Selective Anaplastic Lymphoma Kinase Inhibitor Research Articles

A phase I/II study with a highly selective ALK inhibitor CH5424802 in ALK-positive non-small cell lung cancer (NSCLC) patients: Updated safety and efficacy results from AF-001JP.

8033 Background: Anaplastic lymphoma kinase (ALK) is a constitutively activated tyrosine kinase in a subset of NSCLC with ALK fusion gene derived from chromosomal rearrangement. Previously, CH5424802, a highly selective, second-generation ALK inhibitor, has demonstrated clinically meaningful antitumor activity and a favorable toxicity profile (ESMO 2012). Here we report the updated results of this study. Methods: Patients (Pts) with ALK-positive NSCLC and no prior ALK inhibitor therapy were treated with CH5424802 at 300 mg bid until progressive disease or intolerable toxicity to investigate the efficacy and safety. Results: As of the cut-off date of Dec. 14, 2012, 58 pts have been treated with CH5424802: median age 49.5 years, M/F 25/33, ECOG PS 0/1 26/32, never-smoker 60.3%, ≥2 prior chemotherapy regimens 62%. Among the 46 pts in phase II part of this study, the overall response rate was 93.5% (95% CI: 82.1%, 98.6%) with 2 CRs and 41 PRs. Tumor shrinkage by 30% (PR) was achieved quickly with 65% occurring within 3 weeks of treatment and 87% within 6 weeks. With a median follow-up period of 12.6 months, 47/58 pts (40/46 pts in phase II part) were still on study treatment, and the median treatment duration has passed 10.3 months. The major treatment-related AEs were dysgeusia (21/58, 36%), rash, AST increased, blood bilirubin increased (19/58, 33% each), constipation and blood creatinine increased (17/58, 29% each), mostly grade 1-2. The major treatment-related grade 3 AEs were neutrophil count decreased (4/58, 7%). No grade 4 or fatal AEs were observed. Treatment-related visual disorders, vomiting and nausea were rare and mild. Conclusions: In patients with ALK-positive NSCLC but naïve to any ALK inhibitor therapy, treatment with CH5424802 demonstrated generally mild toxicity and lead to high response rate of 93.5% and durable treatment beyond 10 months, expecting that treatment duration can be longer than that of crizotinib. CH5424802 is therefore a promising new ALK inhibitor for NSCLC. Clinical trial information: JapicCTI-101264.

Abstract 3853: Humanized NOD/Scid/IL2g-/- tumor grafts recapitulate primary anaplastic large cell lymphoma.

Abstract Anaplastic large cell lymphoma (ALCL) is a subtype of peripheral T-cell lymphoma that includes two entities ALK+ and ALK- ALCL based on chromosomal translocations of the anaplastic lymphoma kinase (ALK). ALK- ALCL have very poor clinical outcome with conventional chemotherapy. Lack of representative cell lines and/or models has compromised the development of successful therapy. Toward this end, we report the generation and characterization of a group of novel ALCL tumor grafts. We implanted subcutaneously (s.c.) tumor tissue fragments, fresh or viable cryopreserved, from 11 cases of primary systemic ALCL (3 ALK+ and 8 ALK-) in six- to eight-week old severe immunocombined immunodeficiency NOD Cg-Prkdcscid/Il2rgtm1wjl/SzJ (NSG) mice. A single leukaemic ALCL sample was injected i.v. or s.c. Six cases led to successful lymphoma growth within 6-12 weeks as subcutaneous solid spheroid masses constituted by a uniform population of large pleomorphic cells CD30-positive. All tumor grafts were re-implanted in recipient animals, in same cases up to 21 consecutive implants with an analogous time growth rate. Two cases, in the same way of the original human neoplasia, were early associated with concomitant enlargement of multiple organs. All tumor graft lines eventually showed disseminate disease with involvement of distant lymph nodes and of spleen (with homig in peri-arteriolar spaces), liver (initially observed within the sinusoidal spaces) and kidney. The morphological and immunohistochemical (IHC) evaluation (expression of CD30, cytotoxic proteins and ALK) of the serially propagated ALCL tumor grafts demonstrated a high concordance between primary and derived animal tumors which showed highly stable profile along different passages from the early generation (T1) until the final ones (up to T16). The clonal relationship with primary and corresponding tumor grafts was confirmed by gene TCR rearrangement analyses and by ALK FISH studies. In addition we interrogated the model using gene expression profiling and single nucleotide polymorphism which established a close relationship among primary and derived tumors. Massive parallel sequencing on mRNA transcripts defines the presence of two novel chimeras involving NF1-MAZ and TRAF1-ALK1, which confirmes the evidence of ALK signaling and pinpoint a role of RAS in ALK- ALCL. The tumor grafts displayed, also, therapeutic responses with conventional therapy (CHOP) which reflected that seen in the donor patients: initial partial responses followed invariably by clinical relapses. To overcome this refractory we assayed the sensitivity of two tumorgraft lines, generated from ALK+ ALCL, to a new selective ALK inhibitor (CEP28122) that led a rapid and stable response. This study demonstrated the key role of these libraries of tumor grafts, especially in developing and testing new therapeutic regimens in refractory ALCL patients. Citation Format: Fabrizio Tabbo’, Antonella Barreca, Rodolfo Machiorlatti, Katia Messana, Indira Landra, Francesco Abate, Michela Boi, Maria Todaro, Cristina Abele, Domenico Novero, Alberto Zamo’, Marco Chilosi, Maurilio Ponzoni, Mangeng Cheng, Bruce Ruggeri, Pierpaolo Piccaluga, Stefano Pileri, Enrico Tiacci, Brunangelo Falini, Lenny Shultz, Roberto Piva, Enzo Medico, Francesco Bertoni, Raul Rabadan, Giorgio Inghirami. Humanized NOD/Scid/IL2g-/- tumor grafts recapitulate primary anaplastic large cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3853. doi:10.1158/1538-7445.AM2013-3853

