocytosis.

Protein tyrosine kinases have been recognized as important actors of cell transformation and cancer progression, since their discovery as products of viral oncogenes. SRC-family kinases (SFKs) play crucial roles in normal hematopoiesis. Not surprisingly, they are hyperactivated and are essential for membrane receptor downstream signaling in hematological malignancies such as acute myeloid leukemia (AML) and mastocytosis. The precise roles of SFKs are difficult to delineate due to the number of substrates, the functional redundancy among members, and the use of tools that are not selective. Yet, a large num ber of studies have accumulated evidence to support that SFKs are rational therapeutic targets in AML and mastocytosis. These two pathologies are regulated by two related receptor tyrosine kinases, which are well known in the field of hematology: FLT3 and KIT. FLT3 is one of the most frequently mutated genes in AML, while KIT oncogenic mutations occur in 80–90% of mastocytosis. Studies on oncogenic FLT3 and KIT signaling have shed light on specific roles for members of the SFK family. This review highlights the central roles of SFKs in AML and mastocytosis, and their interconnection with FLT3 and KIT oncoproteins.

HSPG2 overexpression independently predicts poor survival in patients with acute myeloid leukemia

Heparan sulfate proteoglycan 2 (HSPG2), also known as perlecan, is a large multi-domain extracellular matrix proteoglycan, which contributes to the invasion, metastasis and angiogenesis of solid tumor. However, very little is known about the effect of HSPG2 on acute myeloid leukemia (AML). This study aims to investigate the prognostic value of the HSPG2 gene in terms of overall survival and leukemia-free survival in patients with AML. Bone marrow mononuclear cells (BMMCs) from 4 AML patients and 3 healthy controls were processed for RNA-Sequencing (RNA-seq). The mRNA expression level of HSPG2 in BMMCs and CD34+ hematopoietic stem/progenitor cells (HSPC) obtained from enrolled participants and human leukemic cell lines was detected by RT-qPCR. Then the correlations between the expression of HSPG2 and a variety of important clinical parameters, such as median white blood cell (WBC) count and bone marrow (BM) blasts, were further analyzed. The expression level of HSPG2 was significantly upregulated in AML patients at the time of diagnosis, downregulated after complete remission and then elevated again at relapse. Moreover, HSPG2 expression was associated with median WBC count (P < 0.001), median hemoglobin (P = 0.02), median platelet count (P = 0.001), and BM blasts (P < 0.001) in AML patients. Patients with high HSPG2 expression had both worse overall survival (OS) (P = 0.001) and poorer leukemia-free survival (LFS) (P = 0.047). In the multivariate analysis model, HSPG2 was identified as an independent prognostic biomarker of AML. Taken together, these results indicate that HSPG2 overexpression was associated with poor prognosis in AML patients, and may be a prognostic biomarker and therapeutic target of AML.

Apolipoprotein C2 - CD36 Promotes Leukemia Growth and Presents a Targetable Axis in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a devastating hematologic malignancy that affects the hematopoietic stem cells. The 5-year overall survival (OS) of patients with AML is less than 30%, highlighting the urgent need to identify new therapeutic targets. Here, we analyze gene expression datasets for genes that are differentially overexpressed in AML cells compared with healthy hematopoietic cells. We report that apolipoprotein C2 (APOC2) mRNA is significantly overexpressed in AML, particularly in patients with mixed-lineage leukemia rearrangements. By multivariate analysis, high APOC2 expression in leukemia blasts is significantly associated with decreased OS (HR: 2.51; 95% CI, 1.03-6.07; P = 0.04). APOC2 is a small secreted apolipoprotein that constitutes chylomicrons, very-low-density lipoproteins, and high-density lipoproteins with other apolipoproteins. APOC2 activates lipoprotein lipase and contributes to lipid metabolism. By gain and loss of function approaches in cultured AML cells, we demonstrate that APOC2 promotes leukemia growth via CD36-mediated LYN-ERK signaling activation. Knockdown or pharmacological inhibition of either APOC2 or CD36 reduces cell proliferation, induces apoptosis in vitro, and delays leukemia progression in mice. Altogether, this study establishes APOC2-CD36 axis as a potential therapeutic target in AML.

