LKB1 Mutations Research Articles

Abstract B07: Loss of LKB1 promotes CXCL8 production via NF-κB and WNT signaling in human bronchial epithelial cells.

Abstract Loss of LKB1/STK11, a tumor suppressor gene, characterizes 5-39% of primary non-small cell lung cancers (NSCLC). In a murine model of lung carcinogenesis with inducible KRAS activation, concurrent mutation of LKB1 (KrasG12D, Lkb1-/-) yields a higher frequency of NSCLC tumors and metastasis compared to KRAS mutation only. In vitro studies have shown that following LKB1 loss, tumor cells acquire increased migration and invasion through activation of the SRC kinase family. Although these previous findings offer a possible explanation for LKB1-dependent tumor progression, few mechanisms have been proposed to delineate how LKB1 deficiency contributes to pulmonary carcinogenesis. In this study, we utilize human bronchial epithelial cells (HBECs) immortalized in the absence of viral onco-proteins that represent a useful model to study the early events of lung carcinogenesis. Knockdown of LKB1 in HBECs (LKB1-KD HBECs) leads to numerous cancer-associated phenotypes, including increased proliferation (when combined with KRAS mutation), preference for glycolytic metabolism and induction of epithelial-to-mesenchymal transition. More importantly, LKB1-KD HBECs secrete higher levels of multiple inflammatory factors, most prominent of which is the pro-angiogenic chemokine CXCL8. By binding to its membrane receptors CXCR1/2 which are universally expressed by neutrophils, macrophages, endothelial cells and pulmonary epithelial cells, CXCL8 is associated with chemotaxis, angiogenesis, tumorigenicity and metastasis. Our data indicate that knockdown of LKB1 in HBECs leads to transcriptional upregulation and elevated secretion of CXCL8. Moreover, the NF-κB and WNT pathways are activated and mediate the increased release of CXCL8 following LKB1 loss in HBECs. Conversely, re-introduction of wildtype LKB1 in LKB1-null NSCLC tumor cells decreases CXCL8 production and reduces NF-κB and WNT signaling activity. These findings suggest that LKB1 deficiency drives augmentation of CXCL8 in the developing tumor microenvironment via LKB1-dependent NF-κB and WNT signaling in HBECs. In future functional studies, we will determine if the LKB1-CXCL8 axis yields LKB1-dependent angiogenesis and tumorigenesis. Delineating the contribution of LKB1 to these malignant phenotypes may yield a more thorough understanding of the pathogenesis of NSCLC, which is needed to create a pathway for the application of novel early detection and chemoprevention strategies. NIH/NCI #U01CA152751 (SMD, TCW), NCI #U01CA152751-S1 (SMD, TCW), NCI #U01CA152751-AS (SMD, KK), NCI SPORE #P50CA70907 (JDM, JEL) Citation Format: RUI LI, Tonya C. Walser, Kostyantyn Krysan, David Shackelford, Jill E. Larsen, John D. Minna, Steven M. Dubinett. Loss of LKB1 promotes CXCL8 production via NF-κB and WNT signaling in human bronchial epithelial cells. [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 B07.

A genetic screen identifies an LKB1–MARK signalling axis controlling the Hippo–YAP pathway

The Hippo-YAP pathway is an emerging signalling cascade involved in the regulation of stem cell activity and organ size. To identify components of this pathway, we performed an RNAi-based kinome screen in human cells. Our screen identified several kinases not previously associated with Hippo signalling that control multiple cellular processes. One of the hits, LKB1, is a common tumour suppressor whose mechanism of action is only partially understood. We demonstrate that LKB1 acts through its substrates of the microtubule affinity-regulating kinase family to regulate the localization of the polarity determinant Scribble and the activity of the core Hippo kinases. Our data also indicate that YAP is functionally important for the tumour suppressive effects of LKB1. Our results identify a signalling axis that links YAP activation with LKB1 mutations, and have implications for the treatment of LKB1-mutant human malignancies. In addition, our findings provide insight into upstream signals of the Hippo-YAP signalling cascade.

