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High-grade mucinous colorectal cancer (HGM CRC) is particularly aggressive, prone to metastasis and treatment resistance, frequently accompanied by “signet ring” cancer cells. A sizeable fraction of HGM CRCs (20-40%) arises in the context of the Lynch Syndrome, an autosomal hereditary syndrome that predisposes to microsatellite instable (MSI) CRC. Development of patient-derived preclinical models for this challenging subtype of colorectal cancer represents an unmet need in oncology. We describe here successful propagation of preclinical models from a case of early-onset, MSI-positive metastatic colorectal cancer in a male Lynch syndrome patient, refractory to standard care (FOLFOX6, FOLFIRI-Panitumumab) and, surprisingly, also to immunotherapy. Surgical material from a debulking operation was implanted in NOD/SCID mice, successfully yielding one patient-derived xenograft (PDX). PDX explants were subsequently used to generate 2D and 3D cell cultures. Histologically, all models resembled the tumor of origin, displaying a high-grade mucinous phenotype with signet ring cells. For preclinical exploration of alternative treatments, in light of recent findings, we considered inhibition of the proteasome by bortezomib and of the related NEDD8 pathway by pevonedistat. Indeed, sensitivity to bortezomib was observed in mucinous adenocarcinoma of the lung, and we previously found that HGM CRC is preferentially sensitive to pevonedistat in models with low or absent expression of cadherin 17 (CDH17), a differentiation marker. We therefore performed IHC on the tumor and models, and observed no CDH17 expression, suggesting sensitivity to pevonedistat. Both bortezomib and pevonedistat showed strong activity on 2D cells at 72 hours and on 3D organoids at 7 days, thus providing valid options for in vivo testing. Accordingly, three PDX cohorts were treated for four weeks, respectively with vehicle, bortezomib and pevonedistat. Both drugs significantly reduced tumor growth, as compared to the vehicle group. Interestingly, while bortezomib was more effective in vitro, pevonedistat was more effective in vivo. Drug efficacy was further substantiated by a reduction of cellularity and of Ki67-positive cells in the treated tumors. These results highlight proteasome and NEDD8 inhibition as potentially effective therapeutic approaches against Lynch syndrome-associated HGM CRC, also when the disease is refractory to all available treatment options.

Purpose: Regorafenib (REG) is approved for the treatment of metastatic colorectal cancer, but has modest survival benefit and associated toxicities. Robust predictive/early response biomarkers to aid patient stratification are outstanding. We have exploited biological pathway analyses in a patient-derived xenograft (PDX) trial to study REG response mechanisms and elucidate putative biomarkers. Experimental Design: Molecularly subtyped PDXs were annotated for REG response. Subtyping was based on gene expression (CMS, consensus molecular subtype) and copy-number alteration (CNA). Baseline tumor vascularization, apoptosis, and proliferation signatures were studied to identify predictive biomarkers within subtypes. Phospho-proteomic analysis was used to identify novel classifiers. Supervised RNA sequencing analysis was performed on PDXs that progressed, or did not progress, following REG treatment. Results: Improved REG response was observed in CMS4, although intra-subtype response was variable. Tumor vascularity did not correlate with outcome. In CMS4 tumors, reduced proliferation and higher sensitivity to apoptosis at baseline correlated with response. Reverse phase protein array (RPPA) analysis revealed 4 phospho-proteomic clusters, one of which was enriched with non-progressor models. A classification decision tree trained on RPPA- and CMS-based assignments discriminated non-progressors from progressors with 92% overall accuracy (97% sensitivity, 67% specificity). Supervised RNA sequencing revealed that higher basal EPHA2 expression is associated with REG resistance. Conclusions: Subtype classification systems represent canonical “termini a quo” (starting points) to support REG biomarker identification, and provide a platform to identify resistance mechanisms and novel contexts of vulnerability. Incorporating functional characterization of biological systems may optimize the biomarker identification process for multitargeted kinase inhibitors.

Treatment with EGFR-targeted therapies, such as the EGFR monoclonal antibody cetuximab, has improved the outcome of patients with metastatic colorectal cancer (mCRC). However, EGFR inhibition is mainly cytostatic and does not lead to tumor eradication: subsets of drug-tolerant cancer cells often persist even after maximal response to therapy and foster residual disease, leading to relapse. Importantly, drug-tolerant cells are less prone to undergo apoptosis, and thus constitute a reservoir of treatment-refractory persisters that are difficult to extinguish. Here we aimed to investigate the mechanisms by which apoptosis escape mediates survival of cetuximab-tolerant mCRC cells as a means to find new therapeutic strategies for limiting residual disease and delaying disease recurrence. By performing an innovative functional assay named Dynamic BH3 Profiling in cetuximab-sensitive patient-derived organoids (PDOs), we found that antibody treatment increased the “apoptotic priming”, i.e. decreased the distance of cells from the apoptotic threshold without eliciting overt cell death. To start investigating the mechanisms of this increased apoptotic priming, protein levels of the major anti-apoptotic molecules (BCL2, BCL-XL, MCL1) and the pro-apoptotic molecule BIM were evaluated by Western blot in cetuximab-treated PDOs. BIM was consistently upregulated by cetuximab in all tested models. Increased BIM was sequestered by the anti-apoptotic BCL-XL, suggesting that EGFR blockade may render mCRC residual cells dependent on BCL-XL to elude cell death and, consequently, more susceptible to its inhibition. We therefore challenged a panel of 14 PDOs with different BH3 mimetics currently in preclinical or clinical development and found that only the combination of cetuximab with BH3 mimetics against BCL-XL strongly reduced cell viability and induced apoptosis. BCL-XL blockade enhanced cetuximab efficacy also in mCRC patient-derived xenografts, which displayed more durable tumor regression that was maintained also after treatment discontinuation. Notably, the combination of cetuximab with BCL-XL inhibitors was effective in vivo in all tested models except for one, which was characterized by low basal expression of BIM. Mechanistically, unleashed apoptosis by combination therapy was due to cetuximab-induced BIM upregulation concomitantly with its displacement from BCL-XL by the BH3 mimetic. Overall, this study offers new insights into the molecular mechanisms assisting residual disease after cetuximab treatment in mCRC, providing hints to design rational strategies that may convert the cytostatic effect of cetuximab into a cytotoxic, fully apoptotic outcome. Basal expression of BIM could be exploited as a predictive biomarker to enrich for potential responders to the combination of cetuximab and BH3 mimetics. Citation Format: Simonetta Maria Leto, Irene Catalano, Martina Ferri, Valentina Vurchio, Francesca Cottino, Eugenia Rosalinda Zanella, Francesco Sassi, Giorgia Migliardi, Andrea Bertotti, Livio Trusolino. Exploiting patient-derived xenografts and organoids to tackle apoptotic dependencies in EGFR-inhibited colorectal tumours [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3697.

