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We present the case of a 34-year-old Japanese man with anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer and brain metastases. After central nervous system (CNS) disease progression with alecintib and brigatinib, treatment with lorlatinib resulted in a good intracranial response. In this case, we investigated brain penetration ratio of brigatinib using cerebrospinal fluid and paired serum samples, and the ratio was 0.012. Further, we investigated resistance mechanisms via next-generation sequencing (NGS) using lung biopsy at lung cancer diagnosis and brain biopsy sample at progressive disease of brigatinib. No apparent resistance mechanism of known ALK resistance, such as ALK mutations, amplifications, epithelial-mesenchymal transition (EMT) and bypass pathway activation were detected. Taken together, we speculate that the low CNS penetration rate of brigatinib confers CNS progression. Further studies are warranted to reveal the resistance mechanism and propose a treatment strategy for CNS progression in ALK-positive patients., (© 2024 The Author(s). Thoracic Cancer published by John Wiley & Sons Australia, Ltd.)

Introduction: The emergence of anaplastic lymphoma kinase (ALK) rearrangements in non-small cell lung cancer (NSCLC) has revolutionized targeted therapy. This dynamic landscape, featuring novel ALK inhibitors and combination therapies, necessitates a profound understanding of resistance mechanisms for effective treatment strategies. Recognizing two primary categories - on-target and off-target resistance - underscores the need for comprehensive assessment., Areas Covered: This review delves into the intricacies of resistance to ALK inhibitors, exploring complexities in identification and management. Molecular testing, pivotal for early detection and accurate diagnosis, forms the foundation for patient stratification and resistance management. The literature search methodology involved comprehensive exploration of Pubmed and Embase. The multifaceted perspective encompasses new therapeutic horizons, ongoing clinical trials, and their clinical implications post the recent approval of lorlatinib., Expert Opinion: Our expert opinion encapsulates the critical importance of understanding resistance mechanisms in the context of ALK inhibitors for shaping successful treatment approaches. With a focus on molecular testing and comprehensive assessment, this review contributes valuable insights to the evolving landscape of NSCLC therapy.

The CLIP1-LTK fusion was recently discovered as a novel oncogenic driver in non-small cell lung cancer (NSCLC). Lorlatinib, a third-generation ALK inhibitor, exhibited a dramatic clinical response in a NSCLC patient harboring CLIP1-LTK fusion. However, it is expected that acquired resistance will inevitably develop, particularly by LTK mutations, as observed in NSCLC induced by oncogenic tyrosine kinases treated with corresponding tyrosine kinase inhibitors (TKIs). In this study, we evaluate eight LTK mutations corresponding to ALK mutations that lead to on-target resistance to lorlatinib. All LTK mutations show resistance to lorlatinib with the L650F mutation being the highest. In vitro and in vivo analyses demonstrate that gilteritinib can overcome the L650F-mediated resistance to lorlatinib. In silico analysis suggests that introduction of the L650F mutation may attenuate lorlatinib-LTK binding. Our study provides preclinical evaluations of potential on-target resistance mutations to lorlatinib, and a novel strategy to overcome the resistance., (© 2024. The Author(s).)

Background: ROS1 fusion-positive (ROS1+) nonsmall cell lung cancer (NSCLC) patients are highly sensitive to tyrosine kinase inhibitor (TKI) treatments. However, acquired TKI resistance remains the major hurdle preventing patients from experiencing prolonged benefits., Methods: 107 advanced or metastatic ROS1+ NSCLC patients who progressed on crizotinib and lorlatinib were recruited. Tissue and plasma samples were collected at baseline (N = 50), postcrizotinib (N = 91), and postlorlatinib (N = 21), which were all subject to the 139-gene targeted next-generation DNA sequencing. Molecular dynamics modeling was performed to investigate the effects of ROS1 mutations on binding to different TKIs., Results: In patients with postcrizotinib and postlorlatinib samples, an accumulation of on- and off-target resistance alterations after multiple TKI treatments was observed. ROS1 G2032R and MET amplification were the most common on-target and off-target alterations, respectively. Patients with CD74-ROS1 and SLC34A2-ROS1 had longer progression-free survival (PFS) (P < 0.001) and higher rates of resistance mutations (on-target, P = 0.001; off-target, P = 0.077) than other ROS1 fusion variants following crizotinib treatment. Ten distinct on-target resistance mutations were detected after TKI therapies, of which 4 were previously unreported (ROS1 L2010M, G1957A, D1988N, L1982V). Molecular dynamics simulations showed that all 4 mutations were refractory to crizotinib, while G1957A, D1988N, and L1982V were potentially sensitive to lorlatinib and entrectinib., Conclusions: This study provided a comprehensive portrait of TKI-resistance mechanisms in ROS1+ NSCLC patients. Using in silico simulations of TKI activity, novel secondary mutations that may confer TKI resistance were identified and may support clinical therapeutic decision-making., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Anaplastic lymphoma kinase (ALK) is implicated in the genesis of multiple malignant tumors. Lorlatinib stands out as the most advanced and effective inhibitor currently used in the clinic for the treatment of ALK-positive non-small cell lung cancer. However, resistance to lorlatinib has inevitably manifested over time, with double/triple mutations of G1202, L1196, L1198, C1156 and I1171 frequently observed in clinical practice, and tumors regrow within a short time after treatment with lorlatinib. Therefore, elucidating the mechanism of resistance to lorlatinib is paramount in paving the way for innovative therapeutic strategies and the development of next-generation drugs. In this study, we leveraged multiple computational methodologies to delve into the resistance mechanisms of three specific double mutations of ALK G1202R/L1196M , ALK G1202R/L1198F and ALK I1171N/L1198F to lorlatinib. We analyzed these mechanisms through qualitative (PCA, DCCM) and quantitative (MM/GBSA, US) kinetic analyses. The qualitative analysis shows that these mutations exert minimal perturbations on the conformational dynamics of the structural domains of ALK. The energetic and structural assessments show that the van der Waals interactions, formed by the conserved residue Leu1256 within the ATP-binding site and the residues Glu1197 and Met1199 in the hinge domain with lorlatinib, play integral roles in the occurrence of drug resistance. Furthermore, the US simulation results elucidate that the pathways through which lorlatinib dissociates vary across mutant systems, and the distinct environments during the dissociation process culminate in diverse resistance mechanisms. Collectively, these insights provide important clues for the design of novel inhibitors to combat resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Lorlatinib is an active treatment for advanced anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC) pretreated with ALK-tyrosine kinase inhibitors (-TKIs). However, there is paucity of data on the activity of platinum/pemetrexed chemotherapy administered at the time of progression on lorlatinib. In addition, it is uncertain whether continuation of lorlatinib beyond progression (LBP) would provide any additional clinical benefit. Here, we describe two cases experiencing an exceptional response to platinum/pemetrexed chemotherapy plus LBP and make an attempt to identify which patients' characteristics and biologic profiles of the tumor could predict benefit from such an approach. In this report, presence of controlled brain metastases, rapidly progressing extracranial disease, and presence of ALK-dependent mechanisms of resistance were associated with benefit from platinum/pemetrexed chemotherapy plus lorlatinib beyond progression.

Anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor (TKI) often loses effectiveness against non-small cell lung malignancies (NSCLCs) with ALK gene rearrangements (ALK + ). 19 novel imidazo[1,2-b]pyridazine macrocyclic derivatives were designed, synthesized, and tested for their biological activities in an effort to develop ALK inhibitors that would overcome second-generation ALK-TKIs, particularly the G1202R mutation and the lorlatinib-resistant L1196M/G1202R double mutations. Of all the target substances, O-10 had the most effective enzymatic inhibitory activity, with IC 50 values for ALK WT , ALK G1202R , and ALK L1196M/G1202R of 2.6, 6.4, and 23 nM, respectively. O-10, on the other hand, reduced the growth of ALK-positive Karpas299, BaF3-EML4-ALK G1202R , and BaF3-EML4-ALK L1196M/G1202R cells with IC 50 values of 38, 52, and 64 nM, respectively. This was equally effective to the reference drug Repotrectinib (IC 50  = 40, 164, and 208 nM). The kinase selectivity profile, liver microsome stability test and in vivo pharmacokinetic properties in SD rats of compound O-10 were further evaluated. O-10 was regarded as an effective ALK inhibitor for the treatment of mutations overall., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Purpose: ALK-activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1%-2% of cases. Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single-agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data have suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway., Experimental Design: We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX)., Results: Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX, lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth., Conclusions: In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma., (©2023 The Authors; Published by the American Association for Cancer Research.)

Significance: The combined preclinical features of NVL-520 that include potent targeting of ROS1 and diverse ROS1 resistance mutations, high selectivity for ROS1 G2032R over TRK, and brain penetration mark the development of a distinct ROS1 TKI with the potential to surpass the limitations of earlier-generation TKIs for ROS1 fusion-positive patients. This article is highlighted in the In This Issue feature, p. 517., (©2022 The Authors; Published by the American Association for Cancer Research.)

Purpose: Although lorlatinib, the third generation of echinoderm microtubule protein 4-anaplastic lymphoma kinase (EML4-ALK) tyrosine kinase inhibitor (TKI), overcame the previous generation ALK-TKIs' drug resistance problems, but the mechanism of lorlatinib resistance remained unclear. Furthermore, optimal chemotherapy for lorlatinib-resistant non-small cell lung cancer (NSCLC) patients was still unknown., Methods: A lorlatinib-resistant NSCLC cell line SNU-2535LR was generated by gradually increasing dose of lorlatinib to crizotinib-resistant cell line SNU-2535 in vitro. To study the resistance mechanism of SNU-2535LR cells, we applied CCK-8 assay to detect the sensitivity of crizotinib and the reverse effect of APR-246, a p53 activator, on lorlatinib-induced resistance and different chemotherapy drugs to SNU-2535LR cells. We also detected the expressions of EML4-ALK-related proteins of SNU-2535LR cells via western blot.Please confirm that author names have been identified correctly and are presented in the right order.Dear Editor:     I have carefully confirmed that the author names have been identified correctly and are presented in right order.Thank you very much!                                                                     Your sincerely BoXie RESULTS: The sensitivity of SNU-2535LR cells to lorlatinib was decreased significantly than that of SNU-2535 cells. EML4-ALK fusion was decreased both at protein level and DNA level in SNU-2535LR cells. More interesting, the crizotinib-resistant mutation ALK p.G1269A disappeared, while new TP53 mutation emerged in SNU-2535LR cells. APR-246 can reverse the lorlatinib resistance in SNU-2535LR cells, with a reversal index of 4.768. Compared with SNU-2535 cells, the sensitivity of SNU-2535LR cells to gemcitabine, docetaxel and paclitaxel was significantly increased (P < 0.05), but decreased to cisplatin (P < 0.05)., Conclusion: This study demonstrated that the combination of p53 protein agonist and lorlatinib may provide a new therapeutic strategy for NSCLC patients with lorlatinib resistance and TP53 mutation. Furthermore, the results also provide guidance for selecting optimal chemo-regimens for NSCLC patients after ALK-TKIs failure., (© 2022. The Author(s), under exclusive licence to Federación de Sociedades Españolas de Oncología (FESEO).)

Large-cell neuroendocrine lung carcinoma (LCNEC) is a high-grade neoplasm with median survival of 1 year and limited therapeutic options. Here, we report the unusual case of a 47-yr-old female smoker with stage IV LCNEC featuring EML4 - ALK variant 2 (E20:A20), wild-type TP53/RB1 , and low tumor mutational burden of 3.91 mut/Mb. Despite early progression within 3 mo under crizotinib, a durable response was achieved with alectinib. Oligoprogression in the left breast 10 mo later was treated by surgery, followed by a switch to ceritinib upon multifocal progression and detection of ALK :p.V1180L in the mastectomy specimen, but without success. Another rebiopsy revealed ALK :p.L1196M, but the tumor did not respond to brigatinib or carboplatin/pemetrexed, before stabilization under lorlatinib. Diffuse progression 8 mo later with detection of ALK : p.L1196M/p.G1202R and p.L1196M/ p.D1203N evolving from the previous p.L1196M did not respond to chemoimmunotherapy, and the patient succumbed with an overall survival (OS) of 37 mo. This case illustrates the importance of molecular profiling for LCNEC regardless of smoking status, and the superiority of next-generation ALK inhibitors compared to crizotinib for ALK+ cases. Lorlatinib retained efficacy in the heavily pretreated setting, whereas its upfront use could possibly have prevented the stepwise emergence of compound ALK mutations. Furthermore, the disease course was more aggressive and OS shorter compared to the V2/ TP53 wt ALK+ lung adenocarcinoma, whereas crizotinib, ceritinib, and brigatinib did not confer the benefit expected according to next-generation sequencing results, which also underline the need for more potent drugs against ALK in the high-risk setting of neuroendocrine histology., (© 2022 Wiedemann et al.; Published by Cold Spring Harbor Laboratory Press.)

The third-generation anaplastic lymphoma tyrosine kinase inhibitor (ALK-TKI) lorlatinib has a unique side effect profile that includes hypercholesteremia and hypertriglyceridemia in >80% of lung cancer patients. Here, we tested the hypothesis that lorlatinib might directly promote the accumulation of cholesterol and/or triglycerides in human hepatic cells. We investigated the capacity of the hepatoprotectant silibinin to modify the lipid-modifying activity of lorlatinib. To predict clinically relevant drug−drug interactions if silibinin were used to clinically manage lorlatinib-induced hyperlipidemic effects in hepatic cells, we also explored the capacity of silibinin to interact with and block CYP3A4 activity using in silico computational descriptions and in vitro biochemical assays. A semi-targeted ultrahigh pressure liquid chromatography accurate mass quadrupole time-of-flight mass spectrometry with electrospray ionization (UHPLC-ESI-QTOF-MS/MS)-based lipidomic approach revealed that short-term treatment of hepatic cells with lorlatinib promotes the accumulation of numerous molecular species of cholesteryl esters and triglycerides. Silibinin treatment significantly protected the steady-state lipidome of hepatocytes against the hyperlipidemic actions of lorlatinib. Lipid staining confirmed the ability of lorlatinib to promote neutral lipid overload in hepatocytes upon long-term exposure, which was prevented by co-treatment with silibinin. Computational analyses and cell-free biochemical assays predicted a weak to moderate inhibitory activity of clinically relevant concentrations of silibinin against CYP3A4 when compared with recommended (rosuvastatin) and non-recommended (simvastatin) statins for lorlatinib-associated dyslipidemia. The elevated plasma cholesterol and triglyceride levels in lorlatinib-treated lung cancer patients might involve primary alterations in the hepatic accumulation of lipid intermediates. Silibinin could be clinically explored to reduce the undesirable hyperlipidemic activity of lorlatinib in lung cancer patients.

Lorlatinib, a third-generation ALK tyrosine kinase inhibitor, has been approved as a treatment for ALK-positive lung cancer. This review provides information regarding the pharmacology and clinical features of lorlatinib, including its efficacy and associated adverse events. Pivotal clinical trials are discussed along with the current status of lorlatinib as a treatment for ALK-positive lung cancer and future therapeutic challenges.

