Novel Therapeutic Target In Acute Myeloid Leukemia Research Articles

EGFL7 Antagonizes NOTCH Signaling and Represents a Novel Therapeutic Target in Acute Myeloid Leukemia (AML)

Epidermal growth factor-like domain 7 (EGFL7) is a secreted protein. We previously found that EGFL7 is up-regulated in primary AML blasts and that a high mRNA expression correlates with shorter event-free and overall survival in AML patients (Papaioannou et al. PNAS 2017;114(23)). However, the underlying mechanism by which EGFL7 promotes leukemogenesis is largely unknown. To identify EGFL7 binding partners, we took an unbiased high-throughput approach by using an antibody interaction-array to measure the ability of EGFL7 to bind directly ~400 proteins expressed by primary AML blasts. Using this strategy, we found in cell lysates of 3 AML patients that EGFL7 binds several signaling proteins important for normal and malignant hematopoiesis including NOTCH (in all patients P<0.001 compared to control). The direct interaction between EGFL7 and NOTCH1 as well as NOTCH2 receptor was confirmed in co-immunoprecipitation assays in THP1 and primary patient cells. Next, we stimulated AML blasts from 3 patients with recombinant EGFL7 (rEGFL7) and found that it reduced NOTCH intracellular domain (NICD1/2, Figure 1A), indicating that EGFL7 inhibits NOTCH activation. In line with this, we found that stimulation of AML patients samples' with rEGFL7 resulted in a decreased expression of its well-known downstream target gene HES1 (Figure 1B; in all patients P<0.05 compared to unstimulated (unstim) cells). On the other hand, treatment with an anti-EGFL7 blocking antibody (AB) caused increased levels of NICD1/2 (Figure 1C) and expression of HES1 (Figure 1D, in all patients P<0.05 compared to cells treated with IgG) indicating a reactivation of NOTCH signaling. Moreover, the treatment of THP1 cells with the anti-EGFL7 blocking AB resulted in increased apoptosis (1.9 fold increase compared to IgG control, P<0.05) and differentiation (14.9 fold increase in CD11B expression compared to IgG control, P<0.05; 5.6 fold increase in CD14 expression compared to IgG control, P<0.05). To determine whether blocking EGFL7 could provide a new targeted therapy for patients with AML, we treated 3 independent AML models (i.e. an AML cell line based xenotransplant model (n=4 mice per group), a primary murine AML model (n=7 mice per group), and a patient derived xenotransplant model (n=4 mice per group)) with anti-EGFL7 or IgG1 control to determine whether anti-EGFL7 could prolong survival in vivo. In all models, we demonstrated that in vivo treatment with anti-EGFL7 results in prolonged overall survival (cell line model: P<0.01; primary murine AML model: P<0.01; for the patient derived xenotransplant model: P<0.05). In conclusion, 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 EGFL7 might be a novel therapeutic target in AML. Figure 1 A AML patient blasts were cultured in the presence or absence (Unstim) of rEGFL7. Total proteins were extracted for immunoblotting with β-ACTIN as loading control. B Total RNA was extracted for quantitative real time (qRT-PCR) analysis of HES1 mRNA normalized to β-ACTIN control (*P<0.05, **P<0.01, ***P<0.001) stimulated with +rEGFL7 vs Unstim. C Patient blasts were cultured in presence of 100 μg/ml of normal IgG or 100 μg/ml anti-EGFL7 antibody (@EGFL7) for 4 hours. Total protein was extracted for immunoblotting of NICD with b-ACTIN as loading control. D Patient blasts were cultured with 100 μg/ml of normal IgG or anti-EGFL7 antibody for 2 hours and 10 hours. Total RNA was extracted for qRT-PCR analysis of HES1 mRNA with b-ACTIN as internal control. *P<0.05, ***P<0.001 vs IgG control. E Treatment with anti-EGFL7 blocking antibody prolonged survival compared to IgG1 controls in 3 independent mouse models. Disclosures No relevant conflicts of interest to declare.

Exploration of novel therapeutic targets in acute myeloid leukemia via genome-wide CRISPR screening

Acute myeloid leukemia (AML) remains a devasting disease. Progress has been made to define molecular mechanisms underlying disease pathogenesis due, in part, to the near-complete understanding of AML genome. Nonetheless, functional studies are necessary to assess the significance of AML-associated mutations and devise urgently needed therapies. Genome-wide knockout screening, employing CRISPR-Cas9 genome editing, is a powerful tool in functional genomics. In this study, genome-wide CRISPR screening was performed using mouse leukemia cell lines developed in our Center, followed by in vivo screening. Among 20,611 genes, 130 AML essential genes were identified, including clinically actionable candidates. It was shown that mRNA decapping enzyme scavenger (DCPS), an enzyme implicated in mRNA decay pathway, is essential for AML survival. ShRNA-mediated gene knockdown and DCPS inhibitor (RG3039) were employed to validate findings. RG3039 induced cell-cycle arrest and apoptosis in vitro. Furthermore, mass spectrometry analysis revealed an association between DCPS and RNA metabolic pathways, and RNA-Seq showed that RG3039 treatment induced aberrant mRNA splicing in AML cells. Importantly, RG3039 exhibited anti-leukemia effects in PDX models. These findings identify DCPS as a novel therapeutic target for AML, shedding new light on the nuclear RNA metabolic pathway in leukemogenesis.

FIP1L1 Regulates Alternative Polyadenylation of Leukemia-Associated Genes in Acute Myeloid Leukemia

Alternative polyadenylation (APA) is a prevalent mechanism of post-transcriptional gene regulation. APA can impact the protein coding portion of a gene or the length of the 3' untranslated region (UTR), a crucial mRNA region mediating stability, localization, and translation efficiency. Global shifts in APA usage have been reported during development and in cancer transformation; however, the impact of APA in acute myeloid leukemia (AML) is currently unexplored. Therefore, we sought to address whether global shifts in APA patterns can be seen in leukemic blasts and if APA changes contribute to leukemogenesis. Here we report that leukemic blasts exhibit global transcript shortening that contributes to the upregulation of leukemia-associated genes: NRAS, BAALC, and MAPKAPK3. We also implicate FIP1L1 as an important mediator of APA alterations in AML.To identify whether global trends of APA are seen in leukemia patients, we performed a 3'RNA sequencing experiment comparing healthy CD34+ hematopoietic stem and progenitor cells (HSPCs) with CD34+ enriched leukemic blasts from primary patient samples. We observed a trend in whole transcript shortening, with an emphasis on 3'UTR shortening of affected genes. Importantly, genes with altered APA were among pathways associated with oncogenic transformation and specifically AML. We selected three genes for further functional validation (NRAS, BAALC, and MAPKAPK3) that exhibited a correlation of shortened 3'UTRs with higher mRNA expression. Using mRNA stability assays and luciferase assays, we confirmed that 3'UTR shortening contributes to the upregulation of these leukemia-associated genes in blood cells.To probe upstream regulators of APA alterations in leukemia, we performed overexpression and knockdown studies of APA machinery members that have altered expression in leukemia patients. We identified FIP1L1 as a novel regulator of APA in AML. Overexpression of FIP1L1 resulted in 3'UTR lengthening and downregulation of our three selected genes. Conversely, knockdown led to 3' UTR shortening and upregulation. Interestingly, altering FIP1L1 expression (both up and downregulation) is detrimental to leukemia cell lines, suggesting that modest changes in gene expression can have a dramatic impact on hematopoietic cell fitness. We conclude that APA changes represent another dysregulated layer of gene expression that can contribute to AML development. Current studies aimed at identifying the role of FIP1L1 in healthy HSPCs will elucidate the feasibility of FIP1L1 as a novel therapeutic target in AML. DisclosuresNo relevant conflicts of interest to declare.

