Small cell lung cancer: Subtypes and therapeutic implications.

Small cell lung cancer (SCLC) is an extremely aggressive neuroendocrine tumor, accounting for approximated 13% of all lung cancer cases. SCLC is characterized by rapid growth and early metastasis. Despite marked improvements in the number and efficacy of targeted, therapeutic options and overall survival rates in SCLC have remained nearly unchanged for almost three decades. The lack of significant progress can be attributed to our poor understanding of the biology of SCLC. Although immune checkpoint inhibitors were recently approved as front-line therapies for SCLC, we still need to better understand the mechanisms responsible for the selective vulnerability of some SCLCs to these inhibitors. Recent work utilizing sequencing data and single cell analyses identified four distinct subsets of SCLC, based on the expression levels of the transcription factors ASCL1, NEUROD1, POU2F3 and YAP1. Each subset was found to have its own distinct biology and therapeutic vulnerabilities. However, these subsets appear to be phenotypically unstable, representing snapshots in the gradual evolution of a tumor that exhibits significant plasticity. Tumor evolution, a product of this plasticity, results in the emergence of significant intratumoral heterogeneity which plays an important role in multiple aspects of SCLC development and progression, including cell survival and proliferation, metastasis and angiogenesis. The recent paradigm shifting discoveries in the biology of SCLC are now beginning to inform the design of new therapeutic strategies for the management of this intractable disease.

Deletion of histone deacetylase 3 reveals critical roles in S phase progression and DNA damage control.

Histone deacetylases (HDACs) are enzymes that modify key residues in histones to regulate chromatin architecture, and they play a vital role in cell survival, cell-cycle progression, and tumorigenesis. To understand the function of Hdac3, a critical component of the N-CoR/SMRT repression complex, a conditional allele of Hdac3 was engineered. Cre-recombinase-mediated inactivation of Hdac3 led to a delay in cell-cycle progression, cell-cycle-dependent DNA damage, and apoptosis in mouse embryonic fibroblasts (MEFs). While no overt defects in mitosis were observed in Hdac3-/- MEFs, including normal H3Ser10 phosphorylation, DNA damage was observed in Hdac3-/- interphase cells, which appears to be associated with defective DNA double-strand break repair. Moreover, we noted that Hdac3-/- MEFs were protected from DNA damage when quiescent, which may provide a mechanistic basis for the action of HDAC inhibitors on cycling tumor cells.

In-depth proteomic analysis of nonsmall cell lung cancer to discover molecular targets and candidate biomarkers.

Advances in proteomic analysis of human samples are driving critical aspects of biomarker discovery and the identification of molecular pathways involved in disease etiology. Toward that end, in this report we are the first to use a standardized shotgun proteomic analysis method for in-depth tissue protein profiling of the two major subtypes of nonsmall cell lung cancer and normal lung tissues. We identified 3621 proteins from the analysis of pooled human samples of squamous cell carcinoma, adenocarcinoma, and control specimens. In addition to proteins previously shown to be implicated in lung cancer, we have identified new pathways and multiple new differentially expressed proteins of potential interest as therapeutic targets or diagnostic biomarkers, including some that were not identified by transcriptome profiling. Up-regulation of these proteins was confirmed by multiple reaction monitoring mass spectrometry. A subset of these proteins was found to be detectable and differentially present in the peripheral blood of cases and matched controls. Label-free shotgun proteomic analysis allows definition of lung tumor proteomes, identification of biomarker candidates, and potential targets for therapy.

Liver-specific deletion of histone deacetylase 3 disrupts metabolic transcriptional networks.

Histone deacetylase 3 (Hdac3) is an enzymatic component of transcriptional repression complexes recruited by the nuclear hormone receptors. Inactivation of Hdac3 in cancer cell lines triggered apoptosis, and removal of Hdac3 in the germ line of mice caused embryonic lethality. Therefore, we deleted Hdac3 in the postnatal mouse liver. These mice developed hepatomegaly, which was the result of hepatocyte hypertrophy, and these morphological changes coincided with significant imbalances between carbohydrate and lipid metabolism. Loss of Hdac3 triggered changes in gene expression consistent with inactivation of repression mediated by nuclear hormone receptors. Loss of Hdac3 also increased the levels of Ppar gamma2, and treatment of these mice with a Ppar gamma antagonist partially reversed the lipid accumulation in the liver. In addition, gene expression analysis identified mammalian target of rapamycin signalling as being activated after deletion of Hdac3, and inhibition by rapamycin affected the accumulation of neutral lipids in Hdac3-null livers. Thus, Hdac3 regulates metabolism through multiple signalling pathways in the liver, and deletion of Hdac3 disrupts normal metabolic homeostasis.

