ALK Fusion Positive Lung Cancer Research Articles

Negr1-Derived Peptides Trigger ALK Degradation and Halt Neuroblastoma Progression In Vitro and In Vivo.

Neuroblastoma is among the most common childhood cancers. Neuroblastoma in advanced stages is one of the most intractable pediatric cancers, notwithstanding the recent therapeutic advances. ALK mutations are among the leading cause of hereditary neuroblastoma and account for more than 14% of the somatically acquired alterations. ALK kinase activity is currently one of the main targets for pharmacological strategies. However, evidence from ALK fusion-positive lung cancer studies has shown that resistance to ALK inhibition arises during the therapy, causing a relapse within several years. IgLONs are membrane-bound proteins involved in cell-to-cell adhesion. The expression of the IgLON family results altered in different cancers. We found that the IgLON member Negr1 is downregulated in neuroblastoma. The ectopic overexpression of Negr1 impairs neuroblastoma growth in vitro and in vivo. Negr1 exists as a GPI-anchored membrane-bound protein and as a soluble protein released upon metalloprotease cleavage. We generated and characterized a panel of Negr1-derived peptides. The treatment with Negr1 protein and derived peptides induce ALK downregulation and halt neuroblastoma progression in vitro and in vivo.

Longitudinal tracking of ALK-positive lung cancer from plasma using circulating-tumor RNA and circulating-tumor DNA in the BRIGHTSTAR clinical trial.

e21072 Background: While the administration of specific tyrosine-kinase inhibitors (TKIs) in ALK-fusion positive lung cancer has led to significant improvement in clinical outcomes, detection of gene fusions remains challenging, especially from liquid biopsies. In tissue biopsies, assays which incorporate RNA-based detection have demonstrated increased sensitivity for gene fusion detection. We therefore hypothesized that a liquid biopsy assay equally including assessment of gene fusions using circulating-tumor RNA (ctRNA) in addition to circulating-tumor DNA (ctDNA), will improve detection. Furthermore, we hypothesized that detection of gene fusions as well as mutations will also correlate with clinical treatment. Methods: We retrospectively analyzed 89 samples from 35 patients included in the BRIGHTSTAR clinical trial assessing local consolidative therapy (LCT) and brigatinib in patients with stage IV or recurrent Non-small Cell Lung Cancer and confirmed ALK rearrangement (NCT03707938). Samples were included at baseline (N = 31), prior to LCT after 8 weeks of brigatinib treatment (N = 29), after LCT ( < 3 weeks; N = 25) and at progression (N = 4). We used a targeted next generation sequencing (NGS) assay assessing both ctDNA as well as ctRNA (LiquidHALLMARK, Lucence Health, Palo Alto) to detect ALK fusions as well as mutations in 80 genes. Up to 5 ml of plasma was analyzed per sample. Results: At baseline, ALK fusions were detected in 15/31 patients (48%) of which 8 were detected using both ctDNA and ctRNA, while four were exclusively detected in ctDNA and three in ctRNA. Plasma ctDNA concentrations for patients with detectable ALK fusions at baseline were significantly higher than for those without detectable gene fusions (mean 26.1 ng/mL versus 16.6 ng/mL; p = 0.0044). ALK fusions were detected in two patients pre-LCT, exclusively in ctRNA, while ALK fusions cleared completely post-LCT. At progression, ALK fusions were detected in 2/4 samples (50%) in both ctDNA and ctRNA. Of the two negative samples tissue biopsies were available confirming the absence of an ALK rearrangement, including one transformation to squamous cell carcinoma. Overall, including analysis of ctRNA led to a 36% relative additional detection of ALK fusions compared to analyzing ctDNA alone. Furthermore, ctDNA mutation positivity in genes other than ALK was 17/31 (55%), 9/29 (31%), 8/25 (32%) and 3/4 (75%) for baseline, pre-LCT, post-LCT and progression, respectively. Conclusions: We highlight that ALK fusions can be reliably detected in plasma of lung cancer patients and detectability of ALK fusions correlate with exposure to treatment. Analyzing ctRNA in addition to ctDNA improves sensitivity of fusion detection and is a highly promising strategy in this setting.

