Mutant Lung Cancer Cells Research Articles

Cardiac glycosides display selective efficacy for STK11 mutant lung cancer.

Although STK11 (LKB1) mutation is a major mediator of lung cancer progression, targeted therapy has not been implemented due to STK11 mutations being loss-of-function. Here, we report that targeting the Na+/K+-ATPase (ATP1A1) is synthetic lethal with STK11 mutations in lung cancer. The cardiac glycosides (CGs) digoxin, digitoxin and ouabain, which directly inhibit ATP1A1 function, exhibited selective anticancer effects on STK11 mutant lung cancer cell lines. Restoring STK11 function reduced the efficacy of CGs. Clinically relevant doses of digoxin decreased the growth of STK11 mutant xenografts compared to wild type STK11 xenografts. Increased cellular stress was associated with the STK11-specific efficacy of CGs. Inhibiting ROS production attenuated the efficacy of CGs, and STK11-AMPK signaling was important in overcoming the stress induced by CGs. Taken together, these results show that STK11 mutation is a novel biomarker for responsiveness to CGs. Inhibition of ATP1A1 using CGs warrants exploration as a targeted therapy for STK11 mutant lung cancer.

Quercetin induces cell death by caspase-dependent and p38 MAPK pathway in EGFR mutant lung cancer cells

Objectives The aim of this study was whether quercetin induces cell death by caspase and MAPK signaling pathway in EGFR mutant lung cancer cells Methods PC-9 cells, EGFR mutant lung cancer cells, were treated various times and concentrations of quercetin and harvested and measured using MTT assay, DNA fragmentation, Western blotting, and FACS analysis. Results Treatment with quercetin in PC-9 cells resulted in inhibition of cell growth through apoptosis. Quercetin-induced apoptosis was associated with caspase-dependent manner. Quercetin also significantly increased levels of phosphor-p38 and decreased levels of phosphor-ERK, indicating that quercetin induces p38 MAPK signaling pathway in PC-9 cells. Quecetin treatment also generated the release of cytochrome c in PC-9 cells; however, pretreatment with rotenone or z-LEHD-fmk, significantly attenuated quercetin-induced apoptosis. Conclusions Our data indicate that quercetin exhibits EGFR mutant lung cancer effects through apoptosis by caspase dependent and mitochondrial pathway.

Activity-Based Proteomics Reveals Heterogeneous Kinome and ATP-Binding Proteome Responses to MEK Inhibition in KRAS Mutant Lung Cancer.

One way cancer cells can escape from targeted agents is through their ability to evade drug effects by rapidly rewiring signaling networks. Many protein classes, such as kinases and metabolic enzymes, are regulated by ATP binding and hydrolysis. We hypothesized that a system-level profiling of drug-induced alterations in ATP-binding proteomes could offer novel insights into adaptive responses. Here, we mapped global ATP-binding proteomes perturbed by two clinical MEK inhibitors, AZD6244 and MEK162, in KRAS mutant lung cancer cells as a model system harnessing a desthiobiotin-ATP probe coupled with LC-MS/MS. We observed strikingly unique ATP-binding proteome responses to MEK inhibition, which revealed heterogeneous drug-induced pathway signatures in each cell line. We also identified diverse kinome responses, indicating each cell adapts to MEK inhibition in unique ways. Despite the heterogeneity of kinome responses, decreased probe labeling of mitotic kinases and an increase of kinases linked to autophagy were identified to be common responses. Taken together, our study revealed a diversity of adaptive ATP-binding proteome and kinome responses to MEK inhibition in KRAS mutant lung cancer cells, and our study further demonstrated the utility of our approach to identify potential candidates of targetable ATP-binding enzymes involved in adaptive resistance and to develop rational drug combinations.

PARP Inhibition Suppresses Growth of EGFR-Mutant Cancers by Targeting Nuclear PKM2.