Lorlatinib is currently the most advanced, potent and selective anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor for the treatment of ALK-positive non-small cell lung cancer in the clinic; however, diverse compound ALK mutations driving therapy resistance emerge. Here, we determine the spectrum of lorlatinib-resistant compound ALK mutations in patients, following treatment with lorlatinib, the majority of which involve ALK G1202R or I1171N/S/T. We further identify structurally diverse lorlatinib analogs that harbor differential selective profiles against G1202R versus I1171N/S/T compound ALK mutations. Structural analysis revealed increased potency against compound mutations through improved inhibition of either G1202R or I1171N/S/T mutant kinases. Overall, we propose a classification of heterogenous ALK compound mutations enabling the development of distinct therapeutic strategies for precision targeting following sequential tyrosine kinase inhibitors., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Introduction: ROS1-rearranged (ROS1+) non-small-cell lung cancer (NSCLC) is a rare lung cancer with limited treatment options. Phase I-II studies with ROS1-tyrosine kinase inhibitors (TKIs) included small numbers of patients and real-world data are lacking. We investigate the efficacy and safety of lorlatinib, a third-generation TKI targeting ALK and ROS1, in patients with ROS1+ NSCLC treated through an expanded access program., Methods: Consecutive patients with advanced ROS1+ NSCLC treated with lorlatinib between October 2015 and June 2019 were included. Data were collected from medical records. The primary endpoint was progression-free survival., Results: Out of the 80 patients included, 47(59%) were female, 49(62%) never smokers (less than 100 cigarettes over the lifetime), and 68(85%) had stage IV NSCLC at diagnosis. Most frequent histology was adenocarcinoma (95%) and median age was 58.2 years. At the time of lorlatinib initiation, 51(64%) patients had brain metastases and 55(81%) were PS 0-1. Lorlatinib was administered as second/third/fourth/fifth+ line in 29%/28%/18%/26% of patients. All patients previously received at least one ROS1 TKI, and 55(69%) previously received chemotherapy. Median follow-up from lorlatinib initiation was 22.2 months. Median progression-free survival and overall survival from lorlatinib initiation were 7.1 months [95% confidence interval (CI) 5.0-9.9 months] and 19.6 months (95% CI 12.3-27.5 months). Median duration of treatment with lorlatinib was 7.4 months (95% CI 6.5-13.1 months). Overall response and disease control rates were 45% and 82%, respectively. The central nervous system response rate was 72%. Treatment was stopped due to toxicity in 10 patients (13%). The safety profile was consistent with previously published data., Conclusions: Lorlatinib is a major treatment option for advanced refractory ROS1+ NSCLC in treatment strategy., Competing Interests: Role of the funder The funding source had no role in the design, data collection, analysis, or interpretation of the study, or in the preparation of this manuscript. Disclosure SB reports non-financial support from Lilly, GlaxoSmithKline, Roche, Pfizer, personal fees from Roche, Boehringer Ingelheim, grants from Intergroupe Francophone de Cancérologie Thoracique. BB reports grants from Abbvie, Amgen, Aptitude Health, AstraZeneca, BeiGene, Blueprint Medicines, Bristol Myers Squibb (BMS), Boehringer Ingelheim, Celgene, Cergentis, Cristal Therapeutics, Daiichi Sankyo, Eli Lilly, GlaxoSmithKline, Inivata, Janssen, Onxeo, OSE Immunotherapeutics, Pfizer, Roche-Genentech, Sanofi, Takeda, Tolero Pharmaceuticals. DMS reports grants from Pfizer, Roche, AstraZeneca, BMS, Merck Sharp & Dohme (MSD), personal fees from Pfizer, Roche, Takeda, AstraZeneca, Lilly, BMS, MSD, Novartis, Amgen, Abbvie, Becton Dickinson, and non-financial support from Pfizer, Roche, Takeda, AstraZeneca, BMS, MSD. JC reports personal fees from Pfizer, Roche, Takeda, Novartis, AstraZeneca, MSD, BMS, and Boehringer Ingelheim. VW reports honoraria from Roche, AstraZeneca, BMS, MSD and non-financial support from Roche, Pfizer. BR reports grants or contracts from Chugai, consulting fees from BMS, AstraZeneca, Roche, support for attending meetings and/or travel from BMS, Amgen, MSD, Roche. JB reports personal fees for advisory boards and educational symposia from AstraZeneca, Bayer, BMS, MSD, Roche, Daichii, and Servier. All other authors have declared no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Heat shock protein 90 (HSP90) plays an essential role in lung adenocarcinoma, acting as a key chaperone involved in the correct functioning of numerous highly relevant protein drivers of this disease. To this end, HSP90 inhibitors have emerged as promising therapeutic strategies, even though responses to them have been limited to date. Given the need to maximize treatment efficacy, the objective of this study was to use isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic techniques to identify proteins in human lung adenocarcinoma cell lines whose basal abundances were correlated with response to HSP90 inhibitors (geldanamycin and radicicol derivatives). From the protein profiles identified according to response, the relationship between lactate dehydrogenase B (LDHB) and DNA topoisomerase 1 (TOP1) with respect to sensitivity and resistance, respectively, to geldanamycin derivatives is noteworthy. Likewise, rhotekin (RTKN) and decaprenyl diphosphate synthase subunit 2 (PDSS2) were correlated with sensitivity and resistance to radicicol derivatives. We also identified a relationship between resistance to HSP90 inhibition and the p53 pathway by glucose deprivation. In contrast, arginine biosynthesis was correlated with sensitivity to HSP90 inhibitors. Further study of these outcomes could enable the development of strategies to improve the clinical efficacy of HSP90 inhibition in patients with lung adenocarcinoma.

Cancer emergence is associated with cellular adaptations to altered signal transduction mechanisms arbitrated by mutated kinases. Since conventional kinase inhibitors can exhibit certain limitations to such kinase adaptations, overcoming kinase adaptation for cancer treatment gains importance. The cancer chaperone, Hsp90, is implicated in the conformational maturation and functional stabilization of mutated gene products. However, its role in kinase adaptations is not explored in detail. Therefore, the present study aims to understand the mechanisms of Hsp90-dependent kinase adaptation and develop a novel antitumor strategy. We chose malignant human lung cancer cells to demonstrate Hsp90-dependent RAF oncogene adaptation. We show that RAF oncogene adaptations were predominant over wild type RAF and are facilitated by conformation-specific Hsp90. Consequently, the conformation-specific Hsp90 inhibitor, 17AAG, interfered with oncogenic RAF stability and function and inhibited cell proliferation. The enforced cytostasis further triggered premature cellular senescence and acted as an efficient and irreversible tumor suppressor mechanism. Our results also display that oncogenic RAF interactions with Hsp90 require the middle-charged region of the chaperone. Our mice xenografts revealed that 17AAG pretreated tumor cells lost their ability to proliferate and metastasize in vivo. In summary, we demonstrated Hsp90-dependent kinase adaptation in tumor cells and the effect of Hsp90 inhibition in triggering premature senescence to interfere with the tumor progression. Our findings are of both biological relevance and clinical importance., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We have previously identified receptor tyrosine kinase-like orphan receptor 1 (ROR1) as a direct transcriptional target of TTF-1/NKX2-1, a lineage-survival oncogene in lung adenocarcinoma. ROR1 sustains prosurvival signaling from multiple receptor tyrosine kinases including epidermal growth factor receptor, MET, and insulin-like growth factor 1 receptor in part by maintaining the caveolae structure as a scaffold protein of cavin-1 and caveolin-1. In this study, a high throughput screening of the natural product library containing 2560 compounds was undertaken using a cell-based FluoPPI assay detecting ROR1-cavin-1 interaction. As a result, geldanamycin (GA), a known inhibitor of heat shock protein 90 (HSP90), was identified as a potential inhibitor of ROR1. Geldanamycin, as well as two GA derivatives tested in the clinic, 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), decreased ROR1 protein expression. We found that ROR1 physically interacted with HSP90α, but not with other HSP90 paralogs, HSP90β or GRP94. Geldanamycin in turn destabilized and degraded ROR1 protein in a dose- and time-dependent manner through the ubiquitin/proteasome pathway, resulting in a significant suppression of cell proliferation in lung adenocarcinoma cell lines, for which the kinase domain of ROR1, but not its kinase activity or N-glycosylation, was required. Our findings indicate that HSP90 is required to sustain expression of ROR1 crucial for lung adenosarcoma survival, suggesting that inhibition of HSP90 could be a promising therapeutic strategy in ROR1-positive lung adenocarcinoma., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Introduction: Xeroderma pigmentosum complementation group C (XPC) protein is an important DNA damage recognition factor involved in nucleotide excision repair and regulation of non-small-cell lung cancer (NSCLC) cell proliferation and viability. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) blocks ATP binding to heat shock protein 90 (Hsp90), resulting in destabilization of Hsp90-client protein complexes. Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor expressed by many types of tumors. Bevacizumab (Avastin) is a humanized monoclonal antibody against human VEGF used as an antiangiogenesis agent in the therapy of many cancers, proving successful in increasing objective tumor response rate and prolonging overall survival in NSCLC patients., Methods: After the bevacizumab and/or 17-AAG treatment, the expressions of XPC mRNA were determined by quantitative real-time PCR analysis. Protein levels of XPC and phospho-AKT were determined by Western blot analysis. We used specific XPC small interfering RNA and PI3K inhibitor (LY294002) to examine the role of the AKT-XPC signal in regulating the chemosensitivity of bevacizumab and 17-AAG. Cell viability was assessed by the MTS assay and trypan blue exclusion assay., Results: In this study, bevacizumab decreased XPC expression in human lung squamous cell carcinoma H520 and H1703 cells via AKT inactivation. Enhancement of AKT activity by transfection with constitutively active AKT vectors increased XPC expression and cell survival after treatment with bevacizumab. In addition, 17-AAG synergistically enhanced bevacizumab-induced cytotoxicity and cell growth inhibition in H520 and H1703 cells, associated with downregulation of XPC expression and inactivation of AKT., Discussion/conclusion: Together, these results may provide a rationale to combine bevacizumab with Hsp90 inhibitors in future to enhance therapeutic effects for lung cancer., (© 2020 S. Karger AG, Basel.)

