Approved Anaplastic Lymphoma Kinase Inhibitor Research Articles

NVL-655 Is a Selective and Brain-Penetrant Inhibitor of Diverse ALK-Mutant Oncoproteins, Including Lorlatinib-Resistant Compound Mutations.

Three generations of tyrosine kinase inhibitors (TKI) have been approved for anaplastic lymphoma kinase (ALK) fusion-positive non-small cell lung cancer. However, none address the combined need for broad resistance coverage, brain activity, and avoidance of clinically dose-limiting TRK inhibition. NVL-655 is a rationally designed TKI with >50-fold selectivity for ALK over 96% of the kinome tested. In vitro, NVL-655 inhibits diverse ALK fusions, activating alterations, and resistance mutations, showing ≥100-fold improved potency against ALKG1202R single and compound mutations over approved ALK TKIs. In vivo, it induces regression across 12 tumor models, including intracranial and patient-derived xenografts. NVL-655 inhibits ALK over TRK with 22-fold to >874-fold selectivity. These preclinical findings are supported by three case studies from an ongoing first-in-human phase I/II trial of NVL-655 which demonstrate preliminary proof-of-concept clinical activity in heavily pretreated patients with ALK fusion-positive non-small cell lung cancer, including in patients with brain metastases and single or compound ALK resistance mutations. Significance: By combining broad activity against single and compound ALK resistance mutations, brain penetrance, and selectivity, NVL-655 addresses key limitations of currently approved ALK inhibitors and has the potential to represent a distinct advancement as a fourth-generation inhibitor for patients with ALK-driven cancers.

An ultra-fast green ultra-high-performance liquid chromatography-tandem mass spectrometry method for estimating the in vitro metabolic stability of zotizalkib in human liver microsomes.

Zotizalkib (ZTK, TPX-0131) is a fourth-generation highly effective inhibitor of wild-type anaplastic lymphoma kinase (ALK) and ALK-resistant mutations that can penetrate the central nervous system. It exhibited greater potency compared to all five officially approved ALK inhibitors. The aim of this study was to develop a rapid, accurate, eco-friendly, and highly sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for measuring the concentration of ZTK in human liver microsomes (HLMs). The validation aspects of the current UHPLC-MS/MS methodology in the HLMs were conducted in accordance with the bioanalytical method validation standards specified by the US Food and Drug Administration. ZTK and encorafenib were separated using an Agilent C8 column (Eclipse Plus) and an isocratic mobile phase. The calibration curve for the developed ZTK exhibited a linear relationship within the concentration range of 1-3000ng/mL. The results from the Analytical Green-ness Metric Approach program (0.76) suggested that the created method demonstrated a significant degree of environmental sustainability. The in vitro half-life (t1/2) and intrinsic clearance (Clint) of ZTK were determined to be 15.79min and 51.35mL/min/kg, respectively that suggests the ZTK exhibits characteristics similar to those of a medication with a high extraction ratio. These approaches are crucial for the progress of novel pharmaceutical development, especially in improving metabolic stability.

