Targeting mitochondrial oxidative phosphorylation eradicates therapy-resistant chronic myeloid leukemia stem cells

Abstract

Treatment of chronic myeloid leukemia (CML) with imatinib mesylate and other second- and/or third-generation c-Abl-specific tyrosine kinase inhibitors (TKIs) has substantially extended patient survival. However, TKIs primarily target differentiated cells and do not eliminate leukemic stem cells (LSCs). Therefore, targeting minimal residual disease to prevent acquired resistance and/or disease relapse requires identification of new LSC-selective target(s) that can be exploited therapeutically. Considering that malignant transformation involves cellular metabolic changes, which may in turn render the transformed cells susceptible to specific assaults in a selective manner, we searched for such vulnerabilities in CML LSCs. We performed metabolic analyses on both stem cell-enriched (CD34⁺ and CD34⁺CD38^-) and differentiated (CD34^-) cells derived from individuals with CML, and we compared the signature of these cells with that of their normal counterparts. Through combination of stable isotope-assisted metabolomics with functional assays, we demonstrate that primitive CML cells rely on upregulated oxidative metabolism for their survival. We also show that combination treatment with imatinib and tigecycline, an antibiotic that inhibits mitochondrial protein translation, selectively eradicates CML LSCs both in vitro and in a xenotransplantation model of human CML. Our findings provide a strong rationale for investigation of the use of TKIs in combination with tigecycline to treat patients with CML with minimal residual disease.

Figure 1. Primitive CML cells show an increase in oxidative metabolism compared to differentiated counterparts.

(a) Comparative steady-state metabolomics analysis of patient-matched CD34⁺ and CD34^- CML cells measured by LC-MS. Mean, n=4 patients. (b) Representative respirometry output in CD34⁺ and CD34^- CML cells. Mean ± S.D. (c) Basal mitochondrial oxygen consumption rate (OCR) of CD34⁺ and CD34^- CML cells. Mean ± S.E.M. n=4 patient samples. (d) Relative isotopologue distribution of indicated metabolites in CD34⁺ and CD34^- CML cells measured by LC-MS following 24 hours incubation with ¹³C₆-labeled glucose. Acetyl-CoA could not be detected by LC-MS in our experimental conditions. Mean ± S.E.M. n=3 patient samples. FC, fold change of glucose-derived (¹³C ≥ 2) metabolite abundance relative to CD34^- CML cells. PDH, Pyruvate dehydrogenase; PC, Pyruvate carboxylase. P-values were calculated with paired Student’s t-test.

Figure 2. Enhanced mitochondrial metabolic activity in primitive CML cells compared to normal undifferentiated hematopoietic cells.

(a-c) Relative isotopologue distribution of (a) citrate, (b) glutamate and (c) aspartate in CD34⁺ CML and CD34⁺ normal cells measured by LC-MS following 24 hours incubation with ¹³C₆-labeled glucose. Mean ± S.E.M. n=5 patient and normal samples. FC, Fold change of glucose-derived (¹³C ≥ 2) metabolite abundance relative to CD34+ normal cells. P-values were calculated by unpaired Student’s t-test. (d) Representative respirometry output in CD34⁺ CML and CD34⁺ normal cells. Mean ± S.D. (e) Basal mitochondrial OCR. Mean ± S.E.M. n=9 patient samples and n=4 normal samples. P-values were calculated by unpaired Student’s t-test. (f) Representative histograms of Mitotracker Green-labeled CD34⁺CD38^- CML cells (blue) and CD34⁺CD38^- normal cells (red). (g) Mitochondrial content of CD34⁺CD38^- CML cells and CD34⁺CD38^- normal cells was determined from the geometric mean of Mitotracker Green-labeled cells. Mean ± S.E.M. n=3 patient and 3 normal samples. P-values were determined by one sample t-test. (h) Representative histograms of TMRM-labeled CD34⁺CD38^- CML (blue) and CD34⁺CD38^- normal (red) cells. (i) Mitochondrial membrane potential of CD34⁺CD38^- CML cells and CD34⁺CD38^- normal cells was determined from the geometric mean of TMRM-labeled cells. Mean ± S.E.M. n=3 patient and 3 normal samples. FC, fold change relative to normal cells. P-values were determined by one sample t-test. (j-l) Relative isotopologue distribution of (j) citrate, (k) glutamate and (l) aspartate in CD34⁺CD38^- CML and CD34⁺CD38^- normal cells measured by LC-MS following 24 hours incubation with ¹³C₆-labeled glucose. n=1 patient sample.

Figure 3. Inhibition of aberrant oxidative metabolism targets CML progenitors and LSCs.

(a) Protein expression in CD34⁺ CML cells following a 72 hours in vitro treatment with tigecycline (2.5 µM). n=1 patient sample. (b-d) Relative isotopologue distribution of (b) citrate, (c) glutamate and (d) aspartate in CD34⁺ CML cells measured by LC-MS following 24 hours incubation with ¹³C₆-labeled glucose in the presence or absence of tigecycline (2.5 µM). Mean ± S.E.M. n=3 patient samples. (e) Representative flow cytometry histograms obtained from cellular division tracking of CellTrace Violet-stained CD34⁺ CML cells following 72 hours treatment with vehicle only, tigecycline (2.5 µM), imatinib (2 µM) and combination (2.5 µM + 2 µM). (f) Representative images of colonies and (g) colony numbers following 3 days drug treatment of CD34⁺ CML cells. Mean ± S.E.M. n=4 patient samples. (h) Colony number of CD34⁺ normal cells following 72 hours drug treatment with the indicated drugs. Mean ± S.E.M. n=4 normal samples. (i) Number of colonies measured by LTC-IC assay in CD34⁺ CML cells. Mean ± S.E.M. n=5 patient samples. FC, Fold change of glucose-derived (¹³C ≥ 2) metabolite abundance relative to tigecycline-treated CD34⁺ CML cells. TIG, tigecycline; IM, imatinib; Combo, combination. P-values were calculated by paired Student’s t-test.

Figure 4. Inhibition of oxidative metabolism eliminates xenotransplanted human CML LSCs.

(a) Diagram of experimental design. The pre-treatment engraftment levels of CML cells in mice were assessed by monitoring the percentage of human CD45⁺ circulating leukocytes using flow cytometry. (b) Representative analyses of human CD45 and CD34 expression in murine bone marrow was used to assess engrafted CML cells following the indicated treatment. (c) Number of human CD34⁺ and (d) human CD34⁺CD38^- CML cells remaining in the bone marrow following in vivo drug treatment. (e) Number of human CD34⁺ and (f) human CD34⁺CD38^- CML cells remaining in the bone marrow following 2 (experiment 1) or 3 (experiment 2) weeks of drug discontinuation. n≥5 mice per treatment arm. TIG, tigecycline; IM, imatinib. P-values were calculated by unpaired Student’s t-test on logarithmically transformed variables to meet the assumption of normality.