Potent Anaplastic Lymphoma Kinase Inhibitors Research Articles

ALK and ROS1 as a joint target for the treatment of lung cancer: a review.

Rearrangements of the anaplastic lymphoma kinase (ALK) have been described in multiple malignancies, including non-small cell lung cancer (NSCLC). ALK fusions have gain of function properties while activating mutations in wild-type ALK can also occur within the tyrosine kinase domain. ALK rearrangements define a new molecular subtype of NSCLC that is exquisitely sensitive to ALK inhibition. Crizotinib, an orally available small molecule ATP-mimetic compound which was originally designed as a MET inhibitor, was recognized to have "off-target" anti-ALK activity and has been approved in the USA for the treatment of patients with ALK-positive NSCLC. Chromosomal rearrangements involving the ROS1 receptor tyrosine kinase have also been recently described in NSCLC, while crizotinib is currently under clinical trial in this molecular subset of NSCLC patients. The basic approaches of any computer aided drug design work in terms of structure and ligand based drug design. Details of each of these approaches should be covered with an emphasis on utilizing both in order to develop multi-targeted small-molecule kinase inhibitors. Such multi-targeted tyrosine kinase inhibitors can have antiproliferative activity against both ROS1and ALK rearranged NSCLC. Herein, we highlight the importance of targeting these proteins and the advances in optimizing more potent and selective ALK and ROS1 kinase inhibitors.

SY14-3 - Treatment of EML4-ALK Rearranged NSCLC

ABSTRACT Rearrangements in the anaplastic lymphoma kinase (ALK) gene have been detected in ∼3–7% of patients with non-small-cell lung cancer (NSCLC). Crizotinib, an ALK tyrosine kinase inhibitor (TKI), is clinically effective in NSCLC patients with ALK rearrangements. In the phase I clinical trial, a radiographic response rate of 54% was observed. Crizotinib was recently approved by the FDA for the treatment of ALK-positive NSLC. Current clinical trials of crizotinib include two randomized phase III clinical trials. The first compares crizotinib to second-line chemotherapy (either pemetrexed or docetaxel) in ALK-positive NSCLC patients that have failed first-line platinum-based chemotherapy. The second trial compares crizotinib with cisplatin (or carboplatin) and pemetrexed in treatment naive advanced NSCLC ALK positive patients. In addition to ALK TKIs, inhibitors of heat shock protein (HSP) 90 have demonstrated some clinical activity in EML4-ALK NSCL and are undergoing additional clinical evaluation. However, despite the impressive clinical efficacy of crizotinib, it is fully anticipated that acquired drug resistance will emerge. Drug resistance mechanisms can in general be divided into two categories: those that result in secondary mutations in the target itself (i.e. ALK) or those that activate parallel signaling pathways. For ALK-positive cancers both mechanisms have been identified to date. Secondary mutations in ALK to date include L1196M, F1174L, C1156Y and L1152R. One example of an alternative signaling pathway that leads to ALK TKI resistance is EGFR. In vitro, some ALK TKI-resistant cell lines have activated EGFR signaling (via EGFR ligands) that promote crizotinib resistance. The knowledge of a specific drug resistance mechanism is important for choosing the next treatment of ALK positive patients that have developed Crizotinib resistance. In those with evidence of an ALK secondary mutation a more potent ALK inhibitor may be effective. There are several such in preclinical and at least three in phase I clinical development. In cancers harboring activated signaling pathways (such as EGFR) an alternative strategy, a combination of an ALK and EGFR inhibitor, may be effective. A phase I clinical trial is underway combining Crizotinib with the pan-ERBB inhibitor PF299804.

