Prognosis In AML Research Articles

Evaluation of ATG7 and Light Chain 3 (LC3) Autophagy Genes Expression in AML Patients.

Background and aim: Autophagy, known as cell death type II, is a housekeeping pathway that currently has been worked on in matters of tumorigenesis and leukomogenesis. Therefore, expression levels of ATG7 and LC3 as two key genes in AML patients are targeted in this study.Material and method: This study was performed on 55 de novo AML patients against 17 healthy volunteers, acquired samples from bone marrow (BM) and peripheral blood (PB) sources in different ages and gender. The evaluation was executed by mRNA extraction, cDNA synthesis, real-time PCR and data was analyzed by SPSS. Results: Analyzed data indicate a significant decrease between expression of ATG7 and LC3 in AML patients against control (Pv < 0.05). Decrease in both genes expression was detected in most of the patients, 81.81% and 75.55%, respectively. Also LC3 overexpression was detected in 11.33% of AML patients. Moreover, a positive significant correlation between ATG7 and LC3 genes was detected (r = 0.481; Pv = 0.001). Conclusion: This study showed that significant reduction of autophagy genes in de novo AML patients is important to overcome this system and initiate leukomogenesis. It seems a new insight is required for new achievements in diagnosis, prognosis, treatment and monitoring AML patients.

Extracellular Microvesicle Cytokines Secreted from Bone Marrow Mesenchymal Cells Mediate Proliferative and Survival Advantage in Acute Myeloid Leukaemia

BackgroundHaematopoietic stem cell transplantation is still the most effective anti-leukaemic therapy for AML treatment for a large number of patients, but a significant proportion of these will relapse post-transplant and the probability of long term survival is low.The bone marrow microenvironment has been implicated as a major contributor to chemotherapy resistance and relapse through mediating interactions between residual haematopoietic stem cells (HSC), leukaemic stem cells (LSC) and mesenchymal stem cells (MSC) which have been shown to support and maintain the leukaemic niche.Interactions within this malignant niche can be facilitated by exosomes, microvesicles secreted by multiple cell types that function as delivery vehicles of cargo consisting of mRNA, DNA, miRNA, enzymes and cytokines. Exosomes have become the focus of much interest in recent years as there is increasing evidence that they are involved in cancer progression and resistance to therapy, however the role of secreted exosomes in mediating cell communication in the post-transplant microenvironment is relatively unknown.ResultsStromal MSC cultures were derived from diagnostic, post-allogeneic bone marrow transplant (BMT) AML patients (AML-MSC, n=20) and normal bone marrow donors (NBM-MSC, n=5). MSC supernatants were collected from monolayers at passage 3 and exosome preparations were extracted and quantified using NanoSight analysis and western blotting for CD81 and CD63 exosome membrane proteins.Results show that exosome particle number and protein content was significantly increased in diagnostic AML-MSCs samples compared to normal and post-BMT samples (p=0.0028). miRNA yield was also found to be significantly higher in diagnostic samples compared to normal and post-BMT marrow MSC production (p=0.0017).Ex vivo co-culture assays using sucrose cushion derived functional exosome preparations from primary AML-MSCs revealed an exosome induced proliferative effect when cultured with primary AML blasts (p<0.05) and a significant protection against TKI treated co-cultures (n=7, p<0.001) confirming the pro-leukaemic effects of AML-MSC derived exosomes, these effects were absent in NBM-MSC derived exosome preparations. Within stromal co-cultures the addition of exosomes exerted a significantly increased proliferative effect compared to stroma alone demonstrating the additional support exosomes provide (p=0.0079)Secreted cytokine profiling was undertaken using a Luminex bead capture array panel of more than 100 secreted targets in paired AML-MSC exosome and total supernatants.AML-MSC exosome profiles were compared to NBM-MSC fractions and contained significantly higher levels of several inflammatory/angiogenic chemokines including MMP-1 (p=0.0286), CXCL2 (p= 0.0497) and CXCL8 IL-8 (p=0.0284) all of which have been previously associated with poor prognosis in AML. This observation was also reflected in the total supernatant cytokine profile.Within the exosomal fractions the following targets showed increased levels compared to their corresponding supernatant; MMP-3 involved in chemokine signalling, IL-1ra and TRAIL both involved in immunological response and implicated in AML blast proliferation. Profiles of the post-BMT exosomal fraction followed the signature of diagnostic-MSC fractions, indicating the persistence of a diseased phenotype.These results demonstrate significant differences in the exosome production and content of AML-MSC compared to NBM-MSC and whilst both NBM and AML derived MSC exosomes are capable of protecting against drug resistance in co-culture, AML-MSC exosomes produce a considerably stronger proliferative drive and this in part may be mediated through a leukaemic specific MMP/pro-angiogenic signalling axis which has the potential to remodel the surrounding microenvironment. Targeting these pathways warrants further investigation to determine whether suppression of the leukaemic microenvironment may influence transplant outcome for these patients. DisclosuresOttmann:Amgen: Consultancy; Celgene: Consultancy, Research Funding; Fusion Pharma: Consultancy, Research Funding; Pfizer: Consultancy; Incyte: Consultancy, Research Funding; Takeda: Consultancy; Novartis: Consultancy.

Genomic and Proteomic Profiling of AF10-Fusion Oncoproteins Reveal Mechanisms of Leukemogenesis and Actionable Targets