CD45dimCD34+CD38\u2212CD133+ cells have the potential as leukemic stem cells in acute myeloid leukemia

BackgroundLeukemia stem cells (LSCs) in play an important role in the initiation, relapse, and progression of acute myeloid leukemia (AML), and in the development of chemotherapeutic drug resistance in AML. Studies regarding the detection of LSCs and the development of novel therapies for targeting them are extensive. The identification of LSCs and targeting therapies for them has been continuously under investigation.MethodsWe examined the levels of CD45dimCD34+CD38−CD133+ cells in bone marrow samples from patients with hematological malignancies and healthy controls, using four-color flow cytometry.ResultsInterestingly, the CD45dimCD34+CD38−CD133+ cells were highly expressed in the bone marrow of patients with AML compared to that in healthy controls (HC). Moreover, the proportions of CD45dimCD34+CD38−CD133+ cells were also examined in diverse hematological malignancies, including AML, CML, DLBCL, MM, MDS, HL, ALL, and CLL. LSCs were prominently detected in the BMCs isolated from patients with AML and CML, but rarely in BMCs isolated from patients with DLBCL, MM, MDS, ALL, CLL, and HL. Additionally, the high CD45dimCD34+CD38−CD133+ cell counts in AML patients served as a significantly poor risk factor for overall and event free survival.ConclusionsTherefore, our results suggest that CD45dimCD34+CD38−CD133+ cells in AML might potentially serve as LSCs. In addition, this cell population might represent a novel therapeutic target in AML.

The role of the novel LincRNA uc002jit.1 in NF-kB-mediated DNA damage repair in acute myeloid leukemia cells

The roles and therapeutic potential of long noncoding RNAs (lncRNAs) in acute myeloid leukemia (AML) have attracted increased attention. However, many lncRNAs have not been annotated in AML, and their predictive value for AML therapy remains unclear. In this study, we identified a novel large intergenic noncoding RNA uc002jit.1 (D43770) from a lncRNA microarray. We first proved uc002jit.1 is a target gene of nuclear factor kappa B/RELA, RELA regulated uc002jit.1 transcription by binding to its promoter. Additionally, uc002jit.1 knockdown impaired the stability of poly (ADP-ribose) polymerase 1 (PARP1) mRNA, and then reduced PARP1 protein content and PARylation level upon DNA damage, thus inhibiting DNA damage repair in AML cells. Moreover, uc002jit.1 knockdown significantly inhibited AML cells proliferation and increased the sensitivity to chemotherapeutic drugs in vitro as well as in a mouse model in vivo. Overall, our study indicated that uc002jit.1 may be associated with the occurrence and prognosis of AML and could be a new diagnostic/prognostic biomarker and therapeutic target for AML.

Clinical Significance of CD200 and CD56 Expression in Patients with Acute Myeloid Leukemia.

Background:Acute myeloid leukemia (AML) escape from immunosurveillance by immunosuppression. CD200 and CD56 expression represented an independent prognostic factor in many hematological malignancies but its importance in AML patients remains to be identified. Methods: CD200 and CD56 expression were assessed in the bone marrow blasts for Fifty-two (52) newly diagnosed AML by flowcytometry before start of therapy. Results: CD200 + expression was reported in 28.8% of patients while 17.3% of patients showed CD56+ expression. M4 FAB revealed high frequency of both CD200+ and CD56+ expression. The overall survival of CD200+ patients was 19.2% compared to 35.3% in CD200- (P= 0.049). On the other hand, CD56+ patients had the lowest complete remission rate (22.2% vs. 53.4%). In addition, CD56+ population had significant bad influence on overall survival than those of CD56- population (11.1 % vs. 35.5 %, P= 0.047). Conclusions: CD200 and CD56 positive expression by myeloblasts at diagnosis denote poor prognostic indicator and correlated with poor cytogenetic findings. CD200 could be used as therapeutic target in AML.

