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

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

Objectives To identify patient characteristics associated with polypharmacy and inappropriate medication (PIM) use among older patients with newly diagnosed cancer. Materials and Methods This is a cross-sectional study conducted in ambulatory oncology clinics at an academic center. Participants included 117 patients aged ≥65years with newly diagnosed histologically confirmed stage I–IV cancer and were enrolled between April 2008 and September 2009. Medication review, included patient self-report and medical records. Polypharmacy was defined as the concurrent use of ≥five medications, ( Yes / No ). PIM use was defined as use of ≥one medication included in the 2003 update of Beers Criteria, ( Yes / No ). Results The prevalence of polypharmacy and PIM use was 80% and 41%, respectively. Three independent correlates of medication use were identified. An increase in comorbidity count by one, ECOG-PS score by one, and PIM use by one, was associated with an increase in medication use by 0.48 ( P =0.0002), 0.79 ( P =0.01) and 1.22 ( P =0.006), respectively. Two independent correlates of PIM use were identified. The odds of using PIMs decreased by 10% for one unit increase in Body Mass Index [Odds Ratio (OR) 0.90, 95% CI=(0.84, 0.97)], and increased by 18% for each increase in medication count by one [OR 1.18, 95% CI=(1.04, 1.34)]. Conclusion There was a high prevalence of polypharmacy and PIM use in older patients with newly diagnosed cancer. Given the co-occurrence of polypharmacy with poor performance status and multi-morbidity, multi-dimensional interventions are needed in the geriatric-oncology population to improve health and cancer outcomes.

Targeted therapies for cancer are inherently limited by the inevitable recurrence of resistant disease after initial responses. To define early molecular changes within residual tumor cells that persist after treatment, we analyzed drug-sensitive lung adenocarcinoma cell lines exposed to reversible or irreversible epidermal growth factor receptor (EGFR) inhibitors, alone or in combination with MET-kinase inhibitors, to characterize the adaptive response that engenders drug resistance. Tumor cells displaying early resistance exhibited dependence on MET-independent activation of BCL-2/BCL-XL survival signaling. Further, such cells displayed a quiescence-like state associated with greatly retarded cell proliferation and cytoskeletal functions that were readily reversed after withdrawal of targeted inhibitors. Findings were validated in a xenograft model, showing BCL-2 induction and p-STAT3[Y705] activation within the residual tumor cells surviving the initial antitumor response to targeted therapies. Disrupting the mitochondrial BCL-2/BCL-XL antiapoptotic machinery in early survivor cells using BCL-2 Homology Domain 3 (BH3) mimetic agents such as ABT-737, or by dual RNAi-mediated knockdown of BCL-2/BCL-XL, was sufficient to eradicate the early-resistant lung-tumor-cells evading targeted inhibitors. Similarly, in a xenograft model the preemptive cotreatment of lung tumor cells with an EGFR inhibitor and a BH3 mimetic eradicated early TKI-resistant evaders and ultimately achieved a more durable response with prolonged remission. Our findings prompt prospective clinical investigations using BH3-mimetics combined with targeted receptor kinase inhibitors to optimize and improve clinical outcomes in lung-cancer treatment. Cancer Res; 71(13); 4494–505. ©2011 AACR.

In vivo studies suggest that vascular endothelial cells (ECs) can acquire and cross-present exogenous Ag on MHC-I but the cellular mechanisms underlying this observation remain unknown. We tested whether primary female mouse aortic ECs could cross-present exogenous male Ag to the T cell hybridoma, MHH, specific for HYUty plus Db. MHC-I-deficient male spleen cells provided a source of male Ag that could not directly stimulate the MHH cells. Addition of male but not female MHC-I-deficient spleen cells to wild-type syngeneic female EC induced MHH stimulation, demonstrating EC cross-presentation. Lactacystin treatment of the donor male MHC-I-deficient spleen cells, to inhibit proteasome function, markedly enhanced EC cross-presentation showing that the process is most efficient for intact proteins rather than degraded peptide fragments. Additional experiments revealed that this EC Ag-processing pathway is both proteasome and TAP1 dependent. These studies demonstrate that cultured murine aortic ECs can process and present MHC-I-restricted Ag derived from a separate, live cell, and they offer insight into the molecular requirements involved in this EC Ag presentation process. Through this pathway, ECs expressing cross-presented peptides can participate in the effector phase of T cell-mediated inflammatory responses such as autoimmunity, anti-tumor immunity, and transplant rejection.

ARQ-197 is an oral, selective c-Met inhibitor under development by ArQule Inc, in partnership with Daiichi Sankyo Co Ltd and Asian licensee Kyowa Hakko Kirin Co Ltd, for the potential treatment of solid tumors, including NSCLC, hepatocellular carcinoma and pancreatic cancer, as well as microphthalmia transcription factor-driven tumors. c-Met, a key cell surface receptor tyrosine kinase involved in diverse regulatory functions, is often aberrantly activated in human cancers. While the precise mechanism of action of ARQ-197 remains undefined, data from preclinical studies have demonstrated that ARQ-197 inhibits c-Met activation in numerous human tumor cell lines and specifically targets c-Met in various cancer types; uniquely, ARQ-197 inhibits c-Met in a non-ATP-competitive manner. Phase I/II clinical trials demonstrated promise in terms of both tolerability and tumor response. Intriguingly, dose-limiting adverse effects were hematological in nature. Combinational trials are also ongoing to take advantage of the signaling crosstalk between c-Met and other oncogenic signaling systems. Prioritization of the clinical development of c-Met inhibitors, such as ARQ-197, among different tumor disease types is a key challenge at present; an improved understanding of the prediction of molecular determinants in tumors with respect to c-Met kinase as the driver oncogenic receptor, and of the prediction of tumor response, is still urgently needed.

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

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

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....

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...

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.

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

9087 Background: To evaluate the prevalence of polypharmacy and the inappropriate prescribing of medications (IPM), and to determine the relation between polypharmacy, IPM, and functional status in...