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Purpose: Patients with anaplastic lymphoma kinase (ALK) gene rearrangements often manifest dramatic responses to crizotinib, a small-molecule ALK inhibitor. Unfortunately, not every patient responds and acquired drug resistance inevitably develops in those who do respond. This study aimed to define molecular mechanisms of resistance to crizotinib in patients with ALK(+) non-small cell lung cancer (NSCLC)., Experimental Design: We analyzed tissue obtained from 14 patients with ALK(+) NSCLC showing evidence of radiologic progression while on crizotinib to define mechanisms of intrinsic and acquired resistance to crizotinib., Results: Eleven patients had material evaluable for molecular analysis. Four patients (36%) developed secondary mutations in the tyrosine kinase domain of ALK. A novel mutation in the ALK domain, encoding a G1269A amino acid substitution that confers resistance to crizotinib in vitro, was identified in two of these cases. Two patients, one with a resistance mutation, exhibited new onset ALK copy number gain (CNG). One patient showed outgrowth of epidermal growth factor receptor (EGFR) mutant NSCLC without evidence of a persistent ALK gene rearrangement. Two patients exhibited a KRAS mutation, one of which occurred without evidence of a persisting ALK gene rearrangement. One patient showed the emergence of an ALK gene fusion-negative tumor compared with the baseline sample but with no identifiable alternate driver. Two patients retained ALK positivity with no identifiable resistance mechanism., Conclusions: Crizotinib resistance in ALK(+) NSCLC occurs through somatic kinase domain mutations, ALK gene fusion CNG, and emergence of separate oncogenic drivers.

Angiogenesis inhibition is an important therapeutic strategy for advanced stage prostate cancer. Previous work from our laboratory showed that sustained stimulation of Rap1 by 8-pCPT-2'-O-Me-cAMP (8CPT) via activation of Epac, a Rap1 GEF, or by expression of a constitutively active Rap1 mutant (cRap1) suppresses endothelial cell chemotaxis and subsequent angiogenesis. When we tested this model in the context of a prostate tumor xenograft, we found that 8CPT had no significant effect on prostate tumor growth alone. However, in cells harboring cRap1, 8CPT dramatically inhibited not only prostate tumor growth but also VEGF expression and angiogenesis within the tumor microenvironment. Subsequent analysis of the mechanism revealed that, in prostate tumor epithelial cells, 8CPT acted via stimulation of PKA rather than Epac/Rap1. PKA antagonizes Rap1 and hypoxic induction of 1α protein expression, VEGF production and, ultimately, angiogenesis. Together these findings provide evidence for a novel interplay between Rap1, Epac, and PKA that regulates tumor-stromal induction of angiogenesis.

