Faculty Bibliography

Germline mutations in the LKB1 gene (also known as STK11) cause the Peutz-Jeghers Syndrome, and somatic loss of LKB1 has emerged as causal event in a wide range of human malignancies, including melanoma, lung cancer, and cervical cancer. The LKB1 protein is a serine-threonine kinase that phosphorylates AMP-activated protein kinase (AMPK) and other downstream targets. Conditional knockout studies in mouse models have consistently shown that LKB1 loss promotes a highly-metastatic phenotype in diverse tissues, and human studies have demonstrated a strong association between LKB1 inactivation and tumor recurrence. Furthermore, LKB1 deficiency confers sensitivity to distinct classes of anticancer drugs. The ability to reliably identify LKB1-deficient tumors is thus likely to have important prognostic and predictive implications. Previous research studies have employed polyclonal antibodies with limited success, and there is no widely-employed immunohistochemical assay for LKB1. Here we report an assay based on a rabbit monoclonal antibody that can reliably detect endogenous LKB1 protein (and its absence) in mouse and human formalin-fixed, paraffin-embedded tissues. LKB1 protein levels determined through this assay correlated strongly with AMPK phosphorylation both in mouse and human tumors, and with mRNA levels in human tumors. Our studies fully validate this immunohistochemical assay for LKB1 in paraffin-embedded formalin tissue sections. This assay should be broadly useful for research studies employing mouse models and also for the development of human tissue-based assays for LKB1 in diverse clinical settings.

Despite the ongoing "war on cancer," cancer remains one of the major causes of human morbidity and mortality. A new paradigm of targeted therapies holds the most promise for the future, making identification of tumor-specific therapeutic targets of prime importance. ERBB2/HER2, best known for its role in breast cancer tumorigenesis, can be targeted by two types of pharmacological manipulation: antibody therapy against the extracellular receptor domain and small molecule compounds against the intracellular tyrosine kinase domain. Aberrant activation of ERBB2 by gene amplification has been shown to participate in the pathophysiology of breast, ovarian, gastric, colorectal, lung, brain, and head and neck tumors. However, the advent of next-generation sequencing technologies has enabled efficient identification of activating molecular alterations of ERBB2. In this review, we will focus on the functional role of these somatic mutations that cause ERBB2 receptor activation. We will additionally discuss the current preclinical and clinical therapeutic strategies for targeting mutationally activated ERBB2.

Somatic mutation of the tumor suppressor gene LKB1 occurs frequently in lung cancer where it causes tumor progression and metastasis, but the underlying mechanisms remain mainly unknown. Here, we show that the oncogene NEDD9 is an important downstream mediator of lung cancer progression evoked by LKB1 loss. In de novo mouse models, RNAi-mediated silencing of Nedd9 inhibited lung tumor progression, whereas ectopic NEDD9 expression accelerated this process. Mechanistically, LKB1 negatively regulated NEDD9 transcription by promoting cytosolic translocation of CRTC1 from the nucleus. Notably, ectopic expression of either NEDD9 or CRTC1 partially reversed the inhibitory function of LKB1 on metastasis of lung cancer cells. In clinical specimens, elevated expression of NEDD9 was associated with malignant progression and metastasis. Collectively, our results decipher the mechanism through which LKB1 deficiency promotes lung cancer progression and metastasis, and provide a mechanistic rationale for therapeutic attack of these processes.

Patients with lung cancer with activating mutations in the EGF receptor (EGFR) kinase, who are treated long-term with tyrosine kinase inhibitors (TKI), often develop secondary mutations in EGFR associated with resistance. Mice engineered to develop lung adenocarcinomas driven by the human EGFR T790M resistance mutation are similarly resistant to the EGFR TKI erlotinib. By tumor volume endpoint analysis, these mouse tumors respond to BIBW 2992 (an irreversible EGFR/HER2 TKI) and rapamycin combination therapy. To correlate EGFR-driven changes in the lung with response to drug treatment, we conducted an integrative analysis of global transcriptome and metabolite profiling compared with quantitative imaging and histopathology at several time points during tumor progression and treatment. Responses to single-drug treatments were temporary, whereas combination therapy elicited a sustained response. During tumor development, metabolomic signatures indicated a shift to high anabolic activity and suppression of antitumor programs with 11 metabolites consistently present in both lung tissue and blood. Combination drug treatment reversed many of the molecular changes found in tumored lung. Data integration linking cancer signaling networks with metabolic activity identified key pathways such as glutamine and glutathione metabolism that signified response to single or dual treatments. Results from combination drug treatment suggest that metabolic transcriptional control through C-MYC and SREBP, as well as ELK1, NRF1, and NRF2, depends on both EGFR and mTORC1 signaling. Our findings establish the importance of kinetic therapeutic studies in preclinical assessment and provide in vivo evidence that TKI-mediated antiproliferative effects also manifest in specific metabolic regulation.