Upon growth factor stimulation or in some EGFR mutant cancer cells, PKM2 translocates into the nucleus to induce glycolysis and cell growth. Here, we report that nuclear PKM2 binds directly to poly-ADP ribose, and this PAR-binding capability is critical for its nuclear localization. Accordingly, PARP inhibition prevents nuclear retention of PKM2 and therefore suppresses cell proliferation and tumor growth. In addition, we found that PAR level correlates with nuclear localization of PKM2 in EGFR mutant brain and lung cancers, suggesting that PAR-dependent nuclear localization of PKM2 likely contributes to tumor progression in EGFR mutant glioblastoma and lung cancers. In addition, some EGFR-inhibitor-resistant lung cancer cells are sensitive to PARP inhibitors. Taken together, our data indicate that suppression of PKM2 nuclear function by PARP inhibitors represents a treatment strategy for EGFR-inhibitor-resistant cancers.

Pulse Afatinib for ERBB2 Exon 20 Insertion–Mutated Lung Adenocarcinomas

IntroductionGenomic aberrations involving the erb-b2 receptor tyrosine kinase 2 gene (ERBB2) are driver oncogenes in approximately 2% of lung adenocarcinomas. However, the use of daily dosing of ERBB2 tyrosine kinase inhibitors (TKIs)—including afatinib—has been fraught with plasma concentrations that barely achieve preclinical model inhibition, significant patient-reported toxicities, and limited clinical activity. We hypothesized that alternative dosing strategies could improve tolerability and efficacy. MethodsWe profiled lung cancer cell lines against TKIs and retrospectively evaluated the toxicity of and response to pulse afatinib (280 mg once weekly) in lung cancers with ERBB2 mutations. ResultsAn ERBB2 exon 20 insertion–mutated lung cancer cell line had a 50% inhibitory concentration in response to afatinib that was higher than the reported plasma concentration of afatinib, 40 mg daily. Three patients with advanced ERBB2-mutated lung adenocarcinomas were treated with off-label pulse afatinib. The 280-mg weekly dose was well tolerated with no reported rash and minimal diarrhea. One TKI-naive patient achieved a partial response for 5 months and another achieved stable disease for 11 months. ConclusionsPulse afatinib at a weekly dosing scheme induced antitumor activity in ERBB2 exon 20 insertion–mutated lung adenocarcinomas. Future clinical trials of alternative dosing schemes of ERBB TKIs as monotherapy or in combination with other therapies are warranted for ERBB2-mutated tumors.

Oncogenic K-ras confers SAHA resistance by up-regulating HDAC6 and c-myc expression.

Histone deacetylase inhibitors (HDIs) represent a new class of anticancer drugs. Suberoylanilide hydroxamic acid (SAHA), the first HDI approved for the treatment of cutaneous T cell lymphoma (CTCL), is currently being tested in clinical trials for other cancers. However, SAHA has been ineffective against solid tumors in many clinical trials. A better understanding of molecular mechanisms of SAHA resistance may provide the basis for improved patient selection and the enhancement of clinical efficacy. Here we demonstrate that oncogenic K-ras contributes to SAHA resistance by upregulating HDAC6 and c-myc expression. We find that the high levels of HDAC6 expression are associated with activated K-ras mutant in colon cancer patients. And expressions of HDAC6 and c-myc are increased in fibroblasts transformed with activated K-ras. Surprisingly, we find that activated K-ras transformed cells are more resistant to SAHA inhibition on cell growth and anchorage-independent colony formation. We show that a K-ras inhibitor sensitizes K-ras mutated lung cancer cells to SAHA induced growth inhibition. We also find that mutant K-ras induces HDAC6 expression by a MAP kinase dependent pathway. Our study suggests that combined treatment with SAHA and K-ras inhibitors may represent an effective strategy to overcome SAHA resistance.

The apoptotic effect of simvastatin via the upregulation of BIM in nonsmall cell lung cancer cells

