Apoptosis In Acute Myeloid Leukemia Cell Lines Research Articles

Methylsulfonylmethane induces caspase-dependent apoptosis in acute myeloid leukemia cell lines.

Acute myeloid leukemia (AML) is a heterogeneous ailment in both biological and clinical concepts. Numerous efforts have been devoted to discover natural compounds for combating cancer, which showed great potential in cancer management. Methylsulfonylmethane (MSM), an organosulfur dietary supplement, is utilized for improving various clinical conditions, particularly osteoarthritis. MSM can exert antitumor activity in a wide range of cancers. The molecular mechanisms of action underlying antileukemic activity of MSM remain unclear. In this regard, we aimed to investigate the anticancer properties of MSM on human AML cell lines (U937 and HL60) with focus on underlying cell death mechanism. Anticancer activity of the MSM was examined employing MTT assay, Annexin V-PE/7AAD staining, caspase3/7 activity test, and real-time qPCR. Both cell lines were treated with different concentrations (50-400 mM) of MSM for 24 h. Pretreatment of the cells with a caspase inhibitor (i.e., Z-VAD-fmk) was performed for the assessment of apoptosis induction. The results of MTT assay revealed that in both cell lines, the MSM markedly reduced cell viability in comparison to the control cells. Additionally, findings of Annexin V-7AAD staining revealed that MSM induced apoptosis and activated caspase 3/7 in both cell lines markedly. Real-time quantitative PCR results also supported the induction of apoptosis in AML cells. MSM altered the expression levels of various apoptotic genes (BAX, BAD, and BIM). Overall, our results indicated that MSM could induce apoptosis in AML cell lines in a dose-dependent manner, which therefore could be utilized as anantileukemic agent.

A novel iheyamine A derivative L42 suppresses acute myeloid leukemia via dual regulation of the PI3K/AKT/FOXO3a axis and TNF signaling pathway

Acute myeloid leukemia (AML) is one of the most common hematopoietic malignancies and the development of new drugs is crucial for the treatment of this lethal disease. Iheyamine A is a nonmonoterpenoid azepinoindole alkaloid from the ascidian Polycitorella sp., and its anticancer mechanism has not been investigated in leukemias. Herein, we showed the significant antileukemic activity of L42 in AML cell lines HEL, HL-60 and THP-1. The IC50 values were 0.466±0.099 µM, 0.356±0.023 µM, 0.475±0.084 µM in the HEL, HL-60 and THP-1 cell lines, respectively, which were lower than the IC50 (2.594±0.271 µM) in the normal liver cell line HL-7702. Furthermore, L42 significantly inhibited the growth of peripheral blood mononuclear cells (PBMCs) from an AML patient. In vivo, L42 effectively suppressed leukemia progression in a mouse model induced by Friend murine leukemia virus (F-MuLV). Mechanistically, we showed that L42 induced cell cycle arrest and apoptosis in leukemia cell lines. RNA sequencing analysis of L42-treated THP-1 cells revealed that the differentially expressed genes (DEGs) were enriched in the cell cycle and apoptosis and predominantly enriched in the PI3K/AKT pathway. Accordingly, L42 decreased the expression of the phospho-PI3K (p85), phospho-AKT and phospho-FOXO3a. Docking and CETSA analysis indicated that L42 bound to the PI3K isoform p110α (PIK3CA), which was implicated in the suppression of the PI3K/AKT pathway. L42 was also shown to initiate the TNF signaling-mediated apoptosis. Moreover, L42 exhibited stronger anti-leukemia activity and sensitivity in IDH2-mutant HEL cells than in IDH2-wild-type control. In conclusion, L42 effectively suppresses cell proliferation and triggers apoptosis in AML cell lines in part through inhibition of the PI3K/AKT signaling pathway to restore FOXO3a expression and activation of the TNF signaling pathway. Thus, the iheyamine A derivative L42 represents a novel candidate for AML therapy.

Abstract 1829: Brusatol overcomes tyrosine kinase inhibitors resistance in myeloid leukemia cells by inhibiting translation of fast turnover protein cyclin D1 and Mcl-1

Abstract Myeloid leukemia is characterized with fast proliferation and apoptosis evasion, which are controlled by mutant or activated tyrosine kinase signalings. Tyrosine kinase inhibitors (TKI) targeting BCR-ABL in chronic myeloid leukemia (CML) and FLT3-ITD in acute myeloid leukemia (AML) significantly improved the therapy, but not cure, due to the general resistance. Overactivated mTOR protein translation pathway is believed to be one of the mechanisms causing resistance and that targeting protein translation should be one effective approach to overcome resistance. Brusatol is a quassinoid extracted from the fruit of Brucea javanica and an inhibitor of protein translation. We tested its antiproliferation and apoptosis induction in a variety of AML and CML cell lines as well as in TKI resistant cell lines. Brusatol effectively inhibits the growth of seven leukemia cell lines with GI50 values less than 20 nM. Brusatol induces apoptosis in AML cell lines and induces G0/G1 phase arrest in CML cell lines. Brusatol decreases the levels of fast turnover protein cyclin D1 and Mcl-1. In FLT3-ITD inhibitor sorafenib resistant MOLM-13/Sor cell line brusatol exhibits similar growth inhibition and apoptosis induction with the parental MOLM-13 cells, associated with inhibition of Mcl-1 and FLT3-ITD proteins. In BCR-ABL inhibitor imatinib resistant K562/STI cell line brusatol is effective as in parental K562 cells, associated with inhibition of cyclin D1 and BCR-ABL proteins. Brusatol at 2 mg/kg effectively inhibit tumor growth of K562 xenografts without causing toxicity to normal organs. These data indicate that brusatol inhibits oncogenic protein translation and has potential to be used to treat TKI resistant leukemia. Citation Format: Huiming Hua, Ying Yang, Guangfuxin Lin, Jingyi Zhang, Samuel Waxman, Yongkui Jing. Brusatol overcomes tyrosine kinase inhibitors resistance in myeloid leukemia cells by inhibiting translation of fast turnover protein cyclin D1 and Mcl-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1829.

