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Combined targeting of BCL-2 and BCR-ABL tyrosine kinase eradicates chronic myeloid leukemia stem cells.

ABSTRACT: BCR-ABL tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML), but they rarely eliminate CML stem cells. Disease relapse is common upon therapy cessation, even in patients with complete molecular responses. Furthermore, once CML progresses to blast crisis (BC), treatment outcomes are dismal. We hypothesized that concomitant targeting of BCL-2 and BCR-ABL tyrosine kinase could overcome these limitations. We demonstrate increased BCL-2 expression at the protein level in bone marrow cells, particularly in Lin(-)Sca-1(+)cKit(+) cells of inducible CML in mice, as determined by CyTOF mass cytometry. Further, selective inhibition of BCL-2, aided by TKI-mediated MCL-1 and BCL-XL inhibition, markedly decreased leukemic Lin(-)Sca-1(+)cKit(+) cell numbers and long-term stem cell frequency and prolonged survival in a murine CML model. Additionally, this combination effectively eradicated CD34(+)CD38(-), CD34(+)CD38(+), and quiescent stem/progenitor CD34(+) cells from BC CML patient samples. Our results suggest that BCL-2 is a key survival factor for CML stem/progenitor cells and that combined inhibition of BCL-2 and BCR-ABL tyrosine kinase has the potential to significantly improve depth of response and cure rates of chronic-phase and BC CML.

SUBMITTER: Carter BZ

PROVIDER: S-EPMC5111086 | biostudies-other | 2016 Sep

REPOSITORIES: biostudies-other

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Combined targeting of BCL-2 and BCR-ABL tyrosine kinase eradicates chronic myeloid leukemia stem cells.

Carter Bing Z BZ Mak Po Yee PY Mu Hong H Zhou Hongsheng H Mak Duncan H DH Schober Wendy W Leverson Joel D JD Zhang Bin B Bhatia Ravi R Huang Xuelin X Cortes Jorge J Kantarjian Hagop H Konopleva Marina M Andreeff Michael M

Science translational medicine 20160901 355

BCR-ABL tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML), but they rarely eliminate CML stem cells. Disease relapse is common upon therapy cessation, even in patients with complete molecular responses. Furthermore, once CML progresses to blast crisis (BC), treatment outcomes are dismal. We hypothesized that concomitant targeting of BCL-2 and BCR-ABL tyrosine kinase could overcome these limitations. We demonstrate increased BCL-2 expression at the protein lev ...[more]

PMID: 27605552

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Project description:Background In the ongoing battle against BCR-ABL+ leukemia, despite significant advances with tyrosine kinase inhibitors (TKIs), the persistent challenges of drug resistance and the enduring presence of leukemic stem cells (LSCs) remain formidable barriers to achieving a cure. Methods In this study, we demonstrated that Disulfiram (DSF) induces ferroptosis to synergize with TKIs in inhibiting BCR-ABL+ cells, particularly targeting resistant cells and LSCs, using cell models, mouse models, and primary cells from patients. We elucidated the mechanism by which DSF promotes GPX4 degradation to induce ferroptosis through immunofluorescence, co-immunoprecipitation (CO-IP), RNA sequencing, lipid peroxidation assays, and rescue experiments. Results Here, we present compelling evidence elucidating the sensitivity of DSF, an FDA-approved drug for alcohol dependence, towards BCR-ABL+ cells. Our findings underscore DSF's ability to selectively induce a potent cytotoxic effect on BCR-ABL+ cell lines and effectively inhibit primary BCR-ABL+ leukemia cells. Crucially, the combined treatment of DSF with TKIs selectively eradicates TKI-insensitive stem cells and resistant cells. Of particular note is DSF's capacity to disrupt GPX4 stability, elevate the labile iron pool, and intensify lipid peroxidation, ultimately leading to ferroptotic cell death. Our investigation shows that BCR-ABL expression induces alterations in cellular iron metabolism and increases GPX4 expression. Additionally, we demonstrate the indispensability of GPX4 for LSC development and the initiation/maintenance of BCR-ABL+ leukemia. Mechanical analysis further elucidates DSF's ability to circumvent resistance by reducing GPX4 levels through the disruption of its binding with HSPA8, thereby promoting GPX4 ubiquitination and subsequent degradation. Furthermore, the combined treatment of DSF with TKIs effectively targets both BCR-ABL+ blast cells and drug-insensitive LSCs, conferring a significant survival advantage in mouse models. Conclusion In summary, the dual inhibition of GPX4 and BCR-ABL presents a promising therapeutic strategy to synergistically target blast cells and drug-insensitive LSCs in patients, offering potential avenues for advancing leukemia treatment.