Sphingosine-1-Phosphate Receptor 3 (S1PR3) Promotes Myeloid Commitment of Human Hematopoietic and Leukemic Stem Cells

Understanding the mechanisms underlying the abnormal differentiation of human acute myeloid leukemia (AML) may reveal novel therapies to eradicate leukemic stem cells (LSC), which are often resistant to standard treatments and contribute to relapse. Cellular metabolism is recognized as a hallmark of cancer and is known to be distinct between hematopoietic stem cells (HSC) and downstream progenitors. In particular, sphingosine-1-phosphate (S1P) is a bioactive lipid produced from sphingolipid metabolism that regulates proliferation and survival and is implicated in HSC egress, lymphocyte trafficking and mouse lymphoid lineage determination primarily through binding to S1PR1, one of five S1P G-protein coupled receptors. However, sphingolipids are understudied in human LSC and HSC. We recently found that sphingolipid perturbation governs HSC self-renewal and influences lineage outcome. Here, we show that S1PR3 governs myeloid commitment of LSC in a subset of human AML and is thus an attractive therapeutic target.Lipidomic analysis of LSC+ and LSC‒ fractions derived from AML patient samples and validated by xenotransplantation assays showed distinct sphingolipid alterations when compared to each other and to normal human cord blood (CB) CD34+CD38‒ stem cells and CD34+CD38+ progenitor populations. Interestingly, LSC+ fractions have increased S1P levels, prompting us to wonder if S1P signaling contributes to LSC maintenance. To gain a better understanding of the role of S1P in stemness, we examined S1P signaling in normal CB. Analysis of gene expression of S1PR1-5 and S1P transporters (SPNS2 and MFSD2B) within a comprehensive transcriptional roadmap of human hematopoiesis comprising thirteen normal populations from HSC to mature blood lineages demonstrated distinct expression patterns in specific blood lineages. S1PR1 and MFSD2B were most highly expressed in lymphoid and erythroid lineages, respectively, consistent with murine data. Notably, S1PR3 expression is specific to mature monocytes and granulocytes in human CB. S1PR3 protein was absent from the surface of long-term (LT) and short-term (ST)-HSC as determined by flow cytometry. Remarkably, lentiviral overexpression (OE) of S1PR3 was sufficient to promote myelopoiesis at the expense of erythropoiesis from LT- and ST-HSC in vitro in liquid culture, single cell assays and colony forming assays. To ascertain the mechanisms promoting myeloid commitment, we performed gene expression profiling by RNA-seq of LT- and ST-HSC following S1PR3 OE. This yielded a subset of shared genes similarly upregulated following S1PR3 OE relative to controls, including the known myeloid differentiation and AML-associated factors Early Growth Response 1 and 2 (EGR1/2) and Tribbles 1 (TRIB1). Thus, promiscuous expression of S1PR3 promotes a myeloid fate program in human HSC.S1PR3 protein expression was higher in AML patient samples relative to human CB and bone marrow cells by flow cytometry. Bioinformatic analysis of three independent AML cohorts revealed that AML patient samples with high S1PR3 gene expression also had high EGR2 and TRIB1 expression. Limiting dilution xenotransplantation assays of LSC-containing fractions sorted based on surface expression of S1PR3 demonstrated lower LSC frequency in S1PR3high versus S1PR3low/- LSC fractions. Moreover, S1PR3 OE in LSC+ fractions virtually abolished leukemic engraftment in xenotranplantation assays. These results suggest that higher S1PR3 levels in LSC are associated with a more mature myeloid state and that further increasing S1PR3 levels disrupts LSC maintenance. Treatment of mice bearing primary AML xenografts with FTY720, a S1P mimetic that targets S1P receptors including S1PR3, decreased leukemia burden in a subset of patient samples tested, including those obtained from relapsed and treatment-refractory patients. 70% of AML samples tested showed decreased LSC frequency in serial repopulation assays following FTY720 treatment. Importantly, treatment with FTY720 did not alter normal hematopoietic xenografts, demonstrating the existence of a therapeutic window. Collectively, our results provide the first direct evidence that sphingolipids govern myeloid commitment in human HSC and LSC, and demonstrate the potential of S1PR3 as a novel therapeutic target in AML for eradication of LSC while sparing HSC. DisclosuresNo relevant conflicts of interest to declare.

RNA Binding Protein Rbmx Is Required in Acute Myeloid Leukemia By Regulating the Transcriptional Activity of the Heterochromatin Protein HP1α

Acute myeloid leukemia (AML) is characterized by a block in the development of myeloid cells, often due to dysregulation of genes involved in key processes including self-renewal, proliferation, and differentiation. Somatic mutations and aberrant expression of RNA binding proteins (RBPs) have recently been found to be important in hematological malignancies. For example, our group and others have recently determined that increased expression of MUSASHI-2 and SYNCRIP drives aggressive leukemia. To discover novel RBP regulators of leukemia, we performed an in vivo pooled shRNA screen of 127 MSI2 direct protein interactors and associated genes (Vu et al. Nat Gen. 2017). In this screen, shRNAs specific to the RBP RBMX (RNA binding motif protein, X-linked) were selectively depleted in murine MLL-AF9 driven leukemia. RBMX has been implicated in regulating alternative splicing, chromatin cohesion, and DNA-damage response, but its function in hematopoiesis and leukemia is not known. We confirmed that depletion of RBMX with shRNAs in murine MLL-AF9 leukemia cells resulted in reduced myeloid colony formation, increased apoptosis, and increased differentiation as determined by flow analysis of myeloid cell surface markers Gr-1 and Mac-1 (mean of 61-65% shRNA versus mean of 12.95% control). Furthermore, RBMX is highly expressed among human myeloid leukemia cell lines (n=10/11) and primary AML patient samples (n=2/4). Depletion of RBMX with shRNAs led to a dramatic decrease in cell proliferation and 3-fold induction of apoptosis in several human myeloid leukemia cell lines (MOLM-13, THP-1, K562, and KCL-22). Additionally, RBMX depletion in AML cells induced myeloid differentiation and significantly delayed leukemogenesis cells in vivo (median survival of 51.5 days in control versus median ‘not reached’ in shRNA1 and shRNA2). To determine if there is a differential requirement of RBMX in survival of leukemia cells compared to normal hematopoietic stem and progenitor cells (HSPCs), we depleted RBMX with shRNAs in normal murine bone marrow c-Kit+ cells and found no significant changes in colony formation. Depleting RBMX with shRNAs in human cord blood derived CD34+ HSPCs resulted in reduced colony formation but no increase in apoptosis. Thus, these data suggest that there is a differential requirement for RBMX in myeloid leukemia cells compared to normal cells. To uncover the mechanism of RBMX function, we performed RNA-sequencing of human AML cells (MOLM-13) depleted for RBMX. Gene set enrichment analysis demonstrated a loss of cell cycle and DNA repair associated programs in RBMX depleted cells. Complex chromosomal karyotyping analysis of these cells revealed increased metaphases with breaks and gaps (mean of 30.67% shRNA versus mean of 13.33% control) and irregular chromatin compaction (mean of 47.67%shRNA versus mean of 20% control), while cell cycle analysis showed significantly increased S-phase arrest as determined by flow analysis of Hoechst stained cells (mean of 37-40% shRNA versus of 24.18% control). Reanalysis of RBMX transcriptome-wide binding sites in 293T cells revealed that RBMX directly binds to heterochromatin protein HP1α transcripts (Liu et al. Nucleic Acids Res. 2017). HP1α, also called CBX5, is a key heterochromatin protein that binds to histone H3-K9 tri-methylation marks to promote heterochromatin formation, which is critical in chromatin condensation and chromosome segregation. HP1α has also been determined to be required for MLL leukemia stem cell maintenance. We demonstrated that RBMX depletion resulted in a significant decrease of HP1α mRNA expression without affecting its mRNA stability in AML cells. We confirmed that RBMX depletion reduced the protein abundance of HP1α. Moreover, overexpression of HP1α rescued the effect of RBMX depletion on cell growth and apoptosis. Our study finds that RBMX binds to HP1α mRNA and regulates the transcriptional activity of the HP1α locus, which then maintains proper chromatin compaction in leukemia cells. Overall, we determine that RBMX function is critical for myeloid leukemia survival and has potential as a novel therapeutic target in AML. DisclosuresNo relevant conflicts of interest to declare.

