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Background: KRAS G12C oncogenic mutations occur in ~13% of non-small cell lung cancers (NSCLCs) and up to 4% of other solid tumors. JDQ443 is a selective, covalent, orally bioavailable, investigational KRASG12C inhibitor that irreversibly traps KRASG12C in the inactive, GDP-bound state. JDQ443 is structurally unique and forms novel interactions with KRAS in the switch II pocket. Methods: KontRASt-01 (NCT04699188) is a Phase Ib/II, open-label, multicenter, dose-escalation and dose-expansion trial of JDQ443 as monotherapy or in combination with TNO155 (SHP2 inhibitor) and/or tislelizumab (anti-PD-1 monoclonal antibody). Primary objectives of dose escalation are to assess safety and tolerability, and identify the maximum tolerated doses (MTDs) and/or recommended doses (RDs) and regimens for future studies. The primary objective of dose expansion is to assess efficacy. Key inclusion criteria: advanced, KRAS G12C-mutated solid tumors; previous standard-of-care treatment; age ≥18 yrs; ECOG PS 0-1. Key exclusion criteria for the JDQ443 monotherapy arm: active brain metastases, prior KRASG12C inhibitor treatment. Here, we present preliminary results for JDQ443 monotherapy dose escalation. Results: As of Nov 3, 2021, 39 pts were treated with JDQ443 PO continuously across 4 dose levels: 200 mg once daily (QD) (n=10), 400 mg QD (n=11), 200 mg twice daily (BID) (n=11), and 300 mg BID (n=7). Median age was 60 yrs (range 26-76), median prior lines of therapy was 3 (range 1-7), and indications included NSCLC (n=20) and colorectal cancer (CRC) (n=16). Median duration of exposure was 9.1 wks (range 0.9-21), with ongoing treatment in most pts (61.5%) at the time of cut-off. Treatment-related adverse events (TRAEs) occurred in 25 (64.1%) pts. Most TRAEs were Grade (Gr) 1-2. Four Gr 3 TRAEs occurred in 4 (10.3%) separate pts; there were no Gr 4-5 TRAEs. The most common TRAEs (occurring in ≥10% of pts) were fatigue (25.6%), nausea (15.4%), edema (12.8%), pruritus (10.3%), and vomiting (10.3%). There was one DLT (Gr 3 fatigue) and one treatment-related serious AE (Gr 3 photosensitivity reaction), each in separate pts treated at 300 mg BID. TRAEs led to dose reduction in 1 pt and discontinuation in 1 pt. A MTD was not reached. The RD was declared as 200 mg BID. At the RD, PK and PD modeling for JDQ443 predicted average KRASG12C target occupancy of >90% in >82% of pts. Using an efficacy cut-off date of Dec 13, 2021, for the 20 pts with NSCLC among the same 39 pts, the ORR (confirmed complete response or partial response) by RECIST 1.1 was 30.0% (6/20) across dose levels and 43.0% (3/7) at the RD. Additional data will be available at the time of presentation. Conclusions: JDQ443 demonstrates an acceptable safety and tolerability profile, with early signs of clinical activity in pts with NSCLC. Enrollment is ongoing to NSCLC and CRC dose-expansion groups for JDQ443 monotherapy at the RD, and to JDQ443 + TNO155 dose escalation. Citation Format: Daniel S. Tan, Toshio Shimizu, Benjamin Solomon, Rebecca S. Heist, Martin Schuler, Maria J. De Miguel Luken, Anas Gazzah, Martin Wermke, Christophe Dooms, Herbert H. Loong, Neeltje Steeghs, Enriqueta Felip, Conor E. Steuer, Eric van Cutsem, Ross A. Soo, Ashley C. Jaeger, Jaeyeon Kim, Kun Xu, Xueying Chen, Xiaoming Cui, Heather Burks, Anna Farago, Philippe A. Cassier. KontRASt-01: A phase Ib/II, dose-escalation study of JDQ443 in patients (pts) with advanced, KRAS G12C-mutated solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT033.