Abstract 4465: Concurrent roles for IGF1R and EGFR in driving acquired resistance to crizotinib and ability to overcome with a combination of the ALK/IGF1R inhibitor AZD3463 and Iressa.

Abstract Genomic rearrangement of Anaplastic Lymphoma Kinase (ALK) has been observed in several tumor types including 3-6% of non small cell lung cancer (NSCLC). Although the ALK inhibitor crizotinib has clinical efficacy in selected ALK positive NSCLC patients, the majority of patients who show initial responses eventually relapse. Various mechanisms leading to resistance have been proposed and include ALK amplification and resistance mutations, as well as alternative pathway drivers including EGFR and cKIT. To further evaluate potential mechanisms of acquired resistance to crizotinib and potential therapeutic strategies to overcome them, we have developed multiple independently generated cell line derivatives. H3122 cells, a NSCLC line with an EML4-ALK fusion, was selected in increasing concentrations of crizotinib up to 1uM over a period of 4 to 6 months to generate H3122-CR lines. The cell lines generated were confirmed to be resistance to crizotinib as well as the selective ALK inhibitor, CH5424802, in cell proliferation experiments. Evaluation of the levels of potential secondary receptor tyrosine kinase (RTK) drivers by phosphoRTK arrays and western blotting revealed increased levels of total and phosphorylated ALK (pALK), along with increased phosphorylated insulin-like growth factor 1 receptor (pIGF1R). ALK levels were also elevated at the mRNA level. To evaluate potential strategies to most effectively overcome resistance in these cell lines, selective inhibitors of ALK CH5424802, IGF1R OSI-906 and epidermal growth factor receptor (EGFR) Iressa were used alone and in combination to evaluate ability to inhibit growth of two of the H3122-CR lines. In both CR lines, OSI-906 and Iressa had no effect alone on cell growth, however a combination of CH5424802 and OSI-906 or CH5424802 and Iressa increased sensitivity to CH5424802. Most interestingly however, a combination of CH5424802, OSI-906 and Iressa had maximal sensitization effect with complete reversal of resistance observed. A similar finding was obtained with a combination of the ALK/IGF1R inhibitor AZD3463 with Iressa. Further, we were able to demonstrate pathway dependency shifts in the CR lines with OSI-906 inhibiting AKT and Iressa inhibiting ERK and a combination of CH5424802, OSI-906 and Iressa required to effectively repress these main pathways to the level observed with CH5424802 alone in the H3122 parental line. Together, these data support a potential role for both EGFR and IGF1R as secondary RTK drivers occurring concurrently to drive resistance to crizotinib. A combination of approaches inhibiting ALK, IGF1R and EGFR together may therefore be required to most effectively treat crizotinib relapsed patients and delay onset of resistance. This could be achieved by combining multiple selective agents or agents with polypharmacology such as AZD3463 along with Iressa. Citation Format: Lisa Drew, Jeffrey Engelman, Ryohei Katayama, Brenda McDermott, Jamal Saeh, Meghan Scarpitti, Alice Shaw, Minhui Shen, Suping Wang, Graeme Smith. Concurrent roles for IGF1R and EGFR in driving acquired resistance to crizotinib and ability to overcome with a combination of the ALK/IGF1R inhibitor AZD3463 and Iressa. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4465. doi:10.1158/1538-7445.AM2013-4465

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.

