The combination of pralsetinib and bevacizumab enhances antitumor activity against KIF5B-RET lung cancer via PI3K/AKT pathway

Introduction

Lung cancer is the leading cause of cancer-related deaths both in China and worldwide, posing a severe threat to human life and health. A significant proportion (48%) of lung cancer patients present with distant metastases at the time of diagnosis, and these patients have a 5-year relative survival rate of only 8%, thereby highlighting the urgent need for effective therapeutic approaches to improve clinical outcomes in lung cancer patients (1).

Molecularly targeted therapies, particularly tyrosine kinase inhibitors (TKIs), have demonstrated remarkable clinical efficacy in treating various malignancies, such as non-small cell lung cancer (NSCLC) and human epidermal growth factor receptor 2 (HER2)-positive breast cancer. The U.S. Food and Drug Administration (FDA) has approved multiple TKIs (2). Targeting the epidermal growth factor receptor (EGFR) gene, anaplastic lymphoma kinase (ALK), and ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) with TKIs has been shown to significantly extend progression-free survival (PFS) in NSCLC patients, and it has now become the standard first-line treatment for NSCLC patients harboring the respective driver gene mutations (3-6).

Of all human cancers, approximately 2% harbor RET alterations (7). Since the discovery of rearranged during transfection (RET) gene fusions in NSCLC in 2012, at least 12 different fusion variants have been identified, with the KIF5B-RET fusion being the most common and representative mutation (8). Given the approximately 2.5 million new cases of lung cancer diagnosed globally in 2022 (9), treating this rare chromosomal fusion holds significant implications.

In 2020, the U.S. FDA and the National Medical Products Administration (NMPA) of China approved two highly selective RET-TKIs: selpercatinib (LOXO-292) and pralsetinib (BLU-667), for the treatment of NSCLC and thyroid cancer with RET fusions, as well as medullary thyroid cancer with RET mutations (10,11). RET-selective inhibitors selpercatinib and pralsetinib are the preferred first-line treatment options for metastatic NSCLC patients with RET fusions (12). Selpercatinib and pralsetinib have demonstrated durable clinical activity in RET fusion-positive NSCLC, with high objective response rates in both treatment-naive and previously treated patients (13,14).

As well known, resistance is a major challenge in the molecularly targeted therapy using TKIs (15). Some patients may exhibit primary resistance prior to treatment, and even those who initially respond well to therapy will often develop acquired resistance over time (16). Acquired resistance can be developed by secondary mutations of the target, or activation of compensatory signaling pathways (17).

It has been reported that a solvent-front mutation in RET occurs in 10% of patients who develop resistance to RET-TKIs, specifically a glycine-to-arginine substitution at position 810 (G810R), which can interfere with the binding of RET-TKI drugs to the receptor, thus conferring resistance to RET-TKIs (18,19). Studies have shown that MET amplification associated with RET resistance is detected in about 15% of patients (20). A small clinical trial indicated that combining selpercatinib with the MET inhibitor crizotinib can overcome resistance to RET-TKIs and prolong the duration of response in patients (21). Additionally, research suggests that resistance to RET-TKIs can be caused by the activation of KRAS, independent of RET (22). However, understanding of the mechanisms of resistance to RET-TKIs remains limited, and therefore, elucidating the molecular mechanisms of RET-TKI resistance and intervening therapeutically is crucial for improving patient outcomes and extending clinical benefit, holding significant scientific and clinical value.

Vascular endothelial growth factors (VEGFs) are critical factors in inducing tumor microvasculature and are associated with recurrence in NSCLC (23). Drugs targeting the VEGF/VEGF receptor (VEGFR) pathway are commonly used in combination with other therapies to inhibit tumor growth by normalizing the tumor vasculature (24). Some research indicates that inhibition of EGFR can induce the activation of the VEGF pathway through the hypoxia-inducible factor-1 (HIF-1) pathway, significantly increasing the expression of VEGFR-1 and secretion of VEGF, which is notably associated with acquired resistance to EGFR-TKIs (25-29). Moreover, studies have shown that EGFR-TKI treatment in melanoma models can switch the tumor endothelium from EGFR-dependent to VEGFR-dependent, leading to TKI resistance (30).

Numerous clinical studies have demonstrated that combined blockade of VEGFR and EGFR pathways (bevacizumab plus erlotinib) can significantly extend PFS in patients and delay the onset of resistance (31,32). Currently, this “A+T” treatment paradigm has become a standard approach in clinical NSCLC, demonstrating that combined targeted therapy is an effective strategy for overcoming resistance in oncogene-driven NSCLC.