ABSTRACTPurpose: Statins are known to have pleiotropic effects that induce cell death in certain cancer cells. BIM is a member of the bcl-2 gene family, which promotes apoptotic cell death. This study investigated the hypothesis that simvastatin has pro-apoptotic effects in epidermal growth factor receptor (EGFR)-mutated lung cancer cell lines via the upregulation of the expression of the BIM protein. Materials and methods: The cytotoxic effects of simvastatin on gefitinib-sensitive (HCC827, E716-A750del) and -resistant (H1975, T790M + L858R) nonsmall cell lung cancer (NSCLC) cells were compared. Cell proliferation and expression of apoptosis-related and EGFR downstream signaling proteins were evaluated. Expression of BIM was compared in H1975 cells after treatment with simvastatin or gefitinib. SiRNA-mediated BIM depletion was performed to confirm whether the cytotoxicity of simvastatin was mediated by the expression of BIM. Results: H1975 cells showed significantly reduced viability compared with HCC827 cells after treatment with simvastatin (2 μM) for 48 hours. In simvastatin-treated H1975 cells, expression of pro-apoptotic proteins was increased and the phosphorylation of ERK 1/2 (p-ERK 1/2) was reduced. Expression of BIM was suppressed by gefitinib (1 μM) treatment in H1975 cells, but it was significantly increased by treatment with simvastatin. BIM depletion by siRNA transfection enhanced the viability of H1975 cells that received simvastatin treatment and increased their expression of anti-apoptotic proteins. Conclusions: Simvastatin restored the expression of BIM to induce apoptotic cell death in NSCLC cells harboring an EGFR-resistant mutation. Our study suggests the potential utility of simvastatin as a BIM-targeted treatment for NSCLC.

High efficacy of third generation EGFR inhibitor AZD9291 in a leptomeningeal carcinomatosis model with EGFR-mutant lung cancer cells.

Leptomeningeal carcinomatosis (LMC) remarkably decreases the quality of life of EGFR-mutant lung cancer patients. In contrast to the lesions outside the central nervous system (CNS), molecular mechanisms of EGFR tyrosine kinase inhibitor (TKI) resistance in CNS lesions including LMC are largely unknown. In this study, we established an in vivo imaging model for LMC with EGFR mutant lung cancer cell lines harboring an exon 19 deletion in EGFR and evaluated the effect of first generation EGFR-TKIs, erlotinib, second generation afatinib, and third generation AZD9291. In PC-9/ffluc model, erlotinib treatment slowed the development of LMC. Importantly, treatment with afatinib or AZD9291 apparently delayed the development of LMC. Moreover, treatment with a higher dose of AZD9291, also associated with inhibited phosphorylation of EGFR downstream molecule S6, regressed LMC refractory to the aforementioned EGFR-TKI treatments. These observations suggest that the third generation EGFR-TKI AZD9291 may be an effective treatment for first or second generation EGFR-TKI resistant LMC caused by EGFR-mutant lung cancer.

Diet-derived 25-hydroxyvitamin D3 activates vitamin D receptor target gene expression and suppresses EGFR mutant non-small cell lung cancer growth in vitro and in vivo.

Epidemiologic studies implicate vitamin D status as a factor that influences growth of EGFR mutant lung cancers. However, laboratory based evidence of the biological effect of vitamin D in this disease is lacking. To fill this knowledge gap, we determined vitamin D receptor (VDR) expression in human lung tumors using a tissue microarray constructed of lung cancer cases from never-smokers (where EGFR gene mutations are prevalent). Nuclear VDR was detected in 19/19 EGFR mutant tumors. Expression tended to be higher in tumors with EGFR exon 19 deletions than those with EGFR L858R mutations. To study anti-proliferative activity and signaling, EGFR mutant lung cancer cells were treated with the circulating metabolite of vitamin D, 25-hydroxyvitamin D3 (25D3). 25D3 inhibited clonogenic growth in a dose-dependent manner. CYP27B1 encodes the 1α-hydroxylase (1αOHase) that converts 25D3 to the active metabolite, 1,25-dihydroxyvitamin D3 (1,25D3). Studies employing VDR siRNA, CYP27B1 zinc finger nucleases, and pharmacologic inhibitors of the vitamin D pathway indicate that 25D3 regulates gene expression in a VDR-dependent manner but does not strictly require 1αOHase-mediated conversion of 25D3 to 1,25D3. To determine the effects of modulating serum 25D3 levels on growth of EGFR mutant lung tumor xenografts, mice were fed diets containing 100 or 10,000 IU vitamin D3/kg. High dietary vitamin D3 intake resulted in elevated serum 25D3 and significant inhibition of tumor growth. No toxic effects of supplementation were observed. These results identify EGFR mutant lung cancer as a vitamin D-responsive disease and diet-derived 25D3 as a direct VDR agonist and therapeutic agent.

