Clinical Outcome In Acute Myeloid Leukemia Patients Research Articles

GPR68 supports AML cells through the calcium/calcineurin pro-survival pathway and confers chemoresistance by mediating glucose metabolic symbiosis

Accumulating evidence demonstrates that the “Warburg effect” that glycolysis is enhanced even in the presence of oxygen existed in hematopoietic malignancies, contributing to extracellular acidosis. G-protein coupled receptor 68 (GPR68), as a proton sensing GPCR responding to extracellular acidosis, is expected to play a critical role in hematopoietic malignancies. In the present study, we found that GPR68 was overexpressed in acute myeloid leukemia (AML) cells, and GPR68 deficiency impaired AML cell survival in vitro and cell engraftment in vivo. Mechanistic studies revealed that unlike GPR68 regulates Calpain1 in myelodysplastic syndromes (MDS) cells, GPR68 deficiency reduced cytosolic Ca2+ levels and calcineurin (CaN) activity in AML cells through an NFAT-independent mechanism. Moreover, the decreased Ca2+ levels disturbed cellular respiration (i.e., oxidative phosphorylation, OxPhos) by inhibiting isocitrate dehydrogenase (IDH) activity; this was more pronounced when BCL2 was inhibited simultaneously. Interestingly, GPR68 inhibition also decreased aerobic glycolysis in AML cells in a Ca2+-independent manner, suggesting that GPR68 mediated glucose metabolic symbiosis. As glucose metabolic symbiosis and the heterogeneous dependencies on aerobic glycolysis and cellular respiration tremendously impact chemosensitivity, the inhibition of GPR68 potentiated the tumoricidal effect of first-line chemotherapeutic agents, including BCL-2 inhibitors targeting OxPhos and cytarabine (AraC) targeting glycolysis. Consistent with these in vitro observations, higher levels of GPR68 were associated with inferior clinical outcomes in AML patients who received chemotherapies. In short, GPR68 drives the Ca2+/CaN pro-survival pathway and mediates glucose metabolic pathways in AML cells. Targeting GPR68 eradicates AML cells and alleviates chemoresistance, which could be exploited as a therapeutic target.The overexpression of GPR68 drives a Ca2+/CaN pro-survival pathway and mediates glucose metabolic symbiosis in AML cells, suggesting the diagnostic and therapeutic potential of GPR68 in AML. (GPR68, G proton-coupled receptor 68; PLCβ, phospholipase C beta; CaN, Calcineurin; IDH, isocitrate dehydrogenase; HIF-1α, Hypoxia-inducible factor alpha subunit; GLUT1, Glucose transporter type 1; HK-1, Hexokinase 1).

UBC9 Deficiency Accelerates AML Progression By Downregulating PARP1 Sumoylation and Subsequently Activating the NF-ΚB Pathway

Acute myeloid leukemia (AML) is the most common acute leukemia in adults and is a disease of heterogeneous clonal hematopoietic progenitor cells. Despite the relentless development of therapeutic options, including chemotherapy, and identification of many new therapeutic targets, the prognosis of AML is still poor, with a 5-year survival rate of approximately 35% in patients younger than 60 years old and 10% in patients older than 60 years, which may be due to the high degree of AML heterogeneity. Targeting the shared mechanisms of action among subclones has emerged led to find novel strategies, such as targeting protein post post-translational modification. Here we show that the impact of the UBC9 (the only E2 conjugation enzyme) and its related SUMOylation, a critical and reversible posttranslational modification as ubiquitination, on AML progression, to provide novel and potent sight for AML therapy. To identify the role of UBC9 expression in the clinical outcome of AML patients, we measured the expression of key SUMOylation genes, including SAE1, UBA2, UBC9, SUMO1, SUMO2, SUMO3, PIAS1, PIAS2, PIAS3, PIAS4, CBX4, and RANBP2, in the TCGA dataset (n=164) by using a multivariate survival curve based on the online tool SurvExpress. Our results showed that the survival probability curves of the low- and high-risk groups were significantly different due to the differences in the levels of expression of key SUMOylation genes ( p = 0.0011), and we found that the expression level of UBC9 was lower in the high-risk group than in the low-risk group. We next analyzed the UBC9 expression level and the prognosis of AML patients from the Therapeutically Applicable Research To Generate Effective Treatment dataset (n=156), which showed that AML patients with low UBC9 expression showed shorter overall survival ( p = 0.018)(Figure 1). In addition, real-time PCR showed that the UBC9 expression level in bone marrow samples from patients with de novo AML was significantly downregulated in both the high-risk patients based on National Comprehensive Cancer Network guidelines and the liver or spleen enlargement patients ( p = 0.01 and p = 0.001, respectively) (Figure 1). We used RNA interference system to knock out Ubc9 in a mouse model of MLL-AF9 and MLL-ENL induced AML. We observed the survival of AML mice and found that Ubc9 fl/+;Vav-Cre recipient mice showed a significantly shorter survival time than Ubc9 fl/+ mice (Figure 2). In addition, apoptosis experiments revealed reduced apoptosis of leukemia cells and leukemia stem cells in Ubc9 fl/+;Vav-cre mice. HE staining showed significantly higher leukemia cell infiltration in the livers, meninges, and kidneys of Ubc9 fl/+;Vav-cre mice compared to Ubc9 fl/+ mice. Transwell experiments indicated that Ubc9 deficiency resulted in increased migration of leukemia cells. Colony forming assay demonstrated that Ubc9-deficient leukemia cells formed significantly larger colonies. Flow cytometry analysis showed that Ubc9-deficient leukemia cells expressed high levels of c-kit and low levels of Mac1. To further elucidate the role of UBC9 in human AML cells, we constructed UBC9 deficient KG1 and K562 cell lines by using CRISPR-Cas9 technology. The UBC9 KD cell lines presented the same phenotype such as the increase of cell viability and the enhancement of invasiveness. To better understand the role that UBC9 plays in AML cell biology, we performed a system-level approach based on quantitative SUMO proteomic analysis to identify potential UBC9 substrates and then identified PARP1 with SUMOylation site lysine residue 486 (K486) by multiomics analysis. Finally, we validated that UBC9-mediated PARP1 SUMOylation played an important role in AML progression by activating the NF-kappa B pathway via the site-directed mutation of PARP1 (K486R) in UBC9-KO AML cells by using western blot and Co-immunoprecipitation. Together, our study revealed that UBC9 KO accelerated AML progression and enabled us to contrast global SUMOylation profiles of UBC9 in AML cells. In addition, we found that the PARP1 SUMOylation-mediated NF-κB pathway activation is a positive regulatory mechanism in the tumorigenesis of AML. These findings may provide potential clinical implications for AML treatment.

