
==== Front
NPJ Precis Oncol
NPJ Precis Oncol
NPJ Precision Oncology
2397-768X
Nature Publishing Group UK London

39256512
686
10.1038/s41698-024-00686-8
Article
BPI-28592 as a novel second generation inhibitor for NTRK fusion tumors
Sheng Jin 1
Chen Hong 2
Fu Bang 2
Pan Hongming panhongming@zju.edu.cn

1
Wang Jiabing jiabing.wang@bettapharma.com

2
http://orcid.org/0000-0001-7227-3671
Han Weidong hanwd@zju.edu.cn

13
1 grid.13402.34 0000 0004 1759 700X Department of Medical Oncology, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang China
2 grid.510405.5 0000 0004 9156 6009 Betta Pharmaceuticals Co. Ltd., Hangzhou, Zhejiang China
3 https://ror.org/0144s0951 grid.417397.f 0000 0004 1808 0985 Present Address: Department of Colorectal Medical Oncology, Zhejiang Cancer Hospital, Hangzhou, China
11 9 2024
11 9 2024
2024
8 1984 4 2024
28 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Aberrant activation of tropomyosin receptor kinases (TRKs) is a well-defined oncogenic driver for neurotrophic tropomyosin receptor kinase (NTRK)-fusion cancers, and acquired resistant mutations have emerged with clinical use of the first-generation TRK inhibitors. Here we present BPI-28592, a novel second-generation TRK inhibitor with efficacy against TRK fusion-positive cancers, including those with resistant mutations. Docking simulations indicated no steric hindrance between BPI-28592 and TRK mutants, suggesting its potential to overcome drug resistance. Biochemical assays showed strong inhibition and high selectivity against TRKA, TRKB, and TRKC. The inhibitor significantly reduced cell proliferation and blocked TRK signaling. In vivo studies demonstrated effective tumor suppression in xenograft models harboring TRK fusions with or without resistant mutations. Clinically, BPI-28592 achieved a complete response in a patient with malignant melanoma carrying an AP3S2-NTRK3 fusion (Clinicaltrials. gov identifier: NCT05302843).

Subject terms

Drug development
Molecular medicine
https://doi.org/10.13039/501100001809 National Natural Science Foundation of China (National Science Foundation of China) 82173030 81702809 Sheng Jin Han Weidong issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Genetic alterations resulting from the fusion of neurotrophic receptor tyrosine kinase (NTRK) 1, NTRK2, or NTRK3 genes with various partner genes, encoding the tyrosine kinase receptors (TRK) A, TRKB, and TRKC, represent a common mechanism of oncogenic transformation in a variety of malignancies1–3. Rearrangements between the TRK kinase domain and diverse 5’ gene partners have been detected with varying frequencies in different malignancies4–6. These rearrangements generate in-frame fusions that lead to ligand-independent constitutive TRK fusion protein kinase activity. Consequently, TRK fusion proteins can activate downstream signaling pathways related to cell growth and survival, thus promoting malignant transformation both in vitro and in vivo7,8. Moreover, these oncogenic drivers are mutually exclusive from other known oncogenic drivers in human cancers9–11, thus highlighting the potential therapeutic value of TRK fusion inhibitors. These rearrangements can be identified through DNA and RNA sequencing, as well as cell-free DNA profiling6,12,13.

Currently, there is a great deal of focus on the development of new antitumor drugs that specifically target NTRK. The first-generation TRK inhibitors larotrectinib (LOXO-101, Vitrakvi) and entrectinib (Rozlytrek) have shown high response rates in NTRK fusion-positive cancers regardless of histological classification14,15, and have been approved by the Food and Drug Administration (FDA)16,17. The pooled results of three clinical studies (ALKA-372-001, STARTRK-1, and STARTRK-2)18 involving a total of 54 patients with solid tumors harboring NTRK gene fusions demonstrated that entrectinib achieved an overall response rate (ORR) of 57%. The median duration of response (DOR) was 10 months (95% confidence intervals [CI]: 7.1 to not estimable). Overall, these drugs hold great promise for improving the prognosis of patients with NTRK fusion cancers. These agents are generally well tolerated with occasional off-tumor, on-target adverse events such as nausea and fatigue18,19.

