Mutant Non-small Cell Lung Carcinoma Research Articles

Clinicopathological Characteristics and Real-life Data of Patients Receiving Tyrosine Kinase Inhibitor in Metastatic EGFR Mutant Non-small Cell Lung Carcinoma

Clinicopathological Characteristics and Real-life Data of Patients Receiving Tyrosine Kinase Inhibitor in Metastatic EGFR Mutant Non-small Cell Lung Carcinoma

FSP1 confers ferroptosis resistance in KEAP1 mutant non-small cell lung carcinoma in NRF2-dependent and -independent manner

Ferroptosis, a type of cell death induced by lipid peroxidation, has emerged as a novel anti-cancer strategy. Cancer cells frequently acquire resistance to ferroptosis. However, the underlying mechanisms are poorly understood. To address this issue, we conducted a thorough investigation of the genomic and transcriptomic data derived from hundreds of human cancer cell lines and primary tissue samples, with a particular focus on non-small cell lung carcinoma (NSCLC). It was observed that mutations in Kelch-like ECH-associated protein 1 (KEAP1) and subsequent nuclear factor erythroid 2-related factor 2 (NRF2, also known as NFE2L2) activation are strongly associated with ferroptosis resistance in NSCLC. Additionally, AIFM2 gene, which encodes ferroptosis suppressor protein 1 (FSP1), was identified as the gene most significantly correlated with ferroptosis resistance, followed by multiple NRF2 targets. We found that inhibition of NRF2 alone was not sufficient to reduce FSP1 protein levels and promote ferroptosis, whereas FSP1 inhibition effectively sensitized KEAP1-mutant NSCLC cells to ferroptosis. Furthermore, we found that combined inhibition of FSP1 and NRF2 induced ferroptosis more intensely. Our findings imply that FSP1 is a crucial suppressor of ferroptosis whose expression is partially dependent on NRF2 and that synergistically targeting both FSP1 and NRF2 may be a promising strategy for overcoming ferroptosis resistance in cancer.

Silencing of lncRNA CRNDE attenuates nonsmall-cell lung cancer progression by mediating the miR-455-3p/HDAC2 axis.

Nonsmall-cell lung carcinoma (NSCLC) is one of the deadliest malignancies in the world. LncRNAs are confirmed to be involved in the progression of NSCLC. Meanwhile, lncRNA CRNDE is known to be upregulated in NSCLC; however, the mechanism by which CRNDE regulates the tumourigenesis of NSCLC remains unclear. To test the function of CRNDE in NSCLC, cell proliferation, invasion, and migration were investigated by colony formation and Transwell assays, respectively. qPCR and Western blotting were applied to test gene and protein levels. In addition, the relationship among CRNDE, miR-455-3p, and HDAC2 was explored by dual-luciferase and RIP assays. The data revealed that the expression of CRNDE was upregulated in NSCLC tissues, while miR-455-3p was downregulated. CRNDE knockdown inhibited the viability, migration and invasion of NSCLC cells or epidermal growth factor receptor gene (EGFR)-mutant NSCLC cells. Moreover, inhibition of miR-455-3p exhibited the opposite effect. CRNDE bound with miR-455-3p, and HDAC2 was found to be targeted by miR-455-3p. Meanwhile, miR-455-3p downregulation reversed the effect of CRNDE knockdown on NSCLC cell function. Furthermore, miR-455-3p notably inhibited the growth and invasion of NSCLC cells via downregulation of HDAC2. Knockdown of CRNDE attenuated NSCLC progression via modulation of the miR-455-3p/HDAC2 axis. Thus, those findings might provide a novel strategy against NSCLC.

EGFR and PDL1: A Match (Not) Made in Heaven-A Real-World Retrospective Analysis of PDL1 Expression in EGFR-Mutated NSCLC.