Abstract 6642: Rapid tissue donation identifies LAG3 as a potential therapeutic target for ALK fusion positive lung cancer

Abstract Introduction: The rapid tissue donation (RTD) program provides post-therapy primary and metastatic tumor tissue samples at the time of death for the study of resistance mechanisms and the tumor microenvironment to inform therapeutic opportunities. NCCN guidelines (NCCN v5.2022) recommend against PD-1/PD-L1 inhibitor monotherapy in the second-line setting for ALK fusion+ non-small cell lung cancer (NSCLC) due to lack of efficacy, irrespective of tumoral PD-L1 expression. We observed high LAG3 protein expression by multiplex immunofluorescence (mIF) in postmortem ALK fusion-positive tumor tissue samples from 3 RTD donors who were treated with ALK inhibitors. Further studies are recommended to investigate the therapeutic potential of LAG3 inhibitors in this patient population. Case Series: Of the 38 patients with lung cancer tissue collected by RTD, 3 patients had ALK fusion and were treated with ALK inhibitors. Despite the initial response, all 3 patients succumbed to the disease. Tissue samples were collected from primary and multiple metastatic sites after death. Immune checkpoint markers, including LAG3, were quantitated via multiplex immunofluorescence staining, and genomic analyses were performed. Results: Analysis of two tissue microarrays comprised of 130 tumor cores from multiple tissue sites of the first 10 NSCLC RTD donors revealed that 66% of tumor cores had >1% LAG3 expression in CD3+ T cells. Among the three patients with ALK fusion-positive NSCLC, 83% (5 of 6 cores), 85% (6 of 7 cores), and 100% (18 of 18 cores) of the tissue cores had >1% LAG3 expression in T cells. The median LAG3 expression in T cells measured by mIF was 1.8% in 99 tumor cores from the ALK fusion-negative study cohort versus 7.3% in the 31 cores from the ALK fusion-positive subset (p=0.004). Postmortem genomic analyses revealed an ALK p.L1196M resistance mutation in the first patient and an AGK-BRAF fusion in the 2nd patient. No known ALK-TKI resistance mutations were detected in the 3rd patient. Discussion: High-quality post-therapy tumor samples collected by the RTD program enable the investigation of genomic and potential immune-related resistance mechanisms. Such studies can inform potential targets for drug development and the study of new therapies to overcome treatment resistance. Our observation of high LAG3 expression in 3 RTD donors with ALK fusion-positive NSCLC suggests the need for further studies to explore the potential for LAG3 inhibitors in patients with NSCLC whose tumors develop resistance to ALK inhibitors. Citation Format: Hilal Ozakinci, Gina Nazario, Karina Colossi Furlan, Dung-Tsa Chen, Trevor Rose, Matthew B. Schabath, Eric B. Haura, Bruna Pellini, Amer Beg, Theresa A. Boyle. Rapid tissue donation identifies LAG3 as a potential therapeutic target for ALK fusion positive lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6642.

Small cell transformation of ROS1 fusion-positive lung cancer resistant to ROS1 inhibition

Histologic transformation from non-small cell to small cell lung cancer has been reported as a resistance mechanism to targeted therapy in EGFR-mutant and ALK fusion-positive lung cancers. Whether small cell transformation occurs in other oncogene-driven lung cancers remains unknown. Here we analyzed the genomic landscape of two pre-mortem and 11 post-mortem metastatic tumors collected from an advanced, ROS1 fusion-positive lung cancer patient, who had received sequential ROS1 inhibitors. Evidence of small cell transformation was observed in all metastatic sites at autopsy, with inactivation of RB1 and TP53, and loss of ROS1 fusion expression. Whole-exome sequencing revealed minimal mutational and copy number heterogeneity, suggestive of “hard” clonal sweep. Patient-derived models generated from autopsy retained features consistent with small cell lung cancer and demonstrated resistance to ROS1 inhibitors. This case supports small cell transformation as a recurring resistance mechanism, and underscores the importance of elucidating its biology to expand therapeutic opportunities.

TRK inhibitors in TRK fusion-positive cancers.

