Sandra Ortiz-Cuaran

Rationale for co-targeting IGF-1R and ALK in ALK fusion-positive lung cancer

Crizotinib, a selective tyrosine kinase inhibitor (TKI), shows marked activity in patients whose lung cancers harbor fusions in the gene encoding anaplastic lymphoma receptor tyrosine kinase (ALK), but its efficacy is limited by variable primary responses and acquired resistance. In work arising from the clinical observation of a patient with ALK fusion–positive lung cancer who had an exceptional response to an insulin-like growth factor 1 receptor (IGF-1R)-specific antibody, we define a therapeutic synergism between ALK and IGF-1R inhibitors. Similar to IGF-1R, ALK fusion proteins bind to the adaptor insulin receptor substrate 1 (IRS-1), and IRS-1 knockdown enhances the antitumor effects of ALK inhibitors. In models of ALK TKI resistance, the IGF-1R pathway is activated, and combined ALK and IGF-1R inhibition improves therapeutic efficacy. Consistent with this finding, the levels of IGF-1R and IRS-1 are increased in biopsy samples from patients progressing on crizotinib monotherapy. Collectively these data support a role for the IGF-1R–IRS-1 pathway in both ALK TKI–sensitive and ALK TKI–resistant states and provide a biological rationale for further clinical development of dual ALK and IGF-1R inhibitors.

CD74-NRG1 fusions in lung adenocarcinoma

UNLABELLED We discovered a novel somatic gene fusion, CD74-NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74-NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-β3, thereby providing the ligand for ERBB2-ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74-NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K-AKT pathway, and led to increased colony formation in soft agar. Thus, CD74-NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment. SIGNIFICANCE CD74–NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease.

Heterogeneous Mechanisms of Primary and Acquired Resistance to Third-Generation EGFR Inhibitors.

Purpose: To identify novel mechanisms of resistance to third-generation EGFR inhibitors in patients with lung adenocarcinoma that progressed under therapy with either AZD9291 or rociletinib (CO-1686). Experimental Design: We analyzed tumor biopsies from seven patients obtained before, during, and/or after treatment with AZD9291 or rociletinib (CO-1686). Targeted sequencing and FISH analyses were performed, and the relevance of candidate genes was functionally assessed in in vitro models. Results: We found recurrent amplification of either MET or ERBB2 in tumors that were resistant or developed resistance to third-generation EGFR inhibitors and show that ERBB2 and MET activation can confer resistance to these compounds. Furthermore, we identified a KRASG12S mutation in a patient with acquired resistance to AZD9291 as a potential driver of acquired resistance. Finally, we show that dual inhibition of EGFR/MEK might be a viable strategy to overcome resistance in EGFR-mutant cells expressing mutant KRAS. Conclusions: Our data suggest that heterogeneous mechanisms of resistance can drive primary and acquired resistance to third-generation EGFR inhibitors and provide a rationale for potential combination strategies. Clin Cancer Res; 22(19); 4837–47. ©2016 AACR.

Targeting Drug Resistance in EGFR with Covalent Inhibitors: A Structure-Based Design Approach.

Receptor tyrosine kinases represent one of the prime targets in cancer therapy, as the dysregulation of these elementary transducers of extracellular signals, like the epidermal growth factor receptor (EGFR), contributes to the onset of cancer, such as non-small cell lung cancer (NSCLC). Strong efforts were directed to the development of irreversible inhibitors and led to compound CO-1686, which takes advantage of increased residence time at EGFR by alkylating Cys797 and thereby preventing toxic effects. Here, we present a structure-based approach, rationalized by subsequent computational analysis of conformational ligand ensembles in solution, to design novel and irreversible EGFR inhibitors based on a screening hit that was identified in a phenotype screen of 80 NSCLC cell lines against approximately 1500 compounds. Using protein X-ray crystallography, we deciphered the binding mode in engineered cSrc (T338M/S345C), a validated model system for EGFR-T790M, which constituted the basis for further rational design approaches. Chemical synthesis led to further compound collections that revealed increased biochemical potency and, in part, selectivity toward mutated (L858R and L858R/T790M) vs nonmutated EGFR. Further cell-based and kinetic studies were performed to substantiate our initial findings. Utilizing proteolytic digestion and nano-LC-MS/MS analysis, we confirmed the alkylation of Cys797.

Systematic Kinase Inhibitor Profiling Identifies CDK9 as a Synthetic Lethal Target in NUT Midline Carcinoma

Summary Kinase inhibitors represent the backbone of targeted cancer therapy, yet only a limited number of oncogenic drivers are directly druggable. By interrogating the activity of 1,505 kinase inhibitors, we found that BRD4-NUT-rearranged NUT midline carcinoma (NMC) cells are specifically killed by CDK9 inhibition (CDK9i) and depend on CDK9 and Cyclin-T1 expression. We show that CDK9i leads to robust induction of apoptosis and of markers of DNA damage response in NMC cells. While both CDK9i and bromodomain inhibition over time result in reduced Myc protein expression, only bromodomain inhibition induces cell differentiation and a p21-induced cell-cycle arrest in these cells. Finally, RNA-seq and ChIP-based analyses reveal a BRD4-NUT-specific CDK9i-induced perturbation of transcriptional elongation. Thus, our data provide a mechanistic basis for the genotype-dependent vulnerability of NMC cells to CDK9i that may be of relevance for the development of targeted therapies for NMC patients.