Introduction: In the INSIGHT trial primary analysis (NCT01982955; median follow-up: 21.8 months), tepotinib (a potent, highly selective, once daily [QD] MET inhibitor) + gefitinib improved efficacy vs chemotherapy (CTX) in patients with EGFR-mutant NSCLC and resistance to anti-EGFR therapy due to MET amplification (Wu et al, Lancet Respir Med 2020). Here we report final analyses from INSIGHT (data cut-off: September 3, 2021; median follow-up: 57.5 months). Methods: Patients with EGFR-mutant (T790M-negative) NSCLC and anti-EGFR resistance, with MET gene copy number (GCN) ≥5 and/or MET:CEP7 ≥2 by FISH and/or MET IHC 2+/3+, were randomized to tepotinib 500 mg (450 mg active moiety) + gefitinib 250 mg QD or CTX. Primary endpoint was progression-free survival (PFS) per investigator. Preplanned analyses evaluated patients with MET amplification. Results: In the 19/55 randomized patients (34.5%) with MET amplification (GCN ≥5, n=18; MET:CEP7 ≥2, n=13; MET IHC 3+, n=17), median age was 60.4 years, 68.4% were never-smokers, and prior EGFR inhibitors were gefitinib (57.9%), afatinib (21.1%), erlotinib (10.5%) and icotinib (10.5%). Median duration of tepotinib + gefitinib was 11.3 months (range: 1.1-56.5), with treatment duration >1 year in 6 patients (31.6%) and >4 years in 3 patients (15.8%). Two patients continued treatment outside the study. Tepotinib + gefitinib improved PFS (hazard ratio [HR] 0.13; 95% confidence interval [CI] 0.04, 0.43), overall survival (HR 0.10; 95% CI 0.02, 0.36), objective response rate and duration of response vs CTX (Table). Treatment-related Grade ≥3 AEs occurred in 7 patients (58.3%) with tepotinib + gefitinib and 5 (71.4%) with CTX. Most common post-study therapies were kinase inhibitors (n=2 in the tepotinib + gefitinib arm; n=3 in the CTX arm). In patients with MET IHC 3+ (n=34; including 17 patients with MET amplification), tepotinib + gefitinib also markedly improved PFS (HR 0.35; 95% CI 0.17, 0.74) and OS (HR 0.44; 95% CI 0.23, 0.84) vs CTX. Conclusions: Tepotinib + gefitinib greatly improved PFS and OS vs CTX in patients with EGFR-mutant NSCLC with MET amplification. INSIGHT 2 is evaluating tepotinib + osimertinib in this setting. Table. Summary of efficacy and safety data in patients with MET amplification Endpoint Tepotinib + gefitinib (n=12) CTX (n=7) PFS Events, n (%) 7 (58) 7 (100) Median, months (90% CI) 16.6 (8.3, 22.1) 4.2 (1.4, 7.0) HR (90% CI) 0.13 (0.04, 0.43) OS* Events, n (%) 7 (58) 7 (100) Median, months (90% CI) 37.3 (21.1, 52.1) 13.1 (3.3, 22.6) HR (90% CI) 0.10 (0.02, 0.36) ORR n (%) [90% CI] 8 (66.7) [39.1, 87.7] 3 (42.9) [12.9, 77.5] OR (90% CI) 2.67 (0.37, 19.56) DOR Median, months (90% CI) 19.9 (7.0, NE) 2.8 (2.8, NE) Treatment-related Grade ≥3 AEs†, n (%) Amylase increased 4 (33.3) 0 Lipase increased 4 (33.3) 0 Anemia 0 2 (28.6) Neutrophil count decreased 0 2 (28.6) WBC count decreased 0 2 (28.6) *Post-study therapy included an EGFR inhibitor ± a MET inhibitor in two patients in each arm (tepotinib + gefitinib arm: osimertinib ± cabozantinib [n=1], gefitinib + cabozantinib [n=1]; CTX arm: erlotinib, afatinib, and osimertinib ± crizotinib [n=1], osimertinib [n=1]). Post-study CTX was received by one patient in the tepotinib + gefitinib arm and two patients in the CTX arm.†Reported in >20% of patients in either arm. AE, adverse event; CI, confidence interval; CTX, chemotherapy; DOR, duration of response; HR, hazard ratio; NE, not estimable; ORR, objective response rate; OR, odds ratio; OS, overall survival; PFS, progression-free survival; WBC, white blood cell. Citation Format: Chong Kin Liam, Azura Rozila Ahmad, Te-Chun Hsia, Jianying Zhou, Dong-Wan Kim, Ross Andrew Soo, Ying Cheng, Shun Lu, Sang Won Shin, James Chih-Hsin Yang, Yiping Zhang, Jun Zhao, Rolf Bruns, Andreas Johne, Yi-Long Wu. Tepotinib + gefitinib in patients with EGFR-mutant NSCLC with MET amplification: Final analysis of INSIGHT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT538.