Abstract 2363: Enhanced T cell activation and cytotoxicity against AML via targeted anti-CD99-A192 ScFv-ELP nanoparticle treatment

Abstract CD99 is a transmembrane protein overexpressed in acute myeloid leukemia (AML), presenting a potential novel therapeutic target. Our previously developed anti-CD99-A192, comprising scFv and elastin-like polypeptides (ELPs), showed promising anti-leukemic activity by inducing apoptosis in AML cell lines and prolonging survival in AML xenograft models. Considering CD99's expression and role in T cell activation, we proposed that anti-CD99-A192 could have a dual function: targeting leukemic cells while activating T cells. Blood samples were obtained from 20 healthy donors, and peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with PHA and IL-2 cytokines to expand T cells for 72-96 hours. Post-expansion, T cells were treated with either anti-CD99-A192 or control anti-A192. Proliferation was evaluated via cell count and cell trace assays. The percentages of CD4+, CD8+, and CD25+ T cells, and the levels of T cell activation markers CD69 and CD38, were quantified by flow cytometry. Cytokine release profiles were assessed by the Proteome Profiler Human Cytokine Array Kit. The cytotoxic impact of the anti-CD99-A192-activated T cells on MV4-11Luc+ AML cells was measured in co-culture apoptosis and luciferase assays. Statistical comparisons utilized paired and unpaired T-tests with Holm-Sidak correction for multiple comparisons, and P value <0.05 was considered significant. T cells treated with anti-CD99-A192 displayed significant increased proliferation compared with untreated and anti-A192-treated groups at Day 5 (163.3% vs. 97% and 92.4%, p=0.042 and p=0.03, respectively). The percentage of CD4+, CD8+, and CD25+ cells remained unchanged across treated and control groups. Activation marker analysis revealed increase in % CD38+ cells in the anti-CD99-A192 group compared with the control (58.15% vs. 49.15%, p=0.0028). Among the cytokine panel, significant increases in IL-1β/IL-1F2 (11-fold change, p<0.001, adjusted p=0.006) and TNF-α (12-fold change, p=0.002, adjusted p=0.03) were observed, along with a marked decrease in IL-8 (70% reduction, p<0.001, adjusted p=0.001). GM-CSF (20-fold change, p=0.005, adjusted p=0.071) and IL-16 (2-fold change, p=0.003, adjusted p=0.051) levels also increased, though not significantly after adjustment. Co-culture assays showed greater apoptosis in MV4-11 cells at 18 hours (33.6% vs. 41.13%, p=0.015) and a reduction in viable leukemic cells at 18 hours (10.89% vs. 8.05%, p=0.034) and 48 hours (9.69% vs. 6.54%, p=0.065) in the anti-CD99-A192 compared with the control group, results were also validated by the luciferase assay (~32% reduction, p<0.001). Anti-CD99-A192 scFv-ELP nanoparticle stimulates T cell proliferation, elevates activation markers, and increases the release of pro-inflammatory cytokines. Anti-CD99-A192 culminates in heightened cytotoxicity against leukemic cells. Citation Format: Shephali Kadam, Atham Ali, Mateusz Pospiech, Sandra Onyemaechi, Yiting Meng, Kanaka Dhuri, Andrew Mackay, Houda Alachkar. Enhanced T cell activation and cytotoxicity against AML via targeted anti-CD99-A192 ScFv-ELP nanoparticle treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2363.

Enhancing anti-AML activity of venetoclax by isoflavone ME-344 through suppression of OXPHOS and/or purine biosynthesis in vitro

Venetoclax (VEN), in combination with low dose cytarabine (AraC) or a hypomethylating agent, is FDA approved to treat acute myeloid leukemia (AML) in patients who are over the age of 75 or cannot tolerate standard chemotherapy. Despite high response rates to these therapies, most patients succumb to the disease due to relapse and/or drug resistance, providing an unmet clinical need for novel therapies to improve AML patient survival. ME-344 is a potent isoflavone with demonstrated inhibitory activity toward oxidative phosphorylation (OXPHOS) and clinical activity in solid tumors. Given that OXPHOS inhibition enhances VEN antileukemic activity against AML, we hypothesized that ME-344 could enhance the anti-AML activity of VEN. Here we report that ME-344 enhanced VEN to target AML cell lines and primary patient samples while sparing normal hematopoietic cells. Cooperative suppression of OXPHOS was detected in a subset of AML cell lines and primary patient samples. Metabolomics analysis revealed a significant reduction of purine biosynthesis metabolites by ME-344. Further, lometrexol, a purine biosynthesis inhibitor, synergistically enhanced VEN-induced apoptosis in AML cell lines. Interestingly, AML cells with acquired AraC resistance showed significantly increased purine biosynthesis metabolites and sensitivities to ME-344. Furthermore, synergy between ME-344 and VEN was preserved in these AraC-resistant AML cells. In vivo studies revealed significantly prolonged survival upon combination therapy of ME-344 and VEN in NSGS mice bearing parental or AraC-resistant MV4-11 leukemia compared to the vehicle control. This study demonstrates that ME-344 enhances VEN antileukemic activity against preclinical models of AML by suppressing OXPHOS and/or purine biosynthesis.

Effect and Mechanism of NL-101 Combined with Bcl-2 Inhibitor Venetoclax in Acute Myeloid Leukemia