EGFR (epidermal growth factor receptor) mutant NSCLC (non-small cell lung carcinoma) comprises 35-40% of cases in the Asian NSCLC cohort, compared to 15-20% in the rest of the world. Improved response rates have been observed in terms of PFS (progression-free survival) and ORR (overall response rate) when treated with EGFR TKIs (tyrosine kinase inhibitors). However, resistance eventually ensues regardless of the generation of TKI used. Preclinical studies have reported that PDL1 (programmed death ligand1) is a downstream target of EGFR and is interposed by IL-6/JAK/STAT3 (interleukin-6/Janus kinase/signal transducer and activator of transcription3), NF-κB (nuclear factor kappa beta), and p-ERK1/2/p-c-Jun pathways. Hence, it may potentially be repressed by EGFR TKIs. In this retrospective exploratory analysis, we studied whether PDL1 expression affects efficacy of EGFR TKIs and clinical outcome in patients with untreated metastatic EGFR-mutated lung adenocarcinoma. This single-center retrospective, exploratory analysis was performed between January 2015 and December 2019. Among 1350 cases of NSCLC, 470 were EGFR mutant, of which PDL1 expression testing was done in 193 patients who were included in this study. Median age was 60years (range 24-87years). A total of 116 patients (60.1%) had inframe deletion in exon19, 52 (26.9%) had L858R, and 25 (13%) had uncommon mutations. The number of patients with PDL1 tumour proportion score (TPS) < 1% was 109 (56.5%); 1-49%, 57 (29.5%); and ≥ 50%, 27 (14%). Comparing clinical characteristics among various PDL1 groups, there were no statistically significant correlations obtained. However, patients with PDL1 > 50% were smokers, and showed a trend for higher disease burden at diagnosis. Median PFS of PDL1 < 1% was 10.14months, compared to 9.4months in the PDL1 > 1% group; however, the values did not reach statistical significance. The current study was an exploratory retrospective study; however, the results add to the growing body of evidence that PDL1 expression in EGFR-mutated NSCLC does not have any prognostic significance. Also the efficacy of EGFR TKIs is not influenced by variations in PDL1 TPS.

Endothelin-1-Mediated Drug Resistance in EGFR-Mutant Non-Small Cell Lung Carcinoma.

Progression on therapy in non-small cell lung carcinoma (NSCLC) is often evaluated radiographically, however, image-based evaluation of said therapies may not distinguish disease progression due to intrinsic tumor drug resistance or inefficient tumor penetration of the drugs. Here we report that the inhibition of mutated EGFR promotes the secretion of a potent vasoconstrictor, endothelin-1 (EDN1), which continues to increase as the cells become resistant with a mesenchymal phenotype. As EDN1 and its receptor (EDNR) is linked to cancer progression, EDNR-antagonists have been evaluated in several clinical trials with disappointing results. These trials were based on a hypothesis that the EDN1-EDNR axis activates the MAPK-ERK signaling pathway that is vital to the cancer cell survival; the trials were not designed to evaluate the impact of tumor-derived EDN1 in modifying tumor microenvironment or contributing to drug resistance. Ectopic overexpression of EDN1 in cells with mutated EGFR resulted in poor drug delivery and retarded growth in vivo but not in vitro. Intratumoral injection of recombinant EDN significantly reduced blood flow and subsequent gefitinib accumulation in xenografted EGFR-mutant tumors. Furthermore, depletion of EDN1 or the use of endothelin receptor inhibitors bosentan and ambrisentan improved drug penetration into tumors and restored blood flow in tumor-associated vasculature. Correlatively, these results describe a simplistic endogenous yet previously unrealized resistance mechanism inherent to a subset of EGFR-mutant NSCLC to attenuate tyrosine kinase inhibitor delivery to the tumors by limiting drug-carrying blood flow and the drug concentration in tumors. SIGNIFICANCE: EDNR antagonists can be repurposed to improve drug delivery in VEGFA-secreting tumors, which normally respond to TKI treatment by secreting EDN1, promoting vasoconstriction, and limiting blood and drug delivery.

Generation of osimertinib-resistant cells from epidermal growth factor receptor L858R/T790M mutant non-small cell lung carcinoma cell line.

