Important advancements in the treatment of non-small cell lung cancer (NSCLC) have been achieved over the past two decades, increasing our understanding of the disease biology and mechanisms of tumour progression, and advancing early detection and multimodal care. The use of small molecule tyrosine kinase inhibitors and immunotherapy has led to unprecedented survival benefits in selected patients. However, the overall cure and survival rates for NSCLC remain low, particularly in metastatic disease. Therefore, continued research into new drugs and combination therapies is required to expand the clinical benefit to a broader patient population and to improve outcomes in NSCLC.

The biology and management of non-small cell lung cancer

A main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), is effective in a subset of KRAS wild-type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here we show that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance, but resistant cells remained sensitive to combinatorial inhibition of EGFR and mitogen-activated protein-kinase kinase (MEK). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

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Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer

2458 Despite dramatic improvements in treatment over the past 40 years, acute lymphoblastic leukemia (ALL) remains one of the most common causes of death from disease in childhood. Glucocorticoids are among the most effective agents used in the treatment of lymphoid malignancies, and patient response to treatment is an important determinant of long-term outcome in childhood ALL. In spite of its clinical significance, the molecular basis of glucocorticoid resistance is still poorly understood. The aim of this study was to develop an experimental model system to define clinically relevant mechanisms of glucocorticoid resistance in childhood ALL. An in vivo model of childhood ALL has been developed in our laboratory, using patient biopsies established as xenografts in immune-deficient nonobese diabetic severe-combined immunodeficient (NOD/SCID) mice. This model is highly representative of the human disease (Lock et al., Blood, 99: 4100-4108, 2002). The in vivo responses of these xenografts to the glucocorticoid dexamethasone (DEX) correlated significantly with patient outcome (p 1 μM) in xenografts from six patients, five of whom died of their disease. In contrast, four DEX-sensitive xenografts (IC50 values 2-fold in sensitive xenografts within 8 hours of treatment. In contrast, Bim induction was dramatically attenuated in DEX-resistant xenografts. These results have identified a clinically significant and novel mechanism of glucocorticoid resistance in childhood ALL, which occurs downstream of receptor-ligand interactions, but upstream of the signalling pathway resulting in Bim induction and apoptosis.

Novel mechanisms of glucocorticoid resistance in childhood acute lymphoblastic leukemia.

The first cancer-targeted microRNA (miRNA) drug - MRX34, a liposome-based miR-34 mimic - entered Phase I clinical trials in patients with advanced hepatocellular carcinoma in April 2013, and miRNA therapeutics are attracting special attention from both academia and biotechnology companies. Although miRNAs are the most studied non-coding RNAs (ncRNAs) to date, the importance of long non-coding RNAs (lncRNAs) is increasingly being recognized. Here, we summarize the roles of miRNAs and lncRNAs in cancer, with a focus on the recently identified novel mechanisms of action, and discuss the current strategies in designing ncRNA-targeting therapeutics, as well as the associated challenges.

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MicroRNAs and other non-coding RNAs as targets for anticancer drug development

The ErbB/HER family of protein-tyrosine kinases and cancer.

EGFR is a tyrosine kinase that participates in the regulation of cellular homeostasis. Following ligand binding, EGFR stimulates downstream cell signaling cascades that influence cell proliferation, apoptosis, migration, survival and complex processes, including angiogenesis and tumorigenesis. EGFR has been strongly implicated in the biology of human epithelial malignancies, with therapeutic applications in cancers of the colon, head and neck, lung, and pancreas. Accordingly, targeting EGFR has been intensely pursued, with the development of a series of promising molecular inhibitors for use in clinical oncology. As is common in cancer therapy, challenges with respect to treatment resistance emerge over time. This situation is certainly true of EGFR inhibitor therapies, where intrinsic and acquired resistance is now well recognized. In this Review, we provide a brief overview regarding the biology of EGFR, preclinical and clinical development of EGFR inhibitors, and molecular mechanisms that underlie the development of treatment resistance. A greater understanding of the mechanisms that lead to EGFR resistance may provide valuable insights to help design new strategies that will enhance the impact of this promising class of inhibitors for the treatment of cancer.

Understanding resistance to EGFR inhibitors-impact on future treatment strategies.

The proto-oncogene c-Src (Src) is a nonreceptor tyrosine kinase whose expression and activity is correlated with advanced malignancy and poor prognosis in a variety of human cancers. Nine additional enzymes with homology to Src have been identified and collectively are referred to as the Src family kinases (SFKs). Together, SFKs represent the largest family of nonreceptor tyrosine kinases and interact directly with receptor tyrosine kinases, G-protein-coupled receptors, steroid receptors, signal transducers and activators of transcription and molecules involved in cell adhesion and migration. These interactions lead to a diverse array of biological functions including proliferation, cell growth, differentiation, cell shape, motility, migration, angiogenesis, and survival. Studies investigating mutational activation of Src in human cancers suggest this may be a rare event and wild-type Src is weakly oncogenic. Thus, the role of Src in the development and progression of human cancer has remained unclear. Recently, it has been suggested that increased SFK protein levels and, more importantly, SFK tyrosine kinase activity is linked to cancer progression and metastatic disease by facilitating the action of other signaling proteins. This accumulating body of evidence indicates that SFKs may represent a promising therapeutic target for the treatment of solid tumors. This review discusses the role of SFKs in solid tumors and the recent therapeutic advances aimed at targeting this family of tyrosine kinases in cancer.

