Abstract Inhibition of the RAS/RAF/MEK/ERK signaling pathway has emerged as a promising therapeutic strategy for melanoma given the high frequency of BRAF and NRAS mutations in this tumor type. Sensitivity to E6201, an inhibitor of MEK1 and other cancer relevant kinases, was assessed in a panel of 32 cell lines for which the mutation status of common melanoma genes was known. The majority (24/32) of the melanoma cell lines were sensitive to E6201, resulting in G1-cell cycle arrest and cell death in 11 sensitive cell lines and cell cycle arrest but not cell death in two sensitive lines. Xenograft studies revealed that E6201 exhibited a cytotoxic effect in vivo, even in cell lines where only a cytostatic effect was observed in vitro. Among cell lines carrying mutations in BRAF, or NRAS, sensitivity to E6201 was associated with wildtype PTEN status (p<0.03). When the cell lines were classified based on basal pERK levels rather than BRAF mutation status, high levels of pAKT correlated with insensitivity to E6201 (p<0.04). Indeed, high levels of pAKT correlated with E6201 insensitivity independent of BRAF or PTEN mutation status (p<0.03). Together these data demonstrate the cytostatic and cytotoxic activity of E6201 in BRAF mutant melanoma cell lines in vitro and in vivo and, furthermore, indicate that quantification of pAKT levels may have clinical utility as a biomarker of E6201 resistance. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B93.

C86 Endometrial carcinoma is the most common gynecological malignancy in the United States. Although most women present with early disease confined to the uterus, the majority of persistent or recurrent tumors are refractory to current chemotherapies. We identified activating mutations in the Fibroblast growth factor receptor 2 (FGFR2) in 10% (19/187) of endometrial cancers and showed these mutations primarily occurred in tumors of the endometrioid histologic subtype (18/115 cases investigated, 16%). Although endometrial cancers presenting with an endometrioid histology are generally considered 9good actors9, 25% of all endometrial cancer deaths can be attributed to this histotype. FGFRs have been shown to signal through a variety of downstream signaling pathways including MAPK, PI3K/AKT, Src, PLCg and STATs. In tumors with an endometrioid histology, KRAS activation and PTEN inactivation occur in 15-25% and 55-80% of tumors, respectively. For this reason, we sought to determine if FGFR2 activation occurs simultaneously in tumors with downstream activation of the MAPK pathway via KRAS gain-of-function mutations, or downstream activation of the PI3K/AKT pathway due to loss-of-function PTEN mutations. We therefore sequenced exon one of KRAS and all exons of PTEN in the panel of endometrioid tumors, generating data on 109 samples. We identified KRAS mutations in 12% (13/109) tumors and no tumor was identified with activating mutations in both KRAS and FGFR2. Further analysis revealed PTEN mutations in 69% (75/109) of tumors. Of those tumors with FGFR2 mutations, 12/17 also carried a PTEN mutation, suggesting that FGFR2 drives tumorigenesis primarily through the MAPK rather than the PI3K/AKT pathway. Given the known role of the PI3K/AKT pathway in promoting cell survival it was important to determine if inhibiting FGFR2 in the presence of constitutive downstream AKT signaling could result in cell death. To this end we knocked down FGFR2 via lentiviral transduction with two independent shRNAs and demonstrated a reduction in proliferation and an induction of apoptosis in the AN3CA and MFE296 endometrial cancer cell lines, both of which carry the activating N550K FGFR2 mutation. Consistent with the genetic data, Western blot analysis revealed that this induction of apoptosis following knockdown of FGFR2 correlated with inhibition of phospho-ERK and occurred in the presence of constitutively phosphorylated AKT. To verify that inhibition of FGFR2 with a pan-FGFR inhibitor was a viable therapeutic option in this tumor type, four endometrial cancer cell lines (2 with mutant FGFR2 and 2 with wildtype FGFR2) were treated with the pan-FGFR inhibitor, PD173074 (Calbiochem). The two cell lines with mutant FGFR2 (AN3CA and MFE296) were 10-40x more sensitive to inhibition with PD173074. Notably the most sensitive line (IC50=62nM) has loss-of-function mutations on both PTEN alleles. Western blot data confirmed this cell death occurred in the presence of constitutive AKT signaling in both cell lines. These data suggest that endometrial cancer cells with activated FGFR2 demonstrate oncogene addiction. Moreover, we demonstrate that induction of apoptosis following FGFR2 inhibition is due to inhibition of MAPK, and not the simultaneous inhibition of both MAPK and PI3K/AKT signaling as has previously reported for other receptor tyrosine kinases.

The Tat protein has been recently explored as a prospective vaccine candidate against HIV-1 with broad, subtype non-specific action. A truncated version of Tat()Tat) with the basic loop, involved in immunosuppression, removed has been previously demonstrated as efficacious as the full-size Tat protein. We produced both full-size Tat and truncated _Tat in plants, including one edible species – spinach, thus simultaneously addressing problems of an inexpensive Tat production and a direct delivery through the mucosal route. We tested this oral delivery route in a mouse model. Tat and )Tat genes were assembled from a set of synthetic overlapping oligonucleotides, and subsequently cloned into a plant virus-based expression vector. Codon optimization allows production of up to 300–500 mg of Tat or )Tat antigen per 1 g of leaf tissue in spinach. Spinach plants inoculated with the Tat-producing constructs were collected and fed to mice 7–14 days post inoculation with or without mucosal adjuvants. Mice were fed with the Tat-producing or control vector-inoculated spinach. After 3 voluntary feedings, 1 week apart, 1 g per mice, no differences were detected in the growth rate or behavior of the animals fed with these two types of spinach. None of the animals developed measurable Tat antibodies. Challenge DNA vaccination with a homologous Tatexpressing construct was performed using a gene gun. Following DNA vaccination, however, mice previously receiving oral Tat with cholera toxin as an adjuvant, developed higher antibody titers to Tat than did the controls, with the titers peaking at four weeks post-vaccination. Thus, our data suggested that oral Tat primed for the development of Tat antibodies when mice were challengevaccinated with plasmid DNA for expression of Tat. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

The HIV-1 Tat protein has been recently explored as a prospective vaccine candidate with broad, subtype non-specific action. We approached the problem of delivery of Tat through the mucosal route by expressing Tat in an edible plant. The tat gene was assembled from synthetic overlapping oligonucleotides, and was subsequently cloned into a plant virus-based vector tobacco mosaic virus (TMV). Spinach plants inoculated with the Tat-producing constructs were collected and fed to mice 7–14 days post inoculation. DNA vaccinations were performed using a gene gun. Codon optimization of the Tat gene expressed in spinach plants resulted in several-fold yield increase as detected in immunoblots, and did not cause severe symptoms in inoculated plants. Mice were fed with the Tat-producing or control vector-inoculated spinach. After three feedings, 1 week apart, 1 g per mice, no differences were detected in the growth rate or behavior of the animals fed with these three types of spinach. None of the animals developed measurable Tat antibodies. Following DNA vaccination, however, mice having previously received oral Tat developed higher antibody titers to Tat than did the controls, with the titers peaking at 4 weeks post-vaccination. Codon optimization allows production of up to 300–500 μg of Tat antigen per 1 g of leaf tissue in spinach using a plant virus-based expression system. The plant produced Tat does not seem to have any apparent adverse effect on mice growth or behavior, when fed with spinach for 4 weeks. ELISA data suggested that oral Tat primed for the development of Tat antibodies when mice were subsequently vaccinated with plasmid DNA designed for Tat expression.