TUG1 confers Adriamycin resistance in acute myeloid leukemia by epigenetically suppressing miR-34a expression via EZH2

Increasing evidence has suggested the involvement of long non-coding RNA (lncRNA) taurine upregulated gene 1 (TUG1) in chemoresistance of cancer treatment. However, its function and molecular mechanisms in acute myeloid leukemia (AML) chemoresistance are still not well elucidated. In the present study, we investigate the functional role of TUG1 in Adriamycin (ADR) resistance of AML and discover the underlying molecular mechanism. Our study revealed that TUG1 was up-regulated in ADR-resistant AML tissues and cells. High TUG1 expression was correlated with poor prognosis of AML patients. TUG1 knockdown improved the sensitivity of HL60/ADR cells to ADR. Moreover, TUG1 could epigenetically suppress miR-34a expression via recruiting Enhancer of zeste homolog 2 (EZH2). miR-34a overexpression could mimic the functional role of down-regulated TUG1 in ADR resistance. miR-34a knockdown counteracted the inductive effect of TUG1 inhibition on ADR sensitivity of HL60/ADR cells. Furthermore, TUG1 knockdown facilitated ADR sensitivity of ADR-resistant AML cells in vivo. In summary, TUG1 knockdown overcame ADR resistance of AML by epigenetically enhancing miR-34a expression, providing a novel therapeutic target for AML.

Abstract 3340: Identification of CARM1/PRMT4 as a novel therapeutic target for AML

Abstract Chromatin modifying enzymes, and specifically the protein arginine methyltransferases (PRMTs) have emerged as important targets in cancer. PRMT4, also known as CARM1, is overexpressed in a number of cancers, including breast, prostate, pancreatic, and lung cancer. Our lab reported the overexpression of PRMT4 in the context of acute myeloid leukemia (AML), showing that more than 70% of cytogenetically normal AML patients have up-regulation of PRMT4. Here, we investigated the role of PRMT4 in normal hematopoiesis and leukemia development. In order to study the role of PRMT4 in normal hematopoiesis, Prmt4-floxed mice were crossed with Vav1-cre mice purchased from the Jackson Laboratory. Inducible Prmt4 conditional KO mice were generated by crossing Prmt4-floxed mice with Mx1-Cre mice and Prmt4 gene excision was induced by poly(I:C). Using this hematopoietic specific knockout system, we show that loss of PRMT4 has little effect on normal hematopoiesis, but promotes the differentiation of hematopoietic stem and progenitor cells. Next we evaluated the role of PRMT4 in leukemia development using leukemia transplantation models driven by fusion oncoproteins. Strikingly, the knockout of PRMT4 completely abrogates leukemia initiation in fetal liver transplantation models driven by the AML1-ETO or MLL-AF9 fusion proteins. We further characterized the mechanism for the leukemia-specific dependence on PRMT4 using leukemia cell lines and found that knockdown of PRMT4 impairs cell cycle progression, decreases proliferation, and induces rapid apoptosis. To examine PRMT4 dependent changes in gene expression in a leukemia system, we used lentiviral vectors that express RFP and shRNAs directed against PRMT4. We knocked down PRMT4 in four leukemia cell lines or normal human cord-blood derived CD34+ cells. Gene set enrichment analysis showed that all four leukemia cell lines with knockdown of PRMT4 significantly down-regulated E2F target genes compared to the scrambled control. Chromatin immunoprecipitation analysis (ChIP) confirmed the presence of PRMT4 and H3R17 dimethylation at the promoter regions of E2F1 targets. The PRMT4 conditional knockout mice and PRMT4 knockdown experiments both suggest that the loss of PRMT4 protein has a selective effect on leukemia cells compared to normal hematopoietic stem and progenitor cells. Collectively, this work suggests that targeting PRMT4 through chemical inhibition may be an effective therapeutic strategy for AML and other cancers with up-regulation of PRMT4. Citation Format: Sarah M. Greenblatt, Pierre-Jacques J. Hamard, Takashi Asai, Na Man, Concepcion Martinez-Caja, Fan Liu, Stephen Nimer. Identification of CARM1/PRMT4 as a novel therapeutic target for AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3340. doi:10.1158/1538-7445.AM2017-3340

Single-Cell Gene Expression Analysis Identifies Alcam As a Novel Candidate Therapeutic Target in AML

