Loss of cellular identity in common pre-clinical models of serine­threonine kinase 11 (Liver kinase B1) loss.

Nearly 1/3 of lung adenocarcinomas have loss of STK11 (LKB1) function. Herein, a bioinformatics approach was used to determine how accurately preclinical model systems reflect the in vivo biology of STK11 loss in human patients. Hierarchical and K-mean clustering, principle component, and gene set enrichment analyses were employed to model gene expression due to STK11 loss in patient cohorts representing nearly 1000 lung adenocarcinoma patients. K-means clustering classified STK11 loss patient tumors into three distinct sub-groups; positive (54%), neuroendocrine (NE) (35%) and negative (11%). The positive and NE groups are both defined by the expression of NKX2-1. In addition to NKX2-1, NE patients express neuroendocrine markers such as ASCL1 and CALCA. In contrast, the negative group does not express NKX2-1 (or neuroendocrine markers) and is characterized by significantly reduced survival relative to the two other groups. Two gene expression signatures were derived to explain both neuroendocrine features and differentiation (NKX2-1 loss) and were validated through two public datasets involving chemical differentiation (DCI) and NKX2-1 reconstitution. Patients results were then compared with established cell lines, transgenic mice, and patient-derived xenograft models of STK11 loss. Interestingly, all cell line and PDX models cluster and show expression patterns similar with the NKX2-1 negative subset of STK11-loss human tumors. Surprisingly, even mouse models of STK11 loss do not resemble patient tumors based on gene expression patterns. Results suggest pre-clinical models of STK11 loss are pronounced by marked elimination of type II pneumocyte identity, opposite of most in vivo human tumors.

Long noncoding RNA, LINC00460, as a prognostic biomarker in head and neck squamous cell carcinoma (HNSCC).

Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy characterized by frequent mutations and metastasis. Long noncoding RNAs (lncRNAs) have been implicated in tumorigenesis and serve as novel prognostic biomarkers in different cancers. To enhance our understanding of lncRNAs that may have biological significance in HNSCC and may serve as prognostic biomarkers, we globally profiled lncRNAs in HNSCC by analyzing the RNA-seq data from The Atlas of Noncoding RNAs in Cancer (TANRIC) database. Of 3576 lncRNAs, we identified 926 (higher-688, lower-238) lncRNAs with a 2-fold abundance difference among the forty HNSCC and paired adjacent normal tissue. We investigated differential abundance of lncRNAs based on TP53 mutation and p16 status. We found 133 lncRNAs to have differential abundance by 2-fold among the mutant vs wild-type TP53 samples, whereas among p16-negative vs positive samples, we identified 710 lncRNAs with the same criteria. Meanwhile, analysis of the 15 most abundant lncRNAs in the tumor samples identified five lncRNAs whose higher abundance was associated with poor overall patient survival. Among these five, higher abundance of LINC00460 associated with poor patient survival in an independent cohort of 82 HNSCC patients. To further evaluate the potential function of LINC00460, we performed lncRNA-mRNAs co-expression analysis and found that higher abundance of LINC00460 associated with cancer-related biological pathways including EMT and other inflammatory response pathways. In summary, we report LINC00460 is more abundant in tumors compared to adjacent normal tissue and that it may serve as a potential prognostic biomarker in HNSCC.

A Molecular Epidemiological Analysis Of Programmed Cell Death Ligand-1 (PD-L1) Protein Expression, Mutations And Survival In Non-Small Cell Lung Cancer.