Targeting SIRT5 By Succinylating Hadha Synergizes with Venetoclax in Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is genetically diverse, driven by multiple oncogenic factors. Metabolic dysregulation is a critical vulnerability in AML cells for targeted therapy. BCL2 has been previously reported to maintain mitochondrial oxidative phosphorylation (OXPHOS) in AML cells. One of the mechanisms of action of the combination of venetoclax and azacitidine is to inhibit amino acid metabolism, leading to cell death. Although venetoclax-based treatment regimens have shown promising clinical outcomes in AML patients, treatment resistance has become a newly emerged challenge. Leukemia stem cells isolated from venetoclax-resistant patients exhibit distinctive metabolic features, including elevated nicotinamide metabolism. SIRT5 is an NAD+-dependent deacylase enzyme, and its knockout leads to elevated succinylation of various enzymes involved in OXPHOS. Hence, we hypothesize that inhibiting SIRT5 may augment venetoclax's cytotoxic effect by dampening metabolism. To further elucidate this mechanism, we employed proteome analyses of succinylation to identify specific target molecules regulated by SIRT5. Methods: To determine the effects of combination therapy on cell proliferation, apoptosis, and colony formation, AML cell lines were transduced with lentiviruses carrying either shNC or shSIRT5, or treated with NRD167 (a specific inhibitor of SIRT5), followed by venetoclax treatment. To evaluate the clearance of leukemia cells in vivo, NSG mice were transplanted with MOLM-13 cells (carrying shNC or shSIRT5) and then subjected to vehicle or venetoclax treatment (80 mg/kg/day) via oral gavage for 14 days. Weekly live animal in vivo imaging was conducted to monitor tumor engraftment and growth. The synergistic effect on the metabolic level of AML was assessed by measuring ATP, glutathione, mitochondrial superoxide, and oxygen consumption rates (OCR). Electron microscopy was employed to detect changes in cellular mitochondrial structure after SIRT5 knockdown and venetoclax utilization. Furthermore, lysine succinylation proteome analyses were performed to identify proteins and sites with altered succinylation on lysine before and after SIRT5 knockdown in MOLM-13 cells. Immunoprecipitation assays and Western blot were conducted to verify the interaction and altered succinylation levels of the relevant proteins. Results: The combination of SIRT5 inhibition with venetoclax treatment resulted in further suppression of cell proliferation, reduced colony formation, and increased apoptosis in AML cells compared to venetoclax treatment alone.The combination led to alterations in the metabolic profile of leukemia cells, increased the generation of mitochondrial reactive oxygen species (ROS), reduced intracellular glutathione levels, inhibited ATP production, and suppressed OCR. Notably, combination therapy resulted in a significant reduction in the engraftment levels of human leukemia cells in NSG mice. Mechanistically, electron microscopy revealed that combination treatment with SIRT5 knockdown increased mitochondrial damage. Lysine succinylation proteome analyses demonstrated upregulation of succinylation modification on mitochondrial trifunctional enzyme subunit α (HADHA), a protein involved in the fatty acid oxidation (FAO) pathway. Co-immunoprecipitation of SIRT5 confirmed its interaction with HADHA, and the relative succinylation level of precipitated HADHA was upregulated after SIRT5 knockdown. Moreover, the combination of trimetazidine (a specific inhibitor of HADHA) with venetoclax demonstrated a preferential reduction in cell proliferation and induction of apoptosis in AML cells. Conclusion: Targeting of SIRT5 in combination with venetoclax treatment results in synergistic cytotoxic effects in AML cells. We hypothesize that SIRT5 desuccinylase activity promotes FAO and reduces intracellular mitochondrial ROS levels by desuccinylating HADHA. Targeting SIRT5 inhibits fatty acid metabolism in AML cells, thereby enhancing the therapeutic effect of venetoclax. We will further investigate the site(s) of SIRT5 desuccinylation modification of HADHA through lysine site mutation and assess changes in HADHA enzyme activity to elucidate its regulatory mechanism.

Monitoring Changes in Plasma Proteome in AML Patients Receiving Blood Transfusion Products