Lung cancer contributes to high cancer mortality worldwide with 80% of total cases diagnosed as non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) tyrosine kinase (TK) domain serves as a druggable target in NSCLC patients with exon 19 deletion and L858R mutation. However, patients eventually succumbed to resistance to first- and second-generation EGFR-TK inhibitors through activation of T790M mutation. Third-generation EGFR-TKI, Osimertinib exhibits high efficacy in patients with exon 19 deletion/L858R/T790M mutation but they experienced acquired resistance thereafter. Available treatment options in NSCLC patients remains a challenge due to unknown molecular heterogeneity responsible for acquired resistance to EGFR-TKI. In this study, we aim to generate Osimertinib-resistant (OR) cells from H1975 carrying L858R/T790M double mutation which can be used as a model to elucidate mechanism of resistance. OR cells were established via stepwise-dose escalation and limiting single-cell dilution method. We then evaluated Osimertinib resistance potential via cell viability assay. Proteins expression related to EGFR-signalling, epithelial to mesenchymal transition (EMT), and autophagy were analyzed via western blot. OR cell lines exhibited increased drug resistance potential compared to H1975. Distinguishable mesenchymal-like features were observed in OR cells. Protein expression analysis revealed EGFR-independent signaling involved in the derived OR cells as well as EMT and autophagy activity. We generated OR cell lines in-vitro as evidenced by increased drug resistance potential, increased mesenchymal features, and enhanced autophagy activity. Development of Osimertinib resistance cells may serve as in-vitro model facilitating discovery of molecular aberration present during acquired mechanism of resistance.

Small-cell lung carcinoma transformation of lung adenocarcinoma diagnosed by pericardial effusion: A case report.

The present report describes a case of a 68-year-old male patient with epidermal growth factor receptor (EGFR)-mutant non-small cell lung carcinoma (NSCLC). After cytotoxic chemotherapy of three regimens following 22 months of treatment with EGFR-tyrosine kinase inhibitors (EGFR-TKIs), including osimertinib, the patient underwent S-1 treatment. Despite a decrease in carcinoembryonic antigen 1 month after initiating S-1 treatment, the patient developed cardiac tamponade. The evaluation of pericardial effusion confirmed small-cell lung carcinoma (SCLC) transformation. Subsequently, a combination therapy of carboplatin and etoposide was administered, which led to a marked improvement in imaging. In patients with NSCLC who develop pericardial effusion after long-term EGFR-TKI therapy, including osimertinib treatment, it is important to investigate whether SCLC transformation occurs or not as a treatable entity.

Serum CRIPTO does not confer drug resistance against osimertinib but is an indicator of tumor burden in non-small cell lung cancer

BackgroundAdenocarcinoma is the most common subtype of non-small cell lung cancer (NSCLC) and often harbors oncogenic driver mutations in the epidermal growth factor receptor (EGFR). Osimertinib (AZD9291), a third generation EGFR TKI, has replaced earlier generation EGFR TKIs for first line treatment of EGFR mutant lung cancer due to its improved overall survival, longer progression free survival, and better tolerability compared to earlier generation inhibitors. However, like earlier generation EGFR TKIs, only about two thirds of patients respond, indicating an unknown mechanism of intrinsic resistance for the non-responders. We previously identified overexpression of CRIPTO as a potential mechanism of intrinsic resistance to EGFR TKIs of first and second generation. ObjectiveTo determine if CRIPTO could promote drug resistance against the third generation EGFR-TKIs osimertinib. We also wanted to investigate whether this resistance was conferred by both membrane bound and secreted CRIPTO. Finally, we wanted to explore the potential of secreted CRIPTO as a non-invasive biomarker for EGFR-TKI resistance. Materials and methodsHCC827 and H1975, EGFR mutant non-small cell lung carcinoma (NSCLC) cell lines, were transfected with wildtype CRIPTO, two secreted variants of CRIPTO, a membrane only version of CRIPTO, and the mock backbone vector as the control. Western blotting, immunoprecipitation, and in vitro viability experiments were performed.In vivo work was carried out in athymic nude mice; 2 × 106 CRIPTO overexpressing HCC827 cells were implanted per mouse.EGFR mutant NSCLC patient blood samples were collected before treatment with and EGFR-TKI, during response while on treatment, and at progression while on treatment. ResultsAlthough both membrane bound and secreted CRIPTO forms were able to activate downstream pathways such as SRC, CRIPTO was unable to elicit resistance towards osimertinib in vitro or in vivo. CRIPTO serum levels in mice were higher in larger xenograft tumors. Furthermore, CRIPTO serum levels were higher in patients with progressing lung cancer when compared to their CRIPTO serum levels during EGFR-TKI response. ConclusionsCRIPTO does not cause resistance against third generation EGFR-TKI osimertinib. CRIPTO levels in serum might be a potentially useful biomarker for tumor burden in NSCLC patients.