Efficacy of BET Bromodomain Inhibition in Kras-Mutant Non–Small Cell Lung Cancer

Amplification of MYC is one of the most common genetic alterations in lung cancer, contributing to a myriad of phenotypes associated with growth, invasion, and drug resistance. Murine genetics has established both the centrality of somatic alterations of Kras in lung cancer, as well as the dependency of mutant Kras tumors on MYC function. Unfortunately, drug-like small-molecule inhibitors of KRAS and MYC have yet to be realized. The recent discovery, in hematologic malignancies, that bromodomain and extra-terminal (BET) bromodomain inhibition impairs MYC expression and MYC transcriptional function established the rationale of targeting KRAS-driven non-small cell lung cancer (NSCLC) with BET inhibition. We performed functional assays to evaluate the effects of JQ1 in genetically defined NSCLC cell lines harboring KRAS and/or LKB1 mutations. Furthermore, we evaluated JQ1 in transgenic mouse lung cancer models expressing mutant kras or concurrent mutant kras and lkb1. Effects of bromodomain inhibition on transcriptional pathways were explored and validated by expression analysis. Although JQ1 is broadly active in NSCLC cells, activity of JQ1 in mutant KRAS NSCLC is abrogated by concurrent alteration or genetic knockdown of LKB1. In sensitive NSCLC models, JQ1 treatment results in the coordinate downregulation of the MYC-dependent transcriptional program. We found that JQ1 treatment produces significant tumor regression in mutant kras mice. As predicted, tumors from mutant kras and lkb1 mice did not respond to JQ1. Bromodomain inhibition comprises a promising therapeutic strategy for KRAS-mutant NSCLC with wild-type LKB1, via inhibition of MYC function. Clinical studies of BET bromodomain inhibitors in aggressive NSCLC will be actively pursued. Clin Cancer Res; 19(22); 6183-92. ©2013 AACR.

Abstract A41: Inhibition of Lkb1 in cultured embryonic pancreas destabilizes tissue architecture to generate precancerous-like lesions

Abstract The tumor suppressor Lkb1 is a regulator of cellular energy, proliferation, and polarity, yet the mechanism by which it controls tissue morphogenesis or cancer remains poorly defined. By culturing embryonic tissues from mice harboring a mutant Lkb1 that can be rapidly inhibited, we have previously shown that acute loss of Lkb1 results in destabilized tissue architecture and the formation of highly dynamic pancreatic cysts (Lo et al, JCB 2012). Remarkably, the pancreatic cysts evolve within a few days into precancerous-like lesions that we now demonstrate to have a transcriptional profile that is strikingly similar to those reported for mouse and human PanINs (Pancreatic Intraepithelial Neoplasias). Utilizing fluorescence activated cell sorting, we have begun to track gene expression signatures associated with canonical signaling pathways in both single cells and subsets of cells from the embryonic explants. Since our experimental system offers precise temporal control and accessibility to manipulation within a tissue context, we are able to gain unprecedented insight into the earliest events in pancreatic oncogenesis. Citation Format: Bryan Lo, Manuel Viotti, Geraldine Strasser, Ira Mellman. Inhibition of Lkb1 in cultured embryonic pancreas destabilizes tissue architecture to generate precancerous-like lesions. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A41.

The tumor suppressor LKB1 antagonizes WNT signaling pathway through modulating GSK3β activity in cell growth of esophageal carcinoma

The tumor suppressor LKB1 gene encodes a serine-threonine kinase that regulates cell proliferation and polarity. Inactivation of LKB1 by mutations in LKB1 or loss of its expression is highly correlated with lung, ovarian, and pancreatic cancers, and WNT/β-catenin pathway is also known to be involved in many human malignancies. However, the relationship between LKB1 and WNT signaling pathway in esophageal carcinoma remains unknown. The expression of LKB1 in 62 cases of esophageal cancer patients was determined by quantitative real-time PCR. It was found that LKB1 mRNA level was significantly lower than the adjacent normal epithelium and that the LKB1 downregulation was correlating with TNM stages. Moreover, the expression of WNT target genes such as Cyclin D1, C-MYC, MMP2, and FZD2 was significantly upregulated in esophageal cancer tissues. LKB1 overexpression in TE10 cells inhibited TOPFlash luciferase reporter activity and WNT target gene expression even in the presence of WNT3A. Conversely, LKB1 knockdown enhanced WNT signaling activity in esophageal cancer cells. It was also found that LKB1 antagonized WNT signaling pathway through interaction with GSK3β to downregulate β-catenin expression level. Functional investigation revealed that LKB1 suppressed the promotion effects of WNT3A on the cell growth of TE10 cells. The LKB1 functions in regulating cell growth and WNT target genes expression were impaired by GSK3β inhibition, suggesting that LKB1 antagonized WNT-induced cell proliferation through enhancement of GSK3β activity. Together, the interaction between LKB1 and GSK3β upregulates GSK3β activity to suppress WNT-induced cell proliferation in esophageal carcinoma cells. Loss of LKB1 expression may result in the deregulation of WNT/β-catenin pathway to promote malignant progression of esophageal cancer.

