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

Abstract 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. Disclosures Wiley: MEIPharma: Current Employment.

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Voruciclib, an Oral, Selective CDK9 Inhibitor, Enhances Cell Death Induced By the Bcl-2 Selective Inhibitor Venetoclax in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-118372 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1361-1361 ◽

Cited By ~ 2

Author(s):

Daniel A Luedtke ◽

Yongwei Su ◽

Holly Edwards ◽

Lisa Polin ◽

Juiwanna Kushner ◽

...

Keyword(s):

Cell Lines ◽

Myeloid Leukemia ◽

Caspase 3 ◽

Combination Index ◽

Survival Rates ◽

Annexin V ◽

Antileukemic Activity ◽

Induction Of Apoptosis ◽

Induced Apoptosis

Abstract Introduction: Patients with acute myeloid leukemia (AML) face overall 5-year survival rates of 65% and 27% for children and adults, respectively, leaving significant room for improvement. Relapse remains a major contributor to such low overall survival rates, and leukemic stem cells (LSCs) that survive treatment are believed to be responsible for AML relapse. The anti-apoptotic protein Bcl-2 is overexpressed in bulk AML cells and LSCs and is associated with poor clinical outcomes. Thus, Bcl-2 represents a promising therapeutic target for the treatment of AML. Venetoclax (ABT-199) is a selective Bcl-2 inhibitor that has shown great potential for treating a number of malignancies, including AML. Venetoclax inhibits Bcl-2, preventing it from sequestering pro-apoptotic Bcl-2 family protein Bim, leading to Bim activated Bax/Bak, resulting in apoptosis. However, Mcl-1 can also sequester Bim and prevent apoptosis. We previously showed that directly targeting Mcl-1 can enhance the antileukemic activity of venetoclax (Luedtke DA, et al. Signal Transduct Target Ther. Apr 2017). Alternatively, we proposed that indirect targeting of Mcl-1 may preserve or enhance the antileukemic activity of venetoclax, and prevent resistance resulting from Mcl-1. It has been reported that inhibition of CDK9 can downregulate cell survival genes regulated by superenhancers, including Mcl-1, MYC, and Cyclin D1. One CDK9 inhibitor in clinical development, flavopiridol (alvocidib), has progressed to phase II clinical trials in AML. However, off target effects and dose-limiting toxicities remain a concern. Voruciclib is an oral, selective CDK inhibitor differentiated by its potent inhibition of CDK9 as compared to other CDK inhibitors. This selectivity may potentially circumvent toxicities resulting from inhibition of non-CDK targets like MAK and ICK that are inhibited by flavopiridol. Voruciclib has been shown in vitro to promote apoptosis and decrease Mcl-1 expression levels in chronic lymphocytic leukemia (CLL) cells (Paiva C, et al. PLOS One. Nov 2015) and inhibit tumor growth in mouse xenograft models of diffuse large B-cell lymphoma (DLBCL) in combination with venetoclax (Dey J. et al Scientific Reports. Dec 2017). Based on these data, voruciclib may downregulate Mcl-1 in AML cells and therefore synergistically enhance the antileukemic activity of venetoclax. Methods/Results: Culturing AML cell lines (THP-1, U937, MOLM-13, MV4-11, and OCI-AML3) and primary patient samples with various concentrations of voruciclib resulted in a concentration-dependent increase in Annexin V+ cells (2 μM voruciclib induced 13.8-55.8% Annexin V+ cells) along with increased levels of cleaved caspase 3 and PARP, demonstrating that voruciclib induces apoptosis in AML cells. Next, we tested the combination of voruciclib and venetoclax in AML cell lines and primary AML patient samples at clinically achievable concentrations. Annexin V/PI staining, flow cytometry analysis, and combination index calculation (using CalcuSyn software) revealed synergistic induction of apoptosis by voruciclib and venetoclax combination (combination index values for MV4-11, U937, THP-1, and MOLM-13 cells were <0.73; treatment with 2 µM voruciclib and venetoclax for 24 h resulted in >80% apoptosis). Importantly, synergy was observed in both venetoclax sensitive and resistant cell lines. This was accompanied by increased cleavage of caspase 3 and PARP. Lentiviral shRNA knockdown of Bak and Bax partially rescued AML cells from voruciclib-induced apoptosis, showing that voruciclib induces apoptosis at least partially through the intrinsic apoptosis pathway. However, Bak and Bax knockdown had little to no effect on induction of apoptosis by the combination treatment, indicating that there might be other molecular mechanisms underlying the synergistic interaction between the two agents. Treatment with the pan-caspase inhibitor Z-VAD-FMK partially rescued cells from combination treatment induced-apoptosis. Discussion: Collectively, these results demonstrate that voruciclib and venetoclax synergistically induce apoptosis in AML cells in vitro and reverse venetoclax resistance. Further studies to determine the mechanism of action and in vivo efficacy of this promising combination in AML xenografts and PDX models are underway. Disclosures Ge: MEI Pharma: Research Funding.

