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Papers by Valentina Pirazzoli

Purpose: The EGFR tyrosine kinase inhibitors (TKIs), erlotinib and afatinib, have transformed the... more Purpose: The EGFR tyrosine kinase inhibitors (TKIs), erlotinib and afatinib, have transformed the treatment of advanced EGFRmutant lung adenocarcinoma. However, almost all patients who respond develop acquired resistance on average approximately 1 year after starting therapy. Resistance is commonly due to a secondary mutation in EGFR (EGFR T790M). We previously found that the combination of the EGFR TKI afatinib and the EGFR antibody cetuximab could overcome EGFR T790M-mediated resistance in preclinical models. This combination has shown a 29% response rate in a clinical trial in patients with acquired resistance to first-generation TKIs. An outstanding question is whether this regimen is beneficial when used as first-line therapy. Experimental Design: Using mouse models of EGFR-mutant lung cancer, we tested whether the combination of afatinib plus cetuximab delivered upfront to mice with TKI-na€ ve EGFR L858R-induced lung adenocarcinomas delayed tumor relapse and drugresistance compared with single-agent TKIs. Results: Afatinib plus cetuximab markedly delayed the time to relapse and incidence of drug-resistant tumors, which occurred in only 63.6% of the mice, in contrast to erlotinib or afatinib treatment where 100% of mice developed resistance. Mechanisms of tumor escape observed in afatinib plus cetuximab resistant tumors include the EGFR T790M mutation and Kras mutations. Experiments in cell lines and xenografts confirmed that the afatinib plus cetuximab combination does not suppress the emergence of EGFR T790M. Conclusions: These results highlight the potential of afatinib plus cetuximab as an effective treatment strategy for patients with TKI-na€ ve EGFR-mutant lung cancer and indicate that clinical trial development in this area is warranted. Clin Cancer Res; 22(2); 426-35.

INTRODUCTION AIM OF THE WORK UROKINASE-TYPE PLASMINOGEN ACTIVATOR RECEPTOR (UPAR) uPAR expression... more INTRODUCTION AIM OF THE WORK UROKINASE-TYPE PLASMINOGEN ACTIVATOR RECEPTOR (UPAR) uPAR expression uPAR gene uPAR protein Relationship between uPAR and the Ly-6 gene family uPAR structure Species selectivity of the uPA-uPAR interaction VITRONECTIN (VN) Vitronectin expression Vitronectin gene Structure of vitronectin

Clinical Cancer Research, Jan 15, 2014

EGFR mutant lung cancers are highly sensitive to first generation EGFR tyrosine kinase inhibitors... more EGFR mutant lung cancers are highly sensitive to first generation EGFR tyrosine kinase inhibitors (TKIs; gefitinib and erlotinib), but resistance invariably develops. In the majority of patients, disease progression is mediated by a second-site T790M mutation in EGFR. T790M-mediated acquired resistance can be overcome by the combination of the second-generation EGFR TKI, afatinib, with the anti-EGFR monoclonal antibody, cetuximab, in both preclinical models and humans. However, patients still develop acquired resistance to second-line anti-EGFR combination therapy. In order to develop therapeutic strategies for patients whose tumors progress on afatinib/cetuximab, we have modeled resistance using xenografts of PC-9/BRc1 cells (EGFR exon 19 deletion/T790M) treated chronically with the drug combination. Over the course of four months, 4 of 10 animals developed acquired resistance. Xenograft tumors still displayed features of adenocarcinoma only, and mutational analysis did not detect any common secondary hotspot mutations found in lung adenocarcinomas. Analysis of two cell lines derived from resistant tumors by array comparative genomic hybridization (aCGH) suggests that resistant tumors developed additional amplification at the EGFR locus. Fluorescence in situ hybridization (FISH) of all lines confirmed sustained amplification of EGFR in resistant cell lines, and immunoblotting of cell lysates showed increased EGFR protein expression relative to parental cells and vehicle-treated controls. Strikingly, all xenograft-derived cell lines displayed in vitro sensitivity to AZD9291, a third-generation, mutant-specific EGFR TKI. Finally, in vitro comparison of afatinib/cetuximab with AZD9291 in PC-9/BRc1 cells suggests that AZD9291 may even be more potent than afatinib/cetuximab in the setting of T790M-mediated acquired resistance to first and second-generation EGFR TKIs. Confirmatory xenograft experiments are planned, and experiments are ongoing, including with additional cell lines. Collectively, these data suggest that EGFR overexpression may be a mechanism of acquired resistance to afatinib/cetuximab combination therapy, and that resistance may still be overcome by mutant-specific EGFR small molecule inhibitors. Thus, patients with EGFR mutant lung cancers may benefit from sequential lines of therapy (erlotinib->afatinib/cetuximab->AZD9291) targeting EGFR. Further work is needed to discern specific indications for afatinib/cetuximab versus AZD9291 as therapy for patients with acquired resistance to EGFR TKIs. Citation Format: Catherine B. Meador, Hailing Jin, Elisa de Stanchina, Valentina Pirazzoli, Marc Ladanyi, Lu Wang, Xi Chen, Rosana Eisenberg, Darren Cross, William Pao. Acquired resistance to afatinib plus cetuximab in EGFR-mutant lung adenocarcinoma may be mediated by EGFR overexpression and overcome by the mutant-specific EGFR inhibitor, AZD9291. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B10.