Metabolic reprogramming by adenosine antagonism and implications in non-small cell lung cancer therapy.

Non-small cell lung cancer (NSCLC) is a heterogeneous disease with genetic and environmental parameters that influence cell metabolism. Because of the complex interplay of environmental factors within the tumor microenvironment (TME) and the profound impact of these factors on the metabolic activities of tumor and immune cells, there is an emerging interest to advance the understanding of these diverse metabolic phenotypes in the TME. High levels of adenosine are characteristic of the TME, and adenosine can have a significant impact on both tumor cell growth and the immune response. Consistent with this, we showed in NSCLC data from TCGA that high expression of the A2BR leads to worse outcome and that expression of A2BR may be different for different mutation backgrounds. We then investigated the metabolic reprogramming of tumor cells and immune cells (T and dendritic cells) by adenosine. We used A2AR and A2BR antagonism or agonism as well as receptor knockout animals to explore whether these treatments altered specific immune compartments or conferred specific therapeutic vulnerabilities. Using the seahorse assay, we found that an A2BR antagonist modulates oxidative stress homeostasis in NSCLC cell lines. In addition, we found distinct metabolic roles of A2AR and A2BR receptors in T cell activation and dendritic cell maturation. These data suggest potential mechanisms and therapeutic benefits of A2 receptor antagonist therapy in NSCLC.

Comparison of Tumor Microenvironments Between Primary Tumors and Brain Metastases in Patients With NSCLC.

INTRODUCTION: This study investigates the immune profile of the primary lung tumors and the corresponding brain metastasis from patients with NSCLC using multiplex fluorescence immunohistochemistry. METHODS: The study evaluated 34 patients who underwent autopsy or surgical resection for brain metastasis and autopsy, surgical resection, or core biopsy for primary lung cancer. We compared the densities of various immune cells in the primary tumors and the brain metastases by multiplex fluorescence immunohistochemical analysis. RESULTS: The density of CD4-positive (CD4+) T-cells, CD8-positive T-cells, and CD4+ Foxp3-positive T-cells were statistically higher in both tumor and stromal areas in primary lung cancer specimens when compared with brain metastases samples (p < 0.0001). Only CD204-positive cells were statistically higher in the tumor areas of the brain metastases (p = 0.0118). Tumor-infiltrating lymphocytes associated with brain metastases positively correlated with overall survival, but primary lung tumor-infiltrating lymphocytes did not. The density of CD4+ and CD4+ Foxp3-positive T-cells in brain metastases with radiation was statistically higher in the carcinoma and stromal areas compared with those without radiation (p = 0.0343, p = 0.0173). CONCLUSIONS: Our findings that CD204-positive cells were higher in brain metastases may have broader implications for treatment as these macrophages may be immunosuppressive and make the immune environment less reactive. Furthermore, the finding that the density of CD4+ T-cells was higher in cancer and stroma areas of brain metastases after radiotherapy supports the addition of immunotherapy to radiation therapy in the treatment of brain metastases in NSCLC.

Predicting ROR1/BCL2 combination targeted therapy of small cell carcinoma of the lung.

Small cell lung cancer (SCLC) remains a deadly form of cancer, with a 5-year survival rate of less than 10 percent, necessitating novel therapies. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein that is emerging as a therapeutic target and is co-expressed with BCL2 in multiple tumor types due to microRNA coregulation. We hypothesize that ROR1-targeted therapy is effective in small cell lung cancer and synergizes with therapeutic BCL2 inhibition. Tissue microarrays (TMAs) and formalin-fixed paraffin-embedded (FFPE) SCLC patient samples were utilized to determine the prevalence of ROR1 and BCL2 expression in SCLC. Eight SCLC-derived cell lines were used to determine the antitumor activity of a small molecule ROR1 inhibitor (KAN0441571C) alone and in combination with the BCL2 inhibitor venetoclax. The Chou-Talalay method was utilized to determine synergy with the drug combination. ROR1 and BCL2 protein expression was identified in 93% (52/56) and 86% (48/56) of SCLC patient samples, respectively. Similarly, ROR1 and BCL2 were shown by qRT-PCR to have elevated expression in 79% (22/28) and 100% (28/28) of SCLC patient samples, respectively. KAN0441571C displayed efficacy in 8 SCLC cell lines, with an IC50 of 500 nM or less. Synergy as defined by a combination index of <1 via the Chou-Talalay method between KAN0441571C and venetoclax was demonstrated in 8 SCLC cell lines. We have shown that ROR1 inhibition is synergistic with BCL2 inhibition in SCLC models and shows promise as a novel therapeutic target in SCLC.