Studies have shown that RET fusion mutations often co-occur with alterations in other genes, such as EGFR, MET, CTNNB1, MAP2K1, AKT1, TP53, MDM2, and VEGF (33,34). The expression levels of VEGFR-2 protein in vivo correlate positively with RET expression in primary tumors, and VEGF-mediated increases in cell proliferation and viability are RET-dependent (35). VEGFR-2, besides being capable of self-phosphorylation, can also induce phosphorylation of RET-tyr1062 and upregulate the expression of glial-derived neurotrophic factor (GDNF) (36). In addition to the direct interactions between RET and VEGF, there are indirect regulatory effects between the two. MET amplification, which occurs in RET resistance, can enhance tumor invasiveness and metastasis, stimulate angiogenesis, and lead to activation of the VEGF pathway (36). Furthermore, phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin (PI3K/AKT/mTOR) pathway, inducing HIF-1 and thereby modulating the VEGF pathway (37). Therefore, activation of the VEGF pathway represents a predictable mechanism of resistance in RET-TKI treatments, and the combination of RET-TKIs and VEGF-TKIs holds promise for overcoming this resistance. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1-0012/rc).

Methods

Lentiviral packaging for KIF5B-RET gene expression

We use a third-generation lentiviral packaging system for lentiviral packaging, the system consists of four plasmids: pLJM-EGFP, pMDLg-pRRE, pRSV-Rev, and pMD2.G. The packaging cell line was 293T. In the lentiviral vector pLJM-EGFP, the full-length KIF5B-RET gene was inserted to replace the EGFP gene to construct pLJM-KIF5B-RET. Lentiviral particles were obtained by transfecting 293T cells using the lipo2000 transfection reagent (Invitrogen).

Construction of BaF3-KIF5B-RET and BaF3-KIF5B-RET/PST cell lines

In order to construct BaF3-KIF5B-RET cell line, Ba/F3 cells were incubated with 10 mL lentivirus-containing medium of 293T after transfection of 48 h, 2 µg/mL puromycin (Beyotime Biotechnology, Cat. No. ST551, Shanghai, China) was used for screening stable transfected cells. In order to construct pralsetinib resistant BaF3-KIF5B-RET/PST cell line, BaF3-KIF5B-RET cells at the logarithmic growth phase were subjected to a stepwise drug exposure regimen using pralsetinib at concentrations of 0, 1, 5, 10, 25, 50, 100, and 200 nM. The cells were cultured in each concentration until they could grow stably before switch into the next stage. This process continued until the BaF3-KIF5B-RET cells could grow stably in a medium containing 100 nM pralsetinib, which is designated as BaF3-KIF5B-RET/PST cell line.

Cell culture and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

BAF3 cells were cultured in RPMI-1640 medium supplemented with 10 ng/mL IL3 (PeproTech, Cat. No. 213-13, Rocky Hill, NJ, USA). The BaF3-KIF5B-RET and BaF3-KIF5B-RET/PST cell lines were maintained in RPMI-1640 medium without IL3. For the MTT assay, BaF3-KIF5B-RET and BaF3-KIF5B-RET/PST cells (5,000 cells per well) were seeded onto 96-well plates. After incubation for 24 hours in serum-containing medium, the cells were treated with pralsetinib at concentrations of 0, 50, 100, 250, 500, 750, and 1,000 nM in serum-containing medium for 72 hours. The MTT assay solution was added to each well to a final concentration of 0.1 mg/mL, and the plates were incubated at 37 ℃ for 2 hours, and the absorbance was measured at 490 nm with a microplate reader.

Western blot analysis

The BaF3-KIF5B-RET and BaF3-KIF5B-RET/PST cells were collected, whole-cell extracts were prepared for Western blot analysis. RET and p-RET, PI3K and AKT and their phosphorylated forms from Abcam (Cambridge, UK) were used as primary antibody. GAPDH was employed as an internal control. Xenograft tumors were collected at the end of the animal experiment and immediately stored at −80 ℃ until protein extraction. Tumor tissues were homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors, followed by centrifugation at 12,000 rpm for 15 min at 4 ℃. The supernatants were collected, and total protein concentrations were determined using a BCA protein assay. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% non-fat milk, the membranes were incubated overnight at 4 ℃ with primary antibodies against PI3K, p-PI3K, AKT, p-AKT, and GAPDH. After incubation with the corresponding secondary antibodies, protein bands were visualized using an enhanced chemiluminescence detection system. GAPDH was used as the internal loading control, and band intensities were quantified using ImageJ software.