Despite favorable outcomes for the first-generation TRK inhibitors, challenges come with acquired resistance through the development of mutations in the TRK kinase domain20. For instance, TRKAG595R and TRKAG667C resistant mutations were detected in a colorectal cancer patient with LMNA-NTRK1 fusion21,22. Similarly, TRKCG623R were identified in ETV6-NTRK3-positive patients who developed resistance to larotrectinib22,23. In addition, the side effects of these drugs, including fatigue, nausea, vomiting, myalgia, and rashes, significantly affect patients’ quality of life. The development of next generation inhibitors bearing macrocyclic structures, such as repotrectinib (TPX-0005) and selitrectinib (LOXO-195), has been an important step in overcoming resistance-mediated by mutations14,24. Despite relatively slow clinical development of LOXO-19514, repotrectinib has been approved for ROS1-fusion non-small cell lung cancer25.Structural differentiation is a crucial aspect of drug research and development, that contributes to innovative discoveries. A simpler structure can possibly reduce manufacturing costs and alleviate the economic burden on patients. Here, we present the identification, preclinical characterization, and clinical case report of BPI-28592, a novel nonmacrocyclic second-generation TRK inhibitor that has achieved remarkable clinical efficacy in patients with rare malignant cancer.

Results

Modeling of BPI-28592 binding with TRK kinase domain

As previously reported22, TRK mutations can lead to drug resistance. These resistance mutations result in structural changes within the active site that hinder first-generation TRK inhibitor binding. These mutations can manifest at various locations in the active site, including the solvent front (TRKAG595R, TRKCG623R), near the DFG motif (TRKAG667C, TRKCG696A), and at the gatekeeper position (TRKAF589L). Using structural modeling-guided rational drug design, we identified BPI-28592 as a second-generation TRK inhibitor with activity against both wild-type TRKs and multiple drug-resistant TRK mutants, including TRKAG595R, TRKAF589L, TRKCG623R and other mutations. As suggested by the docking simulation model, BPI-28592 binds tightly to ATP-binding pockets in both the wild-type and mutant TRKs, and there is no steric repulsion between BPI-28592 and TRKAG595R or TRKCG623R. This binding provides a reasonable explanation for the observed potent effects on TRKs (Fig. 1).Fig. 1 Modeling of BPI-28592 binding with TRK kinase domain.

Docking simulation of BPI-28592 binding with TRKAWT (A), TRKAG595R (B), TRKCWT (C) and TRKCG623R (D). Black-circles indicate position of BPI-28592 and the mutant residues.

BPI-28592 potently inhibits TRK fusion with or without resistance mutations

As suggested by the enzymatic assay results, BPI-28592 exerted inhibitory effects on wild-type TRKs and TRK fusion proteins comparable to those of larotrectinib and selitrectinib. More importantly, BPI-28592 had greater potency against multiple TRK mutants than larotrectinib, especially for the solvent front drug resistance mutants TRKAG595R and TRKCG623R (Table 1), which had activity similar to selitrectinib.Table 1 Inhibitory activities of BPI-28592 on various TRK kinases

Compound	Kinase inhibition IC50 (nM)	
	Wild type	Fusion	Solvent front mutation	Gatekeeper mutation	xDFG substitution	Other	
	TRKA	TRKB	TRKC	TPM3-TRKA	TPR-TRKA	TRKAG595R	TRKCG623R	TRKAF589L	TRKAG667C	TRKCG696A	TRKAA608D	
Larotrectinib	1.9	0.9	0.3	0.1	0.2	321	133	106	96	2.7	0.9	
BPI-28592	2.1	0.3	0.1	0.2	0.5	5.5	8.2	32	20	2.0	0.9	
Selitrectinib	1.9	0.3	0.1	0.4	0.2	4.0	3.7	10	36	0.6	0.4	

The activity of BPI-28592 was further evaluated at the cellular level using the colon cancer cell line KM12, which naturally carries TPM3-NTRK1, and Ba/F3 cell lines engineered to express various TRK fusions. Overall, the potency of BPI-28592 was superior to that of larotrectinib; notably, BPI-28592 exhibited high activity in multiple Ba/F3 cell lines with resistance mutations that were not sensitive to larotrectinib. As shown in Table 2, the inhibitory potency of this compound was comparable to that of selitrectinib against Ba/F3-TPM3-NTRK1 and Ba/F3-ETV6-NTRK3. For the other tested cell lines, BPI-28592 displayed strong activity against TRK mutants with IC50 values in the two-digit nanomolar range, which was generally similar to that of selitrectinib and significantly superior to that of larotrectinib.Table 2 Proliferative inhibition effects of BPI-28592 on cell lines carrying TRK fusions

Cell line	Compound IC50 (nM)	
	BPI-28592	Larotrectinib	Selitrectinib	
KM12	5.4	10	Not detected	
Ba/F3-TPM3-NTRK1	0.8	4.2	0.9	
Ba/F3-ETV6-NTRK3	0.9	4.4	0.7	
Ba/F3-ETV6-NTRK2	11	23	3.2	
Ba/F3-TPM3-NTRK1 G595R	38	>3000	17	
Ba/F3-LMNA-NTRK1-G595R	41	>300	12	
Ba/F3-LMNA-NTRK1-F589L	73	>300	20	
Ba/F3-ETV6-NTRK3-G623R	16	>300	4	
Ba/F3-ETV6-NTRK2-G639R	94	>300	33	