Oxidative pentose phosphate pathway inhibition is a key determinant of antimalarial induced cancer cell death.

Despite immense interest in employing antimalarials as autophagy inhibitors to treat cancer, it remains unclear if these agents act predominantly via autophagy inhibition or whether other pathways direct their anti-cancer properties. By comparing the treatment effects of the antimalarials chloroquine (CQ) and quinacrine (Q) on KRAS mutant lung cancer cells, we demonstrate that inhibition of the oxidative arm of the pentose phosphate pathway (oxPPP) is required for antimalarial induced apoptosis. Despite inhibiting autophagy, neither CQ treatment nor RNAi against autophagy regulators (ATGs) promote cell death. In contrast, Q triggers high levels of apoptosis, both in vitro and in vivo, and this phenotype requires both autophagy inhibition and p53-dependent inhibition of the oxPPP. Simultaneous genetic targeting of the oxPPP and autophagy is sufficient to trigger apoptosis in lung cancer cells, including cells lacking p53. Thus, in addition to reduced autophagy, oxPPP inhibition serves as an important determinant of antimalarial cytotoxicity in cancer cells.

Characterization of DDR2 Inhibitors for the Treatment of DDR2 Mutated Nonsmall Cell Lung Cancer.

Despite advances in precision medicine approaches over the past decade, the majority of nonsmall cell lung cancers (NSCLCs) are refractory to treatment with targeted small molecule inhibitors. Previous work has identified mutations in the Discoidin Domain Receptor 2 (DDR2) kinase as potential therapeutic targets in NSCLCs. While DDR2 is potently targeted by several multitargeted kinase inhibitors, most notably dasatinib, toxicity has limited the clinical application of anti-DDR2 therapy. Here, we have characterized compound 1 and other tool compounds demonstrating selectivity for DDR2 and show that while these compounds inhibit DDR2 in lung cancer model systems, they display limited antiproliferative activity in DDR2 mutated cell lines as compared to dual DDR2/SRC inhibitors. We show that DDR2 and SRC are binding partners, that SRC activity is tied to DDR2 activation, and that dual inhibition of both DDR2 and SRC leads to enhanced suppression of DDR2 mutated lung cancer cell lines. These results support the further evaluation of dual SRC/DDR2 targeting in NSCLC, and we report a tool compound, compound 5, which potently inhibits both SRC and DDR2 with a distinct selectivity profile as compared to dasatinib.

Abstract 2430: Treatment sequence and molecular biomarker in EGFR mutant lung cancer cells

Abstract Introduction Lung cancer is the leading cause of cancer related death in the US. A subset of patients with lung cancer has a mutation in the Epidermal Growth Factor Receptor (EGFR) which makes them extremely responsive to EGFR tyrosine kinase inhibitor (TKI) erlotinib. The emergence of EGFR TKI resistance is new challenge in the management of these patients. We conducted a study to determine the best treatment sequence after resistance to EGFR TKI, testing the use of chemotherapy Pemetrexed (P), Afatinib (A), Rociletinib (R) and the combination of these agents in lung cancer cell lines with EGFR mutation that were pretreated with erlotinib. Material and methods Lung cancer cell lines with EGFR mutation CRL-2868 (E746 - A750 deletion) and CRL-2871 (L747 - E749 deletion, A750P) mutation were treated with 5 nM erlotinib for 24h and 72 h. The erlotinib resistance cells were then treated with 10nM of A or 10nM of P, or 21.5 nM of R, or the combination AP or the combination of RP. As control, we used lung cancer HTB-177 which does not have EGFR mutation. MTT proliferation assay was performed after 24, 48, 72 and 144h after the treatment. microRNA profiling was done by real time PCR in all cells after erlotinib treatment to find potential biomarkers as hallmark of erlotinib resistance cells. Results Rociletinib induced the maximum inhibition of cell proliferation at 144h in both cell lines with EGFR mutation. The RP also induced decreased cell proliferation in both cell lines with EGFR mutation. RP combination was more effective then R alone in CRL 2871. The use of A alone, P alone or AP combination was not different in CRL2871 but P had antagonistic effect when combined with A or R in CRL 2868. microRNA 10b, 27a and 27b were upregulated in the erlotinib resistant cells. Conclusion. EGFR resistance arises after treatment with erlotinib in lung cancer cell lines with EGFR mutation. Treatment with R and the combination of RP induced greater inhibition of proliferation compared to A, P alone. The combination of AP was antagonistic. This may have implications in the management of patients resistant to EGFR TKI. microRNA analysis showed up-regulation of several microRNA that should be tested in clinical setting. Citation Format: Hannah Motes, Emma Borrego-Diaz Reyes, Jared Kevern, Chao H. Huang, Peter J. Van Veldhuizen. Treatment sequence and molecular biomarker in EGFR mutant lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2430. doi:10.1158/1538-7445.AM2015-2430

