Analysis of adaptive signaling in response to targeted therapies can provide insight into mechanisms of resistance and appropriate combinations of therapeutic agents. In a screen for adaptive resistance mechanisms in BRAF mutant melanomas, we made a serendipitous discovery that may provide a path to targeted therapy for melanomas driven by mutant NRAS, a disease for which there are few options. We found that a combination of a RAF inhibitor (PLX4720) and a HER-family inhibitor (lapatinib) could kill NRAS mutant melanomas that were resistant to either drug alone. The mechanism of killing did not require inhibition of the MAP Kinase or the PI3 Kinase pathways. The data strongly suggest that either RAF or a HER family receptor (or both) are engaged in non-canonical signaling activity that provides a previously undetected cytotoxic vulnerability. This proposal aims to identify the molecular basis for that non-canonical signaling, and determine the most appropriate setting for in vivo studies and a clinical trial.
Aim 1. We will analyze the signaling network that links BRAF, NRAS and HER family members to identify signaling nodes critical for the PLX4720-lapatinib synergy. We will utilize proteomic techniques (Reverse Phase Protein Micro Arrays; RPPA) and gene expression arrays to determine the adaptive responses to drug treatments made in the phosphoproteome and the transcriptome. We also will analyze the mode(s) of cytotoxicity induced by combined therapy (apoptosis, cell cycle arrest, autophagy). These responses will be analyzed using publicly available computational tools as well as integrated pathway analysis tools developed by our collaborators at the University of Virginia. We will explicitly examine our -omic analyses for hypothesized mechanisms including functional interactions of CRAF or BRAF with mitochondria, Protein Kinase A and the spindle assembly checkpoint; and of HER family kinases with the HIPPO pathway, RAC, PAK, Reactive Oxygen, Calcium, and ERK5.
Aim 2. We will test the functional significance of signaling nodes identified in Aim 1. We will perform epistasis experiments, complementing cytotoxicity with RNAi and rescuing inhibited growth with ORFeome expression. We also will determine whether appropriate small molecules can phenocopy the cytotoxic effects of the PLX4720-lapatinib combination. Adaptive homeostatic responses are a major confounder for targeted therapies, and the information obtained here will provide a strong foundation for therapeutic application with FDA-approved drugs and validated targets.
The promise of targeted therapy is that discovery of the 'drivers' of malignancy will enable the development of specific drugs that would be effective and non-toxic. That promise has not been fulfilled in the case of advanced cancers that have developed multiple adaptive mechanisms to escape the toxic effects of targeted drugs. Melanoma presents an excellent example of this dilemma. 50% of cutaneous melanomas are driven by mutated BRAF that can be inhibited by the drug vemurafenib. Most BRAF melanomas respond to vemurafenib treatment, but then come roaring back in a few weeks or months, with lethal consequences. We have been examining the molecular mechanisms of adaptive responses in melanomas because this allows the cancer cell to display its defense mechanisms against therapy. This research has uncovered an unexpected cytotoxic vulnerability in melanomas driven by mutated NRAS. It presents an opportunity for development as a target for melanoma therapy with FDA-approved drugs. 20% of melanomas are driven by NRAS, and there are no good therapeutic options for this group of cancers. We plan to identify this vulnerability at the molecular level, and determine the best path for moving this new knowledge into the clinical setting. The work has the potential for developing treatment for other RAS-driven cancers, which represents 30% of all cancers.
|Roller, Devin G; Capaldo, Brian; Bekiranov, Stefan et al. (2016) Combinatorial drug screening and molecular profiling reveal diverse mechanisms of intrinsic and adaptive resistance to BRAF inhibition in V600E BRAF mutant melanomas. Oncotarget 7:2734-53|
|Axelrod, Mark J; Gordon, Vicki; Mendez, Rolando E et al. (2014) p70S6 kinase is a critical node that integrates HER-family and PI3 kinase signaling networks. Cell Signal 26:1627-35|