Leveraging Systematic Functional Analysis to Benchmark an In Silico Framework Distinguishes Driver from Passenger MEK Mutants in Cancer.

Despite significant advances in cancer precision medicine, a significant hurdle to its broader adoption remains the multitude of variants of unknown significance identified by clinical tumor sequencing and the lack of biologically validated methods to distinguish between functional and benign variants. Here we used functional data on MAP2K1 and MAP2K2 mutations generated in real-time within a co-clinical trial framework to benchmark the predictive value of a three-part in silico methodology. Our computational approach to variant classification incorporated hotspot analysis, three-dimensional molecular dynamics simulation, and sequence paralogy. In silico prediction accurately distinguished functional from benign MAP2K1 and MAP2K2 mutants, yet drug sensitivity varied widely among activating mutant alleles. These results suggest that multifaceted in silico modeling can inform patient accrual to MEK/ERK inhibitor clinical trials, but computational methods need to be paired with laboratory- and clinic-based efforts designed to unravel variabilities in drug response. SIGNIFICANCE: Leveraging prospective functional characterization of MEK1/2 mutants, it was found that hotspot analysis, molecular dynamics simulation, and sequence paralogy are complementary tools that can robustly prioritize variants for biologic, therapeutic, and clinical validation.See related commentary by Whitehead and Sebolt-Leopold, p. 4042.

Combination of alphavirus replicon particle-based vaccination with immunomodulatory antibodies: therapeutic activity in the B16 melanoma mouse model and immune correlates.

Induction of potent immune responses to self-antigens remains a major challenge in tumor immunology. We have shown that a vaccine based on alphavirus replicon particles (VRP) activates strong cellular and humoral immunity to tyrosinase-related protein-2 (TRP2) melanoma antigen, providing prophylactic and therapeutic effects in stringent mouse models. Here, we report that the immunogenicity and efficacy of this vaccine is increased in combination with either antagonist anti-CTL antigen-4 (CTLA-4) or agonist anti-glucocorticoid-induced TNF family-related gene (GITR) immunomodulatory monoclonal antibodies (mAb). In the challenging therapeutic setting, VRP-TRP2 plus anti-GITR or anti-CTLA-4 mAb induced complete tumor regression in 90% and 50% of mice, respectively. These mAbs had similar adjuvant effects in priming an adaptive immune response against the vaccine-encoded antigen, augmenting, respectively, approximately 4- and 2-fold the TRP2-specific CD8(+) T-cell response and circulating Abs, compared with the vaccine alone. Furthermore, while both mAbs increased the frequency of tumor-infiltrating CD8(+) T cells, anti-CTLA-4 mAb also increased the quantity of intratumor CD4(+)Foxp3(-) T cells expressing the negative costimulatory molecule programmed death-1 (PD-1). Concurrent GITR expression on these cells suggests that they might be controlled by anti-GITR mAbs, thus potentially explaining their differential accumulation under the two treatment conditions. These findings indicate that combining immunomodulatory mAbs with alphavirus-based anticancer vaccines can provide therapeutic antitumor immune responses in a stringent mouse model, suggesting potential utility in clinical trials. They also indicate that tumor-infiltrating CD4(+)Foxp3(-)PD-1(+) T cells may affect the outcome of immunomodulatory treatments.

Loss of NF1 in cutaneous melanoma is associated with RAS activation and MEK dependence.