The AF10/MLLT10 gene is recurrently involved in chromosomal rearrangements in human leukemia. AF10 rearrangements are linked to a poor prognosis in AML and T-ALL, underscoring the need to identify targeted therapies for AF10-fusion positive leukemia. Defining the molecular mechanisms of oncogenesis mediated by AF10-fusion proteins (AF10-FPs) may unravel novel actionable targets in leukemias with AF10-gene rearrangements. Towards this end, we established tetracycline (Tet)-inducible models of MLL-AF10 and CALM-AF10 AML and performed RNA-seq in AML cells treated with doxycycline (Dox) compared to vehicle treated counterparts. Since Dox treatment completely abrogates AF10-fusion gene expression from the Tet-regulated promoter, these models can be used to characterize the transcriptional landscape of potential AF10-FP target genes. We observed that among transcripts significantly downregulated upon Dox treatment, 168 genes were common in both the MLL-AF10 Tet-Off or CALM-AF10 Tet-Off conditions, indicating a high overlap between potential transcriptional targets of these distinct AF10-FPs. Expectedly, this list included genes previously implicated in leukemogenesis including Hoxa cluster genes, Meis1, Flt3, Mecom, Cd34, Gfi1b, Eya1 and Nkx2-3. Importantly, in addition to these well-characterized genes, we identified a number of novel pathways that were downregulated in the AF10-FP Tet-Off state. The most striking molecular signature of potential AF10-FP-regulated genes emerging from these analyses were factors involved in innate immunity and pro-inflammatory cytokine signaling. Prominent drivers of these molecular signatures included genes of the Jak/Stat and NFkB signaling pathways as well as Interferon response genes. We confirmed that AF10-FPs strongly activated Jak-Stat and NFkB signaling by performing Western blotting for key factors involved in these pathways. Since pro-inflammatory cytokines have been shown to play a role in AML cell survival, we tested the impact of cytokine depletion on murine AF10-FPs-driven AML cells. Proliferation assays demonstrated that AF10-FP-transformed cells could survive significantly better in cytokine-free medium compared to those transformed with other oncogenes such as MLL-AF9, which were completely dependent on cytokines for survival and proliferation in vitro. These results suggest that activation of cytokine signaling may contribute to increased survival of AF10-FP-driven AML cells.Next, we performed proteomic studies in which affinity-purified epitope-tagged AF10-FPs were evaluated for interacting proteins using Mass Spectrometry (MS). While studies on MLL-AF10 fusion are ongoing, our studies revealed that the strongest interactor of the CALM-AF10 fusion protein was the Janus kinase protein Jak1. We confirmed this finding by immunoprecipitation experiments in CALM-AF10 AML cells using a Jak1-specific antibody. Given the role of JAK1 in cytokine-mediated pro-inflammatory signaling, our findings indicate that CALM-AF10 may activate this pathway through direct recruitment of the Jak1 kinase. We sought to directly test the role of JAK1 in AF10-FP-mediated leukemogenesis. For this, we transformed bone marrow stem and progenitor cells from Jak1 floxed mice with the CALM-AF10 fusion. Deletion of Jak1 using Cre-recombinase in CALM-AF10 AML significantly reduced their proliferation in vitro. Furthermore, Jak1 deletion led to a highly significant reduction in the number of colony forming units (CFUs) from CALM-AF10 AML cells, with a particularly striking decrease in the number of blast-like colonies. We also observed a significant increase in differentiation of CALM-AF10 AML cells following Jak1 deletion, demonstrating that Jak1 activity is important for maintaining the CALM-AF10 leukemia cells in an undifferentiated state. Importantly, these results were recapitulated with two different small-molecule JAK1 inhibitors itacitinib and filgotinib that are being tested in clinical trials for a variety of human diseases. Treatment of CALM-AF10 AML cells with these selective JAK1 inhibitors led to a significant, dose-dependent decrease in proliferation accompanied by growth arrest and apoptosis. Taken together, our studies demonstrate that AF10 fusions activate pro-inflammatory signaling by co-opting the Jak-Stat pathway, presenting a potential therapeutic target in AF10-fusion-driven AML. DisclosuresLevine:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Research Funding; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Prelude: Research Funding; Loxo: Consultancy, Equity Ownership; Imago: Equity Ownership; C4 Therapeutics: Equity Ownership; Novartis: Consultancy; Gilead: Honoraria; Isoplexis: Equity Ownership; Epizyme: Patents & Royalties; Roche: Consultancy, Research Funding. Deshpande:A2A Pharma: Membership on an entity's Board of Directors or advisory committees; Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Bone Marrow Stromal Cells Mediate Adhesion Based Drug Resistance in Acute Myeloid Leukaemia through Reciprocal Feedback of the β-Catenin/CD44 Signalling Axis

Eradication of minimal residual disease is a key goal in AML treatment. It has been found that interaction between leukaemic blasts and different cells of the bone marrow niche contributes to AML drug resistance. Previously we have demonstrated that β-catenin may mediate drug resistance in both short and long-term stromal co-culture assays. β-catenin is known to correlate with poor prognosis in AML. β-catenin has dual roles as both a central effector molecule of the canonical Wnt signalling pathway and a component of adherens junction. The role of Wnt/β-catenin in AML has been well established, but the mechanism through which it mediates adhesion in AML remains unclear.Using a human:human co-culture adhesion model, β-catenin was found to be significantly up-regulated on HS5 and AML derived mesenchymal stromal cell (MSC). Lentivirus mediated β-catenin knock-down in AML cells significantly reduced their adhesion on HS5 and primary normal MSC and AML MSC (n=3, P<0.001). β-catenin knock-down also significantly reduced the proliferation of AML cells either on their own or in suspension from co-culture with primary normal MSC and AML MSC (P<0.001, n=3).In order to dissect the mechanisms underlying β-catenin mediated blast cell adhesion phenotype, KG-1a cells were chosen based on their primitive phenotype, high adhesion to human stroma and β-catenin nuclear translocation ability.KG-1a β-catenin knock-down and control cells were co-cultured with HS5 cells for 24hr and soluble factors in supernatants were harvested and analysed by Luminex bead arrays. β-catenin knock down induced reciprocal feedback signals in both KG-1a cells and HS5 cells. KG-1a catenin knockdown cells displayed increased levels of pro-inflammatory and adhesion pathways including: Galectin-3, N-CAM, NCAM1-1, CEACAM-1, osteonectin, CCL3, C5a, CCL4 and TNFα. Other soluble factors, including S100A8, TIMP-1, CD44, IL-2 and MMP3, were found to be down-regulated in KG-1a cells with β-catenin knock down and co-culture with HS5 abrogated these changes, clearly demonstrating stromal compensation in response to β-catenin knockdown.KG-1a β-catenin knock down and control cells were co-cultured with HS5 cells and suspension and adherent fractions harvested for nuclear and cytoplasmic protein extraction. Proteomic analysis of protein lysates using TMT labelling revealed that significant knock down of catenin target and adhesion molecule CD44 was observed in both cytoplasmic and nuclear fractions in both suspension and adherent KG-1a cells with beta-catenin knock down, suggesting canonical Wnt signalling is universally affected regardless of cellular context. Reduced CD44 production in catenin knockout cell lines was also confirmed in the soluble secretory factors from supernatants analysed by Luminex bead arrays, which also showed a significant reduction in soluble CD44 in β-catenin knock down lines (p<0.01). CD44 was also found to be significantly downregulated on KG-1a cell surface by flow cytometry in both suspension and adherent cells with β-catenin knock down(P<0.01), although adherent KG-1a cells showed considerably higher reduction in CD44 compared with suspension KG-1a cells (P<0.001). Proteomic data further demonstrated the upregulation of nuclear CD44 in adherent fractions, compared with suspension fractions, in both β-catenin knock down cells and control cells, suggesting activated and internalized CD44. Previous publications have shown that nuclear CD44 activates stemness factors, such as Nanog, SOX2 and Oct-4 and also PI3K/AKT and MAPK/ERK signal pathways which might contribute to the stroma mediated drug resistance we observed before.In conclusion, stromal feedback drives an AML blast adhesion phenotype through the Catenin/CD44 signalling axis, which is a contributing mechanism underlying the β-catenin-mediated stromal drug protection in AML. This finding indicates the recent development of novel Wnt inhibitors may be insufficient as a monotherapy to eradicate minimal residual disease in AML and further investigation of stromal-specific Wnt/CD44 targeting combinations is required to tackle this resistance mechanism. DisclosuresOttmann:Fusion Pharma: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy; Incyte: Consultancy, Research Funding; Takeda: Consultancy; Pfizer: Consultancy; Amgen: Consultancy.

