PB0360 - SHP099 inhibits megakaryopoiesis, thrombopoiesis and Mpl signalling

Background: The non-transmembrane protein-tyrosine phosphatase (PTP) Src homology (SH2) domain-containing PTP 2 (Shp2), encoded by the proto-oncogene PTPN11, has been implicated in many cellular processes, including survival, proliferation and differentiation of multiple cell types, including megakaryocytes (MKs). Shp2 has been demonstrated to regulate signalling from a variety of tyrosine kinase-linked receptors, including cytokine and growth factor receptors and is a well-known positive regulator of the Ras-MAPK pathway. Targeting Shp2 pharmacologically, therefore represents a therapeutic strategy for many Ras-driven cancers.

Aims: Investigate the role of Shp2 in mouse and human megakaryocytopoiesis and thrombopoietin (Tpo) receptor Mpl signalling, through the use of a specific allosteric Shp2 inhibitor, SHP099.

Methods: MK maturation, Mpl signalling, proplatelet formation and platelet release were analysed in primary mouse and human haematopoietic progenitors treated with SHP099, by flow cytometer, capillary-based immunoassays and microscopy assays respectively. SHP099 was also tested on human MK cultured ex vivo in an artificial three-dimensional (3D) miniature marrow bioreactor system.

Results: Treatment of primary bone marrow (BM)-derived MKs with 10 microM SHP099 affect slightly cell viability, however it resulted in an inhibition of proliferation and maturation, with decreased number of mature MKs and reduced ploidy, as well as a decrease number of proplatelet-forming MKs. Similar results were observed in human CD34+-derived MKs. SHP099 also inhibited the number of platelet released from CD34+-derived MKs, showing that Shp2 activation is required for platelet formation. Moreover, Tpo-induced Erk1/2 and Akt phosphorylation was significantly decreased in CD34+-derived MKs, demonstrating that Shp2 is a positive regulator of Mpl signalling. Using a 3D ex vivo BM niche model made of silk fibroin, confocal microscopy analysis also revealed a reduction in human proplatelet formation and branching, and a decreased number of platelets released in the presence of SHP099.

Conclusion(s): Shp2 activation is essential for mouse and human megakaryocytopoiesis and in mediating Mpl signal transduction.