Acidic leucine-rich nuclear phosphoprotein-32A expression contributes to adverse outcome in acute myeloid leukemia

BackgroundAcidic leucine-rich nuclear phosphoprotein-32A (ANP32A) is a novel regulator of histone H3 acetylation and promotes leukemogenesis in acute myeloid leukemia (AML). However, its prognostic value in AML remains unclear.MethodsIn this study, we evaluated the prognostic significance of ANP32A expression using two independent large cohorts of cytogenetically normal AML (CN-AML) patients. Multivariable analysis in CN-AML group was also presented. Based on the ANP32A expression, its related genes, dysregulation of pathways, interaction network analysis between microRNAs and target genes, as well as methylation analysis were performed to unveil the complex functions behind ANP32A.ResultsHere we demonstrated overexpression of ANP32A was notably associated with unfavorable outcome in two independent cohorts of CN-AML patients (OS: P=0.012, EFS: P=0.005, n=185; OS: P=0.041, n=232), as well as in European Leukemia Net (ELN) Intermediate-I group (OS: P=0.018, EFS: P=0.045, n=115), National Comprehensive Cancer Network (NCCN) Intermediate Risk AML group (OS: P=0.048, EFS: P=0.039, n=225), and non-M3 AML group (OS: P=0.034, EFS: P=0.011, n=435). Multivariable analysis further validated ANP32A as a high-risk factor in CN-AML group. Multi-omics analysis presented overexpression of ANP32A was associated with aberrant expression of oncogenes and tumor suppressor, up/down-regulation of metabolic and immune-related pathways, dysregulation of microRNAs, and hypomethylation on CpG island and 1st Exon regions.ConclusionsWe proved ANP32A as a novel, potential unfavorable prognosticator and therapeutic target for AML.

EGFL7 Antagonizes NOTCH Signaling and Represents a Novel Therapeutic Target in Acute Myeloid Leukemia.

EGF-like domain 7 (EGFL7) is a secreted protein and recently has been shown to play an important role in acute myeloid leukemia (AML); however, the underlying mechanism by which EGFL7 promotes leukemogenesis is largely unknown. Using an antibody interaction array, we measured the ability of EGFL7 to bind directly approximately 400 proteins expressed by primary AML blasts. Primary patient samples were stimulated in vitro with recombinant EGFL7 (rEGFL7) or anti-EGFL7 blocking antibody to assess alterations in downstream signaling and the ability to effect blast differentiation and survival. We treated three independent AML models with anti-EGFL7 or IgG1 control to determine whether anti-EGFL7 could prolong survival in vivo. We found EGFL7 significantly binds several signaling proteins important for normal and malignant hematopoiesis including NOTCH. Stimulation of AML blasts with rEGFL7 reduced NOTCH intracellular domain and NOTCH target gene expression while treatment with an anti-EGFL7 blocking antibody resulted in reactivation of NOTCH signaling, increased differentiation, and apoptosis. Competitive ligand-binding assays showed rEGFL7 inhibits DELTA-like (DLL) 4-mediated NOTCH activation while anti-EGFL7 combined with DLL4 significantly increased NOTCH activation and induced apoptosis. Using three different AML mouse models, we demonstrated that in vivo treatment with anti-EGFL7 alone results in increased survival. Our data demonstrate that EGFL7 contributes to NOTCH silencing in AML by antagonizing canonical NOTCH ligand binding. Reactivation of NOTCH signaling in vivo using anti-EGFL7 results in prolonged survival of leukemic mice, supporting the use of EGFL7 as a novel therapeutic target in AML.

Oncogenic Long Noncoding RNA DARS-AS1 in Childhood Acute Myeloid Leukemia by Binding to microRNA-425.

Objective:Acute myeloid leukemia (AML) represents a hematological cancer. The aim of the investigation was to probe the regulatory relevance of long non-coding RNA (lncRNA) aspartyl-tRNA synthetase anti-sense 1 (DARS-AS1)/microRNA-425 (miR-425)/transforming growth factor-beta 1 (TGFB1) to the development of AML.Methods:The DARS-AS1 expression in bone marrow tissues was first analyzed in healthy subjects and AML patients. Subsequently, AML cell lines with DARS-AS1 knockdown were constructed, followed by cell proliferation and apoptosis assays. Afterward, downstream miRNA of DARS-AS1 and target mRNA of the miRNA were analyzed by bioinformatics, and their binding relationships were verified. Functional rescue experiments were then implemented. Finally, activation of the Smad2/3 signaling in MV4-11 and BF-24 cells were detected by western blot.Results:DARS-AS1 was overexpressed in bone marrow tissues of AML patients and cells, and DARS-AS1 knockdown suppressed the proliferation of AML cells and induced apoptosis. DARS-AS1 bound to and negatively correlated with miR-425. Further results suggested that TGFB1 might be a target gene of miR-425 and could promote Smad2/3 phosphorylation and nuclear translocation. Finally, DARS-AS1 depletion could diminish the tumor volume in vivo.Conclusion:All in all, we highlighted here that DARS-AS1 enhanced the expression of TGFB1 through binding to miR-425 to modulate AML progression via the Smad2/3 pathway, which might perform as a therapeutic target for AML.

