Résumé
Background: To better understand the mechanism of action of PT-112, we investigated the effects of PT-112 on kinase activity within HCT116 human colorectal cancer cells. PT-112 is a pyrophosphate-conjugated platinum (Pt) containing new chemical entity under clinical development in the US and Taiwan. Designed to reduce certain toxicities associated with Pt agents and drug resistance from DNA-repair pathway activity, PT-112 differs in significant ways from traditional Pt therapies. PT-112 results in potent anti-proliferation effects with lower Pt accumulation in whole cell and DNA extracts. Upregulation of p16 and p21 and, consistently, inhibition of the cell cycle at the G1/S phase transition is observed with PT-112 use, a finding not commonly associated with Pt agents. Further, sensitivity to PT-112 is not significantly affected by the functionality of DNA repair pathways. Finally, the induction of immunogenic cell death by PT-112 has been indicated via release of HMGB1, cell surface localization of calreticulin, and suppression of STAT3.
Methods: A broad analysis of the phosphorylation pattern of kinase targets was achieved by utilizing the Tyrosine Kinase and Serine / Threonine Kinase PamChip® Arrays, each spotted with 144 peptides harboring kinase phosphorylation sites. Data were collected after 1, 6, and 24hr of exposure to 10 μM PT-112, a concentration achievable in human patient plasma at sub-toxic doses, and the degree of phosphorylation of these peptide targets was measured by the PamStation®.
Results: A set of kinase targets was identified as differentially phosphorylated relative to untreated control samples. In total, 4/15/30 targets were more phosphorylated than controls at the 1/6/24hr time points, respectively, while 1/0/10 targets were less phosphorylated. Several of these targets connect to key cancer relevant pathways, including anti-angiogenesis (VGFR1), apoptosis (BAD), cell cycle regulation (CDC2, Rb, RBL2), cell growth (EGFR, MK07, RBL2), and cancer immunology (STAT4, CSF1R).
Conclusions: A narrow set of initial effects on kinase activity from PT-112 exposure, which broaden over time, was observed. While we cannot discriminate direct from indirect effects, these data are consistent with PT-112 having mechanistic targets independent of nuclear DNA. Several observations were consistent with findings from previous experiments (e.g. evidence of impacts on apoptotic, cell cycle, and immunological pathways), while others were novel (e.g. impact on VGFR1, EGFR, MK07). In conclusion, PT-112 at pharmacologically achievable concentrations leads to a unique pattern of kinase disruption in vitro. Further work will be conducted to validate these findings, identify the driver kinases, and connect these observations into a coherent network of kinases and targets affected by PT-112 treatment, and to understand the clinical impact of the unique pharmacodynamic profile of PT-112.
Citation Format: Diego Tosi, Esther Perez-Gracia, Philippe Pourquier, Tyler D. Ames, Richard A. Wing, José Jimeno, Céline Gongora. A kinome analysis of the molecular pharmacodynamics of PT-112 in a human cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2378. doi:10.1158/1538-7445.AM2017-2378