IntroductionAbout 50% of cutaneous melanoma (CM) patients present activating BRAF mutations that can be effectively targeted by BRAF inhibitors (BRAFi). However, 20% of CM patients exhibit intrinsic drug resistance to BRAFi, while most of the others develop adaptive resistance over time. The mechanisms involved in BRAFi resistance are disparate and globally seem to rewire the cellular signaling profile by up-regulating different receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR). RTKs inhibitors have not clearly demonstrated anti-tumor activity in BRAFi resistant models. To overcome this issue, we wondered whether the shared up-regulated RTK phenotype associated with BRAFi resistance could be exploited by using immune weapons as the antibody-dependent cell cytotoxicity (ADCC)-mediated effect of anti-RTKs antibodies, and kill tumor cells independently from the mechanistic roots.Methods and resultsBy using an in vitro model of BRAFi resistance, we detected increased membrane expression of EGFR, both at mRNA and protein level in 4 out of 9 BRAFi-resistant (VR) CM cultures as compared to their parental sensitive cells. Increased EGFR phosphorylation and AKT activation were observed in the VR CM cultures. EGFR signaling appeared dispensable for maintaining resistance, since small molecule-, antibody- and CRISPR-targeting of EGFR did not restore sensitivity of VR cells to BRAFi. Importantly, immune-targeting of EGFR by the anti-EGFR antibody cetuximab efficiently and specifically killed EGFR-expressing VR CM cells, both in vitro and in humanized mouse models in vivo, triggering ADCC by healthy donors’ and patients’ peripheral blood cells. ConclusionOur data demonstrate the efficacy of immune targeting of RTKs expressed by CM relapsing on BRAFi, providing the proof-of-concept supporting the assessment of anti-RTK antibodies in combination therapies in this setting. This strategy might be expected to concomitantly trigger the crosstalk of adaptive immune response leading to a complementing T cell immune rejection of tumors.

Mitogen-activated protein kinase (MAPK) pathway activation is a central step in BRAF V600 -mutant cutaneous melanoma (CM) pathogenesis. In the last years, Spry1 has been frequently described as an upstream regulator of MAPK signaling pathway. However, its specific role in BRAF V600 -mutant CM is still poorly defined. Here, we report that Spry1 knockdown (Spry1 KO ) in three BRAF V600 -mutant CM cell lines markedly induced cell cycle arrest and apoptosis, repressed cell proliferation in vitro, and impaired tumor growth in vivo. Furthermore, our findings indicated that Spry1 KO reduced the expression of several markers of epithelial–mesenchymal transition, such as MMP-2 both in vitro and in vivo. These effects were associated with a sustained and deleterious phosphorylation of ERK1/2. In addition, p38 activation along with an increase in basal ROS levels were found in Spry1 KO clones compared to parental CM cell lines, suggesting that BRAF V600 -mutant CM may restrain the activity of Spry1 to avoid oncogenic stress and to enable tumor growth. Consistent with this hypothesis, treatment with the BRAF inhibitor (BRAFi) vemurafenib down-regulated Spry1 levels in parental CM cell lines, indicating that Spry1 expression is sustained by the MAPK/ERK signaling pathway in a positive feedback loop that safeguards cells from the potentially toxic effects of ERK1/2 hyperactivation. Disruption of this feedback loop rendered Spry1 KO cells more susceptible to apoptosis and markedly improved response to BRAFi both in vitro and in vivo, as a consequence of the detrimental effect of ERK1/2 hyperactivation observed upon Spry1 abrogation. Therefore, targeting Spry1 might offer a treatment strategy for BRAF V600 -mutant CM by inducing the toxic effects of ERK-mediated signaling.