HBO1 is required for the maintenance of leukaemia stem cells.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNApolymeraseII to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.

Notch Signaling Molecules as Prognostic Biomarkers for Acute Myeloid Leukemia.

The role of Notch signaling in acute myeloid leukemia (AML) is still under investigation. We have previously shown that high levels of Notch receptors and ligands could interfere with drug response. In this study, the protein expression of 79 AML blast samples collected from newly diagnosed patients was examined through flow cytometry. Gamma-secretase inhibitors were used in AML mouse xenograft models to evaluate the contribution of Notch pharmacological inhibition to mouse survival. We used univariate analysis for testing the correlation and/or association between protein expression and well-known prognostics markers. All the four receptors (Notch1–4) and some ligands (Jagged2, DLL-3) were highly expressed in less mature subtypes (M0–M1). Notch3, Notch4, and Jagged2 were overexpressed in an adverse cytogenetic risk group compared to good cytogenetic risk patients. Chi-square analysis revealed a positive association between the complete remission rate after induction therapy and weak expression of Notch2 and Notch3. We also found an association between low levels of Notch4 and Jagged2 and three-year remission following allogeneic stem cell transplantation (HSCT). Accordingly, Kaplan–Meier analysis showed improved OS for patients lacking significant expression of Notch4, Jagged2, and DLL3. In vivo experiments in an AML mouse model highlighted both improved survival and a significant reduction of leukemia cell burden in the bone marrow of mice treated with the combination of Notch pan-inhibitors (GSIs) plus chemotherapy (Ara-C). Our results suggest that Notch can be useful as a prognostic marker and therapeutic target in AML.

Side-by-side comparison of BH3-mimetics identifies MCL-1 as a key therapeutic target in AML

Despite advances in the treatment of acute myeloid leukemia (AML), prognosis of AML patients is still dismal and better treatment options are required. B-cell Lymphoma 2 (BCL-2) homology domain 3 (BH3)-mimetics are emerging as a novel class of apoptosis-inducing agents that are currently being tested for the treatment of different hematological malignancies including AML. Particularly, the selective BCL-2 inhibitor ABT-199/Venetoclax is demonstrating clinical responses and has recently been approved in combination for the treatment of AML. Compounds targeting the related protein MCL-1 have recently entered clinical trials, highlighting the urgency to compare the different BH3-mimetics and identify the most promising antiapoptotic target in AML. We performed a side-by-side comparison of different highly selective and potent BH3-mimetics targeting BCL-2 (ABT-199), MCL-1 (S63845) or BCL-xL (A1331852) in a panel of AML cell lines and primary patient cells. Gene knockdown using siRNAs was utilized to investigate the functional relevance of BCL-2 proteins. Western blotting and immunoprecipitations were used to explore the influence of BH3-mimetics on interactions between pro- and antiapoptotic BCL-2 proteins. A1331852 induced apoptosis only in selected cases, indicating that BCL-xL is not a very promising therapeutic target in AML. However, S63845 displayed higher potency than ABT-199, with more cell lines and primary cells responding to S63845 than to ABT-199. MCL-1 dependency in AML cells was confirmed by siRNA-mediated knockdown of MCL-1, which was sufficient to induce apoptosis. S63845-induced cell death was accompanied by a displacement of the BH3-only protein BIM as well as BAK, resulting in BAK-dependent apoptosis. In contrast, ABT-199-induced cell death was mediated by BAX rather than BAK, indicating distinct non-redundant molecular functions of BCL-2 and MCL-1 in AML. Our study reveals that MCL-1 may be a more prevalent therapeutic target than BCL-2 in AML and identifies BIM and BAK as important mediators of S63845-induced apoptosis in AML.