First-line treatment option for patients with ALK-positive metastatic NSCLC

Non-small cell lung cancer (NSCLC) that occupies a leading place in the pattern of cancer incidence and mortality is a highly heterogeneous group of diseases. The presence of a wide spectrum of NSCLC driver mutations has led to a fundamentally different understanding of the treatment strategy for this cohort of patients and a significant improvement in long-term oncological outcomes, even in the metastatic process. Chromosomal rearrangements involving the anaplastic lymphoma kinase (ALK) gene loci on chromosome 2 are found in approximately 3–5% of patients with metastatic NSCLC (mNSCLC) and in most cases are associated not only with a number of specific clinical features, but also with high sensitivity to targeted therapy with tyrosine kinase inhibitors (TKI). Crizotinib was the first approved ALK inhibitor, but although most patients achieved response within the first two years after start of the treatment, disease progression occurred often due to intracranial injury. The development of second-(ceritinib, alectinib), brigatinib and third-generation (lorlatinib) drugs has led to a statistically significant improvement in progression-free survival (PFS) rates, as well as control over intracranial manifestations of the disease and a change in the initial treatment strategy for these patients. In addition, new-generations of TKIs were developed to solve the problem of acquired resistance, as well as to achieve the best outcomes in the presence of such unfavourable factors as the presence of a TP53 mutation and/or ALK inhibitor low-sensitive translocation variants of the intracellular kinase domain of EML4 (echinoderm microtubule‐associated protein‐like 4)‐ALK (anaplastic lymphoma kinase) protein. Thus, advances in the therapeutic options for ALK-positive mNSCLC has completely changed the course of the disease, resulting in a significant increase in overall survival (OS) rates not only with the sequential use of different generation TKIs, but also with the choice of the most effective first-line option. In this article, we present an analysis of data on the efficacy and toxicity of lorlatinib, a third-generation TKI, in the first-line treatment for ALK+ mNSCLC.

Optimal first-line treatment for metastatic ALK+ non-small cell lung cancer-a narrative review.

First-line treatment options for patients with advanced non-small cell lung cancer (aNSCLC) whose tumors harbour anaplastic lymphoma kinase (ALK) gene rearrangements have rapidly evolved from chemotherapy, to the first in class ALK-targeted tyrosine kinase inhibitor (TKI) crizotinib in 2011, and now include no fewer than five Food and Drug Administration (FDA)-approved ALK inhibitors. However, while superiority to crizotinib has been established, head-to-head clinical trials comparing newer generation ALK inhibitors are lacking, and decisions on optimal first-line treatment must be based on analysis of the relevant trials, with attention to systemic and intracranial efficacy, toxicity profile as well as consideration of patient factors and preferences. Here we aim to synthesise findings from review of these trials and to describe options for optimal first-line treatment for ALK+ NSCLC. A literature review of relevant randomised clinical trials was undertaken using Embase database. There were no limitations to time frame or language applied. Crizotinib was established as the standard of care first-line treatment for patients with ALK+ aNSCLC in 2011. Since this time, alectinib, brigatinib, ensartinib and lorlatinib have all demonstrated superiority as first-line treatments compared to crizotinib, based on progression free survival, intra-cranial efficacy, and side-effect profiles. Options for optimal first-line treatment for ALK+ aNSCLC include alectinib, brigatinib and lorlatinib. This review serves as a resource summarizing data from key clinical trials with ALK inhibitors to aid in decision making when tailoring treatment for patients. Future research in the field includes real world analysis of efficacy and toxicity of next-generation ALK-inhibitors, identification of mechanisms of tumor persistence and acquired resistance, development of novel ALK inhibitors, and use of ALK-TKIs in earlier stage disease.

The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain.

The anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase known for its oncogenic potential that is involved in the development of the peripheral and central nervous system. ALK receptor ligands ALKAL1 and ALKAL2 were recently found to promote neuronal differentiation and survival. Here, we show that inflammation or injury enhanced ALKAL2 expression in a subset of TRPV1+ sensory neurons. Notably, ALKAL2 was particularly enriched in both mouse and human peptidergic nociceptors, yet weakly expressed in nonpeptidergic, large-diameter myelinated neurons or in the brain. Using a coculture expression system, we found that nociceptors exposed to ALKAL2 exhibited heightened excitability and neurite outgrowth. Intraplantar CFA or intrathecal infusion of recombinant ALKAL2 led to ALK phosphorylation in the lumbar dorsal horn of the spinal cord. Finally, depletion of ALKAL2 in dorsal root ganglia or blocking ALK with clinically available compounds crizotinib or lorlatinib reversed thermal hyperalgesia and mechanical allodynia induced by inflammation or nerve injury, respectively. Overall, our work uncovers the ALKAL2/ALK signaling axis as a central regulator of nociceptor-induced sensitization. We propose that clinically approved ALK inhibitors used for non–small cell lung cancer and neuroblastomas could be repurposed to treat persistent pain conditions.