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Integrative Analysis of AML Cell Response to Cytarabine Reveals Synergistic Opportunities Centered on Cholesterol Metabolism

Blood ◽

10.1182/blood-2018-99-113634 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2631-2631

Author(s):

Fangli Chen ◽

Xue Wu ◽

Rogozea Adriana ◽

Bin Guo ◽

H. Scott Boswell ◽

...

Keyword(s):

Cell Growth ◽

Cell Lines ◽

Metabolic Stress ◽

Risk Groups ◽

Gene Set Enrichment Analysis ◽

Synergistic Activity ◽

Antileukemic Activity ◽

Cholesterol Levels ◽

Induction Of Apoptosis ◽

Intracellular Cholesterol

Abstract Background: Despite spectacular advances in deciphering the molecular landscape of acute myeloid leukemia (AML), the clinical course of this disease has not improved dramatically for decades. Resistance to therapy and disease recurrence almost invariably occur, highlighting the need for a deeper understanding of these processes. Our general strategy takes into account decreased oxygen (O2) tension, a micro-environmental hallmark which remains understudied in the context of AML. While low O2 compartments such as bone marrow niches are well-recognized hosts of drug resistant AML cells, in vitro studies are still routinely performed in supra-physiologic ambient O2 concentration (21%), with potentially important consequences for clinical translatability. We hereby present an integrated molecular dissection of AML cells response to Cytarabine under O2-controlled conditions and exploit the information to develop novel synergistic/additive treatment combinations. Methods: M14, THP1, OCIAML3 and primary cells (PC) derived from AML patients of different risk groups were incubated in culture medium under 21% O2 or 1% O2 in the presence or absence of Cytarabine (Cy, 1-7.5 µM), Rosuvastatin (Ro, 10-200µM) and/or Pravastatin (Pra, 10-200µM). After 48h, proliferation and apoptosis assays were performed using MTT assays, annexin V/PI staining and FACS analysis. Characterization of gene expression and expression levels of total and phosphorylated proteins were assessed using RNAseq and Reverse Phase Protein Arrays (RPPA), respectively. Gene Set Enrichment Analysis was performed following GSEA User Guide. Quantitative PCR reactions were performed using PowerUp SYBR Green PCR Master Mix (Applied Biosystems, USA) on 7900HT Real-Time PCR System. Intracellular cholesterol (ICC) levels were measured using WAKO Cholesterol assay kit. Results: Treatment with Cy under 1% O2 induced a specific gene signature of metabolic stress responses, including the perturbation of cholesterol homeostasis. Detailed analysis of the cholesterol pathway via RNAseq showed pathway induction by hypoxia itself and severe depletion by Cy. Focused RT-PCR assays on the cholesterol pathway components HMGCS1, MSMO1, LSS and SQLE demonstrated significant induction under 1% compared to 21% O2, and significant downregulation in response to Cy. In line with our RNAseq and PCR data, RPPA showed that critical cholesterol synthesis pathways, such as ACC1, are downregulated in response to Cy. Surprisingly, we observed that ICC levels significantly increased after 48h in response to treatment with Cy (p<.01, n=4 [M14] and [THP1], 21% O2; p<.05, n=4 [M14] and [THP1], 1% O2). In addition, Cy therapy led to strong induction of CD36 mRNA expression (e.g. up to 11.7-fold at 21% O2, p<.05, up to 7.3-fold at 1% O2, p<.01, n=3 [M14]). In order to investigate the effects of cholesterol inhibition against AML cells, we treated several AML cell lines and PCs with Ro or Pra under 21% and 1% O2. We found that Ro exerts dose dependent antileukemic activity at 21% and 1% O2 and, at all doses tested, was significantly more effective than Pra, which was not effective against AML cells at 1% O2. The cytotoxic effects of Ro consisted of lowering ICC levels, induction of apoptosis and inhibition of AML cell growth. Further, Ro combined with Cy significantly enhanced the antileukemic effects of Cy alone and exhibited additive to synergistic activity in AML cell lines (CImax= 0.55, p<.05, n=3) and strong synergy in PCs (CImax= 0.22, n=10) derived from AML patients of different risk groups under 21% and 1% O2. Remarkably, Ro was not toxic against human CD34+ cord blood cells. Summary: Treatment with Cytarabine induces a genetic signature of metabolic stress response, including the perturbation of the cholesterol biosynthesis pathway, in AML cells. Depletion of intracellular cholesterol levels is counterbalanced by upregulation of CD36 expression in Cytarabine-treated AML cells under 21% and 1% O2 conditions. Rosuvastatin exerts antileukemic activity via downregulation of intracellular cholesterol levels, induction of apoptosis and inhibition of AML cell growth. Rosuvastatin displays greater antileukemic effects than pravastatin. Rosuvastatin combined with Cytarabine acts synergistically against AML cells. Conclusions: Further investigation of Rosuvastatin as a component of anti-AML therapy is warranted. Disclosures No relevant conflicts of interest to declare.