Enzyme-linked immunosorbent assay (ELISA)

The BaF3-KIF5B-RET and BaF3-KIF5B-RET/PST cells were seeded at 3×10⁵ cells per well in 6-well plates. The cells were starved in serum-free medium for 24 hours, and after 8 hours, the medium was collected for ELISA analysis. VEGF levels in tumor tissue homogenates were measured using a VEGF ELISA kit validated for mouse tumor samples according to the manufacturer’s instructions. VEGF concentrations were normalized to the total protein concentration of each sample. The VEGF ELISA kit purchased from Beyotime was used and the analysis was performed according to the manufacturer’s instructions.

Animal experiments

Experiments were performed under a project license (No. KYSB20220109) granted by the Laboratory Animal Care and Use Committee of the Jiangsu Province (Suqian) Hospital, in compliance with the institutional guidelines for the care and use of animals. Subcutaneous xenograft models using 6-week-old female BALB/c nude mice were established with BaF3-KIF5B-RET and BaF3-KIF5B-RET/PST cells. Treatment began when the tumor volume reached approximately 100–150 mm³.

For the pralsetinib treatment group, mice received pralsetinib by oral administration at 30 mg/kg per dose. To clarify the dosing schedule, pralsetinib was administered twice daily on days 1–3 and once on day 4, resulting in a total of seven doses. Tumor volume and body weight were monitored until day 16 after treatment initiation to evaluate the durability of tumor control after cessation of dosing, and the bevacizumab group received intraperitoneal injections twice weekly at 5 mg/kg. For the bevacizumab treatment group, mice received intraperitoneal injections of bevacizumab at 5 mg/kg twice weekly, for a total of four injections. Tumor growth was monitored longitudinally to compare the durability of tumor-control effects among the different treatment groups.

Tumor size and tumor tissue

Tumor length and width were measured longitudinally using digital calipers at the indicated time points after treatment initiation. Tumor volume was calculated according to the formula: volume = length × width²/2. When tumor volumes reached approximately 100–150 mm³, mice were randomly allocated to the indicated treatment groups to ensure comparable baseline tumor sizes. Each treatment group included six mice (n=6) in both the BaF3-KIF5B-RET and BaF3-KIF5B-RET/PST xenograft models. Investigators responsible for tumor measurement and data recording were blinded to treatment allocation to minimize potential observation bias. At the end of the in vivo experiment, tumor tissues from the BaF3-KIF5B-RET/PST xenograft model were collected from each treatment group. Tumor tissues were homogenized in ice-cold lysis buffer and centrifuged to obtain the supernatants.

Immunohistochemistry (IHC)

The tumors were placed in 10% (v/v) formaldehyde for fixation, then embedded in paraffin and sliced. Subsequently, the sections were incubated with a CD34 antibody (Abcam) at 4 ℃ for 12 hours, followed by a 1 hour incubation with a secondary antibody (Abcam). The sections were then stained with diaminobenzidine and counterstained with hematoxylin. Images were acquired using a microscope (Leica Microsystems, Wetzlar, Germany).

Statistical analysis

Statistical analyses were performed using GraphPad Prism version 8.0. For data that were approximately normally distributed, Student’s t-test was used to analyze individual variables. Analysis of variance (ANOVA) models were utilized to evaluate continuous data obtained across multiple experimental groups. A P<0.05 was considered statistically significant, whereas ns denotes no statistical significance.

Results

Establishment of resistant cell line BaF3-KIF5B-RET and BaF3-KIF5B-RET/PST

Ba/F3 is a mouse pro-B cell line that depends on interleukin (IL)-3 for survival and proliferation. Initially, we obtained BaF3-KIF5B-RET cells through viral transduction followed by puromycin selection. We first confirmed the expression of the RET protein in the BaF3-KIF5B-RET cell line using Western blot analysis. Compared to the BaF3 cell line, the BaF3-KIF5B-RET cell line stably expressed the RET protein. Treatment of the cell line with pralsetinib led to a significant suppression of p-RET (Figure 1A,1B). Subsequently, the proliferation curve of these cells also demonstrated their ability to survive and proliferate independently of IL-3 (Figure 1C).