The Role of Reactive Oxygen Species in Acute Myeloid Leukaemia.

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with a poor overall survival. Reactive oxygen species (ROS) have been shown to be elevated in a wide range of cancers including AML. Whilst previously thought to be mere by-products of cellular metabolism, it is now clear that ROS modulate the function of signalling proteins through oxidation of critical cysteine residues. In this way, ROS have been shown to regulate normal haematopoiesis as well as promote leukaemogenesis in AML. In addition, ROS promote genomic instability by damaging DNA, which promotes chemotherapy resistance. The source of ROS in AML appears to be derived from members of the “NOX family” of NADPH oxidases. Most studies link NOX-derived ROS to activating mutations in the Fms-like tyrosine kinase 3 (FLT3) and Ras-related C3 botulinum toxin substrate (Ras). Targeting ROS through either ROS induction or ROS inhibition provides a novel therapeutic target in AML. In this review, we summarise the role of ROS in normal haematopoiesis and in AML. We also explore the current treatments that modulate ROS levels in AML and discuss emerging drug targets based on pre-clinical work.

Synthetic Lethality-Based Approach Identified EED and BRD4 As Critical Survival Factorsin Acte Myeloid Leukemia with Monosomy 7

Backgrounds: Monosomy of chromosome 7 (monosomy 7) is observed in about 9% of acute myeloid leukemia (AML) patients and related to resistance to conventional chemotherapy and poor prognosis. It is required to establish an effective treatment strategy based on the specific molecular pathogenesis associated with the chromosomal abnormality. In this study, we aimed to find a novel therapeutic target in AML with monosomy 7. Methods and Results: Chromosome 7 encodes multiple genes regulating histone methylation such as EZH2, KTM2C and KTM2E. Although loss of these genes is considered to contribute to the development of AML with monosomy 7, it may also be required for some leukemia cell functions. We hypothesized that haploinsufficiency of the epigenetic modifiers in the chromosome 7 is compensated by other epigenetic molecules in different chromosomes, and the AML cells with monosomy 7 are vulnerable to inhibition of those molecules. Based on this notion, we performed an RNA-interference (RNAi)-based screening by targeting 53 genes encoding epigenetic enzymes regulating histone methylation by using AML cell lines with monosomy 7: KG-1 and F-36P cells. We found that the siRNA-mediated knockdown of EED, BRD4 or BRDT significantly decreased cell viability in AML cells with monosomy 7 compared to K562 cells as the control without monosomy 7. Stable knockdown of EED expression by retroviral transduction of shRNA markedly attenuated proliferation as well as increased apoptosis specifically in leukemia cells with monosomy 7. Surprisingly, pharmacologic inhibition of EED, which blocks the binding of EED to the polycomb repressive complex 2 (PRC2), was only marginally effective for the monosomy 7 leukemia cells. These results suggest that EED supports survival of the monosomy 7 leukemia cells in a PRC2-independent manner. Recent studies showed that EED is not only a component of PRC2 but also interacts with the polycomb repressive complex 1 (PRC1). In contrast to modest effects of PRC2 inhibition, inhibition of PRC1 activity showed marked efficacy specifically in leukemia cells with monosomy 7. Co-inhibition of PRC1 and PRC2 synergistically suppressed proliferation of the monosomy 7-AML cells. These results collectively suggest that while both PRC1 and PRC2 cooperatively function, the PRC1 rather than PRC2 has a predominant role in the survival of monosomy 7 leukemia cells. In addition to EED, we identified BRD4 as an essential survival factors in AML with monosomy 7. Although inhibition of BET bromodomain proteins have already shown to be effective for a variety of AML, we showed that blockade of bromodomain proteins preferentially affected cellular proliferation of AML cells with monosomy 7. We then explored whether the deletion of specific genes within the chromosome 7 was responsible for increased sensitivity to BET bromodomain protein inhibition. K562 cells were individually treated with the siRNAs against 22 genes in the presence of a BET bromodomain inhibitor JQ1. We found that knockdown of MLL3 or MLL5 resulted in increased apoptosis of the JQ1-treated K562 cells. We also confirmed that pharmacological inhibition of histone methyltransferase activity of MLL3 and MLL5 showed synergistic effects with JQ1 treatment in the leukemia cells. Conclusion: We identified EED and BRD4 as novel promising therapeutic targets in AML with monosomy 7. Disclosures Kagoya: Kyowa Kirin: Speakers Bureau; Nihon Shinyaku: Research Funding. Miyauchi:kyowa Kirin: Research Funding. Kurokawa:Novartis Pharma K.K.: Research Funding; Celgene K.K.: Consultancy, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co.,Ltd.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding; Boehringer Ingelheim: Speakers Bureau; Yakult Honsha Company: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding; Shionogi &amp; Co., Ltd: Consultancy, Honoraria; Bioverativ Japan ltd.: Consultancy; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Conpany: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Chugai Pharmaceutical Company: Consultancy, Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding; MSD K.K.: Consultancy, Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; Shire Japan K.K.: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau.