Background Acute myeloid leukemia (AML) is a kind of malignant clonal disease originating from hematopoietic stem cells with strong heterogeneity,with the highly recurrence rate and mortality rate.Although the application of hematopoietic stem cell transplantation and new targeted drugs has improved the prognosis of some patients in recent years, chemotherapy is still the most important treatment for AML. 50-80% of AML patients eventually relapsed after CR, with a low remission rate after relapse.There are reports that BCL-2 overexpression is a clinical feature of human hematologic malignancies that exacerbates the malignant state and drives apoptosis resistance phenotypes.Therefore, how to reduce treatment-related toxic and side effects and overcome tumor cell drug resistance is a hot and difficult topic in current AML treatment. Venetoclax (ABT-199) is an oral, selective Bcl-2 inhibitor. It showed strong anti-leukemia activity in preclinical stage, but its clinical efficacy as a single drug for AML was limited.Nl-101 (also known as EDE-S101)is a new small molecule compound formed by covalently linking the DNA damage core group of Bendamustine hydrochloride with the inhibition core group of histone deacetylase of the histone inhibitor Volinostat (also known as SAHA).Our previous study found that NL-101 single drug can reduce tumor load and prolong survival of AML mice in vivo.In this study, we used Venetoclax in combination with NL-101 to understand its efficacy and safety in vitro and in vivo, and to explore the synergies and mechanisms of action between the two drugs, providing new effective options for the treatment of AML patients. Purpose To explore the efficacy of Bcl-2 inhibitor Venetoclax (ABT-199) and a novel small molecule compound NL-101 in the treatment of acute myeloid leukemia（AML） and the related mechanisms. Experimental Design AML cell lines and primary patient samples were collected and used for cell proliferation assay. MTS colorimetric assay was used to determine the effect of Venetoclax alone or in combination with NL-101 on the growth of AML cells，the combination index (CI) was calculated to evaluate the synergistic effect of the two drugs. Cell death induced by Venetoclax alone or in combination with NL-101 was analyzed by Annexin V-FITC/ Propyl -iodide staining and flow cytometry. Western blot was used to analyze the protein expression levels of MAPK/ERK, PI3K/AK/GSK pathway members and BCL-2 family members.The MV4-11-derived xenograft mouse model was used to assess in vivo efficacy of the combination of venetoclax and NL-101. Results Venetoclax alone and in combination with NL-101 can inhibit the growth of AML cell lines and primary cells to a certain extent, and the combination of drugs has a synergistic effect. Venetoclax combined with NL-101 effectively synergistically induced apoptosis in AML cell lines and primary patient samples. Venetoclax combined with NL-101 can down-regulate the expression of pro-survival proteins in the Bcl-2 family, such as McL-1, Bcl-2, and Bcl-XL, and up-regulate the expression of pro-apoptotic proteins, such as BIM, BID, and BAX/BAK. Phosphorylated ERK, AKT, and JNK expressions were eliminated by venetoclax in combination with NL-101. In vivo results suggest that Venetoclax combined with NL-101 has the potential to treat AML. Conclusions Venetoclax combined with NL-101 can synergistically inhibit the growth and promote apoptosis of AML cell lines and primary AML cells. In vivo experiments demonstrated that Venetoclax combined with NL-101 can synergistically reduce tumor load and prolong survival time in AML mice. These results suggest that Venetoclax in combination with NL-101 is effective in the treatment of acute myeloid leukemia.

Combined Application of Salinomycin and ATRA Induces Apoptosis and Differentiation of Acute Myeloid Leukemia Cells by Inhibiting WNT/β-Catenin Pathway.

All-trans retinoic acid (ATRA) is only effective in acute promyelocytic leukemia (APL), but not in other subtype of acute myeloid leukemia (AML). Salinomycin targets tumor cells rather than non-tumorigenic cells, and WNT/β-catenin pathway inhibition is one of the mechanisms of its anti-tumor activity. There is a crosstalk between RA and WNT/β-catenin pathway. Here, we investigate the effect of the combination of salinomycin and ATRA (S+RA) in non-APL AML cells. Apoptosis was evaluated by cell viability and Annexin-V assay. Cell differentiation was analyzed by CD11c expression and morphology. To explore the underlying mechanisms, Western blot analysis and mitochondrial transmembrane potentials (ΔΨm) were used. S+RA induced differentiation and apoptosis in AML cell lines and AML primary cells. S+RA inhibited the β-catenin signal pathway as determined by the decreased protein levels of β-catenin, the low-density lipoprotein receptor-related proteins 6 (LRP6), and its downstream proteins such as survivin, c-Myc, caspase-3/7, cdc25A and cyclinD1 and reduced phosphorylation level of GSK3β S9. S+RA also increased the protein levels of CCAAT/enhancer-binding proteins (C/EBPs) and PU.1 and collapsed Δψm. The above molecular and cellular changes induced by S+RA were inhibited by β-catenin specific activator and promoted by β-catenin specific inhibitor. S+RA induced differentiation by β-catenin-inhibition-mediated up-regulation of C/EBPs and PU.1 and suppression of c-Myc. S+RA triggered apoptosis through β-catenin-inhibition-regulated ΔΨm collapse and caspase-3/7 activation. Taken together, our findings may provide novel therapeutic strategies for AML patients by targeting the WNT/β-catenin pathway.

Aging-related genes related to the prognosis and the immune microenvironment of acute myeloid leukemia.

Acute myeloid leukemia (AML), one of the common malignancies of the hematologic system, has progressively increased in incidence. Aging is present in both normal tissues and the tumor microenvironment. However, the relationship between senescence and AML prognosis is still not elucidated. In this study, RNA sequencing data of AML were obtained from TCGA, and prognostic prediction models were established by LASSO-Cox analysis. Differences in immune infiltration between the different risk groups were calculated using the CIBERSORT and ESTIMATE scoring methods. The KEGG and GO gene enrichment and GSEA enrichment were also used to enrich for differential pathways between the two groups. Subsequently, this study collected bone marrow samples from patients and healthy individuals to verify the differential expression of uncoupling protein 2 (UCP2) in different populations. Genipin, a UCP2 protein inhibitor, was also used to examine its effects on proliferation, cell cycle, and apoptosis in AML cell lines in vitro. It showed that aging-related genes (ARGs) expression was correlated with prognosis. And there was a significant difference in the abundance of immune microenvironment cells between the two groups of patients at high risk and low risk. Subsequently, UCP2 expression was found to be elevated in AML patients. Genipin inhibits UCP2 protein and suppresses the proliferation of AML cell lines in vitro. ARGs can be used as a predictor of prognosis in AML patients. Moreover, suppressing UCP2 can reduce the proliferation of AML cell lines, alter their cell cycle, and promote apoptosis in vitro.

Dihydropyrimidinase-like 2 can serve as a novel therapeutic target and prognostic biomarker in acute myeloid leukemia.