Background: Hemato-oncological patients, and in particular patients with acute myeloid leukemia (AML) in the remission induction treatment phase frequently receive transfusion products. These products include platelet- and red blood cell- concentrates and are administered in order to prevent and treat bleeding complications and anemia. During this treatment, patients are constantly monitored by clinical laboratory tests, however, additional studies could add nuanced information about the patients' status. Different studies have shown that plasma proteins change and their patterns' characteristics could be key to illustrate the patients' healthy and/or disease status. Importantly, changes in the plasma proteome in AML patients receiving transfusion products have not yet been studied. Thus, defining protein profiles during this phase is vital to use as a point of reference prior to tracing patients' treatments and their responses. Aim: In light of this, we aim to delineate the baseline plasma proteome in AML patients during their treatment phase. Methods: We employed unbiased mass spectrometry (MS)-based proteomics to gain insight in treatment-induced changes of the plasma proteome (Figure 1A). We included samples derived from 11 healthy donors and 11 AML patients who received chemotherapy and platelet, and red blood cell transfusions. AML patients without a previous transplant or platelet transfusion were included in this study. Results: We identified over 400 proteins in each plasma sample and found associations between proteins and clinical laboratory data, exemplified by a high correlation between Pro-Platelet Basic Protein (PPBP) and platelet incounts (r = 0.7, p-value < 0.001). We observed 169 significantly different proteins between AML patients and healthy controls, amongst the most significant changes we captured a strong signature of inflammation with an increased abundance of proteins involved in the acute-phase response pathway (i.e. SAA1 and CRP) and a decreased abundance of proteins involved in innate immune response (i.e. DEF1 and CAMP). Interestingly, we also found an increased abundance of proteins from the complement pathway (i.e. C9 and CFHR) and decreased abundance in proteins enriching for plasma lipoprotein particle remodeling (i.e. APOA1 and PON1) (Figure 1B). Furthermore, by building correlation networks we analyzed differences in the dynamics of plasma proteins in AML patients compared to healthy controls. This analysis showed several changes in associations between proteins, including alterations in known interactions such as the gain of correlation between APOA1 and GPLD1 as well as a loss of correlation between PLG and KNG1. Conclusion: These results highlight an imbalance in lipid metabolism and innate defense mechanisms, such as the coagulation pathway and complement system, in AML patients during their transfusion-dependent window. The plasma proteome studied in this pilot can serve as the basis to further explore changes induced in specific subgroups of patients, for example patients with and without bleeding complications. Follow up studies to analyze the changes observed here in larger cohorts are currently running (ALPHABET, trial registration NL9204) and they would be key to explore their potential association with clinical outcome in AML patients receiving transfusion products. Figure 1.- Exploring proteome changes in AML patients during their transfusion treatment phase. A) Graphical summary of the study, and B) Proteins with contrasting dynamics when comparing AML patients in treatment phase to healthy controls. Box plots depict median with its upper and lower limits representing the first (25th) and third (75th) percentiles, respectively. Box plot whiskers extend up to the highest or lowest value within 1.5 x IQR.

Venetoclax Overcomes Sorafenib Resistance in Acute Myeloid Leukemia by Targeting BCL2.

Sorafenib, a kinase inhibitor, has shown promising therapeutic efficacy in a subset of patients with acute myeloid leukemia (AML). However, despite its clinical effectiveness, sorafenib resistance is frequently observed in clinical settings, and the mechanisms underlying this resistance as well as effective strategies to overcome it remain unclear. We examined both single-cell and bulk transcription data in sorafenib-resistant and control AML patients and integrated a sorafenib resistance gene signature to predict the sensitivity of AML cells and the clinical outcomes of AML patients undergoing sorafenib therapy. In addition, our drug sensitivity analysis of scRNA-seq data using deconvolution methods showed that venetoclax was effective in targeting sorafenib-resistant AML cells. Mechanistically, sorafenib was found to activate the JAK-STAT3 pathway and upregulate BCL2 expression in sorafenib-resistant AML cells. This upregulation of BCL2 expression rendered the cells vulnerable to the BCL2 inhibitor venetoclax. In conclusion, we developed a platform to predict sorafenib resistance and clinical outcomes in AML patients after therapy. Our findings suggest that the combination of sorafenib and venetoclax could be an effective therapeutic strategy for AML treatment.

FLOT1 knockdown inhibits growth of AML cells through triggering apoptosis and pyroptosis.

Acute myeloid leukemia (AML) is a group of hematological malignancies characterized by clonal proliferation of immature myeloid cells. Lipid rafts are highly organized membrane subdomains enriched in cholesterol, sphingolipids, and gangliosides and play roles in regulating apoptosis through subcellular redistribution. Flotillin1 (FLOT1) is a component and also a marker of lipid rafts and had been reported to be involved in the progression of cancers and played important roles in cell death. However, the role of FLOT1 in AML remains to be explored. In this study, we found that increased expression of FLOT1 was correlated with poor clinical outcome in AML patients. Knockdown of FLOT1 in AML cells not only promoted cell death in vitro but also inhibited malignant cells engraftment in vivo. Mechanically, FLOT1 knockdown triggered apoptosis and pyroptosis. FLOT1 overexpression promoted AML cell growth and apoptosis resistance. Our findings indicate that FLOT1 is a prognostic factor of AML and may be a potential target for AML treatment.

Dysregulated Expression of MiR-19b, MiR-25, MiR-17, WT1, and CEBPA in Patients with Acute Myeloid Leukemia and Association with Graft versus Host Disease after Hematopoietic Stem Cell Transplantation.

Elham JamaliObjectives Acute myeloid leukemia (AML) is a blood malignancy characterized by the proliferation of aberrant cells in the bone marrow and blood that interfere with normal blood cells. We have investigated whether changes in the level of micro-ribonucleic acid (miR)-19b, miR-17, and miR-25, Wilms' tumor (WT1), and CCAAT enhancer-binding protein α (CEBPA) genes expression affect disease prognosis and clinical outcome in AML patients. Materials and Methods The expression level of miR-19-b, miR-17, and miR-25, as well as WT1 and CEBPA genes in a group of patients and controls as well as different risk groups (high, intermediate, and favorite risk), M3 versus non-M3, and graft-versus-host disease (GvHD) versus non-GvHD patients were assessed using a quantitative SYBR Green real-time polymerase chain reaction method. Results When compared with the baseline level at the period of diagnosis before chemotherapy, the expression of miR-19b and miR-17 in AML patients increased significantly after chemotherapy. The level of miR-19b and miR-25 expression in AML patients with M3 and non-M3 French-American-British subgroups differ significantly. MiR-19b and miR-25 expression was elevated in GvHD patients, while miR-19b and miR-25 expression was somewhat decreased in GvHD patients compared with non-GvHD patients, albeit the difference was not statistically significant. Also, patients with different cytogenetic aberrations had similar levels of miR-19-b and miR-25 expression. Conclusion MiR-19b, miR-17, and miR-25 are aberrantly expressed in AML patients' peripheral blood leukocytes, which may play a role in the development of acute GvHD following hematopoietic stem cell transplantation.

