Search

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 EC(50) 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.

Immune checkpoint blockade therapy has revolutionized cancer care, including the treatment of advanced metastatic disease. However, most patients derive little or no clinical benefit from these therapies and many cancer types are notoriously non-responsive. Motivated by (1) the correlation between tumor neoantigen abundance and anti-tumor immunity and (2) that most cancers are characterized by widespread dysregulation of RNA processing, we reasoned that pharmacologic modulation of RNA splicing might increase cancer cell immunogenicity via the generation of splicing-derived neoantigens. We demonstrated that two compounds which modulate RNA splicing via distinct mechanisms, inhibited tumor growth and enhanced response to immune checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Critical for their clinical translatability, therapeutic doses of splicing inhibitors were non-toxic, tolerated by the host immune system, and did not affect T cell activation, proliferation, and anti-cancer killing activities. Mechanistically, splicing modulation induced stereotyped, dose-dependent “splicing failure” — dramatic intron retention, alternative exon skipping, etc. — that was consistent across multiple mouse and human tumor types. By combining RNA-seq-based peptide predictions and mass spectrometry of the MHC I-bound immunopeptidome, we identified drug-induced, splicing-derived peptides that promote the expansion of antigen-specific CD8+ T cells and trigger anti-tumor T cell responses in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic. Citation Format: James D. Thomas, Sydney X. Lu, Emma De Neef, Erich Sabio, Benoit Rousseau, Mathieu Gigoux, David A. Knorr, Benjamin Greenbaum, Yuval Elhanati, Simon J. Hogg, Andrew Chow, Arnab Ghosh, Abigail Xie, Dmitriy Zamarin, Daniel Cui, Caroline Erickson, Michael Singer, Hana Cho, Eric Wang, Bin Lu, Benjamin H. Durham, Harshal Shah, Diego Chowell, Austin M. Gabel, Yudao Shen, Jing Liu, Jian Jin, Matthew C. Rhodes, Richard E. Taylor, Henrik Molina, Jedd D. Wolchok, Taha Merghoub, Luis A. Diaz Jr, Omar Abdel-Wahab, Robert K. Bradley. Pharmacologic modulation of RNA splicing enhances anti-tumor immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5742.

Summary Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic.

Background: Overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-xL and Mcl1 is commonly detected in acute myeloid leukemia (AML) and is correlated with poor patient prognosis. Fortunately, in most AML samples, the expression of pro-apoptotic proteins, including Bax, Bak and Bim, is also increased. This is likely due to a feedback mechanism, which results in AML cells that are particularly vulnerable to treatment with targeted inhibition of anti-apoptotic proteins. However, challenges arise when the important pro-survival proteins named above are either poorly targeted by specific inhibitors (such as BH3-mimetics), as in the case of Mcl1, or when the use of these inhibitors results in severe thrombocytopenia, as in the case of Bcl-xL. Therefore, the development of novel medications to target such molecules with high specificity and low toxicity is of crucial importance. GEX1A, a splicing modulator, induces a large shift in the pattern of exon skipping and intron retention events by inhibiting the SF3B1-PHF5A complex of the U2 snRNP. While GEX1A has shown pre-clinical efficacy in some carcinoma cell lines containing various spiceosomal mutations, the in vitro and in vivo effects of GEX1A in leukemic cells which lack spiceosomal mutations are largely unknown. Experimental procedures: The IC50 of 18 established AML cell lines was determined by treating the cells with GEX1A in vitro using a series of 10-fold dilutions; cell viability was then examined using quantitative colorimetric assays. The in vivo anti-leukemic efficacy of GEX1A was evaluated using xenograft models generated in NSG mice injected with the GEX1A-sensitive human AML cell lines KOPN8, Molm-13, and MV4:11. The molecular mechanism by which GEX1A kills AML cells was then studied. Results: Based on their IC50, 18 established leukemic cell lines can be divided into high (LD50 < 100 nM), moderate (LD50 = 100-500 nM), and low sensitivity (LD50 > 500 nM) groups. We next determined the tolerable dose of GEX1A for in vivo mouse treatment. Intraperitoneal injection of GEX1A significantly improved the survival of leukemic mice compared to the vehicle control group (see Figure). Mechanistically, we found that GEX1A kills leukemic cells by inducing the production of pro-apoptotic Mcl1-S protein at the expense of the pro-survival Mcl1-L protein due to the alternative splicing of the Mcl1 gene. In addition, we found that high levels of Bcl-xL protein predict resistance of leukemic cells to GEX1A treatment. Furthermore, synergistic activity was observed in 2/6 cell lines treated with GEX1A and ABT-263, an inhibitor of Bcl-2/Bcl-xL/Bcl-w, and 6/6 cell lines treated with GEX1A and A-1155463, a highly potent Bcl-xL inhibitor. Conclusion: GEX1A is a novel splicing modulator that shows potent anti-leukemic activity. GEX1A kills leukemic cells by inducing a splicing isoform switch of the Mcl1 gene to produce a pro-apoptotic Mcl1-S protein at the expense of the pro-survival Mcl1-L protein in such cells. GEX1A and a Bcl-xL-specific inhibitor were also combined to show synergistic effects on leukemic cells. Our results indicate that GEX1A may be an effective treatment for leukemic patients when combined with a specific Bcl-xL inhibitor. We are actively evaluating our combination treatment using primary patient samples in in vivo xenograft models. Disclosures No relevant conflicts of interest to declare.