ERK signaling promotes resistance to TRK kinase inhibition in NTRK fusion-driven glioma mouse models.

Pediatric-type high-grade gliomas frequently harbor gene fusions involving receptor tyrosine kinase genes, including neurotrophic tyrosine kinase receptor (NTRK) fusions. Clinically, these tumors show high initial response rates to tyrosine kinase inhibition but ultimately recur due to the accumulation of additional resistance-conferring mutations. Here, we develop a series of genetically engineered mouse models of treatment-naive and -experienced NTRK1/2/3 fusion-driven gliomas. All tested NTRK fusions are oncogenic in vivo. The NTRK variant, N-terminal fusion partners, and resistance-associated point mutations all influence tumor histology and aggressiveness. Additional tumor suppressor losses greatly enhance tumor aggressiveness. Treatment with TRK kinase inhibitors significantly extends the survival of NTRK fusion-driven glioma mice, but fails to fully eradicate tumors, leading to recurrence upon treatment discontinuation. Finally, we show that ERK activation promotes resistance to TRK kinase inhibition and identify MEK inhibition as a potential combination therapy. These models will be invaluable tools to study therapy resistance of NTRK fusion tumors.

Pyrazolo[1,5-a]pyrimidine as a Prominent Framework for Tropomyosin Receptor Kinase (Trk) Inhibitors-Synthetic Strategies and SAR Insights.

Tropomyosin receptor kinases (Trks) are transmembrane receptor tyrosine kinases named TrkA, TrkB, and TrkC and encoded by the NTRK1, NTRK2, and NTRK3 genes, respectively. These kinases have attracted significant attention and represent a promising therapeutic target for solid tumor treatment due to their vital role in cellular signaling pathways. First-generation TRK inhibitors, i.e., Larotrectinib sulfate and Entrectinib, received clinical approval in 2018 and 2019, respectively. However, the use of these inhibitors was significantly limited because of the development of resistance due to mutations. Fortunately, the second-generation Trk inhibitor Repotrectinib (TPX-0005) was approved by the FDA in November 2023, while Selitrectinib (Loxo-195) has provided an effective solution to this issue. Another macrocycle-based analog, along with many other TRK inhibitors, is currently in clinical trials. Two of the three marketed drugs for NTRK fusion cancers feature a pyrazolo[1,5-a] pyrimidine nucleus, prompting medicinal chemists to develop numerous novel pyrazolopyrimidine-based molecules to enhance clinical applications. This article focuses on a comprehensive review of chronological synthetic developments and the structure-activity relationships (SAR) of pyrazolo[1,5-a]pyrimidine derivatives as Trk inhibitors. This article will also provide comprehensive knowledge and future directions to the researchers working in the field of medicinal chemistry by facilitating the structural modification of pyrazolo [1,5-a]pyrimidine derivatives to synthesize more effective novel chemotherapeutics as TRK inhibitors.

Zurletrectinib is a next-generation TRK inhibitor with strong intracranial activity against NTRK fusion-positive tumours with on-target resistance to first-generation agents.

