The use of tyrosine kinase inhibitors (TKI) with activity against ALK, ROS1, or TRKA-C can result in significant clinical benefit in patients with diverse tumors harboring , or rearrangements; however, resistance invariably develops. The emergence of on-target kinase domain mutations represents a major mechanism of acquired resistance. Solvent-front substitutions such as ALK, ROS1 or ROS1, TRKA, and TRKC are among the most recalcitrant of these mechanisms. Repotrectinib (TPX-0005) is a rationally designed, low-molecular-weight, macrocyclic TKI that is selective and highly potent against ROS1, TRKA-C, and ALK. Importantly, repotrectinib exhibits activity against a variety of solvent-front substitutions and As clinical proof of concept, in an ongoing first-in-human phase I/II trial, repotrectinib achieved confirmed responses in patients with or fusion-positive cancers who had relapsed on earlier-generation TKIs due to ROS1 or TRKC solvent-front substitution-mediated resistance. Repotrectinib (TPX-0005), a next-generation ROS1, pan-TRK, and ALK TKI, overcomes resistance due to acquired solvent-front mutations involving , and Repotrectinib may represent an effective therapeutic option for patients with -rearranged malignancies who have progressed on earlier-generation TKIs. .
NTRK chromosomal rearrangements yield oncogenic TRK fusion proteins that are sensitive to TRK inhibitors (larotrectinib, entrectinib) but often mutate, limiting the durability of response for NTRK+ patients. Next-generation inhibitors with compact macrocyclic structures (repotrectinib, selitrectinib) were designed to avoid resistance mutations. Head-to-head potency comparisons of TRK inhibitors and molecular characterization of binding interactions are incomplete obscuring a detailed understanding of how molecular characteristics translate to potency. Larotrectinib, entrectinib, selitrectinib, and repotrectinib were characterized using cellular models of wild-type TRKA/B/C fusions and resistance mutant variants with a subset evaluated in xenograft tumor models. Crystal structures were determined for repotrectinib bound to TRKA (wild-type, solvent front mutant). TKI-naïve and pretreated case studies are presented. Repotrectinib was the most potent inhibitor of wild-type TRKA/B/C fusions and was more potent than selitrectinib against all tested resistance mutations, underscoring the importance of distinct features of the macrocycle structures. Co-crystal structures of repotrectinib with wild-type TRKA and the TRKA G595R SFM variant elucidated how differences in macrocyclic inhibitor structure, binding orientation, and conformational flexibility affect potency and mutant selectivity. The SFM crystal structure revealed an unexpected intramolecular arginine sidechain interaction. Repotrectinib caused tumor regression in LMNA-NTRK1 xenograft models harboring GKM, SFM, xDFG, and GKM+SFM compound mutations. Durable responses were observed in TKI-naïve and -pretreated patients with NTRK+ cancers treated with repotrectinib (NCT03093116). This comprehensive analysis of first-and second-generation TRK inhibitors informs the clinical utility, structural determinants of inhibitor potency, and design of new generations of macrocyclic inhibitors.
Since 2011, with the approval of crizotinib and subsequent approval of four additional targeted therapies, ALK inhibitors have become important treatments for a subset of patients with lung cancer. Each generation of ALK inhibitor showed improvements in terms of CNS penetration and potency against wild-type 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 ATPbinding boundary to inhibit ALK fusion proteins. In cellular assays, TPX-0131 was more potent than all five approved ALK inhibitors against wild-type ALK and many types of ALK resistance mutations, e.g. G1202R, L1196M, and compound mutations. In biochemical assays, TPX-0131 potently inhibited (IC 50 <10 nmol/L) wild-type 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 ~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 wild-type 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.