Acute myeloid leukemia (AML) is the most common acute leukemia in adults, characterized by high rate of relapse even in recent days. AML is organized in a hierarchy of heterogeneous cell populations and relapse is generally attributable to chemoresistant leukemia stem cells (LSCs). Anthracyclins and cytarabine, which cannot eliminate LSCs, have remained the mainstay of AML therapy for almost 40 years and novel drugs targeting LSCs more efficiently are urgently needed. Toward that end, a potential strategy is to identify cell surface markers that can be differentially regulated in LSCs within the bulk of leukemic cells. Here in this study, focusing on heterogeneity of AML cells, we exploited single-cell gene expression analysis to find a novel cell surface antigen differentially expressed in LSCs, which might be missed by analyses as a whole.Firstly, we proposed "single-cell leukemia initiating cell (LIC) score", defined as sum of mRNA expression levels of 15 genes specifically expressed in functionally-determined LICs according to public transcriptome datasets (GSE30375), and graded primary AML samples on a single-cell basis. Single CD34+ CD38- cells (n = 66) and CD34+ CD38+ cells (n = 30) from bone marrow (BM) of two AML patients were analyzed. Single-cell gene expression analysis using Fluidigm technology revealed that within 50 candidate cell surface molecules highly expressed in LICs according to public datasets, expression of 19 genes were positively related to the LIC score. Among them, we focused on ALCAM (Activated Leucocyte Cell Adhesion, CD166), a cell adhesion molecule implicated in tumorigenesis, because survival analyses using public datasets showed that high expression of ALCAM was associated with shorter overall survival in AML.When we examined cell-surface expression of ALCAM in undifferentiated AML cells, ALCAM was highly expressed on CD34+ AML cells compared with CD34+ normal hematopoietic cells. We separated BM cells from AML patients into CD34+ ALCAM high fraction and CD34+ ALCAM low fraction. Colony-forming activities were enriched in CD34+ ALCAM high fraction in vitro (n = 5). And then, we did xenotransplantation model by intravenous injection of human AML BM cells to NSG mice. We show that LICs tended to be enriched within CD34+ ALCAM high fraction in some primary AML samples by using xenotransplantation model.To elucidate whether ALCAM exerts any biological functions and could be a novel therapeutic target in AML, we established human THP-1 AML cells in which expression of ALCAM was silenced by short hairpin RNA (shRNA). Knockdown of ALCAM reduced proliferation and enhanced chemosensitivity of THP-1 cells to cytarabine by curbing genotoxic stress-induced apoptosis in vitro. THP-1 cells expressing shRNA against ALCAM also showed decreased leukomogenic capacity in a xenotransplantation model. When we suppressed activities of ALCAM using a soluble isoform of ALCAM (soluble ALCAM), which binds to ALCAM and inhibits intercellular ALCAM-ALCAM hemophilic interactions, THP-1 cells expressing soluble ALCAM also showed decreased proliferation, enhanced chemosensitivity and decreased leukomogeneic capacity. Furthermore, we transplanted human primary AML cells transduced with a tetracycline-inducible lentiviral construct encoding soluble ALCAM into NSG mice. After engraftment of human cells, expression of soluble ALCAM was induced. BM engraftment of human primary AML cells expressing soluble ALCAM was reduced, which indicated that decreased BM engraftment was not due to impaired homing, but to leukemogenic ability.These findings implicate that LICs with high clonogenic capacity were enriched within CD34+ ALCAM high fraction and ALCAM could be a novel therapeutic target of AML cells. Furthermore, our results also suggests single-cell gene expression analysis and a scoring method such as "LIC score" could be applicable to other hematological malignancies and other cancers to identify and exploit a property of heterogenous cancer cells. DisclosuresNo relevant conflicts of interest to declare.

The Actin Binding Protein Plastin-3 Is Involved in the Pathogenesis of Acute Myeloid Leukemia

Leukemia-initiating cells reside within the bone marrow (BM) in specialized niches where they undergo complex interactions with their surrounding stromal cells. In order to identify genes being implicated in the interaction of acute myeloid leukemia (AML) cells and stromal cells, we performed co-cultures of primary AML cells with primary endothelial cells and osteoblasts. The gene expression of co-cultured AML blasts was compared to AML cells grown without adherent cells using microarray analysis. Amongst those genes being dysregulated upon co-culture was the actin binding protein plastin-3 (PLS3). Further RT-qPCR analysis revealed an endogenous PLS3 expression in about 50% of BM samples from AML patients (n=25). In contrast, expression of PLS3 was only detected in 2 of 12 analyzed AML cell lines with Kasumi-1 showing strong and THP-1 showing only weak expression.Therefore, functional analysis of PLS3 in AML was studied using shRNA knockdown and overexpression of PLS3 in Kasumi-1 cells. We could show that PLS3 has an impact on the colony formation capacity of AML cells in vitro as the knockdown resulted in significantly reduced colony numbers while increased colony growth was observed in the Kasumi-1 cells overexpressing PLS3 (p<0.001 and p<0.001, respectively). To investigate the role of PLS3 in vivo, NSG mice were transplanted with the PLS3 knockdown Kasumi-1 cells. Compared to mice transplanted with Kasumi-1 cells transduced with a vector carrying a scrambled shRNA, the PLS3 knockdown mice survived significantly longer (median survival time 64 vs. 110 days, respectively; p<0.001; n=9 mice per group).Finally, we investigated whether the expression of PLS3 was associated with AML patients' outcome using published microarray-based gene expression data (Verhaak et al, Haematologica 2009;94). Clinical data of 290 AML patients were available. Based on the mean gene expression value, the patient cohort was divided into high vs low PLS3 expressors. The overall survival was analyzed in a multivariate Cox proportional hazards model including PLS3 gene expression and the baseline parameters age, karyotype and FLT3 mutational status. After a stepwise removal of insignificant terms, the patient's age and a high PLS3 expression remained as independent prognostic survival markers (for PLS3: HR 1.58 (CI 1.05 - 2.37) and for age: HR 1.01 (CI 1.00 - 1.03)).In conclusion, our results identify the actin binding protein PLS3 as potential novel therapeutic target in AML. DisclosuresStamm:Astellas: Other: Travel, Accommodation, Expenses. Heuser:BerGenBio: Research Funding; Tetralogic: Research Funding; Novartis: Consultancy, Research Funding; Celgene: Honoraria; Bayer Pharma AG: Research Funding; Pfizer: Research Funding; Karyopharm Therapeutics Inc: Research Funding. Fiedler:Kolltan: Research Funding; Ariad/Incyte: Consultancy; Novartis: Consultancy; Gilead: Other: Travel; Teva: Other: Travel; GSO: Other: Travel; Pfizer: Research Funding; Amgen: Consultancy, Other: Travel, Patents & Royalties, Research Funding. Wellbrock:Astellas: Other: Travel, Accommodation, Expenses.

The N6-Adenine Methyltransferase METTL14 Plays an Oncogenic Role in Acute Myeloid Leukemia