TRK fusions are oncogenic drivers of various adult and paediatric cancers. The first-generation TRK inhibitors, larotrectinib and entrectinib, were granted landmark, tumour-agnostic regulatory approvals for the treatment of these cancers in 2018 and 2019, respectively. Brisk and durable responses are achieved with these drugs in patients, including those with locally advanced or metastatic disease. In addition, intracranial activity has been observed with both agents in TRK fusion-positive solid tumours with brain metastases and primary brain tumours. While resistance to first-generation TRK inhibition can eventually occur, next-generation agents such as selitrectinib (BAY 2731954, LOXO-195) and repotrectinib were designed to address on-target resistance, which is mediated by emergent kinase domain mutations, such as those that result in substitutions at solvent front or gatekeeper residues. These next-generation drugs are currently available in the clinic and proof-of-concept responses have been reported. This underscores the utility of sequential TRK inhibitor use in select patients, a paradigm that parallels the use of targeted therapies in other oncogenic driver-positive cancers, such as ALK fusion-positive lung cancers. While TRK inhibitors have a favourable overall safety profile, select on-target adverse events, including weight gain, dizziness/ataxia and paraesthesias, are occasionally observed and should be monitored in the clinic. These side-effects are likely consequences of the inhibition of the TRK pathway that is involved in the development and maintenance of the nervous system.

P1.16 A Study on Clinicopathological Features and Diagnostic Methods of ALK Fusion-Positive Non-Small Cell Lung Cancer in KR

Lung cancer is one of the most common malignancies and the leading cause of cancer-related death in Korea. A lot of efforts have been put into to develop new, more effective treatments and biological markers to predict the therapeutic responses, leading to the discovery of various genetic changes, the so-called “growth drivers” of carcinogenesis. Some genetic alterations have become the new treatment targets, and there have been suggestions that different mutations are associated with different clinicopathological characteristics and prognosis. In this study we aimed to evaluate the status of the key “driver” mutation ALK fusion in Korean non-small cell lung cancer patients and its association with the clinicopathological characteristics, including the presence of other genetic mutations. We also compared different methods for ALK fusion detection, i.e., fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and next-generation sequencing (NGS) analyses to find out which method would the most effective. A total of 482 NSCLC patients who underwent ALK FISH test were evaluated for clinicopathological features such as age, sex, smoking history, tumor stage, histological subtype, immunohistochemical profile including ALK, EGFR mutation status and survival. Some ALK FISH-positive and -negative cancers were newly submitted to NGS analysis for DNA and RNA alterations. The ALK fusion-positive tumors were associated with younger age, more female patients, frequent nodal metastases and advanced stage, and short survival. The diagnostic agreement rates among ALK FISH, ALK IHC, and NGS ranged from about 60 to 90% and when considering the patient outcome as a measurement of the test efficacy, the ALK FISH may not be the most reliable one in diagnosis of ALK fusion-positive lung cancer. The NGS analysis detected various additional genetic alterations never considered at the time of diagnosis, including NPM3-TRK rearrangement. Comparing the results of ALK FISH, IHC and NGS, we concluded in practice ALK testing should better be diversified concerning FISH and IHC, and NGS analysis would be a good alternative of ALK FISH with an additional advantage of simultaneous detection of different mutations.

Abstract 2600: Analysis of drug resistance mechanisms and strategies for overcoming resistance in cancer therapy using a curated clinical knowledgebase

Abstract Emergence of resistance to targeted therapies is a critical problem in cancer therapy, and understanding both the mechanisms of resistance and strategies for overcoming resistance are crucial to effective treatment of cancer patients. The JAX Clinical Knowledgebase (JAX-CKB), which incorporates data on therapeutic efficacy in the context of molecular alterations, enables rapid analysis of known therapy resistance mechanisms and current data surrounding strategies for overcoming resistance, both in the preclinical and clinical setting. Using the JAX-CKB, we have identified over 1250 lines of evidence corresponding to therapy resistance across tumor types. Of those lines, 147 correspond to therapy resistance in lung cancer. The JAX-CKB contains 22 variants in ALK associated with ALK therapy resistance. Within ALK fusion-positive lung cancer, evidence lines corresponding to ALK inhibitor resistance are associated primarily with complex molecular profiles containing secondary ALK mutations, and mechanisms for overcoming resistance in this setting were associated with use of novel agents. Within EGFR mutation positive lung cancer, lines associated with resistance to EGFR inhibitor therapy included copy number alterations, primary resistance mutations, secondary resistance mutations, and expression level changes. Strategies for overcoming resistance in these settings include novel agents and/or various combination therapies. The JAX-CKB provides a unique global view into current data on resistance to targeted therapies in oncology, which may enable more rapid assessment of effective therapy options and expose opportunities for additional research into strategies for overcoming resistance. Citation Format: Sara E. Patterson, Cara M. Statz, Taofei Yin, Susan M. Mockus. Analysis of drug resistance mechanisms and strategies for overcoming resistance in cancer therapy using a curated clinical knowledgebase [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 2600. doi:10.1158/1538-7445.AM2017-2600

Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma

The success of crizotinib in ALK-positive patients has elicited efforts to find new oncogenic fusions in lung cancer. These efforts have led to the discovery of novel oncogenic fusion genes such as ROS1 and RET. However, the molecular and clinicopathologic characteristics associated with RET or ROS1 fusion, compared with ALK fusion-positive lung cancer, remain unclear. We accordingly analyzed the clinicopathologic characteristics of RET- and ROS1-fusion-positive lung adenocarcinomas. We further performed immunohistochemistry and fluorescence in situ hybridization analysis (FISH) in 15 cases of RET and 9 cases of ROS1 fusion tumors by identified NanoString's nCounter screening. RET fusion-positive patients were younger in age, never-smokers, and in early T stage; ROS1 fusion-positive patients had a higher number of never-smokers compared with patients with quintuple-negative (EGFR−/KRAS−/ALK−/ROS1−/RET−) lung adenocarcinoma. Histologically, RET and ROS1 fusion tumors share the solid signet-ring cell and mucinous cribriform pattern, as previously mentioned in the histology of ALK fusion tumors. Therefore, it can be presumed that fusion gene-associated lung adenocarcinomas share similar histologic features. In immunohistochemistry, the majority of 15 RET and 9 ROS1 fusion-positive cases showed positivity of more than moderate intensity and cytoplasmic staining for RET and ROS1 proteins, respectively. In FISH, the majority of RET and ROS1 rearrangement showed two signal patterns such as one fusion signal and two separated green and orange signals (1F1G1O) and an isolated 3′ green signal pattern (1F1G). Our study has provided not only characteristics of fusion gene-associated histologic features but also a proposal for a future screening strategy that will enable clinicians to select cases needed to be checked for ROS1 and RET rearrangements based on clinicohistologic features.

Rationale for co-targeting IGF-1R and ALK in ALK fusion-positive lung cancer.

The ALK tyrosine kinase inhibitor (TKI), crizotinib, shows significant activity in patients whose lung cancers harbor ALK fusions but its efficacy is limited by variable primary responses and acquired resistance. In work arising from the intriguing clinical observation of a patient with ALK fusion+ lung cancer who had an ‘exceptional response’ to an IGF-1R antibody, we define a therapeutic synergism between ALK and IGF-1R inhibitors. Similar to IGF-1R, ALK fusion proteins bind to the adaptor, IRS-1, and IRS-1 knockdown enhances the anti-tumor effects of ALK inhibitors. In models of ALK TKI resistance, the IGF-1R pathway is activated, and combined ALK/IGF-1R inhibition improves therapeutic efficacy. Consistent with this finding, IGF-1R/IRS-1 levels are increased in biopsy samples from patients progressing on crizotinib therapy. Collectively, these data support a role for the IGF-1R/IRS-1 pathway in both ALK TKI-sensitive and TKI-resistant states and provide biological rationale for further clinical development of dual ALK/IGF-1R inhibitors.

Abstract PR10: Is CNS availability for oncology a no-brainer? Discovery of PF-06463922, a novel small molecule inhibitor of ALK/ROS1 with preclinical brain availability and broad spectrum potency against ALK-resistant mutations.