Abstract Background: About 50% of cutaneous melanoma (CM) harbor the activating BRAFV600E mutation, which exerts most of the oncogenic effects through the activation of the MAPK signaling pathways. BRAF inhibitors (BRAFi) have showed important clinical activities in CM, but their effectiveness is impaired by the emergence of an early drug resistance. Accordingly, a better understanding of the molecular basis of BRAFi resistance may provide insights useful to develop new therapeutic strategies in CM. Spry gene family includes four members (Spry1-4), which differ in their tissue distribution, activity and interaction partners, thus suggesting nonredundant functions. Although interactions between Spry proteins and several MAPK pathway components have been reported, the specific role of Spry1 in CM has yet to be defined. Methods: Bioinformatic analysis on data available on TCGA was performed using the pipeline suggested on the TCGA portal. Spry1 expression levels were measured in BRAFV600 mutant CM cell lines using quantitative real-time PCR (qRT-PCR) and Western blotting. CRISPR based strategy was used to knock out Spry1 gene in BRAFV600 mutant CM cell lines established in our Institution. Transfected cells were sorted into single cells and subsequently expanded. Spry1 knockout (Spry1KO) clones were validated by Sanger sequencing and Western blotting, respectively. Cell viabilities before and after BRAFi treatment were evaluated by MTT and clonogenic assays. Apoptosis was assessed by Annexin V/PI staining. Modulation of MAPK signaling pathways and apoptosis were examined by qRT-PCR and Western blotting. P53 nuclear translocation was evaluated through multispectral imaging flow cytometry analysis. Results: Specific bioinformatic analysis, using TCGA, was performed to provide more information regarding pathways associated with MAPK signaling. Our analysis revealed that Spry proteins are differentially expressed in CM. In line with these findings, Spry1 was found high expressed in a panel of CM cell lines with BRAFV600 mutation both at mRNA and protein level. Preliminary data demonstrated that the expression and/or phosphorylation of key proteins involved in MAPK signaling were modulated in Spry1KO clones with respect to the parental cell line. Furthermore, Spry1 inactivation associated with: i) enhanced activation and nuclear translocation of the tumor suppressor p53, and ii) decreased mRNA and protein levels of several antiapoptotic proteins. Of note, the treatment of Spry1KO clones with BRAFi reduced CM cell survival in clonogenic assays, and induced apoptosis in a dose- and time-dependent manner. Further studies are ongoing in order to determine the molecular changes involved in Spry1KO clones in response to BRAFi treatment. Conclusions: Our results suggest that Spry1 gene may exert oncogenic functions in CM and its upregulation can potentially contribute to BRAFi resistance. Citation Format: Giorgio Giurato, Francesca Colizzi, Aurora Rizzo, Debora Martorelli, Barbara Montico, Katy Mastorci, Dania Benedetti, Alessandro Weisz, Riccardo Dolcetti, Sigalotti Luca, Elisabetta Fratta. Suppression of Spry1 sensitizes cutaneous melanoma to BRAF-targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1839.

Inclusion of new biomarkers to improve a personalized treatment approach for oropharyngeal squamous cell carcinoma (OPSCC) is urgently needed. Hypomethylation of the Long interspersed nucleotide element-1 (LINE-1) repetitive elements, a widely accepted surrogate of overall genomic DNA methylation content, was found to be associated with a poor prognosis in several cancers. At present, no studies have investigated the influence of LINE-1 methylation levels on OPSCC relapse. The main goal of this study was the evaluation of the prognostic value of LINE-1 methylation status in predicting early tumor relapse in locally advanced OPSCC.

As diverse therapeutic options are now available for advanced melanoma patients, predictive markers that may assist treatment decision are needed. A model based on baseline serum lactate dehydrogenase (LDH), peripheral blood relative lymphocyte counts (RLC) and eosinophil counts (REC) and pattern of distant metastasis, has been recently proposed for pembrolizumab-treated patients. Here, we applied this model to advanced melanoma patients receiving chemotherapy (n = 116) or antiCTLA-4 therapy (n = 128). Visceral involvement, LDH and RLC were associated with prognosis regardless of treatment. Instead, when compared to chemotherapy-treated patients with REC < 1.5%, those with REC ≥ 1.5% had improved overall survival when receiving anti-CTLA-4 [Hazard Ratio (HR) = 0.56 (0.4–0.93)] but not chemotherapy [HR = 1.13, (0.74– 1.74)], and the treatment-by-REC interaction was significant for both overall (p = 0.04) and progression free survival (p = 0.009). These results indicate baseline REC ≥ 1.5% as a candidate predictive biomarker for benefit from anti-CTLA-4. Further studies are needed to confirm these findings in patients receiving immune-modulating agents. MELANOMA ACC Annual Meeting 3-5 October 2017 ROME Abstract