Voltage‑dependent anion channel 1 (VDAC1) functions as a porin in the mitochondrial outer membrane (MOM) and plays important roles in mitochondria‑mediated cell apoptosis. VDAC1 interacts with a variety of proteins, such as Bcl‑2 family proteins, hexose kinase (HK), adenine nucleotide translocase (ANT) and α‑tubulin. However, the association between VDAC1 and α‑tubulin, particularly between VDAC1 and acetylated α‑tubulin (Ac‑α‑tubulin), in apoptosis remains unclear. The present study revealed that the heat shock protein 90 inhibitor, tanespimycin, induced VDAC1 upregulation and α‑tubulin acetylation during Calu‑1 cell apoptosis in human lung cancer. Hsp90 mediated the expression level of VDAC1, and the acetylation of α‑tubulin was enhanced in an α‑tubulin acetyltransferase 1 (αTAT1)‑dependent manner following an increase in VDAC1 expression. Docetaxel, as an inhibitor of microtubules, augmented the expression of Ac‑α‑tubulin, VDAC1 and Bax induced by tanespimycin and increased the degree of caspase activation. Immunoprecipitation (IP) experiments revealed that Ac‑α‑tubulin, α‑tubulin and VDAC1 were co‑precipitated in the IP complex, in which α‑tubulin expression was decreased and VDAC1 proteins were oligomerized, and that the p‑AKT/glycogen synthase kinase 3β (GSK3β) signalling pathway mediated the opening of VDAC1. Therefore, it can be asserted that the acetylation of α‑tubulin and VDAC1 upregulation or oligomerization induced by tanespimycin may lead to mitochondrial permeability and consequently induce the apoptosis of lung cancer cells. These findings provide evidence for the use of a combination of drugs that target VDAC1 and tubulin to induce tumour cell apoptosis.

Purpose: Most ALK -positive lung cancers will develop ALK-independent resistance after treatment with next-generation ALK inhibitors. MET amplification has been described in patients progressing on ALK inhibitors, but frequency of this event has not been comprehensively assessed., Experimental Design: We performed FISH and/or next-generation sequencing on 207 posttreatment tissue ( n = 101) or plasma ( n = 106) specimens from patients with ALK-positive lung cancer to detect MET genetic alterations. We evaluated ALK inhibitor sensitivity in cell lines with MET alterations and assessed antitumor activity of ALK/MET blockade in ALK-positive cell lines and 2 patients with MET-driven resistance., Results: MET amplification was detected in 15% of tumor biopsies from patients relapsing on next-generation ALK inhibitors, including 12% and 22% of biopsies from patients progressing on second-generation inhibitors or lorlatinib, respectively. Patients treated with a second-generation ALK inhibitor in the first-line setting were more likely to develop MET amplification than those who had received next-generation ALK inhibitors after crizotinib ( P = 0.019). Two tumor specimens harbored an identical ST7-MET rearrangement, one of which had concurrent MET amplification. Expressing ST7-MET in the sensitive H3122 ALK-positive cell line induced resistance to ALK inhibitors that was reversed with dual ALK/MET inhibition. MET inhibition resensitized a patient-derived cell line harboring both ST7-MET and MET amplification to ALK inhibitors. Two patients with ALK-positive lung cancer and acquired MET alterations achieved rapid responses to ALK/MET combination therapy., Conclusions: Treatment with next-generation ALK inhibitors, particularly in the first-line setting, may lead to MET-driven resistance. Patients with acquired MET alterations may derive clinical benefit from therapies that target both ALK and MET., (©2020 American Association for Cancer Research.)

Introduction : The treatment of lung cancer has changed dramatically with the development of tyrosine kinase inhibitors (TKIs) that target sensitizing somatic (gene) activations including anaplastic lymphoma kinase (ALK)-rearrangements. Despite remarkable initial responses, patients develop progressive disease via various resistance mechanisms, some of which are ALK dependent. Various next-generation ALK TKIs have been developed to improve on central nervous system (CNS) activity and also target the multitude of acquired resistance mechanisms. Of these, lorlatinib has the greatest spectrum of clinical activity against multiple ALK resistance mutations and has also demonstrated promising efficacy in patients with known brain metastases. Areas covered : We discuss the structure, pharmacology and efficacy of lorlatinib and also provide future perspectives in the management of patients with ALK-rearranged non-small cell lung cancer (NSCLC). Expert opinion : Patients invariably develop resistance during treatment with lorlatinib. Unique combinations of ALK resistance mutations may confer sensitivity to alternate ALK TKIs. There is a move toward individualized biomarker-driven treatment strategies to identify the select group of candidates that can benefit from existing therapies.

Purpose: Lorlatinib is a third-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor with proven efficacy in patients with ALK-rearranged lung cancer previously treated with first- and second-generation ALK inhibitors. Beside compound mutations in the ALK kinase domain, other resistance mechanisms driving lorlatinib resistance remain unknown. We aimed to characterize the mechanisms of resistance to lorlatinib occurring in patients with ALK -rearranged lung cancer and design new therapeutic strategies in this setting., Experimental Design: Resistance mechanisms were investigated in 5 patients resistant to lorlatinib. Longitudinal tumor biopsies were studied using high-throughput next-generation sequencing. Patient-derived models were developed to characterize the acquired resistance mechanisms, and Ba/F3 cell mutants were generated to study the effect of novel ALK compound mutations. Drug combinatory strategies were evaluated in vitro and in vivo to overcome lorlatinib resistance., Results: Diverse biological mechanisms leading to lorlatinib resistance were identified. Epithelial-mesenchymal transition (EMT) mediated resistance in two patient-derived cell lines and was susceptible to dual SRC and ALK inhibition. We characterized three ALK kinase domain compound mutations occurring in patients, L1196M/D1203N, F1174L/G1202R, and C1156Y/G1269A, with differential susceptibility to ALK inhibition by lorlatinib. We identified a novel bypass mechanism of resistance caused by NF2 loss-of-function mutations, conferring sensitivity to treatment with mTOR inhibitors., Conclusions: This study shows that mechanisms of resistance to lorlatinib are diverse and complex, requiring new therapeutic strategies to tailor treatment upon disease progression., (©2019 American Association for Cancer Research.)