BRAF molecular testing in cytopathology: Implications for diagnosis, prognosis, and targeted therapeutics.

BRAF molecular testing in cytopathology: Implications for diagnosis, prognosis, and targeted therapeutics.

Efficacy of gefitinib at reduced dose in EGFR mutant non-small cell lung carcinoma.

As a first-generation epidermal growth factor receptor-tyrosine kinase inhibitor, gefitinib was approved by the US Food and Drug Administration for treatment of advanced non-small cell carcinoma with sensitizing EGFR mutations. Gefitinib is known to have adverse effects, which may necessitate dose reduction or even change to alternative preparation of epidermal growth factor receptor-tyrosine kinase inhibitor. There has been concern on dose reduction resulting in reduced dose gefitinib, especially on its efficacy. This was a retrospective single-center cohort study conducted in Queen Mary Hospital in Hong Kong that included 159 Chinese patients with advanced adenocarcinoma of lung that carried sensitizing EGFR mutations and had received gefitinib as first-line treatment. Patients who had reduced dose at 250 mg alternate day were compared with those who were able to maintain on standard dose of gefitinib at 250 mg daily. The primary end-point was progression-free survival. Among the 159 patients, 17 (10.7 %) of them were on reduced dose gefitinib, 14 among the 17 patients (82.4%) because of hepatotoxicity, and 3 (17.6%) because of cutaneous side effects. Patients on reduced dose and standard dose of gefitinib have comparable median progression-free survival. Hazard ratio was 1.121 (95% confidence interval = 0. 655-1.917, P-value = 0.678) for the reduced dose group and 3.385 for the standard dose group (95% confidence interval = 2.181-5.255) respectively (P-value < 0.001). Dose reduction in gefitinib to 250 mg alternate day in response to adverse effects was not associated with inferior outcome for patients on first-line gefitinib for advanced non-small cell carcinoma. Dose reduction is a feasible option for patients who have significant adverse effects with gefitinib.

EGFR exon 20 insertion mutations and response to osimertinib in non-small-cell lung cancer

BackgroundEpidermal growth factor receptor exon 20 insertion (EGFRex20ins) mutations represent approximately 4–12% of EGFR mutations and are generally refractory to the 1st and 2nd generation EGFR tyrosine kinase inhibitors (TKIs). Development of effective therapies for patients with EGFRex20ins mutant non-small-cell lung carcinoma (NSCLC) represents a great unmet need. Preclinical models have shown that osimertinib is active in NSCLC harboring EGFRex20ins, while the antitumor activity of osimertinib remains to be evaluated in patients with EGFRex20ins mutations.MethodsTumor genotyping was performed in 2316 Chinese NSCLC cases with targeted next generation sequencing (NGS) covering the whole exons of EGFR gene. The frequency and genetic characteristics of EGFRexon20ins mutations were analyzed. Furthermore, six patients with specific EGFRexon20ins mutations and receiving osimertinib 80 mg once daily were retrospectively included to assess the antitumor activity and safety of monotherapy osimertinib.ResultsEGFRex20ins mutations were identified in 4.8% (53/1095) of EGFR mutant NSCLC and 2.3% (53/2316) of all NSCLC cases. The most frequently identified EGFRexon20ins is A767_V769dup (17/53,32.1%). We found that the genetic characteristics of EGFRex20ins mutations in Chinese patients with NSCLC were comparable to those reported in Caucasian patients. Four patients with osimertinib therapy achieved partial response and the rest stable disease. Median progression free survival (PFS) was 6.2 months (95% confidence interval 5.0–12.9 months; range 4.9–14.6 months). The most common adverse events (AEs) were diarrhea (2/6), pruritis (2/6), stomatitis (1/6) and nausea (1/6). No grade 3 or more AEs were documented.ConclusionsThis study revealed that the genetic characteristics of EGFRex20ins mutations in Chinese patients with NSCLC were comparable to those reported in Caucasian patients. Furthermore, our study firstly demonstrated promising antitumor activity of osimertinib in certain EGFRex20ins mutant advanced NSCLC patients, indicating that osimertinib treatment for EGFRex20ins positive patients deserves further study.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

