p63 is a transcription factor structurally related to the p53 tumor suppressor. The C-terminal region differs from p53's in that it contains a sterile alpha motif (SAM) domain and is subject to multiple alternative splicings. The N-terminal region is present in the transactivation (TA) and ⌬N configurations, with the latter lacking the transcriptional activation domain 1. Single amino acid substitutions and frameshift mutations of p63 cause the human ankyloblepharon ectodermal dysplasia clefting (AEC) or ectrodactyly ectodermal dysplasia and facial clefting (EEC) syndromes. We have systematically compared the activities of the wild-type p63 isoforms and of the natural mutants in activation and repression assays on three promoters modulated by p53. We found that p63 proteins with an altered SAM domain or no SAM domain-the ␤ isoforms, the EEC frameshift mutant, and the missense AEC mutations-all showed a distinctly higher level of activation of the MDM2 promoter and decreased repression on the HSP70 promoter. Fusion of SAM to the GAL4 DNA-binding domain repressed a heterologous promoter. A second activation domain, TA2, corresponding to exons 11 to 12, was uncovered by comparing the activation of ⌬N isoforms on natural promoters and in GAL4 fusion systems. In colony formation assays, the AEC mutants, but not the EEC frameshift, were consistently less efficient in suppressing growth, in both the TA version and the ⌬N version, with respect to their p63␣ counterparts. These data highlight the modularity of p63, identifying the SAM domain as a dominant transcriptional repression module and indicating that the AEC and EEC frameshift mutants are characterized by a subversion of the p63 transcriptional potential.

Post-translational modification of proteins by the ubiquitin-like molecule SUMO-1 regulates their stability and activity with crucial implications for many cellular processes. Here we show that p63α, but not p63β and γ, is sumoylated in vitro and in vivo at a single lysine residue, K637, in the post-SAM domain. SUMO-1 attachment targets ∆Np63α for proteasome mediated degradation while it does not influence p63α intracellular localization, as wild-type protein and a mutant carring the K637 mutated into arginine (K637R), have the same nuclear localization. Four natural p63 mutations, falling within the SAM and post-SAM domain of p63α, were found to be altered in their sumoylation capacity. The transcriptional activities of the natural mutants and of K637R were strongly increased compared to that of wild type p63, suggesting that sumoylation has a negative effect on p63 driven transcription. The findings that ∆Np63α protein levels are regulated by SUMO-1 and that this regulation is altered in natural p63 mutants, suggest that SUMO conjugation to p63 plays a critical role in regulating the biological activity of p63.

The ARF/MDM2/p53 pathway is a principal defense mechanism to protect the organism from uncontrolled effects of deregulated oncogenes. Oncogenes activate ARF, which interacts with and inhibits the ubiquitin ligase MDM2, resulting in p53 stabilization and activation. Once stabilized and activated, p53 can either induce or repress a wide array of different gene targets, which in turn can regulate cell cycle, DNA repair, and a number of apoptosis-related genes. Here we show that, unlike p53, p63, a member of the p53 family, directly interacts with p14 ARF . Through this interaction ARF inhibits p63-mediated transactivation and transrepression. In p63-transfected cells, ARF, which normally localizes into nucleoli, accumulates in the nucleoplasm. Based on these observations, we suggest that stimuli inducing p14 ARF expression can, at the same time, activate p53 and impair p63 transcriptional activity, altering the pattern of p53 target gene expression. Here we show, for the first time, a physical and functional link between the p14 ARF tumor suppressor protein and p63, a member of the p53 family.

TPS4185 Background: Monopolar Spindle 1 (MPS1) kinase regulates the spindle assembly checkpoint (SAC) which ensures proper division of chromosomes during mitosis. MPS1 is overexpressed in several tumors, including hepatocellular carcinoma (HCC), where it correlates with tumor features and poor overall and disease-free survival. NMS-01940153E is a novel, highly potent and selective small molecule inhibitor of MPS1 kinase with long residence time and strong preclinical anti-tumor activity in different tumor types. In HCC lines, specifically, NMS-01940153E showed ~2-Log higher anti-proliferative activity compared to sorafenib, lenvatinib, and regorafenib. In a previous open-label first-in-human (FIH) study, CL1-81694-001 (EudraCT 2014-002023-10), signs of activity in HCC were detected. Recently, the treatment paradigm for advanced HCC has changed, with immunotherapy combinations in first line and TKIs shifted in later lines. However, the overall prognosis of patients with advanced HCC remains poor, and there is a strong need of new drugs in this setting. NMS-01940153E novel mechanism of action, inhibiting the SAC and interfering with genomic stability in HCC, may offer a new therapeutic option in HCC. Based on the promising FIH results, a Phase I/II study, MPSA-153-001 (EudraCT 2020-001002-26), was initiated in patients with HCC previously treated with more than one systemic therapy. Methods: The Phase II part of the MPSA-293-001 trial is designed as a two-stage study with an interim analysis for futility and safety rules for unacceptable toxicity. The primary objective is to assess the antitumor activity of NMS-01940153E in adult patients with unresectable HCC previously treated with systemic therapy measured as objective response rate (ORR) by investigator-assessed RECIST 1.1. Secondary endpoints are safety, PK, ORR as measured by investigator-assessed mRECIST, DoR, PFS and OS. Exploratory endpoints include biomarkers. NMS-01940153E is administered IV, on days 1, 8 and 15 every 4 weeks at the RP2D of 100 mg/m2/wk, which showed PK in a predicted active range. Key eligibility criteria are 1) diagnosis of HCC; 2) disease progression on standard-of-care treatment including an immune checkpoint inhibitor as first line and at least one TKI; 3) no more than 3 prior systemic treatment lines. Interim evaluation for futility will be undertaken as soon as the first 10 evaluable patients will be enrolled. If at least 1 responder is observed with no safety issues, enrollment will proceed up to 38 evaluable patients. Otherwise, the study will be terminated for futility. An independent DSMB will review the interim results and provide recommendation on the study progress. Recruitment is currently ongoing in Italy and Spain and an FDA “Study May Proceed Notification” was received in January 2023. Clinical trial information: NCT05630937 .