PURPOSE: To characterize programmed cell death ligand-1 (PD-L1) expression in relation to survival and gene mutation status in patients with advanced NSCLC. The study also explored the influence of tumor mutational burden (TMB) on PD-L1 expression and patient characteristics. PATIENTS AND METHODS: Adult patients with histologically or cytologically documented Stage IIIB/Stage IV/recurrent/progressive NSCLC, Eastern Cooperative Oncology Group performance status 0 to 3, and >2 lines of prior systemic treatment regimens were included in this retrospective analysis. Patients were treated from 1997 to 2015 at H. Lee Moffitt Cancer Center and Research Institute, Tampa, or at 7 community centers across the United States. PD-L1 expression level was determined using the VENTANA PD-L1 (SP263) Assay. EGFR and KRAS mutation status and ALK rearrangements were determined by targeted DNA sequencing; these were obtained from clinical records where targeted DNA sequencing was not performed. TMB was calculated as the total number of somatic mutations per sample. RESULTS: From a total of 136 patients included in the study, 23.5% had tumors with high PD-L1 expression (≥25%). There were no significant differences in patient characteristics, overall survival (OS), and progression-free survival (PFS) between patients with high PD-L1 expression (median OS: 39.5 months; median PFS: 15.8 months) vs low PD-L1 expression (<25%; median OS: 38.1 months; median PFS: 18.6 months). PD-L1 expression level correlated (P=0.05) with TMB and was consistent with The Cancer Genome Atlas data. CONCLUSION: In this retrospective analysis, survival outcomes of patients with advanced NSCLC were comparable by PD-L1 expression level. EGFR and KRAS mutation status were not found to be significantly associated with PD-L1 expression level, while TMB was weakly associated with PD-L1 expression level. Overall, PD-L1 expression level was not observed to be an independent prognostic biomarker in this cohort of patients with advanced NSCLC treated with chemotherapy.

MEK Inhibition Modulates Cytokine Response to Mediate Therapeutic Efficacy in Lung Cancer.

Activating mutations in BRAF, a key mediator of RAS signaling, are present in approximately 50% of melanoma patients. Pharmacologic inhibition of BRAF or the downstream MAP kinase MEK is highly effective in treating BRAF-mutant melanoma. In contrast, RAS pathway inhibitors have been less effective in treating epithelial malignancies, such as lung cancer. Here, we show that treatment of melanoma patients with BRAF and MEK inhibitors (MEKi) activated tumor NF-κB activity. MEKi potentiated the response to TNFα, a potent activator of NF-κB. In both melanoma and lung cancer cells, MEKi increased cell-surface expression of TNFα receptor 1 (TNFR1), which enhanced NF-κB activation and augmented expression of genes regulated by TNFα and IFNγ. Screening of 289 targeted agents for the ability to increase TNFα and IFNγ target gene expression demonstrated that this was a general activity of inhibitors of MEK and ERK kinases. Treatment with MEKi led to acquisition of a novel vulnerability to TNFα and IFNγ-induced apoptosis in lung cancer cells that were refractory to MEKi killing and augmented cell-cycle arrest. Abolishing the expression of TNFR1 on lung cancer cells impaired the antitumor efficacy of MEKi, whereas the administration of TNFα and IFNγ in MEKi-treated mice enhanced the antitumor response. Furthermore, immunotherapeutics known to induce expression of these cytokines synergized with MEKi in eradicating tumors. These findings define a novel cytokine response modulatory function of MEKi that can be therapeutically exploited. SIGNIFICANCE: Lung cancer cells are rendered sensitive to MEK inhibitors by TNFα and IFNγ, providing a strong mechanistic rationale for combining immunotherapeutics, such as checkpoint blockers, with MEK inhibitor therapy for lung cancer.See related commentary by Havel, p. 5699.

Characterization of epidermal growth factor receptor (EGFR) P848L, an unusual EGFR variant present in lung cancer patients, in a murine Ba/F3 model.