Background Targeting the epidermal growth factor receptor (EGFR) either alone or in combination with chemotherapy is an effective treatment for patients with RAS wild-type metastatic colorectal cancer (mCRC). However, only a small percentage of mCRC patients receive clinical benefits from anti-EGFR therapies, due to the development of resistance mechanisms. In this regard, HER2 has emerged as an actionable target in the treatment of mCRC patients with resistance to anti-EGFR therapy. Methods We have used SW48 and LIM1215 human colon cancer cell lines, quadruple wild-type for KRAS, NRAS, BRAF and PI3KCA genes, and their HER2–amplified (LIM1215-HER2 and SW48-HER2) derived cells to perform in vitro and in vivo studies in order to identify novel therapeutic strategies in HER2 gene amplified human colorectal cancer. Results LIM1215-HER2 and SW48-HER2 cells showed over-expression and activation of the HER family receptors and concomitant intracellular downstream signaling including the pro-survival PI3KCA/AKT and the mitogenic RAS/RAF/MEK/MAPK pathways. HER2-amplified cells were treated with several agents including anti-EGFR antibodies (cetuximab, SYM004 and MM151); anti-HER2 (trastuzumab, pertuzumab and lapatinib) inhibitors; anti-HER3 (duligotuzumab) inhibitors; and MEK and PI3KCA inhibitors, such as refametinib and pictilisib, as single agents and in combination. Subsequently, different in vivo experiments have been performed. MEK plus PI3KCA inhibitors treatment determined the best antitumor activity. These results were validated in vivo in HER2-amplified patient derived tumor xenografts from three metastatic colorectal cancer patients. Conclusions These results suggest that combined therapy with MEK and PI3KCA inhibitors could represent a novel and effective treatment option for HER2-amplified colorectal cancer. Electronic supplementary material The online version of this article (10.1186/s13046-019-1230-z) contains supplementary material, which is available to authorized users.

The hepatocyte growth factor (HGF) also known as scatter factor activates cancer cell invasion and metastasis. We show that in ovarian cancer cells HGF induced the phosphorylation of the small heat shock protein of 27 kDa (HSP27) by activating the p38MAPK. HSP27 is increased in many cancers at advanced stage including ovarian cancer and associated with cancer resistance to therapy and poor patients' survival. The phosphorylation of HSP27 regulates both its chaperone activity and its control of cytoskeletal stability. We show that HSP27 was necessary for the remodeling of actin filaments induced by HGF and that motility in vitro depended on the p38MAPK-MK2 axis. In vivo, HSP27 silencing impaired the ability of the highly metastatic, HGF-secreting ovarian cancer cells to give rise to spontaneous metastases. This was due to defective motility across the vessel wall and reduced growth. Indeed, HSP27 silencing impaired the ability of circulating ovarian cancer cells to home to the lungs and to form experimental hematogenous metastases and the capability of cancer cells to grow as subcutaneous xenografts. Moreover, HSP27 suppression resulted in the sensitization of xenografts to low doses of the chemotherapeutic paclitaxel, likely because HSP27 protected microtubules from bundling caused by the drug. Altogether, these data show that the HSP27 is required for the proinvasive and prometastatic activity of HGF and suggest that HSP27 might be not only a marker of progression of ovarian cancer, but also a suitable target for therapy.

Background The NEDD8 conjugation pathway modulates the ubiquitination and activity of a wide range of intracellular proteins, and its blockade by pevonedistat is emerging as a promising therapeutic approach in various cancer settings. However, systematic characterization of pevonedistat efficacy in specific tumor types and definition of response predictors are still missing. Methods We investigated in vitro sensitivity to pevonedistat in 122 colorectal cancer (CRC) cell lines by an ATP-based proliferation assay and evaluated apoptosis and DNA content by flow cytometry. Associations between pevonedistat sensitivity and CRC molecular features were assessed by Student's t test. A 184-gene transcriptional predictor was generated in cell lines and applied to 87 metastatic CRC samples for which patient-derived xenografts (PDXs) were available. In vivo reponse to pevonedistat was assessed in PDX models (≥5 mice per group). All statistical tests were two-sided. Results Sixteen (13.1%) cell lines displayed a marked response to pevonedistat, featuring DNA re-replication, proliferative block, and increased apoptosis. Pevonedistat sensitivity did not statistically significantly correlate with microsatellite instability or mutations in KRAS or BRAF and was functionally associated with low EGFR pathway activity. While ineffective on predicted resistant PDXs, in vivo administration of pevonedistat statistically significantly impaired growth of five out of six predicted sensitive models (P < .01). In samples from CRC patients, transcriptional prediction of pevonedistat sensitivity was associated with poor prognosis after surgery (hazard ratio [HR] = 2.49, 95% confidence interval [CI] = 1.34 to 4.62, P = .003) and early progression under cetuximab treatment (HR = 3.59, 95% CI = 1.60 to 8.04, P < .001). Histological and immunohistochemical analyses revealed that the pevonedistat sensitivity signature captures transcriptional traits of poor differentiation and high-grade mucinous adenocarcinoma. Conclusions These results highlight NEDD8-pathway inhibition by pevonedistat as a potentially effective treatment for poorly differentiated, clinically aggressive CRC.