3616 Background: RET fusions/mutations drive oncogenesis in lung and thyroid cancers, and several other malignancies. Selective RET inhibitors (selpercatinib/pralsetinib) are active in patients with these cancers; unfortunately, resistance often occurs. On-target resistance includes the acquisition of solvent front mutations (SFMs i.e. RET G810 substitutions). TPX-0046 is a structurally differentiated RET inhibitor that is potent against a range of RET fusions and mutations including SFMs. Methods: The rationally-designed, compact, macrocyclic RET/SRC inhibitor TPX-0046 was characterized in RET-driven in vitro and in vivo tumor models. Results: In enzymatic assays, TPX-0046 showed low nanomolar potency against wild-type RET and 18 RET mutations/fusions. It was potent against SRC and spared VEGFR2/KDR. TPX-0046 inhibited RET phosphorylation (IC50 < 10 nM) in tumor cell lines (LC2/ad, CCDC6-RET; TT, RET C634W) and Ba/F3 engineered RET models (WT, G810R). In cell proliferation assays, TPX-0046 inhibited KIF5B-RET Ba/F3, LC2/ad, and TT cells with IC50 values ~1 nM. Ba/F3 RET engineered cells with SFMs (e.g. G810C/R/S) were potently inhibited by TPX-0046 (mean proliferation IC50 1–17 nM). TPX-0046 demonstrated marked in vivo anti-tumor efficacy in RET-driven cell-derived and patient-derived xenograft tumor models. In a Ba/F3 KIF5B-RET xenograft model, a single dose of 5 mg/kg TPX-0046 inhibited > 80% of RET phosphorylation (corresponding mean free plasma concentration: 51 nM). At 5 mg/kg BID, tumor regression was observed in RET-dependent xenograft models, including those that harbor RET SFMs: TT, CTG-0838 PDX (NSCLC, KIF5B-RET), CR1520 PDX (CRC, NCOA4-RET), Ba/F3 KIF5B-RET, and Ba/F3 KIF5B-RET G810R. Conclusions: TPX-0046 is a unique next-generation RET inhibitor that possesses potent in vitro and in vivo activity against a diverse range of RET alterations, including SFM-mediated resistance. A phase 1/2 trial for RET inhibitor-resistant and naïve RET-driven cancers is on-going (NCT04161391).
Anaplastic lymphoma kinase (ALK) gene rearrangements occur in up to 7% of patients with non-small cell lung cancer (NSCLC) with the majority as EML4-ALK fusions. Crizotinib (first generation ALK inhibitor) was the first approved ALK inhibitor for the treatment of ALK-positive metastatic non-small cell lung cancer. However, development of resistance to crizotinib caused by secondary kinase domain mutations, bypass signaling, or morphology changes occurs. Second generation ALK inhibitors alectinib, ceritinib, and brigatinib were able to overcome the majority of ALK resistant mutations (L1196M, G1269A and F1174L) acquired with crizotinib. The solvent front mutation (SFM) G1202R is a common resistant mutation to crizotinib and the second generation ALK inhibitors. Lorlatinib, a third generation ALK inhibitor, can overcome G1202R resistance with moderate IC50 values of 40 - 60 nM in cell-based assays. Although, compound mutations such as ones with both gatekeeper and solvent front mutations (L1196M/G1202R) are refractory to lorlatinib, representing an unmet medical need. TPX-0131 is a next generation ALK inhibitor designed with a compact macrocyclic structure that can bind completely within the ATP binding boundary to overcome a variety of ALK resistant mutations, especially SFM G1202R and compound mutations L1196M/G1202R. TPX-0131 potently inhibits wildtype (WT) ALK and over 20 different ALK mutations with IC50 values <5 nM when tested in enzymatic kinase assays in the presence of 10 μM of ATP. In cell proliferation assays, TPX-0131 exhibited comparable antiproliferation activity to the most potent ALK inhibitor lorlatinib in Ba/F3 cells engineered with EML4-ALK WT. Importantly, TPX-0131 is more than 100-fold more potent against G1202R than lorlatinib in cell proliferation assays. Furthermore, TPX-0131 demonstrated antiproliferation IC50 values <2 nM in Ba/F3 cell models engineered with compound mutations including L1196M/G1202R, L1198F/G1202R, L1196M/L1198F, and C1156Y/G1202R, while lorlatinib and other ALK inhibitors are not active (IC50s >1 μM). Taken together, TPX-0131 is a next generation ALK inhibitor that can overcome a broad spectrum of acquired resistance mutations, especially the G1202R solvent front mutation and compound mutations (e.g. L1196M/G1202R). The nonclinical pharmacology profile of TPX-0131 warrants further preclinical investigation. Citation Format: J. Jean Cui, Evan Rogers, Dayong Zhai, Wei Deng, Jane Ung, Vivian Nguyen, Han Zhang, Xin Zhang, Ana Parra, Maria Barrera, Dong Lee, Brion Murray. TPX-0131: A next generation macrocyclic ALK inhibitor that overcomes ALK resistant mutations refractory to current approved ALK inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5226.