BPI-28592 shows excellent kinase selectivity for TRKA, TRKB, and TRKC

To explore the selectivity of BPI-28592, kinome profiling was conducted against a panel of 468 kinases using the KinomeScan approach26 at concentrations up to 1 μM, which is significantly greater than the effective dose. The results of these primary screen binding interactions are reported as “% Ctrl”, where lower numbers indicate stronger hits in the matrix. The top 3 hits were TRKA, TRKB and TRKC, which exhibited almost complete target binding, indicating that TRKA has a dominant interaction with TRKs among various kinases (Fig. 2). For further confirmation, all six hits with % Ctrl lower than 35% were tested for Kd values using the KdELECT platform27. The results once again demonstrated excellent specificity, with Kds values of 0.23 nM, 0.55 nM, and 1.1 nM for TRKA, TRKB and TRKC, respectively. Additionally, Kd values of 30 nM, 19 nM and 100 nM were observed for EPHB6, ROS1 and TNK2, respectively.Fig. 2 BPI-28592 shows excellent kinase selectivity for TRKA, TRKB, and TRKC. Kinome profiling was conducted by KINOMEscan approach.

The results of this primary screen binding interactions are reported as “% Ctrl”, where lower numbers indicate stronger hits in the matrix.

BPI-28592 inhibits the TRK signaling pathway in Ba/F3-TPM3-NTRK1 cells

To evaluate the target engagement of BPI-28592 in a cellular context, the activation of the TRK signaling pathway in engineered Ba/F3 cells expressing the TPM3-NTRK1 fusion protein was evaluated by Western blotting. After 4 hours of treatment, BPI-28592 potently inhibited TRKA phosphorylation, even at a low concentration of 0.1 nM, and exhibited superior activity to that of larotrectinib (Fig. 3). Consistently, the activation of downstream proteins in the TRK signaling pathway was strongly suppressed by BPI-28592, as indicated by reduced phosphorylation levels of Shc, ERK, and AKT. These findings suggest that the inhibitory effect of BPI-28592 on cell proliferation is primarily achieved through the inhibition of TRK activation and its regulation of signaling pathways.Fig. 3 BPI-28592 inhibits TRK signaling pathway in Ba/F3-TPM3-NTRK1 cells.

A Ba/F3 cells expressing TPM3-TRKA were lysed after 4 h of treatment with indicated doses of drugs or DMSO control. Phosphorylation of TRKA (Y490), Shc (Y239/240), ERK (T202/Y204) and AKT (S473) were assessed by antibodies specific to the indicated tyrosine or threonine residues. B Normalized inhibition results on phosphorylation based on gray values of all the protein bands. The experiment was repeated three times and normalized quantification for inhibition rate of phosphorylation was graphed as mean ± standard error. Inhibition rate %=100–100*Tc/Controla. Tc: The relative expression ratio of group phosphorylated proteins treated by the compound. a Relative expression ratio of phosphorylated protein in solvent control group.

BPI-28592 in vivo efficacy in multiple NTRK-mutated xenograft models

Then in vivo efficacy of BPI-28592 was investigated in four CDX models and one PDX model. In CDX models with cell lines carrying the NTRK fusion, including two engineered Ba/F3 cell lines overexpressing TPM3-NTRK1 (Fig. 4A) or ETV6-NTRK3 (Fig. 4B), a human colon cancer cell line KM12 (Fig. 4C) and a colon cancer PDX model that naturally harbors TPM3-NTRK1 (Fig. 4D), BPI-28592 at doses ranging from 1.5 mg/kg to 15 mg/kg displayed superior or comparable efficacy to LOXO-101 (larotrectinib) at 15 mg/kg (Fig. 4A–D), and the efficacy of the high-dose group was similar to that of LOXO-195 (selitrectinib, Fig. 4A, D, and E). In terms of drug resistance, BPI-28592 exhibited stronger tumor suppression than LOXO-101 at the same dose of 75 mg/kg in the Ba/F3 xenograft model with the LMNA-NTRK1G595R mutation (Fig. 4E). All the treatments were well tolerated, with no severe body weight loss up to 200 mg/kg bid (Fig.4F), suggesting a high level of drug safety and thus a large safety margin. Overall, an efficacy study in xenograft models indicated the promising therapeutic potential of BPI-28592 in NTRK fusion cancers, both with and without mutations conferring resistance to first-generation TRK inhibitors. Detailed information about the inhibitory effect of BPI-28592 on tumor growth in multiple xenograft models is listed in Table 3.Fig. 4 BPI-28592 in vivo efficacy in multiple NTRK-mutated xenograft models.

