MErCuRIC Publications


Targeting c-MET in gastrointestinal tumours: rationale, opportunities and challenges

Conor A. Bradley, Manuel Salto-Tellez, Pierre Laurent-PuigAlberto BardelliChristian RolfoJosep TaberneroHajrah A. KhawajaMark LawlerPatrick G. JohnstonSandra Van Schaeybroeck & on behalf of the MErCuRIC consortium

Nature Reviews Clinical Oncology doi:10.1038/nrclinonc.2017.40

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ABSTRACT: c-MET and its ligand HGF are frequently overexpressed in colorectal cancer (CRC) and increased c-MET levels are found in CRC liver metastases. This study investigated the role of the HGF/c-MET axis in regulating migration/invasion in CRC, using pre-clinical models and clinical samples. Pre-clinically, we found marked upregulation of c-MET at both protein and mRNA levels in several invasive CRC cells. Down-regulation of c-MET using RNAi suppressed migration/invasion of parental and invasive CRC cells. Stimulation of CRC cells with rh-HGF or co-culture with HGF-expressing colonic myofibroblasts, resulted in significant increases in their migratory/invasive capacity. Importantly, HGF-induced c-MET activation promoted rapid downregulation of c-MET protein levels, while the MET transcript remained unaltered. Using RNA in situ hybridization (RNA ISH), we further showed that MET mRNA, but not protein levels, were significantly upregulated in tumor budding foci at the invasive front of a cohort of stage III CRC tumors (p < 0.001). Taken together, we show for the first time that transcriptional upregulation of MET is a key molecular event associated with CRC invasion and tumor budding. This data also indicates that RNA ISH, but not immunohistochemistry, provides a robust methodology to assess MET levels as a potential driving force of CRC tumor invasion and metastasis.

Heterogeneity of Acquired Resistance to Anti-EGFR Monoclonal Antibodies in Patients with Metastatic Colorectal Cancer

Filippo Pietrantonio, Claudio Vernieri, Giulia Siravegna, Alessia Mennitto, Rosa Berenato, Federica Perrone, Annunziata Gloghini, Elena Tamborini, Sara Lonardi, Federica Morano, Benedetta Picciani, Adele Busico, Chiara Costanza Volpi, Antonia Martinetti, Francesca Battaglin, Ilaria Bossi, Alessio Pellegrinelli, Massimo Milione, Chiara Cremolini, Maria Di Bartolomeo, Alberto Bardelli and Filippo de Braud

 doi:10.1158/1078-0432.CCR-16-1863

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ABSTRACT:

Purpose: Even if RAS-BRAF wild-type and HER2/MET–negative metastatic colorectal cancer (mCRC) patients frequently respond to anti-EGFR mAbs, acquired resistance almost invariably occurs. Mechanisms of resistance to EGFR blockade include the emergence of KRAS, NRAS, and EGFR extracellular domain mutations as well as HER2/MET alterations. However, these findings derive from retrospective studies that analyzed one single resistance mechanism at a time; moreover, it is still unclear how molecular heterogeneity affects clonal evolution in patients. In this work, we aimed at extensively characterizing and correlating the molecular characteristics of tissue- and blood-based data in a prospective cohort of patients with mCRC who received anti-EGFR antibodies.

Experimental design: Twenty-two RASBRAF wild-type, HER2/MET–negative mCRC patients progressing on anti-EGFR therapy after initial response underwent rebiopsy. Next-generation sequencing and silver in situ hybridization (SISH)/IHC analyses were performed both on archival tumors and postprogression samples. Circulating tumor (ctDNA) molecular profiles were obtained in matched tissue–plasma samples.

Results: RAS mutations and HER2/MET amplification were the most frequently detected resistance mechanisms in both tissue and blood sample analysis. On the other hand, BRAF and EGFR ectodomain mutations were much rarer. Patients with acquired MET amplification showed worse PFS on anti-EGFRs. We detected both intralesion heterogeneity, as suggested by co-occurrence of different resistance mechanisms in the same sample, and interlesion heterogeneity. The combined analysis of tissue and blood (ctDNA) results highlights the complexity of clonal evolution triggered by EGFR blockade.