A phase II trial of erlotinib for EGFR mutant NSCLC to prospectively assess biopsy feasibility and acquired resistance at disease progression.

8076 Background: Erlotinib is a first-line therapy for patients (pts) with advanced EGFR mutant non-small cell lung carcinoma (NSCLC). However, tumors develop acquired resistance; choice of further...

AURKA upregulation plays a role in fibroblast-reduced gefitinib sensitivity in the NSCLC cell line HCC827.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) have been used to treat non-small cell lung carcinoma (NSCLC) patients that have EGFR-activating mutations. EGFR-TKI monotherapy in most NSCLC patients with EGFR mutations who initially respond to EGFR-TKIs results in the development of acquired resistance. We investigated the role of fibroblasts in stromal cell-mediated resistance to gefitinib-induced apoptosis in EGFR-mutant NSCLC cells. While gefitinib induced apoptosis in EGFR-mutant NSCLC cells, apoptosis induction was diminished under stromal co-culture conditions. Protection appeared to be mediated in part by Aurora-A kinase (AURKA) upregulation. The protective effect of stromal cells was significantly reduced by pre-exposure to AURKA-shRNA. We suggest that combinations of AURKA antagonists and EGFR inhibitors may be effective in clinical trials targeting mutant EGFR NSCLCs.

Cationic Lipid-Assisted Polymeric Nanoparticle Mediated GATA2 siRNA Delivery for Synthetic Lethal Therapy of KRAS Mutant Non-Small-Cell Lung Carcinoma

Synthetic lethal interaction provides a conceptual framework for the development of wiser cancer therapeutics. In this study, we exploited a therapeutic strategy based on the interaction between GATA binding protein 2 (GATA2) downregulation and the KRAS mutation status by delivering small interfering RNA targeting GATA2 (siGATA2) with cationic lipid-assisted polymeric nanoparticles for treatment of non-small-cell lung carcinoma (NSCLC) harboring oncogenic KRAS mutations. Nanoparticles carrying siGATA2 (NPsiGATA2) were effectively taken up by NSCLC cells and resulted in targeted gene suppression. NPsiGATA2 selectively inhibited cell proliferation and induced cell apoptosis in KRAS mutant NSCLC cells. However, this intervention was harmless to normal KRAS wild-type NSCLC cells and HL7702 hepatocytes, confirming the advantage of synthetic lethality-based therapy. Moreover, systemic delivery of NPsiGATA2 significantly inhibited tumor growth in the KRAS mutant A549 NSCLC xenograft murine model, suggesting the therapeutic promise of NPsiGATA2 delivery in KRAS mutant NSCLC therapy.

