Hypoxia favors chemoresistance in T-ALL through an HIF1α-mediated mTORC1 inhibition loop

Abstract

Resistance to chemotherapy, a major therapeutic challenge in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), can be driven by interactions between leukemic cells and the microenvironment that promote survival of leukemic cells. The bone marrow, an important leukemia niche, has low oxygen partial pressures that highly participate in the regulation of normal hematopoiesis. Here we show that hypoxia inhibits T-ALL cell growth by slowing down cell cycle progression, decreasing mitochondria activity, and increasing glycolysis, making them less sensitive to antileukemic drugs and preserving their ability to initiate leukemia after treatment. Activation of the mammalian target of rapamycin (mTOR) was diminished in hypoxic leukemic cells, and treatment of T-ALL with the mTOR inhibitor rapamycin in normoxia mimicked the hypoxia effects, namely decreased cell growth and increased quiescence and drug resistance. Knocking down (KD) hypoxia-induced factor 1α (HIF-1α), a key regulator of the cellular response to hypoxia, antagonized the effects observed in hypoxic T-ALL and restored chemosensitivity. HIF-1α KD also restored mTOR activation in low O2 concentrations, and inhibiting mTOR in HIF1α KD T-ALL protected leukemic cells from chemotherapy. Thus, hypoxic niches play a protective role of T-ALL during treatments. Inhibition of HIF-1α and activation of the mTORC1 pathway may help suppress the drug resistance of T-ALL in hypoxic niches.

Graphical abstract

Figure 1.

Hypoxia modulates apoptosis, cell cycle, and metabolism in T-ALL. (A) Growth of T-ALL in conditions of low and high O₂ levels. Shown are the numbers of leukemic cells after 4 days of culture in normoxia and hypoxia expressed as fold variation compared with number of cells plated at day 0 in contact with MS5-DL1 cells. Every dot is the mean of technical triplicates for 1 experiment. T-ALL #1, n = 6exp; T-ALL #2, n = 6exp; T-ALL #3, n = 4exp; T-ALL #4, n = 3exp; and T-ALL #5, n = 2exp. (B) Apoptosis levels for T-ALL in culture at high and low O₂ levels. Shown are fold variation of apoptotic cells relative to control, gated on lymphocyte cells. Every experiment was done in technical triplicate. T-ALL #1, T-ALL #2, T-ALL #3, and T-ALL #4, n = 2exp for each T-ALL. (C) Cell cycle analysis of T-ALL in high and low O₂ levels. (i) Representative plots of Ki67/Hoechst staining of T-ALL #1. Proportion of Ki67⁺ leukemic cells (ii) and proportion of cells in G₀, G₁, S, and G₂/M phases (iii). Shown are mean ± SEM of cultures with T-ALL #1 and T-ALL #3, n = 6exp. (D) Metabolic status of T-ALL in high and low O₂ levels. MTG (i) and TMRE (ii) staining of a representative experiment (T-ALL #2) and MFI from T-ALL cultured in hypoxia reported to controls. Every experiment was done in triplicate. T-ALL #1, T-ALL #2, T-ALL #4, and T-ALL #5, n = 1exp for each T-ALL except for T-ALL #1, which was n = 2exp. (E) Lactate levels in T-ALL medium cultured in hypoxia. The blasts were cultured for 3 days in hypoxia or normoxia. The measure of lactate levels was performed on the culture medium according to the manufacturer protocol (lactate assay kit: 03183700; Roche). Shown are mean ± standard deviation of lactate triplicate values. One experiment was done with each leukemia sample. (F) Relative mRNA expression levels of HIF-1α, Glut3, VEGF, and CXCR4 genes. Tested on T-ALL #1, T-ALL #2, T-ALL #3, and T-ALL #4, n = 9exp. Statistics were done using the Friedman test: *P < .05, **P < .01, ***P < .001. exp, experiment.

Figure 2.

Hypoxia impacts T-ALL cell regrowth activity in vitro and in vivo. (A-B) Growth of T-ALL after hypoxia cultures have been stopped. (A) Leukemic cell numbers of T-ALL precultured in low and high O₂ levels after replating 7 days at 21% of O₂ with MS5-DL1 stromal cells. (B) Same except the secondary cultures were maintained for 28 days. Every experiment was done in triplicate. Shown are mean ± SEM of cultures with each T-ALL. (C) Engraftment levels of T-ALL after cultures in low and high O₂ levels. (i) Representative flow cytometry analysis 8 weeks after transplantation of mouse #1 BM injected with T-ALL #2 precultured in normoxia. (ii) Kinetic of leukemia development in BM after transplantation of 500 cells of T-ALL #2 precultured in normoxia or hypoxia. Shown are percent of leukemic (human CD45⁺CD7⁺) cells. (D) Survival curves of mice transplanted with 500 leukemic cells isolated from cultures in normoxia or in hypoxia (4-5 mice per condition). Statistics were determined with the Friedman test and the log-rank (Mantel-Cox) test for mice survival: **P < .01, ***P < .001.

