Supplementary Methods from MET-Independent Lung Cancer Cells Evading EGFR Kinase Inhibitors Are Therapeutically Susceptible to BH3 Mimetic Agents

Supplementary Figures 1-3 from MET-Independent Lung Cancer Cells Evading EGFR Kinase Inhibitors Are Therapeutically Susceptible to BH3 Mimetic Agents

12020 Background: Despite remarkable upfront response to ALK tyrosine kinase inhibitors (TKIs) in ALK+ lung cancer, acquired resistance eventually develops. Molecular mechanisms of the initial emergence of drug resistance are poorly understood. Methods: ALK+ H3122, H2228 and a patient-derived Ma-ALK001.S NSCLC cell lines were used in our study. In vitro MTS viability and murine in vivo xenograft assays were performed. siRNA knockdown and CRISPR/cas9 gene knockout were used for mechanistic studies. ChIP-qPCR was conducted to analyze HOXB3 epigenetic marks. RNA-seq was also performed for transcriptome analysis. Tumor microarray (TMA) biomarker study was adopted for outcome analysis of the polycomb repressive complex-2 (PRC-2) in NSCLC. Results: Our study revealed a rapid-onset emergence of adaptive drug-resistant ALK+ lung cancer cells within the first 14 days upon TKI treatment initiation. The expression of stem cell transcription factors, most notably HOXB3, was induced adaptively within the tumor cells undergoing drug escape. Similarly, tumoral TGFβ2 autocrine expression both at mRNA and protein levels were significantly elevated, and it regulated the downstream HOXB3 expression and mitochondrial prosurvival BCL-2/BCL-xL signaling, cancer stemness and EMT markers. RNA-seq revealed a rapid-onset adaptive global transcriptome reprogramming in the drug-escaping persister tumor cells. ChIP-qPCR showed resistance emerged via epigenetic regulation of the untreated bivalent HOXB3 promoter closed chromatin state transforming to stem-like open state during drug-escape, based on H3K4me3/H3K27me3 methylation mark balance. Findings were validated via CRISPR/cas9-EZH2 knockout studies. AQUA-TMA biomarker and survival outcome analysis confirmed the clinical relevance of EZH2/UTX of PRC-2. Finally, direct EZH2 inhibition or knock-out promoted ALK-TKI drug resistance, while UTX inhibition enhanced drug sensitivity. Conclusions: ALK+ lung cancer achieves initial rapid-onset emergence of adaptive drug escape through PRC-2 epigenetic chromatin reprogramming to regulate cancer plasticity-EMT/stemness interplay via the TGFβ2-EZH2/UTX-HOXB3-BCL-2/BCL-xL cascade.

The impact of EGFR-mutant NSCLC precision therapy is limited by acquired resistance despite initial excellent response. Classic studies of EGFR-mutant clinical resistance to precision therapy were based on tumor rebiopsies late during clinical tumor progression on therapy. Here, we characterized a novel non-mutational early adaptive drug-escape in EGFR-mutant lung tumor cells only days after therapy initiation, that is MET-independent. The drug-escape cell states were analyzed by integrated transcriptomic and metabolomics profiling uncovering a central role for autocrine TGFβ2 in mediating cellular plasticity through profound cellular adaptive Omics reprogramming, with common mechanistic link to prosurvival mitochondrial priming. Cells undergoing early adaptive drug escape are in proliferative-metabolic quiescent, with enhanced EMT-ness and stem cell signaling, exhibiting global bioenergetics suppression including reverse Warburg, and are susceptible to glutamine deprivation and TGFβ2 inhibition. Our study further supports a preemptive therapeutic targeting of bioenergetics and mitochondrial priming to impact early drug-escape emergence using EGFR precision inhibitor combined with broad BH3-mimetic to interrupt BCL-2/BCL-xL together, but not BCL-2 alone.