STK11/LKB1 Loss of Function Is Associated with Global DNA Hypomethylation and S-Adenosyl-Methionine Depletion in Human Lung Adenocarcinoma.

STK11 (liver kinase B1, LKB1) is the fourth most frequently mutated gene in lung adenocarcinoma, with loss of function observed in up to 30% of all cases. Our previous work identified a 16-gene signature for LKB1 loss of function through mutational and nonmutational mechanisms. In this study, we applied this genetic signature to The Cancer Genome Atlas (TCGA) lung adenocarcinoma samples and discovered a novel association between LKB1 loss and widespread DNA demethylation. LKB1-deficient tumors showed depletion of S-adenosyl-methionine (SAM-e), which is the primary substrate for DNMT1 activity. Lower methylation following LKB1 loss involved repetitive elements (RE) and altered RE transcription, as well as decreased sensitivity to azacytidine. Demethylated CpGs were enriched for FOXA family consensus binding sites, and nuclear expression, localization, and turnover of FOXA was dependent upon LKB1. Overall, these findings demonstrate that a large number of lung adenocarcinomas exhibit global hypomethylation driven by LKB1 loss, which has implications for both epigenetic therapy and immunotherapy in these cancers. SIGNIFICANCE: Lung adenocarcinomas with LKB1 loss demonstrate global genomic hypomethylation associated with depletion of SAM-e, reduced expression of DNMT1, and increased transcription of repetitive elements.

Circulating MicroRNAs and Extracellular Vesicle-Containing MicroRNAs as Response Biomarkers of Anti-programmed Cell Death Protein 1 or Programmed Death-Ligand 1 Therapy in NSCLC.

INTRODUCTION: Anti-programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) antibody therapy is a standard treatment for advanced NSCLC, and PD-L1 immunohistochemistry is used as a predictive biomarker for therapeutic response. However, because not all patients with NSCLC with high PD-L1 respond, and some patients with low PD-L1 expression exhibit durable benefit, more accurate predictive biomarkers are needed. Circulating microRNA (miRNA) and miRNA packaged in extracellular vesicles (EVs) are believed to play a role in intercellular communication among immune cells and between immune cells and tumor cells and may represent a good source of mechanism-related biomarkers. METHODS: Pretreatment plasma of patients with advanced NSCLC treated with single-agent anti-PD-1 or anti-PD-L1 antibody was used in this study. Plasma EVs were isolated using size-exclusion chromatography. Whole plasma and EV-containing RNAs were extracted. The miRNA profile was analyzed with a next-generation sequencing platform. RESULTS: Samples from 14 responders (patients who exhibited partial response or stable disease ≥6 mo) and 15 nonresponders (patients who exhibited progressive disease as per Response Evaluation Criteria in Solid Tumors) were analyzed. In total, 32 miRNAs (p = 0.0030-0.0495) from whole plasma and seven EV-associated miRNAs (p = 0.041-0.0457) exhibited significant concentration differences between responders and nonresponders. The results of some of these circulating miRNAs were validated in a separate cohort with eight responders and 13 nonresponders. The tumor PD-L1 level was also assessed using immunohistochemistry for patients involved in both cohorts. CONCLUSIONS: Specific circulating miRNAs in whole plasma and plasma EVs are differentially expressed between responders and nonresponders and have potential as predictive biomarkers for anti-PD-1/PD-L1 treatment response.

Verification of a Blood-Based Targeted Proteomics Signature for Malignant Pleural Mesothelioma.