IS7-14 - Results of a GlobaL Phase II Study with Crizotinib in Advanced ALK-Positive Non-Small-Cell Lung Cancer (NSCLC)

ABSTRACT Background Anaplastic lymphoma kinase (ALK) is a known oncogenic driver in NSCLC, and approximately 5% of patients harbor ALK gene rearrangements. Crizotinib is a first-in-class, selective small-molecule ALK inhibitor with demonstrated clinical activity in ALK-positive NSCLC. PROFILE 1005 is a global, multicenter, open-label, single-arm phase II study evaluating the safety and efficacy of crizotinib in previously treated patients with ALK-positive advanced NSCLC. Methods Crizotinib 250 mg was administered BID to patients with ALK-positive advanced NSCLC who had received ≥1 chemotherapy for locally advanced/metastatic disease, including those with treated brain metastases. Most patients had centrally confirmed ALK-positivity by break-apart fluorescence in-situ hybridization, defined as ≥15% of tumor cells with split signals. Disease response was evaluated by RECIST 1.1 every 6 weeks. Results As of January 2012, 901 patients had been dosed. The first 261 patients who had enrolled and started crizotinib by February 2011, with a median duration of treatment of 48 weeks, were considered to be the mature population. Demographic, exposure, and efficacy results in both the overall and mature populations are provided in the table. Among all 901 patients, 15% discontinued treatment due to adverse events (AEs) and 10% had a dose reduction due to an AE. The most frequent AEs of any cause were vision disorder (54%), nausea (51%), diarrhea (44%), vomiting (44%), and constipation (37%), which were mostly grade 1/2. Conclusions Crizotinib demonstrated a high response rate that was durable, with a median progression-free survival of 8.1 months. Crizotinib has a favorable tolerability profile. These findings continue to provide strong evidence for crizotinib as a standard of care for advanced ALK-positive NSCLC. Overall population Mature population Total no. of patients, n 901 261 Response-evaluable patients, n 803 259 Demographics: Median age, years Male/female, % ECOG PS 0/1, % Adenocarcinoma, % 53 43/57 82 92 52 46/54 83 94 Duration of treatment (weeks), median (range) 20 ( 48 (1 - 94) ORR, % (95% exact CI) 46 (42–50) 60 (54–66) Duration of response (weeks), median a (95% CI) 47 (36–54) 46 (35–54) Disease control rate, % (95% exact CI): At 6 weeks At 12 weeks 80 (77–83) 61 (58–64) 86 (82–90) 75 (70–80) PFS (months), median a (95% CI) 8.1 (6.9–9.5) 8.1 (6.8–9.7) ECOG PS, Eastern Cooperative Oncology Group performance status; ORR, objective response rate; PFS, progression-free survival. a Kaplan–Meier estimate Disclosure D. Kim: Compensated advisory relationship: Pfizer. Honoraria: Pfizer. S. Lanzalone: Employment: Pfizer. Stock ownership: Pfizer. A. Polli: Employment: Pfizer. Stock ownership: Pfizer. A. Shaw: Compensated advisory relationship: Pfizer, Ariad, Chugai, Novartis, Daiichi-Sankyo. Research funding: AstraZeneca, Novartis. All other authors have declared no conflicts of interest.