SY14-5 - Discovery Research of a New Selective ALK Inhibitor CH5424802

ABSTRACT The recent development of targeted protein kinase inhibitors has provided opportunities in cancer treatment. However, certain factors limit the efficacy of cancer therapies, such as a narrow therapeutic index caused by inhibition of multiple kinases, and the emergence of resistant mutants. Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that is constitutively activated in certain cancers, following gene alterations such as chromosomal translocation, amplification, or point mutation. Especially, EML4-ALK has been identified as a fusion oncogene in non-small-cell lung cancer (NSCLC). We have carried out high-throughput screening of enzyme inhibitors against targets of tyrosine kinases, and we finally found CH5424802, a potent, selective, and orally available ALK inhibitor with a new chemical scaffold. In an enzyme inhibitory assay and a binding assay, CH5424802 had a nanomolar affinity with ATP-competitive inhibition mode. CH5424802 showed preferential antitumor activity against cancers with gene alterations of ALK, such as NSCLC cells expressing EML4-ALK fusion and anaplastic large-cell lymphoma cells expressing NPM-ALK fusion in vitro and in vivo. It had a wide therapeutic index due to higher kinase selectivity. Acquired resistance to kinase inhibitors is one of the most serious problems in the long-term cancer treatment. In particular gatekeeper mutations are reported as one of the common resistant mechanisms of kinase inhibitors, such as ABL, EGFR, and KIT. CH5424802 inhibited ALK L1196M, which corresponds to the gatekeeper mutation conferring common resistance to kinase inhibitors, and blocked EML4-ALK L1196M-driven cell growth. Potent ALK inhibitors effective against the gatekeeper mutants may offer clinical advantages in cancer treatment for patients with ALK-driven tumors.

A Simple, Highly Visual in Vivo Screen for Anaplastic Lymphoma Kinase Inhibitors

Anaplastic lymphoma kinase (ALK) is an important drug target in many cancers, including lymphoma, neuroblastoma, and lung cancer. Here, we demonstrate proof-of-principle for a novel and inexpensive assay for ALK inhibitor activity and identification in zebrafish. We demonstrate that the human oncogenic ALK fusion, NPM-ALK, drives overproduction of iridophores, a highly visible, shiny pigment cell-type in zebrafish. Treatment with the potent ALK inhibitor, TAE684, fully inhibits production of ALK-dependent iridophores. Using our assay, we test multiple properties of TAE684 in vivo, including efficacy, specificity, and toxicity. We note that TAE684 also inhibits the closely related leukocyte tyrosine kinase (Ltk) that is required for endogenous iridophore development. Similar effects are observed with an independent inhibitor, Crizotinib. Our assay can thus be utilized to identify ALK or LTK inhibitors. Importantly, the natural reflectivity of iridophores lends itself to automation for high throughput assessment of ALK and LTK inhibitor compounds in vivo.

440O - Results of a First-In-Human Phase I Study of the Alk Inhibitor LDK378 in Advanced Solid Tumors

ABSTRACT Background Translocations of the anaplastic lymphoma kinase (ALK) gene occur in 3–8% of NSCLC. LDK378 is a novel, potent small molecule ALK inhibitor that produces tumor regressions in ALK-driven (ALK+) NSCLC xenografts. A Phase 1 study is being conducted with the primary objective of determining the MTD and safety profile in patients (pts) with ALK+ cancers. Other objectives are to assess safety, PK, and antitumor activity in pts with ALK+ NSCLC, either previously untreated with ALK inhibitors, or relapsed following ALK inhibitor treatment, and other ALK+ cancers. Methods LDK378 was administered orally once-daily on a continuous 21-day schedule, in adult pts with advanced malignancies harboring a genetic alteration in ALK who progressed on standard therapy or for whom there was no effective therapy. Dose escalation was guided by a Bayesian logistic regression model to determine the MTD, and started at 50 mg/day. Results As of 25 April2012, 56 pts (primary site: lung 50 pts [37 with prior crizotinib]; breast 4 pts; other 2 pts; median age 53 (22–76) years; 88% ECOG PS 0/1) had received LDK378 at doses of 50–750 mg/day. Of 47 pts evaluable for response (per investigator), there were 24 (51%) responses. All responses were in ALK+ NSCLC (FISH positive in ≥15% of tumor cells). In 26 pts with NSCLC who had progressed following crizotinib and were treated at ≥400 mg/day there were 21 (81%) responses. Dose limiting toxicities (DLTs) have occurred in 2/14 pts at 400 mg/day, 2/9 pts at 600 mg/day, and 1/9 pts at 750 mg/day. DLTs included diarrhea, vomiting, nausea, dehydration, and ALT elevation. The MTD was 750 mg/day. At the cutoff date, 36 (64%) pts remain on treatment. Discontinuations were due to adverse events (AEs) in 1 (2%), and disease progression in 19 (34%) pts. The most frequent AEs (all grades) were nausea 33 (59%), vomiting 30 (54%), and diarrhea 27 (48%) pts. The most frequent Grade 3/4 AE was diarrhea (5 [9%] pts). Oral absorption of LDK378 was rapid with a Tmax of 5–6 hours, and half-life was about 36 hours. Conclusions Daily oral LDK378 is well tolerated and the MTD was 750 mg/day. Striking activity was seen in ALK+ NSCLC pts treated at doses ≥400mg, who had previously progressed following crizotinib. Disclosure A.T. Shaw: I have a consultant/advisory role for Pfizer, Novartis, Chugar, Ariad, and Daiichi. D.R. Camidge: I have a compensated consultant/advisory role with Novartis Pharmacuticals Inc. S. Sharma: I have a compensated consultant/advisory role for Novartis (LEAD Summit). I have received honoraria for Novartis LEAD Summit. I have received research funding from Novartis (Phase Ib clinical trial). D.S.W. Tan: I have received research funding from Novartis. M. Goldwasser: Employment or leadership position to disclose: Associate Director Biostatistics: Novartis Pharmaceuticals. D. Dai: Employment or leadership position to disclose. Clinical Pharmacology Expert: Novartis Pharmaceuticals; Stock ownership: Myself; Novartis Pharmaceuticals. A.L. Boral: I am an employee of Novartis Institutes for Biomedical Sciences (Executive Director). I have stock ownership (Novartis). All other authors have declared no conflicts of interest.