Identifying therapeutic targets and prognostic biomarkers significantly contributes to individualized treatment of acute myeloid leukemia (AML). Dihydropyrimidinase-like 2 (DPYSL2) expression was decreased in homoharringtonine (HHT)-resistant AML cells, which were established by our group. DPYSL2 plays an important role in axon growth and has oncogene effect in glioblastoma. However, little research has been conducted to investigate the function of DPYSL2 in AML pathogenesis. Auto-docking was used to reveal the targeting relationship between HHT and DPYSL2. Overall survival (OS), event-free survival (EFS), and relapse-free survival (RFS) were used to evaluate the prognostic impact of DPYSL2 for AML. ShRNA was used to knockdown the expression of SPATS2L. Apoptosis was assessed by flow cytometry. In vivo growth and survival were assessed using a xenotransplantation mice model. RNA sequencing was performed to elucidate the molecular mechanisms underlying the role of SPATS2L in AML and were confirmed by Western blot. We found DPYSL2 was the target of HHT. Next, we found AML cell lines and patients had higher DPYSL2 expression levels than the normal samples. Further multivariate analysis demonstrated that high DPYSL2 expression was an independent poor prognostic factor for OS, EFS, and RFS in AML. Inhibition of DPYSL2 expression suppressed cell growth, induced apoptosis in AML cell lines, and prolonged the survival of AML xenograft NCG mice. Through RNA-seq analysis from TCGA and our data, the JAK2/STAT3/STAT5-PI3K P85/AKT/GSK3b axis was thought to be the critical pathway in regulating DPYSL2 in AML development. We first time confirmed that DPYSL2 was a target of HHT and played an oncogene role in AML by regulating JAK/STAT signaling pathway. Therefore, DPYSL2 could serve as a novel prognostic marker and therapeutic target for AML treatment.

Synergistic activity and mechanism of cytarabine and MCL-1 inhibitor AZD5991 against acute myeloid leukemia.

The 5-year overall survival rate of acute myeloid leukemia (AML) is less than 30%. Improving clinical outcomes is still a clinical challenge for AML treatment. Simultaneous use of chemotherapeutic drugs and targeting of apoptosis pathways has become a first-line clinical treatment for AML. Myeloid cell leukemia 1 (MCL-1) is a candidate target for AML treatment. In this study, we demonstrated that inhibition of the anti-apoptotic protein MCL-1 by AZD5991 synergistically increased chemotherapeutic agent cytarabine (Ara-C)-induced apoptosis in AML cell lines and primary patient samples. Apoptosis induced by a combination of Ara-C and AZD5991 was partially dependent on caspase activity and Bak/Bax. The downregulation of MCL-1 by Ara-C and the enhancement of Ara-C-induced DNA damage through inhibition of MCL-1 are potential mechanisms underlying the synergistic anti-AML activity between Ara-C and AZD5991. Our data support the application of MCL-1 inhibitor in combination with the conventional chemotherapeutic agent for the clinical treatment of AML.

TERRA R-LOOPS ARE INCREASED IN ACUTE MYELOID LEUKEMIA

Telomeric repeat-containing RNA (TERRA) are long non-coding RNAs transcribed from subtelomeric regions towards telomere ends. TERRA binds to subtelomeric and telomeric regions, forming R-loops, structures composed of DNA-RNA hybrids and displaced single-stranded DNA. TERRA R-loops protect telomeres from DNA damage and promote telomere elongation in short telomeres by enabling telomerase or by activating alternative telomere lengthening. However, high or low TERRA levels are associated with DNA damage and telomere dysfunction. RNaseH1 and RNaseH2 cleave R-loops at telomeres, and regulate R-loops levels. The role of TERRA R-loops in telomere maintenance in human cells is not well understood, since conflicting results derive from experiments using diverse organisms and commercial cell lines, with few studies applying primary human cells. Here we describe for the first time the TERRA physiology in hematopoietic cells of patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), telomeropathies (TEL), and healthy subjects. We initially screened 17 patients with AML, 5 with ALL, 9 with TEL, and 20 healthy subjects. Additionally, 88 AML patients were studied. HeLa, H1, THP1, NB4, and VA13 cell lines were used for functional experiments, and lentivirus vectors containing GFP or RNaseH1-GFP motifs were employed. ATRX, TRF2, TERT, RNaseH1, RNaseH2, TERRA 10q, 15q, 20q, XqYq, XpYp, and 17p expression levels were measured by RT-qPCR. Telomere length was determined by qPCR. R-loops were assessed by immunofluorescence using the S9.6 antibody. Cell line viability was defined by MTT assay. NGS panel was designed to evaluate variants in splicing factors and epigenetic regulators. In the screening population, TERRA expression (10q, 15q, 20q, and XqYq) was significantly increased in AML samples (p < 0.05) in comparison to healthy controls and VA-13 cells. Telomeres were very short in AML samples and TEL patients compared to controls. R-loop formation was significantly increased in AML samples compared to controls (p < 0.01). To examine the TERRA physiology in AML, we further eveluated 88 AML samples. Hierarchical cluster algorithm based on TERRA expression defined two AML groups, one with high and the other with low TERRA expression. Both groups dysplayed short telomeres, but the high TERRA AML group exhibited low TERT and RNaseH2 expression levels (p < 0.05). There was no difference between groups on somatic variants or RNaseH1, TRF2, ATRX, and TRF2 expression levels. AML cell lines were transfected RNaseH1-GFP-containing or empty vector lentiviruses and treated with cytarabine. AML cells with exogenous RNAseH1 expression showed more sensitivity to cytarabine in MTT assays (p < 0.05) than cells transfected with the empty vector. These results show for the first time that TERRA is dysregulated in AML. High TERRA levels are unique to a subset of AML cases with low TERT expression. High TERRA correlated with the accumulation of R-loops, which may be therapeutic targets. TERRA upregulation in AML due to short telomeres is unlikely, given the low TERRA levels in TEL patients. TERRA upregulation did not appear to be directly related to neoplastic differentiation, given low TERRA in ALL. TERRA expression and R-loops are dysregulated in AML. TERRA R-loops inhibition increases chemotherapy-induced apoptosis in AML cell lines and may serve as a therapeutic target.

