Venetoclax-Resistant MV4-11 Leukemic Cells Activate PI3K/AKT Pathway for Metabolic Reprogramming and Redox Adaptation for Survival

doi:10.3390/antiox11030461

. 2022 Feb 25;11(3):461.

doi: 10.3390/antiox11030461.

Venetoclax-Resistant MV4-11 Leukemic Cells Activate PI3K/AKT Pathway for Metabolic Reprogramming and Redox Adaptation for Survival

Hind A Alkhatabi^{1

2

3}, Samir F Zohny¹, Mohammed Razeeth Shait Mohammed^{1

4}, Hani Choudhry^{1

4}, Mohd Rehan⁵, Aamir Ahmad⁶, Farid Ahmed^{3

7}, Mohammad Imran Khan^{1

4}

Affiliations

¹ Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
² Department of Biochemistry, College of Science, University of Jeddah, Jeddah 21589, Saudi Arabia.
³ Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
⁴ Centre of Artificial Intelligence for Precision Medicines, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
⁵ King Fahd Medical Research Centre, King Abdulaziz University, P.O. Box 80216, Jeddah 21589, Saudi Arabia.
⁶ Translational Research Institute, Hamad Medical Corporation, Doha 3050, Qatar.
⁷ Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia.

PMID: 35326111
PMCID: PMC8944541
DOI: 10.3390/antiox11030461

Venetoclax-Resistant MV4-11 Leukemic Cells Activate PI3K/AKT Pathway for Metabolic Reprogramming and Redox Adaptation for Survival

Hind A Alkhatabi et al. Antioxidants (Basel). 2022.

. 2022 Feb 25;11(3):461.

doi: 10.3390/antiox11030461.

Authors

Hind A Alkhatabi^{1

2

3}, Samir F Zohny¹, Mohammed Razeeth Shait Mohammed^{1

4}, Hani Choudhry^{1

4}, Mohd Rehan⁵, Aamir Ahmad⁶, Farid Ahmed^{3

7}, Mohammad Imran Khan^{1

4}

Affiliations

¹ Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
² Department of Biochemistry, College of Science, University of Jeddah, Jeddah 21589, Saudi Arabia.
³ Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
⁴ Centre of Artificial Intelligence for Precision Medicines, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
⁵ King Fahd Medical Research Centre, King Abdulaziz University, P.O. Box 80216, Jeddah 21589, Saudi Arabia.
⁶ Translational Research Institute, Hamad Medical Corporation, Doha 3050, Qatar.
⁷ Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia.

PMID: 35326111
PMCID: PMC8944541
DOI: 10.3390/antiox11030461

Abstract

Venetoclax (ABT199) is a selective B-cell lymphoma 2 (BCL-2) inhibitor. The US FDA recently approved it to be used in combination with low-dose cytarabine or hypomethylating agents in acute myeloid leukemia (AML) or elderly patients non-eligible for chemotherapy. However, acquiring resistance to venetoclax in AML patients is the primary cause of treatment failure. To understand the molecular mechanisms inherent in the resistance to BCL-2 inhibitors, we generated a venetoclax-resistant cell line model and assessed the consequences of this resistance on its metabolic pathways. Untargeted metabolomics data displayed a notable impact of resistance on the PI3K/AKT pathway, the Warburg effect, glycolysis, the TCA cycle, and redox metabolism. The resistant cells showed increased NADPH and reduced glutathione levels, switching their energy metabolism towards glycolysis. PI3K/AKT pathway inhibition shifted resistant cells towards oxidative phosphorylation (OXPHOS). Our results provide a metabolic map of resistant cells that can be used to design novel metabolic targets to challenge venetoclax resistance in AML.

Keywords: MV4-11; OXPHOS; PI3K/AKT pathway; acute myeloid leukemia; glycolysis; metabolomics; redox metabolism; venetoclax; venetoclax resistance model.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Figure 1**
ABT199-R cells are highly resistant to venetoclax. (A) MV4-11 and ABT-199R were treated with increasing doses of venetoclax (0–20 µM) for 48 h and assessed with a cell titer blue proliferation assay. The curves indicate the percentage survival of each cell line to increasing doses of venetoclax. (B) MV4-11 and ABT199-R cells were treated with indicated concentrations of venetoclax for 48 h and stained for an Annexin-V/7AAD assay. The percentage values determine the early and late apoptotic populations.

**Figure 2**
Metabolomic analysis of MV4-11 cells vs. ABT199-R cells. (A) The metabolic heatmap profile of differentially accumulated metab olites between sensitive MV4-11 and resistant ABT199-R. (B) Volcano plots of metabolic differentiation between MV4-11 and ABT199-R. (C) ABT199-R cells were treated with several concentrations of PKI-402 for 48 h and assessed with cell titer blue viability assay. The curve indicates the IC50 value.