LKB1 mutant in a KRAS activated adenocarcinoma of the lung associated with Peutz–Jeghers syndrome: A case report

We present a case of a 54-year-old woman who was diagnosed with a KRAS positive adenocarcinoma of the lung on the basis of a Peutz–Jeghers syndrome (PJS), which was unknown before. PJS is a rare hereditary disease, which may be associated with the development of poor outcome adenocarcinomas and LKB1-gene mutations. A very rare type of a LKB1 mutation was found, not previously described in lung cancer. Although seldom screened for LKB-1 mutations are found in up to 30% of lung adenocarcinomas and may be druggable therapeutic targets, in particular in KRAS mutant tumours in the near future as recent preclinical results with nucleotides demonstrate.

Mutation incidence and coincidence in non small-cell lung cancer: meta-analyses by ethnicity and histology (mutMap)

BackgroundMeta-analyses were conducted to characterize patterns of mutation incidence in non small-cell lung cancer (NSCLC). DesignNine genes with the most complete published mutation coincidence data were evaluated. One meta-analysis generated a ‘mutMap’ to visually represent mutation coincidence by ethnicity (Western/Asian) and histology (adenocarcinoma [ADC] or squamous cell carcinoma). Another meta-analysis evaluated incidence of individual mutations. Extended analyses explored incidence of EGFR and KRAS mutations by ethnicity, histology, and smoking status. ResultsGenes evaluated were TP53, EGFR, KRAS, LKB1, EML4-ALK, PTEN, BRAF, PIK3CA, and ErbB2. The mutMap highlighted mutation coincidences occurring in ≥5% of patients, including TP53 with KRAS or EGFR mutations in patients with ADC, and TP53 with LKB1 mutation in Western patients. TP53 was the most frequently mutated gene overall. Frequencies of TP53, EGFR, KRAS, LKB1, PTEN, and BRAF mutations were influenced by histology and/or ethnicity. Although EGFR mutations were most frequent in patients with ADC and never/light smokers from Asia, and KRAS mutations were most frequent in patients with ADC and ever/heavy smokers from Western countries, both were detected outside these subgroups. ConclusionsPotential molecular pathology segments of NSCLC were identified. Further studies of mutations in NSCLC are warranted to facilitate more specific diagnoses and guide treatment.

Molecular pathogenesis of endometrial cancers in patients with Lynch syndrome

The authors hypothesized that Lynch syndrome (LS)-associated endometrial cancer (EC) develops from morphologically normal endometrium that accumulates enough molecular changes to progress through a continuum of hyperplasia to carcinoma, similar to sporadic EC. The primary objective of the current study was to determine whether LS-associated EC involves progression through a preinvasive lesion. The secondary objective was to identify molecular changes that contribute to endometrial carcinogenesis in patients with LS. Women with a confirmed mismatch repair gene mutation for LS who were undergoing a prophylactic or therapeutic hysterectomy were eligible. Cases and controls were matched for EC and hyperplasia based preferentially on age and histology. Mutation status of phosphatidylinositol 3-kinase (PIK3CA); KRAS; AKT; LKB1; catenin (cadherin-associated protein), beta 1, 88kDa (CTNNB1); and phosphatase and tensin homolog (PTEN) protein loss was assessed. Concurrent complex atypical hyperplasia (CAH) was found in EC in 11 cases of LS (39.3%) and 21 sporadic cases (46.6%). Loss of PTEN expression was common in both sporadic (69%) and LS-associated EC (86.2%). There was no significant difference noted with regard to the frequency of KRAS mutations in cases of sporadic EC (10.3%) compared with LS-associated EC (3.4%). AKT and LKB1 mutations were rarely observed. Mutations in PIK3CA and CTNNB1 occurred more frequently in cases of sporadic EC compared with LS-associated EC. Hyperplasia, particularly CAH, is part of the preinvasive spectrum of disease in LS-associated EC, as indicated by the presence of complex hyperplasia and CAH in cases of LS. Although loss of PTEN is common in both LS and sporadic EC cases, there was a lack of additional mutations in LS-associated EC cases. This suggests that in the context of the mismatch repair defects in LS, fewer additional molecular changes are required to progress from preinvasive lesions to cancer.