Heat shock protein 90 (HSP90) is an important chaperone in lung adenocarcinoma, with relevant protein drivers such as EGFR (epidermal growth factor receptor) and EML4-ALK (echinoderm microtubule-associated protein-like protein4 fused to anaplastic lymphoma kinase) depending on it for their correct function, therefore HSP90 inhibitors show promise as potential treatments for lung adenocarcinoma. To study responses to its inhibition, HSP90 was pharmacologically interrupted by geldanamycin and resorcinol derivatives or with combined inhibition of HSP90 plus HSP70 in lung adenocarcinoma cell lines. Two-dimensional electrophoresis was performed to identify proteomic profiles associated with inhibition which will help to understand the biological basis for the responses. HSP90 inhibition resulted in altered protein profiles that differed according the treatment condition studied. Results revealed 254 differentially expressed proteins after treatments, among which, eukaryotic translation initiation factor3 subunit I (eIF3i) and citrate synthase demonstrated their potential role as response biomarkers. The differentially expressed proteins also enabled signalling pathways involved in responses to be identified; these included apoptosis, serine-glycine biosynthesis and tricarboxylic acid cycle. The proteomic profiles identified here contribute to an improved understanding of HSP90 inhibition and open possibilities for the detection of potential response biomarkers which will be essential to maximize treatment efficacy in lung adenocarcinoma., Competing Interests: The authors declare that they have no competing interests

Lung cancer causes the highest number of cancer-related deaths worldwide. Resistance to therapy is a major clinical issue contributing to the poor prognosis of lung cancer. In recent years, targeted therapy has become a concept where subgroups of non-small cell lung cancer (NSCLC) with genetically altered receptor tyrosine kinases are targeted by tyrosine kinase inhibitors (TKIs). One such subgroup harbors a gene fusion of echinoderm microtubule-associated protein-like 4 (EML4) with anaplastic lymphoma kinase (ALK). Although most NSCLC patients with EML4-ALK fusions initially respond to ALK TKI-therapy they eventually develop resistance. While ALK kinase domain mutations contribute to ALK TKI-refractoriness, they are only present in a fraction of all ALK TKI-resistant tumors. In this study we sought to explore a possible involvement of microRNAs (miRNAs) in conferring resistance to ALK TKIs in ALK TKI-refractory NSCLC cell lines. We subjected our ALK TKI-refractory cancer cells along with parental cancer cells to systematic miRNA expression arrays. Furthermore, ALK TKI-refractory cancer cells were exposed to a synthetic miRNA inhibitory Locked Nucleic Acid (LNA)-library in the presence of ALK TKIs Crizotinib or Lorlatinib. The outcome of the combined approaches uncovered miR-100-5p to confer resistance to Crizotinib and Lorlatinib in EML4-ALK NSCLC cells and to be a potential therapeutic target in drug resistance., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction: Approximately 3-5% of patients with non-small cell lung cancer （NSCLC）belonged to anaplastic lymphoma kinase (ALK)-positive NSCLC. The treatment drugs of ALK-positive NSCLC mainly included crizotinib, ceritinib, alectinib, and brigatinib. Although these drugs had some effects, most of them were usually easy to develop drug resistance. Lorlatinib is a new inhibitor of ALK for treating ALK-positive NSCLC,the effect is obvious, and not easy to develop resistance. Areas covered: The main mechanism of action, pharmacokinetics, clinical efficacy and safety of lorlatinib were introduced in this paper. Expert commentary: Lorlatinib is a new, reversible, ATP-competitive small molecule inhibitor of ALK and c-ros oncogene 1 (ROS1). It can inhibit tumor cell growth in ALK- and ROS1-overexpressing tumor cells. Clinical trial indicated that lorlatinib had obvious therapeutic effect for patients with ALK-positive NSCLC. Lorlatinib could also pass through the blood-brain barrier, which had a good effect on patients with brain metastasis. Adverse events of lorlatinib were mostly mild and moderate in severity, and patients were easily tolerated. Most common adverse events were edema, peripheral neuropathy, cognitive effects, dyspnea, fatigue, weight gain, arthralgia, mood effects, and diarrhea.

: Targeted therapy changed the standard of care in ALK-dependent tumors. However, resistance remains a major challenge. Lorlatinib is a third-generation ALK inhibitor that inhibits most ALK mutants resistant to current ALK inhibitors. In this study, we utilize lorlatinib-resistant anaplastic large cell lymphoma (ALCL), non-small cell lung cancer (NSCLC), and neuroblastoma cell lines in vitro and in vivo to investigate the acquisition of resistance and its underlying mechanisms. ALCL cells acquired compound ALK mutations G1202R/G1269A and C1156F/L1198F in vitro at high drug concentrations. ALCL xenografts selected in vivo showed recurrent N1178H (5/10 mice) and G1269A (4/10 mice) mutations. Interestingly, intracellular localization of NPM/ALKN 1178H skewed toward the cytoplasm in human cells, possibly mimicking overexpression. RNA sequencing of resistant cells showed significant alteration of PI3K/AKT and RAS/MAPK pathways. Functional validation by small-molecule inhibitors confirmed the involvement of these pathways in resistance to lorlatinib. NSCLC cells exposed in vitro to lorlatinib acquired hyperactivation of EGFR, which was blocked by erlotinib to restore sensitivity to lorlatinib. In neuroblastoma, whole-exome sequencing and proteomic profiling of lorlatinib-resistant cells revealed a truncating NF1 mutation and hyperactivation of EGFR and ErbB4. These data provide an extensive characterization of resistance mechanisms that may arise in different ALK-positive cancers following lorlatinib treatment. SIGNIFICANCE: High-throughput genomic, transcriptomic, and proteomic profiling reveals various mechanisms by which multiple tumor types acquire resistance to the third-generation ALK inhibitor lorlatinib., (©2018 American Association for Cancer Research.)

Anaplastic lymphoma kinase (ALK) rearrangements were first implicated as driving mutations in non-small cell lung cancer in 2007. Since then, a number of novel, small-molecule inhibitors directed against the ALK receptor have demonstrated superiority over standard chemotherapies in the treatment of ALK rearrangement-positive lung cancer. Of considerable importance when considering such therapies is the ability of each to overcome mutations conferring acquired resistance, as well as penetrate the central nervous system (CNS), the most common site of metastasis and traditionally the most difficult to breach. Herein is a review of the efficacy, indications, and degree of CNS penetration for the ALK-targeting agents crizotinib, ceretinib, alectinib, brigatinib, and lorlatinib, as well as a summary of ongoing clinical trials comparing these drugs., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The cornerstone of treatment for advanced ALK-positive lung cancer is sequential therapy with increasingly potent and selective ALK inhibitors. The third-generation ALK inhibitor lorlatinib has demonstrated clinical activity in patients who failed previous ALK inhibitors. To define the spectrum of ALK mutations that confer lorlatinib resistance, we performed accelerated mutagenesis screening of Ba/F3 cells expressing EML4-ALK. Under comparable conditions, N -ethyl- N -nitrosourea (ENU) mutagenesis generated numerous crizotinib-resistant but no lorlatinib-resistant clones harboring single ALK mutations. In similar screens with EML4-ALK containing single ALK resistance mutations, numerous lorlatinib-resistant clones emerged harboring compound ALK mutations. To determine the clinical relevance of these mutations, we analyzed repeat biopsies from lorlatinib-resistant patients. Seven of 20 samples (35%) harbored compound ALK mutations, including two identified in the ENU screen. Whole-exome sequencing in three cases confirmed the stepwise accumulation of ALK mutations during sequential treatment. These results suggest that sequential ALK inhibitors can foster the emergence of compound ALK mutations, identification of which is critical to informing drug design and developing effective therapeutic strategies. Significance: Treatment with sequential first-, second-, and third-generation ALK inhibitors can select for compound ALK mutations that confer high-level resistance to ALK-targeted therapies. A more efficacious long-term strategy may be up-front treatment with a third-generation ALK inhibitor to prevent the emergence of on-target resistance. Cancer Discov; 8(6); 714-29. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 663 ., (©2018 American Association for Cancer Research.)