Figure 1 Establishment of BaF3 KIF5B-RET cell line and pralsetinib resistant cell line. (A,B) The expression levels of RET and p-RET in BaF3 and BaF3-RET cell line. (C) Relative cell number of BaF3 and BaF3-RET cell line with or without IL-3. (D) Cell viability of BaF3 and BaF3-RET cell line under different concentration of pralsetinib. Data are presented as mean ± SD; ns, not significant; ***, P<0.001. IL-3, interleukin-3; SD, standard deviation.

To obtain a pralsetinib-resistant cell line, we gradually increased the concentration of pralsetinib to treat the BaF3-KIF5B-RET cells and obtained a resistant cell line. The MTT assay showed that this cell strain was resistant to pralsetinib (Figure 1D).

BaF3-KIF5B-RET acquires resistance cell lines via PI3K-AKT signaling pathway (Figure 2)

Figure 2 The features of BaF3-KIF5B-RET and BaF3-KIF5B-RET/PST cell line. (A,B) The expression levels of PI3K, p-PI3K, AKT and p-AKT proteins in BaF3-KIF5B-RET and BaF3-KIF5B-RET/PST with/without pralsetinib. (C) MTT was performed to evaluate cell viability of BaF3-KIF5B-RET and BaF3-KIF5B-RET/PST cell line under pralsetinib or/and bevacizumab. (D) VEGF levels were detected by ELISA at two and four weeks after pralsetinib was added. Data are presented as mean ± SD; ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001. Bev, bevacizumab; ELISA, enzyme-linked immunosorbent assay; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Pra, pralsetinib; SD, standard deviation; VEGF, vascular endothelial growth factor.

Subsequently, we detected protein expression of PI3K-AKT signaling pathway in the BaF3-KIF5B-RET and BaF3-KIF5B-RET/PST cell lines. Western blot results showed that after the addition of pralsetinib, the phosphorylation levels of PI3K and AKT in the pralsetinib-resistant cell line BaF3-KIF5B-RET/PST remained unaffected, whereas the phosphorylation levels of PI3K and AKT in the pralsetinib-sensitive cell line BaF3-KIF5B-RET decreased (Figure 2A,2B). We also detected the VEGF levels at two and four weeks by ELISA, the results demonstrated an increase in VEGF levels in the drug-resistant cell line over time (Figure 2D). We then tested cell viability of BaF3-KIF5B-RET and BaF3-KIF5B-RET/PST cell line under pralsetinib or/and bevacizumab, the BaF3-KIF5B-RET cell line growth were inhibited by pralsetinib, however, both cell line were not inhibited by bevacizumab (Figure 2C).

Combination of pralsetinib and bevacizumab inhibits tumor growth in subcutaneous xenograft mouse models (Figure 3)

Figure 3 Effects of pralsetinib plus bevacizumab on tumor growth in BaF3-KIF5B-RET and BaF3-KIF5B-RET/PST mouse models. (A,B) Tumor volume changes over time after treatment. One-way ANOVA was used to compare tumor volume between group control and pralsetinib and bevacizumab treatment. (C,D) Tumors were weighted at day 16 after treatment. One-way ANOVA was used to compare tumor volume between control and others. (E,F) Images of tumor samples in two mouse models at the end of the experiment. Data are presented as mean ± SD; ns, not significant; ***, P<0.001. ANOVA, analysis of variance; PST + BEV, pralsetinib and bevacizumab treatment; SD, standard deviation.

Given that bevacizumab showed no effect on the two cell lines in vitro, we conducted in vivo animal experiments. Subcutaneous xenograft mouse models were established for both BaF3-KIF5B-RET and BaF3-KIF5B-RET/PST cell lines. The experiment was divided into four groups: control, pralsetinib treatment, bevacizumab treatment, and combined pralsetinib and bevacizumab treatment. For the subcutaneous xenografts of BaF3-KIF5B-RET, pralsetinib, bevacizumab, and the combination of pralsetinib and bevacizumab all significantly inhibited tumor growth (Figure 3A,3C,3E). In contrast, for the subcutaneous xenografts of BaF3-KIF5B-RET/PST only the combined pralsetinib and bevacizumab treatment group significantly inhibited tumor growth (Figure 3B,3D,3F). These results indicate that the BaF3-KIF5B-RET/PST cell line is resistant to single-agent therapy, and that the combination of pralsetinib and bevacizumab is more effective than either agent alone.