Eomes+ T-betint CD8 T Cells Are Functionally Impaired and Associate with Primary Refractory Disease in Patients with Acute Myeloid Leukemia (AML)

Immunotherapy targeting T cell inhibitory mechanisms, such as the PD-1 pathway to unleash the anti-tumor immune response is a promising strategy for cancer treatment. Several studies including ours have demonstrated an involvement of PD-1 and other inhibitory T cell receptors in AML progression. Eomesodermin (Eomes) and T-bet are both T box transcription factors that are crucial in regulating T cell function. Recent studies showed that Eomes and T-bet differentially regulate PD-1 expression and T cell exhaustion in chronic viral infection. Here we examine the effect of these two transcription factors in the pathogenesis of AML using blood samples collected from a large cohort (n=59) of patients with AML. We report that Eomes+T-betintCD8 T cells are increased in AML. Importantly they are functionally impaired and associate with poor clinical outcome.We first performed flow cytometry analyses to examine the intracellular expression of Eomes and T-bet in CD8 T cells of blood samples collected from AML patients at initial diagnosis. Based on the level of Eomes vs. T-bet, three subpopulations were defined among activated CD8 T cells: Eomes- T-bethi, Eomes+ T-bethi, and Eomes+T-betint. We observed a significant increase in the percentage of Eomes+T-betint CD8 T cells from AML patients (n=59) compared to that of healthy donors (n=15) (21.97±1.85% vs. 13.47±2.92%, P=0.0367). In addition, we assessed blood samples from 11 AML patients at initial diagnosis compared to that from the same patients when they are in complete remission (CR). We found that the frequency of Eomes+T-betint CD8 T cells was significantly lower in CR. This finding suggests an association of Eomes+T-betint CD8 T cells with AML progression.We next evaluated the clinical correlation of different Eomes and T-bet expression pattern in AML. Based on the frequency of Eomes+T-betint CD8 T cells, we defined high- (Eomes+T-betint ≥21.72 %) vs. low-(Eomes+T-betint <21.72 %) subgroups in AML patients using the median value of Eomes+T-betint % as the cutoff. Among the 47 AML patients of whom the induction response was able to be evaluated, we found a significantly higher rate of primary refractory disease (failure to achieve CR) in the high-Eomes+T-betint group compared with that of the low- Eomes+T-betint group (7/24 (29.2%) vs. 1/23 (4.3%), P=0.024). Strikingly when the same analysis was applied to Eomes- T-bethi subpopulation cells, we observed an opposite trend, thus low- Eomes- T-bethi expression associates with primary refractory disease. This result indicates a reciprocal effect of Eomes and T-bet on clinical outcome of AML.We further dissected the phenotype and functional status of each T cell subpopulation. The distribution of naïve, central memory, effector memory and terminal differentiated profile was similar among Eomes+T-betint CD8 T cells compared with that of Eomes+ T-bethi and Eomes- T-bethi cells. However, intracellular production of IFN-γ upon in vitro anti-CD3/CD28 stimulation was significantly lower in Eomes+T-betint CD8 T cells compared with that in the other two subpopulations (n=47, P <0.001). In addition, Eomes+T-betint CD8 T cells displayed lower capacity for killing manifested by less expression of Perforin and GranzymeB. Thus consistent with exhaustion, Eomes+T-betint T cells are functionally deficient.To study the effect of Eomes and T-bet in leukemia antigen-specific T cells, we performed a functional assay to detect CD8 T cell response against a WT-1 (a known leukemia-associated antigen) epitope. Purified CD8 T cells derived from HLA-A*0201+ AML patients were co-cultured in vitro with T2 cells (antigen presenting cells) pulsed with HLA-A*0201-binding WT1126-134 or SV40 LT281-289 (as negative control) peptide. Intracellular production of IFN-γ was detected in 1.2% of CD8 T cells after 6 days of stimulation with WT-1 peptide, while essentially 0 CD8 cells responded to control peptide. Importantly Eomes+T-betint leukemia-specific CD8 T cells (gated on IFN-γ+) were less functional as they produced less TNF-α compared with that of the other two subpopulations.Taken together, our study demonstrates a significant association of Eomes+T-betint T cells to immune dysfunction and poor clinical outcome in AML patients. This novel finding provides a great potential to define the prognostic biomarker and therapeutic target for this devastating blood cancer. DisclosuresNo relevant conflicts of interest to declare.

CD33-Single Nucleotide Polymorphism (CD33-SNP) Score Predicts Gemtuzumab Ozogamicin Response in Childhood Acute Myeloid Leukemia: Report from Children's Oncology Group AAML0531 Trial