Genetic and molecular biomarker characterization of KRAS mutant non-small cell lung carcinoma (NSCLC) tumors.

11026 Background: Preclinical studies demonstrated Brahma related gene 1 (BRG1) mutations or loss of expression, and mutations of LKB1 may be associated with lack of sensitivity for MEK inhibitor trametinib in a subset of KRAS mutant NSCLC lines. This study aimed to evaluate the frequency of KRAS, LKB1 and BRG1 mutations in NSCLC tumors; and determine whether KRAS mutations in corresponding plasma samples could be detected by evaluating circulating cell-free DNA (cfDNA). Methods: Human NSCLC FFPE tumor tissue and matched plasma samples were procured from Indivumed GmbH. KRAS mutation status of 101 NSCLC tumors and matched plasma were determined by direct sequencing of genomic DNA (gDNA) from tissue and/or BEAMing on tissue gDNA or plasma cfDNA. Genetic mutations of LKB1 and BRG1 were determined by direct sequencing. Additional mutations were determined using the Ion Torrent AmpliSeq Cancer Panel. BRG1 protein expression was evaluated by IHC. Results: By direct sequencing and BEAMing we found 27/101 (28.4%) NSCLC tissue and/or plasma samples harbored KRAS mutations: G12V (37.0%), G12C (29.6%), G12D (18.5%), G12S, G13C, G13D and Q61H (3.7% each). The KRAS mutation status concordance (mutant or wild-type) between tumor gDNA and plasma cfDNA was 79-81%. Among the KRAS mutant tumors, LKB1 and BRG1 mutations were detected in 10/26 (38%) and 1/26 (3.8%) tumors respectively by direct sequencing. By IHC, loss of BRG1 expression was detected in 1/21 KRAS mutant tumors. The mutation frequency and variants for KRAS and LKB1 in patient samples were comparable with KRAS mutant NSCLC cell lines and COSMIC database. However the frequency of BRG1 mutation and protein loss were much lower in patient tumors. In a subset of 15 KRAS mutant tumors, Ion Torrent confirmed KRAS and LKB1 mutations and provided additional mutations found in TP53, FGFR2, FGFR3, GNAS, KDR, KIT and MET. Conclusions: This study demonstrates that KRAS mutant NSCLC tissues have high frequency of LKB1 mutations along with other mutations. It also supports the feasibility of detection of KRAS mutations in cfDNA from blood of NSCLC patients using BEAMing technology, providing an alternative to invasive biopsy.

AMPK: A Contextual Oncogene or Tumor Suppressor?

The AMP-activated protein kinase (AMPK) functions to monitor and maintain energy homeostasis at the cellular and organism level. AMPK was perceived historically primarily as a component of the LKB1/STK11 tumor suppressor (LKB1 mutations cause the Peutz-Jegher cancer predisposition syndrome) cascade upstream of the TSC1/2/mTOR pathway and thus likely to be a tumor suppressor. However, AMPK has recently been shown to promote cancer cell survival in the face of extrinsic and intrinsic stressors including bioenergetic, growth factor, and oncogene stress compatible with studies showing that AMPK is required for oncogenic transformation. Thus, whether AMPK acts as a bona fide tumor suppressor or a contextual oncogene and, of particular importance, whether AMPK should be targeted for activation or inhibition during cancer therapy, is controversial and requires clarification. We aim to initiate discussions of these critical questions by reviewing the role of AMPK with an emphasis on cancer cell adaptation to microenvironment stress and therapeutic intervention.