Melanoma is a disease characterized by lesions that activate ERK. Although 70% of cutaneous melanomas harbor activating mutations in the BRAF and NRAS genes, the alterations that drive tumor progression in the remaining 30% are largely undefined. Vemurafenib, a selective inhibitor of RAF kinases, has clinical utility restricted to BRAF-mutant tumors. MEK inhibitors, which have shown clinical activity in NRAS-mutant melanoma, may be effective in other ERK pathway-dependent settings. Here, we investigated a panel of melanoma cell lines wild type for BRAF and NRAS to determine the genetic alteration driving their transformation and their dependence on ERK signaling in order to elucidate a candidate set for MEK inhibitor treatment. A cohort of the BRAF/RAS wild type cell lines with high levels of RAS-GTP had loss of NF1, a RAS GTPase activating protein. In these cell lines, the MEK inhibitor PD0325901 inhibited ERK phosphorylation, but also relieved feedback inhibition of RAS, resulting in induction of pMEK and a rapid rebound in ERK signaling. In contrast, the MEK inhibitor trametinib impaired the adaptive response of cells to ERK inhibition, leading to sustained suppression of ERK signaling and significant antitumor effects. Notably, alterations in NF1 frequently co-occurred with RAS and BRAF alterations in melanoma. In the setting of BRAF(V600E), NF1 loss abrogated negative feedback on RAS activation, resulting in elevated activation of RAS-GTP and resistance to RAF, but not MEK, inhibitors. We conclude that loss of NF1 is common in cutaneous melanoma and is associated with RAS activation, MEK-dependence, and resistance to RAF inhibition.

Antibody and CD8+ T cell responses against HER2/neu required for tumor eradication after DNA immunization with a Flt-3 ligand fusion vaccine.

PURPOSE: HER2/neu is frequently overexpressed in breast cancer. In a mouse model, vaccination with HER2/neu DNA elicits antibodies that confer partial protection against tumor challenge. EXPERIMENTAL DESIGN: To enhance antitumor immunity, we fused cDNA encoding Flt-3 ligand (FL) to the rat HER2/neu extracellular domain (neu), generating a chimeric FLneu molecule. FLneu and neu DNA vaccines were compared for immunogenicity and their ability to protect mice from tumor challenge. RESULTS: The neu vaccine generated a HER2/neu-specific antibody response. In contrast, vaccination with FLneu induced CD8+ T cells specific for HER2/neu but a negligible anti-HER2/neu antibody response. The switch from an antibody-mediated to T cell-mediated response was due to different intracellular localization of neu and FLneu. Although the neu protein was secreted, the FLneu protein was retained inside the cell, co-localizing with the endoplasmic reticulum, facilitating processing and presentation to T cells. The neu and FLneu vaccines individually conferred only weak tumor immunity. However, efficient tumor rejection was seen when neu and FLneu were combined, inducing both strong anti-HER2/neu-specific antibody and T cell responses. Adoptive transfer of both immune CD8+ T cells and immune sera from immunized mice was required to confer tumor immunity in naïve hosts. CONCLUSIONS: These results show that active induction of both humoral and cellular immunity to HER2/neu is required for efficient tumor protection, and that neither response alone is sufficient.

Role for dipeptidyl peptidase IV in tumor suppression of human non small cell lung carcinoma cells.

Lung cancer is the leading cause of cancer death. Lung cancers produce a variety of mitogenic growth factors that stimulate tumor cell proliferation and migration. The cell surface protease, dipeptidyl peptidase IV (DPPIV), is involved in diverse biologic functions, including peptide-mediated cellular growth and differentiation. DPPIV is expressed in various normal tissues, including lung tissue, and its expression is lost in many types of human cancers. DPPIV expression and its enzymatic activity are detected in normal bronchial and alveolar epithelium but different histologic subtypes of lung carcinomas lose DPPIV expression. To investigate the role of DPPIV in lung carcinoma, we examined the expression of DPPIV at both mRNA and protein levels in non small cell lung cancer (NSCLC) cell lines and normal human bronchial epithelial cells. DPPIV expression was detectable in normal lung epithelial cells, but was absent or markedly reduced in all NSCLC cell lines at both mRNA and protein levels. Restoration of DPPIV expression in NSCLC cells resulted in profound morphologic changes, inhibition of cell proliferation, anchorage-independent growth, in vitro cell migration and tumorigenicity in nude mice. DPPIV reexpression also correlated with increased p21 expression, leading to induction of apoptosis and cell cycle arrest in G1 stage. These effects were accompanied by increased expression of cell surface proteins, fibroblast-activating protein (Fapalpha) and CD44 that are associated with suppression of tumor growth and metastasis. Thus, DPPIV functions as a tumor suppressor, and its downregulation may contribute to the loss of growth control in NSCLC cells.