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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.

Gene expression levels in H3122, LR, and panobinostat treated LR cells and the promoter or enhancer H3K27 ac gain or loss

Purpose:Although first-line crizotinib treatment leads to clinical benefit in ROS1+ lung cancer, high prevalence of crizotinib-resistant ROS1-G2032R (ROS1G2032R) mutation and progression in the central nervous system (CNS) represents a therapeutic challenge. Here, we investigated the antitumor activity of repotrectinib, a novel next-generation ROS1/TRK/ALK-tyrosine kinase inhibitor (TKI) in ROS1+ patient-derived preclinical models.Experimental Design:Antitumor activity of repotrectinib was evaluated in ROS1+ patient-derived preclinical models including treatment-naïve and ROS1G2032R models and was further demonstrated in patients enrolled in an on-going phase I/II clinical trial (NCT03093116). Intracranial antitumor activity of repotrectinib was evaluated in a brain-metastasis mouse model.Results:Repotrectinib potently inhibited in vitro and in vivo tumor growth and ROS1 downstream signal in treatment-naïve YU1078 compared with clinically available crizotinib, ceritinib, and entrectinib. Despite comparable tumor regression between repotrectinib and lorlatinib in YU1078-derived xenograft model, repotrectinib markedly delayed the onset of tumor recurrence following drug withdrawal. Moreover, repotrectinib induced profound antitumor activity in the CNS with efficient blood–brain barrier penetrating properties. Notably, repotrectinib showed selective and potent in vitro and in vivo activity against ROS1G2032R. These findings were supported by systemic and intracranial activity of repotrectinib observed in patients enrolled in the on-going clinical trial.Conclusions:Repotrectinib is a novel next-generation ROS1-TKI with improved potency and selectivity against treatment-naïve and ROS1G2032R with efficient CNS penetration. Our findings suggest that repotrectinib can be effective both as first-line and after progression to prior ROS1-TKI.

Supplementary table 1

Supplementary Figure S1. Viability of H3122-LR and H2228-LR cells Supplementary Figure S2. Gene set enrichment analysis and western blot Supplementary Figure S3. Effects of AXL inhibition on EMT gene expression in LR cells, related to Fig. 1E. Supplementary Figure S4. Mice with established H3122-derived tumors were treated with four doses (50, 75, 87.5, or 100 mg/kg) of ceritinib to derive ceritinib-resistant tumors Supplementary Figure S5. Enhancer remodeling during ceritinib resistance, related to Fig. 2. Supplementary Figure S6. Genes with H3K27ac loss or gain in their promoters during ceritinib resistance, related to Fig.2.

Supplementary Materials and Methods that contain detailed experimental procedures

Supplementary figure legends S1 to S12

Introduction: Acquired resistance to ALK-tyrosine kinase inhibitors (ALK-TKIs) treatment, particularly target-off resistance, remains a clinical challenge for ALK-rearranged non-small cell lung cancer (NSCLC). To explore novel vulnerabilities of ALK TKI-resistant cancer cells, we focused on their distinct metabolic pathways for growth and survival. Experimental Design: To investigate metabolic pathways in resistance mechanisms, we generated ALK-TKIs -acquired-resistant in vitro/vivo models. We screened metabolite mechanisms using metabolite assay kit, Seahorse Extracellular Flux Analyzer, real-time PCR, western blot, RNA-seq in resistant models. Results: Through an integrated transcriptomic and metabolic assay screening approach, we identified the enhanced reliance on glutamine metabolism in target-off ALK-TKIs-resistant cells. Specifically, resistant cells were characterized by upregulation of glutaminase 1 (GLS1), a mitochondrial enzyme hydrolyzing glutamine into glutamate, simultaneously with downregulation of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this metabolic state intensively accelerates glutaminolysis and subsequent mitochondrial glutamine-derived aspartate synthesis, resulting in TKI resistance by reinforcing antioxidant capacity with increase of NADPH and glutathione. Mechanistically, GLS1 inhibition elicited a marked reduction of cell growth with increase of reactive oxygen species (ROS) in resistant cells, which was restored by supplementation of exogenous aspartate. The antitumor activity of GLS1 inhibition against resistant tumor cells was further validated in in vivo experiments, patient-derived xenograft (PDX) and EML4-ALK transgenic mice. More importantly, glutaminase inhibitor CB-839 enhanced the therapeutic efficacy of anti-PD-L1 treatment in immune checkpoint blockade (ICB)-resistant EML4-ALK transgenic mice. Conclusion: Our findings highlight a new metabolic vulnerability of ALK-TKIs resistant tumors and provide a rationale for targeting GLS1 as a potential treatment option to overcome ALK-TKIs resistance. Citation Format: You Won Lee, Hun Mi Choi, Seung Yeon Oh, Eun Ji Lee, Kyoung-Ho Pyo, Jae Hwan Kim, Youngseon Byeon, Seong Gu Heo, Sun Min Lim, Min Hee Hong, Chang Gon Kim, Hye Ryun Kim, Mi Ran Yun, Byoung Chul Cho. Targeting adaptive metabolic program as a novel treatment approach for TKIs-failed ALK-positive NSCLCs [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 LB544.