Genetic and epigenetic alterations of the LKB1 gene and their associations with mutations in TP53 and EGFR pathway genes in Korean non-small cell lung cancers

IntroductionLiver kinase 1 (LKB1) plays a critical barrier role in lung tumorigenesis by controlling initiation, differentiation and metastasis. We searched for genetic and epigenetic alterations of the LKB1 gene in Korean non-small cell lung cancers (NSCLCs) and correlated the results with clinicopathological features. We also investigated the relationship between genetic and epigenetic alterations of LKB1 and mutations in the TP53 gene and epidermal growth factor receptor (EGFR) pathway genes. MethodsA total of 159 NSCLCs were analyzed for loss of heterozygosity (LOH) at microsatellite loci D19S886, and D19S878. Mutations and methylation status of LKB1 were examined by direct sequencing and a methylation-specific polymerase chain reaction, respectively. ResultsA somatic mutation was found in one of the 159 tumors. LOH and promoter methylation was detected in 19.5% (31/159) and 13.2% (21/159) of the tumors, respectively. Four of the 159 tumors had concomitant LOH and methylation of LKB1. In total, 30.2% of the 159 NSCLCs harbored LKB1 LOH or promoter methylation, which were correlated with down-regulation of gene expression. LKB1 LOH was more frequent in males, smokers, and tumors with a TP53 mutation than in females, never-smokers, and tumors without a TP53 mutation, respectively. However, no significant correlation between LKB1 alterations and mutations in EGFR pathway genes was found. ConclusionThese results suggest that the prevalence of LKB1 genetic and epigenetic alterations in NSCLCs vary depending on patient ethnicity. Our results show that LKB1 alterations often occur simultaneously with mutations in EGFR pathway genes.

Abstract 1277: Utilizing uterine cancer models to study malignant transformation driven by Lkb1 loss.

Abstract The serine-threonine kinase LKB1 is an upstream component of the AMPK-mTOR pathway and regulates cell survival and growth. Inherited germline mutations in LKB1 predispose women to endocervical malignancies whereas somatic mutations in LKB1 promote cervical cancer progression. Our lab has shown that tissue specific knockout of Lkb1 in the endometrial epithelium of mice facilitates growth of highly invasive, lethal tumors that are well differentiated and retain structural polarity. Additionally, stable shRNA mediated knockdown of LKB1 in human immortalized endocervical cells drives anchorage-independent growth. To investigate potential pathways involved in LKB1 mediated oncogenic transformation, we have employed a microarray based approach comparing hits from human immortalized endocervical cell lines and mouse tissue to identify specific genes that are differentially regulated when LKB1 function is absent. Preliminary data suggests that pro-angiogenic and inflammatory mediators are upregulated in LKB1 deficient cells. We are currently assessing the overall contribution of these factors to growth and invasiveness in our model systems and the mechanisms by which Lkb1 loss facilitates their activation. These pathways can be potential therapeutic targets in other cancers characterized by Lkb1 loss of function mutations. Citation Format: Christopher G. Peña, Yuji Nakada, Diego Castrillon. Utilizing uterine cancer models to study malignant transformation driven by Lkb1 loss. [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 1277. doi:10.1158/1538-7445.AM2013-1277

Abstract 2281: LKB1 loss leads to activation of the CREB transcription factor and sensitivity to MEK inhibition in human lung cancer.

Abstract Background: Loss of the LKB1 tumor suppressor is one of the most common genetic events in lung adenocarcinoma, and improved understanding of the cellular phenotypes and molecular pathways activated in these tumors will aid the development of therapeutic strategies targeting LKB1-deficient cancers. Methods: We analyzed gene expression patterns of LKB1 loss among resected lung adenocarcinomas, non-small cell lung cancer (NSCLC) cell lines, and tumors from a murine model of LKB1-deficient, KRAS mutant lung cancer. From this analysis we derived a gene expression signature of LKB1 loss in resected lung adenocarcinomas and assessed its performance in predicting LKB1 loss in multiple clinical and cell line test sets. We identified four ‘nodes’ of transcriptional regulation and compared the resulting gene lists to various collections of gene sets to identify putative biological phenotypes responsible for their expression. Furthermore, our LKB1-deficient signature was then used in conjunction with two compendia of in vitro drug sensitivity data to identify inhibitors with greater efficacy in cell lines that lacked LKB1. We then observed changes in gene expression, pathway activation, and drug sensitivity in three cell lines after stably re-expressing LKB1. Results: Our LKB1-deficient gene expression signature is significantly associated with LKB1 mutations, with sensitivity of 85% and specificity of 82% in independent testing cohorts (p<1E-10). Furthermore, the group of tumors identified by our model has low prevalence of EGFR mutations and never smokers, which recapitulates well-characterized clinical and molecular features of LKB1-deficient tumors. However, the gene expression pattern we observe bears little resemblance to the dysregulated genes observed in murine models of LKB1 loss. Based on analysis of our validated signature, we identify up-regulation of oxidative phosphorylation and activation of NRF2-driven oxidative stress response pathway in tumors lacking LKB1. Furthermore, we find that increased activity of the CREB transcription factor is a defining feature of these tumors, and show that this pathway is associated with in vitro sensitivity to MEK inhibition. Restoring LKB1 in three NSCLC cell lines reverses these phenotypes, with down-regulation of the LKB1-deficient gene expression signature, attenuation of the activity of a CREB-driven luciferase reporter, and increased resistance to the MEK inhibitor selumetinib. Conclusions: We find that the biology of human LKB1-deficient lung adenocarcinomas differs substantially from what is observed in the murine model. Moreover, our analysis of dysregulated pathways in human disease gives insight into pathway activation and biological phenotypes of these tumors, and specifically identifies MEK as a therapeutic target to which LKB1-deficient tumors show increased susceptibility. Citation Format: Jacob M. Kaufman, Joseph Amann, Kyungho Park, David Carbone. LKB1 loss leads to activation of the CREB transcription factor and sensitivity to MEK inhibition in human lung cancer. [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 2281. doi:10.1158/1538-7445.AM2013-2281