// Lucia D’Amico 1, 2 , Dimas Belisario 2 , Giorgia Migliardi 3 , Cristina Grange 4 , Benedetta Bussolati 5 , Patrizia D’Amelio 6 , Timothy Perera 7 , Ettore Dalmasso 8 , Luca Dalle Carbonare 9 , Laura Godio 10 , Paolo Comoglio 3 , Livio Trusolino 3 , Riccardo Ferracini 11 , Ilaria Roato 2 1 Department of Biomedicine, Cancer Immunology, University of Basel, Basel, Switzerland 2 CeRMS, A.O. Citta della Salute e della Scienza, Torino, Italy 3 IRCC, Institute for Cancer Research and Treatment, Candiolo, Torino, Italy 4 Department of Medical Sciences, University of Turin, Torino, Italy 5 Department of Molecular Biotechnology and Health Science, Molecular Biotechnology Center, University of Turin, Torino, Italy 6 Gerontology Section, Department of Medical Sciences, University of Torino, Torino, Italy 7 Janssen Research and Development, Beerse, Belgium 8 Urology Section, A.O. Citta della Salute e della Scienza, Torino, Italy 9 Clinic of Internal Medicine, Section D, Policlinico G.B. Rossi Verona, Verona, Italy 10 Department of Pathology, A.O. Citta della Salute e della Scienza, Torino, Italy 11 Department of Orthopaedic Oncology, CTO Hospital, Torino, Italy Correspondence to: Ilaria Roato, email: ilaria.roato@unito.it Keywords: bone metastasis, renal cancer, cancer stem cells, c-MET, CCL20 Received: May 01, 2016 Accepted: May 29, 2016 Published: June 14, 2016 ABSTRACT Cancer stem cells (CSCs) are key players in bone metastasis. In some renal tumors CSCs overexpress the HGF receptor c-MET, speculating that c-MET targeting could lead to bone metastasis inhibition. To address this hypothesis we isolated renal CD105+/CD24−CSCs, expressing c-MET receptor from a primary renal carcinoma. Then, to study their ability to metastasize to bone, we injected renal CSCs in NOD/SCID mice implanted with a human bone and we tested the effect of a c-MET inhibitor (JNJ-38877605) on bone metastasis development. JNJ-38877605 inhibited the formation of metastases at bone implant site. We showed that JNJ-38877605 inhibited the activation of osteoclasts induced by RCC stem cells and it stimulated osteoblast activity, finally resulting in a reduction of bone turnover consistent with the inhibition of bone metastases. We measured the circulating levels of osteotropic factors induced by RCC stem cells in the sera of mice treated with c-Met inhibitor, showing that IL-11 and CCL20 were reduced in mice treated with JNJ-38877605, strongly supporting the involvement of c-MET in the regulation of this process. To address the clinical relevance of c-MET upregulation during tumor progression, we analysed c-MET in renal cancer patients detecting an increased expression in the bone metastatic lesions by IHC. Then, we dosed CCL20 serum levels resulting significantly increased in patients with bone metastases compared to non-metastatic ones. Collectively, our data highlight the importance of the c-MET pathway in the pathogenesis of bone metastases induced by RCC stem cells in mice and humans.

Purpose: Gene mutations along the Ras pathway (KRAS, NRAS, BRAF, PIK3CA) occur in approximately 50% of colorectal cancers (CRC) and correlate with poor response to anti–EGF receptor (EGFR) therapies. We assessed the effects of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) and phosphoinositide 3-kinase (PI3K)/mTOR inhibitors, which neutralize the major Ras effectors, in patient-derived xenografts from RAS/RAF/PIK3CA-mutant metastatic CRCs (mCRC). Experimental Design: Forty mCRC specimens harboring KRAS, NRAS, BRAF, and/or PIK3CA mutations were implanted in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Each xenograft was expanded into four treatment arms: placebo, the MEK inhibitor AZD6244, the PI3K/mTOR inhibitor, BEZ235, or AZD6244 + BEZ235. Cases initially treated with placebo crossed over to AZD6244, BEZ235, and the anti-EGFR monoclonal antibody cetuximab. Results: At the 3-week evaluation time point, cotreatment of established tumors with AZD6244 + BEZ235 induced disease stabilization in the majority of cases (70%) but did not lead to overt tumor regression. Monotherapy was less effective, with BEZ235 displaying higher activity than AZD6244 (disease control rates, DCRs: AZD6244, 27.5%; BEZ235, 42.5%). Triple therapy with cetuximab provided further advantage (DCR, 88%). The extent of disease control declined at the 6-week evaluation time point (DCRs: AZD6244, 13.9%; BEZ235, 16.2%; AZD6244 + BEZ235, 34%). Cross-analysis of mice harboring xenografts from the same original tumor and treated with each of the different modalities revealed subgroups with preferential sensitivity to AZD6244 (12.5%), BEZ235 (35%), or AZD6244 + BEZ235 (42.5%); another subgroup (10%) showed equivalent response to any treatment. Conclusions: The prevalent growth-suppressive effects produced by MEK and PI3K/mTOR inhibition suggest that this strategy may retard disease progression in patients. However, data offer cautionary evidence against the occurrence of durable responses. Clin Cancer Res; 18(9); 2515–25. ©2012 AACR.

Only a fraction of patients with metastatic colorectal cancer receive clinical benefit from therapy with anti-epidermal growth factor receptor (EGFR) antibodies, which calls for the identification of novel biomarkers for better personalized medicine. We produced large xenograft cohorts from 85 patient-derived, genetically characterized metastatic colorectal cancer samples (“xenopatients”) to discover novel determinants of therapeutic response and new oncoprotein targets. Serially passaged tumors retained the morphologic and genomic features of their original counterparts. A validation trial confirmed the robustness of this approach: xenopatients responded to the anti-EGFR antibody cetuximab with rates and extents analogous to those observed in the clinic and could be prospectively stratified as responders or nonresponders on the basis of several predictive biomarkers. Genotype–response correlations indicated HER2 amplification specifically in a subset of cetuximab-resistant, KRAS/NRAS/BRAF/PIK3CA wild-type cases. Importantly, HER2 amplification was also enriched in clinically nonresponsive KRAS wild-type patients. A proof-of-concept, multiarm study in HER2-amplified xenopatients revealed that the combined inhibition of HER2 and EGFR induced overt, long-lasting tumor regression. Our results suggest promising therapeutic opportunities in cetuximab-resistant patients with metastatic colorectal cancer, whose medical treatment in the chemorefractory setting remains an unmet clinical need. Significance: Direct transfer xenografts of tumor surgical specimens conserve the interindividual diversity and the genetic heterogeneity typical of the tumors of origin, combining the flexibility of preclinical analysis with the informative value of population-based studies. Our suite of patient-derived xenografts from metastatic colorectal carcinomas reliably mimicked disease response in humans, prospectively recapitulated biomarker-based case stratification, and identified HER2 as a predictor of resistance to anti-epidermal growth factor receptor antibodies and of response to combination therapies against HER2 and epidermal growth factor receptor in this tumor setting. Cancer Discovery; 1(6); 508–23. ©2011 AACR. Read the Commentary on this article by Ciardiello and Normanno, p. 472 This article is highlighted in the In This Issue feature, p. 457