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Oxidative phosphorylation safeguards pluripotency via UDP-N-acetylglucosamine

10.1101/2021.07.06.451381 ◽

2021 ◽

Author(s):

Jiani Cao ◽

Meng Li ◽

Kun Liu ◽

Xingxing Shi ◽

Ning Sui ◽

...

Keyword(s):

Oxidative Phosphorylation ◽

Mitochondrial Respiration ◽

Biosynthesis Pathway ◽

Self Renewal ◽

Identity Maintenance ◽

Metabolic Gene Expression ◽

Hexosamine Biosynthesis ◽

Hexosamine Biosynthesis Pathway

The roles of mitochondrial respiration in pluripotency remain largely unknown. We show here that mouse ESC mitochondria possess superior respiration capacity compared to somatic cell mitochondria, and oxidative phosphorylation (OXPHOS) generates the majority of cellular ATP in ESCs. Inhibition of OXPHOS results in extensive pluripotency and metabolic gene expression reprogram, leading to disruption of self-renewal and pluripotency. Metabolomics profiling identifies UDP-N-acetylglucosamine (UDP-GlcNAc) as one of the most significantly decreased metabolites in response to OXPHOS inhibition. The loss of ESC identity induced by OXPHOS inhibition can be ameliorated by directly adding GlcNAc both in vitro and in vivo. This work demonstrates that mitochondrial respiration, but not glycolysis, produces the majority of ATP in ESCs, and uncovers a novel mechanism whereby mitochondrial respiration is coupled with the hexosamine biosynthesis pathway to generate UDP-GlcNAc for ESC identity maintenance.

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Cytotoxicity of Hydrazones of Morpholine Bearing Mannich Bases Towards Huh7 and T47D Cell Lines and Their Effects on Mitochondrial Respiration

Letters in Drug Design & Discovery ◽

10.2174/1570180813666160113002907 ◽

2016 ◽

Vol 13 (8) ◽

pp. 734-741 ◽

Cited By ~ 4

Author(s):

Kaan Kucukoglu ◽

Halise Inci Gul ◽

Mustafa Gul ◽

Rengul Cetin-Atalay ◽

Yosra Baratli ◽

...

Keyword(s):

Cell Lines ◽

Mitochondrial Respiration ◽

T47d Cell ◽

Mannich Bases

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The Interplay between Oxidative Phosphorylation and Glycolysis as a Potential Marker of Bladder Cancer Progression

International Journal of Molecular Sciences ◽

10.3390/ijms21218107 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8107 ◽

Cited By ~ 1

Author(s):

Greta Petrella ◽

Giorgia Ciufolini ◽

Riccardo Vago ◽

Daniel Oscar Cicero

Keyword(s):

Bladder Cancer ◽

Oxidative Phosphorylation ◽

Cell Lines ◽

Cancer Progression ◽

Cancer Genomics ◽

Low Grade ◽

Metabolic State ◽

Extracellular Medium ◽

Potential Marker ◽

Risk Of Progression

Urothelial bladder cancer (UBC) is the most common tumor of the urinary system. One of the biggest problems related to this disease is the lack of markers that can anticipate the progression of the cancer. Genomics and transcriptomics have greatly improved the prediction of risk of recurrence and progression. Further progress can be expected including information from other omics sciences such as metabolomics. In this study, we used 1H-NMR to characterize the intake of nutrients and the excretion of products in the extracellular medium of three UBC cell lines, which are representatives of low-grade tumors, RT4, high-grade, 5637, and a cell line that shares genotypic features with both, RT112. We have observed that RT4 cells show an activated oxidative phosphorylation, 5637 cells depend mostly on glycolysis to grow, while RT112 cells show a mixed metabolic state. Our results reveal the relative importance of glycolysis and oxidative phosphorylation in the growth and maintenance of different UBC cell lines, and the relationship with their genomic signatures. They suggest that cell lines associated with a low risk of progression present an activated oxidative metabolic state, while those associated with a high risk present a non-oxidative state and high glycolytic activity.

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