Abstract Oncogenic fusions of anaplastic lymphoma kinase (ALK) define a subset of human lung adenocarcinoma. The 1st generation ALK inhibitor crizotinib demonstrated impressive clinical benefit in ALK-fusion positive lung cancers and was approved by the FDA for the treatment of ALK-fusion positive NSCLC in 2011. However, as seen with most kinase inhibitors, patients treated with crizotinib eventually develop resistance to therapy. Acquired ALK kinase domain mutations and disease progression in the central nervous system (CNS) are reported as main contributors to patient relapse after ALK inhibitor therapy. Preclinically, crizotinib lacks significant brain penetration and does not potently inhibit activity of ALK kinase domain mutants, so a drug discovery program was initiated aimed to develop a second generation ALK inhibitor that is more potent than existing ALK inhibitors, capable of inhibiting the resistant ALK mutants and penetrating the blood-brain-barrier. These objectives present a considerable challenge in kinase inhibitor chemical space. Here we report that PF-06463922, a novel small molecule ATP-competitive inhibitor of ALK/ROS1, showed exquisite potencies against non-mutant ALK (Ki <0.2 nM; cell IC50 ∼2 nM) and ROS1 kinase (Ki <0.005 nM; cell IC50 ∼0.2 nM), and demonstrated low nanomolar inhibitory activity against a panel of ALK kinase domain mutants representing all of the patient crizotinib resistant mutations reported to date. The more commonly reported L1196M gatekeeper mutant shows significant sensitivity to PF-06463922 (Ki 0.7 nM; cell IC50 16 nM). PF-06463922 is also very selective, and showed >100 fold kinase selectivity against 95% of the kinases tested in a 207 recombinant kinase panel. Specific design considerations were developed leading to novel ATP-competitive kinase inhibitors with desired low efflux in cell lines over-expressing p-glycoprotein and breast cancer resistance protein, providing excellent blood-brain-barrier and cell penetration properties. Efforts to optimize ligand efficiency and lipophilic efficiency leveraging structure based drug design techniques led to ligands with overlapping broad spectrum potency and low efflux. Single and repeat dose preclinical rat in vivo studies of PF-06463922 demonstrated excellent oral bioavailability and CNS availability with free brain exposure approximately 30% of free plasma levels. In addition, CNS-directed safety studies showed no adverse events at predicted efficacious concentrations. It is anticipated that PF-06463922 with its potent activities on non-mutant ALK, ALK kinase domain mutations and CNS metastases would provide great promise for patients with ALK and ROS1 positive cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR10. Citation Format: Ted W. Johnson, Simon Bailey, Benjamin J. Burke, Michael R. Collins, J. Jean Cui, Judy Deal, Ya-Li Deng, Martin P. Edwards, Mingying He, Jacqui Hoffman, Robert L. Hoffman, Qinhua Huang, Robert S. Kania, Phuong Le, Michele McTigue, Cynthia L. Palmer, Paul F. Richardson, Neal W. Sach, Graham L. Smith, Lars Engstrom, Wenyue Hu, Hieu Lam, Justine L. Lam, Tod Smeal, Helen Y. Zou. Is CNS availability for oncology a no-brainer? Discovery of PF-06463922, a novel small molecule inhibitor of ALK/ROS1 with preclinical brain availability and broad spectrum potency against ALK-resistant mutations. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR10.

Abstract C253: PF-06463922, a novel brain-penetrating small molecule inhibitor of ALK/ROS1 with potent activity against a broad spectrum of ALK resistant mutations in preclinical models in vitro and in vivo.

