Small Molecule Inhibitors of USP1 Target ID1 Degradation in Leukemic Cells and Cause Cytotoxicity

ID1 (inhibitor of DNA-binding-1) is a member of the helix-loop-helix family of transcriptional regulatory proteins. The ID-family of proteins (ID1-ID4) inhibit the DNA binding of transcription factors which regulate cellular differentiation and proliferation. Accordingly, deregulation of ID proteins has been observed in many cancer types including leukemia. High levels of ID1 expression are found in primary acute myeloid leukemia (AML) samples and correlate with poor prognosis. ID1 is also identified as a common downstream target of the oncogenic tyrosine kinases, BCR-ABL, TEL-ABL and FLT3-ITD. In addition, Id1 has been shown to promote a myeloproliferative disease in mice, and knockdown of ID1 expression inhibits leukemic cell growth. Therefore, ID1 is an excellent candidate for targeted therapy in leukemia. However, suitable drugs to target ID1 have not been developed to date. 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 screen, 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 nanomolar range, and also targeted the enzyme activity of native USP1. To determine the cellular consequences of USP1 inhibition, we exposed leukemic cells to micromolar concentrations of the inhibitors and evaluated ID1 levels and survival. USP1 inhibitors promoted the degradation of ID1 and, concurrently, inhibited the growth (>90% inhibition in 24 hrs) of chronic myelogenous leukemia (CML) and AML cell lines with induction of apoptosis in a dose dependent manner. The EC50 of the inhibitors for the leukemic cell growth inhibition was approximately 1.07 μM ± 0.08 (95% Confidence Limits). Interestingly, exposure to low doses of USP1 inhibitor for 5 days in culture resulted in erythroid differentiation of K562 leukemic cells. A known USP1 inhibitor, Pimozide, also promoted ID1 degradation and inhibited growth of leukemic cells (>90% inhibition in 48 hrs), though at a higher drug concentrations as compared to the novel USP1 inhibitors. Importantly, the novel USP1 inhibitors promoted ID1 degradation and exhibited cytotoxicity (>90% death in 48 hrs) in primary AML patient-derived leukemic cells. Notably, siRNA-mediated knockdown of USP1 in K562 leukemic cells resulted in growth inhibition, increased apoptosis and cell cycle arrest. Collectively, our results demonstrate 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. Disclosures: No relevant conflicts of interest to declare.