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.
BackgroundDNA double-strand breaks (DSBs) caused by ionizing radiation or by the stalling of DNA replication forks are among the most deleterious forms of DNA damage. The ability of cells to recognize and repair DSBs requires post-translational modifications to histones and other proteins that facilitate access to lesions in compacted chromatin, however our understanding of these processes remains incomplete. UHRF1 is an E3 ubiquitin ligase that has previously been linked to events that regulate chromatin remodeling and epigenetic maintenance. Previous studies have demonstrated that loss of UHRF1 increases the sensitivity of cells to DNA damage however the role of UHRF1 in this response is unclear.ResultsWe demonstrate that UHRF1 plays a critical role for facilitating the response to DSB damage caused by γ-irradiation. UHRF1-depleted cells exhibit increased sensitivity to γ-irradiation, suggesting a compromised cellular response to DSBs. UHRF1-depleted cells show impaired cell cycle arrest and an impaired accumulation of histone H2AX phosphorylation (γH2AX) in response to γ-irradiation compared to control cells. We also demonstrate that UHRF1 is required for genome integrity, in that UHRF1-depleted cells displayed an increased frequency of chromosomal aberrations compared to control cells.ConclusionsOur findings indicate a critical role for UHRF1 in maintenance of chromosome integrity and an optimal response to DSB damage.
Methyl methansulfonate UV sensitive clone 81 (Mus81) and essential meiotic endonuclease 1 (Eme1) form a heterodimeric endonuclease that preserves genomic integrity through the repair of DNA replication‐associated damage. Despite its role as genome guardian and tumour suppressor, our understanding of how this endonuclease is coordinated during the DNA damage response is poor. In order to identify other proteins that interact with Mus81‐Eme1, our laboratory conducted a protein interaction screen to identify and characterize proteins that interact with Eme1. One candidate identified in this screen was found to encode the carboxy‐terminal domain of Np95, a chromatin‐associated ubiquitin ligase. Np95 and Eme1 co‐localize on nuclear chromatin following exposure of cells to camptothecin, an agent that promotes the collapse of replication forks. The observed co‐localization following DNA damage was found to be dependent on an intact RING finger, the structural motif that encodes the E3 ubiquitin ligase activity of Np95. These findings for the first time, link the tumour‐suppressor Mus81‐Eme1 with the replication‐associated chromatin modifier functions of Np95 in the DNA damage response. Supported by grants from the Canadian Cancer Society through the National Cancer Institute of Canada and the Cancer Research Society.
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.
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<p>PDF - 502KB, Supplementary figure 1. USP1 inhibitor SJB2-043 inhibits activity of native USP1 in a dose-dependent manner and causes a decrease in ID1, ID2 and ID3 levels in K562 cells. Supplementary figure 2. Knockdown of USP1 results in growth inhibition, increased apoptosis and cell cycle arrest in K562 cells. Supplementary figure 3. Analogs of USP1 inhibitor lead to ID1 destabilization in leukemic cells. Supplementary figure 4. Caspase inhibitor does not block the degradation of ID1 by USP1 inhibitors. Supplementary figure 5. Effects of USP1 inhibitors on growth of primary human cord blood CD34+ cells. Supplementary figure 6. USP1 inhibitors cause increase in Ub-FANCD2, Ub-PCNA, and p21, and decrease the HR activity.</p>
<p>PDF - 86KB, Additional supplementary methods.</p>
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