Introduction

The tropomyosin receptor kinase (TRK) family includes TRKA, TRKB, and TRKC, which are encoded by NTRK1, NTRK2, and NTRK3 genes, respectively1. TRK fusion proteins involve the 5ʹ of a partner gene and the 3ʹ of the NTRK genes, including their kinase domain coding sequences. The dimerization domain of the partner gene confers ligand-independent activation of the tyrosine kinase, sustaining intracellular signaling, cell proliferation and survival2. TRK fusions have been identified in various cancers, including colorectal, lung, and papillary thyroid carcinomas, melanoma, and sarcoma3.

Also, the RET receptor kinase is rearranged in several types of cancers such as papillary thyroid (PTC), breast, lung, and colorectal (CRC) carcinomas4. In addition, germline RET point mutations are responsible for the Multiple Endocrine Neoplasia type 2 (MEN2), while somatic mutations of RET are associated to about 50% of sporadic medullary thyroid carcinoma (MTC)4.

TRKs and RET derived oncoproteins are attractive therapeutic targets. Several tyrosine kinase inhibitors (TKIs) of TRKs have been developed and tested in clinical trials; among them, Entrectinib (RXDX-101), Selitrectnib (LOXO-195) and Larotrectinib (LOXO-101) have been recently approved by FDA for the treatment of solid tumors with TRK fusion proteins5,6,7. The anti-RET/VEGFR2 TKIs Vandetanib and Cabozantinib were initially approved for MTC patients8,9. More recently, two selective anti-RET drugs (sparing VEGFR2 activity), Pralsetinib and Selpercatinib, have demonstrated clinical efficacy in RET-driven cancers and have been approved as RET-targeted cancer drugs10,11,12,13,14,15.

By a fragment-based medicinal chemistry approach, we discovered the benzimidazole Pz-1 as a potent type II tyrosine kinase inhibitor against RET, VEGFR2 and TRK kinase16,17. Pz-1 was able to inhibit the phosphorylation and downstream signaling of CCDC6-RET and TPM3-TRKA oncoproteins at single-digit nM in TPC-1 and KM12 cells, respectively.

Proteolysis Targeting Chimeras (PROTACs) are chimeric compounds able to target protein of interest (POI) into degradation by the ubiquitin–proteasome pathway. Structurally, PROTACs are bi-functional molecules consisting of one ligand that binds to the POI, conjugated via a linker to another ligand that recruits a E3 ubiquitin ligase18. Thus, by hijacking the E3 ubiquitin ligase, PROTAC brings the POI in its proximity, committing POI to poly-ubiquitination and degradation by the proteasome19. Many proteins ranging from transcription factors to kinases (such as AKT, HER2, EGFR, MET, BCR-ABL) have been successfully targeted by PROTACs20,21,22,23,24. TRKs targeting PROTACs have been recently described against TRKC25 or TRKA; the last ones were based on the scaffold of GNF-8625, a type I pan-TRK kinase inhibitor, and degraded TRKA at a concentration smaller than 0.5 nM26. RET-specific PROTAC have been described based on LOXO-292 scaffold that proved able to induce oncogenic RET proteins degradation at concentrations ranging between 5 and 26 nM27,28,29.

Here, we exploited Pz-1 as a ligand to generate PROTACs directed against RET and TRKA oncoproteins. To this aim, we designed and synthesized a library of PROTACs by conjugating Pz-1 to CRBN binding moieties derived from thalidomide30. Here, we report their generation and characterization.

Materials and methods

Compound preparation

Compounds synthesis is described in detail in the Supplementary information. All compounds were dissolved in DMSO and stored in − 80 °C for future use. Final dosing solution was freshly prepared for each experiment from the stock solution; the equivalent amount of vehicle (DMSO) was used as control. Cycloheximide (C7698), pomalidomide (P0018), and MG132 (M8699) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). The protein neddylation inhibitor MLN4924 (Pevonedistat) was purchased from Selleckchem (Houston, Tx, USA).