Introduction: Gemtuzumab ozogamicin (GO) has re-emerged as a potential agent with promise in acute myeloid leukemia (AML) treatment. GO received accelerated approval in 2000 for treatment of relapsed AML in older patients but was withdrawn from the market due to lack of benefit and increased early mortality in SWOG S0106 trial. Several studies thereafter demonstrated GO improved clinical outcomes with pronounced effects in patients with favorable-risk cytogenetics. In light of new results from several randomized trials, an FDA advisory panel recently determined that GO has a favorable risk/benefit profile as an adjunctive therapy for certain newly-diagnosed AML patients. We recently reported that CD33 splicing polymorphism rs12459419 C>T regulates production of alternatively spliced CD33 isoform lacking exon 2 and hence the IgV domain for CD33(Lamba et al, 2017). Consistent with this, we demonstrated that rs12459419 was significant predictor of cell surface CD33 intensities as well as clinical response in the randomized COG-AAML0531 trial. We now expand our investigation to other potentially significant CD33 SNPs for impact on GO efficacy in de novo AML patients enrolled in the same AAML0531 trial.Methods: We genotyped five CD33 SNPs: rs2455069 (Arg60Gly), rs35112940 (Arg304Gly), rs61736475 (Ser305Pro) and rs1803254 and associated these with CD33 cell surface levels and clinical outcome in AML patients (n=817: 408=No-GO and 409=GO). We further developed a composite CD33 SNP-score ranging from -6 to +2, using genotype information on the 4-CD33 SNPs with significant association with cell surface CD33 levels. A directional genotype score was generated for each SNP by taking into account the direction of association of variant allele with CD33 levels. Genotype score ranged from 0 to -2 for rs12459419, rs1803254 and rs35112940 -with variant alleles associated with lower CD33 intensity. For CD33 SNP rs2455069 -with variant allele associated with high CD33 cell surface intensity, genotype scores ranged from 0 to +2 based on number of variant alleles present. Composite CD33-SNP Score = Σ (individual genotype scores of rs12459419, rs1803254, rs2455069 and rs35112940) followed by grouping into score of ≥0 OR <0.Results: CD33 coding SNPs-rs2455069, rs35112940 and 3'UTR SNP-rs1803254 but not rs61736475 were associated with cell surface CD33 levels on AML blasts and superior response with GO. These SNPs did not show any linkage with each other and the effect seen was independent. The CD33-SNPscore was therefore built using genotype scores for previously reported rs12459419 and the new rs2455069, rs1803254 and rs35112940 CD33 SNPs. Patients with a CD33-SNP score of ≥0 had higher cell surface CD33 levels (Figure 1) and demonstrated improved 5-year disease-free survival of 67±8% vs. 49±9% (hazard ratio [HR]=0.58, [95% confidence interval 0.41-0.84]; p=0.004) as well as reduced risk of relapse (24±7% vs. 48±9%; HR=0.448 [95% CI: 0.30-0.67]; p <0.001, Figure 2) when treated with GO vs. NO-GO. GO did not improve outcomes in patients with a CD33-SNPscore of <0, which was associated with lower CD33 expression levels. Subgroup analyses showed similar results, in low- and intermediate-risk patients, but for high-risk group statistical significance was not reached.Conclusion: In this study to comprehensively evaluate the role of CD33 SNPs on outcome we created a composite CD33-SNP_score using a directional genotype score for these three and our previously reported splicing SNP, rs12459419. Our findings suggest the use of GO with intensive induction chemotherapy in childhood AML should be restricted to patients with CD33-SNP score of ≥0. Although CC genotype for our recently reported splicing SNP rs12459419 was significantly represented in CD33-SNP score of ≥ 0, it is important to realize it occurs with an allele frequency of ~0.3 in European patients but is less frequent in other ethnic groups (ranging from ~0.10-0.15 in African American and Asian and non-existent in Sub-Saharan Africans), whereas rs1803254 is more frequent in black vs. white patient (0.32 vs. 0.15), thus the use of a composite CD33-SNP score would broaden identification of those patients who will benefit from GO or other CD33 targeted agents, in non-European populations. Validation of this CD33-SNP score with GO response remains to be performed and if reproduced, future studies should consider the use of the CD33-SNP score to guide GO therapy. [Display omitted] DisclosuresLoken:Hematologics Inc: Employment, Equity Ownership. Walter:ADC Therapeutics: Research Funding; Aptevo Therapeutics: Research Funding.

Clinical implications of sequential MRD monitoring by NGS at 2 time points after chemotherapy in patients with AML

Next-generation sequencing (NGS) has been applied to measurable/minimal residual disease (MRD) monitoring after induction chemotherapy in patients with acute myeloid leukemia (AML), but the optimal time point for the test remains unclear. In this study, we aimed to investigate the clinical significance of NGS MRD at 2 different time points. We performed targeted NGS of 54 genes in bone marrow cells serially obtained at diagnosis, first complete remission (first time point), and after the first consolidation chemotherapy (second time point) from 335 de novo AML patients. Excluding DNMT3A, TET2, and ASXL1 mutations, which are commonly present in individuals with clonal hematopoiesis of indeterminate potential, MRD could be detected in 46.4% of patients at the first time point (MRD1st), and 28.9% at the second time point (MRD2nd). The patients with detectable NGS MRD at either time point had a significantly higher cumulative incidence of relapse and shorter relapse-free survival and overall survival. In multivariate analysis, MRD1st and MRD2nd were both independent poor prognostic factors. However, the patients with positive MRD1st but negative MRD2nd had a similar good prognosis as those with negative MRD at both time points. The incorporation of multiparameter flow cytometry and NGS MRD revealed that the presence of NGS MRD predicted poorer prognosis among the patients without detectable MRD by multiparameter flow cytometry at the second time point but not the first time point. In conclusion, the presence of NGS MRD, especially after the first consolidation therapy, can help predict the clinical outcome of AML patients.

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

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

TIM-3 levels correlate with enhanced NK cell cytotoxicity and improved clinical outcome in AML patients

ABSTRACT Accumulating evidence indicates that immune checkpoint inhibitors (ICIs) can restore CD8+ cytotoxic T lymphocyte (CTL) functions in preclinical models of acute myeloid leukemia (AML). However, ICIs targeting programmed cell death 1 (PDCD1, best known as PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4) have limited clinical efficacy in patients with AML. Natural killer (NK) cells are central players in AML-targeting immune responses. However, little is known on the relationship between co-inhibitory receptors expressed by NK cells and the ability of the latter to control AML. Here, we show that hepatitis A virus cellular receptor 2 (HAVCR2, best known as TIM-3) is highly expressed by NK cells from AML patients, correlating with improved functional licensing and superior effector functions. Altogether, our data indicate that NK cell frequency as well as TIM-3 expression levels constitute prognostically relevant biomarkers of active immunity against AML.

Clinical values of gene alterations as marker of minimal residual disease in non-M3 acute myeloid leukemia

ABSTRACT Acute myeloid leukemia (AML) is a malignant disease of the hematopoietic system. Residual leukemic cells after treatment are associated with relapse. Thus, detecting minimal residual disease (MRD) is significant. Major techniques for MRD assessment include multiparameter flow cytometry (MFC), polymerase chain reaction (PCR), and next-generation sequencing (NGS). At a molecular level, AML is the consequence of collaboration of several gene alterations. Some of these gene alterations can also be used as MRD markers to evaluate the level of residual leukemic cells by PCR and NGS. However, when as MRD markers, different gene alterations have different clinical values. This paper aims to summarize the characteristics of various MRD markers, so as to better predict the clinical outcome of AML patients and guide the treatment.

