Bone marrow microenvironment drives AML cell OXPHOS addiction and AMPK inhibition to resist chemotherapy

Abstract

The stromal niche plays a pivotal role in AML chemoresistance and energy metabolism reprogramming is a hallmark of a tumor. 5'-Adenosine monophosphate-activated protein kinase (AMPK) is an important energy sensor suppressing mammalian target of rapamycin complex 1 (mTORC1) activity. However, the role of AMPK-mTORC1 pathway on connecting AML cell energy metabolism reprogramming and chemoresistance induced by the bone marrow microenvironment (BMM) is not defined. Here, with a co-culture system that simulates the interaction between BMM and AML cells, it is shown that stromal contact led to a decreased sensitivity to chemotherapy accompanied by an increase of oxidative phosphorylation (OXPHOS) activity and mitochondrial ATP synthesis in AML cells. The increased OXPHOS activity and excessive ATP production promoted chemoresistance of AML cells through inhibiting AMPK activity and in turn activating mTORC1 activity. In an in vivo AML mouse model, depletion of AMPK activity with genetic targeting promoted AML progression and reduced their sensitivity to chemotherapeutic drugs. Collectively, AML cells' acquired increased OXPHOS activity as well as AMPK inhibition could be therapeutically exploited in an effort to overcome BMM-mediated chemoresistance.

Keywords: AMPK-mTORC1 pathway; ATP; OXPHOS; acute myeloid leukemia; chemotherapy resistance.

FIGURE 1

Coculture of AML cells with HS‐5 cells exhibited higher OXPHOS activity and increased ATP synthesis. (A) Sequential injections of oligomycin, FCCP, and rotenone/antimycin were used to obtain mitochondrial respiration dynamics of CD34⁺ cells from the BM of AML patients and healthy donors (n = 10 for AML patients; n = 5 for healthy donors). (B) The mitochondrial basal respiration, maximal respiration, and spare respiratory capacity of CD34⁺ cells from the BM of AML patients and healthy donors were analyzed with cell numbers normalized. (C) Sequential administration of oligomycin, FCCP, and rotenone/antimycin were used to obtain mitochondrial respiration dynamics of AML cells. (D‐E) AML cells were cultured with HS‐5 cells in a contact or non‐contact co‐culture system and then the mitochondrial basal and maximal respiration of AML cells were analyzed with cell numbers normalized. (F‐H) AML cells were cultured with or without HS‐5 cells and then the ATP production Capacity was analyzed with cell numbers normalized. (F) Total ATP production rate, (G) glycolytic, and (H) mitochondrial ATP production rates in AML cells. The data represent the mean ± SD of 3 independent experiments with 3 replicates for each experiment. AML#, AML patients; HD, healthy donors. ns, no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001

FIGURE 2

Coculture of AML cells with HS‐5 cells displayed the decreased sensitivity to chemotherapy due to the increased ATP synthesis. (A) Growth inhibitory effect of 24 h treatment with different concentrations of Ara‐C and DNR on AML cells cultured alone or with HS‐5 cells was measured by CCK‐8 assay. (B) The percentage of apoptotic cells of 24 h treatment with Ara‐C (The concentration of Ara‐C was 50 μM for HL‐60 cells and 5 μM for U937 cells) on AML cells cultured alone or with HS‐5 cells was expressed as mean ± SD from 3 independent experiments. (C) The mitochondrial ATP production rate of AML cells was significantly inhibited by treatment with Oligomycin A. (D) Growth inhibitory effect of 24 h treatment with different concentrations of Ara‐C and DNR on AML cells after treatment with 10 μM Oligomycin A was measured by CCK‐8 assay. (E) The percentage of apoptotic cells of 24 h treatment with Ara‐C (The concentration of Ara‐C was 50μM for HL‐60 cells and 5 μM for U937 cells) on AML cells after treatment with 10 μM Oligomycin A was expressed as mean ± SD from 3 independent experiments. (F) Total ATP production rate and (G) glycolytic ATP production rate in AML cells after treatment with Oligomycin A. The data represent the mean ± SD of 3 independent experiments with 3 replicates for each experiment. AML#, AML patients. ns, no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001

FIGURE 3

AMPK activity was inhibited by increased ATP synthesis in AML cells co‐cultured with HS‐5 cells. (A) Representative western blot analysis of proteins involved in the AMPK‐mTORC1 pathway (total AMPK, p‐AMPK^T172, total p70S6K, p‐p70S6K^T389, total 4E‐BP1, p‐4E‐BP1^S65, and c‐Myc; β‐actin was used as loading control) from AML cells cultured alone or with HS‐5 cells. The expression levels of p‐p70S6K^T389 and p‐4E‐BP1^S65 were used as indicators of mTORC1 activity. (B) Representative western blot analysis of AMPK‐mTORC1 pathway proteins of AML cells after treatment with 10 μM Oligomycin A. The representative images of 3 independent experiments were used

