Abstract A280: Small molecule inhibition of atypical protein kinase C iota reveals a functional basis for oncogenesis.

Background: Atypical protein kinase C iota (PKCι) is a serine/threonine kinase that has been implicated in a variety of cellular processes including polarity, migration, proliferation and survival. PKCι is amplified in many cancers correlating with poor prognosis, however the upstream activating inputs and downstream effectors are not well defined. Experimental Procedures: 3D MDCK epithelial cell spheroids were grown in Matrigel and used to assess polarized morphogenesis and growth. PKCι was manipulated by inhibition with CRT0066854, or transfection with siRNA or cDNA variants. A PKCι binding interface was identified by X-ray crystallography and interrogated by mass spectrometry of wildtype or binding mutant immunocomplexes. Results: To identify potential inputs into PKCι we assessed a panel of transformed MDCK cells; p110α, Src, Ras, ErbB2 and Raf. Parental MDCK cells form polarized cysts in Matrigel, however Ras, ErbB2 and p110α MDCK cells formed dysplastic spheroids. In the case of Ras and ErbB2, but not p110α MDCK spheroids, normal polarized morphogenesis could be rescued by siRNA-PKCι or partial PKCι inhibition with decreased spheroid size seen for all transformed cells lines. An effector binding site (PKCι RIPR motif) identified from the crystal structure of the kinase and characterized as a recruitment site for select targets including LLGL2 and Myosin X, was found to be critical for PKCι to confer polarized morphogenesis. Interestingly this binding motif shows a low frequency of mutation in a subset of human tumors. Conclusions: Downstream of Ras and ErbB2, PKCι activity is enhanced with a resulting dysplasia and loss of polarization, that can be rescued by PKCι inhibition. Mutation of the PKCι-RIPR effector binding motif that is found in cancer, prevents interaction with LLGL2 and its expression also elicits growth as non-polarized, dysplastic epithelial spheroids. This indicates that PKCι may promote cancer, not only by amplification, but by upstream oncogenic inputs and somatic mutation. The oncogene dependent PKCι hyperactivation is pro-proliferative in 3D culture, providing compelling evidence that PKCι is a good target for Ras mutant tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A280. Citation Format: Mark Linch, Marta Sanz-Garcia, Svend Kjaer, Erika Soriano, Philippe Riou, Philip Knowles, Christian Dillon, Jon Roffey, Neil McDonald, Peter Parker. Small molecule inhibition of atypical protein kinase C iota reveals a functional basis for oncogenesis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A280.