Development of PROTACs for targeted degradation of oncogenic TRK fusions

Chromosomal translocations leading to the fusion of tropomyosin receptor kinases (TRK) with diverse partner proteins have been identified as oncogenic drivers in many adult and pediatric cancers. While first-generation TRK kinase inhibitors, such as entrectinib and larotrectinib, have shown positive responses in TRK fusion-positive cancers, resistance mutations against these inhibitors in the kinase domain limit their efficacy. Second-generation inhibitors are in clinical evaluation, highlighting a need for novel therapeutic modalities to achieve durable suppression of the oncogenic activity of TRK fusions. Here, we developed heterobifunctional small molecule degraders (PROTACs) to achieve targeted degradation of TRK fusions. By conjugating entrectinib to thalidomide, we identified JWJ-01-378 as a potent and selective CRBN-recruiting degrader of the TPM3-TRKA fusion. JWJ-01-378 induced TPM3-TRKA degradation through the ubiquitin-proteasome system and proteomics analysis confirmed the acute selectivity of JWJ-01-378 for achieving TPM3-TRKA degradation with minimal off-target effects. While JWJ-01-378 was also able to degrade wild-type TRK, it was unable to degrade TRK inhibitor resistant mutants and ALK fusions. Importantly, TPM3-TRKA degradation by JWJ-01-378 suppressed downstream signaling and reduced cancer cell viability, with improved responses compared to heterobifunctional control compounds that cannot degrade TPM3-TRKA. Together, our study expands the toolbox of compounds for evaluating targeted degradation of TRK fusions in cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/660465v1_ufig13.gif" ALT="Figure 13"> View larger version (19K): org.highwire.dtl.DTLVardef@6c78f7org.highwire.dtl.DTLVardef@179dcdeorg.highwire.dtl.DTLVardef@1939988org.highwire.dtl.DTLVardef@144c718_HPS_FORMAT_FIGEXP M_FIG C_FIG JWJ-01-378 recruits cereblon (CRBN) to induce potent and selective degradation of oncogenic TRK fusions, leading to a collapse in downstream signaling and loss of cancer cell viability. Graphical abstract was created using Biorender.com.