Introduction:Overexpression of MET receptor tyrosine kinase and its ligand hepatocyte growth factor (HGF) and MET gene amplification have been well-documented in non–small-cell lung cancer (NSCLC). Activated MET signaling plays an important role in human cancer tumorigenesis, metastasis, and drug resistance. However, the deregulation of MET/HGF pathway in NSCLC harboring ALK gene rearrangement (ALK[+]), which is sensitive to dual ALK and MET inhibitor Crizotinib, has not been reported.Methods:We performed systematic analysis of MET/HGF expression by immunohistochemistry (IHC) and MET gene amplification by dual color, dual hapten bright field in situ hybridization in 19 ALK(+) and 73 ALK(−) NSCLC tumor tissues from those who had clinical ALK rearrangement test done at the Cleveland Clinic from August 2010 to January 2013. IHC scoring was interpreted on a standard four-tier system.Results:The percentage of MET IHC score 0, 1+, 2+, and 3+ were 5.5%, 27.8%, 50.0%, and 16.7% in ALK(+) group, compared with 28.8%, 33.9%, 23.7%, and 13.6% in ALK(−) group, respectively. The MET high expression (IHC score 2 or 3) was significantly higher in ALK(+) group statistically (66.7% versus 37.3%, p = 0.03). HGF-high expression (IHC score 2 or 3) was 33.3% in ALK(+) and 15.8% in ALK(−) (p = 0.17). We identified eight cases in ALK(−) and one case in ALK(+) tumor who had MET gene amplification (18.4% versus 7.1%, p = 0.43) by dual color, dual hapten bright field in situ hybridization. No significant correlation between MET protein receptor expression and gene amplification was identified.Conclusions:Our study demonstrated for the first time that MET receptor expression, but not MET gene amplification, is significantly increased in ALK(+) NSCLC. MET gene amplification is a relatively rare event in this unique population compared with ALK(−) NSCLC.

Abstract Lung cancer is the second most common malignancy with the highest cancer-mortality rate. EML4-ALK chromosomal translocation is now known to represent a highly actionable unique molecular target in approximately 5% of non-small cell lung cancer (NSCLC) patients. The ALK tyrosine kinase inhibitor (TKI) crizotinib induces remarkably high response rates in molecularly-selected NSCLC patients harboring EML4-ALK driven tumors, and is being used in first-line therapy. Nonetheless, responsive tumors ultimately develop acquired drug resistance. We have recently investigated non-mutational mechanisms of tumor resistant escape emerging under early TKI treatment time-window. Our recent studies in EGFR-mutant NSCLC in early adaptive drug escape against EGFR TKI identified an autocrine upregulation of TGFβ2 within these survivor cells. Here, we found that TGFβ2 was elevated significantly in the EML4-ALK-driven H3122 cells undergoing early adaptive drug escape against ALK TKI (TAE684) at treatment day 14. Interestingly, we found that the augmented TGFβ signaling was specific to TGFβ2, but not TGFβ1 or TGFβ3. We also found correlative enhanced mitochondrial BCL-2/BCL-xL prosurvival signaling in the day 14 TKI surviving H3122 cells. The upregulated TGFβ2-BCL-2/BCL-xL prosurvival mitochondrial priming was further validated in H3122 xenografts by IHC. Exogenous TGFβ2 stimulation in vitro for 7 days induced a dose-dependent upregulated BCL-2 and BCL-xL expression. Inhibiting mitochondrial BCL-2/BCL-xL priming using dual BCL-2/BCL-xL inhibitor ABT-263 as BH3-mimetic led to significant reduction in early adaptive drug escape induced by ALK TKIs. Our study showed that EML4-ALK NSCLC could escape ALK TKI early in treatment through cell plasticity that involved TGFβ2-mitochondrial priming in an upregulated prosurvival state. We also found that the early adaptive drug-escaping cancer cells possessed inherently increased EMT-ness (upregulated vimentin, AXL, GAS6) and cancer stemness (upregulated OCT4, NANOG). These findings provide deeper insights into the process of drug resistance emergence and highlighted potential window-of-opportunities to therapeutically target early adaptive escape to eradicate targeted drug resistance and impact long term survival. Citation Format: Lihong Yin, Wei Zhang, Ivy Shi, Yan Feng, Rakesh Bagai, Daniel Lindner, Patrick C. Ma. Adaptive drug escape of EML4-ALK lung cancer against targeted ALK inhibitor involving TGFβ2-mitochondrial priming. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3718. doi:10.1158/1538-7445.AM2014-3718