FIGURE 4

AMPK inhibition mediated the chemoresistance of AML cells induced by HS‐5 cells. (A) Growth inhibitory effect of 24 h treatment with different concentrations of Ara‐C and DNR on AML cells after treatment with 1 mM 5‐aminoimidazole‐4‐carboxamide ribonucleotide (AICAR) was measured by CCK‐8 assay. (B) The percentage of apoptotic cells of 24 h treatment with Ara‐C (The concentration of Ara‐C was 50 μM for HL‐60 cells and 5 μM for U937 cells) on AML cells after treatment with 1 mM AICAR was expressed as mean ± SD from 3 independent experiments. (C) Growth inhibitory effect of 24 h treatment with different concentrations of Ara‐C and DNR on AML cells after treatment with 5 μM Compound C was measured by CCK‐8 assay. (D) The percentage of apoptotic cells of 24 h treatment with Ara‐C (The concentration of Ara‐C was 50 μM for HL‐60 cells and 5 μM for U937 cells) on AML cells after treatment with 5 μM Compound C was expressed as mean ± SD from 3 independent experiments. (E) Growth inhibitory effect of 24 h treatment with different concentrations of Ara‐C and DNR on U937 cells knocked out for AMPK was measured by CCK‐8 assay. (F) The percentage of apoptotic cells of 24 h treatment with Ara‐C (The concentration of Ara‐C was 50μM for HL‐60 cells and 5 μM for U937 cells) on U937 cells knocked out for AMPK was expressed as mean ± SD from 3 independent experiments. (G) Growth inhibitory effect of 24 h treatment with different concentrations of Ara‐C and DNR on AMPK‐overexpressing U937 cells. (H) The percentage of apoptotic cells of 24 h treatment with Ara‐C on AMPK‐overexpressing U937 cells was expressed as mean ± SD from 3 independent experiments. The data represent the mean ± SD of 3 independent experiments with 3 replicates for each experiment. CON, wide type; NC, empty vector control; AMPK‐OE, AMPK overexpression; AMPK‐sg1/2, AMPK gene was knocked out. ns, no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001

FIGURE 5

AMPK inhibition increases mTORC1 activity and c‐Myc proteins expression. (A) Representative western blot analysis of AMPK‐mTORC1 pathway proteins (total AMPK, p‐AMPK^T172, total p70S6K, p‐p70S6K^T389, total 4E‐BP1, p‐4E‐BP1^S65, and c‐Myc; β‐actin was used as loading control) of AMPK‐overexpressing U937 cells. (B) Representative western blot analysis of AMPK‐mTORC1 pathway proteins in U937 cells knocked out for AMPK expression. (C) Representative western blot analysis of AMPK‐mTORC1 pathway proteins from AML cells after treatment with 1 mM AICAR. (D) Representative western blot analysis of AMPK‐mTORC1 pathway proteins in AML cells after treatment with 5 μM Compound C. WT, wide type; NC, empty vector control; AMPK‐OE, AMPK overexpression; AMPK‐sg1/2, AMPK gene was knocked out. The representative images of 3 independent experiments were used

FIGURE 6

AMPK knockout cells promoted AML progression and chemoresistance in mice. (A) Hematoxylin and eosin (HE) staining of the femurs from NCG mice. Scale bar, 50 μM. (B) Immunofluorescence (IF) staining for human CD45 (green) expression and nuclei marker (DAPI, blue) in the femurs of NCG mice. Scale bar, 50 μM. (C) Representative FCM profiles of human CD45 surface marker expression in AML cells of femurs. (D) Each data point represents the proportion of human CD45⁺ cells in the femurs per mouse (n = 5). (E‐F) Spleen anatomy (E) and weight (F) in the control, U937^WT, U937^NC‐sg, and U937^AMPK‐sg1 mice (n = 5). (G‐H) Each data point represents the proportion of human CD45⁺ cells in the spleens (G) and livers (H) per mouse (n = 4). (I) Each data point represents the proportion of residual human CD45⁺ cells in the femur, spleen, and liver per mouse after Ara‐C treatment (n = 4). (J) The relative number of human CD45⁺ cells after Ara‐C treatment was normalized to vehicle‐treated controls. WT, wide type; NC‐sg, empty vector control; AMPK‐sg1, AMPK gene was knocked out. ns, no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001