Gabriella Nicolini

Clonal expansion of T lymphocytes in human melanoma metastases after treatment with a hapten-modified autologous tumor vaccine.

Metastatic melanoma patients treated with an autologous DNP-modified tumor cell vaccine develop inflammatory responses in metastatic tumors characterized by infiltration of CD8+ T cells. To further define this immune response, we analyzed T cell receptor beta-chain variable (TCRBV) region repertoire in biopsy specimens and peripheral blood lymphocytes of six patients. After administration of DNP vaccine, a restricted set of TCRBV gene families was found to be expanded compared with prevaccine metastases. In several postvaccine lesions of one patient, obtained over a 2-yr period, TCRBV14+ T cells were clonally expanded and identical T cell clonotypes could be detected. Two major recurring clones were biased toward the use of TCRBJ1S5. Furthermore, T cell lines derived from two such infiltrated skin lesions and, enriched in TCRBV14+ T cells, displayed HLA-class I-restricted lysis of the autologous melanoma cells. Clonal expansion of T cells was demonstrated in the T cell-infiltrated, postvaccine metastasis of a second patient as well. These results indicate that vaccination with autologous, DNP-modified melanoma cells can expand selected clones of T cells at the tumor site and that such clones are potentially destructive to the tumor.

Human Cutaneous Melanomas Lacking MITF and Melanocyte Differentiation Antigens Express a Functional Axl Receptor Kinase

Axl, a member of the TAM (Tyro3, Axl, Mer) family of receptor tyrosine kinases, displays an increasingly important role in carcinogenesis. Analysis of 58 cutaneous melanoma lines indicated that Axl was expressed in 38% of them, with significant overrepresentation in NRAS- compared with BRAF-mutated tumors. Axl activation could be induced by autocrine production of its ligand, Gas6, in a significant fraction of Axl-positive tumors. Pearsons correlation analysis on expression data from five data sets of melanoma lines identified several transcripts correlating positively or negatively with Axl. By functionally grouping genes, those inversely correlated were involved in melanocyte development and pigmentation, whereas those positively correlated were involved in motility, invasion, and microenvironment interactions. Accordingly, Axl-positive melanomas did not express microphthalmia transcription factor (MITF) and melanocyte differentiation antigens (MDAs) such as MART-1 and gp100 and possessed a greater in vitro invasive potential compared with Axl-negative ones. Motility, invasivity, and ability to heal a wound or to migrate across an endothelial barrier were inhibited in vitro by Axl knockdown. Pharmacological inhibition of Axl using the selective inhibitor R428 had comparable effects in reducing migration and invasion. These results suggest that targeted inhibition of Axl signaling in the subset of melanomas lacking MITF and MDAs may represent a novel therapeutic strategy.

Peptides with dual binding specificity for HLA-A2 and HLA-E are encoded by alternatively spliced isoforms of the antioxidant enzyme peroxiredoxin 5

Peptides with dual binding specificity for classical HLA class I and non-classical HLA-E molecules have been identified in virus-encoded proteins, but not in cellular proteins from normal or neoplastic cells. Expression screening of a melanoma cDNA library with a CTL clone recognizing an HLA-A2-restricted tumor-specific epitope encoded by mutant peroxiredoxin 5 (Prdx5), a stress-inducible peroxidase, led to the identification of two alternatively spliced isoforms of the same gene. These isoforms, which lack the catalytic cysteine fundamental for enzymatic activity, showed widespread expression in neoplastic and normal tissues but were unstable at the protein level, being detectable, following transient transfection, only after lactacystin treatment to inhibit proteasomal degradation. Isoform-specific sequences which formed, respectively, as result of exon 1 splicing to either exon 3 or 4, encoded two distinct nonapeptides (AMAPIKTHL and AMAPIKVRL, not present in the full-length protein) with anchor residues for HLA-A2 and HLA-E molecules and able to stabilize HLA-A2 and HLA-E cell surface expression. HLA-E+ targets, loaded with these peptides, were not recognized by NK cells expressing CD94/NKG2A inhibitory or CD94/NKG2C activatory receptors. However, both peptides were recognized, although with low avidity, by HLA-E-restricted CD8+ CTL. The nonapeptide AMAPIKVRL was used to elicit HLA-A2-restricted CTL clones that killed peptide-pulsed lymphoblastoid cell lines and melanoma cells expressing the corresponding Prdx5 isoform. Our results suggest that alternatively spliced isoforms of Prdx5, through the generation of HLA-E- and HLA-A2-restricted peptides may be part of immune-mediated stress response contributing to the detection and elimination of damaged normal or neoplastic cells.

Comparative assessment of TCRBV diversity in T lymphocytes present in blood, metastatic lesions, and DTH sites of two melanoma patients vaccinated with an IL-7 gene-modified autologous tumor cell vaccine

A phase I clinical trial using autologous, IL-7 gene-modified tumor cells in patients with disseminated melanoma has been recently completed. Although no major clinical responses were observed, increased antitumor cytotoxicity was measured in postvaccine peripheral blood lymphocytes in a subset of treated patients. To analyze the in situ immune response, the T cell receptor β-chain variable region (BV) repertoire of T cells infiltrating postvaccine lesions was studied in two patients, and compared with that of T cells present in prevaccine ones, in peripheral blood lymphocytes, and, in one patient, in delayed type hypersensitivity (DTH) sites of autologous melanoma inoculum. A relative expansion of T cells expressing few BVs was observed in all postvaccine metastases, and their intratumoral presence was confirmed by immunohistochemistry. Length pattern analysis of the complementarity determining region 3 (CDR3) indicated that the repertoire of T cells expressing some of these BVs was heterogeneous. At difference, CDR3, β-chain joining region usage, and sequence analysis enabled us to demonstrate, within a T-cell subpopulation commonly expanded at DTH sites and at the postvaccine lesion of patient 1, that both DTH sites contained identical dominant T-cell clonotypes. One of them was also expressed at increased relative frequency in the postvaccine lesion compared to prevaccine specimens. These results provide evidence for immunological changes, including in situ clonally expanded T cells, in metastases of patients vaccinated with IL-7 gene-transduced cells.