Association of Clinical Epidemiologic Exposures and Overall Survival with Genome-Wide DNA Methylation Profiles in Acute Myeloid Leukemia: Analysis of the Mayo Clinic AML Epidemiology Cohort

Background: We previously demonstrated that putative clinical epidemiologic exposures associated with leukemia risk are prevalent among AML patients, and some are associated with unique cytogenetic risk group and with clinical phenotype (Finn, Cancer Epidemiol, 2015). Herein, we studied genome-wide DNA methylation in a cohort of AML patients to evaluate the association of individual hyper- and hypo-methylated CpG sites with epidemiologic exposures and overall survival.Methods: The Mayo Clinic AML Epidemiology Cohort is a highly annotated retrospective case series of 295 consecutive patients (pts) with AML diagnosed and treated at Mayo Clinic Florida and Arizona, with central cytogenetics performed in all cases. The prevalence of clinical epidemiologic exposures, past medical and family history as well as medication use and lifestyle was systematically obtained. After IRB approval, we interrogated the cytogenetic database and successfully obtained leukemia DNA from available remnant diagnostic cytogenetic cell pellets in a cohort of 148 AML patients in the Mayo epidemiology case series and performed an assessment of genome-wide DNA methylation using the Infinium HumanMethylation450K BeadChip. Samples were processed using the R Bioconductor package ‘minfi’ using Subset Within Array Quantile Normalization (PMID: 22703947). Individual CpGs that did not reach a detection p-value of <0.05 were filtered out. An internal control was included on each array to control for significant batch effects. To determine differential methylation status of 473,864 individual CpG sites (after exclusion of 11,648 that did not pass detection p-value QC), CpG sites with low interpatient variability, a standard deviation <0.05 of methylation values, and an interquartile range <0.05 of methylation values and X/Y chromosome probes were excluded from analysis. Epidemiologic and important clinical variables occurring in at least 10 pts or previously-identified as of unique interest but in fewer than 10 pts were evaluated, including sex, BMI, performance status and comorbidities, tobacco and alcohol use, family history of hematologic malignancy, medications of interest, history of toxin exposure, and history of immunosuppression and/or solid organ transplant or of secondary and therapy-related AML (t-AML).Associations of differential hypo or hypermethylation at 281,259 CpG sites with epidemiologic exposures/clinical variables were evaluated using Spearman's test of correlation (continuous or ordinal exposures), a Wilcoxon rank sum test (dichotomous exposures), or a Kruskal-Wallis rank sum test (multi-category exposures). A Bonferroni correction was applied for multiple testing, after which p <1.8 x 10-7 was considered as significant. To reduce the likelihood of false-negative findings, we additionally considered p <5 x 10-6 as indicating suggestive evidence of an association. CpG sites and associated gene and function was determined using Illumina manifest file & www.ncbi.nlm.nih.gov/gene.Results: Statistically significant associations (p <1.8 x 10-7) with individual epidemiologic and clinical exposures were identified for 109 unique CpG sites, corresponding to differential methylation in the genes listed in Table. Specifically, obesity (predominantly hypomethylation), specific cytogenetic lesions and risk group, and gender were highly significantly associated with unique CpG methylation status, but not smoking, toxin exposure, family history, comorbidity or performance status, secondary or t-AML, immunosuppression, medication use, of family history in the analysis. A further 353 unique CpG sites had defined suggestive associations (ongoing analysis). We also identified 8 additional CpG sites where differential methylation was associated with overall survival (p <5 x 10-6, Table).Conclusion: Obesity, cytogenetic lesions, and sex are associated with differential methylation of unique CpG sites at AML diagnosis, using stringent univariate statistical criteria. Significant CpG sites were identified in genes previously linked to AML biology and prognosis (DOCK6, HOXB3, MIR10A, FOXN3/CHES1, GPX1, MYST2/KAT7, PTPRD), but some represent novel findings in AML. These preliminary results suggest an association of some AML risk factors and clinical variables with unique gene methylation and will undergo validation in a prospective AML epidemiology dataset. [Display omitted] DisclosuresCerhan:Jannsen: Other: Scientific Advisory Board; Nanostring: Research Funding; Celgene: Research Funding. Foran:Agios: Research Funding; Xencor, Inc.: Research Funding.

Targeting the Epichaperome As an Effective Precision Medicine Approach in a Novel PML-SYK Fusion Acute Myeloid Leukemia

