Enhanced Vulnerability of LKB1-Deficient NSCLC to Disruption of ATP Pools and Redox Homeostasis by 8-Cl-Ado.

Loss-of-function somatic mutations of STK11, a tumor suppressor gene encoding LKB1 that contributes to the altered metabolic phenotype of cancer cells, is the second most common event in lung adenocarcinomas and often co-occurs with activating KRAS mutations. Tumor cells lacking LKB1 display an aggressive phenotype, with uncontrolled cell growth and higher energetic and redox stress due to its failure to balance ATP and NADPH levels in response to cellular stimulus. The identification of effective therapeutic regimens for patients with LKB1-deficient non-small cell lung cancer (NSCLC) remains a major clinical need. Here, we report that LKB1-deficient NSCLC tumor cells displayed reduced basal levels of ATP and to a lesser extent other nucleotides, and markedly enhanced sensitivity to 8-Cl-adenosine (8-Cl-Ado), an energy-depleting nucleoside analog. Treatment with 8-Cl-Ado depleted intracellular ATP levels, raised redox stress, and induced cell death leading to a compensatory suppression of mTOR signaling in LKB1-intact, but not LKB1-deficient, cells. Proteomic analysis revealed that the MAPK/MEK/ERK and PI3K/AKT pathways were activated in response to 8-Cl-Ado treatment and targeting these pathways enhanced the antitumor efficacy of 8-Cl-Ado. IMPLICATIONS: Together, our findings demonstrate that LKB1-deficient tumor cells are selectively sensitive to 8-Cl-Ado and suggest that therapeutic approaches targeting vulnerable energy stores combined with signaling pathway inhibitors merit further investigation for this patient population.

Inhibition of nonsense-mediated decay rescues p53ß/γ isoform expression and activates the p53 pathway in MDM2-overexpressing and select p53-mutant cancers.

Inactivation of p53 is present in almost every tumor, and hence, p53-reactivation strategies are an important aspect of cancer therapy. Common mechanisms for p53 loss in cancer include expression of p53-negative regulators such as MDM2, which mediate the degradation of wildtype p53 (p53α), and inactivating mutations in the TP53 gene. Currently, approaches to overcome p53 deficiency in these cancers are limited. Here, using non-small cell lung cancer and glioblastoma multiforme cell line models, we show that two alternatively spliced, functional truncated isoforms of p53 (p53ß and p53γ, comprising exons 1 to 9ß or 9γ, respectively) and that lack the C-terminal MDM2-binding domain have markedly reduced susceptibility to MDM2-mediated degradation but are highly susceptible to nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. In cancer cells harboring MDM2 overexpression or TP53 mutations downstream of exon 9, NMD inhibition markedly upregulates p53ß and p53γ and restores activation of the p53 pathway. Consistent with p53 pathway activation, NMD inhibition induces tumor suppressive activities such as apoptosis, reduced cell viability, and enhanced tumor radiosensitivity, in a relatively p53-dependent manner. In addition, NMD inhibition also inhibits tumor growth in a MDM2-overexpressing xenograft tumor model. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in p53-deficient tumors bearing MDM2 overexpression or p53 mutations downstream of exon 9, subgroups that comprise approximately 6% of all cancers.

RAD50 Expression Is Associated with Poor Clinical Outcomes after Radiotherapy for Resected Non-small Cell Lung Cancer.

Purpose: Although postoperative radiotherapy is often used to maintain local control after surgical resection and chemotherapy for locally advanced non-small cell lung cancer (NSCLC), both locoregional failure and distant metastasis remain problematic. The mechanisms of therapeutic resistance remain poorly understood.Experimental Design: We used reverse-phase protein arrays (RPPA) to profile the baseline expression of 170 total and phosphorylated proteins in 70 NSCLC cell lines to categorize pathways that may contribute to radiation resistance. Significant markers identified by RPPA were further analyzed in tissue microarrays (TMA) of specimens from 127 patients with NSCLC who had received surgery before receiving postoperative radiotherapy. Cox regression analysis and log-rank tests were used to identify potential predictive factors. We then validated the biological function of the markers in NSCLC cell lines in vitroResults: Of the 170 proteins or phospho-proteins profiled, a subset of 12 proteins was found to correlate with radiation response parameters. TMA analysis of the 12 proteins showing the greatest differences in expression in the RPPA analysis demonstrated that RAD50 had the strongest correlation with distant relapse-free survival, locoregional relapse-free survival, and disease-free survival in patients with NSCLC. We confirmed that knockdown of RAD50 sensitized NSCLC cells to radiation and that upregulation of RAD50 increased radioresistance in in vitro experiments.Conclusions: Upregulated RAD50 may be a predictor of radioresistance in patients with lung cancer who received radiotherapy. Clin Cancer Res; 24(2); 341-50. ©2017 AACR.

