Abstract A2-29: A PDK1 inhibitors that has antitumor activity in a vemurafenib resistant BRAF(V600E)::NRAS(G12V) melanoma PDX
Authors: James Brian P. , Lemos Robert , Bagby Stacey , Robinson Steven , Robinson William , Tentler John , Eckhardt S. Gail , Kirkpatrick D. Lynn , Powis Garth 
Published: November 2015

Phosphatidylinositol 3-kinase/phosphatidylinositide-dependent protein kinase 1 (PDPK1)/Akt signaling is activated in many cancers, and is associated with mutational loss of PTEN function, or activation of Phosphatidylinositide-3-kinase (PI3K). Activation of the PDPK1 signaling pathway contributes to proliferation and survival pathways in tumor cells. In a mouse Braf(V600E)::Pten(-/-) melanoma model genetic inactivation of PDKP1 delays the development of pigmented lesions and melanoma. We have found that the PDPK1 inhibitor PHT-427 has antitumor activity in BRAF(V600E)::NRAS(G12V) melanoma patient derived xenografts (PDX) grown in immune deficient mice.

Pleckstrin homology (PH) domains are 100-120 amino acid domains found in more than 250 human proteins that bind phosphatidylinositide (Ptdlns) lipids in cell membranes. The phosphorylation of Ptdins, and the consequent binding of PH domain containing proteins is found in many signal transduction pathways critical for cell growth, survival, angiogenesis, and metastasis. Though the primary amino acid sequences of PH domains are not highly conserved, PH domains do have a highly conserved tertiary structure. This structural conservation of PH domains combined with the role PH domains play in signal transduction make them promising targets for small molecule inhibitors. PHT-427 was designed to bind PH domains, and experiments show that PHT-427 binds with the highest affinity to the PH domain of PDKP1.

We wanted to test the antitumor activity of PHT-427 in human melanoma PDX models of varying genetic background. About 50% of melanoma tumors are driven by the BRAF(V600E) mutation, and these tumors are effectively targeted with BRAF inhibitors, such as vemurafenib. Unfortunately, these tumors usually develop resistance to vemurafenib, and the disease progresses. Additionally, 40-50% of melanoma tumors do not carry the BRAF(V600E) mutation, indicating the need to target additional pathways in melanoma. We sequenced human PDX samples on the Ion Proton sequencer, using the 409 gene Comprehensive Cancer Panel from Life Technologies. Compared to xenografts, PDXs are closer to the tumor biology of the patient, having a higher degree of molecular subtypes and intratumor heterogeneity, and a mixture of human and mouse stroma. The TMAP alignment program that works with Ion Torrent sequence data cannot handle a combined human-mouse reference genome, we developed a set of scripts we call Graft Extraction Out of REcipient Genome (GEORGE). Sequence reads aligned to the hg19 human reference, and mm10 mouse reference are evaluated by GEORGE and assigned to either the human, or mouse genome. In this project, only human assigned reads were used for variant calling. We sequence 14 melanoma samples, and five samples carried BRAF(V600E). We tested the antitumor activity of PHT-427 in four PDX samples with various BRAF and NRAS mutational statuses. We found that PHT-427 had antitumor activity in a vemurafenib resistant BRAF(V600E)::NRAS(G12V) mutant PDX sample.

Citation Format: Brian P. James, Robert Lemos, Jr., Stacey Bagby, Steven Robinson, William Robinson, John Tentler, S. Gail Eckhardt, D. Lynn Kirkpatrick, Garth Powis. A PDK1 inhibitors that has antitumor activity in a vemurafenib resistant BRAF(V600E)::NRAS(G12V) melanoma PDX. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-29.