CRISPR/Cas9-based genome-wide screening for deubiquitinase subfamily identifies USP1 regulating MAST1-driven cisplatin-resistance in cancer cells

Abstract

Background: Cisplatin is one of the frontline anticancer agents. However, development of cisplatin-resistance limits the therapeutic efficacy of cisplatin-based treatment. The expression of microtubule-associated serine/ threonine kinase 1 (MAST1) is a primary factor driving cisplatin-resistance in cancers by rewiring the MEK pathway. However, the mechanisms responsible for MAST1 regulation in conferring drug resistance is unknown. Methods: We implemented a CRISPR/Cas9-based, genome-wide, dual screening system to identify deubiquitinating enzymes (DUBs) that govern cisplatin resistance and regulate MAST1 protein level. We analyzed K48-and K63-linked polyubiquitination of MAST1 protein and mapped the interacting domain between USP1 and MAST1 by immunoprecipitation assay. The deubiquitinating effect of USP1 on MAST1 protein was validated using rescue experiments, in vitro deubiquitination assay, immunoprecipitation assays, and half-life analysis. Furthermore, USP1-knockout A549 lung cancer cells were generated to validate the deubiquitinating activity of USP1 on MAST1 abundance. The USP1-MAST1 correlation was evaluated using bioinformatics tool and in different human clinical tissues. The potential role of USP1 in regulating MAST1mediated cisplatin resistance was confirmed using a series of in vitro and in vivo experiments. Finally, the clinical relevance of the USP1-MAST1 axis was validated by application of small-molecule inhibitors in a lung cancer xenograft model in NSG mice. Results:The CRISPR/Cas9-based dual screening system identified USP1 as a novel deubiquitinase that interacts, stabilizes, and extends the half-life of MAST1 by preventing its K48-linked polyubiquitination. The expression analysis across human clinical tissues revealed a positive correlation between USP1 and MAST1. USP1 promotes MAST1-mediated MEK1 activation as an underlying mechanism that contributes to cisplatin-resistance in cancers. Loss of USP1 led to attenuation of MAST1-mediated cisplatin-resistance both in vitro and in vivo. The combined pharmacological inhibition of USP1 and MAST1 using small-molecule inhibitors further abrogated MAST1 level and synergistically enhanced cisplatin efficacy in a mouse xenograft model. Conclusions: Overall, our study highlights the role of USP1 in the development of cisplatin resistance and uncovers the regulatory mechanism of MAST1-mediated cisplatin resistance in cancers. Co-treatment with USP1 and MAST1 inhibitors abrogated tumor growth and synergistically enhanced cisplatin efficacy, suggesting a novel alternative combinatorial therapeutic strategy that could further improve MAST1-based therapy in patients with cisplatin-resistant tumors.

Microtubule-Associated Serine/Threonine (MAST) Kinases in Development and Disease

“…The direct binding between MAST1 and hsp90 increases the stabilization of MAST1, as the ubiquitination of lysines 317 and 545 of MAST1 by CHIP is inhibited [45]. A recent study revealed that the stability of MAST1 is also enhanced by the deubiquitinase USP1 in cisplatin-resistant cancer cells [44]. Together, these findings suggest that Hsp90, MAST1 and USP1 may be excellent targets for cisplatin-resistant cancer cells, as their inhibition will enhance cisplatin sensitivity and result in increased cell death.…”

Targeting deubiquitinases for cancer therapy

“…93 Acting as an USP1 inhibitor, pimozide, a diphenyl-butylpiperidine compound, effectively overcomes cancer cell resistance to cisplatin and demonstrates potent inhibition of lung tumor growth both in vitro and in vivo. 94 Similarly, other USP1 inhibitors, including ML323, C527, SJB2-043, SJB3-019A, and KSQ-4279, have shown the ability to induce cell cycle arrest and cell death in a spectrum of malignancies, encompassing esophageal squamous cell carcinoma, leukemia, and multiple myeloma, both in vitro and in vivo settings [95][96][97][98] (Figure 7A). Hence, targeting USP1 presents a promising opportunity for anti-cancer therapy.…”

“…Pimozide, a medication clinically used for Tourette syndrome, inhibits the DUB activity of USP1, resulting in increased levels of PCNA and FANCD2 mono‐ubiquitination 93 . Acting as an USP1 inhibitor, pimozide, a diphenyl‐butylpiperidine compound, effectively overcomes cancer cell resistance to cisplatin and demonstrates potent inhibition of lung tumor growth both in vitro and in vivo 94 . Similarly, other USP1 inhibitors, including ML323, C527, SJB2‐043, SJB3‐019A, and KSQ‐4279, have shown the ability to induce cell cycle arrest and cell death in a spectrum of malignancies, encompassing esophageal squamous cell carcinoma, leukemia, and multiple myeloma, both in vitro and in vivo settings 95–98 (Figure 7A).…”

Nanotechnology‐based CRISPR/Cas9 delivery system for genome editing in cancer treatment

“…[28][29][30][31] Given its promising potential in gene therapy, CRISPR/Cas9 has expanded rapidly in biomedical research of cancer treatment. 24,[32][33][34] Different CRISPR/ Cas9-mediated cancer therapy approaches, such as altering chemo-tolerance genes, changing metabolically relevant genes and tumor stem cell associated genes, as well as cancer immunotherapy, are well-established in diverse cancer types. 32,[35][36][37][38] Although the discovery of CRISPR/Cas9 paved the way for simple, effective, and multiplex manipulation of genes, the off-target impacts, difficulties in targeting specific cells in vivo, and the successful transfer into the nuclei still remain the main obstacles keeping it from realizing its maximum capabilities.…”

“…Given its promising potential in gene therapy, CRISPR/Cas9 has expanded rapidly in biomedical research of cancer treatment 24,32–34 . Different CRISPR/Cas9‐mediated cancer therapy approaches, such as altering chemo‐tolerance genes, changing metabolically relevant genes and tumor stem cell associated genes, as well as cancer immunotherapy, are well‐established in diverse cancer types 32,35–38 .…”