CSH Protocols, Feb 1, 2014

Resistance to targeted therapies has emerged as a major hurdle for the successful use of drugs in... more Resistance to targeted therapies has emerged as a major hurdle for the successful use of drugs in the clinic. Therefore, understanding the underlying molecular mechanisms of drug resistance is crucial for the identification of strategies to prevent and overcome it. Given the defined nature of the oncogenic lesions present in genetically engineered mouse models (GEMMs) and the relative ease of sample collection and analysis, they are ideal systems in which to recapitulate the response and subsequent emergence of resistance to targeted therapies. When agents that are very effective at eradicating tumors are used in GEMMs, obtaining drug-resistant tumors can be a challenge. One approach to generating such tumors is the use of a suboptimal intermittent dosing strategy to treat the animals, which allows for periods of tumor growth and progression in the absence of drug. This intermittent dosing strategy has been used successfully to study resistance to the tyrosine kinase erlotinib in lung cancer models and is described here. Although this protocol is specific for this experimental system, the concepts and general design can be adapted for use with GEMMs of other cancers.

Non-small cell lung cancers (NSCLC) with activating EGFR mutations respond well to the selective ... more Non-small cell lung cancers (NSCLC) with activating EGFR mutations respond well to the selective EGFR kinase inhibitors, gefitinib and erlotinib. Despite initial responses, most patients relapse within one year due to acquired resistance. Approximately half of these cases are associated with a secondary EGFR kinase domain mutation, T790M–the “gatekeeper” mutation. Irreversible EGFR kinase inhibitors have demonstrated pre-clinical efficacy toward EGFR T790M and have been undergoing clinical evaluation. However, the current irreversible inhibitors have yet to yield clinical activity in this setting, possibly due to dose-limiting toxicities. In a cancer cell line screening effort, we identified the PKC inhibitor, Go6976, a staurosporine derivative, as an inhibitor of EGFR mutant-driven NSCLC cells. Further analysis revealed that Go6976 potently suppresses both autophosphorylation and ligand-induced phosphorylation of EGFR mutants including T790M. Among some structurally-related bis-indole-based compounds currently undergoing clinical testing, PKC412, a FLT3 kinase inhibitor, demonstrated -100 fold greater potency against EGFR T790M than Go6976. Interestingly, while irreversible inhibitors are less potent against EGFR T790M than either EGFR WT or EGFR activating mutants, the bis-indole-based compounds show substantially greater potency against EGFR T790M, effectively sparing WT EGFR. Our findings suggest that this class of reversible inhibitors may be more effective and better tolerated than irreversible inhibitors to overcome EGFR T790M-mediated acquired drug resistance in NSCLC. Citation Format: Ho-June Lee, Gabriele Schaefer, Tim Heffron, Shiva Malek, Mark Merchant, Robert L. Yauch, Valentina Pirazzoli, Katerina Politi, Jeff Settleman. Non-covalent wild-type-sparing inhibitors of EGFR T790M . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-309. doi:10.1158/1538-7445.AM2013-LB-309

PDF file - 190K, The effects of combination therapy with afatinib and cetuximab

Supplementary Table 1: Tumor volume measurements during treatment Supplementary Table 2: List of ... more Supplementary Table 1: Tumor volume measurements during treatment Supplementary Table 2: List of mice with drug-resistant tumors Supplementary Table 3: List of mice that did not develop drug resistance. Tables listing mice used in the therapeutic studies showing tumor volumes and tumor volume changes through treatment.

Supplementary Figure 1: MRI images and tumor volume measurements. Supplementary Figure 2: Mouse b... more Supplementary Figure 1: MRI images and tumor volume measurements. Supplementary Figure 2: Mouse body weight during drug treatment. Supplementary Figure 3: Results of molecular studies of resistant tumors.

