Characterization of SF3B1 mutation in chronic lymphocytic leukemia and the role of new splicing modulators

Abstract

Mutations in Splicing Factor 3b Subunit 1(SF3B1) are observed in 15% of patients with chronic lymphocytic leukemia (CLL) and are associated with worse disease prognosis. SF3B1 is involved in pre-mRNA splicing and is the target of anti-tumor drugs. Here, we analyzed RNA-sequencing data from 298 CLL patients and SF3B1WT and SF3B1K700E MEC1 CLL isogenic cell lines and characterized the pre-mRNA sequence features associated with the selection of cryptic 3’splice site (3’ss) upon SF3B1 mutation. In addition, we validated the effects of MAP3K7 alternative splicing events in SF3B1-mutated CLL, and confirmed the activation of NF-κB signaling in this subgroup. Finally, we carried out the first study of the effects of the H3B-8800 splicing modulator in CLL, in vitro in primary CLL samples and in MEC1 WT and SF3B1-mutated isogenic cell lines, as well as in vivo in an immunodeficient NOD-SCID interleukin-2 receptor gamma (IL2Rγ)null (NSG) mice model. We also characterized the transcriptomic effects of H3B-8800 treatment of MEC1 cell lines by RNA-seq. H3B-8800 showed preferential lethality towards SF3B1-mutated cells and synergistic effects with BCL2 inhibitor venetoclax, supporting the use of SF3B1 inhibitors as a possible novel therapeutic strategy in CLL.

El 15% de los pacientes con leucemia linfática crónica (LLC) presentan mutaciones en SF3B1 (Splicing Factor 3b Subunit 1), que les confieren un peor pronóstico. SF3B1 es una proteína implicada en pre-mRNA splicing y es la diana de drogas antitumorales. En este trabajo analizamos resultados de RNA-seq de 298 pacientes con LLC y de líneas celulares de LLC isogénicas MEC1 SF3B1WT y SF3B1K700E caracterizando secuencias del pre-mRNA implicadas en el uso de sitios 3’ de splicing crípticos activados por mutaciones en SF3B1. Adicionalmente, validamos el efecto de uno de estos 3’ss crípticos en el gen MAP3K7, confirmando la activación que produce en la vía de señalización de NF-κB en el subgrupo que presenta mutaciones en SF3B1. Finalmente, probamos por primera vez en LLC los efectos del modulador de splicing H3B-8800, in vitro en células primarias de LLC y en la línea celular isogénica SF3B1K700E MEC1, así como in vivo, en el modelo de ratón inmunodeprimido NOD-SCID interleukin-2 receptor gamma (IL2Rγ)null (NSG). H3B-8800 muestra una letalidad preferencial hacia las células mutadas en SF3B1 y un efecto sinérgico en combinación con el inhibidor de BCL2 venetoclax, sugiriendo que inhibidores de SF3B1 pueden proporcionar una nueva estrategia terapéutica en la LLC.