Therapeutic Agent Development
We are interested in identifying novel molecular targets vital for tumor growth and progression and validating these candidates in pre-clinical models in order to develop rational pharmacological intervention strategies for melanoma and NSCLC patients. To this end, we utilize emerging technologies (RCAS/TVA, Bimolecular Fluorescence Complimentation, inducible CRISPR and high throughput screening) in order to investigate these factors and to identify small molecules that interfere with their function.
An Increase in Melanoma
The incidence of melanoma has been increasing at an alarming rate over the past twenty years. Approximately 76,100 new cases are expected in 2014 with nearly 9,710 resulting in death. It is the most rapidly increasing malignancy among young people in the United States and is the most common form of cancer in young adults 25-29 years old; over half of the patients are younger than 60 years old. Melanoma accounts for the majority of skin cancer deaths, and prognosis is poor for advanced stages of this disease. Patients with metastatic melanoma have limited treatment options and median survival ranges from 6-10 months. Current FDA-approved drugs for advanced melanoma include: Chemotherapy (dacarbazine), Immune modulators (interleukin-2 and ipilimumab), BRAF/MAPK directed agents (vemurafenib, dabrafenib and trametinib), but none offer much hope in the way of providing a lifelong cure for most patients.
Nearly 50% of all melanomas harbor an activating BRAF alteration (typically V600E) that constitutively activates the MAPK signaling pathway. Significant effort has been devoted to developing agents that inhibit BRAF/MEK in this cohort and initial clinical responses have been impressive. Unfortunately most patients develop resistance to these agents and relapse within months. Investigation into the mechanism(s) of resistance has revealed numerous ways to activate alternative survival signaling pathways and/or reactivate MAPK signaling despite continuous dosing. Given the diverse means of overcoming BRAF/MEK inhibition makes it difficult to develop dual targeting strategies. Therefore, additional molecular targets need to be identified which are germane to BRAF driven melanomas as well as NRAS and c-KIT associated melanomas.
To this end we have developed a novel high throughput melanoma mouse model capable of addressing tumor initiation, maintenance, progression and drug resistance based on the RCAS/TVA system. We are currently expanding this versatile model to incorporate a TET-inducible CRISPR (iCRISPR) component capable of deleting any endogenous gene specifically in the tumor at any given time post melanoma formation in vivo. This allows for the identification and validation of vital factors associated with melanoma survival with the ultimate goal of developing rational therapeutic approaches that provide durable responses in all subsets of melanomas.
Non-Small Cell Lung Carcinoma (NSCLC)
Lung cancer remains a significant health burden in the US and worldwide. It is responsible for more deaths than breast, colon, and prostate cancer combined. Lung cancer accounts for nearly 30% of all cancer deaths in the US and remains the leading cause of cancer deaths for both men and women. Presently, the five year overall survival rate for NSCLC remains at 15%, while small cell lung carcinoma survival rates are just six percent. It is clear that current therapies are inadequate and targeted agents may provide a more effective treatment option. RAS mutations (KRAS, HRAS or NRAS) are found in approximately one-third of all human malignancies and are highly prevalent in NSCLC. Notably, alterations in KRAS account for 90% of RAS mutations in lung adenocarcinomas. These mutations promote unregulated GTPase activity, leading to constitutive activation of RAS signaling. Alterations in KRAS promote resistance to receptor tyrosine kinase (RTK) directed inhibitors which target upstream components of RAS signaling. Therefore, KRAS or its downstream effectors are attractive candidates for molecular targeted therapeutic strategies. The primary downstream signaling effectors of RAS constitute the MAPK signaling pathway, the PI3K/AKT signaling cascade and the Ras-like Ral GTPases (RALA and RALB). Developing agents that specifically inhibit mutant RAS have proven elusive to date, therefore much attention has focused on targeting downstream MAPK and AKT signaling effectors. Despite the development and evaluation of numerous specific inhibitors of both signaling pathways none have demonstrated clinical efficacy in NSCLC.
We assessed RTK/KRAS signaling in a large panel of genetically diverse well characterized human NSCLC cell lines and identified that MAPK and/or PI3K signaling was not required for survival in the vast majority of these cells mirroring clinical observations. We did identify high level RALA and RALB signaling in all cell lines evaluated to date. RNAi directed at RALA and RALB induced significant cell death in most of the NSCLC cell lines evaluated regardless of EGFR/KRAS mutation status. Importantly, suppressing RALA/B or KRAS was effective in initiating apoptosis in most NSCLC cell lines. Developing pharmaceutical agents that directly inhibit small GTPases such as KRAS and RAL by traditional means has proven difficult. Therefore, we have developed an in-cell high throughput drug screening assay based on Bimolecular Fluorescence Complimentation (BiFC) in order to identify agents that can impair binding of active RAL or mutant KRAS to upstream activators and/or downstream effectors.
Numerous interactions between cell surface receptors on T-cells and the tumor local environment (TCR/MHC, cytokines and the B7 family members) culminates in tumor-directed immune activation or down regulation (anergy, exhaustion). Historically, ramping up the immune system (via dendritic cells and/or inflammatory cytokines) resulted in few long term responses with significant side effects in patients. Recently, strategies centered on blocking the interaction of B7 family ligands and their receptors on T-cells with blocking antibodies (alone or in combination) has resulted in impressive durable response rates and increased overall survival in melanoma and NSCLC patients.
B7 family immune-regulatory ligands are structurally related cell surface ligands that bind to receptors on T-cells providing co-stimulatory or co-inhibitory signals depending on the context. There are currently 7 known members of the family: B7.1 (CD80), B7.2 (CD86), ICOS-L, PD-L1, PD-L2, B7-H3, and B7-H4. B7 ligands are expressed in lymphoid and non-lymphoid tissues including many cancer types. We are currently investigating a role for each of these family members in melanoma tumor progression in an immune-competent mouse model and are developing novel therapeutic strategies based on blocking these ‘negative’ interactions.