KDR Amplification Is Associated with VEGF-Induced Activation of the mTOR and Invasion Pathways but does not Predict Clinical Benefit to the VEGFR TKI Vandetanib.

PURPOSE: VEGF pathway inhibitors have been investigated as therapeutic agents in the treatment of non-small cell lung cancer (NSCLC) because of its central role in angiogenesis. These agents have improved survival in patients with advanced NSCLC, but the effects have been modest. Although VEGFR2/KDRis typically localized to the vasculature, amplification ofKDRhas reported to occur in 9% to 30% of the DNA from different lung cancers. We investigated the signaling pathways activated downstream ofKDRand whetherKDRamplification is associated with benefit in patients with NSCLC treated with the VEGFR inhibitor vandetanib. METHODS: NSCLC cell lines with or withoutKDRamplification were studied for the effects of VEGFR tyrosine kinase inhibitors (TKI) on cell viability and migration. Archival tumor samples collected from patients with platinum-refractory NSCLC in the phase III ZODIAC study of vandetanib plus docetaxel or placebo plus docetaxel (N= 294) were screened forKDRamplification by FISH. RESULTS: KDRamplification was associated with VEGF-induced activation of mTOR, p38, and invasiveness in NSCLC cell lines. However, VEGFR TKIs did not inhibit proliferation of NSCLC cell lines withKDRamplification. VEGFR inhibition decreased cell motility as well as expression of HIF1α inKDR-amplified NSCLC cells. In the ZODIAC study,KDRamplification was observed in 15% of patients and was not associated with improved progression-free survival, overall survival, or objective response rate for the vandetanib arm. CONCLUSIONS: Preclinical studies suggestKDRactivates invasion but not survival pathways inKDR-amplified NSCLC models. Patients with NSCLC whose tumor hadKDRamplification were not associated with clinical benefit for vandetanib in combination with docetaxel.

Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma with distinct biology, immune profiles, and therapeutic vulnerabilities.

UNLABELLED: The molecular underpinnings that drive the heterogeneity of KRAS-mutant lung adenocarcinoma are poorly characterized. We performed an integrative analysis of genomic, transcriptomic, and proteomic data from early-stage and chemorefractory lung adenocarcinoma and identified three robust subsets of KRAS-mutant lung adenocarcinoma dominated, respectively, by co-occurring genetic events in STK11/LKB1 (the KL subgroup), TP53 (KP), and CDKN2A/B inactivation coupled with low expression of the NKX2-1 (TTF1) transcription factor (KC). We further revealed biologically and therapeutically relevant differences between the subgroups. KC tumors frequently exhibited mucinous histology and suppressed mTORC1 signaling. KL tumors had high rates of KEAP1 mutational inactivation and expressed lower levels of immune markers, including PD-L1. KP tumors demonstrated higher levels of somatic mutations, inflammatory markers, immune checkpoint effector molecules, and improved relapse-free survival. Differences in drug sensitivity patterns were also observed; notably, KL cells showed increased vulnerability to HSP90-inhibitor therapy. This work provides evidence that co-occurring genomic alterations identify subgroups of KRAS-mutant lung adenocarcinoma with distinct biology and therapeutic vulnerabilities. SIGNIFICANCE: Co-occurring genetic alterations in STK11/LKB1, TP53, and CDKN2A/B-the latter coupled with low TTF1 expression-define three major subgroups of KRAS-mutant lung adenocarcinoma with distinct biology, patterns of immune-system engagement, and therapeutic vulnerabilities.

An epithelial-mesenchymal transition gene signature predicts resistance to EGFR and PI3K inhibitors and identifies Axl as a therapeutic target for overcoming EGFR inhibitor resistance.