TPX-0131, a Potent CNS-penetrant, Next-generation Inhibitor of Wild-type ALK and ALK-resistant Mutations.

Since 2011, with the approval of crizotinib and subsequent approval of four additional targeted therapies, anaplastic lymphoma kinase (ALK) inhibitors have become important treatments for a subset of patients with lung cancer. Each generation of ALK inhibitor showed improvements in terms of central nervous system (CNS) penetration and potency against wild-type (WT) ALK, yet a key continued limitation is their susceptibility to resistance from ALK active-site mutations. The solvent front mutation (G1202R) and gatekeeper mutation (L1196M) are major resistance mechanisms to the first two generations of inhibitors while patients treated with the third-generation ALK inhibitor lorlatinib often experience progressive disease with multiple mutations on the same allele (mutations in cis, compound mutations). TPX-0131 is a compact macrocyclic molecule designed to fit within the ATP-binding boundary to inhibit ALK fusion proteins. In cellular assays, TPX-0131 was more potent than all five approved ALK inhibitors against WT ALK and many types of ALK resistance mutations, e.g., G1202R, L1196M, and compound mutations. In biochemical assays, TPX-0131 potently inhibited (IC50 <10 nmol/L) WT ALK and 26 ALK mutants (single and compound mutations). TPX-0131, but not lorlatinib, caused complete tumor regression in ALK (G1202R) and ALK compound mutation-dependent xenograft models. Following repeat oral administration of TPX-0131 to rats, brain levels of TPX-0131 were approximately 66% of those observed in plasma. Taken together, preclinical studies show that TPX-0131 is a CNS-penetrant, next-generation ALK inhibitor that has potency against WT ALK and a spectrum of acquired resistance mutations, especially the G1202R solvent front mutation and compound mutations, for which there are currently no effective therapies.

P84.08 First-line Brigatinib in Anaplastic Lymphoma Kinase-positive Non-small Cell Lung Cancer: A Network Meta-Analysis

Anaplastic Lymphoma Kinase (ALK)-targeted tyrosine kinase inhibitors have shown to be effective as first-line (1L) treatment of ALK-positive non-small cell lung cancer (NCSLC). However, head-to-head comparisons between most available ALK inhibitors are lacking. The objective of this study was to conduct a systematic literature review (SLR) and meta-analyses to estimate the relative efficacy of brigatinib compared to other approved ALK inhibitors or chemotherapy in patients with locally advanced or metastatic ALK inhibitor-naïve ALK-positive NSCLC (≤1 prior chemotherapy regimen was allowed).

Long-term complete response in a patient with postoperative recurrent ALK-rearranged lung adenocarcinoma treated with crizotinib: A case report.

Anaplastic lymphoma kinase (ALK) gene rearrangements are identified in approximately 5% of patients with non-small cell lung cancer (NSCLC). Despite initial dramatic responses to ALK inhibitors, the majority of patients relapse within 1 year, owing to the development of resistance. Herein we present a case of variant type 2 ALK-rearranged lung adenocarcinoma recurrence with multiple lung metastasis that maintained complete response over 5 years with crizotinib, which is the first approved ALK inhibitor. The efficacy of crizotinib may vary among ALK fusion variants and thus, variant type may represent an important factor in guiding the treatment strategy for ALK-rearranged lung adenocarcinoma.

The safety and serious adverse events of approved ALK inhibitors in malignancies: a meta-analysis.