Non-small cell lung cancers (NSCLC) harboring anaplastic lymphoma kinase (ALK) gene rearrangements invariably develop resistance to 2 nd -generation ALK inhibitors. Lorlatinib (PF-06463922) (6) is a 3 rd -generation macrocyclic ALK-TKI that demonstrates many advantages over 2 nd -generation ALK inhibitors. Lorlatinib has demonstrated decent kinase selectivity, promising pharmacokinetic profile, selective brain-penetration and strong antiproliferative activity in several ALK/ROS1-driven tumor models. The current review describes the activity spectrum, key events from discovery to clinical applications and the evidences that lorlatinib acts as an ALK/ROS1 inhibitor in clinical settings., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Advanced, anaplastic lymphoma kinase (ALK)-positive lung cancer is currently treated with the first-generation ALK inhibitor crizotinib followed by more potent, second-generation ALK inhibitors (e.g., ceritinib and alectinib) upon progression. Second-generation inhibitors are generally effective even in the absence of crizotinib-resistant ALK mutations, likely reflecting incomplete inhibition of ALK by crizotinib in many cases. Herein, we analyzed 103 repeat biopsies from ALK-positive patients progressing on various ALK inhibitors. We find that each ALK inhibitor is associated with a distinct spectrum of ALK resistance mutations and that the frequency of one mutation, ALK G1202R , increases significantly after treatment with second-generation agents. To investigate strategies to overcome resistance to second-generation ALK inhibitors, we examine the activity of the third-generation ALK inhibitor lorlatinib in a series of ceritinib-resistant, patient-derived cell lines, and observe that the presence of ALK resistance mutations is highly predictive for sensitivity to lorlatinib, whereas those cell lines without ALK mutations are resistant., Significance: Secondary ALK mutations are a common resistance mechanism to second-generation ALK inhibitors and predict for sensitivity to the third-generation ALK inhibitor lorlatinib. These findings highlight the importance of repeat biopsies and genotyping following disease progression on targeted therapies, particularly second-generation ALK inhibitors. Cancer Discov; 6(10); 1118-33. ©2016 AACRSee related commentary by Qiao and Lovly, p. 1084This article is highlighted in the In This Issue feature, p. 1069., (©2016 American Association for Cancer Research.)

Similarities and differences in the cell cycle components, apoptosis and cytoskeleton-related molecules among mouse skin fibroblast cells (MSFs), mouse squamous cell lung carcinomas (SqCLCs) and mouse embryonic stem cells (mESCs) are important determinants of the behaviour and differentiation capacity of these cells. To reveal apoptotic pathways and to examine the distribution and the role of cell cycle-cell skeleton comparatively would necessitate tumour biology and stem cell biology to be assessed together in terms of oncogenesis and embryogenesis. The primary objectives of this study are to investigate the effects of flavopiridol, a cell cycle inhibitor, and geldanamycin, a heat shock protein inhibitor on mouse somatic, tumour and embryonic stem cells, by specifically focusing on alterations in cytoskeletal proteins, cell polarity and motility as well as cell cycle regulators. To meet these objectives, expression of several genes, cell cycle analysis and immunofluorescence staining of intracellular cytoskeletal molecules were performed in untreated and flavopiridol- or geldanamycin-treated cell lines. Cytotoxicity assays showed that SqCLCs are more sensitive to flavopiridol than MSFs and mESCs. Keratin-9 and keratin-2 expressions increased dramatically whereas cell cycle regulatory genes decreased significantly in the flavopiridol-treated MSFs. Flavopiridol-treated SqCLCs displayed a slight increase in several cell cytoskeleton regulatory genes as well as cell cycle regulatory genes. However, gene expression profiles of mESCs were not affected after flavopiridol treatment except the Cdc2a. Cytotoxic concentrations of geldanamycin were close to each other for all cell lines. Cdkn1a was the most increased gene in the geldanamycin-treated MSFs. However, expression levels of cell cytoskeleton-associated genes were increased dramatically in the geldanamycin-treated SqCLCs. Our results revealing differences in molecular mechanisms between embryogenesis and carcinogenesis may prove crucial in developing novel therapeutics that specifically target cancer cells.

Small-cell lung cancer (SCLC) accounts for nearly 15% of lung cancer cases and exhibits aggressive clinical behavior characterized by rapid growth and metastatic spread to multiple organs. About 70% of patients with SCLC present with extensive disease and distant metastases at diagnosis. HSP90 is a 90-kDa molecular chaperone whose association is required for the stability and function of its numerous "client proteins." Here, we assessed the therapeutic potential of the HSP90 inhibitor 17-DMAG in SCLC. Notably, 17-DMAG hindered the viability of human SCLC cell lines-regardless of their chemosensitivity-via the decreased expression of client proteins, including the proto-oncogene c-Raf (also known as RAF1). In an in vivo imaging model of SCLC multiple-organ metastasis with the human SCLC cell line SBC-5, treatment with 17-DMAG remarkably inhibited the formation of metastatic sites in the liver, but was ineffective in hindering the progression of bone lesions. The latter was likely the result of activation of osteoclasts. IGF-1, which is supposed to be rich in bone environment, preserved c-Raf expression and maintained viability of SBC-5 cells treated with 17-DMAG. Furthermore, the combined use of a bisphosphonate with 17-DMAG significantly attenuated the progression of metastases in both the liver and the bone. These findings suggest that therapeutic effects of HSP90 inhibitors may be organ-specific and should be carefully monitored in SCLC clinical trials., (© 2015 UICC.)

PS-341 is a highly selective and potent proteasome inhibitor that is cytotoxic to various types of cancer. However, no objective response was seen in a clinical trial with PS-341 as a single agent in non-small cell lung cancer. Its antitumor activity is limited by the simultaneously activated antiapoptosis pathway. Recently, PS-341-induced NF-κB activation via IκBα degradation has been suggested to be one of its antiapoptotic effects. In this study, we investigated the effects of a combined application of the heat shock protein (Hsp) 90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) with PS-341 in lung cancer cells. Hsp90 inhibition with 17-AAG was effective in enhancing PS-341-induced lung cancer cell death in vitro and in vivo. 17-AAG pretreatment induced the degradation of upstream regulators of IκB, IL-1R-associated kinase-1 (IRAK-1), and IκB kinases (IKKs), dose and time dependently, which resulted in blocking of PS-341-induced IκBα degradation, p65 nuclear translocation, transcriptional activity, and NF-κB-regulated antiapoptotic gene expressions such as COX-2. The concentrations of 17-AAG used for combinatorial treatment with PS-341 did not change cell viability or the activity of proteasome complex. Moreover, 17-AAG pretreatment decreased the level of phsophorylated Akt at serine 473 residue and suppressed active Akt-dependent inactivation of glycogen synthase kinase 3β. 17-AAG mediated the dissociation of its client proteins (IRAK-1, IKKs, and Akt) from the Hsp90 complex. As a result, it induced degradation of target proteins. Our results suggest that the combination of 17-AAG and PS-341 could be an effective anticancer therapy that overcomes the limited effects of PS-341.