BACKGROUND: We have verified a mass spectrometry (MS)-based targeted proteomics signature for the detection of malignant pleural mesothelioma (MPM) from the blood. METHODS: A seven-peptide biomarker MPM signature by targeted proteomics in serum was identified in a previous independent study. Here, we have verified the predictive accuracy of a reduced version of that signature, now composed of six-peptide biomarkers. We have applied liquid chromatography-selected reaction monitoring (LC-SRM), also known as multiple-reaction monitoring (MRM), for the investigation of 402 serum samples from 213 patients with MPM and 189 cancer-free asbestos-exposed donors from the United States, Australia, and Europe. RESULTS: Each of the biomarkers composing the signature was independently informative, with no apparent functional or physical relation to each other. The multiplexing possibility offered by MS proteomics allowed their integration into a single signature with a higher discriminating capacity than that of the single biomarkers alone. The strategy allowed in this way to increase their potential utility for clinical decisions. The signature discriminated patients with MPM and asbestos-exposed donors with AUC of 0.738. For early-stage MPM, AUC was 0.765. This signature was also prognostic, and Kaplan-Meier analysis showed a significant difference between high- and low-risk groups with an HR of 1.659 (95% CI, 1.075-2.562; P = 0.021). CONCLUSIONS: Targeted proteomics allowed the development of a multianalyte signature with diagnostic and prognostic potential for MPM from the blood. IMPACT: The proteomic signature represents an additional diagnostic approach for informing clinical decisions for patients at risk for MPM.

RANBP9 as potential therapeutic target in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths in the Western world. Despite progress made with targeted therapies and immune checkpoint inhibitors, the vast majority of patients have to undergo chemotherapy with platinum-based drugs. To increase efficacy and reduce potential side effects, a more comprehensive understanding of the mechanisms of the DNA damage response (DDR) is required. We have shown that overexpressby live cell imaging (Incuyion of the scaffold protein RAN binding protein 9 (RANBP9) is pervasive in NSCLC. More importantly, patients with higher levels of RANBP9 exhibit a worse outcome from treatment with platinum-based drugs. Mechanistically, RANBP9 exists as a target and an enabler of the ataxia telangiectasia mutated (ATM) kinase signaling. Indeed, the depletion of RANBP9 in NSCLC cells abates ATM activation and its downstream targets such as pby live cell imaging (Incuy53 signaling. RANBP9 knockout cells are more sensitive than controls to the inhibition of the ataxia and telangiectasia-related (ATR) kinase but not to ATM inhibition. The absence of RANBP9 renders cells more sensitive to drugs inhibiting the Poly(ADP-ribose)-Polymerase (PARP) resulting in a "BRCAness-like" phenotype. In summary, as a result of increased sensitivity to DNA damaging drugs conferred by its ablation in vitro and in vivo, RANBP9 may be considered as a potential target for the treatment of NSCLC. This article aims to report the results from past and ongoing investigations focused on the role of RANBP9 in the response to DNA damage, particularly in the context of NSCLC. This review concludes with future directions and speculative remarks which will need to be addressed in the coming years.

Inhibition of epidermal growth factor receptor signaling elevates 15-hydroxyprostaglandin dehydrogenase in non-small-cell lung cancer.

Evidence indicates that the induction of cyclooxygenase-2 (COX-2) and high prostaglandin E2 (PGE2) levels contribute to the pathogenesis of non-small-cell lung cancer (NSCLC). In addition to overproduction by COX-2, PGE2 concentrations also depend upon the levels of the PGE2 catabolic enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). We find a dramatic down-regulation of PGDH protein in NSCLC cell lines and in resected human tumors when compared with matched normal lung. Affymetrix array analysis of 10 normal lung tissue samples and 49 resected lung tumors revealed a much lower expression of PGDH transcripts in all NSCLC histologic groups. In addition, treatment with the epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) erlotinib increased the expression of 15-PGDH in a subset of NSCLC cell lines. This effect may be due in part to an inhibition of the extracellular signal-regulated kinase (ERK) pathway as treatment with mitogen-activated protein kinase kinase (MEK) inhibitor U0126 mimics the erlotinib results. We show by quantitative reverse transcription-PCR that the transcript levels of ZEB1 and Slug transcriptional repressors are dramatically reduced in a responsive cell line upon EGFR and MEK/ERK inhibition. In addition, the Slug protein, but not ZEB1, binds to the PGDH promoter and represses transcription. As these repressors function by recruiting histone deacetylases to promoters, it is likely that PGDH is repressed by an epigenetic mechanism involving histone deacetylation, resulting in increased PGE2 activity in tumors. This effect is reversible in a subset of NSCLC upon treatment with an EGFR TKI.