EPS8 Inhibition Increases Cisplatin Sensitivity in Lung Cancer Cells

Cisplatin, a commonly used chemotherapeutic, is associated with ototoxicity, renal toxicity and neurotoxicity, thus identifying means to increase the therapeutic index of cisplatin may allow for improved outcomes. A SNP (rs4343077) within EPS8, discovered through a genome wide association study of cisplatin-induced cytotoxicity and apoptosis in lymphoblastoid cell lines (LCLs), provided impetus to further study this gene. The purpose of this work was to evaluate the role of EPS8 in cellular susceptibility to cisplatin in cancerous and non-cancerous cells. We used EPS8 RNA interference to determine the effect of decreased EPS8 expression on LCL and A549 lung cancer cell sensitivity to cisplatin. EPS8 knockdown in LCLs resulted in a 7.9% increase in cisplatin-induced survival (P = 1.98×10−7) and an 8.7% decrease in apoptosis (P = 0.004) compared to control. In contrast, reduced EPS8 expression in lung cancer cells resulted in a 20.6% decrease in cisplatin-induced survival (P = 5.08×10−5). We then investigated an EPS8 inhibitor, mithramycin A, as a potential agent to increase the therapeutic index of cisplatin. Mithramycin A decreased EPS8 expression in LCLs resulting in decreased cellular sensitivity to cisplatin as evidenced by lower caspase 3/7 activation following cisplatin treatment (42.7%±6.8% relative to control P = 0.0002). In 5 non-small-cell lung carcinoma (NSCLC) cell lines, mithramycin A also resulted in decreased EPS8 expression. Adding mithramycin to 4 NSCLC cell lines and a bladder cancer cell line, resulted in increased sensitivity to cisplatin that was significantly more pronounced in tumor cell lines than in LCL lines (p<0.0001). An EGFR mutant NSCLC cell line (H1975) showed no significant change in sensitivity to cisplatin with the addition of mithramycin treatment. Therefore, an inhibitor of EPS8, such as mithramycin A, could improve cisplatin treatment by increasing sensitivity of tumor relative to normal cells.

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.

Abstract 5264: Negative regulation of AMPK-dependent metabolic-stress pathways by MIF and D-DT in non-small cell lung carcinoma.

Abstract AMP-activated protein kinase (AMPK) is a nutrient and metabolic stress sensing enzyme activated by the tumor suppressor kinase, LKB1. Because macrophage migration inhibitory factor (MIF) and its functional homolog, D-dopachrome tautomerase (D-DT), have pro-tumorigenic functions in non-small cell lung carcinoma (NSCLC) but have AMPK activating properties in nonmalignant cell types, we set out to investigate this apparent paradox. Our data now suggest that, in contrast to MIF and D-DT's AMPK activating properties in non-transformed cells, MIF and D-DT cooperatively act to inhibit steady state phosphorylation and activation of AMPK in LKB1 wildtype and LKB1 mutant human NSCLC cell lines. Our data further indicate that MIF and D-DT - acting through their shared cell surface receptor, CD74 - antagonize NSCLC AMPK activation by maintaining glucose uptake, ATP production and redox balance resulting in reduced Ca2+/calmodulin-dependent kinase kinase [[Unsupported Character - Symbol Font &amp;#946;]] (CaMKK[[Unsupported Character - Symbol Font &amp;#946;]])-dependent AMPK activation. Combined, these studies indicate that MIF and D-DT cooperate to inhibit AMPK activation in an LKB1-independent manner. This suggests that MIF and D-DT promote a unique and well conserved adaptation pathway that is usurped by the malignant cells in order to counter metabolic stress-induced, AMPK-dependent, tumor suppression. Importantly, because the LKB1 tumor suppressor locus is mutated in over 30% of NSCLC lesions rendering these tumors insensitive to AMPK-dependent growth suppression, the identification of unique AMPK agonists would be expected to have a tremendous impact on NSCLC disease management for clinicians. Taken together, simultaneous therapeutic targeting of both MIF and D-DT represents an innovative and clinically efficacious approach to re-activating metabolic stress-induced tumor suppression in NSCLC malignant lesions. Citation Format: Stephanie Brock, Beatriz Rendon, Kavitha Yaddanapudi, Robert Mitchell. Negative regulation of AMPK-dependent metabolic-stress pathways by MIF and D-DT in non-small cell lung carcinoma. [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 5264. doi:10.1158/1538-7445.AM2013-5264