Treatment with EGFR monoclonal antibodies, such as cetuximab, has improved the outcome of patients with metastatic colorectal cancer (mCRC). However, EGFR inhibition - even in cases that respond with tumor shrinkage - is cytostatic rather than cytotoxic, with persistence of drug-tolerant cells that appear to be less prone to undergo apoptosis and poised to foster residual disease, which preludes tumor relapse. Our preliminary data, obtained in mCRC patient-derived xenografts (PDXs), indicate that CRC residual cells surviving EGFR blockade display increased expression of the anti-apoptotic proteins BCL-2 and BCL-XL. Inhibition of such proteins together with EGFR prompts apoptosis of PDX-derived organoids, suggesting a pro-survival adaptation triggered by cetuximab and neutralized by pro-apoptotic drugs. On these premises, we plan to investigate how the entire apoptotic machinery is modulated in response to cetuximab in mCRC, with the final goals to: (i) identify and validate new mechanisms sustaining the persistence of drug-tolerant cells, and (ii) find new therapeutic strategies to eradicate residual disease. We first performed a gene expression analysis of the main BCL-2 family members in cetuximab-sensitive PDXs after prolonged EGFR inhibition and found a pronounced reprogramming whereby anti-apoptotic outcomes prevail over cell death signals and apoptosis gets aborted. In order to generate a list of candidates potentially involved in sustaining this anti-apoptotic adaptation, we plan to combine gene expression analysis with “Dynamic BH3 profiling” (DBP), a novel functional approach that enables assessing (i) how cetuximab treatment changes CRC cell propensity to undergo apoptosis and (ii) which anti-apoptotic proteins prevent such priming from switching into overt apoptosis. We performed DBP in two PDX-derived organoid lines and found that exposure to cetuximab induced a strong dependence on BCL-XL in one model. We next examined the effect of combining cetuximab with a specific BCL-XL inhibitor in both lines and found that while either drug alone was insufficient to cause cell death, the combination therapy induced massive apoptosis only in the case featuring cetuximab-induced BCL-XL dependence, as predicted by DBP. This analysis will be extended to a larger number of PDX-derived organoids; then, therapeutic strategies with significant activity in vitro will be validated in vivo in matched PDXs and the underlying mechanistic underpinnings will be further investigated. These preliminary results illustrate how perturbations of the apoptotic machinery can influence the response to EGFR blockade in mCRCs. This information will guide the identification of critical functional nodes involved in sustaining the drug-tolerant state, thus providing new hints to design rational ways to convert the cytostatic effect of cetuximab into a cytotoxic, fully apoptotic outcome. Citation Format: Simonetta M. Leto, Irene Catalano, Valentina Vurchio, Francesca Cottino, Giorgia Migliardi, Livio Trusolino, Andrea Bertotti. Targeting anti-apoptotic reprogramming to counteract drug tolerance in EGFR-inhibited colorectal tumors [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 309.

The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of patients with colorectal cancer. Introduction of the HER2 mutations S310F, L755S, V777L, V842I, and L866M into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutants are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors neratinib and afatinib. HER2 gene sequencing of 48 cetuximab-resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) wild-type (WT) colorectal cancer patient-derived xenografts (PDX) identified 4 PDXs with HER2 mutations. HER2-targeted therapies were tested on two PDXs. Treatment with a single HER2-targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2-targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2-mutated PDXs. Significance: HER2 activating mutations cause EGFR antibody resistance in colorectal cell lines, and PDXs with HER2 mutations show durable tumor regression when treated with dual HER2-targeted therapy. These data provide a strong preclinical rationale for clinical trials targeting HER2 activating mutations in metastatic colorectal cancer. Cancer Discov; 5(8); 832–41. ©2015 AACR. See related commentary by Pectasides and Bass, p. 799. This article is highlighted in the In This Issue feature, p. 783

Purpose: Preclinical studies in HER2-amplified gastrointestinal cancer models have shown that cotargeting HER2 with a monoclonal antibody and a small molecule is superior to monotherapy with either inhibitor, but the underlying cooperative mechanisms remain unexplored. We investigated the molecular underpinnings of this synergy to identify key vulnerabilities susceptible to alternative therapeutic opportunities. Experimental Design: The phosphorylation/activation of HER2, HER3, EGFR (HER receptors), and downstream transducers was evaluated in HER2-overexpressing colorectal and gastric cancer cell lines by Western blotting and/or multiplex phosphoproteomics. The in vivo outcome of antibody-mediated HER2 blockade by trastuzumab, reversible HER2 inhibition by lapatinib, and irreversible HER2 inhibition by afatinib was assessed in patient-derived tumorgrafts and cell-line xenografts by monitoring tumor growth curves and by using antibody-based proximity assays. Results: Trastuzumab monotherapy reduced HER3 phosphorylation, with minor consequences on downstream transducers. Lapatinib alone acutely inhibited all HER receptors and effectors but led to delayed rephosphorylation of HER3 and EGFR and partial restoration of ERK and AKT activity. When combined with lapatinib, trastuzumab prevented HER3/EGFR reactivation and caused prolonged inhibition of ERK/AKT. Afatinib alone was also very effective in counteracting the reinstatement of HER3, EGFR, and downstream signaling activation. In vivo, the combination of trastuzumab and lapatinib—or, importantly, monotherapy with afatinib—resulted in overt tumor shrinkage. Conclusions: Only prolonged inhibition of HER3 and EGFR, achievable by dual blockade with trastuzumab and lapatinib or irreversible HER2 inhibition by single-agent afatinib, led to regression of HER2-amplified gastrointestinal carcinomas. Clin Cancer Res; 21(24); 5519–31. ©2015 AACR.

EGF receptor (EGFR)-targeted monoclonal antibodies are effective in a subset of metastatic colorectal cancers. Inevitably, all patients develop resistance, which occurs through emergence of KRAS mutations in approximately 50% of the cases. We show that amplification of the MET proto-oncogene is associated with acquired resistance in tumors that do not develop KRAS mutations during anti-EGFR therapy. Amplification of the MET locus was present in circulating tumor DNA before relapse was clinically evident. Functional studies show that MET activation confers resistance to anti-EGFR therapy both in vitro and in vivo. Notably, in patient-derived colorectal cancer xenografts, MET amplification correlated with resistance to EGFR blockade, which could be overcome by MET kinase inhibitors. These results highlight the role of MET in mediating primary and secondary resistance to anti-EGFR therapies in colorectal cancer and encourage the use of MET inhibitors in patients displaying resistance as a result of MET amplification. Significance: Amplification of the MET proto-oncogene is responsible for de novo and acquired resistance to anti-EGFR therapy in a subset of colorectal cancers. As multiple anti-MET therapeutic strategies are available, these findings offer immediate novel opportunities to design clinical studies. Cancer Discov; 3(6); 658–73. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 591

