Downregulation of SOX2 by inhibition of Usp9X induces apoptosis in melanoma

Abstract

Melanoma tumors driven by BRAF mutations often do not respond to BRAF/MEK/ERK pathway inhibitors currently used in treatment. One documented mechanism of resistance is upregulation of SOX2, a transcription factor that is essential for tumor growth and expansion, particularly in melanoma tumors with BRAF mutations. Targeting transcription factors pharmacologically has been elusive for drug developers, limiting treatment options. Here we show that ubiquitin-specific peptidase 9, X-linked (Usp9x), a deubiquitinase (DUB) enzyme controls SOX2 levels in melanoma. Usp9x knockdown in melanoma increased SOX2 ubiquitination, leading to its depletion, and enhanced apoptotic effects of BRAF inhibitor and MEK inhibitors. Primary metastatic melanoma samples demonstrated moderately elevated Usp9x and SOX2 protein expression compared to tumors without metastatic potential. Usp9x knockdown, as well as inhibition with DUB inhibitor, G9, blocked SOX2 expression, suppressed in vitro colony growth, and induced apoptosis of BRAF-mutant melanoma cells. Combined treatment with Usp9x and mutant BRAF inhibitors fully suppressed melanoma growth in vivo . Our data demonstrate a novel mechanism for targeting the transcription factor SOX2, leveraging Usp9x inhibition. Thus, development of DUB inhibitors may add to the limited repertoire of current melanoma treatments.

Targeting the Ubiquitin-Proteasome System for Cancer Therapeutics by Small-Molecule Inhibitors

“…Both WP1130 and G9 also partially inhibit another DUB, USP5, but the resultant p53 accumulation only enhances anti-tumor effects in MM [ 265 ]. Recent preclinical results have shown WP1130 and G9 to be effective at inducing apoptosis in other cancers, such as AML [ 78 ] and melanoma [ 76 ]. More recently, FT709 [ 266 ] was identified as a potent inhibitor of USP9X, with greatly extremely improved specificity compared to WP1130.…”

Emerging Role of Deubiquitinating Enzymes (DUBs) in Melanoma Pathogenesis

“…Another important drug target in melanoma is the deubiquitinase USP9X (X-linked ubiquitin-specific peptidase 9), which prevents the degradation of ubiquitin-specific proteins that are essential in various biological pathways involved in the regulation of cell transformation and survival. USP9X activity and expression were found to be elevated in metastases compared to those in the primary tumor [ 52 ]. Thus, enforced expression of USP9X enhances tumor growth in vitro and in vivo [ 52 , 53 ].…”

“…USP9X activity and expression were found to be elevated in metastases compared to those in the primary tumor [ 52 ]. Thus, enforced expression of USP9X enhances tumor growth in vitro and in vivo [ 52 , 53 ]. Particularly for USP9X, its expression and activity are upregulated after ectopic expression of BRAFV600E in 293T cells; conversely, inhibition of the kinase partly suppresses USP9X activity in vemurafenib-sensitive but not resistant cells, indicating that USP9X is one of the first DUBs whose activity is dependent on MAPK signaling [ 52 ].…”

“…Thus, enforced expression of USP9X enhances tumor growth in vitro and in vivo [ 52 , 53 ]. Particularly for USP9X, its expression and activity are upregulated after ectopic expression of BRAFV600E in 293T cells; conversely, inhibition of the kinase partly suppresses USP9X activity in vemurafenib-sensitive but not resistant cells, indicating that USP9X is one of the first DUBs whose activity is dependent on MAPK signaling [ 52 ]. Although depletion of USP9X reduces tumor growth in melanoma cells carrying BRAF or NRAS mutations, its combination with MAPK pathway inhibitors (vemurafenib or MEKi) has been shown to enhance the therapeutic response [ 52 ].…”

“…Particularly for USP9X, its expression and activity are upregulated after ectopic expression of BRAFV600E in 293T cells; conversely, inhibition of the kinase partly suppresses USP9X activity in vemurafenib-sensitive but not resistant cells, indicating that USP9X is one of the first DUBs whose activity is dependent on MAPK signaling [ 52 ]. Although depletion of USP9X reduces tumor growth in melanoma cells carrying BRAF or NRAS mutations, its combination with MAPK pathway inhibitors (vemurafenib or MEKi) has been shown to enhance the therapeutic response [ 52 ]. This improved apoptotic response is mediated by the stability of the transcription factor SOX2, a treatment-induced protein, which, upon depletion of USP9X , leads to SOX2 breakdown, thereby inducing death signals that inhibit growth in vitro and in vivo [ 52 , 53 ].…”