Background: A total of 2%–7% of non-small cell lung cancer (NSCLC) patients have anaplastic lymphoma kinase (ALK) mutations. At present, three or more generations of ALK inhibitors have been used for ALK-positive NSCLC treatment, including crizotinib, alectinib, ceritinib, and brigatinib. Although most adverse events (AEs) of ALK inhibitors are grades 1 to 2 and generally can be well tolerated, serious adverse events (SAEs) of ALK inhibitors lack data analysis, and the lung toxicity of ALK inhibitors needs attention. Thus, we performed this meta-analysis to evaluate the safety of ALK inhibitors, especially in terms of drug-related SAEs.Methods: A total of 19 studies from 4 databases (PubMed, Science Direct, ClinicalTrials.gov and Cochrane Library) were included in this meta-analysis. All statistical analyses in this meta-analysis were performed with the STATA 14.0 software. We analyzed the incidences of total AEs, total SAEs and SAEs for different ALK inhibitors.Results: AEs of the ALK inhibitors occurred in almost all participants, and SAEs occurred in more than 20% of the participants. For ceritinib and brigatinib, SAEs occurred in more than 40% of the participants. Alectinib is most likely the safest of the two generations of ALK inhibitors. Generally, the ALK inhibitors showed significant lung toxicity.Conclusion: In conclusion, attention should be focused on ALK inhibitor-related SAEs, especially lung toxicity. According to this meta-analysis, alxectinib seems to be the safest ALK inhibitor. Physicians should focus on the related SAEs when prescribing ALK inhibitors.

PO-314 ALK inhibitor, ceritinib, abrogates activation of the novel ALK-I1171T mutation in neuroblastoma

IntroductionMutations in the kinase domain of Anaplastic Lymphoma Kinase (ALK) are undoubtedly implicated in the pathogenesis of the childhood cancer, neuroblastoma. However, clinical response of ALK positive neuroblastoma patients to the first generation ALK inhibitor has been rather disappointing.Here we report the appearance of a novel ALK mutation in neuroblastoma together with other chromosomal aberrations that mediate neuroblastoma initiation and progression.Material and methodsGenomic tumour DNA from biopsy samples were extracted and exome sequencing was performed through paired-end sequencing on Illumina plateforms.The novel ALK-I1171T mutant was biochemically analysed by western blot and neurite-outgrowth assay in PC12 cells, and foci formation assay in NIH3T3 cells.Results and discussionsAnalyses of genomic tumour DNA from biopsy samples initially showed an 11q deletion, 17q gain with a mutation of the ALK gene at protein position 1171, which mediate an amino acid change from isoleucine to threonine. We show that mutation of I1171 to threonine generates a potent gain-of-function mutant, as observed in two independent systems. Firstly, in PC12 cell lines expressing ALK-I1171T display ligand independent activation of ALK, neurite outgrowth and further downstream signalling activation. Secondly, ALK-I1171T meditate foci formation in a NIH3T3 transformation analysis. Finally, pharmacological inhibition of ALK-I1171T employing ceritinib, an FDA approved ALK inhibitor show 14-fold better ability to abrogate ALK-I1171T compared with crizotinib.ConclusionThis study suggests that ceritinib presents a viable therapeutic option for ALK-positive neuroblastoma.

Clinical Pharmacokinetics of Anaplastic Lymphoma Kinase Inhibitors in Non-Small-Cell Lung Cancer.

The identification of anaplastic lymphoma kinase rearrangements in 2-5% of patients with non-small-cell lung cancer led to rapid advances in the clinical development of oral tyrosine kinase inhibitors. Anaplastic lymphoma kinase inhibitors are an effective treatment in preclinical models and patients with anaplastic lymphoma kinase-translocated cancers. Four anaplastic lymphoma kinase inhibitors (crizotinib, ceritinib, alectinib, and brigatinib) have recently been approved. Post-marketing studies provided additional pharmacokinetic information on their pharmacokinetic parameters. The pharmacokinetic properties of approved anaplastic lymphoma kinase inhibitors have been reviewed herein. Findings from additional studies on the effects of drug-metabolizing enzymes, drug transporters, and drug-drug interactions have been incorporated. Crizotinib, ceritinib, and alectinib reach their maximum plasma concentrations after approximately 6h and brigatinib after 1-4h. These drugs are primarily metabolized by cytochrome P450 3A with other cytochrome P450 enzymes. They are mainly excreted in the feces, with only a minor fraction being eliminated in urine. Crizotinib, ceritinib, and brigatinib are substrates for the adenosine triphosphate binding-cassette transporter B1, whereas alectinib is not. The different substrate specificities of the transporters play a key role in superior blood-brain barrier penetration by alectinib than by crizotinib and ceritinib. Although the absorption, distribution, and excretion of anaplastic lymphoma kinase inhibitors are regulated by drug transporters, their transporter-mediated pharmacokinetics have not yet been elucidated in detail in patients with non-small-cell lung cancer. Further research to analyze the contribution of drug transporters to the pharmacokinetics of anaplastic lymphoma kinase inhibitors in patients with non-small-cell lung cancer will be helpful for understanding the mechanisms of the inter-individual differences in the pharmacokinetics of anaplastic lymphoma kinase inhibitors.