BACKGROUND: While NTRK fusion-positive cancers can be exquisitely sensitive to first-generation TRK inhibitors, resistance inevitably occurs, mediated in many cases by acquired NTRK mutations. Next-generation inhibitors (e.g., selitrectinib, repotrectinib) maintain activity against these TRK mutant tumors; however, there are no next-generation TRK inhibitors approved by the FDA and select trials have stopped treating patients. Thus, the identification of novel, potent and specific next-generation TRK inhibitors is a high priority. METHODS: In silico modeling and in vitro kinase assays were performed on TRK wild type (WT) and TRK mutant kinases. Cell viability and clonogenic assays as well as western blots were performed on human primary and murine engineered NTRK fusion-positive TRK WT and mutant cell models. Finally, zurletrectinib was tested in vivo in human xenografts and murine orthotopic glioma models harboring TRK-resistant mutations. RESULTS: In vitro kinase and in cell-based assays showed that zurletrectinib, while displaying similar potency against TRKA, TRKB, and TRKC WT kinases, was more active than other FDA approved or clinically tested 1st- (larotrectinib) and next-generation (selitrectinib and repotrectinib) TRK inhibitors against most TRK inhibitor resistance mutations (13 out of 18). Similarly, zurletrectinib inhibited tumor growth in vivo in sub-cute xenograft models derived from NTRK fusion-positive cells at a dose 30 times lower when compared to selitrectinib. Computational modeling suggests this stronger activity to be the consequence of augmented binding affinity of zurletrectinib for TRK kinases. When compared to selitrectinib and repotrectinib, zurletrectinib showed increased brain penetration in rats 0.5 and 2 h following a single oral administration. Consistently, zurletrectinib significantly improved the survival of mice harboring orthotopic NTRK fusion-positive, TRK-mutant gliomas (median survival = 41.5, 66.5, and 104 days for selitrectinib, repotrectinib, and zurletrectinib respectively; P < 0.05). CONCLUSION: Our data identifies zurletrectinib as a novel, highly potent next-generation TRK inhibitor with stronger in vivo brain penetration and intracranial activity than other next-generation agents.

Structural Optimization and Structure-Activity Relationship Studies of 6,6-Dimethyl-4-(phenylamino)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one Derivatives as A New Class of Potent Inhibitors of Pan-Trk and Their Drug-Resistant Mutants.

Tropomyosin receptor kinases (TrkA, TrkB, and TrkC) are attractive therapeutic targets for multiple cancers. Two first-generation small-molecule Trks inhibitors, larotrectinib and entrectinib, have just been approved to use clinically. However, the drug-resistance mutations of Trks have already emerged, which calls for new-generation Trks inhibitors. Herein, we report the structural optimization and structure-activity relationship studies of 6,6-dimethyl-4-(phenylamino)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one derivatives as a new class of pan-Trk inhibitors. The prioritized compound 11g exhibited low nanomolar IC50 values against TrkA, TrkB, and TrkC and various drug-resistant mutants. It also showed good kinase selectivity. 11g displayed excellent in vitro antitumor activity and strongly suppressed Trk-mediated signaling pathways in intact cells. In in vivo studies, compound 11g exhibited good antitumor activity in BaF3-TEL-TrkA and BaF3-TEL-TrkCG623R allograft mouse models without exhibiting apparent toxicity. Collectively, 11g could be a promising lead compound for drug discovery targeting Trks and deserves further investigation.

Design, synthesis and biological evaluation of macrocyclic derivatives as TRK inhibitors.

Tropomyosin receptor kinases (TRKA, TRKB, TRKC) are transmembrane receptor tyrosine kinases, which are respectively encoded by NTRK1, NTRK2, and NTRK3 genes. Herein, we reported the design, synthesis and Structure-Activity Relationship (SAR) investigation of a series of macrocyclic derivatives as new TRK inhibitors. Among these compounds, compound 9e exhibited strong kinase inhibitory activity (TRKG595R IC50 = 13.1 nM) and significant antiproliferative activity in the Ba/F3-LMNA-NTRK1 cell line (IC50 = 0.080 µM) and compound 9e has shown a better inhibitory effect (IC50 = 0.646 µM) than control drug LOXO-101 in Ba/F3-LMNA-NTRK1-G595R cell line. These results indicate that compound 9e is a potential TRK inhibitor for further investigation.

Canadian consensus on TRK-inhibitor therapy for NTRK fusion-positive sarcoma.

Malignant sarcomas are rare accounting for <1% of all adult solid malignancies and approximately 11% to 13% of all pediatric malignancies. TRK-inhibitors have demonstrated robust and long-lasting responses in patients with NTRK fusion-positive solid tumors, including sarcoma. Access to these agents in many jurisdictions such as Canada remains limited. We undertook a modified Delphi consensus to articulate and convey the clinical importance of these agents for the Canadian sarcoma community. A systematic search of published and presented literature was conducted to identify clinical trials reporting outcomes on the use of TRK-inhibitors in relapsed/refractory NTRK fusion-positive sarcoma. Three main consensus questions were identified: (a) is there currently an unmet clinical need for systemic therapy options in relapsed/refractory sarcoma? (b) do TRK-inhibitors confer a clinical benefit to patients with NTRK fusion-positive sarcoma? (c) do phase I/II basket trials provide sufficient evidence to justify funding of TRK-inhibitors in NTRK fusion-positive sarcoma? Response rates to the first and second surveys were 57% (n = 30) and 42% (n = 22), respectively. There was strong agreement among the Canadian sarcoma community that there was unmet clinical need for effective systemic therapy options in relapsed/refractory sarcoma, that TRK-inhibitors are a safe and effective treatment option for patients with NTRK fusion-positive sarcoma, and that available phase I/II basket trials provide sufficient evidence to support funding of these agents in relapsed/refractory NTRK fusion-positive sarcoma. TRK-inhibitors are a safe and effective systemic therapy option for patients with relapsed/refractory NTRK fusion-positive sarcoma.

Discovery of the Next-Generation Pan-TRK Kinase Inhibitors for the Treatment of Cancer.

The neurotrophic receptor tyrosine kinase (NTRK) genes including NTRK1, NTRK2, and NTRK3 encode the tropomyosin receptor kinase (Trk) proteins TrkA, TrkB, and TrkC, respectively. So far, two TRK inhibitors, larotrectinib sulfate (LOXO-101 sulfate) and entrectinib (NMS-E628, RXDX-101), have been approved for clinical use in 2018 and 2019, respectively. To overcome acquired resistance, next-generation Trk inhibitors such as selitrectinib (LOXO-195) and repotrectinib (TPX-0005) have been developed and exhibit effectiveness to induce remission in patients with larotrectinib treatment failure. Herein, we report the identification and optimization of a series of macrocyclic compounds as potent pan-Trk (WT and MT) inhibitors that exhibited excellent physiochemical properties and good oral pharmacokinetics. Compound 10 was identified via optimization from the aspects of chemistry and pharmacokinetic properties, which showed good activity against wild and mutant TrkA/TrkC in in vitro and in vivo studies.

Evolving role of entrectinib in treatment of NTRK-positive tumors.

Targeted therapy has shown to be a very effective treatment in tumors with specific genomic drivers. Trk has proven to be one such target. Efforts to target the Trk fusion with specific inhibitors have shown remarkable responses in a tumor agnostic fashion, with responses seen even in patients with intracranial metastasis. Entrectinib is a first-generation Trk inhibitor with impressive activity in early phase trials performed in patients with NTRK fusion positive solid tumors and ROS1 positive non-small-cell lung cancers with subsequent approval for those indications. Entrectinib was also found to be effective in treatment of brain metastasis and generally well tolerated.

Lay abstract Advances in medical science has allowed us to analyze genes within cancer cells and target abnormal genes more precisely. One such target is called NTRK, which carries genetic information and has been targeted using a medication called entrectinib. This medication is also very effective in patients with cancers that has spread to the brain. This medication can be used in any type of cancer if the cancer cells possess the abnormal DNA. Some of the side effects of entrectinib include weight gain, lightheadedness, throwing up, taste changes, swelling of legs, lack of energy and so on. Based on the benefit of entrectinib seen in clinical trials the medication was approved by the US FDA for treatment of any type of cancer with the NTRK problem. We hope that this new approach to cancer treatment will result in patients having greater benefit and live longer.

FDA Approval Summary: Entrectinib for the Treatment of NTRK gene Fusion Solid Tumors.

The FDA-approved entrectinib on August 15, 2019, for the treatment of adult and pediatric patients 12 years of age and older with solid tumors that have a neurotrophic tyrosine receptor kinase (NTRK) gene fusion without a known acquired resistance mutation, are metastatic or where surgical resection is likely to result in severe morbidity, and have progressed following treatment or have no satisfactory alternative therapy. Approval was based on demonstration of a durable overall response rate of 57% (95% confidence interval: 43-71), including a complete response rate of 7%, among 54 entrectinib-treated patients with 10 different tumor types harboring an NTRK fusion enrolled in one of three single-arm clinical trials. The durations of response ranged from 2.8 months to 26.0+ months; 68% of responses lasted ≥ 6 months. The most serious toxicities of entrectinib are congestive heart failure, central nervous system effects, skeletal fractures, hepatotoxicity, hyperuricemia, QT prolongation, and vision disorders. Adverse reactions were manageable through dose interruptions (46%), dose reductions (29%), or discontinuation of entrectinib (9%). This is the third approval of a cancer drug for treatment of a tissue agnostic, biomarker-defined cancer.

Discovery of First-In-Class Potent and Selective Tropomyosin Receptor Kinase Degraders.

We report compounds 5 (CG416) and 6 (CG428) as two first-in-class tropomyosin receptor kinase (TRK) degraders that target the intracellular kinase domain of TRK. Degraders 5 and 6 reduced levels of the tropomyosin 3 (TPM3)-TRKA fusion protein in KM12 colorectal carcinoma cells and inhibited downstream PLCγ1 signaling at sub-nanomolar concentrations. Both degraders also degraded human wild-type TRKA with similar potency. Interestingly, both degraders, especially 6, showed selectivity for the degradation of endogenous TPM3-TRKA over ectopically expressed ATP/GTP binding protein-like 4 (AGBL4)-TRKB or ETS variant transcription factor 6 (ETV6)-TRKC fusion proteins in KM12 cells. Global proteomic profiling assays demonstrated that 5 is highly selective for the intended target. TPM3-TRKA protein degradation induced by 5 and 6 was further confirmed to be mediated through cereblon and the ubiquitin-proteasome system. Compared with the parental TRK kinase inhibitor, both degraders exhibited higher potency for inhibiting growth of KM12 cells. Moreover, both 5 and 6 showed good plasma exposure levels in mice. Therefore, 5 and 6 are valuable chemical tool compounds for investigating the in vivo function of TRK fusion during tumorigenesis. Our study also paves the way for pharmacological degradation of TRK.

Tropomyosin receptor kinase inhibitors in the management of sarcomas.

PURPOSE OF REVIEW: Genetic aberrations resulting in tropomyosin receptor kinase (TRK) fusion proteins can drive oncogenesis and are postulated to occur in up to 1% of solid tumours. However, TRK fusions in adult sarcomas are rare and there is a significant challenge in identifying patients with sarcomas harbouring TRK fusions in the clinical setting. Despite a recent European Society of Medical Oncology consensus article regarding screening of tumours for TRK fusions, economical and practical limitations present a barrier to widespread screening of sarcomas. RECENT FINDINGS: Larotrectinib and entrectinib are pan-TRK inhibitors which have both received FDA approval for the management of solid tumours harbouring NTRK fusions. Initial results of a number of clinical trials have demonstrated promising efficacy and safety data, including dramatic and durable responses in patients with sarcomas. As such, TRK inhibitors represent a promising treatment option in a small cohort of adult sarcoma patients, where currently treatment options are limited. The emergence of acquired resistance is a concern associated with TRK inhibitor therapy and a number of second-generation agents targeting TRK kinase mutations driving acquired resistance have entered early-phase clinical trials. SUMMARY: With the growing appreciation of the implications of TRK fusions, this review will summarize the emerging clinical trial data of TRK inhibitors in sarcomas. Although in their infancy, clinical trial results are encouraging, and as further results and analyses are released, we will have a greater understanding of their impact on clinical practice and the management of patients with sarcomas.

A newborn with a large NTRK fusion positive infantile fibrosarcoma successfully treated with larotrectinib.

Infantile fibrosarcoma (IFS) is a rare pediatric cancer that typically presents early in life. Surgical resection is commonly curative; however, resection is sometimes not possible requiring additional multimodal treatment. IFS commonly harbors a fusion in one of the neurotrophic receptor tyrosine kinase (NTRK) genes. Larotrectinib, a highly selective inhibitor of tropomyosin receptor kinase (TRK), has been shown to be well tolerated and effective in children as young as 1-month old. We report a case of IFS in a newborn treated with larotrectinib. The patient experienced a rapid clinical and radiographic response demonstrating the potential to treat newborns with larotrectinib.

Larotrectinib, a selective tropomyosin receptor kinase inhibitor for adult and pediatric tropomyosin receptor kinase fusion cancers.

Gene fusions involving NTRK1, NTRK2 and NTRK3 are oncogenic drivers across a wide variety of cancer types. Inhibitors of the chimeric TRKA/B/C protein kinases encoded by these fusions are now available, including larotrectinib, a potent and highly selective oral drug. Integrated data from three trials demonstrate substantial clinical activity of larotrectinib in patients with many different types of cancers harboring NTRK fusions. Larotrectinib has received accelerated approval from both the US FDA and the EMA. Resistance mutations have been observed in the kinase domains of the NTRK fusion genes and development of next-generation tropomyosin receptor kinase inhibitors designed to overcome such resistance mutations is being actively pursued in clinical trials and ongoing drug discovery efforts.

Tropomyosin receptor kinase inhibitors: an updated patent review for 2016-2019.

Introduction: Tropomyosin receptor kinases (Trks) control processes in the fields of growth, survival, and differentiation of neuronal processes. They also play a crucial role in neurodegenerative diseases as well as different types of cancer. Interest in developing Trk inhibitors to target NTRK fusion-driven cancers has escalated in the last decade, leading to the FDA approval of the pan-Trk inhibitors entrectinib and larotrectinib. The development of next-generation inhibitors that overcome resistance mutations arising from treatment with these first generation inhibitors has been the focus in recent years.Area covered: In this updated patent review for 2016-2019, patents covering inhibitors targeting the Trk family are discussed as a continuation of the previous reviews, Tropomyosin receptor kinase inhibitors: an updated patent review for 2010-2016 - Parts 1 & 2. The status of Trk inhibitors in clinical trials is also evaluated. For the identification of relevant patents and clinical trials, Web of Science, Google, Google Patents, and patent referencing were used.Expert opinion: The FDA approval of larotrectinib and entrectinib is a prime example of how basket clinical trial design targeting oncogenic drivers, regardless of tumor histology, is a viable approach to drug discovery and embodies the shift toward personalized medicine.

Entrectinib in patients with advanced or metastatic NTRK fusion-positive solid tumours: integrated analysis of three phase 1-2 trials.