**Figure 3**
Metabolomic analysis of MV4-11, ABT199-R, and ABT199-R treated with PKI-402. (A) Metabolites were extracted and run in LTQ-XL linear ion trap LC-MS, showing their total ion chromatograms. (B) PCA analysis of comprehensive metabolites of MV4-11 cells, ABT199-R cells, and ABT199-R cells treated with PKI-402. (C) Correlation heatmap of ABT-199R cells, MV4-11 cells, and ABT199-R cells treated with PKI-402. (D) Heatmap of differentially expressed metabolites in MV4-11 cells, ABT199-R cells, and ABT199-R cells treated with PKI-402. (E) VIP score based on PCA analysis of principal metabolites. (F) Top pathway enriched in metabolome analysis in MV4-11 cells, ABT199-R cells, and ABT199-R cells treated with PKI-402. (G) Pathway network analysis.

**Figure 4**
p-AKT signaling pathway in ABT199-R cells. (A) Expression of PIP metabolites in MV4-11 cells, ABT199-R cells, and ABT199-R cells treated with PKI-402. (B) Western blot analysis of p-AKT and AKT in MV4-11 cells, ABT199-R cells, and ABT199-R cells treated with PKI-402. (C) Densitometer analysis of Western blot analysis of p-AKT/AKT in MV4-11 cells, ABT199-R cells, and ABT199-R cells treated with PKI-402. ** p < 0.01 and **** p < 0.0001.

**Figure 5**
Venetoclax-resistant ABT199-R cells alter the glycolysis pathway. (**Left**) Quantitative levels of various metabolites involved in glycolysis pathways of MV4-11 cells, ABT199-R cells, and ABT199-R cells treated with PKI-402. (**Right**) Gene expression (RT-PCR) of various genes involved in glycolysis. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

**Figure 6**
TCA cycle metabolism and OXPHOS are altered in ABT199-R cells. (A) The peak intensity of individual metabolites and their quantitative levels involved in the TCA cycle of MV4-11 cells, ABT199-R cells, and ABT199-R cells treated with PKI-402. (B) Gene expression (RT-PCR) of various genes involved in mitochondrial OXPHOS. ns = non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

**Figure 7**
Pentose phosphate pathway (PPP) and energy metabolites modified in ABT199-R cells. (A) The density of metabolic features involved in regulating the energy metabolism of MV4-11 cells, ABT199-R cells, and ABT199-R cells treated with PKI-402. (B) Metabolites involved in PPP. ns = non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

**Figure 8**
Redox homeostasis of venetoclax-resistant cells. (A) Glutathione (metabolite) involved in GSH pathway. (B) GLS-2 (gene) is involved in GSH pathway expression (RT-PCR). (C) Relative glutathione concentrations were measured using GSH assay from total protein. (D) ROS levels using CellRox Green Flow Cytometry assay. The channel Ch05 scans for red, and Ch02 scans for green. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

**Figure 9**
Inhibition of PI3K-AKT induces apoptosis in resistant ABT199-R cells: ABT199-R cells were treated with the IC50 value of PKI-402 for 48 h and stained for an Annexin-V/PI assay. The single-cell images were captured using Amnis^® FlowSight^®. The percentage of apoptosis was calculated based on cell positivity. The channel Ch05 scans for red, and Ch02 scans for green. *** p < 0.001.

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References

1. Mehta S.V., Shukla S.N., Vora H.H. Overexpression of Bcl2 Protein Predicts Chemoresistance in Acute Myeloid Leukemia: Its Correlation with FLT3. Neoplasma. 2013;60:666–675. doi: 10.4149/neo_2013_085. - DOI - PubMed
1. Guerra V.A., DiNardo C., Konopleva M. Venetoclax-Based Therapies for Acute Myeloid Leukemia. Best Pract. Res. Clin. Haematol. 2019;32:145–153. doi: 10.1016/j.beha.2019.05.008. - DOI - PMC - PubMed
1. Gandhi L., Camidge D.R., de Oliveira M.R., Bonomi P., Gandara D., Khaira D., Hann C.L., McKeegan E.M., Litvinovich E., Hemken P.M., et al. Phase I Study of Navitoclax (ABT-263), a Novel Bcl-2 Family Inhibitor, in Patients with Small-Cell Lung Cancer and Other Solid Tumors. J. Clin. Oncol. 2011;29:909–916. doi: 10.1200/JCO.2010.31.6208. - DOI - PMC - PubMed
1. Vandenberg C.J., Cory S. Brief Report LYMPHOID NEOPLASIA ABT-199, a New Bcl-2-Specific BH3 Mimetic, Has in Vivo Efficacy against Aggressive Myc-Driven Mouse Lymphomas without Provoking Thrombocytopenia. Blood J. Am. Soc. Hematol. 2013;121:2285–2288. doi: 10.1182/blood. - DOI - PMC - PubMed
1. Souers A.J., Leverson J.D., Boghaert E.R., Ackler S.L., Catron N.D., Chen J., Dayton B.D., Ding H., Enschede S.H., Fairbrother W.J., et al. ABT-199, a Potent and Selective BCL-2 Inhibitor, Achieves Antitumor Activity While Sparing Platelets. Nat. Med. 2013;19:202–208. doi: 10.1038/nm.3048. - DOI - PubMed