AML Associated Mesenchymal Stroma Cells Support Growth of AML Cells As a Result of Activated Notch Signaling and This Can be Targeted By Dexamethasone

Introduction: Acute myeloid leukemia (AML) is a hematological malignancy characterized by poor prognosis. Interaction with bone marrow mesenchymal stromal cells (BM-MSC) supports leukemic cells to evade chemotherapy-induced apoptosis. Of particular interest, massive parallel transcriptome sequencing of MSCs from mice and patients with preleukemic syndromes identified inflammatory signaling in inducing genotoxic stress, leukemogenesis, and clinical outcome. Recently, Dexamethasone (Dex), an anti-inflammatory drug, demonstrated antileukemic activity and improved clinical outcome in AML patients. These observations intrigued us to further investigate leukemia-BM niche-associated signaling and to identify signaling cascades supporting leukemic cells and BM-MSC interaction. Methods: BM-MSC from AML patients at diagnosis, or in remission were cultured alone or together with human AML cell lines (Kasumi-1 and M-07e) in direct contact or separated by transwells. Following transplantation experiments, BM-MSC were analyzed by microarray analysis. Gene set enrichment analysis (GSEA) was used to identify the hallmark gene sets overrepresented in AML MSC. The functional characterization of BM-MSC and AML cell interaction was investigated using in vitro (cell proliferation and apoptosis) and in vivo (leukemia development, drug response, and survival) analysis. Results: To functionally evaluate the role of BM-MSC, we co-cultured MSCs from AML (AML-MSC) or non-leukemic (non-AML-MSC) patients with Kasumi-1 or M-07e cells. AML-MSCs significantly supported proliferation of AML cells by altering cell cycle distribution from G0/G1 to S phase (30.5 vs 51.3%, p&amp;lt;0.01). Strikingly, the cell cycle profile was unchanged when the cells were separated by a transwell. Consistently, in vivo,MLL-AF9 leukemic cells (Lin-cells transduced with an MLL-AF9 oncogene) and AML-MSCs succumbed to leukemia only when cotransplanted (40 vs 143 days, p&amp;lt;0.05). To gain further insights into leukemic cell-induced changes in MSC, we performed gene expression profiling in MSCs sorted from secondary transplanted leukemic (AML-MSC, n=3) and healthy (HD-MSC, n=3) mice. GSEA analysis identified hallmark-Notch signaling to be significantly overrepresented in AML-MSC as compared to controls (p&amp;lt;0.005, NES: 1.7). Increased Notch-1 and -2 expression could be verified at both the mRNA and protein levels in AML-MSCs. To functionally determine the contribution of Notch activation in MSC, we ectopically expressed the Notch intracellular domain (Notch-ICN) in a murine stromal cell line, MS-5 (Notch-ICN). Underscoring a role for Notch signaling in AML-MSC interaction, Notch-ICN expression significantly supported the proliferation of both MLL-AF9 leukemic cells and MSCs. A role for Notch signaling in the modulation of the inflammatory response has been previously reported. Dex an anti-inflammatory corticosteroid enhanced clinical outcome in AML patients. We, therefore, investigated whether Notch-dependent AML-MSC interaction could be therapeutically targeted using Dex. Interestingly, Dex treatment affected Notch1 expression (52%; p&amp;lt;0.005) and proliferation of leukemia cells (63%; p&amp;lt;0.001). Finally, we evaluated the therapeutic efficacy of Dex by treating mice transplanted with MLL-AF9 leukemic cells. Interestingly, mice treated with Dex exhibited significantly prolonged survival (73 vs 21 days, p&amp;lt;0.01) and had a lower percentage of blasts (50%, p&amp;lt;0.01) and MSC compared to leukemic mice receiving vehicle treatment. Our data thus identified Dex mediated abrogation of AML-MSC interaction is in part mediated by affecting Notch signaling. Conclusion: Our study demonstrated distinct functional differences between AML and non-AML-MSC and direct AML-MSC interaction as an indispensable factor in influencing cell proliferation, leukemogenesis, and survival in mice. We further identified and confirmed the role of Notch signaling in AML-MSC interaction. Therapeutically, Dex abrogated AML-MSC interaction, decreasing AML cell viability, and leukemic mice survival. Further studies are warranted to better understand how aberrant Notch signaling in MSC contributes to AML development and whether the use of Dex alone or in combination with Notch inhibitors could circumvent chemoresistance and relapse in AML patients. Disclosures Lenz: Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy; Agios: Research Funding; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria, Research Funding; AQUINOX: Research Funding; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Verastem: Research Funding; BMS: Consultancy. Khandanpour:Astra Zeneca: Research Funding; Sanofi: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy.

Association between the Wilms tumor-1 rs16754 polymorphism and acute myeloid leukemia: A MOOSE-compliant meta-analysis.

The Wilms tumor-1 (WT1) protein is an important regulator of malignant hematopoiesis and has been implicated in the pathogenesis of acute myeloid leukemia (AML). Recently special attention has been paid to the relationship of the WT1 single nucleotide polymorphism (SNP) rs16754 with AML risk and outcome, but the conflicting results made it difficult to draw definitive conclusions. In the present study, we systematically reviewed the literature and performed a meta-analysis of existing evidence. We searched Embase, Pubmed, Web of Science, Medline, Cochrane Library, Wanfang, and China National Knowledge Infrastructure databases using predefined search methodology for relevant studies. We pooled odd ratio (OR) with 95% confidence intervals (95% CI) to evaluate the association between SNP rs16754 and AML risk. In addition, we analyzed hazard ratio (HR) with 95% CI for overall survive, relapse-free survival, and disease-free survival. Q-statistic was used to assess the homogeneity and Egger test was used to evaluate publication bias. Eleven studies met the inclusion criteria for analysis. The results of fixed-effect meta-analyses revealed no association between SNP rs16754 and AML risk (AA + GA vs GG: OR = 0.92, 95% CI: 0.71–1.19, P = .518; AA vs GA + GG: OR = 1.23, 95% CI: 0.86–1.76, P = .262; AA vs GG: OR = 1.05, 95% CI: 0.68–1.63, P = .820; AG vs AA: OR = 0.77, 95% CI: 0.53–1.13, P = .186; AG vs GG: OR = 0.89, 95% CI: 0.68–1.16, P = .376). In subgroup analysis by race, age, and disease type, we did not find any significant association. However, the presence of rs16754 GA/GG genotype was associated with improved overall survive (HR = 0.48, 95% CI: 0.26–0.91, P = .024) and relapse-free survival (HR = 0.82, 95% CI: 0.68–1.00, P = .048) compared with the rs16754 AA. In summary, the WT1 SNP rs16754 was not associated with AML risk, but it had a significant impact on clinical outcome in AML patients.