Background: ALK gene rearrangement defines a new molecular subtype of non-small-cell lung cancer (NSCLC). In a recent phase 1 clinical trial, the ALK tyrosine-kinase inhibitor (TKI) crizotinib showed marked antitumour activity in patients with advanced, ALK-positive NSCLC. To assess whether crizotinib affects overall survival in these patients, we did a retrospective study comparing survival outcomes in crizotinib-treated patients in the trial and crizotinib-naive controls screened during the same time period., Methods: We examined overall survival in patients with advanced, ALK-positive NSCLC who enrolled in the phase 1 clinical trial of crizotinib, focusing on the cohort of 82 patients who had enrolled through Feb 10, 2010. For comparators, we identified 36 ALK-positive patients from trial sites who were not given crizotinib (ALK-positive controls), 67 patients without ALK rearrangement but positive for EGFR mutation, and 253 wild-type patients lacking either ALK rearrangement or EGFR mutation. To assess differences in overall survival, we assessed subsets of clinically comparable ALK-positive and ALK-negative patients., Findings: Among 82 ALK-positive patients who were given crizotinib, median overall survival from initiation of crizotinib has not been reached (95% CI 17 months to not reached); 1-year overall survival was 74% (95% CI 63-82), and 2-year overall survival was 54% (40-66). Overall survival did not differ based on age, sex, smoking history, or ethnic origin. Survival in 30 ALK-positive patients who were given crizotinib in the second-line or third-line setting was significantly longer than in 23 ALK-positive controls given any second-line therapy (median overall survival not reached [95% CI 14 months to not reached] vs 6 months [4-17], 1-year overall survival 70% [95% CI 50-83] vs 44% [23-64], and 2-year overall survival 55% [33-72] vs 12% [2-30]; hazard ratio 0·36, 95% CI 0·17-0·75; p=0·004). Survival in 56 crizotinib-treated, ALK-positive patients was similar to that in 63 ALK-negative, EGFR-positive patients given EGFR TKI therapy (median overall survival not reached [95% CI 17 months to not reached] vs 24 months [15-34], 1-year overall survival 71% [95% CI 58-81] vs 74% [61-83], and 2-year overall survival 57% [40-71] vs 52% [38-65]; p=0·786), whereas survival in 36 crizotinib-naive, ALK-positive controls was similar to that in 253 wild-type controls (median overall survival 20 months [95% CI 13-26] vs 15 months [13-17]; p=0·244)., Interpretation: In patients with advanced, ALK-positive NSCLC, crizotinib therapy is associated with improved survival compared with that of crizotinib-naive controls. ALK rearrangement is not a favourable prognostic factor in advanced NSCLC., Funding: Pfizer Inc, V Foundation for Cancer Research., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Hypothesis: To explore whether the progression-free survival (PFS) with pemetrexed differs between anaplastic lymphoma kinase (ALK)-positive and other major molecular subtypes of non-small cell lung cancer., Methods: In an ALK-enriched population, patients with metastatic non-small cell lung cancer were screened by ALK fluorescence in situ hybridization and for epidermal growth factor receptor (EGFR) and KRAS mutations. Triple-tested, pemetrexed-treated patients (monotherapy or combination therapy) were identified and PFS with pemetrexed captured retrospectively., Results: Eighty-nine eligible cases were identified (19 ALK fluorescence in situ hybridization positive, 12 EGFR mutant, 21 KRAS mutant, and 37 triple negatives). Eighty-three cases (93%) were adenocarcinomas, two were adenosquamous, one squamous, and three had large cell histology. None of the ALK-positive patients had received crizotinib before pemetrexed. Pemetrexed was first-line therapy in 48% (72% as platinum-based combinations). Median PFS (95% confidence interval) data were EGFR mutant (5.5 months; 1-9), KRAS mutant (7 months; 1.5-10), ALK positive (9 months; 3-12), and triple negative (4 months; 3-5). In a multivariate analysis adjusting for line of therapy, mono- versus platinum and nonplatinum combination therapy, age, sex, histology, and smoking status, the only variable associated with prolonged PFS on pemetrexed was ALK+ (hazard ratio = 0.36 [95% confidence interval: 0.17-0.73], p = 0.0051)., Conclusions: In this exploratory analysis, ALK-positive patients have a significantly longer PFS on pemetrexed compared with triple-negative patients, whereas EGFR or KRAS mutant patients do not. This information should be considered when sizing randomized studies in ALK-positive patients that involve pemetrexed. Pemetrexed should also be prioritized as a cytotoxic to explore further in patients with known ALK-positive disease.

Despite initial and sometimes dramatic responses of specific NSCLC tumors to EGFR TKIs, nearly all will develop resistance and relapse. Gene expression analysis of NSCLC cell lines treated with the EGFR TKI, gefitinib, revealed increased levels of FGFR2 and FGFR3 mRNA. Analysis of gefitinib action on a larger panel of NSCLC cell lines verified that FGFR2 and FGFR3 expression is increased at the mRNA and protein level in NSCLC cell lines in which the EGFR is dominant for growth signaling, but not in cell lines where EGFR signaling is absent. A luciferase reporter containing 2.5 kilobases of fgfr2 5' flanking sequence was activated after gefitinib treatment, indicating transcriptional regulation as a contributing mechanism controlling increased FGFR2 expression. Induction of FGFR2 and FGFR3 protein as well as fgfr2-luc activity was also observed with Erbitux, an EGFR-specific monoclonal antibody. Moreover, inhibitors of c-Src and MEK stimulated fgfr2-luc activity to a similar degree as gefitinib, suggesting that these pathways may mediate EGFR-dependent repression of FGFR2 and FGFR3. Importantly, our studies demonstrate that EGFR TKI-induced FGFR2 and FGFR3 are capable of mediating FGF2 and FGF7 stimulated ERK activation as well as FGF-stimulated transformed growth in the setting of EGFR TKIs. In conclusion, this study highlights EGFR TKI-induced FGFR2 and FGFR3 signaling as a novel and rapid mechanism of acquired resistance to EGFR TKIs and suggests that treatment of NSCLC patients with combinations of EGFR and FGFR specific TKIs may be a strategy to enhance efficacy of single EGFR inhibitors.