Abstract 1379: IKKβ is a potential anti-angiogenic therapeutic target in KRAS-induced lung cancer

Abstract Activating mutations in KRAS are prevalent in cancer, but therapies targeted to oncogenic RAS have been ineffective to date. An alternative route for blocking RAS-driven oncogenic pathways is to target downstream effectors of RAS involved in promoting the oncogenic phenotype. One of the critical characteristics required for tumors to grow and progress is the ability of tumor cells to drive angiogenesis. Interestingly, oncogenic RAS promotes angiogenesis by upregulating the proangiogenic IL-8 cytokine, an NF-kB target gene; and recent studies have shown that oncogenic RAS also activates the NF-κB transcription factor pathway and that KRAS-induced lung tumorigenesis is suppressed by expression of a degradation-resistant form of the IκBα inhibitor or by genetic or pharmacological inhibition of IKKβ or deletion of the RELA/p65 subunit of NF-κB. Therefore, we hypothesized that IKKβ inhibition would reduce KRAS-induced angiogenesis and tumor progression. To test this hypothesis, we used genetic and pharmacological approaches to inhibit IKKβ in K-Ras mutant lung cancer cell lines. Treatment of KRAS-positive A549 e H358 lung cancer cells with the highly specific IKKβ inhibitor Compound A (CmpdA) or siRNA-mediated knockdown of IKKβ in these cells reduced expression and secretion of the proangiogenic IL-8 cytokine. We found that IKKβ targeting also reduced expression and secretion of VEGF, a growth factor involved in promoting angiogenesis, which is also regulated by NF-κB. Moreover, conditioned media from A549 and H358 cells with siRNA-mediated IKKβ knockdown reduced endothelial cell (HUVECS) migration. In order to ascertain whether IKKβ inhibition can directly affect endothelial cell function, we treated HUVECs with CmpdA, which also resulted in reduced HUVEC migration. To evaluate how IKKβ affects endothelial cell function in vivo, we used a mouse model of neonatal retinopathy, where pathological retinal angiogenesis is induced by transient exposure of neonatal mice to hyperoxic conditions. Angiogenesis was significantly reduced in this model when neonatal mice were treated with 3 doses of 10 mg/Kg CmpdA. Finally, IKKβ knockdown in A549 cells also reduced expression of MMP-2 and MMP9 metalloproteases and reduced A549 cell invasion. Taken together, these results suggest that IKKβ inhibition therapy may reduce tumor angiogenesis, as well as invasive properties of KRAS-induced lung tumors, thereby having the potential to result in a sustained therapeutic effect, particularly if combined with other therapeutic approaches. Citation Format: Tatiana C. Lobo, Leila Magalhães, Laura Cardeal, Ricardo Giordano, Albert Baldwin, Daniela Bassères. IKKβ is a potential anti-angiogenic therapeutic target in KRAS-induced lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1379. doi:10.1158/1538-7445.AM2015-1379

Abstract LB-256: Optimal clinical dose-finding strategies: Translational preclinical pharmacokineitcs, pharmacodynamics, and efficacy analysis of HM61713, an orally selective EGFR mutant inhibitor