1230PD - Updated Results of a Global Phase II Study with Crizotinib in Advanced Alk-Positive Non-Small Cell Lung Cancer (NSCLC)

ABSTRACT Background Anaplastic lymphoma kinase (ALK) is a known oncogenic driver in NSCLC, and approximately 5% of patients harbor ALK gene rearrangements. Crizotinib is a first-in-class, selective small-molecule ALK inhibitor with demonstrated clinical activity in ALK-positive NSCLC. PROFILE 1005 is a global, multicenter, open-label, single-arm phase II study evaluating the safety and efficacy of crizotinib in previously treated patients with ALK-positive advanced NSCLC. Methods Crizotinib 250 mg was administered BID to patients with ALK-positive advanced NSCLC who had received ≥1 chemotherapy for locally advanced/metastatic disease, including those with treated brain metastases. Most patients had centrally confirmed ALK-positivity by break-apart fluorescence in-situ hybridization, defined as ≥15% of tumor cells with split signals. Disease response was evaluated by RECIST 1.1 every 6 weeks. Results As of January 2012, 901 patients had been dosed. The first 261 patients who had enrolled and started crizotinib by February 2011, with a median duration of treatment of 48 weeks, were considered to be the mature population. Demographic, exposure, and efficacy results in both the overall and mature populations are provided in the table. Among all 901 patients, 15% discontinued treatment due to adverse events (AEs) and 10% had a dose reduction due to an AE. The most frequent AEs of any cause were vision disorder (54%), nausea (51%), diarrhea (44%), vomiting (44%), and constipation (37%), which were mostly grade 1/2. Conclusions Crizotinib demonstrated a high response rate that was durable, with a median progression-free survival of 8.1 months. Crizotinib has a favorable tolerability profile. These findings continue to provide strong evidence for crizotinib as a standard of care for advanced ALK-positive NSCLC. Overall population Mature population Total no. of patients, n 901 261 Response-evaluable patients, n 803 259 Demographics: Median age, years Male/female, % ECOG PS 0/1, % Adenocarcinoma, % 53 43/57 82 92 52 46/54 83 94 Duration of treatment (weeks), median (range) 20 ( 48 (1 - 94) ORR, % (95% exact CI) 46 (42–50) 60 (54–66) Duration of response (weeks), median a (95% CI) 47 (36–54) 46 (35–54) Disease control rate, % (95% exact CI): At 6 weeks At 12 weeks 80 (77–83) 61 (58–64) 86 (82–90) 75 (70–80) PFS (months), median a (95% CI) 8.1 (6.9–9.5) 8.1 (6.8–9.7) ECOG PS, Eastern Cooperative Oncology Group performance status; ORR, objective response rate; PFS, progression-free survival. a Kaplan–Meier estimate Disclosure D. Kim: Compensated advisory relationship: Pfizer. Honoraria: Pfizer. S. Lanzalone: Employment: Pfizer. Stock ownership: Pfizer. A. Polli: Employment: Pfizer. Stock ownership: Pfizer. A. Shaw: Compensated advisory relationship: Pfizer, Ariad, Chugai, Novartis, Daiichi-Sankyo. Research funding: AstraZeneca, Novartis. All other authors have declared no conflicts of interest.

Anaplastic lymphoma kinase-directed therapy in inflammatory myofibroblastic tumors

Inflammatory myofibroblastic tumors (IMTs) are indolent mesenchymal neoplasms associated with a small risk of aggressive behavior and metastasis. Surgery is the mainstay of treatment and until recently there have been limited effective treatment options for unresectable disease. This review describes the identification of anaplastic lymphoma kinase (ALK) fusion genes in approximately 50% of IMTs and the role of ALK inhibition in the treatment of this disease. A recent phase I dose-escalation trial of the selective MET/ALK inhibitor crizotinib showed a long-term partial response in a patient with IMT carrying an ALK translocation but not in a patient with ALK-negative disease. Emergence of resistance to crizotinib occurs approximately 5-8 months after initiation of therapy and has been shown to be driven by different mechanisms. Multiple second-generation ALK inhibitors are currently being investigated in the preclinical and clinical trial setting. ALK-directed therapy has emerged as a highly effective treatment option for a subset of patients with IMT and pulmonary adenocarcinoma. A number of additional malignancies, including rhabdomyosarcoma, neuroblastoma, anaplastic large cell lymphoma, renal cell carcinoma, and inflammatory breast cancer, have been shown to activate ALK expression by means of ALK fusion proteins, ALK mutations, or increased ALK copy number. Development of more selective ALK inhibitors, which can overcome emergent crizotinib resistance mutations, as well as development of combination treatments with drugs targeting compensatory pathways, will be key to achieving therapeutic success in targeting this potent and prevalent oncogenic driver.

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.