A first-in-human phase I/II study of ALK inhibitor CH5424802 in patients with ALK-positive NSCLC.

7602 Background: Anaplastic lymphoma kinase (ALK) is a tyrosine kinase constitutively activated in a subset of non-small cell lung cancer (NSCLC) following chromosomal gene translocation. CH5424802 was identified as a potent, selective, and oral ALK inhibitor with a unique chemical scaffold, showing preferential antitumor activity against NSCLC cells expressing EML4-ALK fusion in vitro and in vivo (Cancer Cell, 2011). Our results support the potential of CH5424802 as a new therapeutic opportunity for patients (pts) with ALK-positive NSCLC. Methods: Pts with ALK-positive NSCLC received CH5424802 twice daily orally until progressive disease or toxicity was observed. In the phase I portion, dose was escalated using an accelerated titration scheme. The primary objectives were to investigate the safety, tolerability and pharmacokinetic (PK) parameters under fasting conditions, and to determine the recommended dose (RD) for use in the phase II portion. The primary objectives of the phase II portion were to investigate the efficacy and safety at the RD. The phase I portion was amended to include an investigation under non-fasting conditions in addition to fasting conditions. Results: 15 pts (M/F: 9/6) were treated with CH5424802 under fasting conditions in 6 cohorts (20 mg bid to 300 mg bid) and 9 pts (M/F: 2/7) were treated under non-fasting conditions in 2 cohorts (240 mg bid and 300 mg bid). Median age was 42.5 years. The highest dose level defined in the protocol (300 mg bid) did not reach the MTD. Thus, a DLT was not determined. Toxicities were mild to moderate. The most frequent toxicity was grade 1 myalgia. Grade 3 toxicity (cases) occurred as follows; hypophosphatemia (2), neutropenia (2), blood CPK increased (1) and hypermagnesemia (1). PK data from the fasting cohorts showed dose-dependent increases of Cmax and AUC. All pts at all dose levels achieved tumor regression. At dose levels 240 mg bid or more under fasting conditions, all 7 pts with measurable lesions achieved a partial response. So far, 11 pts have been on study treatment ≥ 6 months. Conclusions: CH5424802 was well tolerated with promising efficacy in pts with ALK-positive NSCLC. The phase II portion is ongoing.

Abstract 2916: Discovery of pyrazolimidazopyridine compounds as potent in vitro and in vivo anaplastic lymphoma kinase inhibitors

Abstract Translocations linking anaplastic lymphoma kinase (ALK) to multiple fusion partners were identified in anaplastic large cell lymphoma (ALCL), inflammatory myofibroblastic tumors, esophageal squamous cell carcinomas, neuroblastoma, and in non small cell lung cancer (NSCLC). This fusion leads to constitutive activation of ALK and has potent transforming activity. The recent approval of Crizotinib validated clinically the usefulness of treating EML4-ALK +ve patients with an Alk inhibitor. We report two novel series of compounds that potently inhibit ALK both in vitro and in vivo. Two series of pyrazolimidazolpyridine compounds, with good physical properties, were identified as part of our screening effort as potent ALK inhibitors in ALK enzyme assays. Subsequent testing of the mode of action of these compounds in Del cell lines containing NPM-ALK fusions demonstrated strong inhibition of phospho-ALK. In vivo, oral administration of 10 mg/kg of a pyrazolimidazopyridine compound in a Del xenograft model in SCID mice resulted in greater than 90% inhibition of phospho-ALK over 6 hours. In conclusion, we have identified two series of pyrazolimidazopyridine compounds as potent ALK inhibitors both in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2916. doi:1538-7445.AM2012-2916