Biliary tract carcinoma (BTC) has a poor prognosis due to limited treatment options. There is, therefore, an urgent need to identify new targets and to design innovative therapeutic approaches. Among potential candidate molecules, we evaluated the nonreceptor tyrosine kinase Src, observing promising antitumor effects of its small-molecule inhibitor saracatinib in BTC preclinical models. The presence of an active Src protein was investigated by immunohistochemistry in 19 surgical samples from patients with BTC. Upon saracatinib treatment, the phosphorylation of Src and of its downstream transducers was evaluated in the BTC cell lines TFK-1, EGI-1, HuH28, and TGBC1-TKB. The effect of saracatinib on proliferation and migration was analyzed in these same cell lines, and its antitumor activity was essayed in EGI-1 mouse xenografts. Saracatinib-modulated transcriptome was profiled in EGI-1 cells and in tumor samples of the xenograft model. Src was activated in about 80% of the human BTC samples. In cultured BTC cell lines, low-dose saracatinib counteracted the activation of Src and of its downstream effectors, increased the fraction of cells in G0–G1 phase, and inhibited cell migration. At high concentrations (median dose from 2.26–6.99 μmol/L), saracatinib was also capable of inhibiting BTC cell proliferation. In vivo, saracatinib treatment resulted in delayed tumor growth, associated with an impaired vascular network. Here, we provide a demonstration that the targeted inhibition of Src kinase by saracatinib is of therapeutic benefit in preclinical models of BTC. We propose our results as a basis for the design of saracatinib-based clinical applications. Mol Cancer Ther; 11(7); 1528–38. ©2012 AACR.

Background Activating mutations of the epidermal growth factor receptor (EGFR) confer sensitivity to the tyrosine kinase inhibitors (TKi), gefitinib and erlotinib. We analysed EGFR expression, EGFR mutation status and gene expression profiles of prostate cancer (PC) to supply a rationale for EGFR targeted therapies in this disease. Methods Mutational analysis of EGFR TK domain (exons from 18 to 21) and immunohistochemistry for EGFR were performed on tumour tissues derived from radical prostatectomy from 100 PC patients. Gene expression profiling using oligo-microarrays was also carried out in 51 of the PC samples. Results EGFR protein overexpression (EGFRhigh) was found in 36% of the tumour samples, and mutations were found in 13% of samples. Patients with EGFRhigh tumours experienced a significantly increased risk of biochemical relapse (hazard ratio-HR 2.52, p=0.02) compared with patients with tumours expressing low levels of EGFR (EGFRlow). Microarray analysis did not reveal any differences in gene expression between EGFRhigh and EGFRlow tumours. Conversely, in EGFRhigh tumours, we were able to identify a 79 gene signature distinguishing mutated from non-mutated tumours. Additionally, 29 genes were found to be differentially expressed between mutated/EGFRhigh (n=3) and mutated/EGFRlow tumours (n=5). Four of the down-regulated genes, U19/EAF2, ABCC4, KLK3 and ANXA3 and one of the up-regulated genes, FOXC1, are involved in PC progression. Conclusions Based on our findings, we hypothesize that accurate definition of the EGFR status could improve prognostic stratification and we suggest a possible role for EGFR-directed therapies in PC patients. Having been generated in a relatively small sample of patients, our results warrant confirmation in larger series.

Trastuzumab has changed the prognosis of HER2 positive breast cancers. Despite this progress, resistance to trastuzumab occurs in most patients. Newer anti-HER2 therapies, like the dual tyrosine-kinase inhibitor (TKI) lapatinib, show significant antitumor activity, indicating that HER2 can be still exploited as a target after trastuzumab failure. However, since a high proportion of patients fail to respond to these alternative strategies, it is possible that cell escape from HER2 targeting may rely on HER2 independent pathways. The knowledge of these pathways deserve to be exploited to develop new therapies. We characterized two human HER2 overexpressing breast cancer cell lines resistant to trastuzumab and lapatinib (T100 and JIMT-1) from a molecular and biological point of view. Indeed, we assessed both in vitro and in vivo the activity of the multitarget inhibitor sorafenib. In both cell lines, the previously proposed mechanisms did not explain resistance to HER2 inhibitors. Notably, silencing HER2 by shRNA did not affect the growth of our cells, suggesting loss of reliance upon HER2. Moreover, we identified alterations in two antiapoptotic proteins Mcl-1 and Survivin which are known to be targets of the multikinase inhibitor sorafenib. Moreover, sorafenib, strongly inhibited the in vitro growth of T100 and JIMT-1 cells, through the downregulation of both Mcl-1 and Survivin. Similar results were obtained in JIMT-1 xenografts subcutaneously injected in NOD SCID mice. We provide preclinical evidence that tumor cells resistant to trastuzumab and lapatinib may rely on HER2 independent pathways that can be efficiently inhibited by sorafenib.

Background Advanced biliary tract carcinomas (BTCs) have poor prognosis and limited therapeutic options. Therefore, it is crucial to combine standard therapies with molecular targeting. In this study EGFR, HER2, and their molecular transducers were analysed in terms of mutations, amplifications and over-expression in a BTC case series. Furthermore, we tested the efficacy of drugs targeting these molecules, as single agents or in combination with gemcitabine, the standard therapeutic agent against BTC. Methods Immunohistochemistry, FISH and mutational analysis were performed on 49 BTC samples of intrahepatic (ICCs), extrahepatic (ECCs), and gallbladder (GBCs) origin. The effect on cell proliferation of different EGFR/HER2 pathway inhibitors as single agents or in combination with gemcitabine was investigated on BTC cell lines. Western blot analyses were performed to investigate molecular mechanisms of targeted drugs. Results EGFR is expressed in 100% of ICCs, 52.6% of ECCs, and in 38.5% of GBCs. P-MAPK and p-Akt are highly expressed in ICCs (>58% of samples), and to a lower extent in ECCs and GBCs ( Conclusion These data demonstrate that EGFR and HER2 pathways are suitable therapeutic targets for BTCs. The combination of gemcitabine with drugs targeting these pathways gives encouraging results and further clinical studies could be warranted.