Cell lines

Human medullary thyroid carcinoma (MTC) TT cells were from ATCC and grown in RPMI 1640 (Life Technologies, Carlsbad, CA, USA) supplemented with 20% FBS; human MTC MZ-CRC-1 cells were kindly provided by R.F. Gagel (MD Anderson, Houston, TX, USA) and grown in DMEM (Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS. Human papillary thyroid cancer (PTC) cell lines TPC-1, kindly provided by M. Nagao (National Cancer Center Research Institute, Tokyo, Japan), and B-CPAP were grown in DMEM with 10% FBS. SV40-immortalized human thyroid follicular epithelial Nthy-ori-3-1 cells were obtained from ECACC and were grown in DMEM with 10% FBS. Human colorectal carcinoma (CRC) cell lines KM12 and HCT-116, kindly provided by A. Bardelli (Candiolo Cancer Institute, Torino, Italy) and D. Grieco (Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Naples, Italy) respectively, were grown in RPMI with 10% FBS. Human non-small cell lung cancer (NSCLC) cell lines Lc-2/ad, and A549, were obtained from ECACC. Human lung adenocarcinoma (LUAD) cells Lc-2/ad were grown in RPMI 1640/HAM’S F12 (1:1) (Lonza, Walkersville, MD USA) and A549 cells were grown in DMEM, both supplemented with 10% FBS. Human cervical carcinoma HeLa cells were from American Type Culture Collection (ATCC, Manassas, VA, USA) and were grown in RPMI (Life Technology, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA). Human HEK 293 cells were from American Type Culture Collection (ATCC, Manassas, VA, USA) and were grown in EMEM (Lonza, Walkersville, MD USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA). All cell culture media were supplemented with 100 units/ml penicillin–streptomycin and 2 mM l-glutamine (Life Technologies). Cells were grown in a humidified incubator at 37 °C and with 5% CO2. Authentication of the cell lines was performed at BMR genomics by DNA fingerprint (Aviano, Italy).

Cell transfection

Transient transfections were carried out with Fugene according to the manufacturer’s instructions (Promega, Milano, Italy). HEK 293, HeLa and KM12 cells were allowed to adhere to the 100 mm cell culture plate and transfected with the indicated plasmids (pBABE-TPM3-TRKA, pCDNA-Cullin 4A ΔNEDD8, pBabe-RET/C634R and pBabe-RET/M918T, pCDNA-VEGFR2) when the 70–80% of confluency was reached. Twenty-four hours after transfection, cells were divided into 60 mm plate and treated the next day.

Cell proliferation assay

Cell proliferation was measured by counting cells after 72 or 96 h of treatment with the indicated compounds. Briefly, TPC-1 (10,000/well), KM12 (50,000/well), Nthy-ori 3-1 (20,000/well), B-CAP (20,000/well), A549 (20,000/well) and HCT-116 (20,000/well) were seeded in 6-well plates; the day after plating, the compounds were added to the medium and incubated for 72 (TPC-1, Nthy-ori 3-1, B-CAP, A549, HCT-116) or 96 (KM12) hours. At the end of treatment, cells were counted and IC50 dose was calculated using PRIZM Graphpad software (GraphPad Software Inc).

Growth curves

TPC-1 (5000/well), KM12 (10,000/well), MZ-CRC-1 and Lc-2/ad (100,000/well), and TT (200,000/well) were seeded in 6-well plates. TPC-1 and TT cells were grown in 2% and 20% FBS respectively. All the other cell lines were kept in 10% FBS. The day after plating, cells were counted, and drug or vehicle were added to the medium and changed every 2–3 days. Cells were counted in triplicate every 2–3 days and the number of cells of the last day of the experiment was used to calculate growth inhibition.

Western blotting

Cells were all lysed in a buffer containing 50 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES; pH 7.5), 1% (v/v) Triton X-100, 150 mM NaCl, 5 mM EGTA, 50 mM NaF, 20 mM sodium pyrophosphate, 1 mM sodium vanadate, 2 mM phenylmethanesulfonyl fluoride (PMSF), and 1 μg/mL aprotinin. Lysates were processed as described previously17. Proteins were separated onto 8% polyacrylamide gel, and transferred to nitrocellulose membrane (4℃, overnight). The following day, the membranes were blocked at room temperature for 1 h with a buffer containing 5% BSA in TBS 1X (50 mM Tris–HCl pH 7.5, 150 mM NaCl) and then incubated with specific primary antibodies for 1 h at room temperature. After washing with TBST 1X (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20), membranes were incubated with secondary antibodies for 35 min and then washed for 1 h. Immune complexes were visualized using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) and films were developed using an X-ray film developer. Antibodies for VEGFR2 (2479), phospho-TRKA (Tyr490) (9141), phospho-TRKA (Tyr674/675) (4621), panTRK (A7H6R) (92991) and RET (14698) were from Cell Signaling Technology (Beverly, MA, USA). Anti-tubulin (T6074) was from Sigma-Aldrich. Secondary antibodies coupled to horseradish peroxidase were purchased from Bio-Rad.