N6-methyladenosine (m6A) modification is the most abundant internal RNA modification in eukaryotes. Recent studies have shown that the dynamic and reversible regulation of m6A modifications in mRNAs or non-coding RNAs plays critical roles in tissue development, stem cell self-renewal and differentiation, control of heat shock response, and circadian clock controlling, as well as in RNA metabolism and processing. However, little is known about the functions of m6A and m6A regulators in malignant hematopoiesis.METTL14 is a major m6A writer which together with METTL3 forms the core of the methyltransferase complex that catalyzes the conversion of adenosine (A) to m6A. Through qPCR assays, we found that METTL14 was aberrantly up-regulated in mononuclear cells (MNC) from acute myeloid leukemia (AML) patients with t(11q23), t(15;17), or t(8;21) relative to those from healthy donors.To investigate the pathological role of METTL14 in AML, we transduced lineage negative (Lin-) bone marrow (BM) progenitor cells from Mettl14fl/flCreERT mice with MLL-AF9, AML1-ETO9a, or PML-RARa fusion genes and performed colony-forming/replating assays with or without addition of 4-hydroxytamoxifen (4-OHT). Induction of genetic knockout of Mettl14 by 4-OHT treatment remarkably impaired the colony-forming ability of all these AML-related fusion genes after replating. After the first round of plating, we harvested MLL-AF9-transduced cells that were not treated with 4-OHT and transplanted them into lethally irradiated recipient mice. As expected, tamoxifen (TAM) treatment of transplanted mice significantly delayed leukemogenesis compared to mice treated with vehicle (MLL-AF9+TAM, with median survival of 91 days; MLL-AF9+vehicle, with median survival of 71 days; P=0.0012) (Fig.1A). In addition, specific knockdown of Mettl14 with shRNAs showed similar patterns to Mettl14 knockout. Thus our data demonstrate that Mettl14 is crucial for cell transformation and leukemogenesis. Further, to determine the role of Mettl14 in the maintenance of leukemia, we transduced leukemic BM cells from primary MLL-AF9 leukemic mice with shRNAs against Mettl14 or scramble shRNA and transplanted these cells into lethally irradiated recipient mice. Again, a significantly prolonged survival was observed in Mettl14 knockdown groups compared to that in the control group (MLL-AF9+shRNA1, with median survival of 32 days; MLL-AF9+shRNA2, with median survival of 32 days; MLL-AF9+shScramble, with median survival of 23.5 days; P< 0.001 for both knockdown groups) (Fig.1B). Noticeable, mice in Mettl14 knockdown groups showed less c-kit+ cells in BM than mice in the control group (Fig.1C).In addition to the mouse model, we used human leukemia cell lines to investigate the function of METTL14 in human AML cells. Silencing of METTL14 with shRNAs significantly inhibited cell viability, induced apoptosis as well as terminal differentiation of MONOMAC6 and NB4 cell lines (Fig.1D, E, F). Moreover, xenograft model showed that repression of METTL14 significantly inhibited the engraftment of MONOMAC6 cells and thus delayed the onset of leukemia in NSG-SGM3 (NSGS) immunodeficient mice (Fig.1G). Furthermore, knockdown of METTL14 sensitized MONOMAC cells to ATRA or PMA-induced differentiation. Taken together, our results support the oncogenic role of METTL14 in AML and highlight METTL14 as a novel therapeutic target in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CD99 Is Highly Expressed in Acute Myeloid Leukemia (AML) and Presents a Viable Therapeutic Target

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by clonal proliferation and block of differentiation of myeloid precursors. Overall survival for patients with AML remains dismal (<50% for younger patients and <10% for older patients) due to high relapse rate. In search for novel therapeutic targets in AML, we compared gene expression data of normal hematopoietic vs AML cells from 7 datasets (GSE13159, GSE13164, GSE7186, GSE1159, GSE995, GSE31174 and TCGA Leukemia) available on Oncomine. We identified CD99 to be significantly upregulated in AML cells compared with normal cells in all data sets with available measurements of CD99 expression (median ranking 155, p = 0.013); other genes among the top 10 genes identified in this analysis were FLT3 (median ranking 102, p<0.001) and WT1 (median ranking 120, p<0.001); both are known to play a role in AML. CD99 was significantly over-expressed (p<0.001) in 542 AML patients as compared with PBMCs from 74 healthy donors from the GSE13159 dataset. In the GSE13164 dataset, CD99 was significantly over-expressed in 257 AML patients as compared with PBMCs from 58 healthy donors. Consistently, in the GSE7186 dataset, CD99 was significantly over-expressed (p<0.001) in 23 AML patients as compared with 6 normal bone marrow samples and in the GSE1159 dataset, CD99 was significantly over-expressed (p=0.001) in 285 AML patients as compared with 5 normal bone marrow, and 3 normal blood samples. We also analyzed CD99 expression in cells obtained from 23 patients with AML and sorted according to their CD34 and CD38 expression levels (GSE3077 dataset). We found that CD99 expression was significantly higher (p<0.001) in the CD34+CD38+ and CD34+CD38- subpopulation compared with CD34-CD38- and CD34-CD38+; suggesting a possible role of CD99 in AML stem cells.Interestingly, analysis of three datasets (GSE22848, GSE6891, GSE15434) via R2: Genomics Analysis and Visualization Platform showed a correlation between CD99 expression and the presence of FLT3-ITD mutation. In the GSE22848 dataset, CD99 was significantly over-expressed (p=0.007) in 48 patients with FLT3-ITD as compared with 189 patients with FLT3 wildtype. In the GSE6891 dataset, 126 patients with FLT3-ITD had a significant over-expression of CD99 (p=0.006) as compared with 334 patients with FLT3 wildtype and in the GSE15434 dataset 90 FLT3-ITD positive patients had significantly higher levels of CD99 (p<0.001) as compared with 161 patients with the wildtype gene.CD99 (E2, MIC2), a 32-kD cell surface glycoprotein, is known to be involved in the transendothelium migration of neutrophils, T-cell adhesion, and T-cell death by a caspase-independent pathway. In cancer cells, CD99 was found to be highly expressed on the cell surface of Ewing’s sarcoma tumors and in gliomas. Importantly, CD99 expression levels were found to be correlated with tumor invasiveness and with lower survival rates. In order to examine the role of CD99 in AML, we assessed CD99 expression by flow cytometry in nine AML cell lines (KG-1, KG-1A, MOLM13, MV4-11, Kasumi-1, THP-1, NB4, U937, UOC-M1); we found CD99 to be expressed in all cell lines. To determine whether CD99 is a potential therapeutic target in AML, we treated leukemia cells with anti-CD99 mAb (mAb 0662) at 5 µg/mL and analyzed cell viability 48 hours post-treatment. We found significant decrease in cell viability; 15% in MV4-11 cells (p=0.02), 32% in MOLM13 cells (p=0.002) and 18% in THP-1 cells (p<0.001) as compared with untreated controls measured by Alamar blue assay. Furthermore, inhibiting CD99 led to a decrease in migration of MV4-11, MOLM13 and THP-1 cells when analyzed using a trans-well migration assay.In conclusion, CD99 is highly expressed in AML, and this expression is significantly higher in less differentiated leukemia cells and in patients with FLT3-ITD mutation. Functional studies using CD99 antibodies revealed a possible role of this gene in cell survival and cell migration. Further studies are needed to establish CD99 as a potential therapeutic target and further investigations are ongoing to determine the mechanism by which CD99 regulates cell survival in AML. DisclosuresNo relevant conflicts of interest to declare.

Acid ceramidase is upregulated in AML and represents a novel therapeutic target.