Abstract 1126: Efficacy of BET bromodomain inhibition in Kras-positive non-small cell lung cancer.

Abstract Amplification of MYC is one of the most common genetic alterations in lung cancer, contributing to a myriad of phenotypes associated with growth, invasion and drug resistance. Murine genetics has established both the centrality of somatic alterations of Kras in lung cancer, as well as dependency of Kras-dependent tumors on c-Myc function. Unfortunately, drug-like small-molecule inhibitors of KRAS and c-Myc have yet to be realized. The recent discovery in hematologic malignancies that bromodomain inhibition impairs MYC expression and MYC-dependent transcriptional function prompted the possibility of targeting KRAS-driven NSCLC with a potent, prototypical BET bromodomain inhibitor, JQ1. Here, we report that NSCLC cells harboring the KRAS mutation are sensitive to JQ1 while NSCLC cells with concurrent mutant KRAS and LKB1 mutations are resistant to JQ1. In sensitive NSCLC models, JQ1 treatment results in the coordinate downregulation of the MYC-dependent transcriptional program. Furthermore, we evaluated JQ1 in transgenic mouse lung cancer models expressing mutant kras or concurrent mutant kras and lkb1. We found that JQ1 treatment produces significant tumor regression in mutant kras mice. As predicted, tumors from mutant kras and lkb1 mice did not respond to JQ1. Together, these data provide a compelling rationale for the study of BET bromodomain inhibitors in a common, genetically-defined population of patients with aggressive NSCLC. Citation Format: Takeshi Shimamura, Zhao Chen, Margaret Soucheray, Julian Carretero, Eiki Kikuchi, Jeremy H. Tchaicha, Yandhi Gao, Katherine A. Cheng, Travis J. Cohoon, Jun Qi, Esra A. Akbay, Alec C. Kimmelman, Andrew L. Kung, James E. Bradner, Kwok Kin Wong. Efficacy of BET bromodomain inhibition in Kras-positive non-small cell lung cancer. [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 1126. doi:10.1158/1538-7445.AM2013-1126

Abstract 2796: Patient-derived xenograft models reveal a subset of clinically relevant squamous non-small cell lung cancers that respond to targeted EGFR inhibition.