Figure 3.

Hypoxia enhances T-ALL chemoresistance. (A-B) Effect of vincristine on T-ALL resistance in high and low O₂. (A) Numbers of live cells recovered after treatment during 72 hours with 10 nM (+vincristine) or without (Ø) vincristine. Mean ± SEM of triplicate cultures are represented. Shown are mean ± SEM of cultures with T-ALL #1, T-ALL #, T-ALL #3, T-ALL #4, and T-ALL #5, n = 9 experiments. (B) Same result is presented in percentage of live cells after treatment (+vincristine in panel A) compared with nontreated (Ø in panel A) cells. Every experiment was done in triplicate. Every dot is the mean of those triplicates with T-ALL #1, n = 3exp; T-ALL #2, n = 5exp; T-ALL #3, n = 3exp; T-ALL #4, n = 2exp; and T-ALL #5, n = 1exp. (C) Leukemic cell production after 7 days in normoxia without treatment, following T-ALL treatment with vincristine in normoxia or hypoxia. Every experiment was done in triplicate. Shown are mean ± SEM of cultures with each T-ALL. (D) Propagating activity of T-ALL treated ex vivo with vincristine in high or low O₂ levels. Shown is a kinetic analysis (4, 6, and 8 weeks) of leukemia development in BM after transplantation of 500 leukemic cells of T-ALL #2 recovered from normoxic or hypoxic culture with vincristine treatment, represented in percent of leukemic cells. (i) Representative mouse. (ii) Analysis of 5 mice/group from T-ALL #2. (E) Survival of mice transplanted with 500 cells precultured in normoxia or in hypoxia with vincristine (+Vinc). T-ALL #1, T-ALL #2, and T-ALL #3, 4-5 mice per condition. Statistics were determined using the Friedman test and the log-rank (Mantel-Cox) test for mice survival: *P < .05, **P < .01, ***P < .001. exp, experiment.

Figure 4.

Implication of HIF-1α in hypoxia-related T-ALL chemoresistance. (A) Relative mRNA expression of HIF-1α, Glut3, VEGF, and CXCR4 genes in shHIF-1α/T-ALL reported to levels measured in shCTL/T-ALL controls. Every dot represents gene levels of 1 experiment compared with β2m reporter gene levels. Tested on T-ALL #1, T-ALL #3, T-ALL #5, Jurkat, and DND41 cell lines. Shown are results of a total of 8 experiments. (B) Number of shCTL/T-ALL and shHIF-1α/T-ALL cells after 4 days of coculture with MS5-DL1 stromal cells in normoxia or in hypoxia. Data are expressed as fold variation compared with control (shCTL 21%). Tested in triplicate in every experiment. T-ALL #1, n = 6exp (i); and T-ALL #3, n = 2exp (ii). (C) Decreased HIF-1α modifies T-ALL cycling in hypoxia. Shown is the proportion of shCTL/T-ALL and shHIF-1α/T-ALL cells in G0 phase in normoxia or in hypoxia. Shown are mean ± SEM of cultures with T-ALL #1 and T-ALL #3, n = 4exp. (D) Decreased HIF1α increases chemosensitivity of T-ALL in hypoxia. Shown are the percentages of lived shCTL/T-ALL and shHIF-1α/T-ALL cells recovered after treatment during 72 hours with vincristine compared with nontreated cells. Every experiment was done in triplicate. Shown are mean ± SEM of cultures with T-ALL #1 and T-ALL #3, n = 8exp. (E) Leukemic cell production after 7 days in normoxia without treatment, following shCTL/T-ALL and shHIF-1α/T-ALL cell treatment with vincristine in normoxia or hypoxia. Shown are mean ± SEM of technical triplicates of 1 experiment on T-ALL #1. (F) Survival of mice transplanted with 1000 shCTL/T-ALL or shHIF-1α/T-ALL cells isolated after cultures in normoxia or in hypoxia in presence of 10 nM vincristine. T-ALL #1, 5 mice per condition. Statistics were determined using the Wilcoxon test (A, C) or Friedman test (D-E) and the log-rank (Mantel-Cox) test for mice survival: **P < .01, ***P < .001. exp, experiment.

