Activated ALK and ROS1 tyrosine kinases, resulting from chromosomal rearrangements, occur in a subset of non-small cell lung cancers (NSCLC) as well as other tumor types and their oncogenic relevance as actionable targets has been demonstrated by the efficacy of selective kinase inhibitors such as crizotinib, ceritinib, and alectinib. More recently, low-frequency rearrangements of TRK kinases have been described in NSCLC, colorectal carcinoma, glioblastoma, and Spitzoid melanoma. Entrectinib, whose discovery and preclinical characterization are reported herein, is a novel, potent inhibitor of ALK, ROS1, and, importantly, of TRK family kinases, which shows promise for therapy of tumors bearing oncogenic forms of these proteins. Proliferation profiling against over 200 human tumor cell lines revealed that entrectinib is exquisitely potent in vitro against lines that are dependent on the drug's pharmacologic targets. Oral administration of entrectinib to tumor-bearing mice induced regression in relevant human xenograft tumors, including the TRKA-dependent colorectal carcinoma KM12, ROS1-driven tumors, and several ALK-dependent models of different tissue origins, including a model of brain-localized lung cancer metastasis. Entrectinib is currently showing great promise in phase I/II clinical trials, including the first documented objective responses to a TRK inhibitor in colorectal carcinoma and in NSCLC. The drug is, thus, potentially suited to the therapy of several molecularly defined cancer settings, especially that of TRK-dependent tumors, for which no approved drugs are currently available.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase responsible for the development of different tumor types. Despite the remarkable clinical activity of crizotinib (Xalkori), the first ALK inhibitor approved in 2011, the emergence of resistance mutations and of brain metastases frequently causes relapse in patients. Within our ALK drug discovery program, we identified compound 1, a novel 3-aminoindazole active on ALK in biochemical and in cellular assays. Its optimization led to compound 2 (entrectinib), a potent orally available ALK inhibitor active on ALK-dependent cell lines, efficiently penetrant the blood-brain barrier (BBB) in different animal species and highly efficacious in in vivo xenograft models. Moreover, entrectinib resulted to be strictly potent on the closely related tyrosine kinases ROS1 and TRKs recently found constitutively activated in several tumor types. Entrectinib is currently undergoing phase I/II clinical trial for the treatment of patients affected by ALK-, ROS1-, and TRK-positive tumors.

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.

Growth Factor Receptor. NTRK1 was originally isolated from a colorectal carcinoma (CRC) sample as component of a somatic rearrangement (TPM3-NTRK1) resulting in expression of the oncogenic chimeric protein TPM3-TRKA, but there has been no subsequent report regarding the relevance of this oncogene in CRC. The KM12 human CRC cell line expresses the chimeric TPM3-TRKA protein and is hypersensitive to TRKA kinase inhibition.We report the detailed characterization of the TPM3-NTRK1 genomic rearrangement in KM12 cells and through a cellular screening approach, the identification of NMS-P626, a novel highly potent and selective TRKA inhibitor. NMS-P626 suppressed TPM3-TRKA phosphorylation and downstream signaling in KM12 cells and showed remarkable antitumor activity in mice bearing KM12 tumors.Finally, using quantitative reverse transcriptase PCR and immunohistochemistry (IHC) we identified the TPM3-NTRK1 rearrangement in a CRC clinical sample, therefore suggesting that this chromosomal translocation is indeed a low frequency recurring event in CRC and that such patients might benefit from therapy with TRKA kinase inhibitors.

We have isolated and sequenced partial cDNA clones that encode SO‐6, a ribosome‐inactivating protein from Saponaria officinalis. A cDNA library was constructed from the leaves of this plant and screened with synthetic oligonucleotide probes representing various portions of the protein. The deduced amino acid sequence shows the signal peptide and a coding region virtually accounting for the entire amino acid sequence of SO‐6. The sequence reveals regions of similarity to other ribosome‐inactivating proteins, especially in a region of the molecule where critical amino acid residues might participate in the active site.