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[1] Mehta S.V., Shukla S.N., Vora H.H. Overexpression of Bcl2 Protein Predicts Chemoresistance in Acute Myeloid Leukemia: Its Correlation with FLT3. Neoplasma. 2013;60:666–675. doi: 10.4149/neo_2013_085. - DOI - PubMed

[2] Mehta S.V., Shukla S.N., Vora H.H. Overexpression of Bcl2 Protein Predicts Chemoresistance in Acute Myeloid Leukemia: Its Correlation with FLT3. Neoplasma. 2013;60:666–675. doi: 10.4149/neo_2013_085. - DOI - PubMed

[3] Guerra V.A., DiNardo C., Konopleva M. Venetoclax-Based Therapies for Acute Myeloid Leukemia. Best Pract. Res. Clin. Haematol. 2019;32:145–153. doi: 10.1016/j.beha.2019.05.008. - DOI - PMC - PubMed

[4] Guerra V.A., DiNardo C., Konopleva M. Venetoclax-Based Therapies for Acute Myeloid Leukemia. Best Pract. Res. Clin. Haematol. 2019;32:145–153. doi: 10.1016/j.beha.2019.05.008. - DOI - PMC - PubMed

[5] Gandhi L., Camidge D.R., de Oliveira M.R., Bonomi P., Gandara D., Khaira D., Hann C.L., McKeegan E.M., Litvinovich E., Hemken P.M., et al. Phase I Study of Navitoclax (ABT-263), a Novel Bcl-2 Family Inhibitor, in Patients with Small-Cell Lung Cancer and Other Solid Tumors. J. Clin. Oncol. 2011;29:909–916. doi: 10.1200/JCO.2010.31.6208. - DOI - PMC - PubMed

[6] Gandhi L., Camidge D.R., de Oliveira M.R., Bonomi P., Gandara D., Khaira D., Hann C.L., McKeegan E.M., Litvinovich E., Hemken P.M., et al. Phase I Study of Navitoclax (ABT-263), a Novel Bcl-2 Family Inhibitor, in Patients with Small-Cell Lung Cancer and Other Solid Tumors. J. Clin. Oncol. 2011;29:909–916. doi: 10.1200/JCO.2010.31.6208. - DOI - PMC - PubMed

[7] Vandenberg C.J., Cory S. Brief Report LYMPHOID NEOPLASIA ABT-199, a New Bcl-2-Specific BH3 Mimetic, Has in Vivo Efficacy against Aggressive Myc-Driven Mouse Lymphomas without Provoking Thrombocytopenia. Blood J. Am. Soc. Hematol. 2013;121:2285–2288. doi: 10.1182/blood. - DOI - PMC - PubMed

[8] Vandenberg C.J., Cory S. Brief Report LYMPHOID NEOPLASIA ABT-199, a New Bcl-2-Specific BH3 Mimetic, Has in Vivo Efficacy against Aggressive Myc-Driven Mouse Lymphomas without Provoking Thrombocytopenia. Blood J. Am. Soc. Hematol. 2013;121:2285–2288. doi: 10.1182/blood. - DOI - PMC - PubMed

[9] Souers A.J., Leverson J.D., Boghaert E.R., Ackler S.L., Catron N.D., Chen J., Dayton B.D., Ding H., Enschede S.H., Fairbrother W.J., et al. ABT-199, a Potent and Selective BCL-2 Inhibitor, Achieves Antitumor Activity While Sparing Platelets. Nat. Med. 2013;19:202–208. doi: 10.1038/nm.3048. - DOI - PubMed

[10] Souers A.J., Leverson J.D., Boghaert E.R., Ackler S.L., Catron N.D., Chen J., Dayton B.D., Ding H., Enschede S.H., Fairbrother W.J., et al. ABT-199, a Potent and Selective BCL-2 Inhibitor, Achieves Antitumor Activity While Sparing Platelets. Nat. Med. 2013;19:202–208. doi: 10.1038/nm.3048. - DOI - PubMed

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Venetoclax-Resistant MV4-11 Leukemic Cells Activate PI3K/AKT Pathway for Metabolic Reprogramming and Redox Adaptation for Survival

Affiliations

Venetoclax-Resistant MV4-11 Leukemic Cells Activate PI3K/AKT Pathway for Metabolic Reprogramming and Redox Adaptation for Survival

Authors

Affiliations

Abstract

Conflict of interest statement

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