Rapid Development of Piperidine Carboxamides as Potent and Selective Anaplastic Lymphoma Kinase Inhibitors

Piperidine carboxamide 1 was identified as a novel inhibitor of anaplastic lymphoma kinase (ALK enzyme assay IC(50) = 0.174 μM) during high throughput screening, with selectivity over the related kinase insulin-like growth factor-1 (IGF1R). The X-ray cocrystal structure of 1 with the ALK kinase domain revealed an unusual DFG-shifted conformation, allowing access to an extended hydrophobic pocket. Structure-activity relationship (SAR) studies were focused on the rapid parallel optimization of both the right- and left-hand side of the molecule, culminating in molecules with improved potency and selectivity over IGF1R.

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.

NPM-ALK signals through glycogen synthase kinase 3β to promote oncogenesis

Anaplastic large cell lymphoma (ALCL) is the most common type of pediatric peripheral T-cell lymphoma. In 70-80% of cases, the chromosomal aberration t(2;5)(p23;q35) results in the juxtaposition of anaplastic lymphoma kinase (ALK) with nucleophosmin (NPM) and the subsequent expression of the NPM-ALK fusion protein. NPM-ALK is a chimeric tyrosine kinase, which induces numerous signaling pathways that drive proliferation and abrogate apoptosis. However, the mechanisms that lead to activation of downstream growth regulatory molecules have not been completely elucidated. Using a mass spectrometry-based phosphoproteomic screen, we identified GSK3β as a signaling mediator of NPM-ALK. Using a selective inhibitor of ALK, we demonstrated that the tyrosine kinase activity of ALK regulates the serine-9 phosphorylation of GSK3β. Expression of NPM-ALK in 293T cells led to an increase of pS(9)-GSK3β (glycogen synthase kinase 3 beta) compared with kinase-defective K210R mutant NPM-ALK, but did not affect total GSK3β levels. Phosphorylation of pS(9)-GSK3β by NPM-ALK was mediated by the PI3K/AKT signaling pathway. ALK inhibition resulted in degradation of GSK3β substrates Mcl-1 and CDC25A, which was recovered upon chemical inhibition of the proteasome (MG132). Furthermore, the degradation of Mcl-1 was recoverable with inhibition of GSK3β. ALK inhibition also resulted in decreased cell viability, which was rescued by GSK3β inhibition. Furthermore, stable knockdown of GSK3β conferred resistance to the growth inhibitory effects of ALK inhibition using viability and colony formation assays. pS(9)-GSK3β and CDC25A were selectively expressed in neoplastic cells of ALK+ALCL tissue biopsies, and showed a significant correlation (P<0.001). Conversely, ALK-ALCL tissue biopsies did not show significant correlation of pS(9)-GSK3β and CDC25A expression (P<0.2). Our results demonstrate that NPM-ALK regulates the phosphorylation of S(9)-GSK3β by PI3K/AKT. The subsequent inhibition of GSK3β activity results in accumulation of CDC25A and Mcl-1, which confers the advantage of growth and protection from apoptosis. These findings provide support for the role of GSK3β as a mediator of NPM-ALK oncogenesis.

NPM-ALK Regulated GSK3β Phosphorylation Results in Mcl-1 and CDC25A Accumulation and Enhanced Oncogenic Potential