Clinical Cancer Research, May 15, 2012

The epidermal growth factor receptor (EGFR) T790M mutation confers acquired resistance to tyrosin... more The epidermal growth factor receptor (EGFR) T790M mutation confers acquired resistance to tyrosine kinase inhibitors (TKIs) in approximately 50% of drug-resistant EGFR mutant lung adenocarcinomas. Experiments using genetically engineered mouse models of EGFR mutant lung cancer have revealed that T790M-mediated resistance can be overcome by a second-generation TKI, afatinib, in combination with the anti-EGFR antibody, cetuximab. This drug combination is currently in clinical trials in patients with TKI-resistant tumors and is showing a promising ∼40% response rate. Nevertheless, cases of afatinib+cetuximab resistance are beginning to emerge. To identify the molecular mechanisms that play a role in afatinib+cetuximab resistance and test new therapies to overcome resistance to these drugs, we have developed mouse models of afatinib+cetuximab resistance in mice with inducible expression of the EGFRL858R+T790M mutant in type II pneumocytes. Mice harboring lung tumors were treated five days a week with afatinib and twice a week with cetuximab for four weeks. After the first round of treatment, drug-treatments were interrupted for one month. This on/off drug treatment was repeated until lung tumors no longer responded to treatment, as evidenced by imaging as well as clinical symptoms. Resistance to afatinib+cetuximab treatment was also studied in xenografts. Eight-week-old nu/nu athymic nude mice were injected subcutaneously with human lung adenocarcinoma cells, expressing the erlotinib-resistant EGFRDel19+T790M mutant. Notably, both transgenic and xenografts models harboring EGFRT790M-induced tumors developed resistance to EGFR-dual targeting and preliminary molecular studies on the resistant tumors show the absence of additional mutations in EGFR and in the ERBB2 tyrosine kinase domain in resistant tumors. These findings suggest that an alternative pathway or mechanism is involved in mediating resistance to the afatinib and cetuximab combination. We are further exploring mechanisms of resistance by performing genome-wide and signaling pathway analyses of drug-resistant tumors from these models. Findings from mouse models will be validated in repeat biopsy specimens from patients who have developed resistance to the drug combination. An improved understanding of the molecular mechanisms responsible for acquired resistance to EGFR inhibition will provide new insight into the biology of this subset of lung cancers with immediate therapeutic implications for patients.

PDF file - 248K, PKC412 is a reversible EGFR T790M inhibitor.

Purpose: The EGFR tyrosine kinase inhibitors (TKIs), erlotinib and afatinib, have transformed the... more Purpose: The EGFR tyrosine kinase inhibitors (TKIs), erlotinib and afatinib, have transformed the treatment of advanced EGFRmutant lung adenocarcinoma. However, almost all patients who respond develop acquired resistance on average approximately 1 year after starting therapy. Resistance is commonly due to a secondary mutation in EGFR (EGFR T790M). We previously found that the combination of the EGFR TKI afatinib and the EGFR antibody cetuximab could overcome EGFR T790M-mediated resistance in preclinical models. This combination has shown a 29% response rate in a clinical trial in patients with acquired resistance to first-generation TKIs. An outstanding question is whether this regimen is beneficial when used as first-line therapy. Experimental Design: Using mouse models of EGFR-mutant lung cancer, we tested whether the combination of afatinib plus cetuximab delivered upfront to mice with TKI-na€ ve EGFR L858R-induced lung adenocarcinomas delayed tumor relapse and drugresistance compared with single-agent TKIs. Results: Afatinib plus cetuximab markedly delayed the time to relapse and incidence of drug-resistant tumors, which occurred in only 63.6% of the mice, in contrast to erlotinib or afatinib treatment where 100% of mice developed resistance. Mechanisms of tumor escape observed in afatinib plus cetuximab resistant tumors include the EGFR T790M mutation and Kras mutations. Experiments in cell lines and xenografts confirmed that the afatinib plus cetuximab combination does not suppress the emergence of EGFR T790M. Conclusions: These results highlight the potential of afatinib plus cetuximab as an effective treatment strategy for patients with TKI-na€ ve EGFR-mutant lung cancer and indicate that clinical trial development in this area is warranted. Clin Cancer Res; 22(2); 426-35.