Mouse experiments

In vivo target inhibition studies were conducted at the Dipartimento di Medicina Molecolare e Biotecnologie Mediche in accordance with Italian regulations for experimentation on animals. The study was approved by the Italian Ministry of health (Authorization n. 683/2024-PR). Female BALB/c nu/nu mice were purchased from Charles River Laboratories (Wilmington, MA, USA). All manipulations were performed while the animals were under isoflurane gas anesthesia. No mouse showed signs of wasting or other signs of toxicity. Mice were injected with KM12 cells (10 × 106/mouse) subcutaneously into the right flank; when tumours reached a volume of 400–500 mm3, mice were subjected to intraperitoneal treatment with ZW-6-052 (15 mg/kg) or vehicle. After 12 h mice were subjected to euthanasia by carbon dioxide (CO2) inhalation and tumours were collected and snap frozen in liquid nitrogen. Frozen tumours were homogenized in lysis buffer by using the Mixer Mill MM300 (Qiagen, Germantown, MD, USA). Samples were clarified twice by centrifugation at 10,000×g. Tumour proteins were subjected to standard Western blot.

Statistical analysis

IC50 doses for cell growth were calculated through a curve fitting analysis from last day of growth curves using the PRIZM software (GraphPad Software Inc). Unpaired Student’s t test was performed to compare cell growth using the Instat software program (Graphpad Software Inc, San Diego, CA, USA). P values were two-sided, and differences were statistically significant at P < 0.05.

Results

Design and synthesis of Pz-1-derived PROTACs

We used the RET/TRKA type 2 kinase inhibitor Pz-1 as a binding moiety to generate 22 different PROTAC compounds. The first orally active PROTAC tested in clinical trials was ARV110, containing a CRBN ligand and targeted to the androgen receptor31,32. Therefore, we based our PROTACs on different CRBN ligands (pomalidomide, lenalidomide, or avadomide) which were conjugated through a chemical linker to the solvent-exposure methyl moiety of Pz-1. Only one PROTAC, compound 19, contained a chemical ligand for HL (Fig. 1, Table 1, and Supplementary information). Various chemical linkers have been used to conjugate the the E3 ligase ligands to the target-binding moiety. They can be broadly classified as “flexible”, such as PEG, aliphatic, and triazole-based linear linkers, or “rigid” linkers with ring systems. The first ones are more commonly used, although they are more susceptible to oxidation; the seconds suffer from more challenging synthesis methods33. Thus, here, we used PEG-, aliphatic- as well as triazole-based linkers (Table 1 and Supplementary information). In detail, pomalidomide-based PROTACs were conjugated to either flexible (PEG, alkyl, or triazole) linkers, while for the lenalidomide- or avadomide-based ones we used PEG linkers (Fig. 1, Table 1, and Supplementary information). The precursor compound (ZW-6-020, compound 1; NMR spectra in Supplementary information, Figs. S1 and S2), used as a negative control, was the key intermediate for the generation of the PROTACs by conjugation to the linker via a N-methylformamide (Table 1, and Supplementary information). In addition, starting from compound 9, we generated another negative control, compound 13, by attaching an ethyl moiety onto the glutarimide of pomalidomide to prevent pomalidomide binding to CRBN. Compound 13 is expected to retain the binding to RET/TRKA but not to be able to induce the degradation of target proteins (Supplementary information, Figs. S3–S6).

Fig. 1
figure 1

(A) Mechanism of action of PROTACs. (B) Chemical structure and key elements of PROTAC RET/TRKA degraders. The left end of each chemical linker is attached to Pz-1 and the right end is attached to E3 ligase ligand. The conjugation sites between the chemical linker and Pz-1 or E3 ligase ligand are shown as wavy lines.