The epichaperome is a new cancer target defined, in part, by changes in the interaction strength between chaperone and co-chaperone proteins to form stable hyperconnected networks that support oncoprotein stability and are vital for tumor survival (Nature 2016, Nature Rev Cancer 2018 and Nature Med 2018). Cancers with this altered chaperone configuration may become susceptible to drugs that target the epichaperome, such as the inhibitor PU-H71. We have developed a novel flow cytometry-based test, the PU-FITC binding assay, to evaluate epichaperome levels at the single cell level and identify patients who are most likely to respond to PU-H71 treatment.A 61-year-old woman was diagnosed with an accelerated phase myeloproliferative neoplasm in 2013 after years of leukocytosis, arthritis, urticaria, and vasculitis. Cytogenetic analysis revealed t(9;15) involving the PML gene. Molecular studies were negative for BCR-ABL, PDGFRA/B and FGFR1 rearrangements as well as JAK2 mutation. Mutations in ETV6 and multiple ASXL1 subclones were present. She was treated with hydroxyurea and in Aug 2013 underwent matched unrelated donor allogeneic stem cell transplantation conditioned with Fludarabine/Melphalan, followed by 3 cycles of vidaza in 2014 for early recurrence. In Jan 2016, she relapsed and was treated with hydroxyurea. She also had painful ulcerations of toes, thought to be an atypical presentation of graft versus host disease complicated by her underlying Raynaud's disease and treated successfully with ruxolitinib. During 2017, she had bone marrow (BM) evidence of recurrent disease, skin lesions consistent with recurrent disease, and significant splenomegaly, but since neutrophils and platelets were preserved, she did not require transfusions and she had no bleeding or infections, she was maintained on hydrea, prednisone and ruxolitinib. In 2017, BM biopsy showed progression to AML with fibrosis. She developed progressive splenomegaly and weight loss and was treated with 2 cycles of decitabine without response. The patient was sent for precision medicine evaluation. Whole-exome sequencing and RNA-seq was performed with a control buccal swab. No somatic alterations in clinically relevant genes were found. However, a novel fusion protein, PML-SYK, was detected and validated by FISH and PCR. We found constitutive Syk, Stat5, Erk and ribosomal S6 kinase phosphorylation. Elevated epichaperome levels were found in the cell populations bearing the translocation, suggesting sensitivity to PU-H71. Also, invitro treatment of patient's cells with PU-H71 resulted in cell death and decreased colony formation. These findings confirm preclinical data in AML in which a relationship between a hyperactive signalosome and epichaperome expression was observed (Cell Reports 2015). Based on the poor prognosis, lack of effective therapies, and laboratory data suggesting sensitivity to PU-H71, the patient was granted compassionate access to this medication by the FDA. After 16 doses of PU-H71 at 300 mg/m2 over 3 months, the patient has attained complete remission, with normalization of peripheral blood counts and <5% marrow blasts. Splenomegaly and all constitutional symptoms have completely resolved. Therapy is ongoing. Phosphorylation of SYK in blast as stem cell populations decreased after treatment.In summary, a novel flow cytometry-based PU-FITC binding assay to evaluate epichaperome levels at the single cell level successfully identified a patient predicted to respond to treatment with PU-H71. This poor prognosis AML patient who had relapsed after allogeneic stem cell transplantation is presently in remission and the assay is being used to screen other potential patients. [Display omitted] DisclosuresRoboz:Sandoz: Consultancy; Janssen Pharmaceuticals: Consultancy; AbbVie: Consultancy; Eisai: Consultancy; Celgene Corporation: Consultancy; Sandoz: Consultancy; Celgene Corporation: Consultancy; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Orsenix: Consultancy; Aphivena Therapeutics: Consultancy; Novartis: Consultancy; Orsenix: Consultancy; Roche/Genentech: Consultancy; AbbVie: Consultancy; Argenx: Consultancy; Bayer: Consultancy; Roche/Genentech: Consultancy; Otsuka: Consultancy; Astex Pharmaceuticals: Consultancy; Otsuka: Consultancy; Argenx: Consultancy; Cellectis: Research Funding; Janssen Pharmaceuticals: Consultancy; Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy; Cellectis: Research Funding; Astex Pharmaceuticals: Consultancy; Celltrion: Consultancy; Aphivena Therapeutics: Consultancy; Celltrion: Consultancy; Daiichi Sankyo: Consultancy; Bayer: Consultancy; Eisai: Consultancy; Daiichi Sankyo: Consultancy. Morgan:Samus Therapeutics, Inc: Employment, Equity Ownership. Chiosis:Samus Therapeutics, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Guzman:Cellectis: Research Funding.

The Novel Small Molecule Inhibitor KS99 Targets AML and Inhibits Stemness Markers STAT3 and ALDH

Introduction: Signal transducer and activator of transcription 3 (STAT3) has been shown to be activated in leukemic stem cells (LSCs), especially in Pre-LSCs, and activation has been associated with poor prognosis in multiple cancers including AML. In AML, sorted CD34+/CD38- and aldehyde dehydrogenase-positive (ALDH+) cells have significantly phosphorylated STAT3 when compared to healthy controls. The Isatin analog, KS99 has shown selective anti-cancer activity against Multiple Myeloma (MM) which may, in part, be mediated by inhibition of BTK activation. Here we demonstrate that KS99 selectively targets AML CD34+/CD38- and ALDH+ cells and inhibits STAT3 phosphorylation while sparing normal hematopoietic stem and progenitor cells (HSPCs).Experimental Design: The pro-apoptotic activity of KS99 for primary human AML cells (n=21) and human AML cell lines (n=9) was evaluated using Annexin V and cell proliferation (MTS) assays. Colony forming assays were carried out to demonstrate the specific LSC-targeted effect of KS99. Flow cytometry was used to detect STAT3 phosphorylation and apoptosis in CD34+/CD38- LSCs and ALDH activity in a series of human AMLs. Next, to assess the preclinical efficacy, the human AML cell line U937 was injected intravenously (IV) into NSG mice. Mice with an established leukemic burden were randomized into treatment groups and treated through an intraperitoneal (IP) injection with either vehicle control or KS99 (2.5mg/kg) every other day x8. Two days after the final treatment, leukemic burden (hCD45+) was quantitated in the bone marrow.Results: AML cell lines exhibited dose-dependent sensitivity to KS99 in the nanomolar range (IC50: 150-400nM) as did most primary AML cells (Figure: A). Interestingly, poor prognosis AML subsets, AML with MDS related changes (MDS-RCs), were more sensitive than De Novo AML cases (p= 0.0077) with or without a NMP1 mutation (p= 0.012, p=0.045, respectively). Within De Novo AML, NMP1 wild-type cases were more sensitive than NMP1 mutant cases (p= 0.02) (Figure: B). Furthermore, KS99 treatment selectively reduced the clonogenicity of human AML patient cells vs. normal cord blood mononuclear cells (p<0.001) (Figure: C). KS99 induced apoptosis in the CD34+/CD38- gated cell population of primary AML, leading to reduced blast-like and stem-like cells, but not other cells. It also showed a reduction in ALDH+ cells in an Aldefluor assay. Likewise, KS99 treatment showed reduced pSTAT3 in viable CD34+/CD38- cells (Figure: D). Inhibition of pBTK, BCL-2, and pSTAT3 were seen in Western blot experiments. Animal studies showed 76 % reduction of hCD45+ U937 cells in bone marrow of KS99-treated mice when compared to bone marrow isolated from vehicle-treated animals (p<0.05) (Figure: E).Conclusion: KS99 selectively targets LSCs from poor prognosis AML patients while sparing normal HPSCs. This effect may be mediated via targeting of STAT3 signaling and ALDH, which are critical for proliferating LSCs, but not normal HPSCs. Further studies will aim to demonstrate mechanisms of activity and preclinical efficacy in patient-derived AML xenograft mouse models. [Display omitted] DisclosuresDovat:Elf Zone, Inc.: Membership on an entity's Board of Directors or advisory committees.