VEGF promotes PI3K/AKT activation in pralsetinib-resistant cells, and combination therapy reduces tumor vascular density and PI3K/AKT signaling in vivo (Figure 4)

Figure 4 The synergic effects of pralsetinib and bevacizumab on angiogenesis and PI3K/AKT pathway. (A) IHC staining of tumor with CD34 antibody in BaF3-KIF5B-RET mouse model. (B) IHC staining of tumor with CD34 antibody in BaF3-KIF5B-RET/PST mouse model. (C,D) Western blot analysis of PI3K, p-PI3K, AKT, and p-AKT expression in BaF3-KIF5B-RET and BaF3-KIF5B-RET/PST xenograft tumors after the indicated treatments. (E,F) Quantification of PI3K, p-PI3K, AKT, and p-AKT protein levels in BaF3-KIF5B-RET and BaF3-KIF5B-RET/PST xenograft tumors. (G) Western blot analysis of p-AKT, AKT, p-PI3K, and PI3K expression in BaF3-KIF5B-RET/PST cells treated with recombinant human VEGF-A, recombinant human VEGF-A plus pralsetinib, or recombinant human VEGF-A plus pralsetinib and bevacizumab. (H-K) Quantification of p-AKT, AKT, p-PI3K, and PI3K protein levels in the in vitro VEGF-A stimulation and blockade experiment. Data are presented as mean ± SD; ns, not significant; **, P<0.01; ***, P<0.001. IHC, immunohistochemical; PST + BEV, pralsetinib and bevacizumab treatment; SD, standard deviation; VEGF, vascular endothelial growth factor.

Previous experiments showed that VEGF expression was increased in BaF3-KIF5B-RET/PST pralsetinib-resistant cells. We therefore hypothesized that VEGF-associated signaling may contribute to persistent PI3K/AKT activation and tumor progression in the resistant setting. We firstly evaluate VEGF-associated target engagement in vivo that show VEGF levels were reduced in the bevacizumab-containing groups, with the lowest level observed in the pralsetinib plus bevacizumab combination group from the BaF3-KIF5B-RET/PST xenograft model (Figure S1). In vivo, combination treatment with pralsetinib and bevacizumab reduced tumor vascular density, particularly in the xenograft model (Figure 4A,4B). Consistently, the combination treatment markedly decreased the phosphorylation levels of PI3K and AKT in BaF3-KIF5B-RET/PST tumors, whereas pralsetinib alone had limited inhibitory effects on PI3K/AKT phosphorylation in this resistant model (Figure 4D,4F). In contrast, in the BaF3-KIF5B-RET parental xenograft model, the addition of bevacizumab did not further enhance PI3K/AKT inhibition compared with pralsetinib alone (Figure 4C,4E).

To further examine whether VEGF stimulation could functionally regulate PI3K/AKT signaling in resistant cells, we performed an in vitro VEGF-A stimulation experiment using BaF3-KIF5B-RET/PST cells. Recombinant human VEGF-A increased the phosphorylation levels of PI3K and AKT without substantially altering total PI3K or AKT expression. Pralsetinib did not fully suppress VEGF-A-induced PI3K/AKT phosphorylation, whereas the addition of bevacizumab to pralsetinib attenuated VEGF-A-induced activation of PI3K and AKT (Figure 4G-4K). These results provide functional evidence that VEGF-A stimulation can sustain PI3K/AKT activation in pralsetinib-resistant BaF3-KIF5B-RET/PST cells. Together with the in vivo findings, these data suggest that VEGF-associated signaling contributes to persistent PI3K/AKT activation in pralsetinib-resistant tumors and that combined VEGF blockade enhances PI3K/AKT pathway suppression in the resistant setting.