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Table 1 Pz-1-derived PROTACs with various linkers and E3 ligase ligands.
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Identification of RET/TRKA-targeting Pz-1-derived PROTACs

We used KM12, expressing the TMP3-TRKA chimeric protein, and TPC-1, expressing the CCDC6-RET chimeric protein, cells to assess Pz-1-based PROTACs ability of inducing degradation of TRKA- and RET-derived oncoproteins. Pz-1 and vehicle (DMSO) were used as negative controls.

Eight of the PROTACs (compounds 8, 9, 10, 12, 15, 20, 21 and 22) strongly reduced both CCDC6-RET and TPM3-TRKA protein level already at 10 nM dose, while compound 11 induced TMP3-TRKA degradation but was almost ineffective on CCDC6-RET (Figs. 2 and 3). The remaining compounds (2, 3, 5, 6, 7, 14, 16, 17, 18, and 19) displayed an intermediate activity being able to reduce at least one of the two protein levels at 50 or 250 nM concentration. These data indicated that triazole linker and VHL ligand are unsuitable for degrading TRKA and RET kinases. In the case of pomalidomide-based PROTACs containing PEG or alkyl linkers, compounds with linkers of 7 to 13 carbon and/or oxygen atoms were the most effective against RET; TRKA could adopt also longer linkers. As predicted, the control compounds 1 and 13, described above and lacking CRBN binding ability, were unable to induce degradation of both TPM3-TRKA and CCDC6-RET up to 250 nM concentration (Figs. 2 and 3).

Fig. 2
figure 2

Pz-1-based PROTACs activity on TMP3-TRKA. KM12 cells were treated with the indicated PROTACs or Pz-1 for 12 h at the indicated concentrations. Cells were harvested, and extracts were subjected to Western blotting with anti-panTRK antibody. Tubulin was used as a loading control.

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Fig. 3
figure 3

Pz-1-based PROTACs activity on CCDC6-RET. TPC1 cells were treated with the indicated PROTACs or Pz-1 for 12 h at the indicated concentrations. Cells were harvested, and extracts were subjected to Western blotting with anti-RET antibody. Tubulin was used as a loading control.

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RET/TRKA PROTACs are able to inhibit cancer cell proliferation

We treated KM12 and TPC-1 cells with the ten most potent PROTACs (i.e. compounds 8–12, 15, 18, and 20–22). As shown in Supplementary information (Fig. S7), compounds 9, 11, 20 and 21 were the strongest ones active on both TRKA and RET and displaying a similar IC50 in both KM12 and TPC-1 cells (7.5, 5.5 and 6.3 nM in KM12 cells and 23.3, 14.9 and 15.2 nM in TPC-1 cells, respectively). Among them, compound 9 was selected for further studies.

Inhibition of RET- and TRKA-driven cancer cell proliferation by compound 9

We investigated whether compound 9 was able to hinder proliferation of human cancer cells displaying RET- or TRKA-derived oncoproteins. To this aim, besides KM12 and TPC-1 cells, we exploited TT cells, derived from a MTC harboring RET C634W oncogenic mutant, and Lc-2/ad cells, derived from a LUAD harboring the CCDC6-RET rearrangement. We also used another MTC cell line, MZ-CRC-1, carrying the RET M918T oncogenic mutant.

We performed growth curves of the 5 cell lines by treating them with different doses of the compound. As shown in Fig. 4, the IC50 dose of compound 9 for KM12, TT, TPC-1 and Lc-2/ad cells ranged between 2.6 and 10.4 nM. Accordingly, the compound was able to induce a strong reduction of the concentration of RET- or TRKA-derived oncoproteins at 10 nM (Supplementary information, Fig. S8). Instead, MZ-CRC-1 proliferation was only modestly affected by compound 9 (IC50 600.5 nM) (Fig. 4). Consistently, RET M918T protein levels were reduced only at very high doses (> 1000 nM) of compound 9 (Supplementary information, Fig. S8). Such lack of effect of compound 9 on RET M918T could be due either to an intrinsic resistance of this mutant to PROTAC-mediated degradation or, in alternative, to a defective E3-ligase/proteosome machinery in MZ-CRC-1 cell line. To dissect between these two possibilities, we exogenously expressed RET M918T in HeLa cells, known to be able to sustain degradation mediated by pomalidomide-based PROTACs34. In HeLa cells compound 9 was able to induce RET M918T degradation already at 10 nM, like RET C634R used as a control (Supplementary information, Fig. S8). In addition, RET M918T was also transiently expressed in KM12 cells, that, based on our data are susceptible to TMP3-TRKA degradation upon treatment with compound 9 (Fig. 2 and Supplementary information, Fig. S8). As shown in Supplementary information (Fig. S8), also in this case RET M918T protein was degraded, parallel to endogenous TPM3-TRKA degradation, with a similar potency as RET C634R upon compound 9 treatment. These results indicated that Pz-1 based PROTACs can degrade also RET M918T oncoprotein and that PROTACs activity is cell context dependent. Interestingly, both in HeLa and in KM12 cells a reduction of the degrader activity on RET and TRKA proteins was observed when the PROTAC was used at very high doses (> 1000 nM). This is a typical PROTACs phenomenon, named “hook effect”, and consists in their decreased activity at high concentrations, because of binding sites saturation on either the target protein (POI) or the E3 ligase which prevents the formation of the required ternary complex35.

Fig. 4
figure 4

Compound 9 activity on RET- and TRKA-driven cell proliferation. The indicated cell lines were incubated with vehicle (NT: not treated) or increasing concentrations of compound 9 and counted at the indicated time points. Data are the mean ± SD of a single experiment performed in triplicate. Number of cells plated at Day 0: KM12 (10,000/well); TT (200,000/well); TPC-1 (5000/well); MZ-CRC-1 and Lc-2/ad (100,000/well). IC50 dose was calculated using PRIZM software (GraphPad Software Inc). Confidence intervals are indicated in brackets.

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To compare the ability to induce protein degradation with the potency in inhibiting cell proliferation, we also selected 4 additional PROTACs, one with a similar activity to compound 9 (compound 20), one with no activity (compound 13) and two with an intermediate activity (compound 17 and compound 3) (Fig. 2 and 3). As shown in supplementary information (Figs. S9 and S10), compound 20 was able to inhibit cell proliferation with an IC50 dose (around 1–2 nM) similar to compound 9. On the contrary, the other compounds were far less efficient with IC50 tenfold greater than compound 9.

Finally, to test whether the effect of compounds 9 and 20 was dependent on RET/TRKA inhibition, the two compounds were tested in comparison to Pz-1 in thyroid immortalized and cancer (Nthy-ori 3–1 and B-CPAP, respectively), lung cancer (A549), and colon cancer (HTC-116) cells, all negative for RET or TRKA oncoproteins. As shown in Supplementary information (Fig. S11), these cells were resistant to compound 9 and compound 20 as to Pz-1, featuring a IC50 dose for cell proliferation > 250 nM. Differently from Pz-117, compound 9 and compound 20 were unable to target VEFR2 protein exogenously expressed in Hela cells (Figure S12).

Mechanism of target degradation by compound 9

To characterize the mechanism underlying decrease of protein levels, we measured TPM3-TRKA protein half-life upon treatment of KM12 cells with compound 9 and cycloheximide, a toxin able to block protein synthesis. Cycloheximide alone had no effect on the levels of TPM3-TRKA protein up to 8 h, indicating that TPM3-TRKA half-life is longer than 8 h. In cells co-treated with cycloheximide and compound 9, TPM3-TRKA protein and phosphorylation levels significantly decreased as soon as after 2 h, indicating that compound 9 strongly shortened protein half-life. Specific TRKA autophosphorylation sites (Y674/675 and Y490) were tested (Fig. 5A).

Fig. 5
figure 5

Mechanism of compound 9-induced TPM3-TRKA degradation. (A) KM12 cells were pretreated with 10 μg/ml cycloheximide for 20 min, followed by 50 nM compound 9 for the indicated time points. Cells were harvested, and extracts were subjected to Western blotting with the indicated antibodies. Tubulin was used as a loading control. (B) KM12 cells were pretreated with 25 μM MG132 for 30 min, and then treated with 50 nM Compound 9 for 4 h. Cells were harvested, and extracts were subjected to Western blotting with the indicated antibodies. Tubulin was used as a loading control. (C) HEK 293 cells were transfected with TPM3-TRKA and then, after 48 h from transfection, pretreated with MG132 for 30 min, followed by compound 9 for 4 h. Cells were harvested, and extracts were subjected to immunoprecipitation and Western blotting using the indicated antibodies. Tubulin was used as a loading control. (D) KM12 cells were treated with 50 μM chloroquine and 50 nM compound 9 for 6 h. Cells were then harvested, and extracts were subjected to Western blotting with the indicated antibodies. Tubulin was used as a loading control. (E) KM12 cells were pre-incubated with different concentrations of pomalidomide (µM: 0.5, 1, 5, 10) for 20 min and then treated with 50 nM Compound 9 for 4 h. Cells were then harvested, and extracts were subjected to Western blotting with the indicated antibodies. Tubulin was used as a loading control. (F) HEK293 cells were transfected with TPM3-TRKA alone or together with the myc-tagged Cullin4A ΔNEDD8 truncated construct. 48 h upon transfection cells were treated with compound 9 (50 nM or 250 nM) for 6 h and then harvested. Extracts were subjected to Western blotting with the indicated antibodies. Tubulin was used as a loading control. (G) KM12 cells were treated with the neddylation inhibitor MLN4924 (1 μM) for 2 h, followed by 50 nM compound 9 for 4 h. Cells were harvested, and extracts were subjected to Western blotting with the indicated antibodies. Tubulin was used as a loading control.

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To verify whether the degradation of TPM3-TRKA was mediated by the 26S proteasome, we treated KM12 cells with the proteasome inhibitor MG132. MG132 restored TPM3-TRKA protein levels upon compound 9 treatment, demonstrating that TPM3-TRKA degradation was mediated by the proteasome (Fig. 5B). Interestingly, MG132 was unable to restore TRKA phosphorylation since its inhibition was due to the Pz-1 component of the PROTAC that function as a traditional small molecule kinase inhibitor whose mechanism of action is independent from the proteasome.

Then, to verify whether compound 9 induced TPM3-TRKA poly-ubiquitylation, we transiently expressed TPM3-TRKA in HEK293 cells with or without compound 9 and MG132 (to block protein degradation). TPM-TRKA was immunoprecipitated and the blot stained with anti-ubiquitin antibodies; TPM3-TRKA was poly-ubiquitinated to a greater extent in cells treated with compound 9 and MG132 compared with the cells treated with MG132 alone indicating that the compound promotes TMP3-TRKA poly-ubiquitination (Fig. 5C). Furthermore, we co-treated KM12 cells with compound 9 and chloroquine (50 μM), an autophagy and lysosome inhibitor. Chloroquine had no effect on compound 9 ability to degrade and, consequently, reduce autophosphorylation of TPM3-TRKA, indicating that the autophagic and lysosomal pathways were not involved in compound 9 activity (Fig. 5D).

Next, we explored the role of CRBN in compound 9-mediated TPM3-TRKA polyubiquitination. To this aim, KM12 cells were treated with compound 9 in the presence of an excess of pomalidomide to compete compound 9 binding to CRBN36. In cells treated with compound 9, free pomalidomide blocked protein degradation in a dose-dependent manner; as a control, pomalidomide had no impact on TPM3-TRKA the protein level and, consequently, autophosphorylation (Fig. 5E).

CRBN is part of an E3 ligase complex composed by Cullin 4A, RBX1, DDB1 and CRBN itself, in which Cullin 4A is covalently modified by the attachment of NEDD8, a ubiquitin-like chain. Neddylation of Cullin 4A is necessary to recruit the E2 ubiquitin conjugating enzyme37. Accordingly, in the not-neddlyated state, the Cullin C-terminal domain forms a groove in which the RING domain of Rbx1 is embedded. Such conformation restrains the movements of Rbx1 and sets the ubiquitin E2 away from its substrates38. We transfected HEK293T cells with a myc-tagged Cullin 4A dominant negative construct (Cullin 4A ΔNEDD8), displaying deletion of neddylation site and therefore an impaired ability to recruit E2. Expression of Cullin 4A ΔNEDD8 strongly impaired compound 9 capability to degrade TPM3-TRKA (Fig. 5F). Accordingly, treatment with MLN4924, a neddylation inhibitor, also reduced compound 9 ability of causing TPM3-TRKA downregulation in KM12 cells (Fig. 5G). Autophosphorylation inhibition by compound 9 was not affected by MLN4924 since kinase inhibition was mediated by Pz-1 component of the PROTAC independently from protein polyubiquitination. All together, these results demonstrated that compound 9 degraded TPM3-TRKA by recruiting Cullin 4A/CRBN-containing E3 ligase complex, which, in turn, mediates its polyubiquitination and targeting to the proteasome.