BACKGROUND: Lorlatinib is a selective, potent, brain-penetrant, 3rd-generation (gen) ALK/ROS1 tyrosine kinase inhibitor (TKI) preclinically active against most known ALK resistance mutations. In pts with ALK+ advanced NSCLC with disease progression following 2nd-gen ALK TKIs, lorlatinib has shown robust clinical activity and we found that tumor genotyping for ALK mutations may identify such pts more likely to respond to lorlatinib treatment. Prior analyses have suggested that EML4-ALK variant type may influence ALK TKI treatment benefit. To further identify molecular correlates of response, we performed an exploratory subgroup analysis by EML4-ALK variant type and ALK resistance mutations in pts previously treated with 2nd-gen ALK TKIs and who received the recommended Ph 2 dose of lorlatinib (100 mg once daily). METHODS: Baseline plasma samples were collected from pts with ALK+ NSCLC with ≥1 prior 2nd-gen ALK TKI enrolled in the ongoing registrational Ph 2 study (NCT01970865). Circulating free DNA (cfDNA) was analyzed using Guardant360 (Guardant Health, Inc., CA, USA) to determine EML4-ALK variant and ALK kinase domain mutations. Objective response rate (ORR) and duration of response (DOR), by independent central review, were evaluated according to EML4-ALK variant type and ALK resistance mutation status. RESULTS: ALK fusions were detectable in 64 (41.0%) of 156 pt plasma samples. EML4-ALK variants 1, 2, and 3 were detected at frequency of 17.3%, 2.6% and 15.4%, respectively. Other EML4-ALK variants (including the variant types 4, 5, 7, and 8) were also detected in 3.2% of pts, as well as some other less frequent fusion partners (e.g., KIF5B) in 2.6% of pts. Based on cfDNA, ALK resistance mutations were detected in 40 pts; of whom, 6 pts harbored EML4-ALK variant 1, 1 pt had variant 2 and 18 had variant 3. The G1202R/Del mutation was detected in 23 pts samples; of which, 15 (65.2%) also harbored EML4-ALK variant 3. ORR was 33.3% (95% CI 16.5-54.0), 75.0% (95% CI 19.4-99.4) and 45.8% (95% CI 25.6-67.2) for variants 1, 2 and 3, respectively, while median DOR was similar for pts with variant 1 or 3 (both 6.9 months). Median DOR was not reached for variant 2. Of note, no responses were observed in the pts with other types of ALK rearrangements detected. Finally, ALK fusions were not detected in the cfDNA of 92 pts (59.0%), including 12 pts who had detectable ALK mutations. Among these 92 pts, ORR was 39.1% (95% CI 29.1-49.9) and median DOR was 7.1 months (95% C 5.5-not reached). Confirmation of these results in the tumor tissue is ongoing. CONCLUSION: In this heavily pretreated group of ALK+ NSCLC pts, the presence of an ALK resistance mutation might enrich for EML4-ALK variants 1 and 3. Lorlatinib exhibited antitumor activity irrespective of EML4-ALK variant and across a variety of ALK resistance mutations. Citation Format: Todd M. Bauer, Jean-François Martini, Benjamin Besse, Chia-Chi Lin, Ross A. Soo, Gregory J. Riely, Sai-Hong Ignatius Ou, Francesca Toffalorio, Antonello Abbattista, Holger Thurm, D. Ross Camidge, Steven Kao, Rita Chiari, Shirish Gadgeel, Enriqueta Felip, Alice T. Shaw, Benjamin J. Solomon. Impact of the EML4-ALK fusion variant on the efficacy of lorlatinib in patients (pts) with ALK-positive advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT025.