AML-080 Superior Anti-Leukemic Effects of Combined Acid Ceramidase and Bcl-2 Inhibition in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive, heterogenous disease characterized by clonal expansion of immature myeloid blasts. For the last five decades, intensive induction chemotherapy was the primary treatment option for AML. The 5-year survival rate for younger patients following induction chemotherapy is 40%-50%, which is far superior to the dismal 10%-15% 5-year survival seen in older adult patients, who constitute most AML cases. The specific Bcl-2 antagonist venetoclax was approved in combination with Ara-C or azacitidine for newly diagnosed AML or older adult patients unable to tolerate intensive chemotherapy. However, responses are incomplete and relapse rates remain high, necessitating the development of novel treatment strategies to augment current therapies and improve outcomes. To this end, targeting dysregulated sphingolipid metabolism in AML shows promise. Once overlooked as mere structural membrane components, sphingolipids are now appreciated as important regulators of cell signaling and are involved in many hallmarks of cancer that are of critical importance in AML. Acid ceramidase (AC), a ceramide-catabolizing lipid hydrolase, is upregulated in AML and correlates with poorer patient survival. The breakdown of ceramide, a bona fide pro-death lipid, by AC promotes chemotherapeutic resistance and leukemic survival in part by upregulating Mcl-1, a known venetoclax resistance factor. Our ongoing research aims to characterize the efficacy of venetoclax combined with AC inhibition in AML. SACLAC, a ceramide analog and irreversible AC inhibitor, augmented venetoclax sensitivity and synergistically decreased cell viability and induced apoptosis in AML cell lines with de novo and acquired venetoclax resistance. Remarkably, combinatorial SACLAC and venetoclax treatment was quantitatively more synergistic than standard venetoclax-containing AML treatment regimens (venetoclax+Ara-C and venetoclax+azacitidine) by Bliss analysis in over 60% of primary patient samples tested in vitro. The SACLAC and venetoclax regimen was less toxic to normal peripheral blood mononuclear cells and bone marrow cells relative to leukemic samples and exhibited toxicity towards these normal cell types comparable to venetoclax+Ara-C and venetoclax+azacitidine. Mechanistically, combination treatment potently increased ceramides, synergistically disrupted mitochondrial membrane potential, and depleted several proteins that inhibit apoptosis, including XIAP and cIAP1. Overall, these data provide the rationale for additional mechanistic and preclinical in-vivo studies targeting AC and Bcl-2 in AML.

C-Myc plays a critical role in the antileukemic activity of the Mcl-1-selective inhibitor AZD5991 in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive disease with a low 5-year overall survival rate of 29.5%. Thus, more effective therapies are in need to prolong survival of AML patients. Mcl-1 is overexpressed in AML and is associated with poor prognosis, representing a promising therapeutic target. The oncoprotein c-Myc is also overexpressed in AML and is a significant prognostic factor. In addition, Mcl-1 is required for c-Myc induced AML, indicating that c-Myc-driven AML harbors a Mcl-1 dependency and co-targeting of Mcl-1 and c-Myc represents a promising strategy to eradicate AML. In this study, we investigated the role of c-Myc in the antileukemic activity of Mcl-1 selective inhibitor AZD5991 and the antileukemic activity of co-targeting of Mcl-1 and c-Myc in preclinical models of AML. We found that c-Myc protein levels negatively correlated with AZD5991 EC50s in AML cell lines and primary patient samples. AZD5991 combined with inhibition of c-Myc synergistically induced apoptosis in AML cell lines and primary patient samples, and cooperatively targeted leukemia progenitor cells. AML cells with acquired resistance to AZD5991 were resensitized to AZD5991 when c-Myc was inhibited. The combination also showed promising and synergistic antileukemic activity in vitro against AML cell lines with acquired resistance to the main chemotherapeutic drug AraC and primary AML cells derived from a patient at relapse post chemotherapy. The oncoprotein c-Myc represents a potential biomarker of AZD5991 sensitivity and inhibition of c-Myc synergistically enhances the antileukemic activity of AZD5991 against AML.

P413: THE POTENTIAL USE OF AN ORAL HYPOMETHYLATING AGENT, OR-2100, AS A COMBINATION THERAPY FOR MYELOID MALIGNANCIES

Background: The combination therapy of hypomethylating agents (HMAs), azacytidine (AZA) and decitabine (DAC), with venetoclax (VEN), a selective BCL-2 inhibitor, have revolutionized the outcome of elderly patients with acute myeloid leukemia (AML), which have demonstrated the potential effect of HMAs as a combination therapy with molecular targeting agents for myeloid malignancies. However, there are a few studies on the combined effect of HMAs with tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) and the mechanism by which HMAs enhance the efficacy of molecular targeting agents remain poorly understood. We have developed OR-2100 (OR21), an oral HMA, a 5’-O-trialkylsilylated DAC. OR21 is as effective as DAC, but less myelosuppressive, in xenograft mouse models of solid and hematological malignancies (Hattori et al. Clin Epigenetics 2019, Watanabe et al. Blood 2020, Ureshino et al. Mol Cancer Ther 2021, Kamachi et al. Cancer Letters 2022). We investigated the efficacy and mechanism of OR21 in combination with VEN or TKIs for myeloid malignancies, AML, and CML. Aims: To investigate the efficacy and mechanism of the combination therapy of OR21 for myeloid malignancies. Methods: We investigated the efficacy and mechanism of OR21 in combination with VEN against AML and with TKIs against CML, in vitro and in vivo. Results: In AML, OR21 plus VEN synergistically inhibited cell growth, increased apoptosis in AML cell lines as similar to AZA and DAC, and significantly prolonged survival in a xenograft mouse model using HL60 cell line (p<0.05). Gene expression analysis using AML cell lines (HL60, KG1a) showed that OR21 plus VEN significantly downregulated VAMP7 which regulates mitophagy/autophagy to maintain mitochondrial homeostasis compared to VEN monotherapy. Consistently, OR21 plus VEN enhanced apoptosis by increasing ROS accumulation and attenuated mitophagy/autophagy response triggered by decreased mitochondrial membrane potential. The addition of mitophagy inducers, such as rapamycin and p62-mediated mitophagy inducer, to OR21 plus VEN decreased ROS accumulation and apoptosis, indicating OR21 enhanced ROS accumulation via attenuating VEN-induced mitophagy/autophagy pathway. In CML, OR21 plus TKIs (imatinib, dasatinib, ponatinib) synergistically inhibited cell growth, increased apoptosis in CML cell lines as similar to AZA and DAC, and significantly suppressed the colony-forming capacity of bone marrow CD34+ stem/progenitor cells in CML patients. OR21 plus imatinib significantly suppressed tumor growth compared to imatinib monotherapy in a xenograft mouse model using K562 cell line (p<0.05). In phosphoproteomic, transcriptome and methylome analysis (K562, KBM5), OR21 augmented imatinib-induced apoptosis by dephosphorylating of STAT and Src family proteins by upregulating several tumor suppressor genes, including PTPN6, through demethylation of their promoter CpG regions. Image:Summary/Conclusion: OR21 enhanced the anti-tumor effect in combination with the molecular agents for myeloid malignancies with different mechanisms: OR21 increased ROS accumulation in combination with VEN, and dephosphorylated BCR-ABL1 downstream protein kinases by demethylating effect in combination with TKIs. With its advantage as an oral HMA, OR21 is expected to have future clinical trials as a combination therapy for myeloid malignancies.

