Kinase-Independent Small-Molecule Inhibition of JAK-STAT Signaling

Abstract

Phenotypic cell-based screening is a powerful approach to small-molecule discovery, but a major challenge of this strategy lies in determining the intracellular target and mechanism of action (MoA) for validated hits. Here, we show that the small-molecule BRD0476, a novel suppressor of pancreatic β-cell apoptosis, inhibits interferon-gamma (IFN-γ)-induced Janus kinase 2 (JAK2) and signal transducer and activation of transcription 1 (STAT1) signaling to promote β-cell survival. However, unlike common JAK-STAT pathway inhibitors, BRD0476 inhibits JAK-STAT signaling without suppressing the kinase activity of any JAK. Rather, we identified the deubiquitinase ubiquitin-specific peptidase 9X (USP9X) as an intracellular target, using a quantitative proteomic analysis in rat β cells. RNAi-mediated and CRISPR/Cas9 knockdown mimicked the effects of BRD0476, and reverse chemical genetics using a known inhibitor of USP9X blocked JAK-STAT signaling without suppressing JAK activity. Site-directed mutagenesis of a putative ubiquitination site on JAK2 mitigated BRD0476 activity, suggesting a competition between phosphorylation and ubiquitination to explain small-molecule MoA. These results demonstrate that phenotypic screening, followed by comprehensive MoA efforts, can provide novel mechanistic insights into ostensibly well-understood cell signaling pathways. Furthermore, these results uncover USP9X as a potential target for regulating JAK2 activity in cellular inflammation.

Suppressors and activators of JAK-STAT signaling at diagnosis and relapse of acute lymphoblastic leukemia in Down syndrome

“…5B) (median fluorescence intensity fold-decrease 1.5 ± 0.2). This response was similar to the decrease in STAT1 phosphorylation observed in other biological systems treated with the same agent (46). BRD0476 did not alter STAT3 phosphorylation, supporting a specific effect of the inhibitor on STAT5 (Fig.…”

“…USP9X was also reported to enhance JAK2 signaling, as well as signaling of immune receptors (46,55,56). Thus, the loss of USP9X activity in up to 25% of CRLF2 pos ALLs, a subtype of leukemia believed to be driven by enhanced JAK-STAT signaling, is intriguing.…”

“…S7A), it is likely that the observed effects of USP9X loss would not be restricted merely to JAK2 signaling (46). Activating mutations in IL7RA are associated with CRLF2 rearrangements (Fig.…”

“…Buffering of JAK-STAT Signaling by Loss-of-Function of USP9X. USP9X was recently shown to positively regulate JAK-STAT signaling by binding to JAK2, thereby enhancing JAK phosphorylation via removal of a competing ubiquitin group (46). Consequently, pharmacological inhibition or genetic ablation of USP9X was shown to reduce JAK signaling.…”

“…S6A). The cells were then treated with increasing amounts USP9X specific inhibitor BRD0476 (46) in the presence of the human cytokine TSLP (10 ng/mL). After 2 wk of treatment, most of the cells did not express CRLF2, demonstrating a selective advantage for the nontransduced cells.…”