Abstract The first-generation of EGFR1 inhibitors (Gefitinib and Erlotinib) has significant clinical benefits in NSCLC caused by activating mutations, but the efficacy of these agents is often limited due to the emergence of drug resistance conferred by a gatekeeper residue, T790M. HM61713 is a third-generation EGFR tyrosine kinase inhibitor that has been evaluated as a novel therapeutic agent for the treatment of non-small cell lung cancer (NSCLC) with EGFR mutations. HM61713 is an orally active and a novel EGFR mutant selective inhibitor which is potent on resistance mutation (T790M) without affecting EGFR wild type at efficacious dose level. HM61713 showed an anti-cancer activity in several EGFR mutant lung cancer cell lines including T790M mutation harboring cell line. Integrated pharmacokinetic - pharmacodynamic - xenograft tumor model (PK-PD-XTG) was used to characterize the relationship between HM61713 plasma concentration and tumor growth inhibition (TGI) in H1975 (T790M mutation) xenograft model. Simple one-compartment model applied re-absorption compartment with first-order absorption/elimination was used to describe HM61713 plasma concentration-time profiles. Biophase distribution model with baseline inhibition Emax equation was applied to characterize the PD marker (p-EGFR) and tumor volume shrinkage was explained by michaelis-menten kinetics of p-EGFR. Estimated in vivo IC50 value of p-EGFR inhibition (%) based on plasma free concentration in xenograft mice was 1.14 ng/mL. The human PD marker response curve and the tumor growth inhibition plot were obtained by replacing the mice PK to human PK in our developed model based on the hypothesis thattranslation providesa good relationship of surrogate mice tumor PD to human tumor regression corresponding human PK. According to our simulated curves, we predicted appropriate human active dose from 300 to 800 mg/man and it would be an efficacious dose in patients with NSCLC harboring the EGFR activating and also with T790M resistant mutation. Currently, HM61713 is undergoing in clinical trial phase I/II in NSCLC (ClinicalTrials.gov, NCT01588145). Citation Format: Jooyun Byun, Taehun Song, Donghyun Kim, Junhyeng Son, Kwang-Ok Lee, Jaeho Lee, Yong Hoon Kim, Young-Mi Lee, Kwee Hyun Suh. Optimal clinical dose-finding strategies: Translational preclinical pharmacokineitcs, pharmacodynamics, and efficacy analysis of HM61713, an orally selective EGFR mutant inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-256. doi:10.1158/1538-7445.AM2015-LB-256

Norcantharidin combined with EGFR-TKIs overcomes HGF-induced resistance to EGFR-TKIs in EGFR mutant lung cancer cells via inhibition of Met/PI3k/Akt pathway.

Hepatocyte growth factor (HGF) induces resistance to reversible and irreversible epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) in EGFR mutant lung cancer cells by activating Met and the downstream phosphoinositide 3-kinase (PI3K)/Akt pathway. Moreover, continuous exposure to HGF accelerates the emergence of EGFR-TKI-resistant clones. We examined whether norcantharidin (NCTD), a demethylated analog of cantharidin, could reverse HGF-induced resistance to EGFR-TKIs in mutant lung cancer cells PC-9 and HCC827. MTT assay was used to evaluate cell proliferation of NCTD on PC-9 and HCC827 cells in vitro. Western blotting assays were used to determine the expression of EGFR, p-EGFR (Thr 669), MET, p-MET, AKT, p-AKT (Ser473), PI3kp85, or p-PI3k p85. HGF concentrations were measured by ELISA. HGF-producing cells and PC-9/HGF were established by recombinant adenovirus vectors Ad-GFP-HGF. Xenograft model in SCID mice was used to test the regressive effect of tumor growth on PC-9 cells in vivo. NCTD could reverse resistance to EGFR-TKIs induced by exogenous and endogenous HGF in EGFR mutant lung cancer cells via inhibiting the Met/PI3K/Akt pathway. These results suggested that NCTD may be a promising candidate for developing preventive agents against EGFR-TKIs acquired resistance in NSCLC. In the in vivo model, NCTD plus gefitinib resulted in marked regression of tumor growth associated with inhibition of Akt phosphorylation in cancer cells. NCTD may be a promising candidate for developing preventive agents against EGFR-TKIs acquired resistance in NSCLC.

Inhibition of IGF1R signaling abrogates resistance to afatinib (BIBW2992) in EGFR T790M mutant lung cancer cells.