e19043 Background: Precision therapy for advanced NSCLC with EGFR mutations is the current standard-of-care. Emergence of acquired resistance to therapy invariably occurs despite effective initial response. Classical rebiopsy studies of EGFR-mutant pts at progression have identified diverse resistance mechanisms involving T790M-EGFR, METactivation and AXL upregulation. Our studies focus on tumor cells adaptation early during therapy to map the initial course of therapeutic resistance evolution. Methods: Drug-sensitive model studies were performed usingHCC827 and PC-9 NSCLC cells under erlotinib, and H1975 cells under CL-387,785 inhibition. Affymetrix microarray profiling was performed at 0h, 8h, 9d and 9d TKI followed by 7d drug-washout. Both in vitro and in vivo xenograft analyses were conducted. Mass-spectrometry based metabolomics profiling was also conducted. Results: We identified an early adaptive drug escape in EGFR-mutant cells that emerged as early as 9 days on therapy. The prosurvival cell state was MET-independent with a TKI cytotoxicity escape at 100x higher IC50. Transcriptome profiling revealed a remarkable genome-wide signature adaptive reprogramming, centered on the autocrine TGFβ2 cascade, involving pathways of cell adhesion, cell cycle regulation, cell division, glycolysis, and gluconeogenesis. Corroborating the transcriptome profiling results, metabolomic profiling also revealed an adaptive metabolic reprogramming with suppression of glycolysis, TCA cycle, amino acids metabolism, and lipid bioenergetics during drug escape. Our studies identified a direct link of TGFβ2 within the early adaptive drug escaping cells, with the metabolic-bioenergetics quiescence and mitochondrial BCL-2/BCL-xL priming. Furthermore, this state of cellular plasticity displayed an increased EMT and cancer stem cell signaling as adaptation to the drug treatment and that could be overcome by dual BCL2/BCL-xL BH3 mimetics. Conclusions: We have identified and characterized a cellular plasticity state in early adaptivedrug escaping EGFR-mutant NSCLC cells undergoing remarkable cellular reprogramming, with therapeutic implications.

e19044 Background: ALK inhibitor (TKI) is currently the standard precision therapy for advanced EML4-ALK(+) NSCLC. Acquired clinical resistance unavoidably still develops limiting clinical outcome. Methods: EML4-ALK(+) H3122 and patient-derived TKI acquired resistant biopsied-lung tumor tissue cells were used to investigate drug escape mechanisms. Stem cell transcription factors QPCR array and RNA-sequencing profiling were performed on H3122 cells under ALK TKI up to day 14, compared with untreated and drug-washout controls. Cell viability assays using ALK TKIs, in vivo xenograft IHC analyses and quantitative tumor tissue proteomics assay coupled with laser microdissection were also performed. Results: We identified that H3122 cells can escape ALK TKIs (TAE-684, crizotinib) early after precision therapy initiation, with upregulated prosurvival signaling via upregulated autocrine TGFβ2, but not TGFβ1 or β3, during escape as early as at treatment day 14. We validated both in vitro and in vivo the upregulated TGFβ2-HOX-BCL-2/BCL-xL mitochondrial priming during drug escape. A coordinated reprogramming with adaptive induction of stem cell transcription factor genes, especially HOX family genes, was evident in the early drug escape cell state. RNA-sequencing analysis revealed increased EMT-ness (VIM, AXL, GAS6) and cancer stemness (OCT4, NANOG) in these cells undergoing drug adaptation. Moreover, our RNA-seq and proteomics findings strongly suggest a “reverse Warburg” cell state during drug escape. The adaptive cellular plasticity was verified also in patient-derived bronchoscopic biopsied lung adenocarcinoma tissues (Ma0083) with ALK inhibitor resistance. Interestingly, inhibiting mitochondrial priming using dual BCL-2/BCL-xL BH3-mimetics ABT-263 was effective to suppress early drug escape, but not with the BCL-2-specific agent ABT-199, suggesting BCL-xL is a key target. Conclusions: Our study findings provide novel insights into the initial process of evolution of ALK precision therapy drug resistance and highlighted the unique importance of understanding the role of adaptive tumor cell plasticity in the early drug escape emergence, with therapeutic implications.