Abstract 3910: Lead optimization of a series of 5-aminopyrazol-imidazopyridine compounds as potent anaplastic lymphoma kinase inhibitors active against clinically relevant ALK mutations

Abstract Despite the success of Crizotinib in treating NSCLC patients with EML4-ALK fusions, cancers eventually develop resistance via a variety of mechanisms including mutations in the ATP binding site of the kinase domain. A series of 5-aminopyrazol-imidazopyridine compounds were identified to potently inhibit anaplastic lymphoma kinase and were found to be active against a number of mutations in vitro but suffered from strong Cyp inhibition and were found to be metabolically unstable by incubation in human hepatocytes and microsomes. Installation of a pyrimary alcohol group on the heteroaryl ethyl group of the scaffold consistently improved Cyp3A4 liability, and led to improvement in physical and DMPK properties as well as improvement of their metabolic stability in human Heps. The lead compound from this series was orally administrated to SCID mice in a Del xenograft model and achieved greater than 90% phospho-ALK inhibition over 6 hours post dose at 10 mg/kg dose. The lead compound was subsequently tested against Crizotinib-resistant ALK mutations in enzyme assays, and was shown to inhibit the clinically relevant ALK mutations, including L1196M. In enzyme assays, it was inactive against G1269S mutation, consistent with modeling studies. Subsequent profiling in an FDCP cell line over expressing ALK G1269S mutation showed potent anti-proliferative activity, suggesting off-target activity. Further characterization of this series identified AurB inhibition to be a key cell cycle kinase responsible for the off-target activity in the engineered cell line. The off target activity hinders it from assessing the primary pharmacology responsible for its preclinical efficacy in disease relevant models. In conclusion, we have identified a novel orally bioavailable compound that is a potent dual active ALK and AurB inhibitor that is active against clinically-relevant gatekeeper mutant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3910. doi:1538-7445.AM2012-3910

Design and synthesis of a highly selective, orally active and potent anaplastic lymphoma kinase inhibitor (CH5424802)

Anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is considered an attractive therapeutic target for human cancers, especially non-small cell lung cancer (NSCLC). Our previous study revealed that 8,9-side-chains of 6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazole scaffold crucially affected kinase selectivity, cellular activity, and metabolic stability. In this work, we optimized the side-chains and identified highly selective, orally active and potent ALK inhibitor CH5424802 (18a) as the clinical candidate.

Anaplastic lymphoma kinase as a new target for the treatment of non-small-cell lung cancer

The anaplastic lymphoma kinase (ALK) gene rearrangement identifies a distinct molecular subset in non-small-cell lung cancer (NSCLC) populations susceptible to targeted inhibition. It consists of a small inversion in the short arm of chromosome 2 between exon 20 of the ALK gene and different exons of the echinoderm microtubule-associated protein-like (EML4) gene. This translocation leads to a chimeric protein with constitutive activation of ALK that possesses an oncogenic activity demonstrated both in vitro and in vivo. Other rare translocation partners for ALK other than EML4 may be found in lung cancers, including TRK-fused gene (TFG) and kinesin family member 5B (KIF5B). ALK-positive patients represent 5–6% of all NSCLCs and they seem to have particular clinicopathological and molecular features. Recently, Phase I–II trial results of crizotinib, a potent dual c-MET and ALK inhibitor, demonstrated its dramatic efficacy in ALK-positive patients with advanced NSCLC. This article will present knowledge on the characteristics of ALK-positive patients, discuss the different methods of ALK rearrangement detection and focus on clinical results of crizotinib.

Crizotinib‐Resistant Mutants of EML4‐ALK Identified Through an Accelerated Mutagenesis Screen

Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance.