Significant tumor growth inhibition (TGI) in multiple xenograft models at doses ranging from 1.5 mg/kg, qd~200 mg/kg, bid in a dose-dependent manner. The CDX models included engineered Ba/F3 cell lines expressing TPM3-NTRK1 (A) or ETV6-NTRK3 (B), a human colon cancer cell line KM12 (C), a colon cancer PDX model that naturally harbors TPM3-NTRK1 (D), and a Ba/F3-derived cell over-expressing LMNA-NTRK1G595R resistance mutation (E). All animals tolerated the treatment well, without severe body weight loss than 10% in the model of LMNA-NTRK1G595R (F).

Table 3 Inhibitory effect of BPI-28592 on tumor growth in multiple xenograft models

Models	Treatment	Dose (mg/kg)	TGI (%)	Cmax (ng/mL)	AUC0-last (h*ng/mL)	
Ba/F3-TPM3-NTRK1	Larotrectinib	15	66.0***a	792	1113	
	Selitrectinib	5	74.8***a	3228	9005	
	BPI-28592	1.5	44.5***a	83.6	393	
	BPI-28592	5	60.3***a	156	859	
	BPI-28592	15	70.4***a	397	3810	
Ba/F3-ETV6-NTRK3	Larotrectinib	15	53.8***b	1684	1519	
	BPI-28592	5	47.0***b	292	2000	
	BPI-28592	15	54.6***b	326	2800	
	BPI-28592	50	77.7***b	1460	9250	
KM12	Larotrectinib	15	37.1 c	639	1070	
	BPI-28592	5	21.9 c	257	1840	
	BPI-28592	15	51.9 c	214	2920	
	BPI-28592	50	64.4*c	760	10,500	
CO-04-0284	Larotrectinib	15	104.3***	2065	3114	
	Selitrectinib	5	100.7***	3,680	14,602	
	BPI-28592	1.5	38.6***	129	811	
	BPI-28592	5	81.0***	394	3140	
	BPI-28592	15	101.4***	553	4450	
Ba/F3-LMNA-NTRK1G595R	Larotrectinib	75	49.6**	17,198	41,748	
	Selitrectinib	25	70.5**	14,011	82,681	
	BPI-28592	25	55.4**	1040	10,300	
	BPI-28592	75	66.2**	2600	28,000	
	BPI-28592	200	69.5**	3860	35,600	
Note: One-way ANOVA analysis compared with the vehicle group, *P < 0.05, **P < 0.01, ***P < 0.001.

aCalculated based on the tumor volume on Day 16, considering animal death due to tumor progression in the following days.

bCalculated based on the tumor volume on Day 12, since the average tumor volume could not be obtained with only one mouse survived in the vehicle group on the last day.

cCalculated based on the tumor volume on Day 14, since several animals were euthanized due to excessive tumor volume. LOXO-101 refers to larotrectinib, LOXO-195 refers to selitrectinib.

Clinical response of a novel NTRK fusion sinonasal malignant melanoma (SNMM) patient to BPI-28592

On May 11, 2020, a 60-year-old male underwent endoscopic resection of a tumor in the right paranasal sinus under general anesthesia. The routine histopathological results after surgery suggested sinonasal malignant melanoma (SNMM). Hematoxylin-eosin (HE) and immunohistochemical (IHC) analyses revealed positive results for S-100, Melan-A, MITF, HMB45, and locally positive for tyrosinase (Fig. 5A). The Ki-67 index was approximately 40% but the results for CK-pan, Syn, CD56, and CgA were all negative. From June 4, 2020 to July 23, 2020, the patient received adjuvant radiotherapy at our hospital. Additionally, he received two cycles of intravenous nedaplatin at a dose of 100 mg each on June 5, 2020 and June 26, 2020, followed by intravenous administration of toripalimab (240 mg) once every 3 weeks, for a total of three cycles until August 19, 2020. Enhanced chest CT scans conducted on August 5, 2021, revealed the presence of multiple nodules in the lungs. Percutaneous puncture biopsy of the lung nodules confirmed SNMM metastasis.Fig. 5 Clinical response of BPI-28592 in a NTRK-fusion malignant melanoma of sinus mucosa.

