Attenuation of Graft-Versus-Host-Disease Via Genetic or Pharmacologic Inhibition of the RNA Splicing Factor RBM39

Abstract

Recent studies have identified pharmacologic modulation of RNA splicing via degradation of the splicing factor RBM39 to mediate anticancer effects and enhance responses to immune checkpoint blockade through generation of novel RNA-splicing derived immunogenic peptides in tumors. However rigorous evaluation of the role of RBM39 in immune cells has not been performed. Here we identify that RBM39 modulates exon recognition in T cells to regulate their function. Although essential for the survival of hematopoietic precursors and tumors, RBM39 is dispensable for peripheral T cell survival. Functional evaluation of the T cell alloantigen response in allogeneic hematopoietic stem transplantation (allo-HSCT) revealed that Rbm39 loss attenuated graft-versus-host-disease (GVHD) and improved overall survival while partially sparing graft-versus-tumor (GVT) effects. To rigorously evaluate the role of RBM39 in immune cells, we generated an Rbm39 conditional knockout mouse by floxing Rbm39 exon 7, which encodes its first RNA recognition motif. Pan-hematopoietic deletion of Rbm39 resulted in rapid complete failure of hematopoiesis and impaired hematopoietic stem cell (HSC) self-renewal. We next analyzed the phenotype of animals with T cell specific deletion of RBM39 and compared this with the effects of pharmacologic RBM39 degradation on T cells. Strikingly, in contrast to the profound effects of genetic RBM39 loss in HSCs and the inability of cancer cells to tolerate RBM39 deletion, RBM39 deletion under CD4-cre (deletion early in thymopoiesis) or distal Lck-cre (late in thymopoiesis) yielded modestly fewer viable peripheral T cells, which skewed towards a CD44+ effector/memory phenotype. This observation was also recapitulated with extended treatment of mice with the RBM39 degrader E7820 (in phase II trials currently; clinicaltrials.gov NCT05024994). Deep RNA-seq analysis of splenic CD3+ T cells from distal Lck-cre Rbm39fl/fl mice revealed widespread decreased efficiency of RNA splicing in Rbm39 null T cells compared to controls, indicative of altered RNA splicing in T cells with Rbm39 deletion. Rbm39 KO T cells experienced global increases in exon skipping at genomic regions where upstream introns had reduced GC content. These data identify that RBM39 is required in a cell and tissue-specific manner for cell survival and further highlights the selectivity of therapeutic approaches targeting RBM39. We next assessed the functional consequences of pharmacologic RBM39 degradation or genetic RBM39 deletion in T cells. Consistent with observations in genetic mouse models, T cells could tolerate extensive pharmacologic RBM39 degradation without loss of proliferative capacity in response to PMA/Ionomycin or anti-CD3+CD28 stimulation. Similarly, genetic Rbm39 null T cells had an intact proliferative response to PMA/Ionomycin. Furthermore, when we evaluated the effects of RBM39 loss in the MHC-mismatched C57BL/6 => Balb/c allo-HSCT system, Rbm39 deletion in donor T cells under either CD4- or distal Lck-cre profoundly reduced GVHD severity and improved overall survival in recipients (Fig. A). These genetic findings were recapitulated via pharmacologic degradation of RBM39 in the same HSCT system (Fig. B). Similar experiments performed in the setting of Balb/c hosts engrafted with A20 lymphoma cells revealed partial preservation of GVT activity using Rbm39 KO T cells. Single cell RNA sequencing analyses of CD4-cre Rbm39fl/fl primary T cells revealed upregulation of the integrin beta1 subunit in both CD4 and CD8 T cells. This suggests alteration of T cell trafficking away from GVHD target organs as one mechanism by which RBM39 loss may alter T cell alloreactivity in allo-HSCT. Our results identify cell-type specific roles for RBM39 in hematopoiesis and immune cells and suggest a therapeutic approach for GVHD via direct modulation of RNA splicing. Of note, as the RBM39 degrader E7820 is currently in phase II clinical trials for relapsed myeloid neoplasms, our previous studies and this data identify a treatment for myeloid neoplasms in the post-transplant setting, where RBM39 degraders mediate both direct and immune-mediated anti-tumor effects while also attenuating GVHD via modulation of RNA splicing in T cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal