Brian B. Tuch

An Oncogenic NTRK Fusion in a Patient with Soft-Tissue Sarcoma with Response to the Tropomyosin-Related Kinase Inhibitor LOXO-101

UNLABELLEDnOncogenic TRK fusions induce cancer cell proliferation and engage critical cancer-related downstream signaling pathways. These TRK fusions occur rarely, but in a diverse spectrum of tumor histologies. LOXO-101 is an orally administered inhibitor of the TRK kinase and is highly selective only for the TRK family of receptors. Preclinical models of LOXO-101 using TRK-fusion-bearing human-derived cancer cell lines demonstrate inhibition of the fusion oncoprotein and cellular proliferation in vitro, and tumor growth in vivo. The tumor of a 41-year-old woman with soft-tissue sarcoma metastatic to the lung was found to harbor an LMNA-NTRK1 gene fusion encoding a functional LMNA-TRKA fusion oncoprotein as determined by an in situ proximity ligation assay. In a phase I study of LOXO-101 (ClinicalTrials.gov no. NCT02122913), this patients tumors underwent rapid and substantial tumor regression, with an accompanying improvement in pulmonary dyspnea, oxygen saturation, and plasma tumor markers.nnnSIGNIFICANCEnTRK fusions have been deemed putative oncogenic drivers, but their clinical significance remained unclear. A patient with a metastatic soft-tissue sarcoma with an LMNA-NTRK1 fusion had rapid and substantial tumor regression with a novel, highly selective TRK inhibitor, LOXO-101, providing the first clinical evidence of benefit from inhibiting TRK fusions.

Efficacy of Larotrectinib in TRK Fusion–Positive Cancers in Adults and Children

Background Fusions involving one of three tropomyosin receptor kinases (TRK) occur in diverse cancers in children and adults. We evaluated the efficacy and safety of larotrectinib, a highly selective TRK inhibitor, in adults and children who had tumors with these fusions. Methods We enrolled patients with consecutively and prospectively identified TRK fusion–positive cancers, detected by molecular profiling as routinely performed at each site, into one of three protocols: a phase 1 study involving adults, a phase 1–2 study involving children, or a phase 2 study involving adolescents and adults. The primary end point for the combined analysis was the overall response rate according to independent review. Secondary end points included duration of response, progression‐free survival, and safety. Results A total of 55 patients, ranging in age from 4 months to 76 years, were enrolled and treated. Patients had 17 unique TRK fusion–positive tumor types. The overall response rate was 75% (95% confidence interval [CI], 61 to 85) according to independent review and 80% (95% CI, 67 to 90) according to investigator assessment. At 1 year, 71% of the responses were ongoing and 55% of the patients remained progression‐free. The median duration of response and progression‐free survival had not been reached. At a median follow‐up of 9.4 months, 86% of the patients with a response (38 of 44 patients) were continuing treatment or had undergone surgery that was intended to be curative. Adverse events were predominantly of grade 1, and no adverse event of grade 3 or 4 that was considered by the investigators to be related to larotrectinib occurred in more than 5% of patients. No patient discontinued larotrectinib owing to drug‐related adverse events. Conclusions Larotrectinib had marked and durable antitumor activity in patients with TRK fusion–positive cancer, regardless of the age of the patient or of the tumor type. (Funded by Loxo Oncology and others; ClinicalTrials.gov numbers, NCT02122913, NCT02637687, and NCT02576431.)

A Next-Generation TRK Kinase Inhibitor Overcomes Acquired Resistance to Prior TRK Kinase Inhibition in Patients with TRK Fusion–Positive Solid Tumors

Larotrectinib, a selective TRK tyrosine kinase inhibitor (TKI), has demonstrated histology-agnostic efficacy in patients with TRK fusion-positive cancers. Although responses to TRK inhibition can be dramatic and durable, duration of response may eventually be limited by acquired resistance. LOXO-195 is a selective TRK TKI designed to overcome acquired resistance mediated by recurrent kinase domain (solvent front and xDFG) mutations identified in multiple patients who have developed resistance to TRK TKIs. Activity against these acquired mutations was confirmed in enzyme and cell-based assays and in vivo tumor models. As clinical proof of concept, the first 2 patients with TRK fusion-positive cancers who developed acquired resistance mutations on larotrectinib were treated with LOXO-195 on a first-in-human basis, utilizing rapid dose titration guided by pharmacokinetic assessments. This approach led to rapid tumor responses and extended the overall duration of disease control achieved with TRK inhibition in both patients.Significance: LOXO-195 abrogated resistance in TRK fusion-positive cancers that acquired kinase domain mutations, a shared liability with all existing TRK TKIs. This establishes a role for sequential treatment by demonstrating continued TRK dependence and validates a paradigm for the accelerated development of next-generation inhibitors against validated oncogenic targets. Cancer Discov; 7(9); 963-72. ©2017 AACR.See related commentary by Parikh and Corcoran, p. 934This article is highlighted in the In This Issue feature, p. 920.

Larotrectinib for paediatric solid tumours harbouring NTRK gene fusions: phase 1 results from a multicentre, open-label, phase 1/2 study

BACKGROUNDnGene fusions involving NTRK1, NTRK2, or NTRK3 (TRK fusions) are found in a broad range of paediatric and adult malignancies. Larotrectinib, a highly selective small-molecule inhibitor of the TRK kinases, had shown activity in preclinical models and in adults with tumours harbouring TRK fusions. This study aimed to assess the safety of larotrectinib in paediatric patients.nnnMETHODSnThis multicentre, open-label, phase 1/2 study was done at eight sites in the USA and enrolled infants, children, and adolescents aged 1 month to 21 years with locally advanced or metastatic solid tumours or CNS tumours that had relapsed, progressed, or were non-responsive to available therapies regardless of TRK fusion status; had a Karnofsky (≥16 years of age) or Lansky (<16 years of age) performance status score of 50 or more, adequate organ function, and full recovery from the acute toxic effects of all previous anticancer therapy. Following a protocol amendment on Sept 12, 2016, patients with locally advanced infantile fibrosarcoma who would require disfiguring surgery to achieve a complete surgical resection were also eligible. Patients were enrolled to three dose cohorts according to a rolling six design. Larotrectinib was administered orally (capsule or liquid formulation), twice daily, on a continuous 28-day schedule, in increasing doses adjusted for age and bodyweight. The primary endpoint of the phase 1 dose escalation component was the safety of larotrectinib, including dose-limiting toxicity. All patients who received at least one dose of larotrectinib were included in the safety analyses. Reported here are results of the phase 1 dose escalation cohort. Phase 1 follow-up and phase 2 are ongoing. This trial is registered with ClinicalTrials.gov, number NCT02637687.nnnFINDINGSnBetween Dec 21, 2015, and April 13, 2017, 24 patients (n=17 with tumours harbouring TRK fusions, n=7 without a documented TRK fusion) with a median age of 4·5 years (IQR 1·3-13·3) were enrolled to three dose cohorts: cohorts 1 and 2 were assigned doses on the basis of both age and bodyweight predicted by use of SimCyp modelling to achieve an area under the curve equivalent to the adult doses of 100 mg twice daily (cohort 1) and 150 mg twice daily (cohort 2); and cohort 3 was assigned to receive a dose of 100 mg/m2 twice daily (maximum 100 mg per dose), regardless of age, equating to a maximum of 173% of the recommended adult phase 2 dose. Among enrolled patients harbouring TRK fusion-positive cancers, eight (47%) had infantile fibrosarcoma, seven (41%) had other soft tissue sarcomas, and two (12%) had papillary thyroid cancer. Adverse events were predominantly grade 1 or 2 (occurring in 21 [88%] of 24 patients); the most common larotrectinib-related adverse events of all grades were increased alanine and aspartate aminotransferase (ten [42%] of 24 each), leucopenia (five [21%] of 24), decreased neutrophil count (five [21%] of 24), and vomiting (five [21%] of 24). Grade 3 alanine aminotransferase elevation was the only dose-limiting toxicity and occurred in one patient without a TRK fusion and with progressive disease. No grade 4 or 5 treatment-related adverse events were observed. Two larotrectinib-related serious adverse events were observed: grade 3 nausea and grade 3 ejection fraction decrease during the 28-day follow-up after discontinuing larotrectinib and while on anthracyclines. The maximum tolerated dose was not reached, and 100 mg/m2 (maximum of 100 mg per dose) was established as the recommended phase 2 dose. 14 (93%) of 15 patients with TRK fusion-positive cancers achieved an objective response as per Response Evaluation Criteria In Solid Tumors version 1.1; the remaining patient had tumour regression that did not meet the criteria for objective response. None of the seven patients with TRK fusion-negative cancers had an objective response.nnnINTERPRETATIONnThe TRK inhibitor larotrectinib was well tolerated in paediatric patients and showed encouraging antitumour activity in all patients with TRK fusion-positive tumours. The recommended phase 2 dose was defined as 100mg/m2 (maximum 100 mg per dose) for infants, children, and adolescents, regardless of age.nnnFUNDINGnLoxo Oncology Inc.

Abstract C65: TRK kinase domain mutations that induce resistance to a pan-TRK inhibitor

Background: Gene fusions involving members of the NTRK family have been identified in several types of cancer. The use of TRK inhibitors in vitro and in vivo have demonstrated activity against a number of different NTRK fusions in different tumor types and most recently in a patient with an NTRK1 gene fusion (Doebele et al., Cancer Discovery 2015). Thus, the use of a pan-TRK inhibitor presents a therapeutic opportunity for multiple patient populations. LOXO-101 is an inhibitor of the TRK kinase and is highly selective for the TRKA/B/C family of kinases. Kinase domain (KD) mutations are the most common mechanism of acquired drug resistance found in patients. We therefore undertook a genetic approach to identify candidate resistance mutations in the TRK kinase domain. Methods: We used N-ethyl-N-nitrosourea (ENU)-exposed Ba/F3-MPRIP-NTRK1, Ba/F3-PAN3-NTRK2 and Ba/F3-ETV6-NTRK3 cells to generate mutations that could allow growth of Ba/F3 cells despite the presence of LOXO-101. Mutations identified in the initial screen were validated by cloning the mutation-bearing cDNAs back into Ba/F3 cells to evaluate their sensitivity to LOXO-101 using both proliferation assays and TRK phosphorylation by immunoblot analyses. Modeling of the mutations was performed by predicting the consequences of the amino acid substitutions within the context of a drug-bound TRK kinase domain crystal structure. Results: We have identified three KD mutations within the TRKA kinase domain: V573M, and F589L, G667S. These mutations induce significant drug resistance in BA/F3 cells to LOXO-101 in vitro compared to the unmutated MPRIP-NTRK1. Furthermore, these mutations do not show inhibition of phosphorylation of TRKA at doses that inhibit the native MPRIP-NTRK1. The TRKA F589 position corresponds to the gatekeeper position, homologous to L1196 in ALK or T790 in EGFR, and a common position for drug resistance mutations. The TRKA G667 lies adjacent to the F589 position in the ATP/drug-bind pocket and similar resistance mutations have been observed in the ALK kinase domain following resistance to crizotinib. V573 is also positioned adjacent to both the G667 and F589 residues in the ATP-/drug-binding pocket. Although a similar mutation has been observed in vitro in ALK (V1180M), it has not yet been reported in patient tumor samples. Analysis of additional candidate resistance clones is ongoing. Conclusion: This genetic screen identified several novel mutations in the TRK KD that may confer clinical resistance to LOXO-101. All three mutations identified to date appear to hinder binding by altering the ATP binding site of the kinase. These data should be confirmed in patients who develop clinical resistance, and may assist in the design of next generation TRK inhibitors to potentially overcome acquired resistance in patients treated with LOXO-101 or structurally similar TRK inhibitors. Citation Format: Adriana Estrada-Bernal, Anh T. Le, Brian Tuch, Tatiana Kutateladze, Robert C. Doebele. TRK kinase domain mutations that induce resistance to a pan-TRK inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C65.

Oncogenic TRK fusions are amenable to inhibition in hematologic malignancies

Rearrangements involving the neurotrophic receptor kinase genes (NTRK1, NTRK2, and NTRK3; hereafter referred to as TRK) produce oncogenic fusions in a wide variety of cancers in adults and children. Although TRK fusions occur in fewer than 1% of all solid tumors, inhibition of TRK results in profound therapeutic responses, resulting in Breakthrough Therapy FDA approval of the TRK inhibitor larotrectinib for adult and pediatric patients with solid tumors, regardless of histology. In contrast to solid tumors, the frequency of TRK fusions and the clinical effects of targeting TRK in hematologic malignancies are unknown. Here, through an evaluation for TRK fusions across more than 7,000 patients with hematologic malignancies, we identified TRK fusions in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), histiocytosis, multiple myeloma, and dendritic cell neoplasms. Although TRK fusions occurred in only 0.1% of patients (8 of 7,311 patients), they conferred responsiveness to TRK inhibition in vitro and in vivo in a patient-derived xenograft and a corresponding AML patient with ETV6-NTRK2 fusion. These data identify that despite their individual rarity, collectively, TRK fusions are present in a wide variety of hematologic malignancies and predict clinically significant therapeutic responses to TRK inhibition.

Abstract B192: Identification and characterization of highly potent and selective RET kinase inhibitors for the treatment of RET-driven cancers

Background: Activating mutations and oncogenic fusions of the RET receptor tyrosine kinase have been identified in multiple tumor types, including thyroid, lung, breast and colon carcinoma. Furthermore, tyrosine kinase inhibitors (TKIs) with anti-RET activity have produced clinical responses in patients whose tumors harbor RET alterations. However, currently available RET inhibitors were initially developed to target kinases other than RET and are only moderately potent against RET, inhibit multiple kinases other than RET or poorly inhibit secondary resistance mutations (e.g. gatekeeper mutations) common to other TKIs. We have discovered novel, potent and selective RET inhibitors. The resulting compounds exhibit nanomolar potency against wild type RET and select RET mutants, including the KIF5B-RET fusion and V804M gatekeeper mutation, in both enzyme and cellular assays, with minimal activity against highly related kinases. AR025 is representative of this series; the activity of AR025 and related analogs in relevant in vitro and in vivo models will be presented here. Methods: In vitro and in vivo evaluations, including enzyme and cell-based assays, pharmacokinetic (PK)/pharmacodynamics (PD) correlations, drug metabolism characterization, and non-clinical safety evaluation, were conducted using standard methods. Tumor growth inhibition and PD studies were carried out using subcutaneous allografts of NIH-3T3 cells expressing KIF5B-RET in nude mice. Results: AR025 demonstrated nanomolar potency against both wild type and mutant RET proteins in enzyme and cellular assays. AR025 had minimal activity against an enzyme panel of >200 diverse kinases and demonstrated >50x cellular selectivity against VEGFR, with similar selectivity against other related kinases. AR025 possessed low intravenous clearance and high oral exposure in mice, rats and dogs. Finally, a single oral dose of 30mg/kg produced more than 90% inhibition of phospho-RET in NIH-3T3-KIF5B-RET mouse allografts, while twice-per-day continuous dosing resulted in greater than 90% tumor growth inhibition. Notably, AR025 was minimally toxic at doses up to 100mg/kg per day. Conclusions: We have identified a series of potent and selective RET inhibitors with high oral bioavailability and favorable PK properties in animals. One of these, AR025, demonstrated potent inhibition of RET in enzyme and cellular assays, with minimal activity against highly related kinases. In an NIH3T3-KIF5B-RET allograft model, AR025 effectively inhibited phospho-RET and caused dramatic tumor growth inhibition without significant toxicity. The identification of potent and selective RET inhibitors with significant in vivo activity and minimal toxicity may overcome the limitations of currently available inhibitors with anti-RET activity. Citation Format: Barbara J. Brandhuber, Nisha Nanda, Julia Haas, Karyn Bouhana, Lance Williams, Shannon Winski, Michael Burkard, Brian Tuch, Kevin Ebata, Jennifer Low, Francis Sullivan, Lauren Hanson, Tony Morales, Guy Vigers, Jessica Gaffney, Ross D. Wallace, James Blake, Yutong Jiang, S. Michael Rothenberg, Steven Andrews. Identification and characterization of highly potent and selective RET kinase inhibitors for the treatment of RET-driven cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B192.