There is an urgent unmet need for new therapeutics in acute myeloid leukemia (AML) as standard therapy has not changed in the past three decades and outcome remains poor for most patients. Sphingolipid dysregulation through decreased ceramide levels and elevated sphingosine 1-phosphate (S1P) promotes cancer cell growth and survival. Acid ceramidase (AC) catalyzes ceramide breakdown to sphingosine, the precursor for S1P. We report for the first time that AC is required for AML blast survival. Transcriptome analysis and enzymatic assay show that primary AML cells have high levels of AC expression and activity. Treatment of patient samples and cell lines with AC inhibitor LCL204 reduced viability and induced apoptosis. AC overexpression increased the expression of anti-apoptotic Mcl-1, significantly increased S1P and decreased ceramide. Conversely, LCL204 induced ceramide accumulation and decreased Mcl-1 through post-translational mechanisms. LCL204 treatment significantly increased overall survival of C57BL/6 mice engrafted with leukemic C1498 cells and significantly decreased leukemic burden in NSG mice engrafted with primary human AML cells. Collectively, these studies demonstrate that AC plays a critical role in AML survival through regulation of both sphingolipid levels and Mcl-1. We propose that AC warrants further exploration as a novel therapeutic target in AML.

The Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) Regulates the Function and Migration of Leukemic Blasts through CD63/PI3K/AKT/P21 Axis

Introduction. The Tissue Inhibitor of MetalloProteinases-1 (TIMP-1) is a member of the inflammatory network that dramatically increases in response to inflammation. First described as an inhibitor of MMPs, TIMP-1 exerts pleiotropic effects in the hematopoietic microenviroment. Along with the finding that hematopoietic stem progenitor cells (HSPCs) directly respond to inflammation, a persistent inflammatory stimulation may hinder HSPC functions and eventually lead to hematological malignancies. Specifically, the inflammatory pathways activated by the bone marrow (BM) microenvironment influences the transformation and progression of leukemia. In hematological malignancies, TIMP-1 promotes proliferation in lymphoma cells and increased TIMP-1 serum levels are associated with poor prognosis in a variety of cancers. However, recent studies have revealed the dichotomy of TIMP-1 in cancer progression.Here, in the attempt to provide further evidence for the critical role of inflammation in leukemic microenvironment, we investigated the role of TIMP-1 in leukemic blasts from patients with Acute Myeloid Leukemia (AML) and elucidated the downstream pathway ignited by rhTIMP-1.Methods. Human CD34+ HSPCs were isolated from cord blood (CB) units as control samples, while leukemic cells were collected from AML patients at diagnosis (percentage of blasts >90%; n=36). Cell proliferation was assessed by cell cycle analysis, and CFSE staining. In addition, we performed colony-forming unit assays. TIMP-1's contribution to cell survival was evaluated by AnnexinV/PI staining. The expression of the tetraspannin receptor CD63 (TIMP-1's putative receptor) was assessed by flow cytometry. Leukemic blasts were assayed towards a CXCL12 gradient after exposure to rhTIMP-1 or pre-treated with LY294002 (PI3K inhibitor); migrated cells were counted and characterized for CD63 expression. Leukemic blasts were sorted in two separate fractions, CD63- and CD63+, and their capability to respond to rhTIMP-1 was assessed. Downstream molecular targets of TIMP-1 (such as PI3K, pAkt, p21) were also confirmed by flow cytometry. RT-PCR was employed to detect the expression of hypoxia-inducible factor-1 (HIF-1α). Finally, co-culture system was performed with CFSE-positive leukemic blasts and mesenchymal stromal cells (MSCs) from normal or AML patients in the presence of rhTIMP-1.Results. We firstly found that TIMP-1 levels are increased in the plasma of BM of AML patients at diagnosis. Similarly to CD34+ HSPCs from CB, the clonogenic potential of primary human AML blasts is increased upon exposure to rhTIMP-1. Moreover, rhTIMP-1 promotes in vitro AML blast survival, possibly due to anti-apoptotic effects and a slight but significant increase in the proportion of leukemic cells entering S-phase of the cell cycle. Such effect is related to the downregulation of cyclin-dependent kinase inhibitor p21. As for the dissection of TIMP-1 signalling pathway, our data revealed that the tetraspannin CD63 receptor is required for TIMP-1's cytokine functions. Indeed, about half of leukemic blasts expresses CD63, whose activation leads to PI3K recruitment and Akt phopshorylation, key modulators of survival/proliferation pathways. Exposure of AML blasts to rhTIMP-1 resulted in the induction of HIF-1α, which is known to play an important role in survival of leukemic stem cells. Moreover, rhTIMP-1 increased the SDF1-driven migration of leukemic blasts through the activation of PI3K. Interestingly, migrating AML blasts are highly enriched in CD63+ cells, whereas the CD63-negative pre-sorted fraction of leukemic cells showed reduced response to migration and pAkt expression after exposure to rhTIMP-1.Finally, in the co-culture system between AML cells and normal versus leukemic MSCs, we defined the particular role of TIMP-1 in normal and leukemic microenviroment. In particular, our results suggest that a defective capacity of AML-MSCs in sustaining the proliferation and migration of AML blasts may be restored by rhTIMP-1.Conclusions. In our study TIMP-1 emerges as an important factor in the leukemic BM microenvironment, modulating leukemic blasts survival, migration and function. Specifically, our work suggests that TIMP-1 promotes survival and migration via CD63/PI3K/AKT/p21 signalling. Taken together, our findings indicates TIMP-1 pathway as a potential novel therapeutic target in AML. DisclosuresCavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

The dual epigenetic role of PRMT5 in acute myeloid leukemia: gene activation and repression via histone arginine methylation.

Changes in the enzymatic activity of protein arginine methyltransferase (PRMT) 5 have been associated with cancer; however, the protein's role in acute myeloid leukemia (AML) has not been fully evaluated. Here, we show that increased PRMT5 activity enhanced AML growth in vitro and in vivo while PRMT5 downregulation reduced it. In AML cells, PRMT5 interacted with Sp1 in a transcription repressor complex and silenced miR-29b preferentially via dimethylation of histone 4 arginine residue H4R3. As Sp1 is also a bona fide target of miR-29b, the miR silencing resulted in increased Sp1. This event in turn led to transcription activation of FLT3, a gene that encodes a receptor tyrosine kinase. Inhibition of PRMT5 via sh/siRNA or a first-in-class small-molecule inhibitor (HLCL-61) resulted in significantly increased expression of miR-29b and consequent suppression of Sp1 and FLT3 in AML cells. As a result, significant antileukemic activity was achieved. Collectively, our data support a novel leukemogenic mechanism in AML where PRMT5 mediates both silencing and transcription of genes that participate in a 'yin-yang' functional network supporting leukemia growth. As FLT3 is often mutated in AML and pharmacologic inhibition of PRMT5 appears feasible, the PRMT5-miR-29b-FLT3 network should be further explored as a novel therapeutic target for AML.