Discussion

Pralsetinib has been approved for the treatment of NSCLC patients with RET fusions. Although it demonstrates good antitumor activity and safety in RET fusion-positive NSCLC, resistance limits its efficacy. Bevacizumab targets VEGF but is not a standard RET-directed therapy for RET fusion-positive NSCLC (38). Our study investigated whether the addition of bevacizumab would enhance the antitumor activity of pralsetinib in vitro and in vivo, particularly in overcoming tumor resistance. Initially, we found that in vitro, the combination of pralsetinib and bevacizumab did not show a stronger inhibitory effect compared to pralsetinib alone. This might be due to the fact that VEGFR is expressed on vascular endothelial cells rather than on malignant cells in human solid tumors (39). However, in animal experiments, the combination therapy showed better results. Ba/F3 cell survival and proliferation are dependent on IL-3. After transduction with driver genes, Ba/F3 cells become independent of IL-3 and rely instead on the transduced driver gene, thus, the Ba/F3 cell line has become a popular system for studying TKI resistance (40). We first constructed a Ba/F3 cell line dependent on RET and selected a cell line with high VEGF expression by gradually increasing the concentration of pralsetinib. We observed that in the resistant cell line, the downstream AKT/PI3K pathway was activated and not inhibited by pralsetinib alone, but responded more strongly to combination therapy, which was confirmed in animal experiments. To further clarify the mechanistic relationship between VEGF signaling and PI3K/AKT activation in the pralsetinib-resistant setting, we performed additional in vitro functional experiments using BaF3-KIF5B-RET/PST cells. Exogenous VEGF-A stimulation increased the phosphorylation levels of PI3K and AKT without substantially affecting total PI3K or AKT expression, indicating that VEGF signaling can promote activation of this pathway in resistant cells. Pralsetinib alone did not fully suppress VEGF-A-induced PI3K/AKT phosphorylation, whereas the addition of bevacizumab to pralsetinib attenuated this activation. These findings provide functional support for the hypothesis that VEGF-associated signaling contributes to sustained PI3K/AKT activation after acquisition of pralsetinib resistance. Together with the in vivo reduction in tumor vascular density and PI3K/AKT phosphorylation observed in the combination treatment group, these results suggest that the benefit of combining bevacizumab with pralsetinib may be mediated by both anti-angiogenic effects and suppression of VEGF-associated bypass signaling in resistant tumors.

These findings suggest that combination therapy may be a better option for NSCLC patients with RET fusions. VEGF plays a central role in angiogenesis and is essential for the survival of tumor endothelial cells, with the expression of VEGF protein depending on the type of cancer (41). Bevacizumab, by reducing VEGF levels, can block angiogenesis, temporarily normalize tumor vasculature, making the tumor more sensitive to radiotherapy and chemotherapy, improving tumor oxygenation, lowering interstitial pressure, and restoring drug delivery into the tumor (42,43). Thus, the combination of bevacizumab with pralsetinib may enhance antitumor effects by increasing the intratumoral concentration of pralsetinib. Indeed, in our animal experiments, mice in the combination therapy group showed lower levels of VEGF and a decrease in tumor-associated microvessel density compared to the monotherapy groups. On the other hand, when RET-TKI resistance occurs, whether through mutations in RET or activation of downstream pathways, it leads to increased expression of VEGF. Combining VEGF-TKI with RET-TKI simultaneously blocks both upstream and downstream signaling, which can suppress both pre-existing resistant tumor cells and those that express high levels of VEGF due to selective pressures imposed by treatment.

In a recent Case Report, a patient with a KIF13A-RET fusion experienced recurrence after treatment with pralsetinib, the addition of bevacizumab to the original treatment regimen was beneficial (44), which also demonstrates the efficacy of this combination strategy.

Our results indicate that combination therapy can enhance the treatment efficacy in RET fusion-positive NSCLC. However, this effect is applicable only in cases where resistance is VEGF-dependent, and there might be other mutations after RET resistance. Compared to combination therapies targeting a single pathway, targeting different signaling pathways can yield better therapeutic outcomes. Therefore, in addition to angiogenesis, novel combinations with immune checkpoint inhibitors such as programmed death-1 (PD-1) blockers could provide broader applicability. However, in clinical practice, caution should be exercised, especially concerning the associated side effects.

Conclusions

In conclusion, pralsetinib combined with bevacizumab showed enhanced antitumor activity in KIF5B-RET-positive NSCLC models, particularly in the pralsetinib-resistant setting. The combination reduced tumor vascular density and suppressed persistent PI3K/AKT signaling associated with increased VEGF levels. These findings suggest that VEGF-dependent bypass signaling may contribute to pralsetinib resistance and that dual RET and VEGF blockade represents a potential therapeutic strategy for RET fusion-positive NSCLC. Further clinical validation is warranted.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1-0012/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1-0012/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1-0012/prf

Funding: This work was supported by the Suqian Science and Technology Program (No. SY202221).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1-0012/coif). All authors report grant support from the Suqian Science and Technology Program (No. SY202221). The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license (No. KYSB20220109) granted by the Laboratory Animal Care and Use Committee of the Jiangsu Province (Suqian) Hospital, in compliance with the institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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