Non-small cell lung cancer (NSCLC) patients with an epidermal growth factor receptor (EGFR) mutation have benefited from treatment of reversible EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib. Acquisition of a secondary mutation in EGFR T790M is the most common mechanism of resistance to first generation EGFR TKIs, resulting in therapeutic failure. Afatinib is a second generation of EGFR TKI that showed great efficacy against tumors bearing the EGFR T790M mutation, but it failed to show the improvement on overall survival of lung cancer patients with EGFR mutations possibly because of novel acquired resistance mechanisms. Currently, there are no therapeutic options available for lung cancer patients who develop acquired resistance to afatinib. To identify novel resistance mechanism(s) to afatinib, we developed afatinib resistant cell lines from a parental human-derived NSCLC cell line, H1975, harboring both EGFR L858R and T790M mutations. We found that activation of the insulin-like growth factor 1 receptor (IGF1R) signaling pathway contributes to afatinib resistance in NSCLC cells harboring the T790M mutation. IGF1R knockdown not only significantly sensitizes resistant cells to afatinib, but also induces apoptosis in afatinib resistance cells. In addition, combination treatment with afatinib and linsitinib shows more than additive effects on tumor growth in in vivo H1975 xenograft. Therefore, these finding suggest that IGF1R inhibition or combination of EGFR-IGF1R inhibition strategies would be potential ways to prevent or potentiate the effects of current therapeutic options to lung cancer patients demonstrating resistance to either first or second generation EGFR TKIs.

O10.2 - HM61713, an EGFR-mutant selective inhibitor

ABSTRACT EGFR tyrosine kinase inhibitors (TKIs) are effective for EGFR mutation positive non-small cell lung cancer (NSCLC) patients. However, acquired resistance occurs in most cases and EGFR T790M mutation is a major (>50%) resistance mechanism of first-generation EGFR TKIs. HM61713 is an orally active, EGFR-mutant selective inhibitor showed strong anti-cancer activity in EGFR mutant lung cancer cell lines including T790M mutation positive one. In addition, lower activity to wild-type EGFR inhibition suggested the possibility of decreased clinical toxicities such as skin rash and diarrhea. A phase 1/2 trial is ongoing to evaluate the safety, pharmacokinetics, and efficacy of HM61713 in patients with advanced EGFR mutation positive NSCLC who had failed to previous EGFR-TKIs (NCT01588145). In dose escalation part, the 3 + 3 dose-escalation scheme was used and the maximum tolerated dose was determined as 800 mg qd. An early expansion cohort was implemented at the dose 300 mg qd (N = 83) and the preliminary result was reported in ASCO 2014; the objective response rate was 29.2% in T790M positive patients (N = 48) and 11.8% in T790M negative population (N = 34). The disease control rate was 75.0% and 55.9%, respectively. Commonly reported adverse events (AEs) included skin exfoliation, nausea, diarrhea, rash, decreased appetite and pruritus. Most AEs were typically Gr 1/2, easily manageable and reversible without interruption of dosing. HM61713 showed good safety profile and promising anti-tumor activity in pts with EGFR mutated NSCLC who failed to EGFR-TKIs, especially in patients with T790M mutation. The phase 2 part is ongoing at the dose of 800 mg qd.

Effect of EGFR-TKI on lymphangiogenesis of lung cancer with EGFR mutation

背景与目的探索表皮生长因子受体酪氨酸激酶抑制剂（epidermal growth factor receptor-tyrosine kinase inhibitor, EGFR-TKI）对EGFR突变肺癌新生淋巴管的影响，探讨靶点治疗对新生淋巴管的抑制作用及其在肺癌治疗中所发挥的作用。方法采用EGFR双位点突变的NCI-H1975肺癌细胞株构建小鼠移植瘤模型。设立溶剂对照组和EGFR-TKI给药组，每组5只小鼠，观察EGFR-TKI对小鼠移植瘤的生长抑制作用；运用淋巴管内皮特异性抗体D2-40，采用免疫组织化学的方法，观察新生淋巴管的密度、面积、最大径，探讨EGFR-TKI对于肺癌组织淋巴管新生的影响。结果EGFR-TKI给药组小鼠肿瘤重量、肿瘤相对体积小于溶剂对照组。EGFR-TKI给药组小鼠平均新生淋巴管密度为6.44个/例，溶剂对照组小鼠平均新生淋巴管密度为10.70个/例，EGFR-TKI给药组小鼠平均新生淋巴管密度较低（P=0.023）。EGFR-TKI给药组小鼠新生淋巴管面积、最长径小于溶剂对照组。而EGFR-TKI对新生淋巴管的肿瘤细胞侵犯没有明显影响（P=0.519）。结论EGFR-TKI可以抑制EGFR突变肺癌组织淋巴管的新生，抑制新生淋巴管管径及面积的扩大。