Pharmacological targeting of miR-155 via the NEDD8-activating enzyme inhibitor MLN4924 (Pevonedistat) in FLT3-ITD acute myeloid leukemia

High levels of microRNA-155 (miR-155) are associated with poor outcome in acute myeloid leukemia (AML). In AML, miR-155 is regulated by NF-κB, the activity of which is, in part, controlled by the NEDD8-dependent ubiquitin ligases. We demonstrate that MLN4924, an inhibitor of NEDD8-activating enzyme presently being evaluated in clinical trials, decreases binding of NF-κB to the miR-155 promoter and downregulates miR-155 in AML cells. This results in the upregulation of the miR-155 targets SHIP1, an inhibitor of the PI3K/Akt pathway, and PU.1, a transcription factor important for myeloid differentiation, leading to monocytic differentiation and apoptosis. Consistent with these results, overexpression of miR-155 diminishes MLN4924-induced antileukemic effects. In vivo, MLN4924 reduces miR-155 expression and prolongs the survival of mice engrafted with leukemic cells. Our study demonstrates the potential of miR-155 as a novel therapeutic target in AML via pharmacologic interference with NF-κB-dependent regulatory mechanisms. We show the targeting of this oncogenic microRNA with MLN4924, a compound presently being evaluated in clinical trials in AML. As high miR-155 levels have been consistently associated with aggressive clinical phenotypes, our work opens new avenues for microRNA-targeting therapeutic approaches to leukemia and cancer patients.

Inhibition Of Telomerase Is a Novel and Effective Therapy In MLL-Rearranged Acute Myeloid Leukemia (AML)

Telomerase is activated to maintain the long-term replicative potential in many human cancers including AML and novel inhibitors of telomerase have recently entered clinical trials for a variety of malignancies. We investigated the therapeutic potential of telomerase inhibition on MLL-rearranged AML using genetic and pharmacological approaches in murine and humanized models.Telomerase-deficient AML was generated by retroviral transduction of G3 Terc-/- LKS+ (Lin-Kit+Sca1+, enriched for hematopoietic stem cells) with pMIG-MLL-AF9 and compared to wild type (WT) controls. Transformed Terc-/- LKS+ colony-forming units (CFU) were mildly reduced at early passage (week 1 Terc-/- 13.1 ± 1 vs. WT 32.7 ± 4 per 1000 cells input, p < 0.01) but became progressively extinguished with serial replating (week 6 Terc-/- 3.8 ± 0.4 vs. WT 27.0 ± 2.7 per 1000 input, p < 0.01). Loss of CFU correlated with enforced differentiation (reduced Kit, increased Gr1), cell cycle arrest and preferential apoptosis of Kit+ cells. In vivo, AML developed with delayed latency, but was fully penetrant in recipients of G3 Terc-/- and WT cells (Terc-/- 64 days vs. WT 45 days, p < 0.01). Leukemic burden and leukemia stem cell (LSC, GFP+Lin-Sca1-Kit+FcgR+CD34-) frequency were similar between Terc-/- and WT AML.To determine the consequences of telomerase loss on AML LSCs, we performed gene expression profiling of purified LSCs. MLL-AF9-Terc-/- LSCs revealed enrichment of pathways controlling DNA damage/repair, cell cycle and apoptosis. Upstream analysis predicted activation of p53, Rbl1 and Cdkn2a, and inhibition of E2f1 in Terc-/- LSCs. Functionally, shRNA-mediated knockdown of p53 in Terc-/- LSCs partially rescued in vitro CFU, differentiation, cell cycle arrest and apoptosis.The phenotypic changes in Terc-/- AML were amplified by serial passage, suggesting that replicative stress may exacerbate the deleterious effects of telomerase loss on LSC function. To enforce replicative stress in vivo, we performed serial transplantation of Terc-/- AML vs. WT AML. Terc-/- LSCs were unable to generate secondary AML (survival Terc-/- not reached vs. WT 28 days, p < 0.01) and this was confirmed by limiting dilution analysis (Terc-/- LSC frequency 1:224,000 vs. WT 1:184 p < 0.001). Initial engraftment was similar between Terc-/- and WT LSCs. In vivo leukemogenesis was prevented by cell cycle arrest, DNA damage and massive apoptosis (Terc-/- 76.8% ± 3.6% vs. WT 22.49% ± 2.3%, p < 0.0001). Together, these findings demonstrate that in this murine model, telomerase loss eradicates LSC in vivo.To validate these findings in human AML we performed lentiviral shRNA knockdown of hTERT in the MLL-AF9-containing AML cell line Monomac6, followed by transplantation into NSGS (NOD/SCID/IL2Rgamma-/- transgenic for hSCF/hIL3/hGMCSF) xenograft recipients. Two independent shTERT constructs revealed significantly increased survival compared to non-transduced and non-targeting controls (sh-hTERT 149.5 days and 146 days vs. non-transduced 47 days, non-targeting 53.5 days, p< 0.01 for both sh-hTERT). hTERT knockdown correlated with reduced hCD45 engraftment, induction of DNA damage, cell cycle arrest and apoptosis compared to non-transduced or non-targeting shRNA controls. Pharmacological inhibition of telomerase (Telomerase Inhibitor IX, T-IX, Sigma) reduced growth of multiple human AML cell lines in vitro. Treatment with T-IX followed by transplantation of equal numbers of Monomac6 cells prolonged NSGS xenograft survival (T-IX 96.5 days vs. DMSO 59 days, p < 0.05).Finally, we examined the prognostic impact of telomerase-regulated genes in a large cohort of patients with AML. The top 140 differentially expressed genes (p < 0.001) between murine Terc-/- and WT LSCs predicted survival in 2 independent AML clinical trial cohorts. Computational modelling using a random forest approach was able to identify 10 key telomerase-regulated human homologues that could cluster AML patients into prognostically relevant groups and was reproducible in multiple independent datasets.These findings provide new mechanistic understanding into the effects of telomerase inhibition on MLL-rearranged AML and identify the telomerase complex as a novel therapeutic target for AML. Disclosures:No relevant conflicts of interest to declare.

Targeting the Mammalian Mitochondrial Clpp (mClpP) As a Novel Therapeutic Strategy for Acute Myeloid Leukemia