A The hematoxylin-eosin (HE) and immunohistochemical (IHC) series results were positive for S-100, Melan-A, MITF, HMB45, locally positive for Tyrosinase. Bar=100μm. B Intra-chromosomal NTRK3-AP3S2 fusion t(15;15)(q25;q26). NTRK3-AP3S2 included exons 1–14 of NTRK3 and exon 1 of AP3S2. C The Integrative Genomics Viewer (IGV) snapshot of NTRK3-AP3S2 fusion. D Course of the treatment history with serial CT images during the treatment of BPI-28592.

Next-generation sequencing (NGS) analysis using DNA-based hybrid capture was performed on samples of formalin-fixed and paraffin-embedded tumor tissue and matched whole blood in an effort to determine the optimal therapy. A novel fusion of AP3S2-NTRK3 was detected, with breakpoints between the AP3S2 downstream gene region and exon 14 of the NTRK3 (Fig. 5B, C). The entire NTRK3 kinase domain was retained in this fusion, which could lead to abnormal NTRK3 activation and thus could become a potential oncogene. Other common oncogenic mutations such as KIT, BRAF, or RAS were not observed. The PD-L1 cancer program score (CPS) was zero, and the tumor mutational burden was 6.2 Muts/Mb. The detailed NGS data are presented in the Supplementary Table 1. On September 1, 2021, the patient signed an informed consent form to participate in an “open phase I dose-increasing study evaluating the safety, tolerability, pharmacokinetics, and efficacy of BPI-28592 tablets in patients with advanced solid tumors”. As shown in Fig. 5D, the patient took 100 mg BPI-28592 twice daily orally, achieved marked tumor regression, and achieved confirmed complete response without disease progression or adverse events for more than two years, indicating that BPI-28592 was effective and safe. As of the last follow-up on February 26, 2024, the patient was still receiving BPI-28592 and was in good health (ECOG PS = 0).

Discussion

TRK oncogenic gene fusions are rare in solid cancers but are commonly found in rare tumors, including childhood malignancies. The first generation of TRK inhibitors, such as larotectinib and entrectinib, have successfully targeted cancers harboring NTRK fusions, with excellent efficacy. However, the effectiveness of these inhibitors may ultimately be limited by the development of resistance mutations, such as TRKAG595R and TRKCG623R21,28. Currently, several second-generation of TRK inhibitors have been designed to combat on-target resistance mutations while retaining potency against wild-type TRKA/B/C22,29. Selitrectinib and repotrectinib, exhibit increased activity against wild-type TRKA/B/C compared with first-generation TRK inhibitors. Specifically, the IC50 of selitrectinib and repotrectinib in enzymatic assays for solvent front substitutions are 2.0–2.3 nM and 2.7–4.5 nM, respectively22,24,29. Repotrectinib is a multitarget inhibitor of TRK, ALK, ROS1 and Src29, and has been approved by the U.S. FDA for the treatment of ROS1-positive non-small cell lung cancer25,30. Both selitrectinib and repotrectinib have compact macrocyclic structures, which may cause problems in CMCs, as the formation of large rings of macrocycles has been historically challenging31,32. The compound BPI-28592, which we present here, is a novel nonmacrocyclic second-generation TRK inhibitor designed under the guidance of structural modeling. Moreover, significant architectural variations could engender distinct action profiles, potentially contributing to overcoming resistance mechanisms divergent from those of existing TKIs.

First, BPI-28592 was predicted to bind to TRK mutants with high affinity for the ATP-binding pocket and no stereo-rejection due to mutations such as TRKAG595R and TRKCG623R, which conferred extensive adaptability to pathological structural mutations for overcoming on-target resistance mutations. These findings were objectively confirmed by kinase inhibition and cell proliferation assays. The results support further evaluation of BPI-28592 as a potential clinical TRK inhibitor. Selectivity is of utmost importance when developing small molecule inhibitors that target specific kinases, as it can greatly influence the safety and efficacy of a drug. The robust binding affinity and outstanding selectivity of BPI-28592 for the TRK family underscore its low off-target risks, which implies the possibility of a more favorable safety profile. In addition, activation of downstream proteins in the TRK signaling pathway was strongly inhibited by BPI-28592. We observed varying degrees of inhibition of TRKA, Shc, ERK, and ERK phosphorylation. BPI-28592 inhibits the growth of Ba/F3-TPM3-NTRK1 cells by inhibiting TRKA and ERK. It also significantly inhibited the activation of Shc or AKT signaling at higher concentrations.