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors is limited by the development of drug resistance. The irreversible EGFR kinase inhibitor WZ4002 is effective against the most common mechanism of drug resistance mediated by the EGFR T790M mutation. Here, we show, in multiple complementary models, that resistance to WZ4002 develops through aberrant activation of extracellular signal-regulated kinase (ERK) signaling caused by either an amplification of mitogen-activated protein kinase 1 (MAPK1) or by downregulation of negative regulators of ERK signaling. Inhibition of MAP-ERK kinase (MEK) or ERK restores sensitivity to WZ4002 and prevents the emergence of drug resistance. We further identify MAPK1 amplification in an erlotinib-resistant EGFR-mutant non-small cell lung carcinoma patient. In addition, the WZ4002-resistant MAPK1-amplified cells also show an increase both in EGFR internalization and a decrease in sensitivity to cytotoxic chemotherapy. Our findings provide insights into mechanisms of drug resistance to EGFR kinase inhibitors and highlight rational combination therapies that should be evaluated in clinical trials.

PURPOSE: We describe the anticancer activity of ganetespib, a novel non-geldanamycin heat shock protein 90 (HSP90) inhibitor, in non-small cell lung cancer (NSCLC) models. EXPERIMENTAL DESIGN: The activity of ganetespib was compared with that of the geldanamycin 17-AAG in biochemical assays, cell lines, and xenografts, and evaluated in an ERBB2 YVMA-driven mouse lung adenocarcinoma model. RESULTS: Ganetespib blocked the ability of HSP90 to bind to biotinylated geldanamycin and disrupted the association of HSP90 with its cochaperone, p23, more potently than 17-AAG. In genomically defined NSCLC cell lines, ganetespib caused depletion of receptor tyrosine kinases, extinguishing of downstream signaling, inhibition of proliferation and induction of apoptosis with IC(50) values ranging 2 to 30 nmol/L, substantially lower than those required for 17-AAG (20-3,500 nmol/L). Ganetespib was also approximately 20-fold more potent in isogenic Ba/F3 pro-B cells rendered IL-3 independent by expression of EGFR and ERBB2 mutants. In mice bearing NCI-H1975 (EGFR L858R/T790M) xenografts, ganetespib was rapidly eliminated from plasma and normal tissues but was maintained in tumor with t(1/2) 58.3 hours, supporting once-weekly dosing experiments, in which ganetespib produced greater tumor growth inhibition than 17-AAG. However, after a single dose, reexpression of mutant EGFR occurred by 72 hours, correlating with reversal of antiproliferative and proapoptotic effects. Consecutive day dosing resulted in xenograft regressions, accompanied by more sustained pharmacodynamic effects. Ganetespib also showed activity against mouse lung adenocarcinomas driven by oncogenic ERBB2 YVMA. CONCLUSIONS: Ganetespib has greater potency than 17-AAG and potential efficacy against several NSCLC subsets, including those harboring EGFR or ERBB2 mutation.

We assessed somatic alleles of six receptor tyrosine kinase genes mutated in lung adenocarcinoma for oncogenic activity. Five of these genes failed to score in transformation assays; however, novel recurring extracellular domain mutations of the receptor tyrosine kinase gene ERBB2 were potently oncogenic. These ERBB2 extracellular domain mutants were activated by two distinct mechanisms, characterized by elevated C-terminal tail phosphorylation or by covalent dimerization mediated by intermolecular disulfide bond formation. These distinct mechanisms of receptor activation converged upon tyrosine phosphorylation of cellular proteins, impacting cell motility. Survival of Ba/F3 cells transformed to IL-3 independence by the ERBB2 extracellular domain mutants was abrogated by treatment with small-molecule inhibitors of ERBB2, raising the possibility that patients harboring such mutations could benefit from ERBB2-directed therapy.

Tyrosine kinase inhibitors (TKI) that target the EGF receptor (EGFR) are effective in most non-small cell lung carcinoma (NSCLC) patients whose tumors harbor activating EGFR kinase domain mutations. Unfortunately, acquired resistance eventually emerges in these chronically treated cancers. Two of the most common mechanisms of acquired resistance to TKIs seen clinically are the acquisition of a secondary "gatekeeper" T790M EGFR mutation that increases the affinity of mutant EGFR for ATP and activation of MET to offset the loss of EGFR signaling. Although up to one-third of patient tumors resistant to reversible EGFR TKIs harbor concurrent T790M mutation and MET amplification, potential therapies for these tumors have not been modeled in vivo. In this study, we developed a preclinical platform to evaluate potential therapies by generating transgenic mouse lung cancer models expressing EGFR-mutant Del19-T790M or L858R-T790M, each with concurrent MET overexpression. We found that monotherapy targeting EGFR or MET alone did not produce significant tumor regression. In contrast, combination therapies targeting EGFR and MET simultaneously were highly efficacious against EGFR TKI-resistant tumors codriven by Del19-T790M or L858R-T790M and MET. Our findings therefore provide an in vivo model of intrinsic resistance to reversible TKIs and offer preclinical proof-of-principle that combination targeting of EGFR and MET may benefit patients with NSCLC.