Purpose: Anaplastic lymphoma kinase (ALK) rearrangements, associated with sensitivity to an experimental ALK/MET inhibitor, occur in 3% to 5% of non-small cell lung cancers. Intratumoral fluorescence in situ hybridization (FISH) heterogeneity has been reported. We explored the heterogeneity basis, the requirements for accurately determining ALK FISH positivity, and the effect of enriching the tested population using clinical and molecular factors., Experimental Design: Lung cancer patients were screened by ALK and MET FISH and for EGFR and KRAS mutations., Results: Thirteen ALK-positive cases were identified from 73 screened patients. Gene copy number increases occurred together with classic rearrangements. All positive cases were adenocarcinomas, 12 were EGFR/KRAS wild-type, and 1 had a coexistent EGFR exon 20 mutation. No association with MET amplification occurred. ALK positivity was associated with <10-pack-year smoking status (P = 0.0004). Among adenocarcinomas, without KRAS or EGFR mutations, with <10-pack-year history, 44.8% of cases were ALK positive. ALK FISH positivity was heterogeneous, but mean values in tumor areas from ALK-positive patients (54% of cells; range, 22-87%) were significantly higher than in adjacent normal tissue or tumor/normal areas from ALK-negative patients (mean, 5-7%). Contiguous sliding field analyses showed diffuse heterogeneity without evidence of focal ALK rearrangements. One hundred percent sensitivity and specificity occurred when four or more fields (∼60 cells) were counted., Conclusions: Intratumoral ALK FISH heterogeneity reflects technique, not biology. The clinical activity of ALK/MET inhibitors in ALK-positive patients probably reflects ALK, but not MET, activity. Prescreening by histology, EGFR/KRAS mutations, and smoking status dramatically increases the ALK-positive hit rate compared with unselected series., (©2010 AACR.)

The epidermal growth factor receptor (EGFR), a member of the HER family of receptors, has become a well-established target for the treatment of patients with non-small cell lung cancer (NSCLC). Several EGFR-targeted agents produce objective responses in a minority of unselected patients, but a majority of those with EGFR-activating mutations; however, all responders eventually develop resistance. The modest activity of agents that target only EGFR may be due, in part, to the complexity and interdependency of HER family signaling. The interdependent signaling that occurs between EGFR and HER2 provides a rationale for the simultaneous inhibition of these receptors with reversible and irreversible inhibitors. Several agents with activity against both EGFR and HER2 are currently under development. Irreversible EGFR/HER2 tyrosine kinase inhibitors (TKIs) (e.g., BIBW 2992, HKI-272) and pan-HER TKIs (e.g., PF00299804) comprise a novel class of agents in clinical development that may prevent and overcome inherent and acquired resistance to first-generation reversible EGFR TKIs. Other agents in development include the monoclonal antibody pertuzumab, and XL-647, which inhibits EGFR and HER2, as well as multiple vascular endothelial growth factor receptor family members. Here we briefly review the currently available EGFR-targeted agents, discuss the rationale for extending inhibition to other HER family members, weigh the merits of irreversible HER family inhibition, and summarize preclinical and clinical data with EGFR/HER2 and pan-HER inhibitors under clinical development., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