Abstract Oncogenic fusions of Anaplastic Lymphoma Kinase (ALK) define a subset of human lung adenocarcinomas. The 1st generation ALK inhibitor XALKORI ® (crizotinib) demonstrated impressive clinical benefit in ALK-fusion positive lung cancers and was approved by the FDA for the treatment of ALK-fusion positive NSCLC in 2011. However, as seen with most kinase inhibitors, patients treated with XALKORI eventually developed resistance to therapy. Acquired ALK kinase domain mutations and brain metastases are significant contributors to the relapse after XALKORI therapy. To date, multiple types of ALK kinase domain mutations have been identified in XALKORI refractory patients including ALKG1269A, ALKL1196M, ALKC1156Y, ALKL1152R, ALKF1174L, ALKS1206Y, ALK1151Tins and ALKG1202R, accounting for about 1/3 of patient samples tested. Currently, a number of 2nd generation ALK inhibitors are under development aiming to overcome XALKORI resistant mutations. Even though in preclinical models, some ALK mutants such as ALKG1202R and ALK1151Tins confer high-levels of resistance to almost all of the 2nd generation ALK inhibitors tested. Here we report PF-06463922, a novel ATP competitive small molecule inhibitor of ALK/ROS1, with potent and selective inhibitory activity against all known acquired XALKORI resistant mutations identified in patients. PF-06463922 is also capable of penetrating the blood brain barrier in preclinical animal models. In vitro, PF-06463922 demonstrated potent inhibition in catalytic activities of ALK and 8 different ALK mutant kinases in recombinant enzyme and cell based assays (cell IC50s = 1 to 65 nM). PF-06463922 also showed potent growth inhibitory activity and induced apoptosis in the NSCLC cells harboring either non-mutant ALK or mutant ALK fusions (IC50s = 1 to 30 nM). In vivo, PF-06463922 demonstrated marked cytoreductive activity in mice bearing tumor xenografts that express EML4-ALK, EML4-ALKL1196M, EML4-ALKG1269A, EML4-ALKG1202R or NPM-ALK at low nM free plasma concentrations. These effects were associated with significant inhibition in cellular Ki67 and increased cleaved-caspase3 levels in tumors. In addition, PF-06463922 achieved brain exposure of 20-30% of its plasma levels in mice, and significantly regressed the brain tumors and prolonged survival of mice bearing orthotopic EML4-ALK and EML4-ALKL1196M positive brain tumor implants. The antitumor efficacy of PF-06463922 was dose dependent and strongly correlated with inhibition of ALK phosphorylation and downstream signaling. Our data indicate that PF-06463922 is the most potent ALK inhibitor reported to date (to our knowledge, against both non-mutant or mutant ALK in cell assays), and it demonstrates great potential for treating ALK fusion positive cancers including patients who relapsed from XALKORI therapy due to various ALK kinase domain mutations and/or brain metastases. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C253. Citation Format: Helen Y. Zou, Lars R. Engstrom, Qiuhua Li, Melissa West Lu, Ruth Wei Tang, Hui Wang, Konstantinos Tsaparikos, Jinwei Wang, Sergei Timofeevski, Dac M. Dinh, Hieu Lam, Justine Lam, Shinji Yamazaki, Wenyue Hu, Timothy Affolter, Patrick B. Lappin, Hovhannes Gukasyan, Nathan Lee, Jennifer M. Tursi, Ted W. Johnson, Valeria Fantin, Tod Smeal. PF-06463922, a novel brain-penetrating small molecule inhibitor of ALK/ROS1 with potent activity against a broad spectrum of ALK resistant mutations in preclinical models in vitro and in vivo. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C253.

Abstract 1788: Preclinical development of a selective, potent small molecule ALK inhibitor

Abstract A chromosomal inversion [inv (2)(p21p23)] resulting in the expression of an oncogenic kinase fusion protein known as EML4-ALK [echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase] has been implicated in the pathogenesis of a subset of lung adenocarcinomas. An agent (PF-02341066), originally described as a MET tyrosine kinase inhibitor (TKI) but with “off-target” ALK activity, has demonstrated significant activity in ALK-fusion positive lung cancer patients and is currently in phase II/III clinical trials. In this study, we describe the development and characterization of a novel and highly selective ALK TKI. X-276 (racemic mixture) is an orally bioavailable ATP mimetic that has high selectivity for ALK as assessed by Ambit assays (binding constant 2nM). Treatment of H3122 lung adenocarcinoma cells, which harbor the EML4-ALK E13;A20 fusion product, resulted in potent inhibition of cell growth with an IC50 of 10nM, approximately 10 fold less than the IC50 of H3122 cells treated with a racemic mixture of PF-02341066. Immunoblot analysis confirmed that X-276 inhibited ALK phosphorylation in H3122 cells. Importantly, the IC50 of X-276 in H1993 lung cancer cells, known to be MET-dependent, was >100 fold higher. In addition, the selectivity index of H3122 cells vs. hepatocyte HepG2 cells was >135. Finally, compared to vehicle controls, X-276 delayed the growth of H3122 xenografts in established nude mice by >60%. Collectively, these data show that TKIs can be developed with greater specificity against ALK. Such compounds may lead to the development of ‘second-generation’ ALK TKIs with more potency against ALK-mutant cancers. Future studies will focus on determining the mechanism of acquired resistance to ALK TKIs using these more potent ALK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1788.