Primary cross-resistance to BRAFV600E-, MEK1/2- and PI3K/mTOR-specific inhibitors in BRAF-mutant melanoma cells counteracted by dual pathway blockade

Intrinsic cross-resistance to inhibition of different signaling pathways may hamper development of combinatorial treatments in melanoma, but the relative frequency of this phenotype and the strategies to overcome this hurdle remain poorly understood. Among 49 BRAF-mutant melanoma cell lines from patients not previously treated with target therapy, 21 (42.9%) showed strong primary resistance (IC50 > 1 μM) to a BRAFV600E inhibitor. Most of the BRAF-inhibitor-resistant cell lines showed also strong or intermediate cross-resistance to MEK1/2- and to PI3K/mTOR-specific inhibitors. Primary cross-resistance was confirmed in an independent set of 23 BRAF-mutant short-term melanoma cell cultures. MEK1/2 and PI3K/mTOR co-targeting was the most effective approach, compared to BRAF and PI3K/mTOR dual blockade, to counteract primary resistance to BRAF inhibition and the cross-resistant phenotype. This was shown by extensive drug interaction analysis, tumor growth inhibition assays in-vivo, p-ERK and p-AKT inhibition, promotion of melanoma apoptosis, apoptosis-related protein modulation, activation of effector caspases and selective modulation of genes involved in melanoma drug resistance and belonging to the ERK/MAPK and PI3K/AKT canonical pathways. Compared to co-targeting of mutant BRAF and PI3K/mTOR, the association of a MEK1/2 and a PI3K/mTOR inhibitor was more effective in the activation of Bax and of caspase-3 and in the induction of caspase-dependent melanoma apoptosis. Furthermore Bax silencing reduced the latter effects. These results suggest that intrinsic resistance to BRAF inhibition is frequently associated with primary cross-resistance to MEK and PI3K/mTOR blockade in BRAF-mutant melanoma and provide pre-clinical evidence for a combinatorial approach to counteract this phenotype.

Abstract 226: The focal adhesion kinase is phosphorylated on serine732 by a Growth factor-dependent pathway and contributes to the proliferation of tumor cells

Focal adhesion kinase (FAK) is a tyrosine kinase localized at the site of focal adhesions (FA). FAK is identified as a key mediator of signaling by integrins, the major family of cell surface receptors for extracellular matrix, as well as by other receptors in both normal and cancer cells. FAK plays a prominent role in tumor progression and metastasis regulating cellular processes including migration, invasion, epithelial to mesenchymal transition, and angiogenesis. Integrin-FAK signaling has been shown to activate a number of biological mechanisms through phosphorylation and protein-protein interactions promoting tumorigenesis. FAK seems also to have a role in the proliferation of tumor cells by its biochemical and biological association to cytoplasmic kinases such as src and ERK. However, the mechanisms that involve FAK in those processes are not yet clarify. The best characterized FAK phosporylation event is the auto-phosphorylation at the tyrosine397 (Tyr397) which creates a motif that is recognized by various SH2 domain-containing proteins, such as src which phosphorylates FAK on Tyr576. The c-terminal domain of FAK undergoes to several serine-phosphorylation events whose role is not well-understood. Previous studies in neural and endothelial cells (EC) have shown a new role for FAK in the regulation of centrosome functions during mitosis dependent on Ser732 phosphorylation. We have found that EGF stimulation of selected starved melanoma, thyroid and high stage ovarian tumor cells induced, in dividing cells, accumulation of phosphorylated (P-)FAK on Ser732. Inhibition of cell/substrate adhesion with specific anti-integrin antibodies demonstrated that P-FAKSer732 activation is not induced by integrin clustering. Confocal immunofluorescence and immunoprecipitation showed that, in these cells, P-FAKSer732 is localized in a perinuclear site independently from FA and P-FAKTyr397. Treatment of an high proliferative melanoma cell line with the Rock inhibitor Y27632 partially decreased the levels of P-FAKSer732, the cellular proliferation and impaired the mitotic spindle formation. FACS analysis showed that the P-FAKSer732 decreased upon treatment with the MEK inhibitor UO126 in a dose-dependent manner together with the levels of P(Ser 10) -Histone H3 thus demonstrating that FAK is involved in the mitotic process regulated by MEK/ERK activation. Indeed, P-FAKSer732 co-localized with microtubules of the mitotic spindle in proliferating cells and immunoprecipitated with the motor protein dynein and with the acetylated tubulin of polymerized microtubules. The finding of a novel role for FAK in tumor cells might provide the underpinning for therapeutic strategies in selected solid tumors. Partially supported by Italian Ministery of Health and Associazione Italiana per la Ricerca sul Cancro (AIRC). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 226. doi:1538-7445.AM2012-226