RAS/RAF activation, functions and targeted inhibition in platelets

• Applicant: Professorin Dr. Kerstin Jurk
• Subject Area: Hematology, Oncology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 562818402

The canonical signaling pathway RAS-RAF-MEK-ERK controls cell growth, differentiation and survival. Its pathologic upregulation occurs in cancer due to mostly somatic mutations affecting RAS and RAF isoforms. Several small molecule protein kinase inhibitors, targeting BRAF and MEK are clinically approved, for instance for mono- and combination-therapy of advanced melanoma. As key players in thrombosis-related diseases and triggers of vascular inflammation, platelets promote cancer metastasis. However, the signaling/functional roles of RAS and RAF in human platelets are surprisingly unexplored. My preliminary data indicate that in human platelets 1) relevant components of the RAF-signaling module are expressed, 2) clinically relevant concentrations of MEK and RAF inhibitors reduce MEK1/2-ERK1/2 signaling after GPVI stimulation and 3) RAF isoforms may be involved in degranulation and aggregation. With biochemical, pharmacological, genetic and in vivo approaches I aim to elucidate the RAS-RAF-MEK-ERK pathway in human and murine platelets. FDA-approved (or in late-stage development) inhibitors targeting RAS, RAF and effectors will serve as reliable tools to decipher platelet RAS-RAF-MAPK pathways and functional readouts. Immunoblotting-based pull-down and kinase assays will be complemented by PamChip microarray and G-LISA to demonstrate that RAF kinases are active and activated by RAS in response to immunoreceptor tyrosine-based activating motif-associated receptors compared to G-protein coupled receptors (GPCR). Utilizing label-free quantitative mass spectrometry, I plan to show that RAF-mediated signaling involves crosstalk with other MAPK and Ser/Thr-PK-signaling pathways. To test the hypothesis that RAF sensitizes and triggers platelet degranulation and thrombus formation, advanced platelet function analysis will be applied after submaximal and threshold activation. This also includes machine learning-assisted characterization of platelet-populations by multiparameter flow cytometry to establish RAF-related functional signatures. Platelets from patients with nonsense RASGRP2 mutations will be used to explore the role of RASGRP2/CalDAG-GEFI as RAS-GEF. BRAF- and KRAS/CRAF-driven mouse models of lung metastasis will be established for functional in vivo validation of the mechanistic in vitro data. As clinical proof-of-concept approach, the inhibition of the platelet RAF-MEK-ERK pathway will be studied in melanoma patients receiving MEK and/or RAF-inhibitor therapy. I expect novel insights into platelet RAS-RAF-MEK signaling/function, which will be clinically very important. New, signaling-guided combinations of both anticancer and antiplatelet drugs could have an improved therapeutic window. This project may extend the basis to address the role of platelets as therapeutic target for novel protein kinase inhibitors in cancer and inflammation.

• DFG Programme: Research Grants