9-Substituted 6,6-Dimethyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazoles as Highly Selective and Potent Anaplastic Lymphoma Kinase Inhibitors

9-Substituted 6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazoles were discovered as highly selective and potent anaplastic lymphoma kinase (ALK) inhibitors by structure-based drug design. The high target selectivity was achieved by introducing a substituent close to the E(0) region of the ATP binding site, which has a unique amino acid sequence. Among the identified inhibitors, compound 13d showed highly selective and potent inhibitory activity against ALK with an IC(50) value of 2.9 nM and strong antiproliferative activity against KARPAS-299 with an IC(50) value of 12.8 nM. The compound also displayed significant antitumor efficacy in an established ALK fusion gene-positive anaplastic large-cell lymphoma (ALCL) xenograft model in mice without body weight loss.

Design, Synthesis, and Biological Activity of Urea Derivatives as Anaplastic Lymphoma Kinase Inhibitors

In anaplastic large-cell lymphomas, chromosomal translocations involving the kinase domain of anaplastic lymphoma kinase (ALK), generally fused to the 5' part of the nucleophosmin gene, produce highly oncogenic ALK fusion proteins that deregulate cell cycle, apoptosis, and differentiation in these cells. Other fusion oncoproteins involving ALK, such as echinoderm microtubule-associated protein-like 4-ALK, were recently found in patients with non-small-cell lung, breast, and colorectal cancers. Recent research has focused on the development of inhibitors for targeted therapy of these ALK-positive tumors. Because kinase inhibitors that target the inactive conformation are thought to be more specific than ATP-targeted inhibitors, we investigated the possibility of using two known inhibitors, doramapimod and sorafenib, which target inactive kinases, to design new urea derivatives as ALK inhibitors. We generated a homology model of ALK in its inactive conformation complexed with doramapimod or sorafenib in its active site. The results elucidated why doramapimod is a weak inhibitor and why sorafenib does not inhibit ALK. Virtual screening of commercially available compounds using the homology model of ALK yielded candidate inhibitors, which were tested using biochemical assays. Herein we present the design, synthesis, biological activity, and structure-activity relationships of a novel series of urea compounds as potent ALK inhibitors. Some compounds showed inhibition of purified ALK in the high nanomolar range and selective antiproliferative activity on ALK-positive cells.

Discovery of novel tetracyclic compounds as anaplastic lymphoma kinase inhibitors

Anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is considered a promising therapeutic target for human cancers. We identified novel tetracyclic derivatives as potent ALK inhibitors. Among them, compound 27 showed strong cytotoxicity against KARPAS-299 with an IC(50) value of 21 nM and significant antitumor efficacy in ALK fusion-positive blood and solid cancer xenograft models in mice without body weight loss.

Discovery of 3,5-Diamino-1,2,4-triazole Ureas as Potent Anaplastic Lymphoma Kinase Inhibitors.

A series of novel 3,5-diamino-1,2,4-triazole benzyl ureas was identified as having potent anaplastic lymphoma kinase (ALK) inhibition exemplified by 15a, 20a, and 23a, which exhibited antiproliferative IC50 values of 70, 40, and 20 nM in Tel-ALK transformed Ba/F3 cells, respectively. Moreover, 15a and 23a potently inhibited the growth and survival of NPM-ALK positive anaplastic large cell lymphoma cell (SU-DHL-1) and neuroblastoma cell lines (KELLY, SH-SY5Y) containing the F1174L ALK mutation. These compounds provide novel leads for the development of small-molecule ALK inhibitors for cancer therapy.

Abstract LB-298: AP26113, a potent ALK inhibitor, overcomes mutations in EML4-ALK that confer resistance to PF-02341066 (PF1066)