INTRODUCTION AIM OF THE WORK UROKINASE-TYPE PLASMINOGEN ACTIVATOR RECEPTOR (UPAR) uPAR expression... more INTRODUCTION AIM OF THE WORK UROKINASE-TYPE PLASMINOGEN ACTIVATOR RECEPTOR (UPAR) uPAR expression uPAR gene uPAR protein Relationship between uPAR and the Ly-6 gene family uPAR structure Species selectivity of the uPA-uPAR interaction VITRONECTIN (VN) Vitronectin expression Vitronectin gene Structure of vitronectin

Clinical Cancer Research, Jan 15, 2014

EGFR mutant lung cancers are highly sensitive to first generation EGFR tyrosine kinase inhibitors... more EGFR mutant lung cancers are highly sensitive to first generation EGFR tyrosine kinase inhibitors (TKIs; gefitinib and erlotinib), but resistance invariably develops. In the majority of patients, disease progression is mediated by a second-site T790M mutation in EGFR. T790M-mediated acquired resistance can be overcome by the combination of the second-generation EGFR TKI, afatinib, with the anti-EGFR monoclonal antibody, cetuximab, in both preclinical models and humans. However, patients still develop acquired resistance to second-line anti-EGFR combination therapy. In order to develop therapeutic strategies for patients whose tumors progress on afatinib/cetuximab, we have modeled resistance using xenografts of PC-9/BRc1 cells (EGFR exon 19 deletion/T790M) treated chronically with the drug combination. Over the course of four months, 4 of 10 animals developed acquired resistance. Xenograft tumors still displayed features of adenocarcinoma only, and mutational analysis did not detect any common secondary hotspot mutations found in lung adenocarcinomas. Analysis of two cell lines derived from resistant tumors by array comparative genomic hybridization (aCGH) suggests that resistant tumors developed additional amplification at the EGFR locus. Fluorescence in situ hybridization (FISH) of all lines confirmed sustained amplification of EGFR in resistant cell lines, and immunoblotting of cell lysates showed increased EGFR protein expression relative to parental cells and vehicle-treated controls. Strikingly, all xenograft-derived cell lines displayed in vitro sensitivity to AZD9291, a third-generation, mutant-specific EGFR TKI. Finally, in vitro comparison of afatinib/cetuximab with AZD9291 in PC-9/BRc1 cells suggests that AZD9291 may even be more potent than afatinib/cetuximab in the setting of T790M-mediated acquired resistance to first and second-generation EGFR TKIs. Confirmatory xenograft experiments are planned, and experiments are ongoing, including with additional cell lines. Collectively, these data suggest that EGFR overexpression may be a mechanism of acquired resistance to afatinib/cetuximab combination therapy, and that resistance may still be overcome by mutant-specific EGFR small molecule inhibitors. Thus, patients with EGFR mutant lung cancers may benefit from sequential lines of therapy (erlotinib->afatinib/cetuximab->AZD9291) targeting EGFR. Further work is needed to discern specific indications for afatinib/cetuximab versus AZD9291 as therapy for patients with acquired resistance to EGFR TKIs. Citation Format: Catherine B. Meador, Hailing Jin, Elisa de Stanchina, Valentina Pirazzoli, Marc Ladanyi, Lu Wang, Xi Chen, Rosana Eisenberg, Darren Cross, William Pao. Acquired resistance to afatinib plus cetuximab in EGFR-mutant lung adenocarcinoma may be mediated by EGFR overexpression and overcome by the mutant-specific EGFR inhibitor, AZD9291. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B10.

CSH Protocols, Feb 1, 2014

Resistance to targeted therapies has emerged as a major hurdle for the successful use of drugs in... more Resistance to targeted therapies has emerged as a major hurdle for the successful use of drugs in the clinic. Therefore, understanding the underlying molecular mechanisms of drug resistance is crucial for the identification of strategies to prevent and overcome it. Given the defined nature of the oncogenic lesions present in genetically engineered mouse models (GEMMs) and the relative ease of sample collection and analysis, they are ideal systems in which to recapitulate the response and subsequent emergence of resistance to targeted therapies. When agents that are very effective at eradicating tumors are used in GEMMs, obtaining drug-resistant tumors can be a challenge. One approach to generating such tumors is the use of a suboptimal intermittent dosing strategy to treat the animals, which allows for periods of tumor growth and progression in the absence of drug. This intermittent dosing strategy has been used successfully to study resistance to the tyrosine kinase erlotinib in lung cancer models and is described here. Although this protocol is specific for this experimental system, the concepts and general design can be adapted for use with GEMMs of other cancers.