Conclusions: Our results indicate that it may be extremely challenging to target the complex landscape of molecular heterogeneity associated with emergence of resistance to targeted therapies in patients with mCRC.

Efficacy of NEDD8 Pathway Inhibition in Preclinical Models of Poorly Differentiated, Clinically Aggressive Colorectal Cancer

J Natl Cancer Inst (2017) 109 (2): 1-12. doi:10.1093/jnci/djw209

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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.

Molecular Landscape of Acquired Resistance to Targeted Therapy Combinations in BRAF-Mutant Colorectal Cancer

Daniele Oddo, Erin M. Sennott, Ludovic Barault, Emanuele Valtorta, Sabrina Arena, Andrea Cassingena, Genny Filiciotto, Giulia Marzolla, Elena Elez, Robin M.J.M. van Geel, Alice Bartolini, Giovanni Crisafulli, Valentina Boscaro, Jason T. Godfrey, Michela Buscarino, Carlotta Cancelliere, Michael Linnebacher, Giorgio Corti, Mauro Truini, Giulia Siravegna, Julieta Grasselli, Margherita Gallicchio, René Bernards, Jan H.M. Schellens, Josep Tabernero, Jeffrey A. Engelman, Andrea Sartore-Bianchi, Alberto Bardelli, Salvatore Siena, Ryan B. Corcoran and Federica Di Nicolantonio

Cancer Res (2017) 76(15); 4504–15. doi:10.1158/0008-5472.CAN-16-0396

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Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF-mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF-mutant resistant cell line models across seven different clinically relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental-sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS, and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1. These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. To identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF-mutant colorectal cancer.

Transcriptional upregulation of c-MET is associated with invasion and tumor budding in colorectal cancer

Conor A. Bradley, Philip D. Dunne, Victoria Bingham, Stephen McQuaid, Hajrah Khawaja, Stephanie Craig, Jackie James, Wendy L. Moore, Darragh G. McArt, Mark Lawler, Sonali Dasgupta, Patrick G. Johnston, Sandra Van Schaeybroeck

Oncotarget. 2016; 7:78932-78945. doi: 10.18632/oncotarget.12933

Details

c-MET and its ligand HGF are frequently overexpressed in colorectal cancer (CRC) and increased c-MET levels are found in CRC liver metastases. This study investigated the role of the HGF/c-MET axis in regulating migration/invasion in CRC, using pre-clinical models and clinical samples. Pre-clinically, we found marked upregulation of c-MET at both protein and mRNA levels in several invasive CRC cells. Down-regulation of c-MET using RNAi suppressed migration/invasion of parental and invasive CRC cells. Stimulation of CRC cells with rh-HGF or co-culture with HGF-expressing colonic myofibroblasts, resulted in significant increases in their migratory/invasive capacity. Importantly, HGF-induced c-MET activation promoted rapid downregulation of c-MET protein levels, while the MET transcript remained unaltered. Using RNA in situ hybridization (RNA ISH), we further showed that MET mRNA, but not protein levels, were significantly upregulated in tumor budding foci at the invasive front of a cohort of stage III CRC tumors (p < 0.001). Taken together, we show for the first time that transcriptional upregulation of MET is a key molecular event associated with CRC invasion and tumor budding. This data also indicates that RNA ISH, but not immunohistochemistry, provides a robust methodology to assess MET levels as a potential driving force of CRC tumor invasion and metastasis.

Blood circulating tumor DNA for non-invasive genotyping of colon cancer patients

Giulia Siravegna, Alberto Bardelli
Molecular Oncology (2016) 10(3), 475–480. doi:10.1016/j.molonc.2015.12.005

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Most solid tumors, including colorectal cancers, shed cell-free DNA (ctDNA) in the blood. ctDNA can be analyzed to generate molecular profiles which capture the heterogeneity of the disease more comprehensively then tumor tissue biopsies. This approach commonly called ‘liquid biopsy’ can be applied to monitor response to therapy, to assess minimal residual disease and to uncover the emergence of drug resistance. This review will discuss current and future developments of ctDNA analysis in the clinical management of colorectal cancer patients.