Tumors depend upon angiogenesis for growth and metastasis. It is therefore critical to understand the inhibitory signaling mechanisms in endothelial cells that control angiogenesis. Epac is a cyclic adenosine 5'-monophosphate-activated guanine nucleotide exchange factor for Rap1. In this study, we show that activation of Epac or Rap1 leads to potent inhibition of angiogenesis in vivo. Epac/Rap1 activation down-regulates inhibitor of differentiation 1 (Id1), which negatively regulates thrombospondin-1 (TSP1), an inhibitor of angiogenesis. Consistent with this mechanism, activation of Epac/Rap 1 induces expression of TSP1; conversely, depletion of Epac reduces TSP1 levels in endothelial cells. Blockade of TSP1 binding to its receptor, CD36, rescues inhibition of chemotaxis or angiogenesis by activated Epac/Rap1. Mitogen-activated protein kinase kinase 5, a downstream mediator of vascular endothelial growth factor, antagonizes the effects of Epac/Rap1 by inducing Id1 and suppressing TSP1 expression. Finally, TSP1 is also secreted by fibroblasts in response to Epac/Rap1 activation. These results identify Epac and Rap1 as inhibitory regulators of the angiogenic process, implicate Id1 and TSP1 as downstream mediators of Epac/Rap1, and highlight a novel interplay between pro- and antiangiogenic signaling cascades involving multiple cell types within the angiogenic microenvironment.

Angiogenesis involves the assembly of endothelial cells into capillaries from a pre-existing vasculature. Because abnormal angiogenesis is a hallmark of many cancers, it is critical to find factors that control this process. Endothelial cells are enriched in the anthrax receptor; we therefore determined the effect of anthrax edema toxin (ET), an adenylyl cyclase, on chemotaxis. cAMP generated by ET does not block proliferation or survival but causes cytoskeletal changes and inhibits chemotaxis by primary human microvascular endothelial cells (HMVECs). These effects are due to the action of a downstream cAMP effector, Epac, a guanine nucleotide exchange-activating protein for Rap1 (RAP1-GEF). ET induces transcription of Epac-related activators of Rap1, Epac2 (RapGEF4), and MR-GEF/RapGEF5. Similar to ET, activated Epac or Rap1 induces cytoskeletal changes and blocks chemotaxis in human endothelial cells. These results identify Epac and Rap1 as key regulators of signaling cascades leading to endothelial cell chemotaxis.

HLA-DM (DM) edits major histocompatibility complex class II (MHCII)-bound peptides in endocytic compartments and stabilizes empty MHCII molecules. Crystal structures of DM have revealed similarity to MHCII but not how DM and MHCII interact. We used mutagenesis to map a MHCII-interacting surface on DM. Mutations on this surface impair DM action on HLA-DR and -DP in cells and DM-dependent peptide loading in vitro. The orientation of DM and MHCII predicted by these studies guided design of soluble DM and DR molecules fused to leucine zippers via their beta chains, resulting in stable DM/DR complexes. Peptide release from the complexes was fast and only weakly sequence dependent, arguing that DM diminishes the selectivity of the MHCII groove. Analysis of soluble DM action on soluble DR/peptide complexes corroborates this conclusion.

During maturation of MHC II molecules, newly synthesized and assembled complexes of MHC II alphabeta dimers with invariant chain (Ii) are targeted to endosomes, where Ii is proteolyzed, leaving remnant class II-associated Ii peptides (CLIP) in the MHC II peptide binding groove. CLIP must be released, usually with assistance from the endosomal MHC II peptide exchange factor, HLA-DM, before MHC II molecules can bind endosomal peptides. Structural factors that control rates of CLIP release remain poorly understood, although peptide side chain-MHC II specificity pocket interactions and MHC II polymorphism are important. Here we report that mutations betaS11F, betaS13Y, betaQ70R, betaK71E, betaK71N, and betaR74Q, which map to the P4 and P6 pockets of the groove of HLA-DR3 molecules, as well as alphaG20E adjacent to the groove, are associated with elevated CLIP in cells. Most of these mutations increase the resistance of CLIP-DR3 complexes to dissociation by SDS. In vitro, the groove mutations increase the stability of CLIP-DR3 complexes to dissociation. Dissociation rates in the presence of DM, as well as coimmunoprecipitation of some mutant DR3 molecules with DM, are also diminished. The profound phenotypes associated with some of these point mutations suggest that the need to maintain efficient CLIP release represents a constraint on naturally occurring MHC II polymorphism.