In vivo TMP3-TRKA target inhibition by compound 20

We ought to verify whether Pz-1-based degraders were able to act on TMP3-TRKA in living animals. Due to the potential metabolic liability at the amide bond in the linker of compound 9, we selected compound 20, which does not contain it, for the in vivo testing (NMR spectra are reported in Supplementary information, Figs. S13, S14). We treated mice xenografted with KM12 cells with a single intraperitoneal dose of compound 20 (15 mg/kg) and verified its ability to induce TMP3-TRKA degradation after 12 h. Compound 20 demonstrated a robust efficacy in inducing degradation of TRKA, with a concomitant decrease of protein autophosphorylation (Fig. S15).

Discussion

Chromosomal aberrations causing the fusion of TRKA and RET tyrosine kinase domains to the N-ter of heterologous proteins, able to mediate dimerization, is a common mechanism of oncogenic conversion of these receptors. These fusions have been identified in various types of cancers, including thyroid, lung, and colorectal carcinomas. In addition, point mutations of RET are a major driver of sporadic and familial medullary thyroid carcinoma.

Therefore, medicinal chemistry efforts have been made to target the abnormal activation of RET and TRKA fusions and of RET point mutants with small molecule drugs2,5,11. Small molecule kinase inhibitors bind to the kinase domain, thereby preventing enzymatic function and signal transduction.

Heterobifunctional degraders (PROTACs) have attracted attention due to their capability of erasing, rather than simply inhibiting, the targeted protein, pending that the E3 ligase and the POI are expressed in the same cells and are able to form a stable ternary complex with the PROTAC20. With this rationale, we aimed to generate small molecules capable of inducing degradation of RET and TRKA derived oncoproteins. By conjugating pomalidomide, lenalidomide, or avadomide (specific ligands of CRBN, a component of a CRL E3 ligase) with Pz-1 (a RET/TRKA small molecule tyrosine kinase inhibitor), we synthesized a series of RET/TRKA-targeted PROTAC compounds.

Among these compounds, we identified compound 9 as a potent RET-TRKA degrader. Mechanistically, compound 9 caused reduced half-life of TPM3-TRKA by promoting its ubiquitylation and proteasome-mediated degradation. CRBN recruitment was critical for such compound 9 activity. Indeed, ethylation of the NH group in the pomalidomide glutarimide ring (compound 13), which is essential for CRBN binding, abrogated compound activity30. Compound 9 activity was also impaired by either using the neddylation inhibitor MLN4924 or a Cullin 4A dominant negative mutant, consistent with the essential role played by Cullin 4A neddylation in the function of the CRBN-based E3 ligase. Finally, the replacement of a CRBN ligand with a VHL ligand resulted in an inactive compound (compound 19), consistent with recent studies showing that protein degradation of some specific POI may occur only by using a CRBN ligand39. Though Pz1 is active against VEGFR2, its PROTAC derivatives selectively degrade TRKA and RET, but not VEGFR2. In addition, compound 20 was able to induce TMP3-TRKA degradation also in vivo in animal-based experiments.

In summary, this study provides the proof-of-concept of a new medicinal chemistry strategy aimed at targeting the degradation of RET- and TRKA-derived oncoproteins and highlights compound 20, with its derivatives, as promising lead to develop new RET/TRKA directed anti-neoplastic agents. We posit that these compounds may be effective at inhibiting both RET- and TRKA-mutant cancers. It is also possible that the dual activity may find application in specific contexts. Indeed, although RET and TRKA mutations are in general mutually exclusive, it has been reported that secondary TRK mutation may mediate resistance to RET inhibition; in these cases, a dual RET/TRKA inhibitor may prove advantageous40. Finally, the reported functional interaction between RET and TRKA suggests that in some contexts their dual blockade may result in a synergistic effect41,42.