Myeloid sarcoma is associated with poor clinical outcome in pediatric patients with acute myeloid leukemia.

The impact of myeloid sarcoma (MS) on clinical outcome of pediatric acute myeloid leukemia (AML) patients remains controversial. Moreover, little is known about the role of stem cell transplantation (SCT) in such patients. Clinical data of patients with AML under 18years of age were retrieved from the TARGET dataset. We analyzed the prevalence, clinical profile, molecular characteristics, and prognosis of MS in these patients. Among 884 pediatric patients with AML, the frequency of MS was 12.3%. Pediatric AML with MS was associated with age under 1-year, abnormal cytogenetics, and KMT2A rearrangement. Moreover, MS was associated with a low complete remission rate, high induction death, poor 5-year EFS, and OS. KMT2A rearrangement had a negative impact on clinical outcome in AML patients with MS. In addition, SCT had no significant effect on the survival of AML patients with MS. Multivariate analysis revealed that MS was an unfavorable prognostic factor in pediatric AML in terms of EFS (Hazard ratio 1.670, P < 0.001) and OS (Hazard ratio 1.623, P = 0.004). The presence of MS at diagnosis of pediatric AML is associated with poor clinical outcomes, particularly when associated with KMT2A rearrangements. Moreover, pediatric patients with AML and MS may not benefit from SCT.

Genetic Characteristics and Outcomes By Mutation Status in a Phase 3 Study of CPX-351 Versus 7+3 in Older Adults with Newly Diagnosed, High-Risk/Secondary Acute Myeloid Leukemia (AML)

Lindsley: Jazz Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Consultancy; Medlmmune: Research Funding. Stone:AbbVie, Actinium, Agios, Argenx, Arog, Astellas, AstraZeneca, Biolinerx, Celgene, Cornerstone Biopharma, Fujifilm, Jazz Pharmaceuticals, Amgen, Ono, Orsenix, Otsuka, Merck, Novartis, Pfizer, Sumitomo, Trovagene: Consultancy; Argenx, Celgene, Takeda Oncology: Other: Data and Safety Monitoring Board/Committee: ; Novartis, Agios, Arog: Research Funding. Cortes:Astellas Pharma: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; BiolineRx: Consultancy; Sun Pharma: Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding; Merus: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Immunogen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Biopath Holdings: Consultancy, Honoraria; Forma Therapeutics: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding. Uy:GlycoMimetics: Consultancy; Astellas: Consultancy; Curis: Consultancy; Pfizer: Consultancy. Lin:Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria. Ritchie:Celgene, Incyte, Novartis, Pfizer: Consultancy; Ariad, Celgene, Incyte, Novartis: Speakers Bureau; AStella, Bristol-Myers Squibb, Novartis, NS Pharma, Pfizer: Research Funding; Celgene, Novartis: Other: travel support; Jazz Pharmaceuticals: Research Funding; Genentech: Other: Advisory board; Celgene: Other: Advisory board; Pfizer: Other: Advisory board, travel support; agios: Other: Advisory board; Tolero: Other: Advisory board. Prebet:novartis: Honoraria; pfizer: Honoraria; pfizer: Honoraria; novartis: Honoraria; Boehringer Ingelheim: Research Funding. Ryan:Jazz Pharmaceuticals: Employment, Equity Ownership. Faderl:Jazz Pharmaceutics: Employment, Equity Ownership. Lancet:Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Other: fees for non-CME/CE services ; Agios, Biopath, Biosight, Boehringer Inglheim, Celator, Celgene, Janssen, Jazz Pharmaceuticals, Karyopharm, Novartis: Consultancy.

Abstract A072: Calreticulin exposures by malignant blasts correlate with robust anticancer immunity and improved clinical outcome in AML patients

Abstract Cancer cell death can be perceived as immunogenic by the host only when malignant cells emit immunostimulatory signals (so-called “damage-associated molecular patterns,” DAMPs), as they die in the context of failing adaptive responses to stress. Accumulating preclinical and clinical evidence indicates that the capacity of immunogenic cell death to (re-)activate an anticancer immune response is key to the success of various chemo- and radiotherapeutic regimens. Malignant blasts from patients with acute myeloid leukemia (AML) exposed multiple DAMPs, including calreticulin (CRT), heat-shock protein 70 (HSP70), and HSP90 on their plasma membrane irrespective of treatment. In these patients, high levels of surface-exposed (ecto)-CRT correlated with an increased proportion of natural killer cells and effector memory CD4+ and CD8+ T-cells in the periphery. Moreover, CRT exposure on the plasma membrane of malignant blasts positively correlated with the frequency of circulating T-cells specific for leukemia-associated antigens, indicating that ecto-CRT favors the initiation of anticancer immunity in patients with AML. Finally, although the levels of ecto-HSP70, ecto-HSP90, and ecto-CRT were all associated with improved relapse-free survival, only CRT exposure significantly correlated with superior overall survival. Thus, CRT exposure represents a novel powerful prognostic biomarker for patients with AML, reflecting the activation of a clinically relevant AML-specific immune response. Citation Format: Jitka Fucikova. Calreticulin exposures by malignant blasts correlate with robust anticancer immunity and improved clinical outcome in AML patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A072.