Abstract B40: A novel activating I1171T ALK mutation in neuroblastoma responds better to ceritinib compare to the first generation inhibitor crizotinib

Abstract Mutations in the kinase domain of Anaplastic Lymphoma Kinase (ALK) have been suggested to be important players in the genetics and progression of the childhood tumor neuroblastoma. Here we report the appearance of a novel ALK mutation in neuroblastoma together with other chromosomal aberrations that mediate neuroblastoma initiation and progression. Analyses of genomic tumor DNA from biopsy samples initially showed an 11q minus, 17q gain with a mutation of the ALK gene at protein position 1171, which mediate an amino acid change from isoleucine to threonine. We show that mutation of I1171 to threonine generates a potent gain-of-function mutant, as observed in two independent systems. Firstly, in PC12 cell lines expressing ALKI1171T display ligand independent activation of ALK, neurite outgrowth and further downstream signaling activation. Secondly, ALKI1171T meditate foci formation in a NIH3T3 transformation analysis. Finally, pharmacological inhibition of ALKI1171T employing ceritinib, an FDA approved ALK inhibitor show 14-fold better ability to abrogate ALKI1171T compared with crizotinib. Citation Format: Joachim T. Siaw, Jikui Guan, Tommy Martinsson, Magnus Hansson, Per Kogner, Ruth Palmer. A novel activating I1171T ALK mutation in neuroblastoma responds better to ceritinib compare to the first generation inhibitor crizotinib [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B40.

Spotlight on brigatinib and its potential in the treatment of patients with metastatic ALK-positive non-small cell lung cancer who are resistant or intolerant to crizotinib.

In the last decade, there have been major therapeutic advances in the management of patients with anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer. Crizotinib was the first approved ALK inhibitor with significant benefits over chemotherapy. However, patients inevitably develop disease progression especially in central nervous system and acquire resistance to crizotinib. Several next-generation ALK inhibitors have been developed to overcome these resistance mechanisms and have demonstrated clinical benefits in crizotinib-refractory non-small cell lung cancer including in central nervous system. Brigatinib is a second-generation ALK inhibitor with high level of activity against ALK and several other targets. It is active in vitro against many ALK kinase domain mutations including L1196M, E1210K, and G1202R which may mediate acquired resistance to other ALK inhibitors. In Phase I/II and ALTA clinical studies, brigatinib has demonstrated substantial and durable responses and intracranial responses after progression on crizotinib. It has acceptable safety profile, but early pulmonary toxicity has been observed prompting to pursue daily dosing of 180 mg (with lead-in). Overall, 180 mg (with lead-in) has showed consistently better efficacy than 90 mg. In this review, we will discuss in detail these two pivotal trials that led to the accelerated approval for brigatinib and its future directions.