Abstract Emergence of drug resistance is the major cause of failure of BRAF inhibitors (BRAFi) treatment in cutaneous melanoma (CM). Epigenetic modifications are known to physiologically trigger massive modifications in cellular commitment and several studies report a correlation between the drug-resistant phenotype and epigenetic alterations of tumor cells. In this setting, long noncoding RNAs (lncRNAs) represent a class of gene regulators acting at epigenetic, transcriptional and post-transcriptional level. Several studies have implicated lncRNAs in chemoresistance through their ability to impair cell cycle arrest and apoptosis, but also to induce and modulate epithelial-mesenchymal transition and cell adhesion-associated signaling pathways. LncRNAs interact with histone modifying complexes and/or DNA methyltransferases, being also targets of these epigenetic mediators. Furthermore, epigenetic drugs have been recently identified as modulators for lncRNAs function as well as their related targeting signals. Starting from these evidences, we asked the question whether epigenetic drugs could differentially affect the survival of BRAFi-resistant (VR) and -sensitive CM cells, investigating the mechanistic network involved, with a specific focus on the role of lncRNA. A panel of BRAFi-sensitive and VR CM cell lines was treated with the FDA-approved HDAC inhibitor vorinostat (SAHA). FACS analysis of annexin V-FITC/propidium iodide stained cells showed that SAHA cytotoxic activity was more pronounced on VR CM cells than on their parental counterparts. RNA-Seq analysis revealed that a large number of differentially expressed lncRNAs was modulated in VR CM cells treated with SAHA. Intriguingly, the expression of several VR up-regulated lncRNAs was decreased to levels similar to those observed in the matched parental cells. Functional analysis indicated these lncRNAs were statistically enriched in pathways involving cellular growth and proliferation, but also cellular assembly and organization. Though additional studies are required, epigenetic modulation of VR-associated lncRNAs promises to have significant therapeutic potential to restore BRAFi sensivity in CM, being concomitantly effective in killing VR cells as monotherapy. Based on our preliminary data, we could anticipate that the combined use of epigenetic and targeted drugs would increase therapeutic efficacy in CM patients relapsing to BRAFi. Citation Format: Barbara Montico, Giorgio Giurato, Katy Mastorci, Aurora Rizzo, Maria Ravo, Francesca Rizzo, Alessandro Weisz, Riccardo Dolcetti, Francesca Colizzi, Luca Sigalotti, Elisabetta Fratta. Epigenetic drugs modulate long noncoding RNAs expression in BRAF inhibitor-resistant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1196. doi:10.1158/1538-7445.AM2017-1196

Local irradiation of cancer through radiotherapy can induce spontaneous regression of non-directly irradiated lesions, suggesting the involvement of systemic antitumor immune responses. In oligometastatic breast cancer (BC) patients, the use of stereotactic body radiotherapy (SBRT) favors the local control of treated lesions and may contribute to break local tolerance and release tumor-associated antigens (TAAs), improving host antitumor immunity. We performed a detailed immunomonitoring of BC patients undergoing SBRT to verify its ability to "switch on" the anti-tumor immunity both systemically, in peripheral blood, and locally, employing in vitro BC models. Twenty-one BC patients with ≤6 metastases were treated with 3 daily doses of 10 Gy with SBRT. Blood samples for immune profiling were collected before and after treatment. One month after treatment a third of patients displayed the boosting or even the de novo appearance of polyfunctional CD4+ and CD8+ T cell responses against known BC TAAs (survivin, mammaglobin-A, HER2), through intracellular staining in flow cytometry. Half of patients showed increased numbers of activated natural killer (NK) cells, measured with multispectral flow cytometry, immediately after the first dose of SBRT. Interestingly, high levels of activated NK cells at diagnosis correlated with a longer progression-free survival. BC in vitro models, treated with the same SBRT modality, showed enhanced expression of MHC class-I and class-II, major histocompatibility complex class I-related chain A/B, and Fas molecules, and increased release of pro-inflammatory cytokines, such as IL-1β and TNF-α. Consistently, we noticed enhanced production of perforin by CD4+ T cells when patients' lymphocytes were cultured in the presence of irradiated BC cell line, compared to untreated targets. Besides immunogenic effects, SBRT also enhanced the percentages of circulating regulatory T cells, and increased indoleamine 2,3 dioxygenase and PD-L1 expression in BC in vitro models. These results suggest that SBRT may boost host antitumor immune responses also in an advanced disease setting such as oligometastatic BC, by inducing immunomodulating effects both locally and systemically. However, the concomitant induction of immunosuppressive pathways suggests that a combination with immunotherapy could further enhance the in situ vaccination ability of radiotherapy, possibly further improving the curative potential of SBRT in this subset of patients.