Co-Expression Analysis Reveals Mechanisms Underlying the Varied Roles of NOTCH1 in NSCLC.

INTRODUCTION: Notch receptor family dysregulation can be tumor promoting or suppressing depending on cellular context. Our studies shed light on the mechanistic differences that are responsible for NOTCH1's opposing roles in lung adenocarcinoma and lung squamous cell carcinoma. METHODS: We integrated transcriptional patient-derived datasets with gene co-expression analyses to elucidate mechanisms behind NOTCH1 function in subsets of NSCLC. Differential co-expression was examined using hierarchical clustering and principal component analysis. Enrichment analysis was used to examine pathways associated with the underlying transcriptional networks. These pathways were validated in vitro and in vivo. Endogenously epitope-tagged NOTCH1 was used to identify novel interacting proteins. RESULTS: NOTCH1 co-expressed genes in lung adenocarcinoma and squamous carcinoma were distinct and associated with either angiogenesis and immune system pathways or cell cycle control and mitosis pathways, respectively. Tissue culture and xenograft studies of lung adenocarcinoma and lung squamous models with NOTCH1 knockdown showed growth differences and opposing effects on these pathways. Differential NOTCH1 interacting proteins were identified as potential mediators of these differences. CONCLUSIONS: Recognition of the opposing role of NOTCH1 in lung cancer, downstream pathways, and interacting proteins in each context may help direct the development of rational NOTCH1 pathway-dependent targeted therapies for specific tumor subsets of NSCLC.

The Effect of LKB1 Activity on the Sensitivity to PI3K/mTOR Inhibition in Non-Small Cell Lung Cancer.

INTRODUCTION: Liver kinase B1 (LKB1), also called serine/threonine kinase 11 (STK11), is a tumor suppressor that functions as master regulator of cell growth, metabolism, survival, and polarity. Approximately 30% to 35% of patients with NSCLC possess inactivated liver kinase B1 gene (LKB1), and these patients respond poorly to anti-programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) immunotherapy. Therefore, novel therapies targeting NSCLC with LKB1 loss are needed. METHODS: We used a new in silico signaling analysis method to identify the potential therapeutic targets and reposition drugs by integrating gene expression data with the Kyoto Encyclopedia of Genes and Genomes signaling pathways. LKB1 wild-type and LKB1-deficient NSCLC cell lines, including knockout clones generated by clustered regularly interspaced short pallindromic repeats-Cas9, were treated with inhibitors of mechanistic target of rapamycin kinase (mTOR) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and a dual inhibitor. RESULTS: In silico experiments showed that inhibition of both mTOR and PI3K can be synergistically effective in LKB1-deficient NSCLC. In vitro and in vivo experiments showed the synergistic effect of mTOR inhibition and PI3K inhibition in LKB1-mutant NSCLC cell lines. The sensitivity to dual inhibition of mTOR and PI3K is higher in LKB1-mutant cell lines than in wild-type cell lines. A higher compensatory increase in Akt phosphorylation after rapamycin treatment of LKB1-deficient cells than after rapamycin treatment of LKB1 wild-type cells is responsible for the synergistic effect of mTOR and PI3K inhibition. Dual inhibition of mTOR and PI3K resulted in a greater decrease in protein expression of cell cycle-regulating proteins in LKB1 knockout cells than in LKB1 wild-type cells. CONCLUSION: Dual molecular targeted therapy for mTOR and PI3K may be a promising therapeutic strategy in the specific population of patients with lung cancer with LKB1 loss.

Identification of Recurrent Activating HER2 Mutations in Primary Canine Pulmonary Adenocarcinoma.