Abstract 3454: Establishing role of KRAS mutation on NSCLC radio-sensitivity

Abstract Lung cancer is the leading cause of cancer related mortality in the US of which more than 75% cases are that of non-small cell lung carcinoma (NSCLC). Mutations in the proto-oncogene KRAS have been linked with poor prognosis for NSCLC patients. In this study, we aimed at analyzing the relationship between specific KRAS mutations and NSCLC cell radiosensitivity and protein expression patterns. We analyzed 22 NSCLC cell lines and stratified them according to their KRAS status. Our results show that NSCLC cells harbouring G12C mutations are more sensitive to radiation compared to cells with other KRAS status (SF2 = 0.35 ± 0.16 for G12C vs. 0.63 ± 0.17 for other KRAS mutants vs 0.54 ± 0.15 for wt KRAS). Our protein expression data suggests that G12C mutants have reduced expression of DNA-repair proteins such as ATM, Rad 50, Ku 80 and XRCC1 (p &amp;lt; 0.05) which may be responsible for their radiosensitive nature. Further we found that G12C mutant NSCLC cell lines have reduced expression of pAkt compared to cells with other KRAS status (p =0.009). This suggests that NSCLC cells harboring G12C mutation would be less susceptible to PI3K inhibition induced radio-sensitization. Our clonogenic assay results confirm our finding when 1-hour pretreatment with 5 µM LY294002 (PI3K inhibitor) sensitized H460 and A549 (non-G12C mutated NSCLC cells) but not H1792 and H23 (G12C mutant NSCLC). Our results strongly suggests that G12C KRAS mutant cells are radio-sensitive compared to NSCLC cells of other KRAS status and PI3K inhibitor therapy along with radiation could be beneficial for patients harboring non-G12C mutational KRAS status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3454. doi:1538-7445.AM2012-3454

Abstract 3244: Compensatory function of the homologous proteins MIF and D-DT in regulation of AMPK-dependent metabolic-stress pathways in non-small cell lung carcinoma

Abstract Macrophage migration inhibitory factor (MIF) is over-expressed in most solid malignancies and is important for many pro-tumorigenic pathways. Several studies reveal that genetic deletion or inhibition of MIF results in anti-cancer phenotypes, but these phenotypes are not nearly as profound as one would anticipate given its reported activities. We recently discovered that MIF's only related family member, D-dopachrome tautomerase (D-DT), functionally compensates for MIF-dependent promotion of angiogenic potential in non-small cell lung carcinoma (NSCLC). This finding not only provides an explanation for why MIF individual targeting has not provided more efficacious anti-cancer activities, but also suggests that therapeutic targeting of MIF and D-DT simultaneously may provide a highly effective and novel new class of anti-lung cancer pharmacologic agents. Here we present further evidence of the important cooperative function of these two homologous proteins in NSCLC cells. We reveal a strong collaborative effect of MIF and D-DT on the negative regulation of AMP-activated protein kinase (AMPK) activation. AMPK is phosphorylated by the tumor suppressor, LKB1, in response to metabolic stress, resulting in AMPK-dependent p53 activation and mTOR inhibition. We show that while siRNA-mediated knockdown of endogenous MIF or D-DT individually has modest effects on AMPK activation in NSCLC cells, combined knockdown of both MIF and D-DT results in a significant activation of AMPK-dependent regulation of p53 and mTOR pathways. Importantly, this re-activation of AMPK by MIF and D-DT occurs in LKB1 mutant NSCLC cell lines. In addition, overexpression of MIF or D-DT alone by adenoviral delivery causes a substantial reduction in p53 expression, with a dramatic increase in mTOR pathway activation. Our findings suggest that MIF and D-DT promote a unique and well conserved adaptation pathway that is usurped by the malignant cells in order to counter metabolic stress-induced, AMPK-dependent, tumor suppression. Because the LKB1 tumor suppressor locus is mutated in over 30% of NSCLC lesions rendering these tumors insensitive to AMPK-dependent growth suppression, the identification of unique AMPK agonists would be expected to have a tremendous impact on NSCLC disease management for clinicians. Taken together, simultaneous therapeutic targeting of both MIF and D-DT represents an innovative and clinically efficacious approach to re-activating metabolic stress-induced tumor suppression in NSCLC malignant lesions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3244. doi:1538-7445.AM2012-3244