Prediction of Competing Endogenous RNA Coexpression Network as Prognostic Markers in AML

Background: Recently, competing endogenous RNAs (ceRNAs) hypothesis composed of long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and messenger RNAs (mRNAs), has gained a great interest in the study of molecular biological mechanisms of cancer occurrence and progression. However, studies on leukemia are limited, and there is still a lack of comprehensive analysis of lncRNA-miRNA-mRNA ceRNA regulatory network of AML based on high -throughput sequencing and large-scale sample size. Methods: We obtained RNA-Seq data and compared the expression profiles between 407 normal whole bloods (GTEx dataset) and 151 bone marrows of AML (TCGA dataset). R package (edgeR) was used to identify differentially expressed genes in AML and normal samples. ClusterProfiler was applied for Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA). The similarity between two sets of genes with trait in the network was measured by weighted correlation network analysis (WGCNA). MiRcode, starBase, miRTarBase, miRDB and TargetScan was used to predict interactions between lncRNAs, miRNAs and target mRNAs. The univariate and multivariate cox proportional hazard regression analysis were conducted to verify the association of clinicopathological variables and the expression of genes with patients' survival. Findings: Significantly different expressed mRNAs and lncRNAs between the samples from normal tissues and AML patients by WGCNA were explored to enrich modules which were most related with AML. MiRNA dataset was applied to predict target mRNAs. Multivariate cox proportional hazard regression analysis was utilized to set up a survival model with 8 target mRNAs: HOXA9 INSR KRIT1 MYB SPRY2 UBE2V1 WEE1 ZNF711, of which the area under receiver operating characteristic curve (AUC) was 0.831, indicating its sensitivity and specificity in AML prognostic prediction. At last, we identified 108 lncRNAs, 10 miRNAs and 8 mRNAs to construct the lncRNA -miRNA-mRNA ceRNA network, which might act as biomarkers of AML. Interpretation: CeRNA networks could open an important stratum of study on gene regulation, development and prognosis of AML. Funding Statement: This study was supported by the Science and Technology Planning Project of Guangdong Province (No. 2016A020215078 to Z.-J. Long), the Natural Science Foundation of Guangdong Province (No. 2018A030313071 to Z.-J. Long) and the Fundamental Research Funds for the Central Universities (No. 18ykzd01 to Z.-J. Long). Declaration of Interests: All authors declare that they have no competing interests.

Abstract 1892: Discovery of targeted therapies for acute myeloid leukemia patients with NUP98-NSD1 and FLT3-ITD

Abstract NUP98-NSD1 fusion resulting from the t(5;11)(q35;p15.5) is a clinically significant indicator of adverse prognosis in AML. While the understanding of NUP98-NSD1-driven leukaemogenesis has increased over the years, these patients still suffer from highly progressive disease with no effective therapies. To address this challenge, we analyzed primary patient samples and experimental cell models by high-throughput drug screening and RNA sequencing. Primary samples included bone marrow mononuclear cells (BM MNCs) from three NUP98-NSD1/FLT3-ITD AML patients and ten healthy donors. Experimental cells included lineage-depleted balb/c BM cells transduced with chimeric NUP98-NSD1 and FLT3-ITD retroviruses alone or together, and Ba/F3 cells transduced with human-cloned NUP98-NSD1 lentivirus. The cells were seeded on 384-well plates containing up to 306 approved and investigational drugs in five concentrations over a 10,000-fold range and incubated for 72h at +37°C. Cell viabilities were measured using CellTiter-Glo®. Drug sensitivity scores (DSS) were calculated from area under the dose response curve and select DSS by subtracting DSS of mock-transduced parental cells from the experimental model. We discovered 14 drugs with highly significant mean DSS difference (p &amp;lt; 0.001) between patients and healthy controls. Further analysis revealed that only two of these drugs (dasatinib and navitoclax) had positive correlation between DSS and BM blast percentage (Pearson's R: 0.83-0.98) as well as higher mean DSS in the context of other AML patients with (n = 9) and without (n = 38) FLT3-ITD. Experimental balb/c cells expressing NUP98-NSD1 had significantly increased sensitivity to BCL2 inhibitors (n = 3) (p &amp;lt; 0.05), while cells co-expressing NUP98-NSD1 and FLT3-ITD had significantly increased sensitivity to FLT3 (n = 11) and MEK inhibitors (n = 6) in comparison to cells expressing FLT3-ITD alone. In pilot combination screen, select efficacy of FLT3 and MEK inhibitors was further increased in the dual positive cells by dasatinib (100 nM). In synergy screens, navitoclax-dasatinib and navitoclax-quizartinib combinations were highly synergistic in primary cells and balb/c cells co-expressing NUP98-NSD1 and FLT3-ITD. Gene expression analysis showed significant up-regulation of dasatinib target genes LCK and FGR, and navitoclax target gene BCL2A1 in the primary AML cells. Interestingly, BCL2A1 was also upregulated in the balb/c BM cells expressing NUP98-NSD1 alone or with FLT3-ITD, but not in cells expressing FLT3-ITD alone. Taken together, we have identified potential candidate drugs and drug combinations for t(5;11) positive AML. Although the sample size was small, our data suggests that further research into BCL2 inhibitors in combination with TKIs may eventually translate to better survival outlook of AML patients with t(5;11). Citation Format: Jarno Kivioja, Angeliki Thanasopoulou, Mika Kontro, Ashwini Kumar, Bhagwan Yadav, Muntasir Mamun Majumder, Kimmo Porkka, Juerg Schwaller, Caroline A. Heckman. Discovery of targeted therapies for acute myeloid leukemia patients with NUP98-NSD1 and FLT3-ITD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1892.

Abstract 1429: MYH8 R1292X: A novel mutation in relapsed AML induces EMT features and poor prognosis