Anaplastic lymphoma kinase (ALK) inhibitors are highly effective in patients with ALK fusion-positive lung cancer, but acquired resistance invariably emerges. Identification of secondary mutations has received considerable attention, but most cases cannot be explained by genetic causes alone, raising the possibility of epigenetic mechanisms in acquired drug resistance. Here, we investigated the dynamic changes in the transcriptome and enhancer landscape during development of acquired resistance to ALK inhibitors. Histone H3 lysine 27 acetylation (H3K27ac) was profoundly altered during acquisition of resistance, and enhancer remodeling induced expression changes in both miRNAs and mRNAs. Decreased H3K27ac levels and reduced miR-34a expression associated with the activation of target genes such as AXL. Panobinostat, a pan-histone deacetylase inhibitor, altered the H3K27ac profile and activated tumor-suppressor miRNAs such as miR-449, another member of the miR-34 family, and synergistically induced antiproliferative effects with ALK inhibitors on resistant cells, xenografts, and EML4-ALK transgenic mice. Paired analysis of patient samples before and after treatment with ALK inhibitors revealed that repression of miR-34a or miR-449a and activation of AXL were mutually exclusive of secondary mutations in ALK. Our findings indicate that enhancer remodeling and altered expression of miRNAs play key roles in cancer drug resistance and suggest that strategies targeting epigenetic pathways represent a potentially effective method for overcoming acquired resistance to cancer therapy.Significance: Epigenetic deregulation drives acquired resistance to ALK inhibitors in ALK-positive lung cancer. Cancer Res; 78(12); 3350-62. ©2018 AACR.

The discovery of somatic mutations in EGFR and development of EGFR tyrosine kinase inhibitors (TKI) have revolutionized treatment for lung cancer. However, resistance to TKIs emerges in almost all patients and currently no effective treatment is available. Here, we show that β-catenin is essential for development of EGFR-mutated lung cancers. β-Catenin was upregulated and activated in EGFR-mutated cells. Mutant EGFR preferentially bound to and tyrosine phosphorylated β-catenin, leading to an increase in β-catenin–mediated transactivation, particularly in cells harboring the gefitinib/erlotinib-resistant gatekeeper EGFR-T790M mutation. Pharmacologic inhibition of β-catenin suppressed EGFR-L858R-T790M mutated lung tumor growth, and genetic deletion of the β-catenin gene dramatically reduced lung tumor formation in EGFR-L858R-T790M transgenic mice. These data suggest that β-catenin plays an essential role in lung tumorigenesis and that targeting the β-catenin pathway may provide novel strategies to prevent lung cancer development or overcome resistance to EGFR TKIs. Cancer Res; 74(20); 5891–902. ©2014 AACR.

Background MET amplification (amp) is a resistance mechanism to EGFR tyrosine kinase inhibitors (TKIs). Tepotinib (TEP), an oral, selective MET TKI, has shown promising antitumor activity in combination with gefitinib (GEF) in patients with MET+ EGFR-mutant NSCLC and acquired resistance to 1st-line EGFR TKIs in a phase 1b/2 study (NCT01982955). In the phase 1b part of the study, 4/6 patients with METamp had an objective response with TEP+GEF (67%). Methods In the phase 2 part of the study, Asian patients with advanced MET+ (MET2+ or 3+ by immunohistochemistry and/or METamp by in-situ hybridization) EGFR-mutant T790M- NSCLC and acquired resistance to 1st-line EGFR TKIs were randomized to receive oral TEP 500 mg + GEF 250 mg once daily or platinum + pemetrexed chemotherapy (CTx). The primary endpoint was investigator-assessed PFS. Secondary endpoints included PFS by independent review committee (IRC), overall response rate (ORR), and safety. Data from a minimum follow-up of 6-months are presented (cut-off 26 Mar 2018) for the pre-planned analysis of patients with METamp, defined as gene copy number (GCN) ≥5 and/or MET/CEP7 ratio ≥2. Results Low recruitment halted enrolment into phase 2. From 24 Apr 2015 to 12 Jun 2017, 55/156 planned patients were enrolled; 19 had METamp (TEP+GEF, 12; CTx, 7) and are included in this analysis. Median age of METamp patients was 60.4 years (range 42-74), 6 were men, 13 were never smokers, 5 had ECOG PS 0 and 14 had PS 1. Median GCN was 8.8 (range 4.8-29.5); 18 patients