MM-151 overcomes acquired resistance to cetuximab and panitumumab in colorectal cancers harboring EGFR extracellular domain mutations

Sabrina ArenaGiulia SiravegnaBenedetta MussolinJeffrey D. KearnsBeni B. WolfSandra MisaleLuca LazzariAndrea BertottiLivio TrusolinoAlex A. AdjeiClara MontagutFederica Di NicolantonioRachel NeringAlberto Bardelli

Science Translational Medicine 8(324) 324ra14. doi:10.1126/scitranslmed.aad5640

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The anti–epidermal growth factor receptor (EGFR) antibodies cetuximab and panitumumab are used to treat RAS wild-type colorectal cancers (CRCs), but their efficacy is limited by the emergence of acquired drug resistance. After EGFR blockade, about 20% of CRCs develop mutations in the EGFR extracellular domain (ECD) that impair antibody binding and are associated with clinical relapse. We hypothesized that EGFR ECD–resistant variants could be targeted by the recently developed oligoclonal antibody MM-151 that binds multiple regions of the EGFR ECD. MM-151 inhibits EGFR signaling and cell growth in preclinical models, including patient-derived cells carrying mutant EGFR. Upon MM-151 treatment, EGFR ECD mutations decline in circulating cell-free tumor DNA (ctDNA) of CRC patients who previously developed resistance to EGFR blockade. These data provide molecular rationale for the clinical use of MM-151 in patients who become resistant to cetuximab or panitumumab as a result of EGFR ECD mutations.

Tumor Heterogeneity and Lesion-Specific Response to Targeted Therapy in Colorectal Cancer

Mariangela Russo, Giulia Siravegna, Lawrence S. Blaszkowsky, Giorgio Corti, Giovanni Crisafulli, Leanne G. Ahronian, Benedetta Mussolin, Eunice L. Kwak, Michela Buscarino, Luca Lazzari, Emanuele Valtorta, Mauro Truini, Nicholas A. Jessop, Hayley E. Robinson, Theodore S. Hong, Mari Mino-Kenudson, Federica Di Nicolantonio, Ashraf Thabet, Andrea Sartore-Bianchi, Salvatore Siena, A. John Iafrate, Alberto Bardelli and Ryan B. Corcoran

Cancer Discov (2015); 6(2); 147–53. doi:10.1158/2159-8290.CD-15-1283

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How genomic heterogeneity associated with acquired resistance to targeted agents affects response to subsequent therapy is unknown. We studied EGFR blockade in colorectal cancer to assess whether tissue and liquid biopsies can be integrated with radiologic imaging to monitor the impact of individual oncogenic alterations on lesion-specific responses. Biopsy of a patient’s progressing liver metastasis following prolonged response to cetuximab revealed a MEK1K57Tmutation as a novel mechanism of acquired resistance. This lesion regressed upon treatment with panitumumab and the MEK inhibitor trametinib. In circulating tumor DNA (ctDNA), mutant MEK1 levels declined with treatment, but a previously unrecognized KRASQ61H mutation was also identified that increased despite therapy. This same KRAS mutation was later found in a separate nonresponding metastasis. In summary, parallel analyses of tumor biopsies and serial ctDNA monitoring show that lesion-specific radiographic responses to subsequent targeted therapies can be driven by distinct resistance mechanisms arising within separate tumor lesions in the same patient.

Significance: Molecular heterogeneity ensuing from acquired resistance drives lesion-specific responses to subsequent targeted therapies. Analysis of a single-lesion biopsy is inadequate to guide selection of subsequent targeted therapies. ctDNA profiles allow the detection of concomitant resistance mechanisms residing in separate metastases and assessment of the effect of therapies designed to overcome resistance.