Background: Because anaplastic lymphoma kinase (ALK) is dependent on Hsp90 for protein stability, Hsp90 inhibitors are effective in controlling growth of lung cancer cells with ALK rearrangement. We investigated the mechanism of acquired resistance to 17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), a geldanamycin analogue Hsp90 inhibitor, in H3122 and H2228 non-small cell lung cancer cell lines with ALK rearrangement., Methods: Resistant cell lines (H3122/DR-1, H3122/DR-2 and H2228/DR) were established by repeated exposure to increasing concentrations of 17-DMAG. Mechanisms for resistance by either NAD(P)H/quinone oxidoreductase 1 (NQO1), previously known as a factor related to 17-DMAG resistance, or P-glycoprotein (P-gp; ABCB1/MDR1) were queried using RT-PCR, western blot analysis, chemical inhibitors, the MTT cell proliferation/survival assay, and cellular efflux of rhodamine 123., Results: The resistant cells showed no cross-resistance to AUY922 or ALK inhibitors, suggesting that ALK dependency persists in cells with acquired resistance to 17-DMAG. Although expression of NQO1 was decreased in H3122/DR-1 and H3122/DR-2, NQO1 inhibition by dicumarol did not affect the response of parental cells (H2228 and H3122) to 17-DMAG. Interestingly, all resistant cells showed the induction of P-gp at the protein and RNA levels, which was associated with an increased efflux of the P-gp substrate rhodamine 123 (Rho123). Transfection with siRNA directed against P-gp or treatment with verapamil, an inhibitor of P-gp, restored the sensitivity to the drug in all cells with acquired resistance to 17-DMAG. Furthermore, we also observed that the growth-inhibitory effect of 17-DMAG was decreased in A549/PR and H460/PR cells generated to over-express P-gp by long-term exposure to paclitaxel, and these cells recovered their sensitivity to 17-DMAG through the inhibition of P-gp., Conclusion: P-gp over-expression is a possible mechanism of acquired resistance to 17-DMAG in cells with ALK rearrangement.

Hsp90 inhibitors have become well-studied antitumor agents for their selective property against tumors versus normal cells. The combined treatment of Hsp90 inhibitor and conventional photon radiation also showed more effective tumor growth delay than radiation alone. However, little is known regarding the combined treatment of Hsp90 inhibitor and heavy-ion irradiation. In this study, SQ5 human lung tumor cells were used in vitro for clonogenic cell survival and in vivo for tumor growth delay measurement using a mouse xenograft model after 17-allylamino-17-demethoxygeldanamycin (17AAG) pretreatment and carbon ion irradiation. Repair of DNA double strand breaks (DSBs) was also assessed along with expressions of DSB repair-related proteins. Cell cycle analysis after the combined treatment was also performed. The combined treatment of 17AAG and carbon ions revealed a promising treatment option in both in vitro and in vivo studies. One likely cause of this effectiveness was shown to be the inhibition of homologous recombination repair by 17AAG. The more intensified G2 cell cycle delay was also associated with the combined treatment when compared with carbon ion treatment alone. Our findings indicate that the combination of Hsp90 inhibition and heavy-ion irradiation provides a new effective therapeutic alternative for treatment of solid tumors., (© 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

The large tumour suppressor 1 (LATS1) signalling network has been proved to be an essential regulator within the cell, participating in multiple cellular phenotypes. However, it is unclear concerning the clinical significance of LATS1 and the regulatory mechanisms of 17-Allylamino-17- demethoxygeldanamycin (17-AAG) in lung adenocarcinoma (LAC). The aim of the present study was to investigate the correlation of LATS1 and yes-associated protein (YAP) expression with clinicopathological characteristics in LAC patients, and the effects of 17-AAG on biological behaviours of LAC cells. Subcutaneous LAC tumour models were further established to observe the tumour growth in nude mice. The results showed that the positive expression of LATS1 was significantly lowered (26.7% versus 68.0%, P < 0.001), while that of YAP was elevated (76.0% versus 56.0%, P = 0.03) in LAC tissues compared to the adjacent non-cancerous tissues; LAST1 expression was negatively correlated with YAP expression (r = 0.432, P < 0.001) and lymphatic invasion of the tumour (P = 0.015). In addition, 17-AAG inhibited proliferation and invasion, and induced cell apoptosis and cycle arrest in LAC cells together with increased expression of E-cadherin and p-LATS1, and decreased expression of YAP and connective tissue growth factor. Tumour volumes and weight were much smaller in 17-AAG-treated groups than those in untreated group (P < 0.01). Taken together, our findings indicate that decreased expression of LATS1 is associated with lymphatic invasion of LAC, and 17-AAG suppresses growth and invasion of LAC cells via regulation of the LATS1/YAP pathway in vitro and in vivo, suggesting that we may provide a promising therapeutic strategy for the treatment of human LAC., (© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

HSP90 inhibitors are currently undergoing clinical evaluation in combination with antimitotic drugs in non-small cell lung cancer (NSCLC), but little is known about the cellular effects of this novel drug combination. Therefore, we investigated the molecular mechanism of action of IPI-504 (retaspimycin HCl), a potent and selective inhibitor of HSP90, in combination with the microtubule targeting agent (MTA) docetaxel, in preclinical models of NSCLC. We identified a subset of NSCLC cell lines in which these drugs act in synergy to enhance cell death. Xenograft models of NSCLC demonstrated tumor growth inhibition, and in some cases, regression in response to combination treatment. Treatment with IPI-504 enhanced the antimitotic effects of docetaxel leading to the hypothesis that the mitotic checkpoint is required for the response to drug combination. Supporting this hypothesis, overriding the checkpoint with an Aurora kinase inhibitor diminished the cell death synergy of IPI-504 and docetaxel. To investigate the molecular basis of synergy, an unbiased stable isotope labeling by amino acids in cell culture (SILAC) proteomic approach was employed. Several mitotic regulators, including components of the ubiquitin ligase, anaphase promoting complex (APC/C), were specifically down-regulated in response to combination treatment. Loss of APC/C by RNAi sensitized cells to docetaxel and enhanced its antimitotic effects. Treatment with a PLK1 inhibitor (BI2536) also sensitized cells to IPI-504, indicating that combination effects may be broadly applicable to other classes of mitotic inhibitors. Our data provide a preclinical rationale for testing the combination of IPI-504 and docetaxel in NSCLC.

Lung cancer remains the most common cause of cancer-related death worldwide. This malignancy is a complex disease, and it is important to identify potential biological targets, the blockade of which would affect multiple downstream signaling cascades. A growing number of reports recognize novel therapeutic targets in the protein homeostasis network responsible for generating and protecting the protein fold. The heat shock protein 90 (Hsp90) is an essential molecular chaperon involved in the posttranslational folding and stability of proteins. It is required for conformational maturation of multiple oncogenic kinases that drive signal transduction and proliferation of cancer cells. However, in the case of unfolded protein accumulation endoplasmic reticulum (ER) stress is induced and several response pathways such as proteasome functions are activated. The ubiquitin-proteasome system orchestrates the turnover of innumerable cellular proteins. Here, we suggest that the therapeutic efficacy of Hsp90 inhibition may be augmented by coadministering proteasome inhibitor on human non-small-cell lung cancer (NSCLC) cell lines. Indeed, we showed that coadministration of the Hsp90 inhibitor 17-demethoxygeldanamycin (17-DMAG) and proteasome inhibitor (velcade) induced ER stress evidenced by increased unfolded protein response markers. The consequences were evident in multiple aspects of the NSCLC phenotype: reduced viability and cell count, increased apoptotic cell death, and most profoundly, synergistically decreased cell motility. Our findings provide proof-of-concept that targeting ER homeostasis is therapeutically beneficial in NSCLC cell lines.

