Small-Molecule Inhibitors of USP1 Target ID1 Degradation in Leukemic Cells

Abstract

Inhibitor of DNA-binding-1 (ID1) transcription factor is essential for the proliferation and progression of many cancer types including leukemia. However, the ID1 protein has not yet been therapeutically targeted in leukemia. ID1 is normally polyubiquitinated and degraded by the proteasome. Recently, it has been shown that USP1, a ubiquitin specific protease, deubiquitinates ID1 and rescues it from proteasome degradation. Inhibition of USP1 therefore offers a new avenue to target ID1 in cancer. Here, using a Ubiquitin-Rhodamine-based high throughput screening, we identified small molecule inhibitors of USP1 and investigated their therapeutic potential for leukemia. These inhibitors blocked the deubiquitinating enzyme activity of USP1 in vitro in a dose-dependent manner with an IC50 in the high nanomolar range. USP1 inhibitors promoted the degradation of ID1 and, concurrently, inhibited the growth of leukemic cell lines in a dose dependent manner. A known USP1 inhibitor, Pimozide, also promoted ID1 degradation and inhibited growth of leukemic cells. In addition, the growth of primary Acute Myeloid Leukemia (AML) patient-derived leukemic cells was inhibited by a USP1 inhibitor. Collectively, these results indicate that the novel small molecule inhibitors of USP1 promote ID1 degradation and are cytotoxic to leukemic cells. The identification of USP1 inhibitors therefore opens up a new approach for leukemia therapy.

PDGF Engages an E2F-USP1 Signaling Pathway to Support ID2-Mediated Survival of Proneural Glioma Cells

“…S3B). Importantly, USP1 can be therapeutically targeted by pimozide (23,27), an antipsychotic drug of the diphenylbutylpiperidine class that has limited toxicities, has been approved for use in humans, and can cross the blood-brain barrier (34). These characteristics make USP1 a possible therapeutic target in glioblastoma.…”

“…These findings show that PDGF signaling can upregulate Usp1 expression. Inhibition of USP1 has been previously shown to decrease proliferation of leukemia cells and spheroid formation of human glioblastoma cells (22,27). To examine whether Usp1 affects the survival of PDGF-GSCs, we infected these cells with lentiviruses encoding Usp1-targeting shRNAs (short hairpin RNAs) or a nontarget control (ShControl) and performed Annexin V/PI staining.…”

“…ID proteins are known posttranslational targets of USP1 (26,27), hence we examined whether they were regulated by PDGF signaling in a manner that parallels USP1 regulation. Inhibition of PDGF signaling by doxycycline hyclate led to a decrease in ID2 protein, and an inconsistent decrease in ID1 protein levels (Fig.…”

“…Inhibition of USP1 by multiple strategies has been suggested as a potential strategy to enhance the therapeutic efficacy of chemotherapy and radiation in treating cancer (22)(23)(24). USP1, upon forming a molecular complex with UAF1 (WDR48), deubiquitinates substrates including PCNA, FANCI, FANCD2, and the short-lived Inhibitor of DNA Binding (ID) proteins ID1, ID2, and ID3 that each have a half-life of about 10 minutes (19,20,(25)(26)(27)(28). Members of the Id gene family, including Id2, have been proposed as mediators of glioblastoma pathology and are known to be highly expressed in a number of different tumor types (28)(29)(30).…”

A novel role for the deubiquitinase USP1 in the control of centrosome duplication

“…11 USP1 has been identified as a key regulator in the DNA repair processes, mainly in the Fanconi anemia pathway and Translesion DNA synthesis by regulating ubiquitination status of FANCD2 and PCNA, 12,13 Mono-ubiquitinated FANCD2 and PCNA serve as a platform to recruit DNA repair proteins to DNA damage sites, [14][15][16] and USP-induced deubiquitination of FANCD2 and PCNA is crucial for the correct function of DNA repair pathway. 17,18 Indeed, Usp1 gene deletion in mice 18 and USP1 inhibition by small molecules displayed chemosensitizing effects against DNA damaging agents, [19][20][21] indicating that USP1 is required for an efficient DNA repair activity. Besides DNA repair-related function, recent study reveals that increased level of USP1 is found in human osteosarcoma cell lines.…”