The recombinant monoclonal antibody trastuzumab has antiproliferative effect on breast cancer (BC) cells with ErbB2 overexpression. We postulated that a mechanism able to modify ErbB2 expression enhances the antitumor effect of trastuzumab. We analyzed whether granulocyte-colony stimulating factor (G-CSF), widely used in adjuvant cancer therapy to alleviate chemotherapy-induced myelotoxicity, could influence ErbB2 expression in BC cells and patients. The expression of ErbB2 (Herceptest) was analyzed in four BC cell lines (BT474, SKBR3, ZR75.1, and T47D) treated with G-CSF and in five samples biopsies from BC patients subjected to G-CSF rescue after chemotherapy. The effects of G-CSF and trastuzumab alone or their combination on cell growth and apoptosis were investigated. G-CSF receptor was detected on all cell lines and BC patients. G-CSF induced upregulation of ErbB2 in SKBR3, ZR75, and T47D cells. This modulation was not associated with an increase in tumor cell growth in vitro. Trastuzumab alone inhibited colony formation in soft agar but did not induce apoptosis on BC cells with no or low ErbB2 genomic amplification. The combination of trastuzumab and G-CSF enhanced the inhibition of tumor colony formation and induced apoptosis on these cells. This effect was further increased by G-CSF pretreatment. Five of nine BC patients showed an increase of Herceptest score after G-CSF administration. G-CSF treatment increases ErbB2 expression in vitro and in vivo enhancing the activity of trastuzumab on BC cell lines inducing apoptosis of BC cells with low or no ErbB2 genomic amplification.

Purpose: Preclinical studies in HER2-amplified gastrointestinal cancer models have shown that co-targeting HER2 with a monoclonal antibody and a small molecule is superior to monotherapy with either inhibitor, but the underlying cooperative mechanisms remain unexplored. We investigated the molecular underpinnings of this synergy to identify key vulnerabilities susceptible to alternative therapeutic opportunities. Experimental Design: The signaling consequences of HER2, HER3 and EGFR (HERs) blockade were evaluated in HER2-overexpressing colorectal and gastric cancer cell lines by Western blotting. HERs’ canonical downstream transducers were quantitatively gauged by multiplex phospho-proteomics. The in vivo outcome of antibody-mediated HER2 blockade by trastuzumab, reversible HER2 inhibition by lapatinib, and irreversible HER2 inhibition by afatinib was assessed in patient-derived tumorgrafts and cell-line xenografts by monitoring tumor growth curves and by using antibody-based proximity assays for phosphoprotein analysis. Results: Trastuzumab monotherapy reduced HER3 phosphorylation, with minor consequences on downstream transducers. Lapatinib alone acutely inhibited all HER receptors and effectors but led to delayed rephosphorylation of HER3 and EGFR and partial restoration of ERK and AKT activity. When combined with lapatinib, trastuzumab prevented HER3/EGFR reactivation and caused prolonged inhibition of ERK/AKT. Afatinib alone was as effective as trastuzumab plus lapatinib in counteracting the reinstatement of HER3, EGFR, and downstream signaling activation. In vivo, the combination of trastuzumab and lapatinib - or monotherapy with afatinib - resulted in overt tumor shrinkage. Conclusions: Only prolonged inhibition of HER3 and EGFR, achievable by dual blockade with trastuzumab and lapatinib or irreversible HER2 inhibition by single-agent afatinib, led to regression of HER2-amplified gastrointestinal carcinomas. Citation Format: Simonetta Maria Leto, Francesco Sassi, Irene Catalano, Giorgia Migliardi, Eugenia Rosalinda Zanella, Andrea Bertotti, Livio Trusolino. Sustained inhibition of HER3 and EGFR is necessary to induce regression of HER2-amplified gastrointestinal carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 659. doi:10.1158/1538-7445.AM2015-659

Only 10% of genetically unselected patients with chemorefractory metastatic colorectal cancer (mCRC) receive clinical benefit from therapy with the anti-EGFR antibodies cetuximab and panitumumab. Among non-responsive patients, 70% bear tumors harboring at least one alteration in KRAS, NRAS, BRAF and PIK3CA genes; therefore, the remaining 30% of “quadruple negative” cases display still-unidentified features that sustain primary resistance to EGFR-targeted therapies. By performing genotype-response correlations in a preclinical platform of patient-derived mCRC xenografts (xenopatients), our group has recently identified HER2 amplification as a new biomarker of resistance to cetuximab within a quadruple negative population. We found that the anti-HER2 monoclonal antibody trastuzumab, despite its demonstrated efficacy in patients with HER2-positive breast and gastric cancers, was completely ineffective in HER2-amplified mCRC xenopatients. Importantly, while the dual EGFR/HER2 small molecule inhibitor lapatinib induced disease stabilization, the combination of lapatinib with trastuzumab was synergistic, achieving rapid and long lasting tumor regressions. When the same regimens where applied to HER2-positive gastric cancer cells in vivo, trastuzumab and lapatinib as monotherapies induced disease stabilization, while the combination of both – similar to CRC – had stronger therapeutic effect. The aim of this study was to better characterize the molecular mechanisms underlying the synergistic effect of the combination therapy in HER2-positive gastric and colorectal cancers. We first generated three CRC cell lines (NCI-H508, DiFi and HDC-142) stably transfected with the HER2 oncogene and provided proof-of-concept of the causative role of HER2 in installing cetuximab resistance in mCRC. Indeed, while parental controls proved to be extremely sensitive to cetuximab, HER2 overexpressors displayed overt resistance, both in viability assays in vitro and in xenografts experiments in vivo. Moreover, NCI-H508-HER2 cells in vivo were poorly responsive to trastuzumab, while lapatinib induced disease stabilization and the combination of both achieved the strongest therapeutic effect. Overall, these responses were very similar to those observed for HER2-positive mCRC xenopatients, attesting to the reliability of this artificial cellular model for further mechanistic studies. We next analyzed the activation status of EGFR, HER2 and HER3 upon treatment with trastuzumab, lapatinib and combo and explored the signaling consequences of receptor blockade. Dose-response curves revealed that trastuzumab did not affect activation of EGFR and HER2, but was able to reduce HER3 phosphorylation; at variance, lapatinib and combo potently inhibited phosphorylation of all HERs in a similar manner, with no additive effect by combo. Interestingly, time-course experiments highlighted that treatment with lapatinib resulted in a paradoxical increase of HER3 expression and phosphorylation both in HER2-positive gastric cancer cells and in colorectal cancer cells. Importantly, adding trastuzumab to lapatinib completely neutralized lapatinib-induced HER3 hyper-phosphorylation, without affecting HER3 levels. Possibly, up-regulated HER3 could provide an alternate route to activate a “bypass track” signaling, attenuating the antitumor effects of lapatinib as monotherapy in these tumor settings. Full inhibition of a residual HER2 activity and consequent abolishment of HER3 phosphorylation achieved by the addition of trastuzumab could provide a mechanistic explanation of the synergic effect obtained with the combination therapy. Overall, these results provide new mechanistic insights into resistance and response to EGFR and HER2 targeted therapies and deserve further investigations to explore the key pathways involved in HER3 feedback up-regulation in the context of HER2-positive cancers. Citation Format: Simonetta Maria Leto, Francesco Sassi, Giorgia Migliardi, Eugenia Zanella, Irene Catalano, Andrea Bertotti, Livio Trusolino. HER2-amplified gastric and colorectal cancers: Mechanistic insights into HER2 targeting approaches. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B43.