Abstract 586Anaplastic large cell lymphoma (ALCL) is the most common type of pediatric peripheral T-cell lymphoma. ALCLs are frequently characterized by translocations involving the anaplastic lymphoma kinase (ALK) gene. In 70–80% of these cases, the chromosomal aberration t(2;5)(p23;q35) results in the juxtaposition of ALK with nucleophosmin (NPM) and the subsequent expression of the NPM-ALK tyrosine kinase. NPM-ALK is an oncogenic tyrosine kinase which induces numerous signaling pathways that drive proliferation and abrogate apoptosis. However, the mechanisms that lead to activation of downstream growth regulatory molecules in ALCLs have not been completely elucidated. Using a mass spectrometry-based phosphoproteomic screen, we identified GSK3β, as a potential signaling mediator of NPM-ALK. Using a selective inhibitor of ALK, we demonstrated that the tyrosine kinase activity of ALK regulates the phosphorylation of GSK3β at serine-9 (pS9-GSK3β) in a time and dose-dependent manner. Expression of NPM-ALK in 293T cells led to increased phosphorylation of GSK3β at serine-9 compared to kinase-defective K210R mutant NPM-ALK but did not affect total GSK3β levels. Inducible knockdown of ALK in SU-DHL-1 cells also led to decreased phosphorylation of GSK3β at serine-9 without affecting total GSK3β levels. Selective inhibition of PI3Kδ by CAL-101 resulted in decreased phosphorylation of GSK3β at serine-9 in a dose dependent manner without affecting total GSK3β or NPM-ALK activity. Ablation of ALK kinase activity resulted in proteasomal degradation of GSK3β substrates Mcl-1 and CDC25A. This degradation was recovered upon chemical inhibition of GSK3β (GSK3 IX). To address the role of GSK3β in ALCL proliferation, WST-1 assay revealed that ALK inhibition resulted in a decrease in cell viability which was rescued by GSK3β inhibition. ALK inhibition resulted in significant cellular apoptosis as detected by propidium iodide and Annexin V staining followed by flow cytometry. This apoptotic response was rescued by GSK3β inhibition. The effect of GSK3β on cellular oncogenic potential was addressed by colony formation assay using ALCL-derived cell lines. While ALK inhibition resulted in decreased colony numbers, simultaneous ALK and GSK3β inhibition recovered colony numbers compared to ALK inhibition alone. Additionally, stable GSK3β knockdown conferred resistance to growth inhibitory effect of ALK inhibition as determined by colony formation assay. Furthermore, pS9-GSK3β and its known substrate, CDC25A were selectively expressed in neoplastic cells of ALK+ALCL tissue biopsies and showed a significant correlation (p<0.001). Conversely, ALK-ALCL tissue biopsies showed no significant correlation of pS9-GSK3β and CDC25A expression (p<0.2). Our results demonstrate that NPM-ALK signals through PI3K/AKT to phosphorylate GSK3β at serine-9. This phosphorylation inhibits GSK3β kinase activity which results in the accumulation of CDC25A and Mcl-1 thus providing growth advantage and protection from apoptosis. These findings provide support for the role of GSK3β as a novel mediator of NPM-ALK oncogenesis. Disclosures:No relevant conflicts of interest to declare.

Abstract 3559: A new selective ALK inhibitor CH5424802 shows potent efficacy against ALK-positive cancers including the gatekeeper mutant-driven tumors

Abstract The recent development of targeted protein kinase inhibitors has provided new opportunity 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. Thus, kinase inhibitors with more potent and selective properties and effectiveness against resistant mutants need to be developed. 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. Here, we identified CH5424802, a potent, selective, and orally available ALK inhibitor with a new chemical scaffold, showing preferential antitumor activity against cancers with gene alterations of ALK, such as non-small cell lung cancer (NSCLC) cells expressing EML4-ALK fusion and anaplastic large-cell lymphoma (ALCL) cells expressing NPM-ALK fusion in vitro and in vivo. CH5424802 inhibited ALK L1196M, which corresponds to the gatekeeper mutation conferring common resistance to kinase inhibitors, and blocked EML4-ALK L1196M-driven tumors. Our results support the potential for the clinical evaluation of CH54242802 for long-term treatment of patients with ALK-driven tumors. CH5424802 is currently being investigated in Phase I/II clinical trials for patients with ALK-positive NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3559. doi:10.1158/1538-7445.AM2011-3559

Comparison of 2 Cell-Based Phosphoprotein Assays to Support Screening and Development of an ALK Inhibitor

Anaplastic lymphoma kinase (ALK) when expressed as a fusion protein with nucleophosmin (NPM) has been implicated as a driving oncogene in a subset of lymphomas. Recent reports of ALK expression in a number of other cancers have raised the possibility that an ALK inhibitor may benefit patients with these diseases as well. In a campaign to identify and develop a selective ALK inhibitor, 2 assays were devised to measure the phosphorylation of tyrosine residue 1604 of ALK (pY(1604) ALK). Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen(®)) and phosflow platforms were used to detect modulation of pY(1604) ALK to determine the relative potency of a set of small-molecule inhibitors. Prior to making use of these assays in diverse settings, the authors attempted to ensure their equivalence with a direct comparison of their performance. The pY(1604) ALK assays correlated well both with each other and with assays of ALK enzyme activity or ALK-dependent cell proliferation. The AlphaScreen(®) assay was amenable to automation and enabled rapid, high-throughput compound assessment in an NPM-ALK-driven cell line, whereas the phosflow assay enabled the authors to characterize the activity of compounds with respect to their impact on targeted enzymes and pathways. Results show that both AlphaScreen(®) and phosflow ALK assays exhibited diverse characteristics that made them desirable for different applications but were determined to be equally sensitive and robust in the detection of inhibition of pY(1604) ALK.

