Entrectinib is a fi rst-in-class pan-TRK kinase inhibitor currently undergoing clinical testing in colorectal cancer and other tumor types. A patient with metastatic colorectal cancer harboring an LMNA-NTRK1 rearrangement displayed a remarkable response to treatment with entrectinib, which was followed by the emergence of resistance. To characterize the molecular bases of the patient's relapse, circulating tumor DNA (ctDNA) was collected longitudinally during treatment, and a tissue biopsy, obtained before entrectinib treatment, was transplanted in mice (xenopatient), which then received the same entrectinib regimen until resistance developed. Genetic profi ling of ctDNA and xenopatient samples showed acquisition of two point mutations in the catalytic domain of NTRK1 , p.G595R and p.G667C. Biochemical and pharmacologic analysis in multiple preclinical models confi rmed that either mutation renders the TRKA kinase insensitive to entrectinib. These fi ndings can be immediately exploited to design next-generation TRKA inhibitors. SIGNIFICANCE:We provide proof of principle that analyses of xenopatients (avatar) and liquid biopsies allow the identifi cation of drug resistance mechanisms in parallel with clinical treatment of an individual patient. We describe for the fi rst time that p.G595R and p.G667C TRKA mutations drive acquired resistance to entrectinib in colorectal cancers carrying NTRK1 rearrangements. Cancer Discov; 6(1);[36][37][38][39][40][41][42][43][44]
Here, we describe the dramatic response of a patient with an ETV6-NTRK3-driven mammary analogue secretory carcinoma to treatment with a pan-Trk inhibitor, and the development of acquired resistance linked to a novel NTRK3 mutation that interferes with drug binding. This case emphasizes how molecular profiling can identify therapies for rare diseases and dissect mechanisms of drug resistance.
Neuroblastoma (NB) is one of the most common and deadly childhood solid tumors. These tumors are characterized by clinical heterogeneity, from spontaneous regression to relentless progression, and the Trk family of neurotrophin receptors plays an important role in this heterogeneous behavior. We wanted to determine if entrectinib (RXDX-101, Ignyta, Inc.), an oral Pan-Trk, Alk and Ros1 inhibitor was effective in our NB model. In vitro effects of entrectinib, either as a single agent or in combination with the chemotherapeutic agents Irinotecan (Irino) and Temozolomide (TMZ), were studied on an SH-SY5Y cell line stably transfected with TrkB. In vivo growth inhibition activity was studied in NB xenografts, again as single agent or in combination with Irino-TMZ. Entrectinib significantly inhibited the growth of TrkB-expressing NB cells in vitro, and it significantly enhanced the growth inhibition of Irino-TMZ when used in combination. Single agent therapy resulted in significant tumor growth inhibition in animals treated with entrectinib compared to control animals [p<0.0001 for event-free survival (EFS)]. Addition of entrectinib to Irino-TMZ also significantly improved the EFS of animals compared to vehicle or Irino-TMZ treated animals [p<0.0001 for combination vs. control, p=0.0012 for combination vs. Irino-TMZ]. We show that entrectinib inhibits growth of TrkB expressing NB cells in vitro and in vivo, and that it enhances the efficacy of conventional chemotherapy in in vivo models. Our data suggest that entrectinib is a potent Trk inhibitor and should be tested in clinical trials for NBs and other Trk-expressing tumors.