had GCN ≥5, 13 had MET/CEP7 ratio ≥2. Median TEP+GEF treatment duration was 37 weeks (range 4.6-91.9) and 4 patients are still receiving treatment, ongoing for ≥18 months. Investigator-assessed PFS improved with TEP+GEF vs CTx (mPFS 21.2 vs 4.2 months; HR 0.17 [90% CI 0.05, 0.57]), as well as IRC-assessed PFS (19.8 vs 5.5 months; HR 0.25 [90% CI 0.07, 0.85]). ORR also improved with TEP+GEF (investigator: 66.7% vs 42.9%; OR 2.67 [90% CI 0.37, 19.56] and IRC: 75.0% vs 42.9%; OR 4.00 [90% CI 0.51, 31.38]). Disease control was achieved in 11 (91.7%) patients receiving TEP+GEF vs 5 (71.4%) patients receiving CTx. Related grade ≥3 treatment-emergent adverse events (TEAEs) were reported in 6 (50.0%) patients receiving TEP+GEF and 5 (71.4%) patients receiving CTx. The most common related TEAEs in the TEP+GEF arm were diarrhea (TEP+GEF 50.0% vs CTx 14.3%) and amylase increased (TEP+GEF 41.7% vs CTx 0%) and in the CTx arm were anemia (TEP+GEF 0% vs CTx 57.1%), white blood cell count decreased (TEP+GEF 8.3% vs CTx 57.1%), neutrophil count decreased (TEP+GEF 0% vs CTx 57.1%), and nausea (TEP+GEF 8.3% vs CTx 42.9%). Conclusions Promising antitumor activity with TEP+GEF was reported in patients with METamp, EGFR-mutant T790M- NSCLC and acquired resistance to 1st-line EGFR TKIs. No new safety signals were observed. METamp can be considered a biomarker for tepotinib that should be further explored. Citation Format: James Chih-Hsin Yang, Jianying Zhou, Dong-Wan Kim, Azura Rozila Ahmad, Ross Andrew Soo, Rolf Bruns, Josef Straub, Andreas Johne, Jürgen Scheele, Keunchil Park, Yi-Long Wu. Tepotinib + gefitinib vs chemotherapy in MET-amplified EGFR-mutant non-small cell lung cancer (NSCLC): Predefined subgroup analysis of a Phase Ib/II study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT193.

Background: Being able to capture circulating tumor cells (CTCs) and characterize them at a molecular level promises novel insights into the mechanisms that drive metastasis, tumor recurrence and drug resistance. However, next generation sequencing (NGS) of CTCs has been challenged by antibody-enrichment bias, low CTC purity and nucleic acid yields commonly derived from existing technologies. Here, we describe the results of a new label-free protocol for CTC enrichment and NGS analysis. Methods: The lung adenocarcinoma cell line, NCI-H1975, with EGFR T790M and L858R mutations, and colorectal cancer cell line HCT-116, with KRAS G13D mutation, were fluorescently labelled and spiked in concentrations of 250, 50, 10 and 0 cells per 7.5 mL healthy volunteer blood in duplicate. CTCs were enriched using a modified protocol of the label-free spiral microfluidics-based ClearCell® FX system. DNA was extracted by QIAamp Micro kit and sequenced using the Ion AmpliSeq Cancer Hotspot Panel and Ion Torrent PGM system. Results: Imaging analysis indicated that CTCs were efficiently enriched (40-70% recovery) with extremely low leukocyte background (650-1300 cells). Despite a low DNA quantity ( Conclusion: The new protocol generated sufficient label-free CTC purity for NGS analysis, overcoming current limitations in the field, and facilitating discovery and clinical application. Citation Format: Nur Lina Mohd Salleh, Yi Fang Lee, Mei Hui Tan, Michelle A. Rosario, Ross A. Soo, Ali Asgar Bhagat, Richie Chuan Teck Soong. High purity isolation of circulating tumor cells for next-generation sequencing. [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 1603. doi:10.1158/1538-7445.AM2015-1603

Lung cancer is the leading cause of cancer deaths in the United States with an estimated >160,000 deaths and >200,000 newly diagnosed cases each year. Over the past 20 years, the survival rate for patients remains extremely poor (∼15% over 5 years). Discovery of novel pathway involved in the pathogenesis of the lung cancer can provide new therapeutic opportunities. Stem cell factor SALL4 has recently merged to be a diagnostic tool and therapeutic target for acute myeloid leukemia and liver cancer. However, very little is known on its role in lung cancer. In order to investigate the functional role of SALL4 in lung cancer tumorigenesis and progression, we first performed gene expression profile analysis and revealed that SALL4 expression level was significant higher in all types of non-small cell lung cancer samples compared to normal lung tissue control, which was validated