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The chromosomal translocation t(2;5)(p23;q35) involving the ALK tyrosine kinase gene results in expression of the NPM-ALK fusion protein which represents the driving force for survival and proliferation of a subset of Anaplastic Large Cell Lymphoma. More recently, a distinct chromosomal rearrangement of the ALK gene leading to a new fusion variant EML4-ALK, has been identified as a low frequency event, mutually exclusive with respect to EGFR and K-ras mutation, in Non Small Cell Lung cancer patients. As previously found for NPM-ALK, this new fusion variant has constitutively active ALK kinase and was demonstrated to have strong oncogenic potential. Taken together these findings support the hypothesis that ALK represents an innovative and valuable target for cancer therapy both in ALCL and NSCLC patients whose tumors harbor translocated ALK. Here we further describe the preclinical characterization of NMS-E628, an orally available small-molecule inhibitor of ALK kinase activity. Proliferation profiling on a wide panel of human tumor cell lines demonstrated that the compound selectively blocks proliferation of ALK-dependent cell lines and potently inhibits ALK-dependent signaling. In vivo, NMS-E628 induced complete tumor regression when administered orally for ten consecutive days to SCID mice bearing Karpas-299 or SR-786 xenografts, with ex vivo analyses demonstrating dose-dependent target modulation that was maintained for up to 18 hours after single treatment. NMS-E628 was also highly efficacious in a transgenic mouse leukemia model in which human NPM-ALK expression was targeted to T cells. In this latter model, which faithfully recapitulates pathological features of human ALCL, treatment of NPM-ALK transgenic mice with NMS-E628 for as little as 3 consecutive days induced complete regression of tumor masses observed in the thymus and in lymph nodes. NMS-E628 was also highly efficacious in inhibiting the in vitro and in vivo growth of the NSCLC cell line NCI-H2228, which bears the EML4-ALK rearrangement. Complete regressions were also achieved in this model, and prolonged inhibition of ALK phosphorylation and downstream effector activation were observed at active doses. NMS-E628 has favorable pharmacokinetic and toxicological properties and biodistribution analysis revealed that it is able to cross the blood-brain barrier in different animal species. To confirm that therapeutic doses are reached in the brain, NCI-H2228 cells were injected intracranially in nude mice and NMS-E628 was administered orally with different schedules. Dose-dependent increase in survival, together with inhibition of tumor growth as assessed by MRI, confirmed that NMS-E628 does indeed possess antitumor activity in this setting, an important finding considering that a significant proportion of NSCLC patients develop brain metastases. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A244.

The Anaplastic Lymphoma Kinase (ALK) is a receptor tyrosine kinase with a recognized role in the pathogenesis of different tumors. There is strong preclinical evidence that inhibition of ALK kinase activity results in anti-tumor efficacy and the approval of the ALK inihibitor Xalkori (crizotinib) for treatment of NSCLC patients bearing ALK positive tumor represents the most recent success of targeted therapy. Nevertheless, clinical data show that after initial response to Xalkori, patients experience relapse, the mechanisms of which are still under study, but which at least in some cases appear due to emergence of mutations that confer resistance to the drug. Thus, the development of next-generation ALK inihibitors, able to overcome ALK-dependent Xalkori resistance is needed. We have previously presented the identification of NMS-E628, a potent and selective small-molecule inhibitor of ALK kinase activity. The compound displays strong anti-tumor efficacy in several models of ALK dependent tumors after oral administration, with complete and durable regression observed in treated animals. NMS-E628 passes the blood brain barrier in all the species tested and was found able to effectively control the growth of intracranial tumors. Being structurally distinct from Xalkori, NMS-E628 might be able to overcome mutations in the ALK kinase domain which confer resistance by affecting Xalkori binding. To test this hypothesis, the activity of NMS-E628 on WT ALK and on two mutants identified in Xalkori-relapsed patients, L1196M and C1156Y, was investigated using different approaches. Ki determination revealed that at the biochemical level, NMS-E628 is ca. 7–8 fold more potent than Xalkori on both L1196M and C1156Y. In Ba/F3 cells made dependent upon mutated ALK forms, NMS-E628 was found superior than Xalkori in inhibiting the proliferation of both L1196M ALK and C1156Y ALK-driven cells in vitro. Mechanism of action studies confirmed that of the two drugs, NMS-E628 is better able to downmodulate ALK phosphorylation, having similar potency on wt ALK and mutated forms. To assess antitumor activity in vivo, Ba/F3 xenografts driven by different ALK mutants were generated. As expected from in vitro results, Xalkori showed poor efficacy on L1196M and C1156Y mutants when tested at 100 and 200 mg/kg, while NMS-E628 retains significant anti-tumor activity in both mutant models. Taken together, these data support the idea that NMS-E628 might represent a valid therapeutic opportunity for Xalkori-relapsed patients that experience acquired resistance to specific ALK mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A232.