Discovery of a potent dual ALK and EGFR T790M inhibitor

The mutational activations of anaplastic lymphoma kinase (ALK) and epidermal growth factor receptor (EGFR) are validated oncogenic events and the targets of approved drugs to treat non-small cell lung cancer (NSCLC). Here we report highly potent dual small molecule inhibitors of both ALK and EGFR, particularly the T790M mutant which confers resistance to first generation EGFR inhibitors. Dual ALK/EGFR inhibitors may provide an efficient approach to prevent resistance that arises as a consequence of clinically reported reciprocal activation mechanisms. Our lead compound 7c displayed remarkable inhibitory activities against both ALK and EGFR in enzymatic and cellular assays. We demonstrate that 7c is capable of recapitulating the signaling effects and antiproliferative activity of combined treatment with the approved ALK inhibitor ceritinib and T790M EGFR inhibitor osimertinib against patient-derived non-small cell lung cancer cell line, DFCI032 which harbors both EML4-ALK and activated EGFR.

Optimizing treatment for patients with anaplastic lymphoma kinase-positive lung cancer.

In the 9 years since the initial discovery of anaplastic lymphoma kinase (ALK) gene rearrangements in non-small cell lung cancer (NSCLC), there has been tremendous progress, culminating in an ever-expanding repertoire of agents that have activity in this disease. This review article provides an overview of currently approved ALK inhibitors, other ALK inhibitors in development, and commonly described mechanisms of resistance to ALK inhibitors. We also discuss emerging controversies in treatment of patients with ALK-positive lung cancer, including the choice of first-line ALK inhibitor and the role of tyrosine kinase inhibitors in the treatment of central nervous system metastases.

Aberrant expression of anaplastic lymphoma kinase in lung adenocarcinoma: Analysis of circulating free tumor RNA using one-step reverse transcription-polymerase chain reaction.

Lung adenocarcinoma patients harboring anaplastic lymphoma kinase (ALK) gene rearrangements respond well to approved ALK inhibitors. However, to date, limited evidence is available regarding whether using circulating free tumor mRNA to identify aberrant ALK expression is possible, and its feasibility remains to be clearly addressed. The present study evaluated ALK expression by a one-step reverse transcription‑polymerase chain reaction (PCR) assay on the circulating free tumor mRNA from12lung adenocarcinoma patients. Additionally, the present study tested for ALK rearrangements by fluorescence insitu hybridization (FISH) and immunohistochemistry. A molecular genetic characterization was performed on tumor tissues and plasma samples. Aberrant ALK expression was detected in2/12patients using mRNA purified from plasma specimens and the results agreed with the FISH and immunohistochemistry findings of solid biopsy samples. The detection of aberrant ALK expression on circulating free tumor RNA may be feasible using a one‑step real‑time PCR assay and may be particularly helpful when a solid biopsy sample is not available.

Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK-positive neuroblastoma cells, Drosophila and mice.

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor which has been implicated in numerous solid and hematologic cancers. ALK mutations are reported in about 5-7% of neuroblastoma cases but the ALK-positive percentage increases significantly in the relapsed patient population. Crizotinib, the first clinically approved ALK inhibitor for the treatment of ALK-positive lung cancer has had less dramatic responses in neuroblastoma. Here we investigate the efficacy of a second-generation ALK inhibitor, brigatinib, in a neuroblastoma setting. Employing neuroblastoma cell lines, mouse xenograft and Drosophila melanogaster model systems expressing different constitutively active ALK variants, we show clear and efficient inhibition of ALK activity by brigatinib. Similar abrogation of ALK activity was observed in vitro employing a set of different constitutively active ALK variants in biochemical assays. These results suggest that brigatinib is an effective inhibitor of ALK kinase activity in ALK addicted neuroblastoma that should be considered as a potential future therapeutic option for ALK-positive neuroblastoma patients alone or in combination with other treatments.