Non-small cell lung cancers (NSCLC) with activating EGFR mutations respond well to the selective ... more Non-small cell lung cancers (NSCLC) with activating EGFR mutations respond well to the selective EGFR kinase inhibitors, gefitinib and erlotinib. Despite initial responses, most patients relapse within one year due to acquired resistance. Approximately half of these cases are associated with a secondary EGFR kinase domain mutation, T790M–the “gatekeeper” mutation. Irreversible EGFR kinase inhibitors have demonstrated pre-clinical efficacy toward EGFR T790M and have been undergoing clinical evaluation. However, the current irreversible inhibitors have yet to yield clinical activity in this setting, possibly due to dose-limiting toxicities. In a cancer cell line screening effort, we identified the PKC inhibitor, Go6976, a staurosporine derivative, as an inhibitor of EGFR mutant-driven NSCLC cells. Further analysis revealed that Go6976 potently suppresses both autophosphorylation and ligand-induced phosphorylation of EGFR mutants including T790M. Among some structurally-related bis-indole-based compounds currently undergoing clinical testing, PKC412, a FLT3 kinase inhibitor, demonstrated -100 fold greater potency against EGFR T790M than Go6976. Interestingly, while irreversible inhibitors are less potent against EGFR T790M than either EGFR WT or EGFR activating mutants, the bis-indole-based compounds show substantially greater potency against EGFR T790M, effectively sparing WT EGFR. Our findings suggest that this class of reversible inhibitors may be more effective and better tolerated than irreversible inhibitors to overcome EGFR T790M-mediated acquired drug resistance in NSCLC. Citation Format: Ho-June Lee, Gabriele Schaefer, Tim Heffron, Shiva Malek, Mark Merchant, Robert L. Yauch, Valentina Pirazzoli, Katerina Politi, Jeff Settleman. Non-covalent wild-type-sparing inhibitors of EGFR T790M . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-309. doi:10.1158/1538-7445.AM2013-LB-309

PDF file - 190K, The effects of combination therapy with afatinib and cetuximab

Supplementary Table 1: Tumor volume measurements during treatment Supplementary Table 2: List of ... more Supplementary Table 1: Tumor volume measurements during treatment Supplementary Table 2: List of mice with drug-resistant tumors Supplementary Table 3: List of mice that did not develop drug resistance. Tables listing mice used in the therapeutic studies showing tumor volumes and tumor volume changes through treatment.

Supplementary Figure 1: MRI images and tumor volume measurements. Supplementary Figure 2: Mouse b... more Supplementary Figure 1: MRI images and tumor volume measurements. Supplementary Figure 2: Mouse body weight during drug treatment. Supplementary Figure 3: Results of molecular studies of resistant tumors.

Clinical Cancer Research, May 15, 2012

The epidermal growth factor receptor (EGFR) T790M mutation confers acquired resistance to tyrosin... more The epidermal growth factor receptor (EGFR) T790M mutation confers acquired resistance to tyrosine kinase inhibitors (TKIs) in approximately 50% of drug-resistant EGFR mutant lung adenocarcinomas. Experiments using genetically engineered mouse models of EGFR mutant lung cancer have revealed that T790M-mediated resistance can be overcome by a second-generation TKI, afatinib, in combination with the anti-EGFR antibody, cetuximab. This drug combination is currently in clinical trials in patients with TKI-resistant tumors and is showing a promising ∼40% response rate. Nevertheless, cases of afatinib+cetuximab resistance are beginning to emerge. To identify the molecular mechanisms that play a role in afatinib+cetuximab resistance and test new therapies to overcome resistance to these drugs, we have developed mouse models of afatinib+cetuximab resistance in mice with inducible expression of the EGFRL858R+T790M mutant in type II pneumocytes. Mice harboring lung tumors were treated five days a week with afatinib and twice a week with cetuximab for four weeks. After the first round of treatment, drug-treatments were interrupted for one month. This on/off drug treatment was repeated until lung tumors no longer responded to treatment, as evidenced by imaging as well as clinical symptoms. Resistance to afatinib+cetuximab treatment was also studied in xenografts. Eight-week-old nu/nu athymic nude mice were injected subcutaneously with human lung adenocarcinoma cells, expressing the erlotinib-resistant EGFRDel19+T790M mutant. Notably, both transgenic and xenografts models harboring EGFRT790M-induced tumors developed resistance to EGFR-dual targeting and preliminary molecular studies on the resistant tumors show the absence of additional mutations in EGFR and in the ERBB2 tyrosine kinase domain in resistant tumors. These findings suggest that an alternative pathway or mechanism is involved in mediating resistance to the afatinib and cetuximab combination. We are further exploring mechanisms of resistance by performing genome-wide and signaling pathway analyses of drug-resistant tumors from these models. Findings from mouse models will be validated in repeat biopsy specimens from patients who have developed resistance to the drug combination. An improved understanding of the molecular mechanisms responsible for acquired resistance to EGFR inhibition will provide new insight into the biology of this subset of lung cancers with immediate therapeutic implications for patients.

PDF file - 248K, PKC412 is a reversible EGFR T790M inhibitor.