Abstract B01: Improve MEK/ERK targeting in mutant KRAS lung cancer

Abstract KRAS activating mutations are detected in 30% Non-Small Cell Lung Cancer (NSCLC) cases, and represent the subset of patients with the worst prognosis. Current efforts to develop treatments for this type of lung cancer are partially focused on inhibiting the Raf/MEK/ERK signaling cascade downstream of KRAS. Inhibition of the Raf/MEK/ERK pathway results in death of many mutant KRAS-driven lung cancer cell lines in vitro, and we have previously reported a murine active Kras lung cancer model that showed significant tumor regression upon treatment with the MEK inhibitor, selumetinib, when it was combined with other agents, such as PI3K/mTOR dual inhibitor BEZ235 or the cytotoxic agent docetaxel. The activity of the latter combination was validated in a recent phase II clinical trial in NSCLC patients harboring KRAS mutations. However, the responses were not durable in either human patients or mice, as resistance develop rapidly following chronic MEK inhibition. We have developed two approaches to reduce or delay the emergence of resistant clones. First, we have identified a number of novel treatment strategies that would directly improve the initial efficacy of MEK/ERK inhibition to dramatically enhance the initial response to MEK inhibition. Second, we have designed chemical screening strategies that identify compounds that act against active Kras lung cancer independently from the Raf/MEK/ERK pathway. We are using the first approach to delay the development of chronic resistance, while using the second approach to overcome resistance to MEK/ERK inhibition. Citation Format: Zhao Chen, Ellen Weisberg, Katherine Cheng, Andrew L. Kung, James Bradner, James Griffin, Kwok-Kin Wong. Improve MEK/ERK targeting in mutant KRAS lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B01. doi: 10.1158/1557-3125.RASONC14-B01

Metabolic and transcriptional profiling reveals pyruvate dehydrogenase kinase 4 as a mediator of epithelial-mesenchymal transition and drug resistance in tumor cells.

BackgroundAccumulating preclinical and clinical evidence implicates epithelial-mesenchymal transition (EMT) in acquired resistance to anticancer drugs; however, mechanisms by which the mesenchymal state determines drug resistance remain unknown.ResultsTo explore a potential role for altered cellular metabolism in EMT and associated drug resistance, we analyzed the metabolome and transcriptome of three lung cancer cell lines that were rendered drug resistant following experimental induction of EMT. This analysis revealed evidence of metabolic rewiring during EMT that diverts glucose to the TCA cycle. Such rewiring was at least partially mediated by the reduced expression of pyruvate dehydrogenase kinase 4 (PDK4), which serves as a gatekeeper of the TCA cycle by inactivating pyruvate dehydrogenase (PDH). Overexpression of PDK4 partially blocked TGFβ-induced EMT; conversely, PDK4 inhibition via RNAi-mediated knockdown was sufficient to drive EMT and promoted erlotinib resistance in EGFR mutant lung cancer cells. We identified a novel interaction between PDK4 and apoptosis-inducing factor (AIF), an inner mitochondrial protein that appears to play a role in mediating this resistance. In addition, analysis of human tumor samples revealed PDK4-low as a predictor of poor prognosis in lung cancer and that PDK4 expression is dramatically downregulated in most tumor types.ConclusionsTogether, these findings implicate PDK4 as a critical metabolic regulator of EMT and associated drug resistance.Electronic supplementary materialThe online version of this article (doi:10.1186/2049-3002-2-20) contains supplementary material, which is available to authorized users.