Mark Sellin1, Ryan Mack1, Matthew C Rhodes2, Lei Zhang1,3, Stephanie Berg1,4, Kanak Joshi1, Shanhui Liu1,5, Wei Wei1, Peter Breslin S J1,6,7, Peter Larsen8, Richard E Taylor9, Jiwang Zhang10,11,12. 1. Department of Cancer Biology, Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL, USA. 2. Department of Chemistry and Biochemistry, the Harper Cancer Research Institute, and the Warren Family Research Center for Drug Discovery & Development, University of Notre Dame, Notre Dame, IN, USA. 3. Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, National Clinical Research Center for Hematologic Diseases, Soochow University, Suzhou, China. 4. Department of Internal Medicine, Division of Hematology and Oncology, Loyola University Medical Center, Maywood, IL, USA. 5. Lanzhou University Second Hospital, Lanzhou University, Lanzhou, China. 6. Department of Biology, Loyola University Chicago, Chicago, IL, USA. 7. Department of Molecular/Cellular Physiology, Loyola University Chicago Stritch School of Medicine, Maywood, IL, USA. 8. Loyola Genomics Facility, Center for Translational Research & Education, Loyola University Chicago Stritch School of Medicine, Maywood, IL, USA. 9. Department of Chemistry and Biochemistry, the Harper Cancer Research Institute, and the Warren Family Research Center for Drug Discovery & Development, University of Notre Dame, Notre Dame, IN, USA. rtaylor@nd.edu. 10. Department of Cancer Biology, Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL, USA. jzhang@luc.edu. 11. Department of Pathology, Loyola University Medical Center, Maywood, IL, USA. jzhang@luc.edu. 12. Department of Radiology, Loyola University Medical Center, Maywood, IL, USA. jzhang@luc.edu.
Abstract
INTRODUCTION: Splice modulators have been assessed clinically in treating haematologic malignancies exhibiting splice factor mutations and acute myeloid leukaemia. However, the mechanisms by which such modulators repress leukaemia remain to be elucidated. OBJECTIVES: The primary goal of this assessment was to assess the molecular mechanism by which the natural splice modulator GEX1A kills leukaemic cells in vitro and within in vivo mouse models. METHODS: Using human leukaemic cell lines, we assessed the overall sensitivity these cells have to GEX1A via EC50 analysis. We subsequently analysed its effects using in vivo xenograft mouse models and examined whether cell sensitivities were correlated to genetic characteristics or protein expression levels. We also utilised RT-PCR and RNAseq analyses to determine splice change and RNA expression level differences between sensitive and resistant leukaemic cell lines. RESULTS: We found that, in vitro, GEX1A induced an MCL-1 isoform shift to pro-apoptotic MCL-1S in all leukaemic cell types, though sensitivity to GEX1A-induced apoptosis was negatively associated with BCL-xL expression. In BCL-2-expressing leukaemic cells, GEX1A induced BCL-2-dependent apoptosis by converting pro-survival BCL-2 into a cell killer. Thus, GEX1A + selective BCL-xL inhibition induced synergism in killing leukaemic cells, while GEX1A + BCL-2 inhibition showed antagonism in BCL-2-expressing leukaemic cells. In addition, GEX1A sensitised FLT3-ITD+ leukaemic cells to apoptosis by inducing aberrant splicing and repressing the expression of FLT3-ITD. Consistently, in in vivo xenografts, GEX1A killed the bulk of leukaemic cells via apoptosis when combined with BCL-xL inhibition. Furthermore, GEX1A repressed leukaemia development by targeting leukaemia stem cells through inhibiting FASTK mitochondrial isoform expression across sensitive and non-sensitive leukaemia types. CONCLUSION: Our study suggests that GEX1A is a potent anti-leukaemic agent in combination with BCL-xL inhibitors, which targets leukaemic blasts and leukaemia stem cells through distinct mechanisms.
INTRODUCTION: Splice modulators have been assessed clinically in treating haematologic malignancies exhibiting splice factor mutations and acute myeloid leukaemia. However, the mechanisms by which such modulators repress leukaemia remain to be elucidated. OBJECTIVES: The primary goal of this assessment was to assess the molecular mechanism by which the natural splice modulator GEX1A kills leukaemic cells in vitro and within in vivo mouse models. METHODS: Using human leukaemic cell lines, we assessed the overall sensitivity these cells have to GEX1A via EC50 analysis. We subsequently analysed its effects using in vivo xenograft mouse models and examined whether cell sensitivities were correlated to genetic characteristics or protein expression levels. We also utilised RT-PCR and RNAseq analyses to determine splice change and RNA expression level differences between sensitive and resistant leukaemic cell lines. RESULTS: We found that, in vitro, GEX1A induced an MCL-1 isoform shift to pro-apoptotic MCL-1S in all leukaemic cell types, though sensitivity to GEX1A-induced apoptosis was negatively associated with BCL-xL expression. In BCL-2-expressing leukaemic cells, GEX1A induced BCL-2-dependent apoptosis by converting pro-survival BCL-2 into a cell killer. Thus, GEX1A + selective BCL-xL inhibition induced synergism in killing leukaemic cells, while GEX1A + BCL-2 inhibition showed antagonism in BCL-2-expressing leukaemic cells. In addition, GEX1A sensitised FLT3-ITD+ leukaemic cells to apoptosis by inducing aberrant splicing and repressing the expression of FLT3-ITD. Consistently, in in vivo xenografts, GEX1A killed the bulk of leukaemic cells via apoptosis when combined with BCL-xL inhibition. Furthermore, GEX1A repressed leukaemia development by targeting leukaemia stem cells through inhibiting FASTK mitochondrial isoform expression across sensitive and non-sensitive leukaemia types. CONCLUSION: Our study suggests that GEX1A is a potent anti-leukaemic agent in combination with BCL-xL inhibitors, which targets leukaemic blasts and leukaemia stem cells through distinct mechanisms.
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