e19025 Background: Targeted therapy with tyrosine kinase inhibitor (TKI) crizotinib against EML4-ALK (ALK+) non-small cell lung cancer (NSCLC) have impacted cancer therapy as a new paradigm. However, there is still an unmet need to develop affordable and robust technology platforms to assay circulating tumor cells (CTCs), which may form the basis of distant hematogenous metastases. There are relative advantages and pitfalls with various CTC platforms. Methods: We performed pilot studies utilizing two different platforms of CTC detection and enrichment. First, we adopted the CellSearch “positive-selection” platform to profile 11 pts with ALK(+) NSCLC who were treated with crizotinib prospectively. Blood samples were collected (i) pretreatment, (ii) on TKI with CR/PR/SD, and (iii) at disease progression. Second, we evaluated a novel “negative selection” CTCs capture-isolation platform, based on magnetic separation of pan-leukocyte marker CD45+ cells coupled with RBC lysis, to avoid potential bias of predefined CTC criteria. Pilot studies utilizing ALK+ H3122 cell line and ALK+ patients’ blood samples were performed for assay optimization and comparison. Results: Using ALK+ NSCLC patients peripheral blood samples, we demonstrated the presence of the EML4-ALK fusion (variant 1) in QPCR assay from the enriched CTC isolated using the CellSearch platform. Also, CellSearch enumeration in our pilot ALK+ cohort revealed a trend of correlation between the CTC numbers and disease status. The spike-in experiment in “negative selection” CTC platform enriched the spiked H3122 cells by 10,000 fold from the nucleated blood cells. Applying our “negative-selection” CTC assay to a patient with known EML4-ALK variant 1 (EML4-ex13/ALK-ex20) fusion lung cancer during disease progression on crizotinib, we detected the specific EML4-ALK variant 1 fusion in QPCR assay from the CTC enriched “eluate” fraction, but not in the “feed” fraction, whether the CellSearch platform revealed any CTCs or not. Conclusions: Taken together, our pilot CTC study results support the high sensitivity of the unbiased “negative selection” enrichment platform and its potential to empower molecular and genomic determinations in lung cancer.

Abstract Background. Advanced NSCLC with sensitizing EGFR mutation is being treated with EGFR inhibitor first-line. Despite initial tumor response, targeted therapy invariably fails due to acquired resistance. Classical studies of acquired drug resistance focusing on late clinical events in rebiopsy studies have identified some resistance mechanisms including T790M-EGFR, MET amplification and AXL upregulation. Methods. HCC827 and PC-9 NSCLC cells (sensitizing EGFR exon 19-del) were used in vitro and in vivo drug-sensitive models. MTS viability assay, TLVM cell mobility assay, immunoblot and immunofluorescence and in vivo xenograft IHC analysis were performed. Transcriptome analysis of HCC827 cells under erlotinib inhibition time-course was performed using Affymetrix microarray gene expression profiling (Human GeneChip 1.0 ST arrays) in triplicate at 0 hr, 8 hr, 9 days of erlotinib-treatment, and 9 days pretreatment with erlotinib followed by 7 days drug-washout. Data analysis was performed using PCA, heatmap, BAMarray and PathwayStudio 6.0 analysis. We also performed mass-spectrometry-based global profiling analysis of cellular metabolome in 5 replicate. Results. We analyzed drug-sensitive HCC827 and PC-9 cells under erlotinib inhibition to characterize the adaptive response that engenders drug resistance. We identified an early adaptive drug-escape that emerged after 9 days of erlotinib, characterized with MET-independent mitochondrial BCL-2/BCL-xL prosurvival priming to result in 100-fold IC50 increase towards resistance. These cells displayed a quiescence state associated with retarded cell proliferation/cytoskeletal functions. Transcriptome profiling analysis of the early adaptive resistant HCC827 cells revealed a remarkable genome-wide adaptive reprogramming of gene expression signature, involving pathways of cell adhesion, cell cycle regulation, cell division/mitosis, glycolysis and gluconeogenesis, response to DNA damage stimulus, and DNA repair. Metabolomic profiling revealed a global adaptive metabolic reprogramming also, with resultant suppression of glucose and TCA cycle metabolism, and lipid bioenergetics. Our studies also identified autocrine TGFβ2 signaling pathway in mediating the early adaptive resistance in the mutated-EGFR lung cancer cells that escaped erlotinib inhibition through a combination of quiescence-state and