Purpose: Although first-line crizotinib treatment leads to clinical benefit in ROS1+ lung cancer, high prevalence of crizotinib-resistant ROS1-G2032R (ROS1G2032R) mutation and progression in the central nervous system (CNS) represents a therapeutic challenge. Here, we investigated the antitumor activity of repotrectinib, a novel next-generation ROS1/TRK/ALK-tyrosine kinase inhibitor (TKI) in ROS1+ patient-derived preclinical models. Experimental Design: Antitumor activity of repotrectinib was evaluated in ROS1+ patient-derived preclinical models including treatment-naïve and ROS1G2032R models and was further demonstrated in patients enrolled in an on-going phase I/II clinical trial (NCT03093116). Intracranial antitumor activity of repotrectinib was evaluated in a brain-metastasis mouse model. Results: Repotrectinib potently inhibited in vitro and in vivo tumor growth and ROS1 downstream signal in treatment-naïve YU1078 compared with clinically available crizotinib, ceritinib, and entrectinib. Despite comparable tumor regression between repotrectinib and lorlatinib in YU1078-derived xenograft model, repotrectinib markedly delayed the onset of tumor recurrence following drug withdrawal. Moreover, repotrectinib induced profound antitumor activity in the CNS with efficient blood–brain barrier penetrating properties. Notably, repotrectinib showed selective and potent in vitro and in vivo activity against ROS1G2032R. These findings were supported by systemic and intracranial activity of repotrectinib observed in patients enrolled in the on-going clinical trial. Conclusions: Repotrectinib is a novel next-generation ROS1-TKI with improved potency and selectivity against treatment-naïve and ROS1G2032R with efficient CNS penetration. Our findings suggest that repotrectinib can be effective both as first-line and after progression to prior ROS1-TKI.

Clinical benefit of ALK tyrosine kinase inhibitors (ALK‐TKIs) in ALK‐rearranged lung cancer has been limited by the inevitable development of acquired resistance, and bypass‐molecular resistance mechanisms remain poorly understood. We investigated a novel therapeutic target through screening FDA‐approved drugs in ALK‐TKI‐resistant models. Cerivastatin, the rate‐limiting enzyme inhibitor of the mevalonate pathway, showed anti‐cancer activity against ALK‐TKI resistance in vitro/in vivo, accompanied by cytoplasmic retention and subsequent inactivation of transcriptional co‐regulator YAP. The marked induction of YAP‐targeted oncogenes (EGFR, AXL, CYR61, and TGFβR2) in resistant cells was abolished by cerivastatin. YAP silencing suppressed tumor growth in resistant cells, patient‐derived xenografts, and EML4‐ALK transgenic mice, whereas YAP overexpression decreased the responsiveness of parental cells to ALK inhibitor. In matched patient samples before/after ALK inhibitor treatment, nuclear accumulation of YAP was mainly detected in post‐treatment samples. High expression of YAP in pretreatment samples was correlated with poor response to ALK‐TKIs. Our findings highlight a crucial role of YAP in ALK‐TKI resistance and provide a rationale for targeting YAP as a potential treatment option for ALK‐rearranged patients with acquired resistance to ALK inhibitors., Acquired resistance to targeted therapy is a major challenge in treatment for ALK‐rearranged NSCLC. The transcriptional activation of YAP induces multiple resistance factors, conferring acquired resistance to ALK‐TKIs. Statins represent proxy inhibitors to overcome YAP‐mediated drug resistance.