Figure 5.

Role of HFI-1α in the development and sensitivity of T-ALL in vivo. (A) Protocol of the experimental settings. T-ALL#1 was transduced with shCTL (Cherry⁺) or shHIF-1α (GFP⁺) vectors. Sorted shHIF-1α (GFP⁺) cells were mixed at a 1:1 ratio with sorted shCTL (Cherry⁺) cells. A total of 10 000 mixed leukemic cells were transplanted into NSG recipients. (B) Plots of the flow cytometry analysis of the GFP⁺/Cherry⁺ cell mixture before mice injection. (C) The NSG mouse recipients were monitored for T-ALL development by BM cell samplings 9 weeks after transplantation. Shown are representative plots from mouse #3. GFP⁺/Cherry⁺ percentage are given relative to gated human CD45⁺ T-ALL cells before and after chemotherapeutic treatments. (D) Efficacy of chemotherapy in 6 mice measured before and after 1 week of drug treatment. The percentage of human CD45⁺ leukemic cells in the BM is shown for each mouse. (E) Percentage of shHIF-1α/GFP⁺ cells and (F) of shCTL/Cherry⁺ cells in human CD45⁺ leukemic cells before and after treatment of mice. (G) HIF1α expression in T-ALL#1 after transplantation in immune-deficient mice. Shown are results from the BM of 2 mice. BM cells were recovered after 1 week of chemotherapy treatment. Statistics were determined using the Wilcoxon test: *P < .05.

Figure 6.

Relationship between hypoxia, HIF-1α expression, mTOR activation, and chemoresistance in T-ALL. (A) Phosphorylation of mTOR (pS2448) and (B) 4EBP1 and S6R. Shown are representative histograms (i) and MFI ratio obtained with leukemic cells harvested from hypoxia compared with normoxia cultures (Aii,Bi,Bii). Every dot is the mean of technical triplicates. In panel A, T-ALL #1, n = 4exp; T-ALL #3, n = 4exp; and T-ALL #5, n = 1exp. In panel B, T-ALL#1, n = 2exp; T-ALL#3, n = 1exp. (C) Effect of rapamycin (Rapa) on T-ALL growth in normoxia. Absolute leukemic cell number recovered from every culture condition. The data are expressed as fold variation between the number of cells recovered after 4 days of coculture in presence or absence of rapamycin compared with cells plated at day 0. Every dot is the mean of technical triplicates. T-ALL #1, n = 5exp; T-ALL #2, n = 3exp; T-ALL #3, n = 3exp; T-ALL #4, n = 1exp; and T-ALL #5, n = 3exp. (D) Rapamycin decreases T-ALL cell cycle progression. Shown are proportions of leukemic cells in G₀, G₁, S, and G₂/M phases, during culture at 21% of O₂ in presence or absence of rapamycin. Shown are mean ± SEM of triplicate cultures performed with T-ALL #1, T-ALL #2, and T-ALL #4, n = 6exp. (Ei) Rapamycin protects T-ALL from vincristine in normoxia. Shown are the absolute numbers of cells recovered after vincristine treatment in normoxia in presence or absence of rapamycin. (Eii) Same results are presented as percentage of live cells after vincristine treatment in presence (+Rapa) or in absence (Ø) of rapamycin compared with nontreated cells. Every dot is the mean of technical triplicates from T-ALL #1, n = 2exp; T-ALL #2, n = 2exp; T-ALL #3, n = 3exp; and T-ALL #5, n = 3exp. (F) Leukemic cell number recovered 7 days after replating T-ALL pretreated with vincristine ± rapamycin. Shown are mean ± SEM of technical triplicate cultures. (G) HIF-1α impacts on mTOR phosphorylation in low and high O₂ concentration. (i) Phosphorylation of mTOR (pS2448) in a representative experiment with T-ALL #1. (ii) Ratio of MFI from shCTL/T-ALL or shHIF-1α/T-ALL cells cultured in hypoxia compared with those cultured in normoxia. Every dot is the mean of technical triplicates obtained with T-ALL #1, n = 2exp; and T-ALL #3, n = 1exp. Statistics were determined using the Friedman test (A), Wilcoxon test (B-E,G), or Mann-Whitney test (F): *P < .05, **P < .01, or ***P < .001. (H) Percentages of live shCTL/T-ALL and shHIF-1α/T-ALL cells recovered after treatment during 72 hours in hypoxia with vincristine in presence (+Rapa) or in absence (Ø) of rapamycin compared with nontreated cells. (I) Leukemic cell production after 7 days in normoxia without treatment after T-ALL. exp, experiment.