EMT, cellular metabolic reprogramming and STAT3/BCL-2/BCL-xL mitochondrial prosurvival priming. Conclusion. Early adaptive drug-escape emerges within a subpopulation of EGFR-mutant NSCLC under erlotinib inhibition. This constitutes an early-stage minimal residual disease leading to eventual resistant tumor relapse. These early drug-escaping tumor cells undergo global cellular reprogramming with upregulated mitochondrial prosurvival priming, representing attractive therapeutic targets to eradicate drug resistance. Citation Format: Patrick C. Ma, Praveena S. Thiagarajan, Patrick Leahy, Rakesh Bagai, Ivy Shi, Wei Zhang, Yan Feng, Martina L. Veigl, Daniel Lindner, David Danielpour, Lihong Yin. Transcriptome and metabolome reprogramming in EGFR-mutant NSCLC early adaptive drug-escape against erlotinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1835. doi:10.1158/1538-7445.AM2014-1835

Abstract Background: Recent studies revealed that ALK (anaplastic lymphoma kinase) 2p23 translocation, most commonly fused with echinoderm microtubule-associated protein-like 4 (EML4), is a novel driver oncogene in about 5% of non-small cell lung cancer (NSCLC). ALK 2p23 fusion (ALK+) can be diagnosed by dual color break-part ALK FISH assay to inform personalized targeted therapy using the ALK inhibitor crizotinib. However, the underlying mechanisms of ALK oncogenic signaling and more importantly ALK inhibitor crizotinib resistance are not well understood. Materials and Methods: To understand the mechanism of oncogenic ALK signaling, an ALK+ NSCLC microarray dataset was retrieved from NCBI-GEO data repository (GSE31210) and analyzed using data-mining bioinformatics approaches. This dataset includes the gene expression profiles of 11 ALK+ NSCLC and 20 normal lung tissue samples. Statistical analysis was carried out to identify altered genes in ALK+ NSCLC and Ingenuity Pathway Analysis (IPA) software was deployed to uncover their network interactions. To study the resistance mechanism of the ALK+ H3122 human NSCLC cell line against the specific ALK inhibitor TAE684, real-time polymerase chain reaction (qRT-PCR), cell viability assay and immunoblotting were performed using standard techniques. Results: We identified a set of 1,656 genes that were significantly altered in ALK+ NSCLC, consisting of 662 upregulated and 994 downregulated. The principal component analysis (PCA) based on the top 51 of these dysregulated genes evidently separated ALK+ and normal lung tissue samples, implicating that these genes could represent potential genomic signature of ALK+ NSCLC. The IPA analysis revealed 35 canonical pathways linked to ALK signaling, including beta-adrenergic and angiopoietin signaling pathways. The results of qRT-PCR, cell viability assay and immunoblotting showed that in translocated ALK+ H3122 human NSCLC cell line, HGF (hepatocyte growth factor) overexpression could activate ALK downstream signaling via activation of MET receptor kinase pathway, thus potentially bypassing the oncogenic ALK signaling under ALK inhibitor TAE684 treatment. Hence, alternative direct activation of ALK downstream signaling molecules via MET/HGF axis activation may potentially contribute to ALK inhibitor drug resistance. Conclusion: Using bioinformatics data-mining, we have identified dysregulated genes and pathways in ALK+ NSCLC, many of which are novel biomarkers not previously reported. We also verified the relevance and potential key role of MET/HGF signaling axis in ALK inhibitor resistance. Taken together, our results highlighted the cross-talk between ALK and MET signaling in NSCLC and shed new insights into possible pitfalls of potential specific ALK targeting inhibitor drugs. Citation Format: Ivy Shi, Lihong Yin, Patrick C. Ma. Integrative study of dysregulated genes in translocated ALK-positive non-small cell lung cancer and personalized targeted therapy resistance. [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 4466. doi:10.1158/1538-7445.AM2013-4466