MiR-338-3p Plays a Significant Role in Casticin-Induced Suppression of Acute Myeloid Leukemia via Targeting PI3K/Akt Pathway.

Objective Casticin is generally used in traditional herbal medicine for its anti-inflammatory and anticarcinogenic pharmacological properties. Also, microRNAs are indispensable oncogenes or cancer suppressors being dysregulated in various diseases. In this study, we aimed to elucidate the mechanisms underlying effects of casticin on the progression of acute myeloid leukemia (AML). Methods CCK-8 and flow cytometry were utilized to measure the proliferation and apoptosis of AML cell lines, respectively, after treatment with different concentrations of casticin. The alteration of several microRNA expressions in response to casticin treatment was detected by performing qRT-PCR, and the activity of PI3K/Akt pathways was evaluated through immunoblotting. Afterwards, the potential target gene of miR-338-3p was investigated by dual-luciferase reporter assay. In order to evaluate the role of miR-338-3p in the casticin-induced cellular phenotype changes, AML cells were transfected with miR-338-3p mimics or inhibitor and then subjected to proliferation and apoptosis analysis. Finally, a mouse xenograft model system was employed to investigate the role of casticin in AML progression in vivo. Results Suppressed cellular proliferation and enhanced apoptosis were observed in HL-60 and THP-1 cells after exposure to casticin, accompanied by remarkable upregulation of the miR-338-3p expression as well as a decline in the phosphorylation of PI3K and Akt proteins. RUNX2 was identified as a direct target molecular of miR-338-3p, which might account for the findings that miR-338-3p knockdown enhanced the PI3K/Akt pathway activity, whereas the miR-338-3p overexpression inactivated this signaling pathway. In addition, the inhibition of the miR-338-3p expression attenuated severe cell apoptosis and suppressions of PI3K/Akt pathway induced by casticin. Furthermore, casticin treatment retarded tumor growth rate in mouse models, whilst elevating miR-338 expression and repressing the activity of PI3K/Akt pathway in vivo. However, miR-338-3p depletion could also abolish the phenotypic alterations caused by casticin treatment. Conclusion Casticin promotes AML cell apoptosis but inhibits AML cell proliferation in vitro and tumor growth in vivo by upregulating miR-338-3p, which targets RUNX2 and thereafter inactivates PI3K-Akt signaling pathway. Our results provide insights into the mechanisms underlying the action of casticin in the control of AML progression.

Bioengineered PLEKHA7 nanodelivery regularly induces behavior alteration and growth retardation of acute myeloid leukemia

Acute myeloid leukemia (AML) is the most lethal leukemia with an extremely poor prognosis and high relapse rates. In leukemogenesis, adhesion abnormalities can readily guide an imbalance between hematopoietic progenitor cells and bone marrow stromal cells, altering the normal hematopoietic bone marrow microenvironment into leukemic transformation that enhances leukemic proliferation. Here, we have firstly studied the PLEKHA7 expression in leukemic cells to assess their growth capability affected by the restoration of PLEKHA7 in the cells. The efficacy of PLEKHA7-loaded cRGD-mediated PEGylated cationic lipid nanoparticles for efficient PLEKHA7 delivery in leukemic cells as well as the effect of PLEKHA7 on the regulated induction of AML behavior and growth alterations were investigated. PLEKHA7 re-expression diminished colony-forming ability and reinforced the incidence of growth retardation without apoptosis in AML cell lines. PLEKHA7 regulated the restoration of cell surface adhesion and integrity during normal homeostasis. Our findings revealed that PLEKHA7 functions as a behavior and growth modulator in AML. To our knowledge, the role of PLEKHA7 in AML had not been studied previously and our data could be exploited for further mechanistic studies and insights into altering human AML behavior and growth.

The Inhibitory Effect of Sulforaphane on The Proliferation of Acute Myeloid Leukemia Cell Lines through Controlling miR-181a.

Objective The present study investigated the role of miR-181a as a small non-coding RNA molecule in acute myeloid leukemia (AML) pathogenesis and reflected on the effects of Sulforaphane (SFN) on AML progression. Materials and Methods This experimental study had two parts. In vivo study, the miR-181a levels was measured in patients with symptoms of AML and compared to healthy controls (HCs) to investigate its role in AML pathogenesis. Afterward, an in vitro study was performed to examine the effects of SFN on the growth, apoptosis and proliferation rate of AML cell lines. Finally, the effect of SFN on miR-181a was evaluated as a major miRNA involved in hematopoiesis. Results The results of this study showed an increasing trend (2.9-fold, P=0.0019) in miR-181a expression levels in AML patients as compared with HCs. The data associated with MTT assay and flow cytometry (FCM) additionally demonstrated the anti-proliferative effects of SFN against AML cell lines, with a reduction in miR-181a levels. As well, no significant difference was noted between 24 hours and 48 hours treatments by SFN. It was deduced that modulation of miR-181a expression levels could be one of the mechanisms associated with the anti-proliferative effects of SFN against AML. ConclusionMiR-181a levels contribute to AML pathogenesis and thus they can be considered as a strategy in controlling AML progression in patients. Accordingly, SFN can arrest cell proliferation and induce apoptosis in AML cell lines through retardation expression of miR-181a and affecting miR-181a pathway, which already clarified its role in the differentiation of hematopoietic stem cells and indicates another mode of anti-cancer action of sulforaphane.