BACKGROUND: Entrectinib is a potent inhibitor of tropomyosin receptor kinase (TRK) A, B, and C, which has been shown to have anti-tumour activity against NTRK gene fusion-positive solid tumours, including CNS activity due to its ability to penetrate the blood-brain barrier. We present an integrated efficacy and safety analysis of patients with metastatic or locally advanced solid tumours harbouring oncogenic NTRK1, NTRK2, and NTRK3 gene fusions treated in three ongoing, early-phase trials. METHODS: An integrated database comprised the pivotal datasets of three, ongoing phase 1 or 2 clinical trials (ALKA-372-001, STARTRK-1, and STARTRK-2), which enrolled patients aged 18 years or older with metastatic or locally advanced NTRK fusion-positive solid tumours who received entrectinib orally at a dose of at least 600 mg once per day in a capsule. All patients had an Eastern Cooperative Oncology Group performance status of 0-2 and could have received previous anti-cancer therapy (except previous TRK inhibitors). The primary endpoints, the proportion of patients with an objective response and median duration of response, were evaluated by blinded independent central review in the efficacy-evaluable population (ie, patients with NTRK fusion-positive solid tumours who were TRK inhibitor-naive and had received at least one dose of entrectinib). Overall safety evaluable population included patients from STARTRK-1, STARTRK-2, ALKA-372-001, and STARTRK-NG (NCT02650401; treating young adult and paediatric patients [aged ≤21 years]), who received at least one dose of entrectinib, regardless of tumour type or gene rearrangement. NTRK fusion-positive safety evaluable population comprised all patients who have received at least one dose of entrectinib regardless of dose or follow-up. These ongoing studies are registered with ClinicalTrials.gov, NCT02097810 (STARTRK-1) and NCT02568267 (STARTRK-2), and EudraCT, 2012-000148-88 (ALKA-372-001). FINDINGS: Patients were enrolled in ALKA-372-001 from Oct 26, 2012, to March 27, 2018; in STARTRK-1 from Aug 7, 2014, to May 10, 2018; and in STARTRK-2 from Nov 19, 2015 (enrolment is ongoing). At the data cutoff date for this analysis (May 31, 2018) the efficacy-evaluable population comprised 54 adults with advanced or metastatic NTRK fusion-positive solid tumours comprising ten different tumour types and 19 different histologies. Median follow-up was 12.9 months (IQR 8·77-18·76). 31 (57%; 95% CI 43·2-70·8) of 54 patients had an objective response, of which four (7%) were complete responses and 27 (50%) partial reponses. Median duration of response was 10 months (95% CI 7·1 to not estimable). The most common grade 3 or 4 treatment-related adverse events in both safety populations were increased weight (seven [10%] of 68 patients in the NTRK fusion-positive safety population and in 18 [5%] of 355 patients in the overall safety-evaluable population) and anaemia (8 [12%] and 16 [5%]). The most common serious treatment-related adverse events were nervous system disorders (three [4%] of 68 patients and ten [3%] of 355 patients). No treatment-related deaths occurred. INTERPRETATION: Entrectinib induced durable and clinically meaningful responses in patients with NTRK fusion-positive solid tumours, and was well tolerated with a manageable safety profile. These results show that entrectinib is a safe and active treatment option for patients with NTRK fusion-positive solid tumours. These data highlight the need to routinely test for NTRK fusions to broaden the therapeutic options available for patients with NTRK fusion-positive solid tumours. FUNDING: Ignyta/F Hoffmann-La Roche.

TrkC-Targeted Kinase Inhibitors And PROTACs.

A small molecule motif (IY-IY), which binds the tropomyosin receptor kinase C (TrkC), was used to deliver the promiscuous kinase inhibitor (KI) dasatinib into breast cancer. Conjugates with noncleavable (1) and cleavable (2) linkers were compared in cellular assays and shown to have more impact on the cell viabilities of TrkC+ breast cancer cells over TrkC- epithelial cells. The IY-IY fragment was also used to recruit the E3 ligase cereblon, giving a potent proteolysis targeting chimeric (PROTAC) for TrkC degradation in metastatic breast cancer cells.

Testing algorithm for identification of patients with TRK fusion cancer.

The neurotrophic tyrosine receptor kinase (NTRK) gene family encodes three tropomyosin receptor kinases (TRKA, TRKB, TRKC) that contribute to central and peripheral nervous system development and function. NTRK gene fusions are oncogenic drivers of various adult and paediatric tumours. Several methods have been used to detect NTRK gene fusions including immunohistochemistry, fluorescence in situ hybridisation, reverse transcriptase polymerase chain reaction, and DNA- or RNA-based next-generation sequencing. For patients with TRK fusion cancer, TRK inhibition is an important therapeutic target. Following the FDA approval of the selective TRK inhibitor, larotrectinib, as well as the ongoing development of multi-kinase inhibitors with activity in TRK fusion cancer, testing for NTRK gene fusions should become part of the standard diagnostic process. In this review we discuss the biology of NTRK gene fusions, and we present a testing algorithm to aid detection of these gene fusions in clinical practice and guide treatment decisions.