Liquidambaric lactone is a potent inhibitor of TRAF2 for cancer therapy

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is an oncogenic regulator of Wnt/β-catenin signaling in colon cancer. Previously we have reported a pentacyclic triterpenoid compound liquidambaric acid (LDA) inhibiting Wnt/β-catenin signaling via targeting TRAF2, however, LDA inhibition of cancer cells was observed only at high doses. It is important to identify small molecules targeting TRAF2 in colon cancer with higher potency . Microscale thermophoresis (MST) assay was used to screen a panel of pentacyclic triterpenoids. Cellular thermal shift assay (CETSA) was used to confirm target engagment in living cells. Drug affinity responsive target stability (DARTS) coupled with mass spectrometry (MS) was used to identify the potential targets of candidate TRAF2 binder. Wnt signaling inhibition was evaluated by 7TGC reporter assay, TOPFlash assay, reverse transcription and real-time quantitative PCR, as well as zebrafish eyeless assay. Cancer inhibition was assessed by cell viability assay, colony formation assay and xenograft studies. CRISPR/Cas9 knockout HCT116 cells was used to assess the requirement of TRAF2 for liquidambaric lactone (LDL) inhibition of cancer. A LDA analogue LDL selectively targeted TRAF2 with higher binding affinity. Both LDL and LDA targeted the TRAF-C domain of TRAF2 with slightly different binding sites. Of note, LDL showed much stronger inhibition of colon cancer cells than LDA in colony formation assay. LDL inhibition of cancer was also comfirmed in xenograft mice. Depletion of TRAF2 totally blocked LDL inhibition of Wnt signaling, TNF signaling and growth of cancer cells. A natural product LDL is a potent inhibitor of TRAF2 for cancer therapy, and TRAF2 is required for LDL inhibition of cancer.