Structural insight into selectivity and resistance profiles of ROS1 tyrosine kinase inhibitors

Oncogenic ROS1 fusion proteins are molecular drivers in multiple malignancies, including a subset of non-small cell lung cancer (NSCLC). The phylogenetic proximity of the ROS1 and anaplastic lymphoma kinase (ALK) catalytic domains led to the clinical repurposing of the Food and Drug Administration (FDA)-approved ALK inhibitor crizotinib as a ROS1 inhibitor. Despite the antitumor activity of crizotinib observed in both ROS1- and ALK-rearranged NSCLC patients, resistance due to acquisition of ROS1 or ALK kinase domain mutations has been observed clinically, spurring the development of second-generation inhibitors. Here, we profile the sensitivity and selectivity of seven ROS1 and/or ALK inhibitors at various levels of clinical development. In contrast to crizotinib's dual ROS1/ALK activity, cabozantinib (XL-184) and its structural analog foretinib (XL-880) demonstrate a striking selectivity for ROS1 over ALK. Molecular dynamics simulation studies reveal structural features that distinguish the ROS1 and ALK kinase domains and contribute to differences in binding site and kinase selectivity of the inhibitors tested. Cell-based resistance profiling studies demonstrate that the ROS1-selective inhibitors retain efficacy against the recently reported CD74-ROS1(G2032R) mutant whereas the dual ROS1/ALK inhibitors are ineffective. Taken together, inhibitor profiling and stringent characterization of the structure-function differences between the ROS1 and ALK kinase domains will facilitate future rational drug design for ROS1- and ALK-driven NSCLC and other malignancies.

Abstract 2827: Discovery of AP26113, a potent, orally active inhibitor of anaplastic lymphoma kinase and clinically relevant mutants

Abstract Phosphorus, despite its abundance in the human body, is rarely found in drug molecules, with clinical utility limited to a few phosphonic or bisphosponic acid-based medicines and several phosphonate or phosphate-containing prodrugs. Concerns about poor cell penetration, low oral bioavailability, or biological instability have limited application of these functional classes in drug design. In our efforts to discover pharmaceuticals with novel functionality, we introduced a neutral, stable phosphine oxide moiety as a unique hydrogen-bond acceptor in the design of anaplastic lymphoma kinase (ALK) inhibitors. ALK is a receptor tyrosine kinase first identified as a chromosomal rearrangement (NPM-ALK fusion gene) in anaplastic large cell lymphoma (ALCL) and subsequently detected as an alternate fusion oncogene (EML4-ALK) in a subset of non-small cell lung cancers (NSCLC). Crizotinib, the first approved ALK inhibitor, has demonstrated impressive clinical benefit in EML4-ALK (ALK+) NSCLC patients. Drug resistance, however, emerges rapidly and point mutations within the kinase domain have been identified as a major resistance mechanism. With the specific objective to identify more potent ALK inhibitors with pan-inhibitory activity against crizotinib-resistant ALK mutants, we designed and tested a series of phosphine oxide-based compounds culminating in the identification of the clinical candidate AP26113. AP26113 exhibited 10-fold greater potency than crizotinib against ALK-positive ALCL and NSCLC cell lines, and effectively inhibited clinically relevant crizotinib-resistant mutants. AP26113 displayed ∼100-fold increased selectivity for ALK-positive cells over ALK-negative cell lines while maintaining selectivity over insulin receptor tyrosine kinase receptors. Consistent with the in vitro profile, AP26113 demonstrated oral efficacy in multiple ALK+ mouse models including Karpas-299 (ALCL), H3122 (NSCLC), and Ba/F3 cells expressing crizotinib resistant mutants including G1269S and L1196M. Finally, AP26113 exhibited excellent drug-like properties including high cell permeability and solubility, moderate cross-species protein binding, and inactivity toward major cytochrome P450 iso-enzymes and hERG ion channels. Medicinal chemistry efforts leading to the discovery of this potent pan-ALK inhibitor, including design strategy, chemical series evolution, and DMPK optimization will be presented. AP26113 is currently in a global phase 2 registration trial (NCT02094573) in patients with locally advanced or metastatic NSCLC who test positive for the ALK oncogene and were previously treated with crizotinib. Citation Format: Wei-Sheng Huang, Feng Li, Lisi Cai, Yongjin Xu, Sen Zhang, Scott D. Wardwell, Yaoyu Ning, Anna Kohlmann, Tianjun Zhou, Emily Y. Ye, Xiaotian Zhu, Narayana I. Narasimhan, Tim Clackson, Victor M. Rivera, David Dalgarno, William C. Shakespeare. Discovery of AP26113, a potent, orally active inhibitor of anaplastic lymphoma kinase and clinically relevant mutants. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2827. doi:10.1158/1538-7445.AM2015-2827