PURPOSE: Epithelial-mesenchymal transition (EMT) has been associated with metastatic spread and EGF receptor (EGFR) inhibitor resistance. We developed and validated a robust 76-gene EMT signature using gene expression profiles from four platforms using non-small cell lung carcinoma (NSCLC) cell lines and patients treated in the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) study. EXPERIMENTAL DESIGN: We conducted an integrated gene expression, proteomic, and drug response analysis using cell lines and tumors from patients with NSCLC. A 76-gene EMT signature was developed and validated using gene expression profiles from four microarray platforms of NSCLC cell lines and patients treated in the BATTLE study, and potential therapeutic targets associated with EMT were identified. RESULTS: Compared with epithelial cells, mesenchymal cells showed significantly greater resistance to EGFR and PI3K/Akt pathway inhibitors, independent of EGFR mutation status, but more sensitivity to certain chemotherapies. Mesenchymal cells also expressed increased levels of the receptor tyrosine kinase Axl and showed a trend toward greater sensitivity to the Axl inhibitor SGI-7079, whereas the combination of SGI-7079 with erlotinib reversed erlotinib resistance in mesenchymal lines expressing Axl and in a xenograft model of mesenchymal NSCLC. In patients with NSCLC, the EMT signature predicted 8-week disease control in patients receiving erlotinib but not other therapies. CONCLUSION: We have developed a robust EMT signature that predicts resistance to EGFR and PI3K/Akt inhibitors, highlights different patterns of drug responsiveness for epithelial and mesenchymal cells, and identifies Axl as a potential therapeutic target for overcoming EGFR inhibitor resistance associated with the mesenchymal phenotype.

Frame-disrupting mutations elicit pre-mRNA accumulation independently of frame disruption.

The T-cell receptor (TCR) and immunoglobulin (Ig) genes are unique among vertebrate genes in that they undergo programmed rearrangement, a process that allows them to generate an enormous array of receptors with different antigen specificities. While crucial for immune function, this rearrangement mechanism is highly error prone, often generating frameshift or nonsense mutations that render the rearranged TCR and Ig genes defective. Such frame-disrupting mutations have been reported to increase the level of TCRbeta and Igmicro pre-mRNA, suggesting the hypothesis that RNA processing is blocked when frame disruption is sensed. Using a chimeric gene that contains TCRbeta sequences conferring this upregulatory response, we provide evidence that pre-mRNA upregulation is neither frame- nor translation-dependent; instead, several lines of evidence suggested that it is the result of disrupted cis elements necessary for efficient RNA splicing. In particular, we identify the rearranging VDJ(beta) exon as being uniquely densely packed with exonic-splicing enhancers (ESEs), rendering this exon hypersensitive to mutational disruption. As the chimeric gene that we developed for these studies generates unusually stable nuclear pre-mRNAs that accumulate when challenged with ESE mutations, we suggest it can be used as a sensitive in vivo system to identify and characterize ESEs.

Nonsense codons trigger an RNA partitioning shift.

T-cell receptor-beta (TCRbeta) genes naturally acquire premature termination codons (PTCs) as a result of programmed gene rearrangements. PTC-bearing TCRbeta transcripts are dramatically down-regulated to protect T-cells from the deleterious effects of the truncated proteins that would otherwise be produced. Here we provide evidence that two responses collaborate to elicit this dramatic down-regulation. One is rapid mRNA decay triggered by the nonsense-mediated decay (NMD) RNA surveillance pathway. We demonstrate that this occurs in highly purified nuclei lacking detectable levels of three different cytoplasmic markers, but containing an outer nuclear membrane marker, suggesting that decay occurs either in the nucleoplasm or at the outer nuclear membrane. The second response is a dramatic partitioning shift in the nuclear fraction-to-cytoplasmic fraction mRNA ratio that results in few TCRbeta transcripts escaping to the cytoplasmic fraction of cells. Analysis of TCRbeta mRNA kinetics after either transcriptional repression or induction suggested that this nonsense codon-induced partitioning shift (NIPS) response is not the result of cytoplasmic NMD but instead reflects retention of PTC(+) TCRbeta mRNA in the nuclear fraction of cells. We identified TCRbeta sequences crucial for NIPS but found that NIPS is not exclusively a property of TCRbeta transcripts, and we identified non-TCRbeta sequences that elicit NIPS. RNA interference experiments indicated that NIPS depends on the NMD factors UPF1 and eIF4AIII but not the NMD factor UPF3B. We propose that NIPS collaborates with NMD to retain and degrade a subset of PTC(+) transcripts at the outer nuclear membrane and/or within the nucleoplasm.