Comprehensive in vivo evaluations demonstrated that BPI-28592 is effective at suppressing NTRK-driven tumors, including those with drug resistance mutations. We found that BPI-28592 showed superior or at least comparable efficacy to larotrectinib at the same doseacross all xenograft models tested. Importantly, all the treatments were well tolerated without significant weight loss even at a high dose, indicating that BPI-28592 has a large safety margin. Moreover, as shown in Fig. 4 and Table 3, the efficacy of the high-dose group of BPI-28592 (15 ~ 200 mg/kg) was similar to that of selitrectinib (5–25 mg/kg), which is mainly attributed to increased exposure to selitrectinib instead of higher potency. However, due to different PK profiles across species, dose differences in animal models do not necessarily mean that BPI-28592 requires a more efficacious dose than selitrectinib in humans. In fact, the dose in case reports of selitrectinib escalated to 100 mg BID33, which is the same dose that was utilized in our BPI-28592 case report in this paper. These findings suggest that BPI-28592 has the potential to treat NTRK-fused cancers with or without resistance mutations, and may offer advantages over existing first generation TRK inhibitors.

The activity of BPI-28592 was clinically confirmed in a patient with NTRK3 fusion-positive SNMM. In this case, exons 1–14 of the NTRK3 gene were rearranged with exon 1 of the AP3S2 gene, resulting in formation of the AP3S2-NTRK3 fusion. The rearrangement breakpoint belongs to the region where the NTRK3 gene is frequently rearranged, thus conserving the NTRK3 kinase domain, which has been previously reported as an oncogenic driver16,34–36. The patient enrolled in the phase I clinical trial of BPI-28592, and showed a significant response after two cycles of 100 mg oral treatment twice daily. Treatment continued for two years with no progression or adverse events, demonstrating the safety and efficacy of BPI-28592. To the best of our knowledge, this is the first report of this novel AP3S2 (intergenic)-NTRK3 fusion in patients with malignancies. Although NTRK fusion-positive malignancies are relatively rare, previous studies have demonstrated clinical benefit of NTRK inhibitor therapy33,37. Our research expanded the spectrum of NTRK3 arrangement types and provided a promising NTRK-TKI for consideration.

The Human PK study of BPI-28592 revealed adequate PK profile for clinical efficacy. Taking IC50 for Ba/F3-TPM3-NTRK1 as an example, the converted free drug concentration in human plasma can be covered by the clinical exposure of 100 mg BID. It should be noted that the phase 1 study has not been completed, and RP2D has not been determined yet, 100 mg BID is only one of the dose groups during escalation. Given the high safety of BPI-28592, DLT has not yet observed at higher doses, so RP2D is very likely to be higher than 100 mg BID.

Although the safety, appropriate dosage, and clinical efficacy of BPI-28592 are still being thoroughly studied, these preliminary results suggest that it may offer a potential treatment for NTRK-arranged malignancies. It is imperative to further evaluate the clinical use of BPI-28592 for NTRK-positive patients with additional research and clinical trials. Future research should focus on exploring the efficacy of BPI-28592 in addressing acquired resistance following treatment with larotrectinib or entrentinib in clinical setting.

In conclusion, BPI-28592, a novel second-generation TRK inhibitor, has the potential to overcome resistance mutations commonly identified in patients treated with first generation TRK inhibitors, and thus may prolong the duration of response. This compound represents an important step towards improving the outcomes of patients with NTRK-fusion-driven cancers.

Methods

Structural modeling

Crystal structure of the TRKA (PDBID: 4YNE) and TRKC (PDBID:4YMJ) were used. Both the side chain conformation and homology model for mutant TRK protein were carried out using Prime module (Schrodinger Release 2022-2). The molecular docking was performed with Glide module (Schrodinger Release2022-2).

Enzymatic assays

Freshly prepared reaction buffer: 20 mM HEPES, pH 7.5; 10 mM MgCl2; 1 mM EGTA; 0.02% Brij35; 0.02 mg/ml BSA; 0.1 mM Na3VO4; 2 mM DTT; 1% DMSO; ATP 10 μM. Delivered kinases (TRKAA608D, TRKAF589L, TRKAG595R, TRKAG667C, TPM3-TRKA, TPR-TRKA, TRKB, TRKC, TRKCG623R) into the compound solution and mix gently. Delivered compounds in DMSO into the kinase reaction mixture. Delivered 33P-ATP (specific activity 0.01 μCi/μl final) into the reaction mixture to initiate the reaction, and incubated for 120 minutes at room temperature. Reactions were spotted onto P81 ion exchange paper (Whatman # 3698-915). Washed filters extensively in 0.75% phosphoric acid. Measured the radioactive phosphorylated substrate remaining on the filter paper. Kinase activity data were expressed as the percent remaining kinase activity in test samples compared to DMSO reactions. IC50 values and curve fits were obtained using Graphpad Prism 7 (GraphPad, prism software).