In recent years, recurrent somatic mutations in epigenetic regulators have been identified in patients with hematological malignancies. Furthermore, chromosomal translocations in which the fusion protein partners are themselves epigenetic regulators or where epigenetic regulators are recruited/targeted by oncogenic fusion proteins have also been described. Evidence has accumulated showing that "epigenetic drugs" are likely to provide clinical benefits in several hematological malignancies, granting their approval for the treatment of myelodysplastic syndromes and cutaneous T-cell lymphomas. A large number of pre-clinical and clinical trials evaluating epigenetic drugs alone or in combination therapies are ongoing. The aim of this review is to provide a comprehensive summary of known epigenetic alterations and of the current use of epigenetic drugs for the treatment of hematological malignancies.

Background: Epigenetic remodelling of cancer cells is an attractive therapeutic strategy and distinct DNA hypomethylating agents (DHA) are being actively evaluated in patients with hemopoietic or solid tumours. However, no studies have investigated the modulation of gene expression profiles (GEP) induced by DHA in transformed and benign tissues. Such information is mandatory to clarify the fine molecular mechanism(s) underlying the clinical efficacy of DHA, to identify appropriate therapeutic combinations, and to address safety issues related to their demethylating potential in normal tissues. Thus, utilising a syngeneic mouse model, we investigated the remodelling of GEP of neoplastic and normal tissues induced by systemic administration of DHA. Methods: The murine mammary carcinoma cells TS/A were injected s.c. into female BALB/c mice that were treated i.p. with four cycles of the DHA 5-aza-2′-deoxycytidine (5-AZA-CdR) at a fractioned daily dose of 0.75 mg kg −1 (q8 h × 3 days, every week). Whole mouse transcriptomes were analysed by microarrays in neoplastic and normal tissues from control and treated mice. Results were processed by bioinformatic analyses. Results: In all, 332 genes were significantly ( P ⩽0.05; FC⩾4) modulated (294 up and 38 downregulated) in neoplastic tissues from 5-AZA-CdR-treated mice compared with controls. In decreasing order of magnitude, changes in GEP significantly ( P ⩽0.05) affected immunologic, transport, signal transduction, spermatogenesis, and G–protein–coupled receptor protein signalling pathways. Epigenetic remodelling was essentially restricted to tumour tissues, leaving substantially unaltered normal ones. Conclusion: The ability of 5-AZA-CdR to selectively target tumour GEP and its major impact on immune-related genes, strongly support the clinical use of DHA alone or combined with immunotherapeutic agents.

Maffé A, Toschi B, Circo G, Giachino D, Giglio S, Rizzo A, Carloni A, Poletti V, Tomassetti S, Ginardi C, Ungari S, Genuardi M. Constitutional FLCN mutations in patients with suspected Birt–Hogg–Dubé syndrome ascertained for non‐cutaneous manifestations.

Cutaneous melanoma is a very aggressive neoplasia of melanocytic origin with constantly growing incidence and mortality rates world-wide. Epigenetic modifications (i.e., alterations of genomic DNA methylation patterns, of post-translational modifications of histones, and of microRNA profiles) have been recently identified as playing an important role in melanoma development and progression by affecting key cellular pathways such as cell cycle regulation, cell signalling, differentiation, DNA repair, apoptosis, invasion and immune recognition. In this scenario, pharmacologic inhibition of DNA methyltransferases and/or of histone deacetylases were demonstrated to efficiently restore the expression of aberrantly-silenced genes, thus re-establishing pathway functions. In light of the pleiotropic activities of epigenetic drugs, their use alone or in combination therapies is being strongly suggested, and a particular clinical benefit might be expected from their synergistic activities with chemo-, radio-, and immuno-therapeutic approaches in melanoma patients. On this path, an important improvement would possibly derive from the development of new generation epigenetic drugs characterized by much reduced systemic toxicities, higher bioavailability, and more specific epigenetic effects.