The Hepatocyte Growth Factor (HGF) also known as Scatter Factor, activates cancer cell invasion and metastasis. We show that in ovarian cancer cells HGF induced the phosphorylation of the small heat shock protein of 27KDa (HSP27) by activating the p38MAPK. HSP27 is increased in many cancers at advanced stage including ovarian cancer and associated with cancer resistance to therapy and poor patients' survival. The phosphorylation of HSP27 regulates both its chaperone activity and its control of cytoskeletal stability. We show that HSP27 was necessary for the motility in vitro and the remodeling of actin filaments induced by HGF. In vivo, HSP27 silencing impaired the ability of the highly metastatic, HGF-secreting ovarian cancer cells to give rise to spontaneous metastases. This was due to defective motility across the vessel wall and reduced growth. Indeed, HSP27 silencing impaired both the ability of ovarian cancer cells to form experimental haematogenous metastases and to grow as subcutaneous xenografts. Moreover, HSP27 suppression resulted in the sensitization of xenografts to low doses of the chemotherapeutic Paclitaxel, likely because HSP27 protected microtubules from bundling caused by the drug. Altogether, these data show that the HSP27 is required for the pro-invasive and pro-metastatic activity of HGF and suggest that HSP27 might be not only a marker of progression of ovarian cancer, but also a suitable target for therapy. Grant sponsors: This work has been supported by grants to M.F.D.: 2012 IG grant and 2010 Special Program Molecular Clinical Oncology 5xMille of the Italian Association of Cancer Research (AIRC), Project n° 9970, and Grant of the CARIPLO Foundation. Citation Format: Simona Pavan, Daniele Musiani, Erica Torchiaro, Giorgia Migliardi, Jessica Erriquez, Martina Olivero, Maria Flavia Di Renzo. HSP27 is required for invasion and metastasis triggered by hepatocyte growth factor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B3.

11005 Background: The anti EGFR monoclonal antibodies cetuximab and panitumumab are used to treat metastatic colorectal cancer patients but their clinical efficacy is limited by the development of acquired resistance. We recently reported that secondary KRAS mutations are responsible for acquired resistance in approximately 50% of the patients who initially respond to cetuximab or panitumumab (Misale et al., Nature 2012; Diaz et al., Nature 2012). Here we studied the molecular bases of relapse in CRC patients who do not develop KRAS mutations during the course of anti-EGFR therapy. Methods: Next generation sequencing was applied to tumor biopsies to identify genetic alterations associated with relapse to cetuximab and panitumumab in mCRC patients. Detection and quantitation of genetic alterations in circulating tumor DNA was used to monitor the occurrence of KRAS mutations and MET amplification in blood samples. Results: Molecular analyses of tumor biopsies from patients who did not develop KRAS mutations during anti-EGFR therapy revealed high level of amplification of the MET proto-oncogene in 3/5 cases. Quantitative PCR, FISH and IHC analysis confirmed high level of MET amplification in the post-therapy samples but not in the matched pre-treatment tissues. We developed a PCR based assay to detect the presence of the MET amplicon in circulating, cell-free, DNA. We found that MET amplification could be detected in the blood as early as 3 months after initiation of anti EGFR therapy. To functionally evaluate the role of MET amplification on resistance to anti EGFR antibody therapies we exploited patient-derived CRC xenografts (‘xenopatients). We found that (2/2) xenopatients established from MET amplified tumors were completely refractory to cetuximab but showed sensitivity to the Met inhibitor crizotinib. Conclusions: Amplification of the MET proto-oncogene is responsible for acquired acquired resistance to anti-EGFR antibody therapy in a subset of CRCs. The emergence of MET amplification in circulating, cell-free, DNA may be used to select patients most likely to benefit from anti MET therapies.

Results Xenopatients retained the histological and genomic features of the original counterparts, responded to the anti-EGFR antibody cetuximab similarly to clinical observations, and could be prospectively stratified as responders or nonresponders based on predictive biomarkers. Genotype-response correlations indicated HER2 amplification specifically in a subset of cetuximab-resistant, KRAS/ NRAS/BRAF/PIK3CA wild-type cases. In this subset, combined HER2/EGFR inhibition induced long-lasting tumor regression. We also assessed the effects of MEK and PI3K/ mTor inhibitors (AZD6244 and BEZ235 respectively) in 40 specimens harboring KRAS/ NRAS/BRAF/PIK3CA mutations. Cotreatment of xenografts with AZD6244 +BEZ235 induced disease stabilization in the majority of cases (70%) but did not lead to tumor regression. Monotherapy was less effective, with BEZ235 displaying higher activity than AZD6244. Triple therapy with cetuximab provided further advantage. The extent of disease control declined upon prolonged treatment.

HER2 targeting with trastuzumab has changed the prognosis of breast cancer patients carrying amplification and/or overexpression of this oncogene. Despite this progress, however, resistance to trastuzumab occurs in the vast majority of patients. Newer anti-HER2 therapies, like the dual tyrosine-kinase inhibitor (TKI) lapatinib, show antitumor activity in a limited proportion of patients, indicating that HER2 can be still exploited as a target after trastuzumab failure. However, due to the high proportion of patients that fail to respond to these alternative strategies, it is reasonable to assume that cells escaping HER2 targeting may rely on alternative pathways not involving HER2 to sustain their growth. Their knowledge is relevant for exploiting new therapies. To investigate this hypothesis we generated a human HER2 overexpressing breast cancer cell line resistant to trastuzumab and lapatinib (T100) and we have characterized it genetically and molecularly. In T100 cells, the previously proposed mechanisms of resistance to HER2 inhibitors did not explain cell escape. Notably, silencing HER2 by shRNA did not affect the growth of T100 cells, suggesting loss of reliance upon HER2. Among the alternative pathways that we explored, altered levels of the antiapoptoptic proteins Mcl-1 and Survivin were observed. This suggested the possibility of targeting resistant cells with the multitarget inhibitor sorafenib. Moreover, sorafenib, nearly inactive in parental cells, strongly inhibited the in vitro growth of T100 cells. In conclusion, we provide a new model where the activation of HER2 independent mechanisms sustains the growth of tumor cells, significantly increasing the sensitivity to sorafenib. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A82.