Project description:Background:Imatinib and second-generation tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib have statistically significantly improved the life expectancy of chronic myeloid leukemia (CML) patients; however, resistance to TKIs remains a major clinical challenge. Although ponatinib, a third-generation TKI, improves outcomes for patients with BCR-ABL-dependent mechanisms of resistance, including the T315I mutation, a proportion of patients may have or develop BCR-ABL-independent resistance and fail ponatinib treatment. By modeling ponatinib resistance and testing samples from these CML patients, it is hoped that an alternative drug target can be identified and inhibited with a novel compound. Methods:Two CML cell lines with acquired BCR-ABL-independent resistance were generated following culture in ponatinib. RNA sequencing and gene ontology (GO) enrichment were used to detect aberrant transcriptional response in ponatinib-resistant cells. A validated oncogene drug library was used to identify US Food and Drug Administration-approved drugs with activity against TKI-resistant cells. Validation was performed using bone marrow (BM)-derived cells from TKI-resistant patients (n?=?4) and a human xenograft mouse model (n?=?4-6 mice per group). All statistical tests were two-sided. Results:We show that ponatinib-resistant CML cells can acquire BCR-ABL-independent resistance mediated through alternative activation of mTOR. Following transcriptomic analysis and drug screening, we highlight mTOR inhibition as an alternative therapeutic approach in TKI-resistant CML cells. Additionally, we show that catalytic mTOR inhibitors induce autophagy and demonstrate that genetic or pharmacological inhibition of autophagy sensitizes ponatinib-resistant CML cells to death induced by mTOR inhibition in vitro (% number of colonies of control[SD], NVP-BEZ235 vs NVP-BEZ235+HCQ: 45.0[17.9]% vs 24.0[8.4]%, P = .002) and in vivo (median survival of NVP-BEZ235- vs NVP-BEZ235+HCQ-treated mice: 38.5 days vs 47.0 days, P = .04). Conclusion:Combined mTOR and autophagy inhibition may provide an attractive approach to target BCR-ABL-independent mechanism of resistance.

Project description:Previously, it has been stated that the BCR-ABL fusion-protein is sufficient to induce Chronic Myeloid Leukemia (CML), but additional genomic-changes are required for disease progression. Hence, we profiled control and tyrosine kinase inhibitors (TKI) alone or in combination with other drug-treated CML-samples in different phases, categorized as drug-sensitive and drug-resistant on the basis of BCR-ABL transcripts, the marker of major molecular-response. Molecular-profiling was done using the molecular-inversion probe-based-array, Human Transcriptomics-Array2.0, and Axiom-Biobank genotyping-arrays. At the transcript-level, clusters of control, TKI-resistant and TKI-sensitive cases were correlated with BCR-ABL transcript-levels. Both at the gene- and exon-levels, up-regulation of MPO, TPX2, and TYMS and down-regulation of STAT6, FOS, TGFBR2, and ITK lead up-regulation of the cell-cycle, DNA-replication, DNA-repair pathways and down-regulation of the immune-system, chemokine- and interleukin-signaling, TCR, TGF beta and MAPK signaling pathways. A comparison between TKI-sensitive and TKI-resistant cases revealed up-regulation of LAPTM4B, HLTF, PIEZO2, CFH, CD109, ANGPT1 in CML-resistant cases, leading to up-regulation of autophagy-, protein-ubiquitination-, stem-cell-, complement-, TGF?- and homeostasis-pathways with specific involvement of the Tie2 and Basigin signaling-pathway. Dysregulated pathways were accompanied with low CNVs in CP-new and CP-UT-TKI-sensitive-cases with undetectable BCR-ABL-copies. High CNVs (previously reported gain of 9q34) were observed in BCR-ABL-independent and -dependent TKI, non-sensitive-CP-UT/AP-UT/B-UT and B-new samples. Further, genotyping CML-CP-UT cases with BCR-ABL 0-to-77.02%-copies, the identified, rsID239798 and rsID9475077, were associated with FAM83B, a candidate for therapeutic resistance. The presence of BCR-ABL, additional genetic-events, dysregulated-signaling-pathways and rsIDs associated with FAM83B in TKI-resistant-cases can be used to develop a signature-profile that may help in monitoring therapy.

Dataset Information

Combined targeting of BCL-2 and BCR-ABL tyrosine kinase eradicates chronic myeloid leukemia stem cells.

Publications

Combined targeting of BCL-2 and BCR-ABL tyrosine kinase eradicates chronic myeloid leukemia stem cells.

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