Background Preclinical models that can better predict therapeutic activity in clinical trials are needed in this era of personalized cancer treatment. Herein, we established genomically and clinically annotated patient-derived xenografts (PDXs) from non-small-cell lung cancer (NSCLC) patients and investigated whether these PDXs would faithfully recapitulate patient responses to targeted therapy. Methods Patient-derived tumors were implanted in immunodeficient mice and subsequently expanded via re-implantation. Established PDXs were examined by light microscopy, genomic profiling, and in vivo drug testing, and the successful engraft rate was analyzed with the mutation profile, histology, or acquisition method. Finally, the drug responses of PDXs were compared with the clinical responses of the respective patients. Results Using samples from 122 patients, we established 41 NSCLC PDXs [30 adenocarcinoma (AD), 11 squamous cell carcinoma (SQ)], among which the following driver mutation were observed: 13 EGFR-mutant, 4 ALK-rearrangement, 1 ROS1-rearrangement, 1 PIK3CA-mutant, 1 FGFR1-amplification, and 2 KRAS-mutant. We rigorously characterized the relationship of clinical features to engraftment rate and latency rates. The engraft rates were comparable across histologic type. The AD engraft rate tended to be higher for surgically resected tissues relative to biopsies, whereas similar engraft rates was observed for SQ, irrespective of the acquisition method. Notably, EGFR-mutants demonstrated significantly longer latency time than EGFR-WT (86 vs. 37days, P = 0.007). The clinical responses were recapitulated by PDXs harboring driver gene alteration (EGFR, ALK, ROS1, or FGFR1) which regressed to their target inhibitors, suggesting that established PDXs comprise a clinically relevant platform. Conclusion The establishment of genetically and clinically annotated NSCLC PDXs can yield a robust preclinical tool for biomarker, therapeutic target, and drug discovery.

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.

Crizotinib is highly effective in patients with non-small cell lung cancer (NSCLC) harboring the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion. However, its efficacy has been limited by the development of acquired resistance, and the mechanisms of such resistance remain largely unknown. Herein, we investigated a possible candidate for circumventing the acquired resistance to crizotinib. We established a model of acquired resistance to crizotinib (H3122-CR) by exposing EML4-ALK-positive H3122 lung cancer cells to increasing doses of crizotinib, and performed MTT screening using a library of FDA (Food and Drug Administration)-approved drugs composed of a collection of 640 clinically used compounds. Our MTT screening identified that cerivastatin, a drug targeting 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, significantly inhibited the cell growth in H3122-CR cells, which was completely restored by addition of geranylgeranyl pyrophosphate (GGPP), a key metabolite of mevalonate pathway. We also found that yes-associated protein (YAP), a major effector of the Hippo tumor suppressor pathway, was robustly accumulated in nucleus with a concomitant decrement of YAP phosphorylation in H3122-CR cells compared to parental cells. Importantly, inhibition of geranylgeranylation (GGylation) by GGTI-298 or cerivastatin markedly increased YAP phosphorylation, led to cytoplasmic translocation of YAP, and subsequently induced YAP inactivation. Moreover, the enrichment of EGFR signaling pathway and cell cycle signature including transcriptional targets of YAP was enhanced in H3122-CR cells, whose induction contributed YAP-mediated resistance to crizotinib. Inhibition of YAP function with siRNA or verteporfin as a YAP inhibitor greatly abrogated cell growth of H3122-CR cells by modulating cell cycle regulators and EGFR activation. Finally, we confirmed up-regulation of nuclear YAP expression in crizotinib-acquired resistant patient derived tumor xenograft (PDTX) models established from EML4-ALK positive NSCLC patients. Collectively, our findings define the GGylation-mediated YAP transcriptional activation as a new mechanism of resistance to crizotinib, providing a rationale for further exploring statins as a possible anticancer agent to overcome the acquired resistance to crizotinib in EML4-ALK positive NSCLC cells. Citation Format: Miran Yun, Hun Mi Choi, Kyoung Ho Pyo, Byoung Chul Cho. Cerivastatin overcomes the acquired resistance to crizotinib in EML4-ALK positive non-small cell lung cancer by controlling YAP activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2054. doi:10.1158/1538-7445.AM2017-2054

