H3B-8800, an orally available small-molecule splicing modulator, induces lethality in spliceosome-mutant cancers

Abstract

Genomic analyses of cancer have identified recurrent point mutations in the RNA splicing factor-encoding genes SF3B1, U2AF1, and SRSF2 that confer an alteration of function. Cancer cells bearing these mutations are preferentially dependent on wild-type (WT) spliceosome function, but clinically relevant means to therapeutically target the spliceosome do not currently exist. Here we describe an orally available modulator of the SF3b complex, H3B-8800, which potently and preferentially kills spliceosome-mutant epithelial and hematologic tumor cells. These killing effects of H3B-8800 are due to its direct interaction with the SF3b complex, as evidenced by loss of H3B-8800 activity in drug-resistant cells bearing mutations in genes encoding SF3b components. Although H3B-8800 modulates WT and mutant spliceosome activity, the preferential killing of spliceosome-mutant cells is due to retention of short, GC-rich introns, which are enriched for genes encoding spliceosome components. These data demonstrate the therapeutic potential of splicing modulation in spliceosome-mutant cancers.

The cryo-EM structure of the SF3b spliceosome complex bound to a splicing modulator reveals a pre-mRNA substrate competitive mechanism of action

“…Moreover, the different pre-mRNA substrates in turn help further classify the chemical probes as strong or weak modulators. Importantly, tight binding compounds that are nonselective toward strong and weak substrates in these IVS assays are expected to be more pleiotropic, while weaker binding compounds show preferential inhibition toward short and GC-rich introns (Seiler et al 2018). …”

“…Therefore, the degree to which any splicing event is affected depends on the relative strength of the compound, the affinity of the particular pre-mRNA substrate, and their relative concentrations. This explains, at least in part, why these compounds do not behave as universal splicing inhibitors but rather as modulators of certain splice junctions, as exemplified in global RNA-seq studies in which only a subset of junctions is impacted (Seiler et al 2018). Further structural and biochemical studies will be needed to better characterize the interactions between different premRNA substrates and with different splicing modulators.…”

“…The Ad2 variant (Ad2.2) (Seiler et al 2018) was cloned into pcDNA3.1(+) vector (Promega) using the same restriction sites. The plasmids were linearized using XbaI and used as DNA templates in the in vitro transcription reactions.…”

“…Together, these may account for the "spliceosome sickness" observed in these genetic backgrounds (Darman et al 2015) and present a potential vulnerability that could be targeted by splicing modulators in a therapeutic setting. The first splicing modulator to enter the clinic was E7107 (Hong et al 2014), and, recently, H3B-8800 has entered phase 1 clinical trials (Seiler et al 2018).…”

Mutations in spliceosome genes and therapeutic opportunities in myeloid malignancies

“…These mutations are presumed to contribute to oncogenic transformation, but the underlying mechanisms remain elusive and are currently an area of intense research. The high frequency of spliceosomal mutations in myeloid malignancies has generated enthusiasm for their therapeutic targeting and preclinical studies have provided evidence that they can serve as an Achilles' heel in these cancers when using small molecule spliceosomal inhibitors . There is anticipation that a deeper understanding of the oncogenic mechanisms of these mutations might shed light on other therapeutic avenues as well.…”