KinomeScan™ profiling

Kinase-tagged T7 phage strains were grown in parallel in E. coli (BL21). E. coli was infected with T7 phage and incubated at 32 °C until lysis. Cell debris were removed from the lysates by centrifuge (6000× g) and filter (0.2 μm). The kinases were produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection. For kinase assays, streptavidin-coated beads were incubated with biotinylated ligands for 30 minutes to generate affinity resins, blocked with excess biotin, and washed with blocking buffer (SeaBlock[Pierce], 1% BSA, 0.05% Tween 20, 1 mM DTT). Reactions were performed in 384-well plates with kinases, prepared beads, test compounds in binding buffer (20% SeaBlock, 0.17× PBS, 0.05% Tween 20, 6 mM DTT). The plates were shaken for 1 hour, and the beads were washed and re-suspended in elution buffer (1× PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand). The kinase concentration in eluates was measured by qPCR.

The results for primary screen binding interactions are reported as ‘% Ctrl’, which calculated by ([test compound signal-positive control signal)/(DMSO control signal-positive control signal]) ×100.

Binding constants (Kds) were calculated with a standard dose-response curve using the Hill equation: response=background + (signal-background)/((1 + [Kdhill slope/dosehill slope]), where the hill slope was set to −1. Curves were fitted using a non-linear least square fit with the Levenberg-Marquardt algorithm.

Cell lines and cell proliferative assay

KM12 cell line was purchased from Otwo Biotech (Shenzen, China) and cultured in RPMI 1640 supplied with 10% fetal bovine serum (FBS). Ba/F3-TPM3-NTRK1 G595R, Ba/F3-ETV6-NTRK3, and Ba/F3 TPM3-NTRK1 cell lines were provided by Pharmaron Inc. (Beijing, China). Ba/F3-ETV6-NTRK2, Ba/F3 LMNA-NTRK1-G595R, Ba/F3 LMNA-NTRK1-F589L, Ba/F3 EVT6-NTRK3-G623R and Ba/F3 ETV6-NTRK2-G639R cell lines were provided by Kyinno Bio (Beijing, China). All the Ba/F3-derived cells were cultured in RPMI 1640 supplied with 10% fetal bovine serum (FBS), penicillin G (100 units/mL), streptomycin (100 µg/mL) and puromycin (2 μg/mL). All cells were incubated in a humidified incubator with 5% CO2 at 37 °C. Cells were seeded in culture plate and kept in incubator overnight, then DMSO diluted compounds were added into the plate at indicated concentrations and cultured for another 72 h. Cell viability was detected by CellTiter-Glo® Luminescent Cell Viability Assay (Promega) following manufacture’s instruction. Antibodies against phospho-TRKA (Y490), TRKA, phospho-Shc (Y239/240), shc, phospho-AKT (S473), AKT, Phospho-ERK (T202/Y204) and ERK, were obtained from Cell Signaling Technology. Uncropped scans of western blots were provided in Supplementary Fig. 1.

Xenograft models

All animal studies were conducted following the guidelines set out by the Guide for the Care and Use of Laboratory Animals and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). All the procedures related to animal handling, care and the treatment in the study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec (Shanghai) Co., Ltd. and IACUC of Phenotek Biotechnology (Shanghai) Co.,Ltd. The corresponding IACUC numbers are ON01-003-2017v1.0, ON01-003-2019v1.0, and IACUC-1909-002.

All mice weighed 18–22 g and were aged 6–8 weeks. Animals were introduced into the test environment about 1 week before the start of experiment for acclimatization and were kept in individual ventilation cages (IVC) in SPF-grade animal room at temperature between 20 °C and 26 °C and humidity between 40% and 70%, with 4–6 animals in each cage. All cages, bedding, and water were sterilized before use and changed twice a week. The environment condition was 12-hour light-dark cycle with light onset at 8:00 a.m. and off at 8:00 p.m. Animals had free access to sterilized food and water.

For cell-derived xenograft (CDX) models, cells were harvested in an exponential growth phase, and each female Balb/c nude mouse (Beijing Vital River Laboratory Animal Technology Co., Ltd.) was inoculated subcutaneously at the right flank with Ba/F3-TPM3-NTRK1, Ba/F3-ETV6-NTRK3, or KM12 cells (5 × 106) in 0.1 mL of PBS, mice were randomized according to the tumor volume and body weight when the average tumor size reached 104 ~ 139 mm3. The mice with tumors that were either too large or too small were excluded before grouping, therefore, 56/90 mice (n = 8), 60/110 mice (n = 12), and 40/110 (n = 8) were included in experimental groups for Ba/F3-TPM3-NTRK1, Ba/F3-ETV6-NTRK3, and KM12 model respectively.

