There is clear and irrefutable evidence that the Notch developmental signaling pathway is an important driver in many cancers, specifically in breast cancer. However, because of the ubiquity of this pathway in normal tissues, therapeutics targeting Notch have been associated with significant deleterious side effects. Herein, we provide evidence that USP9x selectively drives Notch in breast cancer and that therapeutic USP9x inhibition prevents tumor progression without collateral damage to normal tissues.

Triple-negative breast cancer (TNBC) is a breast cancer subtype that lacks targeted treatment options. The activation of the Notch developmental signaling pathway, which is a feature of TNBC, results in the secretion of proinflammatory cytokines and the recruitment of protumoral macrophages to the tumor microenvironment. While the Notch pathway is an obvious therapeutic target, its activity is ubiquitous, and predictably, anti-Notch therapies are burdened with significant on-target side effects. Previously, we discovered that, under conditions of cellular stress commonly found in the tumor microenvironment, the deubiquitinase USP9x forms a multiprotein complex with the pseudokinase tribbles homolog 3 (TRB3) that together activate the Notch pathway. Herein, we provide preclinical studies that support the potential of therapeutic USP9x inhibition to deactivate Notch. Using a murine TNBC model, we show that USP9x knockdown abrogates Notch activation, reducing the production of the proinflammatory cytokines, C-C motif chemokine ligand 2 (CCL2) and interleukin-1 beta (IL-1). Concomitant with these molecular changes, a reduction in tumor inflammation, the augmentation of antitumor immune response, and the suppression of tumor growth were observed. The pharmacological inhibition of USP9x using G9, a partially selective, small-molecule USP9x inhibitor, reduced Notch activity, remodeled the tumor immune landscape, and reduced tumor growth without associated toxicity. Proving the role of Notch, the ectopic expression of the activated Notch1 intracellular domain rescued G9-induced effects. This work supports the potential of USP9x inhibition to target Notch in metabolically vulnerable tissues like TNBC, while sparing normal Notch-dependent tissues.

Author contributions: A.J., K.M., and M.R. designed research; A.J., K.M., A.E., Y.S., S.J.D., and P.S.O. performed research; S.A.W. and N.J.D. contributed new reagents/analytic tools; A.J., K.M., A.E., Y.S., S.J.D., P.S.O., and M.R. analyzed data; and A.J. and M.R. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at

All study data are included in the article and/or SI Appendix.

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Therapeutic inhibition of USP9x-mediated Notch signaling in triple-negative breast cancer -

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