Novel CrkL Adaptor Protein Inhibitors Suppress Tumor Growth and Reverse NK Cell Exhaustion 2257879

Abstract

Abstract Introduction Crk adaptor proteins (CrkI/II and CrkL) integrate oncogenic and immune signals. Phosphorylated CrkL (pCrkL, Y207) enforces an autoinhibited state linked to metastasis and NK/T‑cell dysfunction. We posited that blocking CrkL phosphorylation could suppress triple‑negative breast cancer (TNBC) while sparing normal immunity. Methods We screened >2,000 compounds across five libraries with an AlphaScreen assay for CrkL Y207 phosphorylation. Hits were triaged by thermal shift, in‑vitro kinase, and single‑molecule FRET. Cell assays used TNBC lines and human PBMCs (proliferation, phospho‑flow/Western, apoptosis). Orthotopic and PDX TNBC models assessed in‑vivo efficacy and tolerability with daily oral dosing. Results Seventy‑one primary hits were refined to two natural products, NC5 and NC11, that bind CrkL. smFRET showed NC5 counters Abl‑driven CrkL phosphorylation, shifting to an open, low‑FRET state. NC5 reduced TNBC proliferation (IC50 ∼1.3—3.6 µM) while sparing PBMCs (IC50 >18 µM) and increased Annexin V+ apoptosis. Pathway readouts in TNBC showed ↓pCrkL and ↓pERK1/2 with ↑pJNK and ↑BIM, with minimal PBMC effects. In vivo, low‑dose NC5 (5 mg/kg/day, p.o.) suppressed tumor growth and prolonged survival versus vehicle and imatinib (MDA‑MB‑231 median 41 vs 25 and 28 days; PDX 53 vs 39 days). Serum chemistries remained within reference ranges, indicating favorable tolerability. Conclusion Targeting CrkL phosphorylation with the natural product NC5 delivers dual benefits: direct TNBC suppression and reinvigorates anti-tumor immunity. These data indicate NC5 as a first‑in‑class pCrkL inhibitor and support further development, including combination strategies and IND‑enabling studies. Funding Source R01AI130197 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)