Investigating the Molecular Mechanism of H3B-8800: A Splicing Modulator Inducing Preferential Lethality in Spliceosome-Mutant Cancers

Abstract

The SF3B1 protein, part of the SF3b complex, recognizes the intron branch point sequence of precursor messenger RNA (pre-mRNA), thus contributing to splicing fidelity. SF3B1 is frequently mutated in cancer and is the target of distinct families of splicing modulators (SMs). Among these, H3B-8800 is of particular interest, as it induces preferential lethality in cancer cells bearing the frequent and highly pathogenic K700E SF3B1 mutation. Despite the potential of H3B-8800 to treat myeloid leukemia and other cancer types hallmarked by SF3B1 mutations, the molecular mechanism underlying its preferential lethality towards spliceosome-mutant cancer cells remains elusive. Here, microsecond-long all-atom simulations addressed the binding/dissociation mechanism of H3B-8800 to wild type and K700E SF3B1-containing SF3b (K700ESB3b) complexes at the atomic level, unlocking that the K700E mutation little affects the thermodynamics and kinetic traits of H3B-8800 binding. This supports the hypothesis that the selectivity of H3B-8800 towards mutant cancer cells is unrelated to its preferential targeting of K700ESB3b. Nevertheless, this set of simulations discloses that the K700E mutation and H3B-8800 binding affect the overall SF3b internal motion, which in turn may influence the way SF3b interacts with other spliceosome components. Finally, we unveil the existence of a putative druggable SF3b pocket in the vicinity of K700E that could be harnessed in future rational drug-discovery efforts to specifically target mutant SF3b.

Establishing the catalytic and regulatory mechanism of RNA‐based machineries

“…Cross‐correlation and PCA analysis of wt Hsh155 disclosed a spring‐like motion of this protein, with two hinge points located at the BPA binding pocket and at the H5–H7 region, where most mutations cluster 110 . Consistently, the same movement was also observed in later simulations with human SF3B1 110,124 . When a cancer‐associated mutation was introduced into Hsh155, the binding of pre‐mRNA was destabilized and/or the functional dynamics of Hsh155 was affected 110 .…”

“…110 Consistently, the same movement was also observed in later simulations with human SF3B1. 110,124 When a cancer-associated mutation was introduced into Hsh155, the binding of pre-mRNA was destabilized and/or the functional dynamics of Hsh155 was affected. 110 Interestingly, the cancer-associated mutations (N295D and K335E) induced these changes only upon binding of a non-consensus BPS, in accordance with experimental evidence.…”

“…As to the second field, RNA-only and RNA/protein complexes are emerging as an appealing alternative drug target, 166 particularly for combating diseases linked to undruggable proteins. While targeting the protein and the protein-RNA interface in RNPs with small molecules has been already successfully done, 124,167,168 interfering with aberrant RNA/RNA interactions occurring in different pathologies represents an exciting opportunity for creating novel therapeutics. 169 However, RNA molecules are extremely flexible and conformationally versatile.…”

All-Atom Simulations Elucidate the Impact of U2AF2 Cancer-Associated Mutations on Pre-mRNA Recognition

“…Owing to the overwhelming implications of the mutated SFs in the onset of cancer, this study advances our understanding of the recognition mechanism of a major class of splice site signals mediated by a key splicing factor. 15,34,41…”

“…Notably, the cooperative recognition mechanism presented here may be shared by other RNA binding proteins, which, like U2AF2, are characterized by multidomain architecture and high plasticity. Owing to the overwhelming implications of the mutated SFs in the onset of cancer, this study advances our understanding of the recognition mechanism of a major class of splice site signals mediated by a key splicing factor. ,, …”