Targeting NMNAT1 in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is primarily a disease of older adults with poor treatment outcomes. Despite years of intensive research, the standard induction therapy for AML has remained largely unchanged for decades. Thus, the development of new and efficacious therapeutic targets for AML is urgently needed. Leukemia cells exhibit multiple metabolic aberrations that may be therapeutically targeted. Here, we show that nicotinamide adenine dinucleotide (NAD+) promotes leukemogenesis and causes chemotherapy treatment resistance through fueling energetic metabolism, and pinpoints nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) is a novel therapeutic target for AML. To identify novel genes essential for AML, we performed a whole genome CRISPR dropout screen by using MOLM13 cell line and identified 1,951 essential genes (Fig. A). By searching druggable targets among these genes, we narrowed down to 345 genes, among which we found two genes, NMNAT1 (nicotinamide nucleotide adenylyltransferase 1) and NAMPT (nicotinamide phosphoribosyltransferase), both involved in key steps in NAD+ biosynthesis. We comprehensively analyzed dependency scores for all genes involved in the NAD+ biosynthetic pathways (de novo synthesis pathway, the Preiss-Handler pathway and the salvage pathway) across a broad panel of cancer cell lines from the Dependency Map database (https://depmap.org/portal/). The results showed that NMNAT1 and NAMPT are both strongly selective and uniquely required for hematological malignancies compared to other cancers (Fig. B). Since little success has been achieved for NAMPT inhibitors in clinical trials, our attention was drawn to NMNAT1, which encodes a nuclear localized enzyme catalyzing the final step in NAD+ biosynthesis. We confirmed that deletion of NMNAT1 in AML cells significantly reduced nuclear NAD+ level and cell viability over time while sparing normal hematopoietic progenitor cells, suggesting that NMNAT1 is targetable to AML. Overexpression of wild-type Nmnat1 but not the enzymatically inactive forms rescued NMNAT1-KO AML, indicating that the catalytic activity of NMNAT1 is required for AML. To study the role of NAD+ in AML, we first measured NAD+ levels in leukemic and normal cells, and found higher NAD+ levels in leukemia-initiating cells from a murine MLL-AF9-induced AML model compared to normal cells. Supplementation of NAD+ metabolites (NMN, NAM and NR) increased AML proliferation, enhanced glycolysis (lactate production) and oxidative phosphorylation (ATP production), resulting in chemotherapy resistance (Fig. C). Deletion of NMNAT1 sensitized AML cell to chemotherapy treatment. To study the role of NMNAT1 in leukemogenesis in vivo, we genetically deleted NMNAT1 in murine or human leukemia cells, transplanted them into recipient mice, and found that deletion of NMNAT1 reduced leukemic burden and extended leukemia-free survival (Fig. D). Finally, to reveal the molecular mechanisms underlying NMNAT1 KO-mediated cell death (increased levels of gamma-H2AX), RNA-seq and functional assay of NAD+ dependent enzymes were performed. We found that the reduction of nuclear NAD+ resulting from NMNAT1 deletion upregulated genes involved in DNA repair pathway, which may be linked to impaired PARPs and Sirtuins activity. Our findings reveal the important function of NAD+ in leukemogenesis and chemoresistance, and identify NMANT1 as a novel therapeutic target for AML. Figure Disclosures No relevant conflicts of interest to declare.