Identification of Gene Networks Associated with the Anti-Leukemic Effect of Anti-Inflammatory Drugs on Acute Myeloid Leukemia Cell Lines

IntroductionDespite recent advances in therapy, acute myeloid leukemia (AML) remain a medical challenge with high morbidity and mortality rates. For most patients, allogeneic hematopoietic stem cell transplantation remain the only curative option, but due to the advanced age at diagnosis, a significant proportion of patients are not elegible to this form of therapy. Nevertheless, novel therapies are warranted. There is preclinical evidence that anti-inflammatory compounds, such as COX-2 inhibitors and steroids, may have anti-neoplastic activity in different tumor types, including AML; nevertheles the mechanisms associated with its anti-neoplastic activity are not clear. Therefore, the aim of this work was to evaluate the anti-leukemic effect of the anti-inflammatory compounds nimesulide and prednisolone in AML cell lines and to identify genes and molecular pathways associated with cytotoxicity through transcriptome analysis.MethodsThe leukemic cell lines HL-60, THP-1, OCI-AML2 and OCI-AML3 were treated with nimesulide and prednisolone at 100 µM alone and in combination and with cytarabine at 2.5 µM. Twenty four hours after treatment , we measured the amount of cell death using Annexin V Apoptosis Detection Kit FITC (ThermoFisher) and the cell cycle was analized after fixing the cells with 70% alcohol and incubation with propidium iodide (1mg/ml) and RNAse (10mg/ml). In another experiment, we harvested the cells after 4 hours of treatment for transcriptome analysis. RNA was extracted from control (DMSO) and treatment groups (1 - nimesulide, 2 - prednisolone , 3 - nimesulide and prednisolone) with RNeasy Mini Kit (Qiagen). The Illumina® NEBNext® Ultra II Directional RNA Library Prep Kit was used for library preparation, following the manufacturer protocol using Poly(A) mRNA Magnetic Isolation Module. Equimolar amount of libraries was sequenced using an Illumina NextSeq 500, following the manufacturer's instructions, on the Oklahoma Medical Research Foundation Genomics Core (USA). The sequences obtained with the RNA-Seq technique were aligned in the human genome of reference GRCh37.75 by the software Spliced Transcripts Alignment to a Reference (STAR) v2.5 and to obtain normalized counts in FPKM, the software Expectation-Maximization (RSEM) v1.3.0 was used. To identify network of genes correlated with the treatment (modules), we used the Weighted Correlation Network Analysis (WGCNA). Functional enrichment analysis of the WGCNA differentially expressed modules was performed using the Integrated Annotation, Visualization and Discovery Database (DAVID) v6.8 in order to correlate with biological processes.ResultsIn the cell cycle analysis, we observed a significant increase (p < 0.05) in the sub-G0 phase (cell death) after treatment with nimesulide alone, and in combination with prednisolone (figure 1). No effect was observed in the prednisolone only group. The cell cycle effect induced by nimesulide on HL-60 and OCI-AML2 was similar to the induced by cytarabine, a standard chemotherapy agent for AML that in known to induce arrest in the S phase. In addition, the cell line arrest in THP-1 was greater with nimesulide than with cytarabine, while OCI-AML3 was less sensitive to both nimesulide and cytarabine. Regarding cell death mechanism, treatment with nimesulide induced predominantly an increase in late apoptosis that was potentiated after combined treatment with nimesulide and cytarabine (figure 2). After the demonstration of cell cycle arrest and apoptosis induction after treatment with nimesulide, we performed whole transcriptome sequencing followed by WGCNA analysis. We have identified gene modules that were significantly correlated with anti-inflammatory treatments, being 1 module down-regulated (lightyellow with p = 0.00052) and 2 modules up-regulated (lightcyan with p = 0 .00025 and tan with p = 0.000038). Analysis of functional enrichment using DAVID showed up-regulation of gene networks associated with apoptotic processes and autophagy and down- regulation of gene networks associated with cell cycle and RNA splicing pathwaysConclusionsThe COX-2 inhibitor nimesulide caused cell cycle arrest and apoptosis in AML cell lines and potentiated the cytotoxic effects of cytarabine. This treatment was associated with up- regulation of autophagy and apoptosis and down-regulation of cell cycle and RNA splicing gene networks. [Display omitted] DisclosuresSantos: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Campregher: Astellas: Consultancy.

Validation of NMT1 and NMT2 As Novel Drug Targets in Adult Acute Myeloid Leukemia: Rationale for N-Myristoyltransferase Inhibition with Pclx-001 for Clinical Trials

Background: N-terminal myristoylation is the transfer of the saturated fourteen carbon fatty acid myristate to an N-terminal glycine residue. This co- or post-translational protein modification promotes protein-protein and protein-lipid interactions and is essential for proper membrane localization and/or activity of up to 600 human intracellular proteins. N-myristoyltransferases (NMTs) are the enzymes responsible and two isoforms are found in humans. NMT1 (ubiquitous and essential for cell survival) and NMT2 (more variably expressed) differ in activity level and substrate specificities. NMT expression levels vary in some cancers, and with myristoylation being essential for activity of certain oncogenes including Src Family Kinases (SFKs). NMTs have therefore been proposed as an anti-cancer target. Dysregulation and oncogenic activity of SFKs occurs frequently in acute myeloid leukemia (AML), suggesting NMT inhibition could provide therapeutic benefit. PCLX-001 is a low nanomolar small molecule pan-NMT inhibitor with high oral bioavailability in clinical trials as once daily oral therapy for lymphoma and solid tumors.Methods and Results: Data from the TCGA Transcriptome database showed high NMT1 and low NMT2 were associated with reduced overall and event-free survival in adult AML, and high NMT1 - but not NMT2 - expression is associated with proliferative gene sets in AML cell lines. AML cell lines treated with PCLX-001 showed a significant reduction in total protein myristoylation, as well as reduced levels of SFK proteins and SFK phosphorylation. PCLX-001 induced apoptosis in AML cell lines and patient blasts at concentrations which spared a large proportion of peripheral blood lymphocytes and monocytes from healthy individuals. AML cell lines showed significant increase in BIP protein and ER stress in response to PCLX-001, along with caspase 3 cleavage. In an AML cell line derived xenograft (CDX) and two AML patient derived xenograft (PDX) series (n=1 DX for MV-4-11 and n=2 PDX), PCLX-001 monotherapy had dose-dependent anticancer activity and resulted in complete remissions in subcutaneous AML cell deposits. In tail-vein injection PDX models, PCLX-001 treatment resulted in up to 95% reduction of human CD45+ cells in peripheral blood and bone marrow.Conclusions: These findings validate NMT inhibition as a novel therapeutic strategy for AML. PCLX-001 preferentially targeted AML cells that rely on oncogenic activity of myristoylated proteins, inducing apoptosis and reducing leukemic burden. PCLX-001 warrants evaluation in clinical trials for adult AML. DisclosuresGamma: Pacylex Pharmaceuticals: Current holder of individual stocks in a privately-held company. Yap: Pacylex Pharmaceuticals: Current holder of individual stocks in a privately-held company, Patents & Royalties. Beauchamp: Pacylex Pharmaceuticals: Current Employment, Current holder of individual stocks in a privately-held company, Patents & Royalties. Mackey: Pacylex Pharmaceuticals, Inc.: Current holder of individual stocks in a privately-held company. Pemmaraju: Dan's House of Hope: Membership on an entity's Board of Directors or advisory committees; Abbvie Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Aptitude Health: Consultancy; Sager Strong Foundation: Other; Celgene Corporation: Consultancy; LFB Biotechnologies: Consultancy; Plexxicon: Other, Research Funding; MustangBio: Consultancy, Other; Roche Diagnostics: Consultancy; Daiichi Sankyo, Inc.: Other, Research Funding; DAVA Oncology: Consultancy; Cellectis S.A. ADR: Other, Research Funding; Springer Science + Business Media: Other; Stemline Therapeutics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; HemOnc Times/Oncology Times: Membership on an entity's Board of Directors or advisory committees; ASCO Leukemia Advisory Panel: Membership on an entity's Board of Directors or advisory committees; Samus: Other, Research Funding; ASH Communications Committee: Membership on an entity's Board of Directors or advisory committees; CareDx, Inc.: Consultancy; Novartis Pharmaceuticals: Consultancy, Other: Research Support, Research Funding; Incyte: Consultancy; Affymetrix: Consultancy, Research Funding; Protagonist Therapeutics, Inc.: Consultancy; Clearview Healthcare Partners: Consultancy; Blueprint Medicines: Consultancy; Bristol-Myers Squibb Co.: Consultancy; ImmunoGen, Inc: Consultancy; Pacylex Pharmaceuticals: Consultancy. Borthakur: Astex: Research Funding; Ryvu: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Protagonist: Consultancy; ArgenX: Membership on an entity's Board of Directors or advisory committees; University of Texas MD Anderson Cancer Center: Current Employment; Takeda: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy. Brandwein: AbbVie: Honoraria; Jazz: Honoraria; Taiho: Honoraria; Astellas: Honoraria; Bristol Myers Squibb: Honoraria; Roche: Honoraria; Pfizer: Honoraria; Amgen: Honoraria. Berthiaume: Pacylex Pharmaceuticals, Inc.: Current holder of individual stocks in a privately-held company.