LSD1 modulates the non-canonical integrin β3 signaling pathway in non-small cell lung carcinoma cells

The epigenetic writer lysine-specific demethylase 1 (LSD1) is aberrantly upregulated in many cancer types and its overexpression correlates with poor survival and tumor progression. In this study, we analysed LSD1 function in non-small cell lung cancer adenocarcinomas. Expression profiling of 182 cases of lung adenocarcinoma proved a significant correlation of LSD1 overexpression with lung adenocarcinoma progression and metastasis. KRAS-mutated lung cancer cell clones were stably silenced for LSD1 expression. RNA-seq and comprehensive pathway analysis revealed, that genes related to a recently described non-canonical integrin β3 pathway, were significantly downregulated by LSD1 silencing. Hence, invasion and self-renewal capabilities were strongly decreased. Notably, this novel defined LSD1/integrin β3 axis, was also detected in human lung adenocarcinoma specimens. Furthermore, the linkage of LSD1 to an altered expression pattern of lung-lineage specific transcription factors and genes, which are involved in alveolar epithelial differentiation, was demonstrated. Thus, our findings point to a LSD1-integrin β3 axis, conferring attributes of invasiveness and tumor progression to lung adenocarcinoma.

Prognostic and predictive role of CD8 and PD-L1 determination in lung tumor tissue of patients under anti-PD-1 therapy

BackgroundNo study has evaluated the predictive and prognostic role of CD8 and PD-L1 coexpression in non–small-cell lung cancer (NSCLC).MethodsWe analyzed RNA sequencing and/or immunohistochemistry staining in NSCLC patients from The Cancer Genome Atlas (n = 1016), and 34 metastatic NSCLC samples not treated by immunotherapy as prognostic cohorts. As predictive aspect of CD8 and PD-L1, we used 85 NSCLC patients treated with anti-PD-1. Two validation cohorts were used including 44 NSCLC patients treated with anti-PD-1 and an external cohort with different tumor types.ResultsIn prognostic cohorts, high CD8A expression was associated with longer OS (p = 0.02), while high CD274 mRNA was associated with poor prognosis (p = 0.05). In predictive cohort, high CD8 expression and CD8A mRNA were associated with longer progression-free survival (PFS) (p = 0.0002). There was no significant association between PD-L1 expression and PFS while high CD274 mRNA was associated with longer PFS (p = 0.009). A combination of CD8A and CD274 was highly predictive of outcome. These results were confirmed in the validation cohorts. This two-genes signature demonstrated similar results compared to gold standard signatures.ConclusionCD8 represents both a prognostic and predictive factor of outcomes, while PD-L1 share different prognostic and predictive roles.

Abstract 956: Elucidating the mechanisms of acquired resistance in lung adenocarcinomas

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In lung adenocarcinomas, targeted therapy with the EGFR tyrosine kinase inhibitors (TKIs) erlotinib, gefitinib and afatinib is associated with longer progression free survival (PFS) and higher radiographic response (RR) rates when compared to standard first-line chemotherapy. In ALK rearranged lung cancers, targeted therapy with crizotinib is associated with PFS of approximately 9,7 months and RR of 60.8%. However, despite the initial success of these agents, all patients progress with a median PFS of 7 to 16 months. Acquired resistance in EGFR mutant tumors is driven by the occurrence of a secondary EGFR mutation (T790M) in about 50% of the cases and by MET amplification in 5 to 10 % of the cases. Other mechanisms include HER2 amplification, PTEN loss, phenotypic change to small cell histology, rare mutations in BRAF and AXL activation. Resistance to crizotinib, on the other hand, is caused by secondary mutations in the ALK kinase domain, by ALK or cKIT amplification or by alterations in EGFR and KRAS. Here, we made use of next generation sequencing techniques to better understand the mechanisms that drive resistance in lung adenocarcinomas treated with erlotinib or crizotinib. For this purpose, we used transbronchial or CT-guided rebiopsies from patients that had either prolonged stable disease or partial response to therapy, and developed radiographic progression under TKI therapy. Samples were analyzed by FISH and sequenced on a benchtop Illumina platform (MiSeq) in order to evaluate the presence of known mechanisms of resistance. Samples that were negative for any of the reported mechanisms were analyzed by genome, exome or trascriptome sequencing. From the sequencing output of the pan-negative samples, filtering of mutation candidates included: absence of the mutation in the pre-treatment sample (when available), expression of the candidate gene in lung adenocarcinomas, absence of the mutation in primary lung adenocarcinomas, high impact of the mutation at protein level (Polyphen), mutant allelic fraction in the tumor higher than 10%, among other factors. After filtering, validation of mutation calls was performed by Sanger sequencing. Sequencing of the erlotinib resistant samples revealed mutations in members of a functionally wide spectrum of protein families including the proteoglycan family, the ATP-binding cassette (ABC) transporters family, an Fms-related tyrosine kinase receptor and a member of the transforming growth factor beta family of cytokines. On the other hand, crizotinib resistant samples showed mutations in a cell surface receptor for macrophage-stimulating protein with tyrosine kinase activity, in a C2H2 type zinc finger gene, a semaphorin, a mitogen-activated protein kinase and a member of the SWI/SNF family of proteins. Our results evidence the possible contribution of a wide range of cellular pathways in the process of acquired resistance to EGFR and ALK inhibitors in lung adenocarcinomas. Citation Format: Sandra Ortiz-Cuaran, Lynnette Fernandez-Cuesta, Marc Bos, Lukas Heukamp, Christine M. Lovly, Martin Peifer, Masyar Gardizi, Matthias Scheffler, Ilona Dahmen, Christian Muller, Katharina Konig, Kerstin Albus, Alexandra Florin, Sascha Ansen, Reinhard Buettner, Jurgen Wolf, William Pao, Roman K. Thomas. Elucidating the mechanisms of acquired resistance in lung adenocarcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 956. doi:10.1158/1538-7445.AM2014-956