Crystal Structures of Anaplastic Lymphoma Kinase in Complex with ATP Competitive Inhibitors

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase involved in the development of several human cancers and, as a result, is a recognized target for the development of small-molecule inhibitors for the treatment of ALK-positive malignancies. Here, we present the crystal structures of the unphosphorylated human ALK kinase domain in complex with the ATP competitive ligands PHA-E429 and NVP-TAE684. Analysis of these structures provides valuable information concerning the specific characteristics of the ALK active site as well as giving indications about how to obtain selective ALK inhibitors. In addition, the ALK-KD-PHA-E429 structure led to the identification of a potential regulatory mechanism involving a link made between a short helical segment immediately following the DFG motif and an N-terminal two-stranded beta-sheet. Finally, mapping of the activating mutations associated with neuroblastoma onto our structures may explain the roles these residues have in the activation process.

Abstract 3623: Efficacy and pharmacodynamic analysis of AP26113, a potent and selective orally active inhibitor of Anaplastic Lymphoma Kinase (ALK)

Abstract Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified in anaplastic large cell lymphoma (ALCL; NPM-ALK) and non-small cell lung cancer (NSCLC; EML4-ALK). The dual Met/ALK inhibitor PF-02341066 (PF1066) has demonstrated promising clinical activity against tumors carrying activating ALK gene rearrangements (Kwak ASCO 2009: #3509) validating ALK as a therapeutic target. Previously, AP26113 was identified as a novel, potent, orally bioavailable ALK inhibitor with demonstrated selectivity over related receptor tyrosine kinase family members IGF-1R and InsR and no inhibition of Met. Here the efficacy and exposure/activity relationship of AP26113 was further characterized in preclinical models and compared to that of PF1066. In a panel of 7 EML4-ALK or NPM-ALK positive NSCLC and ALCL cell lines, the concentration of AP26113 that inhibited growth by 50% (GI50) ranged from 4.2 - 30.8 nM. In each cell line the GI50 for PF1066 was ∼10-fold greater (range 62 - 309 nM). In 4 cells lines tested, the IC50 for inhibition of ALK phosphorylation tracked with potency in cell proliferation assays and was 10-fold greater for PF1066 than AP26113. Across 3 ALK-negative NSCLC and ALCL cell lines the GI50s for AP26113 (503 - 2387 nM) and PF1066 (928 - 1773 nM) were similar. Overall, AP26113 exhibited ∼100-fold selectivity for ALK-positive lines compared with a ∼10-fold selectivity for PF1066. The in vivo activities of daily oral dosing of AP26113 (10, 25 and 50 mg/kg) and PF1066 (25, 50 and 100 mg/kg) were examined in Karpas-299 ALCL (2 week dosing) and H3122 NSCLC (3 week dosing) xenograft models. At the highest doses tested, strong regressions were achieved with AP26113, but not PF1066. Tumor growth inhibition by 25 mg/kg and 10 mg/kg doses of AP26113 in the ALCL and NSCLC models, respectively, was similar to that of 100 mg/kg PF1066. In a PK/PD study in the ALCL model, inhibition of ALK phosphorylation after administration of 100 mg/kg PF1066 was intermediate between that observed after administration of 10 or 25 mg/kg AP26113. Results from the analysis of plasma levels of each drug showed that AP26113 had equivalent efficacy to PF1066 at 4- to 10-fold lower levels of exposure (AUC and 24 h trough plasma levels). AP26113 demonstrated favorable properties including moderate in vitro plasma protein binding (&amp;#8804;77% in mouse, rat, monkey, and human plasma), negligible inhibition of major CYP isoforms (IC50 &amp;gt; 10 &amp;#956;M for 3A4, 2C9, 2D6), and good oral bioavailability (multiple animal species). In animal models, AP26113 was well-tolerated at and above predicted clinically effective plasma levels. In conclusion, these data demonstrate that AP26113 is a highly potent and selective inhibitor of ALK and support the clinical evaluation of AP26113 in patients with ALK-driven tumors. 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 3623.