ITK is organized in modular domains that play critical roles in its activation (47). Upon T-cell engagement, ITK colocalizes with the TCR, a process dependent on the pleckstrin homology (PH) domain of ITK and its interaction with PIP3 at the plasma membrane (11,19). Activation of ITK also requires interaction with adaptor proteins, such as SLP-76 and LAT (8, 10). The SH2 domain of ITK appears to be critical for its interaction with LAT, whereas both the SH2 and SH3 domains are required for interaction with 10). In vitro studies have demonstrated that the SH3 domain of ITK interacts with the proline-rich (PR) region of SLP-76, and it has been speculated that this interaction is critical for the activation of ITK (6,8). However, the biological significance of the interaction has not been demonstrated in live cells. In the present investigation, we used a cell-permeable peptide as a competitive inhibitor of the interaction between ITK and SLP-76. To this end, we synthesized a 12-amino-acid peptide, which represents the PR region of SLP-76 that binds to the ITK-SH3 domain, and rendered it cell permeable by the addition of nine arginines at its N-proximal end. Here, we show that this cellpermeable peptide, henceforth called R9-QQP, is readily taken up by both Jurkat T cells and murine splenocytes and disrupts events that are mediated by the engagement of the TCR. Thus, association of ITK and SLP-76, recruitment of ITK and actin polarization at the T-cell contact site, LCKmediated transphosphorylation of ITK on tyrosine 511, and production of Th2 cytokines are inhibited by R9-QQP in a dose-dependent and peptide-specific manner. The data presented here are novel and significant because they provide the first demonstration of the biological relevance of the specific interaction between the ITK-SH3 domain and the SLP-76 PR region in live cells. Furthermore, the data underscore the potential of cell-permeable peptides as useful probes for dissecting signal transduction pathways in live cells, and in view of the regulatory role that ITK plays in Th2 cytokine production, they have implications for the pharmacological manipulation of ITK in disease situations. MATERIALS AND METHODSCell lines, mice, antibodies, and other reagents. Wild-type Jurkat T cells (E6.1) were obtained from the American Type Culture Collection (ATCC). The SLP-76-deficient mutant, J14, was a kind gift from Art Weiss (University of California-San Francisco). The cells were cultured as previously described (10). Male C57BL/6 mice were purchased from Harlan Sprague Dawley or Jackson Laboratories and were used between the ages of 6 and 12 weeks. All experimental protocols using animals were approved by the
Gene rearrangements involving NTRK1, NTRK2, NTRK3, ROS1 and ALK result in oncogenic fusion proteins that have been identified in many types of cancer, including lung, colorectal, salivary gland, sarcoma, papillary thyroid, glioblastoma, melanoma and other histologies. Entrectinib (RXDX-101) is an orally available, highly potent and selective ATP-competitive pan-Trk, ROS1 and ALK inhibitor. In preclinical studies, entrectinib effectively inhibits target kinase activity and cancer cell proliferation and in vivo tumor growth across various fusion partners and cancer types. More importantly, entrectinib's activity has been validated clinically in patients across multiple fusion partners and tissue histologies. Trk inhibitors, including entrectinib, have shown promising clinical activity in molecularly selected patients. Predictably, potential resistance mechanisms have also begun to emerge. For example, mutations in the Trk kinase domain were identified as one of the in vitro induced resistance mechanisms to the Trk inhibitor, Loxo-101. The three reported resistance mutations in the Ba/F3-MPRIP-NTRK1 cell line model treated with Loxo-101 were F589, G667 and V573. The F589 location on TrkA is equivalent to the gatekeeper mutations, L1196 location on ALK and L2026 location on ROS1. These gatekeeper mutations often arise as resistance mechanisms in patients treated with ALK and ROS1 inhibitors. To test the activity of entrectinib against these three reported NTRK1 mutations, we introduced mutated Trk proteins into Ba/F3 and cancer cell lines and performed dose-dependent proliferation studies. Entrectinib was able to inhibit proliferation of cells harboring each of these three mutations that confer resistance to other Trk inhibitors. Particularly, the IC50 values of entrectinib against kinase domain wildtype and gatekeeper mutated (F589) are essentially unchanged (low single-digit nM), which is consistent with the observation that entrectinib is also able to inhibit the gatekeeper mutation in ALK (L1196) in both cell based assays and in vivo tumor growth inhibition studies. In conclusion, our preclinical data suggest that entrectinib is an effective treatment for patients with NTRK-rearranged tumors, including cancers that harbor certain resistance mutations to other Trk inhibitors. Citation Format: Ge Wei, Elena Ardini, Roopal Patel, Nicholas Cam, Jason Harris, Jean-Michel Vernier, Nanqun Zhu, Litain Yeh, Robert Shoemaker, Pratik Multani, Zachary Hornby, Robert Wild, Gary G. Li. Entrectinib is effective against the gatekeeper and other emerging resistance mutations in NTRK-, ROS1- and ALK- rearranged cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2136.
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