Abstract 3603The mammalian mitochondrial caseinolytic protease (mClpP) is a nuclear-encoded enzyme complex responsible for degrading excess proteins in the mitochondria. It is functionally similar to the proteasome complex in the cytoplasm and structurally homologous to the bacterial ClpP enzyme complex. While the proteasome has been well characterized as a therapeutic target for the treatment of hematologic malignancy, little is known regarding mClpP as a potential therapeutic target in malignant cells. Therefore, we evaluated the expression of mClpP in acute myeloid leukemia (AML) and normal hematopoietic cells by immunoblotting. Strikingly, mClpP was robustly expressed in AML cell lines (n=3/4) and primary AML patients (n=4/4), but was undetectable in normal bone marrow samples (n = 3) and G-CSF-mobilized peripheral blood mononuclear cells from consenting volunteers donating stem cells for allotransplant (n = 5). We also demonstrated over-expression of mClpP mRNA in these AML samples compared to normal by Q-RT-PCR. Next, we knocked down mClpP and its regulatory chaperone protein mClpX in OCI-AML2 and TEX human leukemia cells using 3 independent shRNA in lentiviral vectors. Target knockdown was confirmed by Q-RT-PCR and immunoblotting. Compared to cells infected with control sequences, knockdown of mClpP reduced the growth and viability of these leukemia cells by > 90%. Knockdown of the chaperone protein mClpX also reduced the growth and viability of these cells, but with less potency than mClpP knockdown.Rho-zero cells have been depleted of mitochondrial DNA by treatment with ethidium bromide and therefore lack mitochondrial protein synthesis. Through feedback mechanisms, expression of nuclear encoded proteins that contribute to oxidative metabolism are also reduced. Thus, these cells have lower rates of mitochondrial protein accumulation and turnover. We demonstrated that wild type and rho-zero 143B rhabdomyosarcoma cells both express mClpP protein, but levels were slightly lower in the rho-zero cells. We then tested the effects of mClpP knockdown in rho-zero cells and demonstrated that knockdown of mClpP reduced the growth and viability of wild type 143B cells, but had little effect on their rho-zero counterparts.As a chemical approach to evaluate the effects of mClpP inhibition on AML and normal hematopoietic cells, we synthesized a derivative of recently reported beta-lactone bacterial ClpP inhibitor and generated (3RS,4RS)-3-(non-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one that we termed A2–32–01. We confirmed that A2–32–01 inhibited the enzymatic activity of recombinant bacterial ClpP similar to the activity of the reported beta-lactone inhibitors. A2–32–01 induced cell death in TEX, OCI-AML2, and K562 leukemia cells that express mClpP as measured by trypan blue staining. We also isolated mitochondria from these cells after treatment with A2–32–01 and demonstrated that the compound reduced the enzymatic activity of the mClpP protease as measured by cleavage of the fluorogenic substance N-succinyl-Leu-Tyr-7-amidomethylcoumarin (Suc-LY-AMC). In contrast, A2–32–01 was not cytotoxic to HL60 cells that had undetectable mClpP. Likewise, A2–32–01 induced death in 143B wild type cells, but not the rho-zero counterparts.Finally, we evaluated the effects of mClpP inhibition on primary AML and normal hematopoietic cells. Primary AML and normal hematopoietic cells were treated with increasing concentrations of A2–32–01 and cell viability was measured after 48 hours incubation by Annexin V/PI staining followed by flow cytometry. A2–32–01 did not kill normal hematopoietic cells (n = 3 samples), but was cytotoxic to 5/6 tested primary AML cells.In summary, we have used genetic and chemical approaches to highlight the mitochondrial protease, mClpP, as a novel therapeutic target for AML. Disclosures:No relevant conflicts of interest to declare.

Small Molecule Inhibition of SIRT Activity: A Novel Therapeutic Approach for Acute Myeloid Leukemia

Abstract 84New therapeutic strategies are urgently needed to improve clinical outcomes for patients with acute myeloid leukemia (AML), which is an extremely aggressive disease with very few long-term survivors. The sirtuin deacetylases (SIRTs) are critical regulators of genes that are essential for longevity, cell growth, tumor suppression, and apoptosis. Elevated SIRT expression has been reported in several types of cancer and may promote pathogenesis and drug resistance by increasing the lifespan and survival capacity of malignant cells. Our preliminary analysis of SIRT expression indicated that SIRT1 was consistently expressed at significantly higher levels in AML cell lines and primary AML blasts as compared with normal controls. In order to investigate the potential role of SIRT1 as a regulator of AML pathogenesis, we utilized shRNA to stably knockdown its expression in MV4-11 and KG-1 AML cells. Cells with targeted SIRT1 knockdown displayed an altered gene expression profile as compared with non-targeted controls. Moreover, antagonizing SIRT1 expression significantly impeded the progression of AML in a xenograft mouse model. A number of deacetylase inhibitors have been clinically evaluated for cancer therapy. However, disrupting SIRT function as an anticancer strategy remains to be rigorously investigated as none of these previously studied drugs significantly inhibit the activity of this class of NAD+-dependent deacteylases. Tenovin-6 is a novel small molecule SIRT inhibitor. We investigated the efficacy and pharmacodynamic effects of tenovin-6 in AML cell lines, primary blasts from patients with AML, and mouse models. Treatment with tenovin-6 induced apoptosis and dramatically diminished AML clonogenic survival. Tenovin-6 promoted a dose-dependent increase in the acetylated levels of the SIRT-regulated gene p53 in AML cells and triggered the induction of several p53 transcriptional targets including p21 and PUMA. Targeted knockdown of PUMA with shRNA significantly reduced the pro-apoptotic effects of tenovin-6, indicating that it is a critical mediator of its anti-leukemic activity. Notably, administration of tenovin-6 to mice implanted with AML cells was well-tolerated and led to a highly significant reduction in disease burden and increase in overall survival. Our collective findings demonstrate that SIRT1 is a promising novel therapeutic target in AML. Further investigation aimed to elucidate the safety, efficacy, and mechanism of action of tenovin-6 is warranted. Disclosures:No relevant conflicts of interest to declare.

PP2A impaired activity is a common event in acute myeloid leukemia and its activation by forskolin has a potent anti-leukemic effect

Protein phosphatase 2A (PP2A) is a human tumor suppressor that inhibits cellular transformation by regulating the activity of several signaling proteins critical for malignant cell behavior. PP2A has been described as a potential therapeutic target in chronic myeloid leukemia, Philadelphia chromosome-positive acute lymphoblastic leukemia and B-cell chronic lymphocytic leukemia. Here, we show that PP2A inactivation is a recurrent event in acute myeloid leukemia (AML), and that restoration of PP2A phosphatase activity by treatment with forskolin in AML cells blocks proliferation, induces caspase-dependent apoptosis and affects AKT and ERK1/2 activity. Moreover, treatment with forskolin had an additive effect with Idarubicin and Ara-c, drugs used in standard induction therapy in AML patients. Analysis at protein level of the PP2A activation status in a series of patients with AML at diagnosis showed PP2A hyperphosphorylation in 78% of cases (29/37). In addition, we found that either deregulated expression of the endogenous PP2A inhibitors SET or CIP2A, overexpression of SETBP1, or downregulation of some PP2A subunits, might be contributing to PP2A inhibition in AML. In conclusion, our results show that PP2A inhibition is a common event in AML cells and that PP2A activators, such as forskolin or FTY720, could represent potential novel therapeutic targets in AML.

C/EBPα regulated microRNA-34a targets E2F3 during granulopoiesis and is down-regulated in AML with CEBPA mutations

AbstractThe transcription factor, CCAAT enhancer binding protein alpha (C/EBPα), is crucial for granulopoiesis and is deregulated by various mechanisms in acute myeloid leukemia (AML). Mutations in the CEBPA gene are reported in 10% of human patients with AML. Even though the C/EBPα mutants are known to display distinct biologic function during leukemogenesis, the molecular basis for this subtype of AML remains elusive. We have recently showed the significance of deregulation of C/EBPα-regulated microRNA (miR) in AML. In this study, we report that miR-34a is a novel target of C/EBPα in granulopoiesis. During granulopoiesis, miR-34a targets E2F3 and blocks myeloid cell proliferation. Analysis of AML samples with CEBPA mutations revealed a lower expression of miR-34a and elevated levels of E2F3 as well as E2F1, a transcriptional target of E2F3. Manipulation of miR-34a reprograms granulocytic differentiation of AML blast cells with CEBPA mutations. These results define miR-34a as a novel therapeutic target in AML with CEBPA mutations.