Abstract BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer cases, carries a poor prognosis and remains an area of high unmet need. Despite recent advances in treating NSCLC adenocarcinomas with targeted epidermal growth factor receptor (EGFR) kinase inhibitors, few advances have been made in the treatment of squamous cell carcinoma (SCC), which accounts for 25% of NSCLC. Although the incidence of EGFR mutations in SCC is low, a retrospective clinical study of erlotinib in SCC suggested that a proportion of SCC patients would benefit from treatment with an EGFR inhibitor (Tseng et al., 2012). METHODS: NSCLC samples obtained from untreated patients undergoing surgery were collected with ethical consent, disaggregated and implanted subcutaneously in MF-1 nude mice (Harlan UK) admixed with a human stromal cell component to generate patient-derived xenograft (PDX) models (LION PDX models). Tumour material was diagnosed on the basis of operative histology and immunohistochemistry (IHC) used to confirm histological subtype. IHC was also used to evaluate EGFR expression and tumours characterised for mutations in p53, LKB1, EGFR and K-RAS by direct DNA sequencing. Patient-derived xenografts were maintained in vivo and study cohorts of mice dosed when tumours reached 100-200mm3 with either erlotinib or gefitinib or vehicle placebo. Response to treatment was evaluated by tumour growth inhibition and waterfall plots. RESULTS: We have identified a subset of tumours with squamous histology in our LION panel of patient-derived NSCLC xenografts that respond to treatment with a targeted EGFR inhibitor. Four out of 11 SCC models tested (36%) responded to treatment with either erlotinib or gefitinib with greater than 50% growth inhibition and two SCC models responded with ∼30% growth inhibition. Responses correlated well with EGFR expression. Work to characterise a further 8 LION SCC PDX models is also underway. CONCLUSIONS: These data support the proposition that a significant subset of NSCLC patients with squamous histology may benefit from treatment with targeted EGFR inhibitors. Our LION SCC models could be valuable for biomarker studies to identify the basis of the EGFR inhibitor responder subset and for evaluating new agents in a more clinically relevant setting. Citation Format: Aaron Cranston, Andrew Mckenzie, Phil Mallinder, Simon Jiang, Kerry Moakes, Yinfei Yin, Alex Reece-Smith, Anna Grabowska, Martin Page, Rajendra Kumari. Patient-derived xenograft models reveal a subset of clinically relevant squamous non-small cell lung cancers that respond to targeted EGFR inhibition. [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 2796. doi:10.1158/1538-7445.AM2013-2796

Abstract 3105: Orphan nuclear receptor NR4A2 exhibits oncogenic activity in lung cancer cells.

Abstract LKB1 (also known as STK11) is a frequent target for mutations in lung cancer, however, the role of LKB1 loss in tumorigenesis is undefined. NR4A2, together with NR4A1, and NR4A3, constitute the nuclear receptor (NR) subfamily 4A (NR4A), which are orphan NRs lacking identified ligands. NR4A family has been implicated in cell cycle regulation, apoptosis, inflammation, metabolism and more recently in carcinogenesis. However, there are conflicting data for tumorigenesis as NR4A1 and NR4A3 act as tumor suppressors in leukemia mouse models, while NR4A2 appears to stimulate tumor cell growth in vitro. Our laboratory discovered that the CRTC1 gene was aberrantly activated in LKB1-null cancer cells, stimulating the transcription of cAMP/CREB targets including NR4A gene members. The aim of this study is to examine NR4A2 as a potential oncogene and therapeutic target under the regulation of CRTC1 in LKB1 wildtype and null lung cancer cells. We demonstrated here that somatic loss of LKB1 in tumor cells was associated with underphosphorylation and nuclear localization of CRTC1, this resulted in upregulated expression and stronger transcriptional activity of NR4A2 in LKB1 mutant lung cancer cells and clinical primary lung cancer samples. We confirmed that forskolin, which can activate adenylate cyclase then catalyze the transformation of ATP to cAMP, could induce CRTC1 dephosphorylation resulting in enhanced NR4A2 expression. We identified that activation of the CRTC gene family induced NR4A2 expression, whereas knockdown of CRTCs decreased the expression of NR4A2. Furthermore, short hairpin RNA-mediated down-regulation of NR4A2, results in attenuated lung cancer cell proliferation and colony formation in vitro and decreased the tumor sizes in vivo in nude mice. In summary, our data suggested a CRTC-mediated transcriptional regulatory mechanism for NR4A2 activity, and proposed an oncogenic role of NR4A2 in LKB1 null lung cancer cell lines. Citation Format: Chunxia Cao, Min Zhang, Ruli Gao, Douglas Cress, Zirong Chen, Lizi Wu, Maria Zajac Kaye, Frederic Kaye. Orphan nuclear receptor NR4A2 exhibits oncogenic activity in lung cancer cells. [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 3105. doi:10.1158/1538-7445.AM2013-3105

Abstract 1589: LKB1 overexpression regulates DNA repair pathway and sensitivity to radiation in LKB1 mutant non-small cell lung cancer cell lines .