Acquired Resistance to the TRK Inhibitor Entrectinib in Colorectal Cancer

Mariangela Russo, Sandra Misale, Ge Wei, Giulia Siravegna, Giovanni Crisafulli, Luca Lazzari, Giorgio Corti, Giuseppe Rospo, Luca Novara, Benedetta Mussolin, Alice Bartolini, Nicholas Cam, Roopal Patel, Shunqi Yan, Robert Shoemaker, Robert Wild, Federica Di Nicolantonio, Andrea Sartore Bianchi, Gang Li, Salvatore Siena and Alberto Bardelli

Cancer Discov (2015); 6(1); 36–44. doi:10.1158/2159-8290.CD-15-0940

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Entrectinib is a first-in-class pan-TRK kinase inhibitor currently undergoing clinical testing in colorectal cancer and other tumor types. A patient with metastatic colorectal cancer harboring an LMNA–NTRK1 rearrangement displayed a remarkable response to treatment with entrectinib, which was followed by the emergence of resistance. To characterize the molecular bases of the patient’s relapse, circulating tumor DNA (ctDNA) was collected longitudinally during treatment, and a tissue biopsy, obtained before entrectinib treatment, was transplanted in mice (xenopatient), which then received the same entrectinib regimen until resistance developed. Genetic profiling of ctDNA and xenopatient samples showed acquisition of two point mutations in the catalytic domain of NTRK1, p.G595R and p.G667C. Biochemical and pharmacologic analysis in multiple preclinical models confirmed that either mutation renders the TRKA kinase insensitive to entrectinib. These findings can be immediately exploited to design next-generation TRKA inhibitors.

Significance: We provide proof of principle that analyses of xenopatients (avatar) and liquid biopsies allow the identification of drug resistance mechanisms in parallel with clinical treatment of an individual patient. We describe for the first time that p.G595R and p.G667C TRKA mutations drive acquired resistance to entrectinib in colorectal cancers carrying NTRK1 rearrangements.

Acquired Resistance to the TRK Inhibitor Entrectinib in Colorectal Cancer

Mariangela Russo, Sandra Misale, Ge Wei, Giulia Siravegna, Giovanni Crisafulli, Luca Lazzari, Giorgio Corti, Giuseppe Rospo, Luca Novara, Benedetta Mussolin, Alice Bartolini, Nicholas Cam, Roopal Patel, Shunqi Yan, Robert Shoemaker, Robert Wild, Federica Di Nicolantonio, Andrea Sartore Bianchi, Gang Li, Salvatore Siena and Alberto Bardelli

Cancer Discov (2015); 6(1); 36–44. doi:10.1158/2159-8290.CD-15-0940

Details

Entrectinib is a first-in-class pan-TRK kinase inhibitor currently undergoing clinical testing in colorectal cancer and other tumor types. A patient with metastatic colorectal cancer harboring an LMNA–NTRK1 rearrangement displayed a remarkable response to treatment with entrectinib, which was followed by the emergence of resistance. To characterize the molecular bases of the patient’s relapse, circulating tumor DNA (ctDNA) was collected longitudinally during treatment, and a tissue biopsy, obtained before entrectinib treatment, was transplanted in mice (xenopatient), which then received the same entrectinib regimen until resistance developed. Genetic profiling of ctDNA and xenopatient samples showed acquisition of two point mutations in the catalytic domain of NTRK1, p.G595R and p.G667C. Biochemical and pharmacologic analysis in multiple preclinical models confirmed that either mutation renders the TRKA kinase insensitive to entrectinib. These findings can be immediately exploited to design next-generation TRKA inhibitors.

Significance: We provide proof of principle that analyses of xenopatients (avatar) and liquid biopsies allow the identification of drug resistance mechanisms in parallel with clinical treatment of an individual patient. We describe for the first time that p.G595R and p.G667C TRKA mutations drive acquired resistance to entrectinib in colorectal cancers carrying NTRK1 rearrangements.

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