Abstract Introduction: Recent advances in tumor genomic analysis have led to the discovery of NPM1, FLT3, CEBPA, MLL, NRAS, and RUNX1 alterations as the cause of AML. Nevertheless, there is a limit to the treatment and clarification of AML, and research for the identification of novel genetic alterations that cause AML is actively underway. Materials and methods: In this study, we performed Whole exome sequencing (WES) with 53 AML patient's samples and conducted targeted re-sequencing using 391 AML patient's samples based on locus of somatic mutation that were found by WES. For functional validation of novel oncogenic mutations, we used CRISPR-Cas9 system to generate knock-in (KI) cell line. For characterization of mutant cells, we performed proliferation assay, cell cycle assay, adhesion assay, and wound healing assay. Epithelial to mesenchymal transition (EMT) markers were checked by western blotting. Results: Using WES and targeted resequencing, we could identify MYH8 R1292X novel mutation as recurrent potentially oncogenic mutation. Additional validation using separate AML cohort revealed MYH8 R1292X variants in four AML patients, suggesting that MYH8 R1292X is potential oncogenic mutation. In functional validation using KI cell line, we could not find change in morphology of KI cells. However, there was a difference in proliferation – the rate of proliferation was faster in KI cells than in cells without mutation. In the cell cycle assay, the mutant cells showed more S phase DNA than the non-mutant cells. Wound healing assay showed that the mutant cells had higher migration ability and lowered the ability of adhesion in comparison. PCR and western blot showed that EMT markers except vimentin increased in mutant cells.Survival analysis based on TCGA data showed that both the overall survival and the disease-free survival curves were significantly different according to MYH8 alterations. Conclusion: Taken together, we conclude that the novel alteration MYH8 R1292X is associated with recurrent AML and poor prognosis by increasing migration, and inducing an increase in EMT markers. Citation Format: Hyejoo Park, Daeyoon Kim, Dongchan Kim, Yungyeong Park, Youngil Koh, Sung-Soo Yoon. MYH8 R1292X: A novel mutation in relapsed AML induces EMT features and poor prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1429.

Study of DNA methyl transferase 3A mutation in acute myeloid leukemic patients

BackgroundRecent studies have shown that somatic mutations in DNA methyltransferase (DNMT3A) might affect the prognosis of AML. ObjectiveThe aim of this work was to investigate the frequency and prognostic impact of the most frequent mutation of DNMT3A, R882H mutation in AML using simple and rapid molecular techniques. Patients and methodsWe have used allele-specific blocker (ASB-PCR) and endonuclease restriction for the detection of DNMT3A R882H mutation in 56 adult patients with AML. ResultsDNMT3A R882H mutation was detected in 7/56 (12.5%) of patients. R882H mutation positive patients were older compared to the wild-type AML (p = 0.08). No association was found with initial laboratory parameters including white blood cells (WBC), hemoglobin (HGB) and Bone marrow (BM) blasts (p > 0.05). Thirty-two patients (57.1%) achieved complete remission (CR), 11/56 (19.6%) died before day 28 induction death (ID) and 13/56 (23.2%) had resistant disease (RD). DNMT3A R882H positive patients were not different regarding the response to induction chemotherapy (CR) compared to the negative group (wild-type) (p > 0.05). Median follow-up period for all patients was 1.6 months, Overall survival (OS) was 65%, and the median was 9.89 months. OS of DNMT3A positive patients was not statistically significant compared to wild-type patients (p = 0.09). Disease free survival (DFS) was 54.6% for all patients, with no difference between wild and mutants (0.59) patients. ConclusionDNMT3A R882H is a frequent mutation in adult de novo AML. The frequency of the mutation tends to increase with age. The two methods used in the study are easy to interpret and are recommended for rapid detection of the mutation required for risk stratification.

Gene mutational pattern and expression level in 560 acute myeloid leukemia patients and their clinical relevance

BackgroundCytogenetic aberrations and gene mutations have long been regarded as independent prognostic markers in AML, both of which can lead to misexpression of some key genes related to hematopoiesis. It is believed that the expression level of the key genes is associated with the treatment outcome of AML.MethodsIn this study, we analyzed the clinical features and molecular aberrations of 560 newly diagnosed non-M3 AML patients, including mutational status of CEBPA, NPM1, FLT3, C-KIT, NRAS, WT1, DNMT3A, MLL-PTD and IDH1/2, as well as expression levels of MECOM, ERG, GATA2, WT1, BAALC, MEIS1 and SPI1.ResultsCertain gene expression levels were associated with the cytogenetic aberration of the disease, especially for MECOM, MEIS1 and BAALC. FLT3, C-KIT and NRAS mutations contained conversed expression profile regarding MEIS1, WT1, GATA2 and BAALC expression, respectively. FLT3, DNMT3A, NPM1 and biallelic CEBPA represented the mutations associated with the prognosis of AML in our group. Higher MECOM and MEIS1 gene expression levels showed a significant impact on complete remission (CR) rate, disease free survival (DFS) and overall survival (OS) both in univariate and multivariate analysis, respectively; and an additive effect could be observed. By systematically integrating gene mutational status results and gene expression profile, we could establish a more refined system to precisely subdivide AML patients into distinct prognostic groups.ConclusionsGene expression abnormalities contained important biological and clinical informations, and could be integrated into current AML stratification system.

FDA approves liposome-encapsulated combination of daunorubicin-cytarabine for adults with some types of poor prognosis AML

FDA approves liposome-encapsulated combination of daunorubicin-cytarabine for adults with some types of poor prognosis AML

BCRP mRNA and FLT3‐ITD are independent poor risk factors in adult patients with acute myeloid leukemia and intermediate or normal karyotype

FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is aberration associated with poor prognosis in AML. We have analyzed the expression of MDR-1, MRP-1, and BCRPmRNA in relation to FLT3-ITD in 100 AML adult patients with normal and intermediate karyotype. The RQ-PCR method was performed to assess the expression of MDR-1, MRP-1, and BCRPmRNA, and the results were presented as coefficients calculated using an intermediate method according to Pfaffl's rule. According to univariate analysis, the following pretreatment variables negatively influenced disease-free survival (DFS): WBC count ≥25×109 /L (P=.037), MRP-1 mRNA ≥1.6818 (P=.028), BCRPmRNA ≥1.1892 (P=.004), FLT3-ITD (P=.005) and overall survival (OS): WBC count ≥25×109 /L (P=.031), MRP-1 mRNA ≥1.6818 (P=.01), BCRPmRNA ≥1.1892 (P=.01), FLT3-ITD (P=.001). When all prognostic variables were pooled into a multivariate model, we found that WBC count ≥25×109 /L (P=.026) and BCRPmRNA ≥1.1892 (P=.011). We observed trend in negative influence of FLT3-ITD on DFS (P=.057). BCRPmRNA ≥1.1892 (P=.035) and FLT3-ITD (P=.006) negatively, independently influenced the OS. The high expressions of BCRPmRNA calculated with Pfaffl's rule and FLT3-ITD are independent poor risk factors in adult patients with AML and intermediate or normal karyotype.