Abstract Background. MET is the tyrosine kinase receptor for the ligand hepatocyte growth factor (HGF). MET kinase has been identified as a novel promising target in non-small cell lung lung cancer (NSCLC). Crizotinib, a recently FDA-approved ALK inhibitor for NSCLC harboring ALK 2p23 fusion (ALK+), was initially developed as a MET inhibitor. MET expression level is a potential biomarker for response to MET inhibitors. The role of MET/HGF pathway in ALK+ NSCLC is unknown. Methods. A cohort of 51 NSCLC patients tested for ALK fusion at Cleveland Clinic (2000 to 2012), including 15 ALK(+) and 36 ALK(-) subjects, were included in the study. Immunohistochemistry (IHC) stain against MET was performed using a monoclonal CONFIRM anti-Total c-MET antibody (SP44, Ventana). Staining was performed on a Ventana Benchmark XT automated immunostainer (Ventana Medical Systems). A four-tier (0, 1+, 2+, 3+) scoring system and H-score (the sum of each intensity score x %) were used. Thirteen ALK(+) patients treated on crizotinib therapy were prospectively followed and a total of 32 blood plasma samples were collected (1) prior to crizotinib initiation, (2) during responding period and (3) at disease progression, for HGF level measurement using HGF ELISA kit (R&D). Statistical analysis was performed using Fisher exact test and Wilcoxon rank sum test in JMP. Results. Of the 51 tumor tested for MET IHC, 39 were adenocarcinoma (15 ALK+ and 24 ALK-), 6 squamous cell, 4 large cell carcinoma and 2 other NSCLC. None received any MET inhibitor when tissue was collected. The percentage of negative MET IHC (score 0 or 1+ in 50% tumor cells) was not statistically significant between ALK(+) and ALK(-) groups (40% vs 30%, p=0.5). The plasma HGF levels in ALK(+) patients varies from undetectable to 14000pg/ml. The median (25th-75th quantiles) level in patients prior to crizotinib therapy (n=5), during responding period (CR/PR/SD, n=12) and at disease progression (n=5) were 2137(767-12100)pg/ml, 1629(785-3430)pg/ml and 4064(1108-7308)pg/ml. The plasma HGF level showed a trend of decreasing during clinical response to crizotinib and elevation upon disease progression, albeit not reaching statistical significance in this small study cohort (p>0.05). Conclusions. MET expression is commonly seen in NSCLC, and no significant difference were observed between fusion ALK(+) and ALK(-) NSCLC. The baseline plasma HGF concentration varied significantly among ALK(+) NSCLC patients, but showed a trend correlating with therapeutic response and resistant progression, to crizotinib therapy. Prospective study with larger cohorts is warranted to further evaluate the role of MET/HGF as biomarker in ALK targeted therapy. Citation Format: Yan Feng, Eugen Minca, Wei Zhang, Lihong Yin, Nathan Pennell, Carol Farver, Raymond Tubbs, Angen Liu, Patrick Ma. Tumoral MET expression and plasma HGF level in patients with ALK 2p23 fusion driven lung cancer. [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 2374. doi:10.1158/1538-7445.AM2013-2374

e22032 Background: Lung cancer targeted therapy is largely limited by inevitable recurrent resistant disease after initial response to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), typically accompanied with divergent late acquired resistance mechanisms. We now focused on studying the emergence of early adaptive resistance to uncover attractive therapeutic targets to overcome drug resistance. Methods: HCC827 cells were treated with EGFR-TKI (0-9 days) with apoptosis pathway-specific QPCR array and TLVM analysis performed. MTS and crystal violet assays were performed. Western blot analysis was performed to examine prosurvival signaling developed against erlotinib, alone or in combination with MET inhibitor SU11274. IHC was performed on lung cancer tumor microarray (TMA) using BCL-2 and caspase-recruitment domain-containing protein 8 (CARD8) antibodies and graded (4 tier scoring system). NSCLC cell lines and murine xenograft models (HCC827, H1975) were developed for resistance biomarkers expression analysis in pre-/post-TKI treatment using anti-human CARD8, p-STAT3 and BCL-2 antibodies. Results: We characterized the emergence of early resistant lung cancer cells in escape against targeted TKIs with 100-fold higher IC50 in adaptive drug resistance. The resistant cells that evaded EGFR-TKI based targeted inhibition exhibited MET-independent induction of CARD8 and STAT3/BCL-2 mitochondrial prosurvival signaling in cellular quiescence, and inhibited cytoskeletal functions. Expression analysis studies demonstrated common tumor-associated expression of CARD8 but relatively low BCL-2 level in NSCLC. In vitro cell line studies suggest that CARD8 induction was preceded by a resurgence of STAT3 activation. In vivo xenograft model (HCC827/erlotinib; H1975/erlotinib+ SU11274) also verified upregulated CARD8/BCL-2 activation within early resistant cells. Conclusions: Resistant tumor cells that evaded EGFR inhibitors, alone or in combination with MET inhibitors, exhibited increased expression of CARD8-STAT3/BCL-2 prosurvival signaling cascade. Further studies to define the mechanism of CARD8 in promoting adaptive tumor drug resistance would be warranted.