A Novel Isoflavone, ME-344, Enhances Venetoclax Antileukemic Activity Against AML Via Suppression of Oxidative Phosphorylation and Purine Biosynthesis

The 5-year survival rate for adult patients with acute myeloid leukemia (AML) treated with cytarabine-based chemotherapy remains less than 30%, due to drug resistance and disease relapse. Recently, a selective inhibitor of anti-apoptotic Bcl-2, venetoclax, was approved by the FDA in combination with low dose cytarabine or hypomethylating agents for treating newly diagnosed AML patients who are 75 years of age or older or for those who are unfit for standard chemotherapy, providing more treatment options for this group of patients. Although the response rate to these newly approved combination therapies is reported to be 70%, the median overall survival is only 10-18 months showing that the duration of response is limited. Therefore, novel therapeutic agents are in demand to enhance venetoclax activity against AML and to combat AML resistant to cytarabine-based chemotherapy.Cytarabine-resistant AML cells lead to relapse and rely on oxidative phosphorylation (OXPHOS) for survival. In addition, it has been reported that targeting OXPHOS can enhance venetoclax activity against preclinical models of AML. Thus, we hypothesize that OXPHOS suppressing agents would be good candidates to combine with and enhance venetoclax antileukemic activity against newly diagnosed AML and those with resistance to cytarabine.A novel isoflavone, ME-344, has been shown to suppress OXPHOS in cell lines derived from solid tumors by inhibiting Complex I of the electron transport chain. We hypothesized that combining ME-344 with venetoclax would result in synergistic antileukemic activity against AML. Consistent with our hypothesis, combining ME-344 with venetoclax resulted in synergistic induction of apoptosis in AML cell lines, including those with acquired cytarabine resistance. The combination of these two agents also resulted in synergistic antileukemic activity in one primary AML patient sample, as determined by MTT assay. The combination of ME-344 and venetoclax prolonged the median survival of MV4-11 leukemia- bearing NSGS mice by 37% (median survival of 48 days compared to 35 days for vehicle control treated mice, n=5 per arm, p<0.0001). This is in contrast to the venetoclax combination with cytarabine, which prolonged median survival of the same xenograft model by 7.5% (Luedtke et al., Signal Transduction and Targeted Therapy, 2020; 5:17). ME-344 alone (9-hour treatment) reduced basal mitochondrial respiration in AML cells by 10% prior to induction of apoptosis. When treated with ME-344 for 8-hours followed by combined ME-344 and venetoclax for an additional 1-hour, basal mitochondrial respiration was reduced by 18% (again prior to detection of apoptosis initiation). This sequential combination regimen also decreased the mitochondrial membrane potential (by JC-1 staining and flow cytometry analysis) when compared to untreated control and single treatment. Additionally, apoptosis induction by the combination of ME-344 and venetoclax or ME-344 alone was significantly enhanced when AML cells were forced to utilize OXPHOS by replacing glucose with galactose in the culture medium. Further investigation revealed that apoptosis induced by ME-344 was partially attenuated when Mcl-1 was overexpressed, Bak was knocked down, or caspase activation was inhibited. This suggests a mechanism that involves components of the intrinsic apoptosis pathway. Targeted metabolomics analyses of MV4-11 cells treated with ME-344 for 8 h revealed a significant reduction of essential metabolites involved in the de novo purine biosynthesis pathway, specifically AICAR (p=0.001) and IMP (p=0.004). Given the critical role of purine in cell proliferation and survival, suppression of purine biosynthesis by ME-344 may represent a novel mechanism underlying its enhancement on the antileukemic activity of venetoclax against AML. Interestingly, inhibition of this pathway by the purine biosynthesis inhibitor lometrexol, also synergistically enhanced apoptosis in AML cells induced by venetoclax. Taken together, these results suggest that ME-344 suppresses OXPHOS and the purine biosynthesis pathway to enhance the antileukemic activity of venetoclax against AML. Further in-depth mechanistic studies into the suppression of purine biosynthesis and OXPHOS, as well as studies of ME-344 and venetoclax against cytarabine-resistant AML in a mouse model are warranted. DisclosuresWiley: MEIPharma: Current Employment.