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Cellular and Molecular Biology

Molecular mechanisms by which splice modulator GEX1A inhibits leukaemia development and progression

British Journal of Cancer volume 127, pages 223–236 (2022)Cite this article

Subjects

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.

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Fig. 1: GEX1A demonstrated potent anti-leukaemic activity in certain cell types as a single-treatment agent in vitro and in vivo.
Fig. 2: GEX1A induces a pro-apoptotic switch in splicing of MCL-1, but not BCL-xL.
Fig. 3: Bcl-xL predicts GEX1A resistance in leukaemic cells.
Fig. 4: GEX1A exhibits strong synergistic anti-leukaemic activity with Bcl-x inhibitors, but antagonised the effect of Bcl-2 inhibitor, in vitro.
Fig. 5: GEX1A induces XPO-1 mediated nuclear exportation of NR4A proteins which can be inhibited by XPO-1 inhibitor selinexor.
Fig. 6: GEX1A + Bcl-xL inhibitor combination additively represses leukaemia development in vivo.
Fig. 7: GEX1A inhibits LSCs by inducing intron retention of the FASTK gene and repressing m-FASTK expression.
Fig. 8: GEX1A represses FLT3-ITD signalling by inducing intron retention in the FLT3-ITD gene.

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Data availability

RNA sequence data have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) and is available for public access (GEO Submission GSE166591).

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Acknowledgements

The authors thank the staff of the Department of Comparative Medicine of Loyola University Medical Center for the excellent animal care services they provided. We appreciate laboratory support in the form of FACS sorting and analysis assistance by Patricia Simms.

Funding

This work was supported by NIH grants R01 HL133560-01 and R01 CA223194-01 through Loyola University Chicago, as well as Loyola programme development funds to Jiwang Zhang. This work was also partially supported by a grant from the National Institutes of General Medical Sciences (R01-GM129465), as well as the Walther Cancer Foundation through the Harper Cancer Research Institute at the University of Notre Dame.

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Authors and Affiliations

  1. Department of Cancer Biology, Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL, USA

    Mark Sellin, Ryan Mack, Lei Zhang, Stephanie Berg, Kanak Joshi, Shanhui Liu, Wei Wei, Peter Breslin S. J. & Jiwang Zhang

  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

    Matthew C. Rhodes & Richard E. Taylor

  3. Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, National Clinical Research Center for Hematologic Diseases, Soochow University, Suzhou, China

    Lei Zhang

  4. Department of Internal Medicine, Division of Hematology and Oncology, Loyola University Medical Center, Maywood, IL, USA

    Stephanie Berg

  5. Lanzhou University Second Hospital, Lanzhou University, Lanzhou, China

    Shanhui Liu

  6. Department of Biology, Loyola University Chicago, Chicago, IL, USA

    Peter Breslin S. J.

  7. Department of Molecular/Cellular Physiology, Loyola University Chicago Stritch School of Medicine, Maywood, IL, USA

    Peter Breslin S. J.

  8. Loyola Genomics Facility, Center for Translational Research & Education, Loyola University Chicago Stritch School of Medicine, Maywood, IL, USA

    Peter Larsen

  9. Department of Pathology, Loyola University Medical Center, Maywood, IL, USA

    Jiwang Zhang

  10. Department of Radiology, Loyola University Medical Center, Maywood, IL, USA

    Jiwang Zhang

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  1. Mark Sellin
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Contributions

MS, RM, MR and KJ, LZ, SL, SB, WW, PL, PB and JZ conducted the experiments and analysed the data. MS drafted the first version of the paper. PB, RT and JZ contributed to the writing and editing of this manuscript.

Corresponding authors

Correspondence to Richard E. Taylor or Jiwang Zhang.

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Sellin, M., Mack, R., Rhodes, M.C. et al. Molecular mechanisms by which splice modulator GEX1A inhibits leukaemia development and progression. Br J Cancer 127, 223–236 (2022). https://doi.org/10.1038/s41416-022-01796-5

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  • DOI: https://doi.org/10.1038/s41416-022-01796-5

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