Abstract Lung cancer is the second most common malignancy in the United States with the highest cancer-mortality rate. Molecular targeted therapy in oncogene addicted non-small cell lung cancer (NSCLC) has gain landmark proof-of-principle success in clinical utility in recent years. Prime examples are EGFR targeted therapy against mutated EGFR tumors using tyrosine kinase inhibitors (TKIs) such as erlotinib/gefitinib, and also ALK targeted therapy against EML4-ALK tumors using ALK inhibitor crizotinib. Superior response rates and progression free survival outcomes were observed with the use of targeted therapeutics even in first line setting. Nonetheless, targeted therapeutics against mutant-EGFR and EML4-ALK tumors invariably develop an acquired tumor resistance to the TKIs. Mutational resistant mechanism has been well-established. Alternative signal pathway activation such as MET/HGF in both EGFR and ALK TKI resistance, or HER family signaling activation in EML4-ALK resistance have also been reported. We have recently begun studies focusing on the non-mutational mechanisms of tumor resistance under early time window of TKI treatment. Using HCC827 and PC9 cells as mutant EGFR addiction model, as well as H3122 and H2228 NSCLC cells as EML4-ALK addicted model, we have uncovered a novel early adaptive TKI resistance that can be evident as early as 7-14 days under initial TKI treatment. The early adaptive tumor cells displayed remarkable tumor resistance against the TKIs with 100-fold higher IC50. In mutant-EGFR against erlotinib, there was an upregulated mitochondrial antiapoptotic/prosurvival BCL-2/BCL-xL signaling dependence that is MET-independent. The early resistant cells were adaptive, highly reversible and adopted a quiescent state during TKI-induced resistance. On the other hand, our studies of early TKI resistance in EML4-ALK fusion cells revealed that while the adaptive resistance to TAE684 was also reversible, it appears to be not dependent on BCL-2/BCL-xL upregulation. We found that in both targeted TKI early resistance, the tumor survivor cells exhibited epithelial-mesenchymal transition (EMT) markers changes with decreased E-cadherin expression and increased vimentin expression. Our studies also provided in vitro and in vivo evidence that these early adaptive TKI resistant tumor cells can be targetable for prevention or eradication of tumor resistance emergence, such as utilizing BCL-2/BCL-xL pathway inhibitors, such as ABT-737, in combination with EGFR TKI in mutant-EGFR addicted NSCLC. Further preclinical model studies will focus on investigating the molecular and genomic changes and gene network regulation in the early adaptive resistant tumor cells, to enable a new paradigm of novel therapeutic targets discovery to overcome early tumor resistance emergence and improve long term outcome of targeted therapy in lung cancer. Citation Format: Lihong Yin, Ivy Shi, Wei Zhang, Rakesh Bagai, Yan Feng, Patrick C. Ma. Studies of early adaptive resistance in mutant-EGFR and EML4-ALK addicted non-small cell lung cancer in escape against small molecule inhibitors. [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 4455. doi:10.1158/1538-7445.AM2013-4455

Activating somatic mutations in the epidermal growth factor receptor (EGFR) gene are frequently seen in the tumors of light- and never-smoking patients with non–small-cell lung cancer (NSCLC), and the presence of these mutations predicts for objective responses to EGFR tyrosine kinase inhibitors such as erlotinib.1Sequist LV Bell DW Lynch TJ Haber DA Molecular predictors of response to epidermal growth factor receptor antagonists in non-small-cell lung cancer.J Clin Oncol. 