An alternative branch of the nonsense-mediated decay pathway.

The T-cell receptor (TCR) locus undergoes programmed rearrangements that frequently generate premature termination codons (PTCs). The PTC-bearing transcripts derived from such nonproductively rearranged genes are dramatically downregulated by the nonsense-mediated decay (NMD) pathway. Here, we show that depletion of the NMD factor UPF3b does not impair TCRbeta NMD, thereby distinguishing it from classical NMD. Depletion of the related factor UPF3a, by itself or in combination with UPF3b, also has no effect on TCRbeta NMD. Mapping experiments revealed the identity of TCRbeta sequences that elicit a switch to UPF3b dependence. This regulation is not a peculiarity of TCRbeta, as we identified many wild-type genes, including one essential for NMD, that transcribe NMD-targeted mRNAs whose downregulation is little or not affected by UPF3a and UPF3b depletion. We propose that we have uncovered an alternative branch of the NMD pathway that not only degrades aberrant mRNAs but also regulates normal mRNAs, including one that participates in a negative feedback loop controlling the magnitude of NMD.

RNA splicing promotes translation and RNA surveillance.

Aberrant mRNAs harboring premature termination codons (PTCs or nonsense codons) are degraded by the nonsense-mediated mRNA decay (NMD) pathway. mRNAs transcribed from genes that naturally acquire PTCs during lymphocyte development are strongly downregulated by PTCs. Here we show that a signal essential for this robust mRNA downregulatory response is efficient RNA splicing. Strong mRNA downregulation can be conferred on a poor NMD substrate by either strengthening its splicing signals or removing its weak introns. Efficient splicing also strongly promotes translation, providing a molecular explanation for enhanced NMD and suggesting that efficient splicing may have evolved to enhance both protein production and RNA surveillance. Our results suggest simple approaches for increasing protein expression from expression vectors and treating human genetic diseases caused by nonsense and frameshift mutations.

Boundary-independent polar nonsense-mediated decay.

Nonsense-mediated decay (NMD) is an RNA surveillance mechanism that degrades mRNAs containing premature termination (nonsense) codons. The second signal for this pathway in mammalian cells is an intron that must be at least approximately 55 nucleotides downstream of the nonsense codon. Although the functional significance of this '-55 boundary rule' is not known, it is widely thought to reflect the important role of an exon junction protein complex deposited just upstream of exon-exon junctions after RNA splicing. Here we report that a T-cell receptor (TCR)-beta gene did not conform to this rule. Rather than a definitive boundary position, nonsense codons had a polar effect, such that nonsense codons distant from the terminal downstream intron triggered robust NMD and proximal nonsense codons caused modest NMD. We identified a region of the TCR-beta gene that conferred this boundary-independent polar expression pattern on a heterologous gene. Collectively, our results suggest that TCR-beta transcripts contain one or more sequence elements that elicit an unusual NMD response triggered by a novel second signal that ultimately causes boundary-independent polar regulation. TCR genes may have evolved this unique NMD response because they frequently acquire nonsense codons during normal development.

T-cell receptor sequences that elicit strong down-regulation of premature termination codon-bearing transcripts.

The nonsense-mediated decay (NMD) RNA surveillance pathway detects and degrades mRNAs containing premature termination codons (PTCs). T-cell receptor (TCR) and immunoglobulin transcripts, which commonly harbor PTCs as a result of programmed DNA rearrangement during normal development, are down-regulated much more than other known mammalian gene transcripts in response to nonsense codons. Here, we demonstrate that this is not because of promoter or cell type but instead is directed by regulatory sequences within the rearranging VDJ exon and immediately flanking intron sequences of a Vbeta8.1 TCR-beta gene. Insertion of these sequences into a heterologous gene elicited strong down-regulation (>30-fold) in response to PTCs, indicating that this region is sufficient to trigger robust down-regulation. The rearranging Vbeta5.1 exon and the flanking intron sequences from another member of the TCR-beta family also triggered strong down-regulation, suggesting that down-regulatory-promoting elements are a conserved feature of TCR genes. Importantly, we found that the Vbeta8.1 down-regulatory-promoting element was position dependent, such that it failed to function when positioned downstream of a PTC. To our knowledge, this is the first class of down-regulatory elements identified that act upstream of nonsense codons.