Abstract LB-081: Novel and effective RUNX1-targeted therapy for AML expressing RUNX1 mutation

Abstract RUNX1 is a transcription factor involved in normal and malignant hematopoiesis. Somatic mutations in RUNX1 (mtRUNX1) have been documented in de novo and secondary AML (10%), MDS (~10%) and CMML (up to 37%). Germ-line, mono-allelic mutations and deletions in RUNX1 cause the highly-penetrant (~40%) autosomal dominant, Familial Platelet Disorder with a propensity to Myeloid Malignancy (FPD-MM). The majority of mtRUNX1 are missense mutations, large deletions or truncation-mutations in the DNA-binding ‘RUNT’ homology domain, or in the C-terminal transactivation domain. These mutations behave mostly as loss of function mutations, associated with relative resistance to standard chemotherapy and an unfavorable prognosis in AML. This highlights an unmet need to develop and test novel targeted therapies for AML due to germ-line or somatic mtRUNX1. In the present studies, we demonstrate that shRNA-mediated knockdown of mutant and wild-type RUNX1 repressed its target genes MYC, PU.1 and MPO (myeloperoxidase), as well as inhibited growth and induced apoptosis of AML cells expressing mtRUNX1 (OCI-AML5 and Mono-Mac-1). Ex vivo depletion of RUNX1 abrogated the leukemia initiating potential of OCI-AML5 cells. After engraftment, tetracycline-inducible shRNA-mediated in vivo knockdown of RUNX1, as compared to the non-induced controls, enhanced survival of the immune-depleted (NSG) mice engrafted with OCI-AML5 cells. RUNX1 transcription is driven by the BET protein BRD4-occupied super enhancer in the first intron of the RUNX1 gene. A heat map of Hi-C interaction scores within the RUNX1 TAD (topology-associated domain) showed triangle-shaped regions of high interaction, with CTCF binding sites defining the TAD boundaries, anchoring a loop that separates the RUNX1 TAD from other TADs. Consistent with this, our findings show that, shRNA-mediated depletion of BRD4 or treatment with BET protein (BETP) inhibitor (BETi) OTX015 reduced BRD4 occupancy at the enhancer and promoter of RUNX1. This was associated with depletion of RUNX1 and its target-gene expressions and apoptosis of cultured and patient-derived (PD), primary AML blast progenitor cells (BPCs). Additionally, treatment of NSG mice engrafted with luciferase-transduced OCI-AML5 cells with OTX015 (50 mg/kg/day X 5 days, for 3 weeks) reduced the AML burden and significantly improved their survival (p &amp;lt; 0.01). Co-treatment with the BETi and BCL2-antagonist venetoclax or CDK4/6 antagonist palbociclib or decitabine synergistically induced apoptosis of OCI-AML5 and PD AML BPCs (combination index values of &amp;lt; 1.0). Results describing the in vivo efficacy of BETi and BETP-PROTAC (proteolysis targeting chimera), alone and in combinations against AML expressing mtRUNX1 will be updated at the AACR meetings. Collectively, these findings highlight novel and effective, mechanistically-targeted, single agent or combination, BETP antagonist-based therapy of AML expressing mtRUNX1. Citation Format: Christopher P. Mill, Courtney DiNardo, Warren C. Fiskus, Dyana T. Saenz, Baohua Sun, Agnieszka J. Nowak, Misun Kim, Mark Routbort, Koichi Takahashi, Kapil N. Bhalla. Novel and effective RUNX1-targeted therapy for AML expressing RUNX1 mutation [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 LB-081. doi:10.1158/1538-7445.AM2017-LB-081

Abstract 994: Diagnosing leukemia structural variations via a novel genome mapping approach

Abstract Identification of structural variants (SVs) is critical to the prognosis of AML. SVs are not readily identified by next generation sequencing methods and are currently diagnosed by cytogenetics, an intrinsically low resolution tool. We have adapted a new method for determining large SVs as a clinical tool for evaluating AML and other cancer genomes. The Bionano Irys images in microfluidic nanochannels large (&amp;gt;150 Kb) segments of genomic DNA that have been bar coded by site specific nicking and local incorporation of fluorescent label. The complement of individual molecules can be assembled through matching bar codes to create a de novo map of the genome from which, by matching to a reference genome, identifies and catalogs structural variants at resolution of ca. 1 Kb. With this technology, we characterized structural variants signatures in breast cancer, renal clear cell carcinoma, and CML cell lines. Structural variants were then validated via PCR/Sanger sequencing, fluorescent in situ hybridization and whole genome sequencing. We identified a thorough compendium of consistent false positive rearrangements identified by this technology, most of which reflect errors in the reference human genome assembly. We then applied this methodology to ten AML patient samples, seven from archived blood samples and three directly obtained from newly diagnosed patients. Karyotyping on one archived sample identified a t(6:9) translocation in the majority of cells. We identified that rearrangement at a much higher resolution than cytogenetics, which allowed us to map the breakpoint via PCR/Sanger sequencing to nucleotide resolution and confirm a fusion of NUP214 to DEK, a known oncogene. We also discovered the reciprocal t(9:6) translocation, which had not been identified by cytogenetics. In the six other cases from archived samples, we have confirmed the translocation identified by cytogenetic karyotyping and in several cases found additional rearrangements. Moreover, in the first pediatric case, which we completed within five days of obtaining the sample, we have identified four distinct translocations, all of which have been associated previously with AML. Overall, this is technology has the ability to quickly and accurately identify SVs in cancer cell lines and AML patient samples with higher resolution than cytogenetics, demonstrating its clinical usefulness in the diagnosis and subsequent treatment of leukemia. Citation Format: Christopher Pool, Jie Xu, Darrin Bann, David Goldenberg, Arati Sharma, David Claxton, Feng Yue, James Broach. Diagnosing leukemia structural variations via a novel genome mapping approach [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 994. doi:10.1158/1538-7445.AM2017-994

Effect of cytarabine/anthracycline/crenolanib induction on minimal residual disease (MRD) in newly diagnosed FLT3 mutant AML.