Although epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have been introduced for the treatment of non-small cell lung cancer (NSCLC), the emergence of secondary T790M mutation in EGFR or amplification of the Met proto-oncogene restrain the clinical success of EGFR-TKIs. Since heat shock protein-90 (Hsp90) stabilizes various oncoproteins including EGFR and c-Met, the inhibition of Hsp90 activity appears as a rational strategy to develop anticancer drugs. Despite preclinical efficacy of geldanamycin-anasamycin (GA)-derivatives containing benzoquinone moiety as Hsp90 inhibitors, the hepatotoxicity of these GA-derivatives restricts their therapeutic benefit. We have prepared WK-88 series of GA-derivatives, which lack the benzoquinone moiety. In this study, we have examined the anticancer effects of WK88-1 in Met-amplified- and gefitinib-resistant (HCC827GR) NSCLC cells and its parental HCC827 cells. Treatment with WK88-1 reduced the cell viability in both HCC827 and HCC827GR cells, which was associated with marked decrease in the constitutive expression of Hsp90 client proteins, such as EGFR, ErbB2, ErbB3, Met and Akt. Moreover, WK88-1 attenuated phosphorylation of these Hsp90 client proteins and reduced the anchorage-independent growth of HCC827GR cells. Administration of WK88-1 did not cause hepatotoxicity in animals and significantly reduced the growth of HCC827GR cells xenograft tumors in nude mice. Our study provides evidence that ErbB3 might be a client for Hsp90 in Met-amplified NSCLCs. In conclusion, we demonstrate that inhibition of Hsp90 dampens the activation of EGFR- or c-Met-mediated survival of Met-amplified NSCLCs and that WK88-1 as a Hsp90 inhibitor alleviates gefitinib resistance in HCC827GR cells., (© 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

Background: Up-regulation of hsp90 gene expression occurs in numerous cancers such as lung cancer. D,L-lactic-co-glycolic acid-poly ethylene glycol-17-dimethylaminoethylamino-17-demethoxy geldanamycin (PLGA-PEG-17DMAG) complexes and free 17-DMAG may inhibit the expression. The purpose of this study was to examine whether nanocapsulating 17DMAG improves the anti cancer effect over free 17DMAG in the A549 lung cancer cell line., Materials and Methods: Cells were grown in RPMI 1640 supplemented with 10% FBS. Capsulation of 17DMAG is conducted through double emulsion, then the amount of loaded drug was calculated. Other properties of this copolymer were characterized by Fourier transform infrared spectroscopy and H nuclear magnetic resonance spectroscopy. Assessment of drug cytotoxicity on the grown of lung cancer cell line was carried out through MTT assay. After treatment, RNA was extracted and cDNA was synthesized. In order to assess the amount of hsp90 gene expression, real-time PCR was performed., Results: In regard to the amount of the drug load, IC50 was significant decreased in nanocapsulated(NC) 17DMAG in comparison with free 17DMAG. This was confirmed through decrease of HSP90 gene expression by real-time PCR., Conclusions: The results demonstrated that PLGA-PEG-17DMAG complexes can be more effective than free 17DMAG in down-regulating of hsp90 expression by enhancing uptake by cells. Therefore, PLGA-PEG could be a superior carrier for this kind of hydrophobic agent.

Inhibition of heat shock protein 90 (Hsp90) can lead to degradation of multiple client proteins, which are involved in tumor progression. Epidermal growth factor receptor (EGFR) is one of the most potent oncogenic client proteins of Hsp90. Targeted inhibition of EGFR has shown clinical efficacy in the treatment of patients with non-small-cell lung cancer (NSCLC). However, primary and acquired resistance to the existing EGFR inhibitors is a major clinical problem. In the present study, we investigated the effect of the novel Hsp90 inhibitor CH5164840 on the antitumor activity of erlotinib. The NSCLC cell lines and xenograft models were treated with CH5164840 and erlotinib to examine their mechanisms of action and cell growth inhibition. We found that CH5164840 showed remarkable antitumor activity against NSCLC cell lines and xenograft models. The addition of CH5164840 enhanced the antitumor activity of erlotinib against NCI-H292 EGFR-overexpressing xenograft models. Phosphorylation of Stat3 increased with erlotinib treatment in NCI-H292 cells, which was abrogated by Hsp90 inhibition. Furthermore, in a NCI-H1975 T790M mutation erlotinib-resistant model, CH5164840 enhanced the antitumor activity of erlotinib despite the low efficacy of erlotinib treatment alone. In addition, ERK signaling was effectively suppressed by combination treatment with erlotinib and CH5164840 in a NCI-H1975 erlotinib-resistant model. Taken together, these data indicate that CH5164840 has potent antitumor activity and is highly effective in combination with erlotinib against NSCLC tumors with EGFR overexpression and mutations. Our results support the therapeutic potential of CH5164840 as a Hsp90 inhibitor for combination therapy with EGFR-targeting agents against EGFR-addicted NSCLC., (© 2013 Japanese Cancer Association.)

Metabolic reprogramming is an important driver of tumor progression; however, the metabolic regulators of tumor cell motility and metastasis are not understood. Here, we show that tumors maintain energy production under nutrient deprivation through the function of HSP90 chaperones compartmentalized in mitochondria. Using cancer cell lines, we found that mitochondrial HSP90 proteins, including tumor necrosis factor receptor-associated protein-1 (TRAP-1), dampen the activation of the nutrient-sensing AMPK and its substrate UNC-51-like kinase (ULK1), preserve cytoskeletal dynamics, and release the cell motility effector focal adhesion kinase (FAK) from inhibition by the autophagy initiator FIP200. In turn, this results in enhanced tumor cell invasion in low nutrients and metastatic dissemination to bone or liver in disease models in mice. Moreover, we found that phosphorylated ULK1 levels were correlated with shortened overall survival in patients with non-small cell lung cancer. These results demonstrate that mitochondrial HSP90 chaperones, including TRAP-1, overcome metabolic stress and promote tumor cell metastasis by limiting the activation of the nutrient sensor AMPK and preventing autophagy.

Objective: The tumor necrosis factor (TNF) and the cellular NF-κB pathway protein IKKβ play important roles in various cellular processes such as cell proliferation, survival, differentiation, and apoptosis. A heat shock protein 90 inhibitor, 17-DMAG, can induce apoptosis of some tumor cells. This study is to determine the combined effects of 17-DMAG and TNF on malignant cells and the related mechanisms., Methods: We have determined effects of 17-DMAG, an Hsp90 inhibitor, and TNF treatments on the small cell lung cancer cell line (MS-1), the adenocarcinoma cell line (A549), the squamous-cell carcinoma cell line (LK-2), and the normal human bronchial epithelium cell line (NuLi-1) by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrozolium bromide assay. To determine if 17-DMAG inhibit the expression of IKKβ in the normal human NuLi-1 cells, and the malignant MS-1, A549, and LK-2 cells, immunoblotting assays and luciferase assays were performed., Results: It was found that the combined treatments resulted in synergistic killing of malignant cells, which was confirmed by the apoptosis determination using a fluorescence microscopic assay following staining of the drug-treated cells with Hoescht 33258. The immunoblotting results indicated that the synergistic killing due to 17-DMAG and TNF treatments may be related to the decreases in IKKβ levels in the presence of 17-DMAG., Conclusions: The results suggest that combination of 17-DMAG and TNF treatments might be useful for treating malignancies upon further study in the further., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2041198513886824.