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The role of Src in solid tumors.

Epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase involved in the etiology of several human cancers. Cetuximab is an EGFR-blocking antibody that has been approved for the treatment of patients with head and neck squamous cell carcinoma and metastatic colorectal cancer. Previous reports have shown that EGFR translocation to the nucleus is associated with cell proliferation. Here we investigated mechanisms of acquired resistance to cetuximab using a model derived from the non-small cell lung cancer line H226. We demonstrated that cetuximab-resistant cells overexpress HER family ligands including epidermal growth factor (EGF), amphiregulin, heparin-binding EGF and β-cellulin. Overexpression of these ligands is associated with the nuclear translocation of the EGFR and this process was mediated by the Src family kinases (SFK). Treatment of cetuximab-resistant cells with the SFK inhibitor, dasatinib, resulted in loss of nuclear EGFR, increased membrane expression of the EGFR and resensitization to cetuximab. In addition, expression of a nuclear localization sequence-tagged EGFR in cetuximab-sensitive cells increased resistance to cetuximab both in vitro and in mouse xenografts. Collectively, these data suggest that nuclear expression of EGFR may be an important molecular determinant of resistance to cetuximab therapy and provides a rationale for investigating nuclear EGFR as a biomarker for cetuximab response. Further, these data suggest a rationale for the design of clinical trials that examine the value of treating patients with cetuximab-resistant tumors with inhibitors of SFKs in combination with cetuximab.

Nuclear EGFR contributes to acquired resistance to cetuximab.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a major role in oncogenesis. Cetuximab is an EGFR-blocking antibody that is FDA approved for use in patients with metastatic colorectal cancer (mCRC) and head and neck squamous cell carcinoma (HNSCC). Although cetuximab has shown strong clinical benefit for a subset of cancer patients, most become refractory to cetuximab therapy. We reported that cetuximab-resistant NSCLC line NCI-H226 cells have increased steady-state expression and activity of EGFR secondary to altered trafficking/degradation and this increase in EGFR expression and activity lead to hyper-activation of HER3 and down stream signals to survival. We now present data that Src family kinases (SFKs) are highly activated in cetuximab-resistant cells and enhance EGFR activation of HER3 and PI(3)K/Akt. Studies using the Src kinase inhibitor dasatinib decreased HER3 and PI(3)K/Akt activity. In addition, cetuximab-resistant cells were resensitized to cetuximab whe...

Epidermal growth factor receptor cooperates with Src family kinases in acquired resistance to cetuximab.

KRAS mutation is a predictive biomarker for resistance to cetuximab (Erbitux) in metastatic colorectal cancer (mCRC) This study sought to determine if KRAS mutant CRC lines could be sensitized to cetuximab using dasatinib (BMS-354825, Sprycel), a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src family kinases (SFKs) We analyzed 16 CRC lines for: (1) KRAS mutation status, (2) dependence on mutant KRAS signaling and (3) expression level of epidermal growth factor receptor (EGFR) and SFKs From these analyses, we selected three KRAS mutant (LS180, LoVo and HCT116) cell lines and two KRAS wild-type cell lines (SW48 and CaCo2) In vitro, using poly-D-lysine/laminin plates, KRAS mutant cell lines were resistant to cetuximab, whereas KRAS wild-type lines showed sensitivity to cetuximab Treatment with cetuximab and dasatinib showed a greater antiproliferative effect on KRAS mutant lines when compared with either agent alone in vitro and in vivo To investigate potential mechanisms for this antiproliferative response in the combinatorial therapy, we performed Human Phospho-Kinase Antibody Array analysis, measuring the relative phosphorylation levels of 39 intracellular proteins in untreated, cetuximab, dasatinib or the combinatorial treatment in the KRAS mutant lines LS180, LoVo and HCT116 cells The results of this experiment showed a decrease in a broad spectrum of kinases centered on the β-catenin pathway, the mitogen-activated protein kinase (MAPK) pathway, AKT/mammalian target of rapamycin (mTOR) pathway and the family of signal transducers and activators of transcription (STATs) when compared with the untreated control or monotherapy treatments Next, we analyzed tumor growth with cetuximab, dasatinib or their combination in vivo KRAS mutant xenografts showed resistance to cetuximab therapy, whereas KRAS wild type demonstrated an antitumor response when treated with cetuximab KRAS mutant tumors exhibited minimal response to dasatinib monotherapy However, as in vitro, KRAS mutant lines exhibited a response to the combination of cetuximab and dasatinib Combinatorial treatment of KRAS mutant xenografts resulted in decreased cell proliferation, as measured by Ki67, and higher rates of apoptosis, as measured by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) The data presented in this study indicate that dasatinib can sensitize KRAS mutant CRC tumors to cetuximab and may do so by altering the activity of several key signaling pathways Furthermore, these results suggest that signaling via EGFR and SFKs may be necessary for cell proliferation and survival of KRAS mutant CRC tumors These data strengthen the rationale for clinical trials combining cetuximab and dasatinib in the KRAS mutant CRC genetic setting

Emily F. Dunn

Papers

Understanding resistance to EGFR inhibitors-impact on future treatment strategies.

The role of Src in solid tumors.

Nuclear EGFR contributes to acquired resistance to cetuximab.

Epidermal growth factor receptor cooperates with Src family kinases in acquired resistance to cetuximab.

Dasatinib sensitizes KRAS mutant colorectal tumors to cetuximab