2007; 25: 587-595Crossref PubMed Scopus (560) Google Scholar This report describes the first known case of a mother and son who both developed metastatic lung adenocarcinoma; both of them were responsive to tyrosine kinase inhibitor therapy and have been stable for 3 to 5 years. Patient 1 is a 62-year-old white man, never smoker, who presented with right-sided pleuritic chest pain in January 2006. Chest radiography showed a right lower lobe (RLL) infiltrate, and he was treated with antibiotics without improvement. Chest computed tomography (CT) showed a RLL consolidation with bilateral ground-glass opacities. Transthoracic needle biopsy of the RLL lesion was positive for adenocarcinoma, lepidic predominant. He had no exposure to asbestos or significant second-hand smoke. His family history was significant for a maternal grandmother with pancreatic cancer and his mother who had breast cancer at age 53. The patient was treated with carboplatin and paclitaxel for eight cycles with a partial response (PR) and, upon progression in July 2006, was started on single-agent erlotinib at 150 mg daily. He had a PR with disappearance of the small bilateral ground-glass lesions and significant shrinkage of his RLL lesion. As of October 2011, he remained on erlotinib after more than 5 years without progression. Allele-specific polymerase chain reaction testing of his archived tumor sample showed an exon 19 deletion mutation in the EGFR gene (Clarient Diagnostic Services, Aliso Viejo, CA). Patient 2 is the mother of patient 1, and also presented in 2006 at the age of 78 years with moderate dyspnea on exertion. A chest radiograph showed bilateral pulmonary nodules, and a CT scan of the chest confirmed bilateral mixed solid and ground-glass opacities. She had a history of breast cancer in 1981 and had been treated with mastectomy followed by adjuvant chemotherapy and tamoxifen. She had a 5-year history of smoking, quitting more than 25 years prior. She has another son who is healthy. She underwent a bronchoscopy, which was nondiagnostic. In 2008 there was radiographic progression of two of the lesions (Fig. 1A and 1C), and repeat transbronchial biopsy was positive for lepidic predominant adenocarcinoma. There was insufficient tissue for EGFR mutation testing, and she was empirically started on first-line erlotinib at a dose of 150 mg. She had a grade 3 acneiform rash and the dose was reduced to 100 mg daily. She also had a PR in the bilateral lesions, and the lesions remained stable on treatment as of January 2012 (Fig. 1B and 1D). Although little is known about the inherited risk of developing EGFR mutant NSCLC, there is evidence that germline EGFR mutations may be a risk factor. Two groups of investigators2Bell DW Gore I Okimoto RA et al.Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR.Nat Genet. 2005; 37: 1315-1316Crossref PubMed Scopus (415) Google Scholar,3Tibaldi C Giovannetti E Vasile E et al.Inherited germline T790M mutation and somatic epidermal growth factor receptor mutations in non-small cell lung cancer patients.J Thorac Oncol. 2011; 6: 395-396Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar have identified families with germline T790M EGFR mutations that have multiple cases of lung adenocarcinoma with secondary somatic sensitizing EGFR mutations. Girard et al.4Girard N Lou E Azzoli CG et al.Analysis of genetic variants in never-smokers with lung cancer facilitated by an Internet-based blood collection protocol: a preliminary report.Clin Cancer Res. 2010; 16: 755-763Crossref PubMed Scopus (71) Google Scholar examined germline DNA from 369 nonsmoking NSCLC patients and found two, both of whom had sensitizing somatic EGFR mutations, who also had germline T790M and a family history of lung cancer. Given this data, germline DNA was isolated from our patients’ peripheral blood and examined using the Human Lung Cancer qBiomarker Somatic Mutation PCR Array (Qiagen-SABiosciences, Frederick, MD) to screen for germline genetic variants in common lung cancer genes5Ding L Getz G Wheeler DA et al.Somatic mutations affect key pathways in lung adenocarcinoma.Nature. 2008; 455: 1069-1075Crossref PubMed Scopus (2169) Google Scholar including AKT1, BRAF, CTNNB1/beta-catenin, EGFR, ERBB2, HRAS, KRAS, NRAS, PIK3CA, LKB1/STK11, and P53. The germline DNA from both patients were found to be wild-type for both EGFR and all other genes tested. Hence, a mechanism other than germline mutations in EGFR may be responsible for any potential inherited suceptibility.