Abstract 4698: Combination of ceritinib (LDK378) with PI3K inhibitors (buparlisib [BKM120] and alpelisib [BYL719]) demonstrates synergistic preclinical antitumor activity in ALK-rearranged non-small cell lung cancer (NSCLC)

Abstract Introduction: NSCLCs harboring rearrangements of the anaplastic lymphoma kinase (ALK) gene are highly sensitive to ALK inhibition. Ceritinib is a novel ALK inhibitor that demonstrates significant preclinical and clinical antitumor potency, including in tumors that have developed resistance to the approved ALK inhibitor crizotinib. Upregulation of the phosphatidylinositol 3-kinase (PI3K) pathway may be involved in ALK inhibitor resistance, suggesting that combining ceritinib with a PI3K inhibitor may provide synergistic antitumor activity in ALK-rearranged NSCLC. Here, we assess the synergy and antiproliferative activity of PI3K inhibitor/ceritinib combinations in crizotinib-naïve primary preclinical models of NSCLC. Methods: In vitro experiments used the EML4−ALK-translocated lung cancer cell line H2228 to screen for synergistic combinations of ceritinib with 18 other compounds (PI3K, MEK, and CDK4/6 inhibitors, among others). In vivo experiments used a human-mouse primary xenograft lung cancer model with similar EML4−ALK translocation (LUF1656). Tumor fragments (diameter 2−3 mm) from stock mice inoculated with LUF1656 lung cancer tissue were harvested and used for inoculation into nu/nu mice for tumor development. Once mean tumor size had reached ∼150 mm3, treatment was initiated with single-agent or combination regimens of buparlisib (pan-PI3K inhibitor; 35 mg/kg QD), alpelisib (PI3Kα inhibitor; 30 mg/kg QD), or ceritinib (25 mg/kg [low dose] or 50 mg/kg [full dose] QD). All doses used were equivalent to known therapeutic doses in patients. Results: In vitro experiments revealed the strongest antiproliferative activity when ceritinib was combined with either buparlisib or alpelisib versus the other compounds tested. In vivo, low-dose ceritinib in combination with buparlisib improved tumor growth delay over single-agent, full-dose ceritinib. Full-dose ceritinib plus alpelisib showed no significant difference in tumor growth delay versus full-dose ceritinib alone. Low-dose ceritinib plus alpelisib appeared to be better tolerated than full-dose ceritinib plus alpelisib, but with similar efficacy to low-dose ceritinib alone. A delayed tumor growth rate after treatment interruption was noted in all ceritinib combinations. Conclusion: Synergy was observed between ceritinib and PI3K inhibitors in a crizotinib-naïve ALK-translocated lung model in vitro. In the in vivo EML4−ALK lung preclinical cancer models, low-dose ceritinib (25 mg/kg) combined with buparlisib showed improved efficacy versus full-dose ceritinib (50 mg/kg) alone. Preclinical experiments exploring combinations of PI3K- and ALK-targeted therapies in crizotinib-resistant ALK-rearranged tumors are also ongoing. Citation Format: Emmanuelle Di Tomaso, Ronald Linnartz, Fang Li, Cristian Massacesi, Samit Hirawat. Combination of ceritinib (LDK378) with PI3K inhibitors (buparlisib [BKM120] and alpelisib [BYL719]) demonstrates synergistic preclinical antitumor activity in ALK-rearranged non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4698. doi:10.1158/1538-7445.AM2015-4698