Abstract Background: LKB1 is a serine/threonine kinase which is mutated in 20-30% of non-small cell lung cancers (NSCLC).In general, DNA repair pathway activation leads cellular resistant to DNA damaging agents. The loss of LKB1 leads NSCLC to be more invasive and resistant to DNA damage based therapies. We hypothesize that loss of LKB1 may contribute to DNA damage response. We investigated LKB1- mediated signaling pathways by stably overexpressing LKB1 in its mutated NSCLC cell lines, A549 and H460. Methods: Expression of 139 proteins was analyzed by reverse phase protein array (RPPA) in LKB1 overexpressing cell lines along with the appropriate vector control lines. Differences in protein expression at baseline in clones with LKB1 versus clones with vector control were assessed by unpaired T-test with Welch's correction. Ionizing radiation sensitivity (reproductive cell death) was assessed by clonogenic cell survival assay (CSA). Results: LKB1 overexpression in A549 and H460 showed a significant downregulation of genes involved in DNA damage and repair pathway, such as RAD50 (1.4-fold relative expression, p< 0.05 and 1.5-fold relative expression p<0.02 respectively), XRCC1 (1.2-fold relative expression p<0.005 and 1.1-fold relative expression p<0.05, respectively), and DNA-PKCS (1.2-fold relative expression p≈0.067 and 1.2-fold relative expression p<0.01. Also, CSA showed that A549 with LKB1 overexpression led to enhanced sensitization of cells to radiation (DER = 2) which is in consistent with the downregulation of RAD50, XRCC1 and DNA-PKCS. Furthermore, expression of CHK2 and phosphorylation of CHK1 are downregulated, H460 with LKB1 overexpression showed a significant decreasing of CHK1 phosphorylation level (p<0.05). Conclusion: Our results suggest that LKB1 overexpression modulates DNA repair capacity through downregulation of RAD50, XRCC1, DNA-PKCS, CHK1 and CHK2. LKB1 loss may lead to the resistance to DNA damaging induced cell death. Citation Format: Chao Yang, Uma Giri, Maria Angelica Cortez, Jayanthi Gudikote, YouHong Fan, Lixia Diao, Lauren A. Byers, John V. Heymach. LKB1 overexpression regulates DNA repair pathway and sensitivity to radiation in LKB1 mutant non-small cell lung cancer cell lines . [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 1589. doi:10.1158/1538-7445.AM2013-1589

LKB1 Mutation Sensitizes NSCLC Cells to Phenformin

Phenformin, a metformin analogue, selectively induces apoptosis in LKB1-mutant NSCLC models.

LKB1 Inactivation Dictates Therapeutic Response of Non-Small Cell Lung Cancer to the Metabolism Drug Phenformin

The LKB1 (also called STK11) tumor suppressor is mutationally inactivated in ∼20% of non-small cell lung cancers (NSCLC). LKB1 is the major upstream kinase activating the energy-sensing kinase AMPK, making LKB1-deficient cells unable to appropriately sense metabolic stress. We tested the therapeutic potential of metabolic drugs in NSCLC and identified phenformin, a mitochondrial inhibitor and analog of the diabetes therapeutic metformin, as selectively inducing apoptosis in LKB1-deficient NSCLC cells. Therapeutic trials in Kras-dependent mouse models of NSCLC revealed that tumors with Kras and Lkb1 mutations, but not those with Kras and p53 mutations, showed selective response to phenformin as a single agent, resulting in prolonged survival. This study suggests phenformin as a cancer metabolism-based therapeutic to selectively target LKB1-deficient tumors.

GNAS is not involved in gastrointestinal tumour formation in Peutz-Jeghers syndrome

Peutz-Jeghers syndrome (PJS), caused by germ-line mutations in LKB1, is characterized by the development of hamartomatous polyps in the gastrointestinal (GI) tract. McCune Albright syndrome (MAS), caused by somatic activating mutations in GNAS, presents with cutaneous, skeletal, and endocrine manifestations. Recently, hamartomatous GI polyps with histological features similar to those in PJS were observed in MAS patients, suggesting a role for GNAS in the pathogenesis of PJS. This study reports the first somatic GNAS mutation analysis in GI tumours of PJS patients. No mutations were observed, suggesting that GNAS is not involved in the pathogenesis of GI tumours in PJS.