Osteosarcoma (OS) is the most common primary bone tumor in children and young adults. Despite improved prognosis, metastatic or relapsed OS remains largely incurable and no significant improvement has been observed in the last 20 years. Therefore, the search for alternative agents in OS is mandatory. We explored phospho-ERK 1/2, MCL-1, phospho-Ezrin/Radixin/Moesin (P-ERM) as potential therapeutic targets. The activation of these pathways was shown by immunohistochemistry in about 70% of cases and in all OS cell lines analyzed. Mutational analysis revealed no activating mutations in KRAS. While, BRAF gene was found mutated in 4/30 OS samples from patients. Based on these results we tested the multikinases inhibitor sorafenib (BAY 43-9006) in preclinical models of OS. Sorafenib inhibited OS cell lines proliferation, induced apoptosis and downregulated P-ERK1/2, MCL-1, P-ERM in a dose dependent manner. ERM dephosphorylation is not due to ERK inhibition. The downregulation of MCL-1 increased apoptosis in OS cell lines. In chick embryo chorioallantoic membrane the angiogenesis induced by OS supernatants was blocked by sorafenib treatments. Indeed, sorafenib reduced VEGF and MMP2 production. Sorafenib treatment dramatically reduced tumor volume of OS xenografts and lung metastasis in SCID mice. In conclusion, ERK1/2, MCL-1 and ERM pathways are active in OS. Sorafenib is able to interrupt their routes in vitro and in vivo displaying antitumoral activity, antiangiogenic effect and reducing metastasis colonies formation in lungs. These data support the testing of sorafenib as a potential therapeutic option in metastatic or relapsed OS patients failing standard treatments. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C213.

4582 Background: Biliary tract carcinomas express EGFR and are potential candidates to EGFR target therapies. We recently described somatic mutations of EGFR that can enhance MAPK or Akt activation (Clin Cancer Res, 2006). Some of them are identical to those previously reported to confer sensitivity to some tyrosine kinase inhibitors (TKIs) like erlotinib or gefitinib in lung cancer. Here we report a molecular analysis of EGFR transducers potentially involved in TKI response. Methods: In 49 samples of biliary tract carcinoma we performed mutational analysis of exons from 18 to 21 of EGFR, exons 9 and 20 of phosphatidylinositol 3’-kinase (PI3K), exon 2 of K-Ras, exon 15 of B-Raf and exons from 5 to 8 of PTEN. Nuclear PTEN expression was analyzed by immunohistochemistry and the expression in cancer cells was compared to that of normal cholangiocites. Results: Mutations of EGFR have been detected in 7 out of 49 samples (14.3%). One of them was a new stop-codon mutation. Five hotspot mutations of PI3K (codon 545, 546, 1048 and 1059) were found in 4 cases (8.2%); 3 cases (6.1%) had single mutations in K-Ras and 4 (8.2%) had the V599E mutation in B-Raf. In some samples, mutations of multiple trasducers were present simultaneously. PI3K mutations were significantly more frequent in EGFR mutated samples compared to wild type (28% vs. 4.7% respectively, p No significant financial relationships to disclose.

e14654 Background: Metastatic colorectal cancer (mCRC) is currently considered incurable. Recent evidences support the importance of micro-environment and angiogenesis on survival and propagation of mCRC. Lenalidomide is a derivative of thalidomide, effective in several hematologic disorders, whose activity includes modulation on tumor microenvironment and inhibition of angiogenesis. We investigated the preclinical activity of lenalidomide against mCRC in vitro and in vivo. Methods: CRC cell lines and primary mCRC cells were treated with scalar concentrations of lenalidomide (10 to 150 µM). Two cohorts of NOD/SCID mice (n=8) implanted with mCRC xenografts derived from two different patients were treated orally for 21 consecutive days with Lenalidomide (50 mg/Kg/die). Results: In vivo a significant reduction of tumor growth was observed in one cohort compared to untreated controls (n=8), 247±118 mm3 vs. 567±216 mm3 (P=0.02), while no significant differences occurred in the second cohort. Pathology review on tumor samples removed from the mice revealed a trend toward the restoration of normal colon tissue morphology and architecture in lenalidomide treated samples compared to untreated controls. Quantitative evaluation of tumor angiogenesis on the responsive cohort, by immunohistochemistry for factor VIII, revealed a tumor-vessel reduction of 49% with lenalidomide compared to controls. Moreover, necrotic-hypoxic areas were reduced by 6 fold in lenalidomide treated xenografts compared to untreated controls (P

Background Osteosarcoma (OS) is the most common primary bone tumour in children and young adults. Despite improved prognosis, metastatic or relapsed OS remains largely incurable and no significant improvement has been observed in the last 20 years. Therefore, the search for alternative agents in OS is mandatory. Results We investigated phospho-ERK 1/2, MCL-1, and phospho-Ezrin/Radixin/Moesin (P-ERM) as potential therapeutic targets in OS. Activation of these pathways was shown by immunohistochemistry in about 70% of cases and in all OS cell lines analyzed. Mutational analysis revealed no activating mutations in KRAS whereas BRAF gene was found to be mutated in 4/30 OS samples from patients. Based on these results we tested the multi-kinase inhibitor sorafenib (BAY 43-9006) in preclinical models of OS. Sorafenib inhibited OS cell line proliferation, induced apoptosis and downregulated P-ERK1/2, MCL-1, and P-ERM in a dose-dependent manner. The dephosphorylation of ERM was not due to ERK inhibition. The downregulation of MCL-1 led to an increase in apoptosis in OS cell lines. In chick embryo chorioallantoic membranes, OS supernatants induced angiogenesis, which was blocked by sorafenib and it was also shown that sorafenib reduced VEGF and MMP2 production. In addition, sorafenib treatment dramatically reduced tumour volume of OS xenografts and lung metastasis in SCID mice. Conclusion In conclusion, ERK1/2, MCL-1 and ERM pathways are shown to be active in OS. Sorafenib is able to inhibit their signal transduction, both in vitro and in vivo, displaying anti-tumoural activity, anti-angiogenic effects, and reducing metastatic colony formation in lungs. These data support the testing of sorafenib as a potential therapeutic option in metastatic or relapsed OS patients unresponsive to standard treatments.