PURPOSE: Naturally occurring primary canine lung cancers share clinicopathologic features with human lung cancers in never-smokers, but the genetic underpinnings of canine lung cancer are unknown. We have charted the genomic landscape of canine lung cancer and performed functional characterization of novel, recurrent HER2 (ERBB2) mutations occurring in canine pulmonary adenocarcinoma (cPAC). EXPERIMENTAL DESIGN: We performed multiplatform genomic sequencing of 88 primary canine lung tumors or cell lines. Additionally, in cPAC cell lines, we performed functional characterization of HER2 signaling and evaluated mutation-dependent HER2 inhibitor drug dose-response. RESULTS: We discovered somatic, coding HER2 point mutations in 38% of cPACs (28/74), but none in adenosquamous (cPASC, 0/11) or squamous cell (cPSCC, 0/3) carcinomas. The majority (93%) of HER2 mutations were hotspot V659E transmembrane domain (TMD) mutations comparable to activating mutations at this same site in human cancer. Other HER2 mutations were located in the extracellular domain and TMD. HER2 V659E was detected in the plasma of 33% (2/6) of dogs with localized HER2 V659E tumors. HER2 V659E cPAC cell lines displayed constitutive phosphorylation of AKT and significantly higher sensitivity to the HER2 inhibitors lapatinib and neratinib relative to HER2-wild-type cell lines (IC50 < 200 nmol/L in HER2 V659E vs. IC50 > 2,500 nmol/L in HER2 WT). CONCLUSIONS: This study creates a foundation for molecular understanding of and drug development for canine lung cancer. These data also establish molecular contexts for comparative studies in dogs and humans of low mutation burden, never-smoker lung cancer, and mutant HER2 function and inhibition.

Mtgr1 is a transcriptional corepressor that is required for maintenance of the secretory cell lineage in the small intestine.

Two members of the MTG/ETO family of transcriptional corepressors, MTG8 and MTG16, are disrupted by chromosomal translocations in up to 15% of acute myeloid leukemia cases. The third family member, MTGR1, was identified as a factor that associates with the t(8;21) fusion protein RUNX1-MTG8. We demonstrate that Mtgr1 associates with mSin3A, N-CoR, and histone deacetylase 3 and that when tethered to DNA, Mtgr1 represses transcription, suggesting that Mtgr1 also acts as a transcriptional corepressor. To define the biological function of Mtgr1, we created Mtgr1-null mice. These mice are proportionally smaller than their littermates during embryogenesis and throughout their life span but otherwise develop normally. However, these mice display a progressive reduction in the secretory epithelial cell lineage in the small intestine. This is not due to the loss of small intestinal progenitor cells expressing Gfi1, which is required for the formation of goblet and Paneth cells, implying that loss of Mtgr1 impairs the maturation of secretory cells in the small intestine.

Histone Deacetylase Inhibition Enhances the Antitumor Activity of a MEK Inhibitor in Lung Cancer Cells Harboring RAS Mutations.

Non-small cell lung cancer (NSCLC) can be identified by precise molecular subsets based on genomic alterations that drive tumorigenesis and include mutations in EGFR, KRAS, and various ALK fusions. However, despite effective treatments for EGFR and ALK, promising therapeutics have not been developed for patients with KRAS mutations. It has been reported that one way the RAS-ERK pathway contributes to tumorigenesis is by affecting stability and localization of FOXO3a protein, an important regulator of cell death and the cell cycle. This is through regulation of apoptotic proteins BIM and FASL and cell-cycle regulators p21Cip1 and p27Kip1 We now show that an HDAC inhibitor affects the expression and localization of FOXO proteins and wanted to determine whether the combination of a MEK inhibitor with an HDAC inhibitor would increase the sensitivity of NSCLC with KRAS mutation. Combined treatment with a MEK inhibitor and an HDAC inhibitor showed synergistic effects on cell metabolic activity of RAS-mutated lung cancer cells through activation of FOXOs, with a subsequent increase in BIM and cell-cycle inhibitors. Moreover, in a mouse xenograft model, the combination of belinostat and trametinib significantly decreases tumor formation through FOXOs by increasing BIM and the cell-cycle inhibitors p21Cip1 and p27Kip1 These results demonstrate that control of FOXOs localization and expression is critical in RAS-driven lung cancer cells, suggesting that the dual molecular-targeted therapy for MEK and HDACs may be promising as novel therapeutic strategy in NSCLC with specific populations of RAS mutations. Mol Cancer Ther; 17(1); 17-25. ©2017 AACR.

RANBP9 affects cancer cells response to genotoxic stress and its overexpression is associated with worse response to platinum in NSCLC patients.