7016 Background: Baseline characteristics such as age &gt;60, WBC&gt;100,000/µL and FLT3/NPM1/DNMT3A+ve are known to be associated with a poor prognosis in AML. Ivey et al. (NEJM 2016) reported that FLT3-ITD+ve patients (pts) who were MRD+ve after 2 cycles of induction chemotherapy were more likely to relapse as compared to those who became MRD-ve (92% vs 35%). Eradication of FLT3+veclones may lead to reduced relapse rates. Crenolanib is a type I FLT3 TKI, which inhibits both FLT3-ITD and TKD mutations. We here report that a single induction cycle of cytarabine/anthracycline/crenolanib leads to MRD negativity by multiparameter flow cytometry (MPF), and low rate of early relapse in pts with newly diagnosed FLT3+veAML. Methods: This abstract includes 29 consecutively treated, newly diagnosed, FLT3+ve AML pts, who achieved CR1 after one course of cytarabine/anthracycline/crenolanib. Pts received 7+3 induction with cytarabine 100 mg/m2/d for 7d and either daunorubicin (&lt;60 y: 90 mg/m2; ≥60 y: 60 mg/m2) or idarubicin 12 mg/m2 for 3d. Crenolanib (100 mg TID) was started on day 9 until 72 h prior to next chemotherapy. Results: 29 pts (15M, 14F), median age 55y (10pts ≥60y) are included. MRD at time of count recovery was assessed by MPF in 25/29 pts. 20/25 (80%) became MRD–ve. With a median follow up of 7mth, 4/25 pts have relapsed (2/5 MRD+ve, 2/20 MRD-ve).Age ≥60 was a risk factor for MRD+ve and relapse. All 4 pts with WBC&gt;100K as well as 5 pts with FLT3/NPM1/DNMT3A+ve AML became MRD-ve after one induction cycle and none have relapsed. Conclusions: These data suggest, in the context of an ongoing trial (NCT02283177), crenolanib in combination with standard induction is associated with a high rate of achieving an MRD negative state by MPF and a low rate of relapse in previously untreated adults with mutant FLT3. Longer follow-up and comparison of MRD data with similar pts treated with standard chemo alone will be necessary to reach more definitive conclusions. Clinical trial information: NCT02283177. [Table: see text]

Clinical observations of AML expressing mutant RUNX1 and pre-clinical studies of RUNX1-targeted novel therapy of AML.

7030 Background: Runt-related transcription factor 1 ( RUNX1) is critically involved in normal and malignant hematopoiesis. Somatic mutations in RUNX1 occur in ~10% of AML, especially in older patients with history of radiation or antecedent hematologic disorder. Presence of mRUNX1 is reported to confer relative resistance to therapy and poorer prognosis in AML, and there are no mRUNX1-targeted or specific therapies available. Methods: We retrospectively analyzed outcomes of 94 mRUNX1 and 444 wild-type RUNX1 AML patients treated at our institution from 9/2013 to 12/2016. We also determined the pre-clinical efficacy of a targeted therapy against cultured and primary AML cells expressing mRUNX1. Results: 67% of mRUNX1 patients were &gt; 65 years of age. Co-occurring mutations with mRUNX1 were ASXL1 (33%), N/KRAS (20%), FLT3 (20%), IDH2 (18%), IDH1 (13%) and TET2 (10%). In patients &gt; 65 years treated with hypomethylating agent-based therapy, the presence or absence of mRUNX1 did not impact response rate (42% vs 46% CR/CRp, p = 0.67), median event-free survival (3.4 vs 4.7 mo, p = 0.82) or overall survival (11.5 vs 9.3 mo, p = 0.97). In mRUNX1 expressing AML OCI-AML5 and MonoMac1 cells, knockdown of RUNX1 by shRNA repressed its targets, e.g., MYC and PU.1, inhibited growth and induced apoptosis. Ex vivo knockdown of RUNX1 abrogated in vivo leukemia initiation by OCI-AML5 cells. After engraftment, inducible shRNA-mediated in vivo knockdown of RUNX1 restored survival of immune-depleted NSG mice engrafted with OCI-AML5 cells. RUNX1 transcription is driven by a super enhancer occupied by the bromodomain extraterminal protein (BETP), BRD4. Accordingly, BRD4 knockdown by shRNA or treatment with the BRD4-inhibitor OTX015 depleted RUNX1 and its targets, and induced apoptosis of AML cells. Treatment of OCI-AML5 cell-engrafted NSG mice with OTX015 (50 mg/Kg/day X 5, for 3 wks) reduced AML burden and improved survival (p &lt; 0.01). Co-treatment with the BETi and BCL2 inhibitor venetoclax or CDK4/6 antagonist palbociclib or decitabine synergistically induced apoptosis of OCI-AML5 and primary AML blasts. Conclusions: These findings highlight a novel, promising, BETP antagonist-based therapy of AML expressing mtRUNX1.

Decitabine-Vorinostat combination treatment in acute myeloid leukemia activates pathways with potential for novel triple therapy.

Despite advancements in cancer therapeutics, acute myeloid leukemia patients over 60 years old have a 5-year survival rate of less than 8%. In an attempt to improve this, epigenetic modifying agents have been combined as therapies in clinical studies. In particular combinations with Decitabine and Vorinostat have had varying degrees of efficacy. This study therefore aimed to understand the underlying molecular mechanisms of these agents to identify potential rational epi-sensitized combinations.Combined Decitabine-Vorinostat treatment synergistically decreased cell proliferation, induced apoptosis, enhanced acetylation of histones and further decreased DNMT1 protein with HL-60 cells showing a greater sensitivity to the combined treatment than OCI-AML3. Combination therapy led to reprogramming of unique target genes including AXL, a receptor tyrosine kinase associated with cell survival and a poor prognosis in AML, which was significantly upregulated following treatment. Therefore targeting AXL following epi-sensitization with Decitabine and Vorinostat may be a suitable triple combination. To test this, cells were treated with a novel triple combination therapy including BGB324, an AXL specific inhibitor. Triple combination increased the sensitivity of OCI-AML3 cells to Decitabine and Vorinostat as shown through viability assays and significantly extended the survival of mice transplanted with pretreated OCI-AML3 cells, while bioluminescence imaging showed the decrease in disease burden following triple combination treatment.Further investigation is required to optimize this triple combination, however, these results suggest that AXL is a potential marker of response to Decitabine-Vorinostat combination treatment and offers a new avenue of epigenetic combination therapies for acute myeloid leukemia.

High level of miR-196b at newly diagnosed pediatric acute myeloid leukemia predicts a poor outcome.

Differential expression of microRNAs (miRNAs) has been implicated in leukemogenesis. We investigate the expression pattern of miR-196b. Using quantitative real-time PCR (qRT-PCR), we detected the expression of miR-196b and its correlated genes (SMC1A/MLH1) in initial pediatric AML. A significant association was observed between overexpression of miR-196b and inferior overall survival of pediatric AML (Log Rank P<0.0001). AML M4/5 subtype, high white blood cell (WBC) count at presentation, MLL rearrangement, or FLT3-ITD mutation at diagnosis and non-remission group after the first induction chemotherapy possessed higher miR-196b expression. Furthermore, a positive relationship was found between the expression of miR-196b and SMC1A/MLH1 (Spearman's r=0.37 and 0.44, P=0.001 and <0.0001, respectively). Taken together, these findings suggest that differentially high expression of miR-196b in diagnostic marrow samples of pediatric AML is associated with unfavorable outcome, and miR-196b potentially can be a novel biomarker for the diagnosis, prognosis and treatment in pediatric AML.