Lung cancer patients with mutations in epidermal growth factor receptor (EGFR) benefit from treatments targeting tyrosine kinase inhibitors (TKIs). However, both intrinsic and acquired resistance of tumors to TKIs are common, and EGFR variants have been identified that are resistant to multiple TKIs. In the present study, we characterized selected EGFR variants previously observed in lung cancer patients and expressed in a murine bone marrow pro-B Ba/F3 cell model. Among these EGFR variants, we report that an exon 20 deletion/insertion mutation S768insVGH is resistant to erlotinib (a first-generation TKI), but sensitive to osimertinib (a third-generation TKI). We also characterized a rare exon 21 germline variant, EGFR P848L, which transformed Ba/F3 cells and conferred resistance to multiple EGFR-targeting TKIs. Our analysis revealed that P848L (a) does not bind erlotinib; (b) is turned over less rapidly than L858R (a common tumor-derived EGFR mutation); (c) is not autophosphorylated at Tyr 1045 [the major docking site for Cbl proto-oncogene (c-Cbl) binding]; and (d) does not bind c-Cbl. Using viability assays including 300 clinically relevant targeted compounds, we observed that Ba/F3 cells transduced with EGFR P848L, S768insVGH, or L858R have very different drug-sensitivity profiles. In particular, EGFR P848L, but not L858R or S768insVGH, was sensitive to multiple Janus kinase 1/2 inhibitors. In contrast, cells driven by L858R, but not by P848L, were sensitive to multikinase MAPK/extracellular-signal-regulated kinase (ERK) kinase and ERK inhibitors including EGFR-specific TKIs. These observations suggest that continued investigation of rare TKI-resistant EGFR variants is warranted to identify optimal treatments for cancer.

Somatic Mutations and Ancestry Markers in Hispanic Lung Cancer Patients.

INTRODUCTION: To address the lack of genomic data from Hispanic/Latino (H/L) patients with lung cancer, the Latino Lung Cancer Registry was established to collect patient data and biospecimens from H/L patients. METHODS: This retrospective observational study examined lung cancer tumor samples from 163 H/L patients, and tumor-derived DNA was subjected to targeted-exome sequencing (>1000 genes, including EGFR, KRAS, serine/threonine kinase 11 gene [STK11], and tumor protein p53 gene [TP53]) and ancestry analysis. Mutation frequencies in this H/L cohort were compared with those in a similar cohort of non-Hispanic white (NHW) patients and correlated with ancestry, sex, smoking status, and tumor histologic type. RESULTS: Of the adenocarcinomas in the H/L cohort (n = 120), 31% had EGFR mutations, versus 17% in the NHW control group (p < 0.001). KRAS (20% versus 38% [p = 0.002]) and STK11 (8% versus 16% [p = 0.065]) mutations occurred at lower frequency, and mutations in TP53 occurred at similar frequency (46% versus 40% [p = 0.355]) in H/L and NHW patients, respectively. Within the Hispanic cohort, ancestry influenced the rate of TP53 mutations (p = 0.009) and may have influenced the rate of EGFR, KRAS, and STK11 mutations. CONCLUSIONS: Driver mutations in H/L patients with lung adenocarcinoma differ in frequency from those in NHW patients associated with their indigenous American ancestry. The spectrum of driver mutations needs to be further assessed in the H/L population.

cAMP/CREB-regulated LINC00473 marks LKB1-inactivated lung cancer and mediates tumor growth.

The LKB1 tumor suppressor gene is frequently mutated and inactivated in non-small cell lung cancer (NSCLC). Loss of LKB1 promotes cancer progression and influences therapeutic responses in preclinical studies; however, specific targeted therapies for lung cancer with LKB1 inactivation are currently unavailable. Here, we have identified a long noncoding RNA (lncRNA) signature that is associated with the loss of LKB1 function. We discovered that LINC00473 is consistently the most highly induced gene in LKB1-inactivated human primary NSCLC samples and derived cell lines. Elevated LINC00473 expression correlated with poor prognosis, and sustained LINC00473 expression was required for the growth and survival of LKB1-inactivated NSCLC cells. Mechanistically, LINC00473 was induced by LKB1 inactivation and subsequent cyclic AMP-responsive element-binding protein (CREB)/CREB-regulated transcription coactivator (CRTC) activation. We determined that LINC00473 is a nuclear lncRNA and interacts with NONO, a component of the cAMP signaling pathway, thereby facilitating CRTC/CREB-mediated transcription. Collectively, our study demonstrates that LINC00473 expression potentially serves as a robust biomarker for tumor LKB1 functional status that can be integrated into clinical trials for patient selection and treatment evaluation, and implicates LINC00473 as a therapeutic target for LKB1-inactivated NSCLC.