by IHC. Furthermore, we performed loss of function study using multiple shRNAs. Our data showed that knockdown of SALL4 could induce dramatic cell growth arrest in multiple lung cell lines including large cell carcinoma H661 and adenocarcinoma H522, H292 and PC9, but not in HCC827 cells with none/low SALL4 level. In order to elucidate the downstream pathway of SALL4 in lung cancer, we first conducted gene expression microarray using SALL4 knockdown H661 cells and cells treated with scramble control vectors. Combination of one-way ANOVA analysis and pathway analysis using Pathway Interaction Database (PID) showed that both the EGFR and IGF1R signaling pathways were affected by SALL4 knockdown in H661 lung cancer cells. Using western blot, we further validated that SALL4 depletion could lead to a decrease in both EGFR and IGF1R protein expressions in lung cancer cells. However, we did not observe any changes at the mRNA level for these two receptors upon SALL4 depletion. This leads us to wonder whether SALL4 could affect these two molecules indirectly. It has been well established that receptor tyrosine kinase (RTK) including EGFR and IGF1R can be negatively regulated by Cbl RING ubiquitin ligases. Of interest, our microarray data also showed that loss of SALL4 led to a dramatic upregulation of Cbl-b, a member of Cbl RING ligases, which was further validated by using real-time RT-PCR. In addition, SALL4 downregulation also dramatically increased Cbl-b protein expression in these lung cancer cells. Therefore, we propose that SALL4 can indirectly positively affect the expression of EGFR and IGF1R proteins through Cbl-b. In summary, we have demonstrated that SALL4 expression is aberrantly elevated in non-small cell lung cancer cells. Furthermore our data suggested that SALL4 plays an essential role in lung cancer tumor growth and maintenance by affecting both EGFR and IGFR signaling pathways via regulating Cbl-b expression. Citation Format: Ailing Li, Wenbin Ou, Jonathan Fletcher, Kol Jia Yong, Henry Yang, Ross Soo, Daniel Tenen, Li Chai. Targeting SALL4 in lung cancer through a novel Cbl-b/IGF1R&EGFR axis. [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 3389. doi:10.1158/1538-7445.AM2014-3389

Acquired resistance to EGFR inhibitors can be mediated through PI3K/mTOR pathway activation. This has prompted the consideration of PI3K/mTOR inhibitors for treatment of EGFR inhibitor-refractory non-small cell lung cancer (NSCLC). This study aimed to generate and elucidate mechanisms of acquired resistance to BEZ235, a PI3K/mTOR inhibitor, in H1975 NSCLC cells containing the EGFR T790M resistance mutant. IC50 values of compounds were determined at 72 hours using the MTS assay. Acquired resistant clones of H1975 were generated by continual exposure of H1975 cells to parental IC50 concentrations of BEZ235, followed by monoclonal selection. Protein levels, cell invasion/migration, and cell cycle analysis were evaluated using standard techniques. Apoptosis was measured using Roche Cell Death kit. Two resistant clones were generated with stably higher IC50 values (Clone 5 2.29uM, Clone 6 1.94uM) to parental H1975 cells (0.32uM) after 6 months. The clones were resistant to other PI3K/mTOR inhibitors but not other cytotoxic compounds or targeted agents such as EGFR/MEK inhibitors. Following BEZ235 treatment, acquired resistant clones underwent less cell cycle G1 arrest and displayed greater invasion and migration compared to parental cells. No significant differences were seen in basal levels of pRTKs, pAKT, pmTOR, pS6 and p4EBP1, and apoptosis. In summary, EGFR inhibitor-refractory cells with acquired resistance to PI3K/mTOR inhibition have been generated and characterized with phenotypes suggestive of a class-mediated mechanism of acquired resistance. Possible mediators of acquired resistance have emerged following RNA-sequencing and reverse phase protein array analyses and are currently being validated. Citation Format: King Xin Koh, Bhaskar Bhattacharya, Joanne Loh, Hong Kiat Ng, Ross Soo, Barry Iacopetta, Richie Soong. Determinants of acquired resistance to PI3K/mTOR inhibition in cells refractory to EGFR inhibition. [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 5650. doi:10.1158/1538-7445.AM2013-5650