For Ba/F3-LMNA-NTRK1-G595R cell, 2 × 107 cells in 0.1 mL of PBS mixed with Matrigel (1:1) were inoculated to each male NPSG mouse (ViewSolid Biotech). When the average tumor volume reached 137 mm3, mice with tumor volume <70 mm3 or >189 mm3 were excluded, and the remaining 72/90 mice were randomly grouped into six groups (12 mice each group) according to the tumor volume and body weight.

For patient-derived xenograft (PDX) model, patient-derived colon cancer tissue (CO-04-0284) passaged to FP9 was inoculated subcutaneously to female Balb/c nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.), When the average tumor volume reached 114 mm3, mice with tumor volume <79 mm3 or >184 mm3 were excluded, and the remaining 48/100 mice were randomly grouped (8 mice each group) according to the tumor volume and body weight.

All mice were orally administered with indicated treatment when conscious. Animals will be checked daily for morbidity and mortality after tumor injection. Once the animals showed signs of heavily decreased body condition or were found to be moribund, they were humanely sacrificed by CO2.

The major endpoint was to see if the tumor growth could be delayed or mice could be cured. Tumor size was measured twice weekly in two dimensions using a caliper, and tumor volume was calculated by 1/2×L×W2, where L is the tumor length, W is the tumor width. One-way ANOVA was performed to compare three or more groups. Comparisons between groups were carried out with Dunnet (2-sided) test. All data were analyzed using GraphPad Prism 6.0. p < 0.05 was considered to be statistically significant.

Clinical treatment design and conduct

Patients were screened and enrolled in an “Open Phase I dose-increasing study evaluating the safety, tolerability, pharmacokinetics, and efficacy of BPI-28592 tablets in patients with advanced solid tumors (ClinicalTrials.gov under the identifier NCT05302843)” at Sir Run Run Shaw Hospital (SRRSH), School of Medicine, Zhejiang University. The trail registration date was March 10, 2022. The study complies with the principles of the Declaration of Helsinki. The Institutional Review Board of SRRSH has approved the study (approval number 20200814-4), and the patients provided informed consent prior to screening and enrollment. The phase I clinical trial schema was depicted in Supplementary Fig. 2. An “accelerated titration” design was used in this study, and five doses were initially designed, which were 50 mg/d, 100 mg/d, 100 mg/bid, 150 mg/bid, and 200 mg/bid, respectively. Based on the pharmacokinetic and safety data obtained, the dosing setting (including but not limited to the magnitude, interval, frequency and/or interval of dosing) may be adjusted. Up to date, 100 mg BID, 150 mg BID, and 200 mg BID cohorts were added.

Blood samples were collected at 0, 0.5, 1.0, 1.5, 2, 4, 6, 8, 12, and 24 hours after dosing during the steady state after multiple dosing. The plasma was separated for subsequent drug concentration detection using validated LC-MS/MS methods.

Clinical treatment and response assessment

In this study, BPI-28592 was given to patients in the form of a tablet. The frequency and duration of dose modifications and interruptions was determined by a predefined algorithm. Blood samples were collected at 0, 0.5, 1.0, 1.5, 2, 4, 6, 8, 12, and 24 hours after dosing during the steady state after multiple dosing. The plasma was separated for subsequent drug concentration detection using validated LC-MS/MS methods. Data that provides context for the case and preliminary PK results of patients received BPI-28592 were listed as Supplementary Fig. 3. Adverse events were recorded using the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. The response to the treatment was evaluated using the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Supplementary information

SUPPLEMENTAL MATERIAL

Reporting summary

Supplementary information

The online version contains supplementary material available at 10.1038/s41698-024-00686-8.

Acknowledgements

This work was supported by National Natural Science Foundation of China (No. 81702809, 82173030). The funders played no role in study design, data collection, data analysis, data interpretation, or writing of the report. Hong Chen, Bang Fu and Jiabing Wang are employees of Betta Pharmaceuticals Co., Ltd. Weidong Han is currently an employee of Department of Colorectal Medical Oncology, Zhejiang Cancer Hospital, but was an employee of Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University at the time of study completion. The remaining authors have no conflicts of interest to declare.

Author contributions

Conception and design: J.Wang, W.Han, J.S. Compound design and synthesis: B.F. Biological evaluation in preclinical development: H.C. Clinical treatment design and conduct: J.S. and W.Han. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.S., H.C., B.F. Writing, review, and/or revision of the manuscript: J.S., H.C., B. F. Study supervision: J.Wang, W.Han, H.Pan.

Data availability

The datasets generated and/or analyzed during the current study are not publicly available for the following reason: data contain information that could compromise research participant privacy. However, the data can be made available upon reasonable request to the corresponding author with permission from the Institutional Review Board of SRRSH.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Jin Sheng, Hong Chen, Bang Fu.
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