Iron Overload in Patients Aged 60 Years and Older with Acute Myeloid Leukemia May Impact Adversely on Overall Survival

Background: Analyses in myelodysplastic syndromes (MDS) and other acquired anemias suggest an association between iron overload and inferior clinical outcomes. There are minimal data examining iron levels in patients with acute myeloid leukemia (AML) and its relation to clinical outcomes. Patients with AML aged 60 years or older have inferior outcomes in general and no studies examine iron load and clinical outcomes in these patients. We wished to determine whether iron levels might contribute to the prognosis of these patients.Methods: We performed a retrospective analysis of patients with AML aged ≥60 years diagnosed from 2002-2018. Patients were identified from the clinical database and charts reviewed. Clinical data extracted included baseline characteristics (demographics; AML presentation; ECOG performance status [PS]; comorbidities [Charlson Comorbidity Index (CI) and Hematopoietic Stem Cell Transplantation CI]; predisposing conditions; prior transfusions; blood counts; bone marrow findings; cytogenetic analysis) AML treatment received, status at last follow up and cause of death (COD). Serum ferritin level (SF) and bone marrow iron stores (BMIS) at AML diagnosis were recorded. BMIS were: absent, 0; reduced, 1; normal, 2; increased, 3; and markedly increased, 4. Elevated iron was: SF>750ng/mL or BMIS ≥3. Statistical analyses were performed using SPSS for Windows, version 25.Results: Of 369 AML patients, 101 were ≥60 years and had adequate data for analysis. The median age at AML diagnosis was 70 (range 60-93) years and 60 (59.4%) were male. ECOG PS was 0, 1, 2, 3 and 4 in 7 (6.9%), 17 (16.8%), 48 (47.5%), 23 (22.8%) and 4 (4%) patients, respectively. 51 (50.5%) were de novo AML, and 49 (48.5%) had predisposing conditions, including MDS (n=39; 38.6%), MPN (n=7; 6.9%), and prior chemotherapy (CT) or radiation (n=3; 3.0%). Cytogenetic risk group was intermediate in 47 (46.5%) and adverse in 33 (32.7%). Treatment received included supportive care in 41 (40.6%), low dose chemotherapy (hydroxyurea, n=18; low dose cytarabine, n=4) in 22 (21.8%), induction CT in 18 (17.8%), and azacitidine in 20 (19.8%). Infection or inflammation were documented in 8 (7.9%). Comorbidities were present in 43 (42.6%) patients, and the median (range) number of comorbidities per patient was 1 (1-3). SF was available in 55 (54.5%), BMIS in 68 (67.3%), and both in 22 (21.8%). There was a significant correlation between SF >750ng/mL and BMIS ≥3 (r=0.555, p=0.008). A composite score including SF and BMIS revealed elevated iron stores (ELFE) in 39 (38.6%) and normal to decreased iron stores in 62 (61.4%). In univariate analysis, factors significant for overall survival (OS) included ECOG PS, p<0.0001; de novo versus secondary AML, p=0.02; neutrophil count, p<0.0001; platelet count, p<0.0001; bone marrow blast %, p=0.04; cytogenetic risk group, p=0.02; and AML treatment received, p<0.0001. Factors with a trend toward significance included the presence of dysplasia, p=0.07; and ELFE, p=0.07. In pairwise comparisons, dysplasia and AML treatment received lost significance, p=0.09. Factors remaining significant for OS when paired with ELFE included ECOG PS, p=0.02; and AML treatment (trend), p=0.05. In a multivariate analysis including ELFE, ECOG PS and AML treatment, ELFE and ECOG PS were significant for OS: p=0.046, hazard ratio (HR) for death 2.5, 95% confidence intervals (CI) 1.02-6.14, and p=0.006, HR=4.1, 95% CI=1.50-11.36, respectively. The median OS for patients receiving induction CT with no ELFE was 18 (range 0.2-82) months versus 10.1 (0.1-18.2) months with ELFE (p=0.05, see Figure). Baseline characteristics in the subgroup receiving induction CT were not significantly different between ELFE non-ELFE patients, with the exception of hemoglobin (p=0.008). COD in all patients were: AML progression, n=72 (71.3%); infection, n=4 (4.0%); and bleeding, n=4 (4.0%); with no significant difference in COD between patients with and without ELFE (p=0.4).Conclusions: In this retrospective, non-controlled analysis, for patients aged ≥60 years with a new diagnosis of AML, elevated iron appears to be associated with inferior overall survival, particularly in those receiving induction chemotherapy. To our knowledge, these are the first data examining clinical outcomes in AML patients ≥60 years of age according to iron status. These results should be confirmed in larger, prospective analyses. [Display omitted] DisclosuresLeitch:AbbVie: Research Funding; Alexion: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau.

Novel combination of histone methylation modulators with therapeutic synergy against acute myeloid leukemia in vitro and in vivo

Acute myeloid leukemia (AML) is a hematological malignancy with rapid disease progression and often becomes lethal without treatment. Development of effective new therapies is essential to improve the clinical outcome of AML patients. Enhancer of zeste homolog 2 (EZH2) and lysine specific demethylase 1 (LSD1) play important roles in epigenetic regulation and their altered expressions have been observed in cancer. Although EZH2 and LSD1 have opposite histone methylation functions, we found that both enzymes were paradoxically up-regulated in AML cells. Importantly, a combined inhibition of EZH2 and LSD1 resulted in a synergistic activity against AML in vitro and in vivo. Such synergy was mechanistically correlated with up-regulation of H3K4me1/2 and H3K9Ac and down-regulation of H3K27me3, leading to a decrease of anti-apoptotic protein Bcl-2. These epigenetic alterations also compromised the mitochondrial respiration capacity and glycolytic activity and resulted in ATP depletion, a key event contributing to the potent cytotoxic effect of the drug combination. Taken together, our work identified a novel therapeutic approach against AML by combining two small molecules that inhibit different histone methylation-modulating proteins with apparently opposite enzyme activities. Such a new drug combination strategy likely has significant clinical implications since epigenetic modulators are currently in clinical trials.