Oncogenic TRKA/B/C fusions are identified in multiple cancer types in adults and children. TRK inhibitors (TRKis) have demonstrated marked efficacy in patients with these cancers, however, acquired on-target resistance mediated by kinase domain mutations can occur. Next-generation TRKis targeting both wildtype and mutant TRK fusions can address this unmet need. Repotrectinib was designed to potently inhibit wildtype (WT) TRKs and overcome resistance mutations. The anti-proliferative activity of 1st generation (larotrectinib/entrectinib) and next-generation (repotrectinib/LOXO-195) TRKis were compared using engineered Ba/F3 cells expressing WT or mutated TRKs (Table). Repotrectinib was over 10-fold more potent than LOXO-195 against WT TRK fusions and solvent front mutations (SFMs), and more than 100-fold more potent against the gatekeeper mutations TRKA F589L and TRKC F617I. Furthermore, repotrectinib was the only TRKi active against the compound mutation TRKA G595R/F589L in cis in preclinical Ba/F3 cells. In xenograft tumor models, repotrectinib led to significant tumor regression in tumors carrying WT or mutated TRK fusions. In the ongoing TRIDENT-1 phase 1 clinical trial of repotrectinib (NCT03093116), the SFMs TRKA G595R, TRKC G623R and TRKC G623E and the gatekeeper mutation TRKA F589L were detected in plasma cfDNA samples at baseline from three TRKi-resistant patients. Repotrectinib was active against ETV6-TRKC G623E in an entrectinib-resistant patient with a salivary gland tumor (-82%, confirmed partial response, RECIST v1.1). Tumor regression (-33%) was achieved in a larotrectinib-resistant cholangiocarcinoma patient with LMNA-TRKA G595R and F589L mutations in trans. TRIDENT-1 is currently enrolling NTRK fusion-positive patients with advanced solid tumors. Ba/F3 Cell Proliferation Assay IC50 (nM)LMNA-TRKAETV6-TRKBETV6-TRKCTRK InhibitorWTG595RG667CF589LG595R/F589LWTG639RWTG623RG623EF617IRepotrectinib<0.10.19.2<0.117.4<0.13.2<0.10.40.9<0.2LOXO-1958.613.194.931.6531.11.028.41.724.649.153Larotrectinib15.918981863625.31000013.1300023.2699921625000Entrectinib0.3614186.7<0.220000.220000.41340171261.7 Citation Format: Alexander Drilon, Dayong Zhai, Wei Deng, Xin Zhang, Dong Lee, Evan Rogers, Jeffrey Whitten, Zhongdong Huang, Armin Graber, Juliet Liu, Shanna Stopatschinskaja, J. Jean Cui, Dong-Wan Kim, Byoung Chul Cho, Robert C. Doebele, Sai-Hong Ignatius Ou, Jeeyun Lee, Alice T. Shaw. Repotrectinib, a next generation TRK inhibitor, overcomes TRK resistance mutations including solvent front, gatekeeper and compound mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 442.
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