Abstract AP26113 is a potent and selective inhibitor of anaplastic lymphoma kinase (ALK) (AACR 2010; #3623). Activating gene rearrangements of ALK, such as EML4-ALK, have been identified as driver mutations in NSCLC and other cancers. There is strong precedence for the development of resistance to targeted therapies that inhibit driver mutations. Kinase domain mutations that confer resistance in patients have been successfully predicted by in vitro mutagenesis screens in BaF3 cells (e.g. BCR-ABL in CML). Here, the BaF3 system was used to identify mutations in ALK that confer resistance to PF1066, a clinically validated dual Met/ALK inhibitor (ASCO 2009; #3509), or AP26113. PF1066-resistant mutations were identified at all concentrations tested (up to 2000 nM). In contrast, 1000 nM AP26113 completely suppressed emergence of resistance. Six mutations, all in the kinase domain, were identified that confer some degree of resistance to 1 or both compounds (Table). AP26113 inhibited viability of BaF3 cells expressing these mutants with IC50s of 23 - 269 nM. PF1066 inhibited viability with IC50s of 311 -1419 nM, with 3 mutants having sensitivity indistinguishable from parental BaF3 cells, which lack EML4-ALK. The 2 mutations that confer the greatest resistance to PF1066 were examined in a BaF3 xenograft model in which compounds were administered daily by oral dosing. A 200 mg/kg dose of PF1066 induced regression of tumors expressing native EML4-ALK but was completely inactive against G1269S or L1196M (gatekeeper) mutants. In contrast, AP26113 induced regression of tumors expressing native EML4-ALK and the G1269S and L1196M mutants at 25, 50 and 50 mg/kg, respectively. Analysis of ALK phosphorylation in tumors demonstrated strong inhibition of the mutants by 50 mg/kg AP26113 but not 200 mg/kg PF1066. These results identify several mutations that may confer resistance to PF1066 in patients and suggest that more potent compounds such as AP26113 may be required to overcome such resistance.Sensitivity of BaF3 cells expressing native and mutant EML4-ALK to PF-02341066 and AP26113BaF3 cell linePF-02341066 IC50 ± SD AP26113 IC50 ± SD BaF3 cell line(nM)TI**(nM)TI**Parental*1116 ±436—2169 ±655—Native EML4-ALK153 ± 29714 ±2151EML4-ALK mutants: F1174C497 ± 202104 ± 1921L1196M1157 ± 401166 ±733S1206R838 ± 2521269 ± 688E1210K311 ± 80488 ± 1225F1245C366 ±8373 ± 1130G1269S1419 ±618123 ± 594*Parental BaF3 cells lack EML4-ALK and therefore require IL-3 for growth**Therapeutic Index = Parental IC50 / EML4-ALK IC50 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 LB-298.

Functional validation of the anaplastic lymphoma kinase signature identifies CEBPB and Bcl2A1 as critical target genes

Anaplastic large cell lymphomas (ALCLs) represent a subset of lymphomas in which the anaplastic lymphoma kinase (ALK) gene is frequently fused to the nucleophosmin (NPM) gene. We previously demonstrated that the constitutive phosphorylation of ALK chimeric proteins is sufficient to induce cellular transformation in vitro and in vivo and that ALK activity is strictly required for the survival of ALK-positive ALCL cells. To elucidate the signaling pathways required for ALK-mediated transformation and tumor maintenance, we analyzed the transcriptomes of multiple ALK-positive ALCL cell lines, abrogating their ALK-mediated signaling by inducible ALK RNA interference (RNAi) or with potent and cell-permeable ALK inhibitors. Transcripts derived from the gene expression profiling (GEP) analysis uncovered a reproducible signature, which included a novel group of ALK-regulated genes. Functional RNAi screening on a set of these ALK transcriptional targets revealed that the transcription factor C/EBPbeta and the antiapoptotic protein BCL2A1 are absolutely necessary to induce cell transformation and/or to sustain the growth and survival of ALK-positive ALCL cells. Thus, we proved that an experimentally controlled and functionally validated GEP analysis represents a powerful tool to identify novel pathogenetic networks and validate biologically suitable target genes for therapeutic interventions.

Structural Insights into the ATP Binding Pocket of the Anaplastic Lymphoma Kinase by Site-Directed Mutagenesis, Inhibitor Binding Analysis, and Homology Modeling

Anaplastic lymphoma kinase (ALK) is a valid target for anticancer therapy; however, potent ALK inhibitors suitable for clinical use are lacking. Because the majority of described kinase inhibitors bind in the ATP pocket of the kinase domain, we have characterized this pocket in ALK using site-directed mutagenesis, inhibition studies, and molecular modeling. Mutation of the gatekeeper residue, a key structural determinant influencing inhibitor binding, rendered the fusion protein, NPM/ALK, sensitive to inhibition by SKI-606 in the nanomolar range, while PD173955 inhibited the NPM/ALK mutant at micromolar concentrations. In contrast, both wild type and mutant NPM/ALK were insensitive to imatinib. Computer modeling indicated that docking solutions obtained with a homology model representing the intermediate conformation of the ALK kinase domain reflected closely experimental data. The good agreement between experimental and virtual results indicate that the ALK molecular models described here are useful tools for the rational design of ALK selective inhibitors. In addition, 4-phenylamino-quinoline compounds may have potential as templates for ALK inhibitors.