Abstract 1690: Attacking EGFR mutant lung cancer by combined EGFR and c-Met inhibition

In lung adenocarcinomas, targeted therapy with the EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib is associated with longer progression free survival (PFS). However, despite the initial success, all patients progress with a median PFS of 12 to 16 months. Acquired resistance is driven by the occurrence of a secondary EGFR mutation (T790M) in about 50% of the cases and by c-Met amplification in 5 to 10 % of the cases. We report the activity of a new reversible and ATP-competitive c-Met inhibitor, EMD1214063, in the setting of primary lung adenocarcinomas harboring EGFR activating mutations and in two models of acquired resistance. EMD1214063 showed to be specifically active in c-Met amplified and c-Met dependent cells in a large panel of genotypically characterized lung cancer cell lines. In the context of acquired resistance, we studied the effect of EMD1214063 alone and in combination of afatinib, an irreversible EGFR TKI, in PC9 cells (EGFR Exon19del), H1975 cells (EGFR L858R and T790M), HCC827 (EGFR Exon19del, not c-Met amplified) and in HCC827GR cells (EGFR Exon19del and c-Met amplified). Treatment of HCC827GR cells with EMD1214063 resulted in substantial growth inhibition and induced apoptosis. The in vitro results showed a significant synergistic effect of the combination of EMD121039 and afatinib in the induction of growth inhibition in the context of c-Met amplification and EGFR T790M mutation. Combination treatment robustly suppressed expression of downstream pErk and pAkt, showing efficient suppression of PI3K and MAPK signaling. To determine whether the antitumor activity of the combination observed in vitro might also be apparent in vivo, we injected these cells in nude mice to elicit the formation of solid tumors. Mice were treated with EMD121043 (25mg/kg), afatinib (10mg/kg) or the combination of both. In H1975 xenografts only the combination therapy could significantly reduce tumor growth, thus confirming the results obtained in vitro. In the c-Met amplified setting, we treated mice harboring HCC827, HCC827GR and mixed xenografts. HCC827-driven tumors strongly responded to both afatinib monotherapy and to the combination, leading to complete remission within 40 days of therapy. HCC827/HCC827GR mixed xenografts containing 0.1%, 1% and 10% of HCC827GR cells partially responded to afatinib monotherapy; however, growth was completely abolished by combination therapy. Treatment with EMD1214063 led to significant growth reduction in mice harboring HCC827GR tumors. In this model, only the combination therapy led to massive tumor shrinkage. Assessment of cell proliferation in vivo by [18F]FLT-PET showed a decline in FLT-uptake of 40% in both the combination therapy and the EMD1214063 monotherapy. In conclusion, the combination of EMD1214063 and afatinib promote tumor regression in erlotinib acquired resistant lung cancers driven by EGFR L858R/T790M or EGFR Exon19 del/c-Met amplification. Citation Format: Sandra Ortiz-Cuaran, Jakob Schottle, Ilona Dahmen, Martin Peifer, Caroline Wieczoreck, Mirjam Koker, Michaela A. Ihle, Alexandra Florin, Berit Pinther, Lukas C. Heukamp, Roland T. Ullrich, Roman K. Thomas. Attacking EGFR mutant lung cancer by combined EGFR and c-Met inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1690. doi:10.1158/1538-7445.AM2014-1690