Although limited by severe side effects and development of resistance, platinum-based therapies still represent the most common first-line treatment for non-small cell lung cancer (NSCLC). However, a crucial need in the clinical management of NSCLC is represented by the identification of cases sensitive to DNA damage response (DDR)-targeting drugs, such as cisplatin or PARP inhibitors. Here, we provide a molecular rationale for the stratification of NSCLC patients potentially benefitting from platinum compounds based on the expression levels of RANBP9, a recently identified player of the cellular DDR. RANBP9 was found overexpressed by immunohistochemistry (IHC) in NSCLC compared to normal adjacent tissues (NATs) (n = 147). Moreover, a retrospective analysis of 132 platinum-treated patients from the multi-centric TAILOR trial showed that RANBP9 overexpression levels are associated with clinical response to platinum compounds [Progression Free Survival Hazard Ratio (RANBP9 high vs low) 1.73, 95% CI 1.15-2.59, p = 0.0084; Overall Survival HR (RANBP9 high vs low) 1.99, 95% CI 1.27-3.11, p = 0.003]. Accordingly, RANBP9 KO cells showed higher sensitivity to cisplatin in comparison with WT controls both in vitro and in vivo models. NSCLC RANBP9 KO cells were also more sensitive than control cells to the PARP inhibitor olaparib alone and in combination with cisplatin, due to defective ATM-dependent and hyper-activated PARP-dependent DDR. The current investigation paves the way to prospective studies to assess the clinical value of RANBP9 protein levels as prognostic and predictive biomarker of response to DDR-targeting drugs, leading to the development of new tools for the management of NSCLC patients.

Selective profiling of proteins in lung cancer cells from fine-needle aspirates by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PURPOSE: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to analyze tumor sections and can determine tumor type, nodal involvement, and survival, and shows promise in predicting therapeutic response. Our purpose was to develop a method compatible with MALDI-TOF MS that allows selective analysis of cancer cells in mixed clinical samples such as fine-needle aspirates. EXPERIMENTAL DESIGN: Lung cancer cell lines were cytocentrifuged onto metal-coated, transparent glass slides and used for optimization of fixation, staining, and RBC lysis protocols. Fine-needle aspirates from human tumors and mouse model tumors were used to provide fresh tissue samples for determining the feasibility of this method. RESULTS: The MALDI-TOF MS compatible fixation and staining techniques provided high-resolution cellular morphology, which allowed identification and selective spotting of tumor cells. The RBC lysis step efficiently removed contaminating RBC yielding spectra nearly free from hemoglobin peaks. Protein profiles of fine-needle aspirates were found highly reproducible and similar to the profiles of the tissue from which they were obtained. Using this method, we were able to differentiate between xenograft tumors derived from two different human cell lines, A549 and H460. CONCLUSION: This procedure results in the production of high-quality, cancer cell-specific protein profiles. This highly reproducible technique could be applied to many other types of mixed clinical samples and has the potential to be very useful in the clinical diagnosis, classification, and, potentially, the individualized treatment of cancer patients.

A Transcriptional Signature Identifies LKB1 Functional Status as a Novel Determinant of MEK Sensitivity in Lung Adenocarcinoma.

LKB1 is a commonly mutated tumor suppressor in non-small cell lung cancer that exerts complex effects on signal transduction and transcriptional regulation. To better understand the downstream impact of loss of functional LKB1, we developed a transcriptional fingerprint assay representing this phenotype. This assay was predictive of LKB1 functional loss in cell lines and clinical specimens, even those without detected sequence alterations in the gene. In silico screening of drug sensitivity data identified putative LKB1-selective drug candidates, revealing novel associations not apparent from analysis of LKB1 mutations alone. Among the candidates, MEK inhibitors showed robust association with signature expression in both training and testing datasets independent of RAS/RAF mutations. This susceptibility phenotype is directly altered by RNA interference-mediated LKB1 knockdown or by LKB1 re-expression into mutant cell lines and is readily observed in vivo using a xenograft model. MEK sensitivity is dependent on LKB1-induced changes in AKT and FOXO3 activation, consistent with genomic and proteomic analyses of LKB1-deficient lung adenocarcinomas. Our findings implicate the MEK pathway as a potential therapeutic target for LKB1-deficient cancers and define a practical NanoString biomarker to identify functional LKB1 loss. Cancer Res; 77(1); 153-63. ©2016 AACR.