Purpose: Patient-derived cells (PDCs) established from malignant effusions can represent an excellent tool for the study of non-small cell lung cancer (NSCLC) with oncogenic driver mutations. However, because of cellular paucity and low tumor purity, PDCs have a limited utility for in-depth molecular analysis. In this study, we developed a high-purity patient-derived cell (HP-PDC) method for primary culture of malignant effusions. Experimental design: HP-PDCs were established by a multistep tumor cell isolation protocol as follows. 1) Initial cultures of malignant effusions were observed under a light microscope to identify spheroid-forming cells. 2) Tumor purity of PDCs was analyzed by Immunofluorescence (IF) and flow cytometry. 3) Mutation status of EGFR or ALK in PDCs was screened by direct or PCR-based sequencing and RT-PCR and then was compared with driver mutations detected in matching patient tumor samples. 4) HP-PDCs were tested for drug efficacies by a cell viability assay, a colony-forming assay, and western blot. 5) HP-PDCs were screened with a panel of 84 kinase inhibitors to identify effective drugs. Results: A consecutive series of 45 malignant effusion samples was collected from 36 patients with advanced NSCLC. Spheroid-forming cells were detected in 19 samples (42.2%) which were cultured in AR5 media to establish PDCs. IF analysis revealed that TTF-1 was upregulated in 9 PDCs. These 9 PDCs with high TTF-1 expression also showed EpCAM-positive cell population in flow cytometry with high purity of over 70%. Using Sanger sequencing, EGFR exon 19 deletion was detected 3 cases, EGFR T790M with L858R in 2 cases, and ALK rearrangements in 4 cases. Two of PDCs established from patients, who carried EGFR mutations and progressed on gefitinib, showed resistance to gefitinib in vitro. One of gefitinib-resistant PDCs was sensitive to AZD9291 with IC50 value of 47 nM. Three of PDCs established from patients, who carried ALK fusion genes and progressed on ALK inhibitors (crizotinib, alectinib, and ceritinib respectively), also recapitulated patient’s response to the ALK inhibitors. In an alectinib-resistant PDC, crizotinib and ceritinib exhibited potency with IC50 values of 321 nM and 215 nM, respectively. Colony-forming assay and western blot results were correlated with the cell viability assay data. Time spent for establishment of HP-PDCs ranged from 26 days to 132 days. Conclusions: Nine HP-PDCs established by multistep tumor cell isolation protocol had high tumor purity, and recapitulated patient’s genomic profile and drug responses. HP-PDC is a powerful preclinical model which predicts responses to targeted drugs in a short period of time and may provide an excellent platform for personalized therapies in patients with advanced NSCLC. Citation Format: Seok-Young Kim, Hwan Kim, Sung-Moo Kim, Hye Ryun Kim, Mi Ran Yun, Ji Min Lee, Hun Mi Choi, Byoung Chul Cho. High-purity patient-derived cells: An advanced cell culture model for precision cancer medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3835. doi:10.1158/1538-7445.AM2017-3835