C/EBPα gene expression is frequently lost in non-small cell lung cancer, suggesting it acts as a tumor suppressor. Here, we generated an inducible lung-specific mouse model of C/EBPα deletion (C/EBPαLung-Δ mice) that develops lung adenocarcinomas. We observed that C/EBPα excision results in upregulation of Bmi-1, and that tumor initiation strictly depends on Bmi-1 gene dosage, as C/EBPαLung-Δ mice carrying only one functional Bmi-1 allele escape tumorigenesis. Accordingly, C/EBPα negative human adenocarcinomas show elevated Bmi-1 expression, in line with our observation that C/EBPα acts as tumor suppressor in lung cells by directly inhibiting Bmi-1 transcription. Furthermore, pharmacological inhibition of Bmi-1 impairs the ability of C/EBPα null adenocarcinoma cells to form tumors in xenografts. Overall, we have identified Bmi-1 as a critical therapeutic target in those patients carrying abnormal C/EBPα function. Citation Format: Elena Levantini, Daniela S. Basseres, Wen Cai Zhang, Robert S. Welner, Meritxell Alberich-Jorda’, Kol Jia Yong, Bhavin M. Thakkar, Junyan Zhang, Chiara Battelli, Christopher J. Hetherington, Min Ye, Karen O'Brien, Maria Cristina Magli, Marie Loh, Min En Nga, Yin Huei Pang, Alain C. Borczuk, Lyuba Varticovski, Olivier Kocher, Pu Zhang, Ross A. Soo, Bing Lim, Balazs Halmos, Daniel G. Tenen. C/EBPα acts as tumor suppressor in lung cancer by inhibiting the proto-oncogene Bmi-1. [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 LB-46. doi:10.1158/1538-7445.AM2013-LB-46

The use of tyrosine kinase inhibitors (TKI) to target oncogenic kinases has led to remarkable responses in patients with chronic myeloid leukemia (CML) and EGFR-mutated non-small cell lung cancer (EGFR NSCLC). However, a significant subset of patients have a minimal or very brief response. It has been suggested that germline polymorphisms may account for this upfront TKI resistance, and that identifying such polymorphisms will allow personalization of targeted therapy to achieve optimal responses in patients. Using paired-end DNA sequencing, we discovered a common (12.3% carrier rate) deletion polymorphism in intron 2 of the BIM gene. BIM is a pro-apoptotic member of the BCL2 family of proteins, and is required for TKIs to induce apoptosis in many cancers. We investigated the effects of the polymorphism on BIM function and TKI resistance in CML and EGFR NSCLC. Inspection of BIM gene structure suggested the polymorphism would result in mutually exclusive splicing of exon 3 (E3) and 4 (E4). Importantly, such an event is predicted to affect TKI sensitivity, since the pro-apoptotic BH3 domain is found only in E4. Using minigenes, we confirmed the deletion favored splicing of E3 over E4 by 5-fold (p=0.008), and that the deletion contained a cis-acting splicing suppressor. Next, using Zn finger nuclease-editing, we recreated the polymorphism in TKI sensitive CML (K562) and EGFR NSCLC (PC9) cell lines. Polymorphism-containing subclones had increased E3/E4 transcript ratios, decreased expression of BH3-containing BIM protein and defective apoptotic signaling, and were intrinsically TKI resistant. Importantly, while manipulation of E3-containing transcript levels did not alter the resistance phenotype, pharmacologic restoration of BH3 function (using a BH3 mimetic ABT-737) restored apoptotic signaling as well as TKI-sensitivity. Finally, we determined if the polymorphism predicted for inferior clinical responses in TKI-treated CML and EGFR NSCLC patients. In 203 CML patients, the polymorphism predicted inferior imatinib responses (defined by EuropeanLeukemiaNet criteria) among those with the polymorphism vs those without (odds ratio=2.94, p=0.02, 95% CI 1.17-7.43). In 141 EGFR NSCLC patients, the polymorphism predicted a shorter PFS of 6.6 vs 11.9 months (p=0.0027), and was independently prognostic for poorer PFS (hazard ratio=2.14, p=0.0026, 95% CI 1.30-3.50). In summary, by altering BIM splicing, the BIM polymorphism is sufficient to cause intrinsic TKI resistance in vitro, and predicts inferior TKI responses in patients. Upfront testing of CML and EGFR NSCLC patients for the BIM polymorphism may identify individuals at risk for developing clinical TKI resistance. Our results also offer an explanation for the heterogeneity of TKI responses among CML and EGFR NSCLC patients, and suggest the possibility of personalizing therapy with BH3 mimetics to improve TKI responses. 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 1911. doi:1538-7445.AM2012-1911