Abstract C196: Identification of first-in-class, highly potent FGFR kinase inhibitors that spare FGFR1

Background: The fibroblast growth factor receptor (FGFR1-4) family of tyrosine kinases plays an important role in normal physiologic processes, including angiogenesis, wound healing and regulation of calcium and phosphate metabolism. In addition, dysregulation of FGFR signaling through genetic alterations or altered expression of individual receptors and their ligands has been frequently observed in human tumors. While tyrosine kinase inhibitors (TKIs) with anti-FGFR activity have produced clinical responses in patients whose tumors harbor FGFR alterations, currently available FGFR TKIs inhibit multiple other kinases, including multiple FGFRs. As a result, dose-limiting toxicities have been frequently observed in patients, including hyperphosphatemia which may arise from the inhibition of FGFR1 in the kidney. These toxicities may ultimately limit the efficacy of pan-FGFR inhibitors. However, the ability to develop inhibitors that spare individual FGFRs has been hampered by the high degree of structural similarity between FGFR1, FGFR2, and FGFR3. The development of these tools to distinguish the functional contributions of the individual FGF receptors not only advances the biological understanding of the individual receptors in the context of their expression but may also provide therapeutic agents that have an improved therapeutic index. Methods: In vitro and in vivo evaluations including both enzyme and cell-based assays, pharmacokinetic (PK) studies, measurement of drug metabolism and non-clinical safety evaluation were conducted using standard methods. Tumor growth inhibition and pharmacodynamics (PD) measurements were carried out using subcutaneous xenografts of RT112/84 (which harbor an FGFR3-TACC fusion) bladder cancer cells in nude mice. Results: We have developed compounds with nanomolar FGFR3 enzyme and cell potency, but relatively spare FGFR1 and have minimal activity against an enzyme panel of >200 diverse kinases. This series was further optimized to provide high oral exposure in rodent species. One representative compound was evaluated in a single dose mouse PK at doses of 10, 30 and 100 mg/kg and provided predicted pharmacokinetic coverage of the FGFR3 cell IC50 for >8, >12 and >24 hrs respectively, which correlated with phospho-FGFR3 reduction. The compound demonstrated greater than 40% and greater than 65% tumor regressions of RT112/84 subcutaneous xenografts at doses of 30 and 45 mg/kg/day x 14 day, respectively. These doses were well tolerated and minimal hyperphosphatemia was observed. Conclusions: We have identified potent and selective FGFR inhibitors that spare FGFR1 and other related kinases, and possess high oral bioavailability and favorable PK properties in animals. This next-generation class of compounds will be able to examine selectively the biology of selective FGFR inhibition. The identification of potent and selective FGFR inhibitors with minimal activity against FGFR1 may improve the efficacy and tolerability compared to the currently available pan-FGFR inhibitors. Citation Format: Shannon Winski, Nisha Nanda, Eric Brown, Tony Tang, Barbara Brandhuber, Robyn Hamor, Brian Tuch, Kevin Ebata, Jennifer Low, Francis Sullivan, Darin Smith, Guy Vigers, Megan Strough, Rob Rieger, James Blake, David Moreno, David Chantry, S. Michael Rothenberg, Steven Andrews. Identification of first-in-class, highly potent FGFR kinase inhibitors that spare FGFR1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C196.