Regulatory Role of the Sprouty Gene Family on PDGFβR Fusion Oncogenes.

Constitutively activated fusion tyrosine kinases of the Platelet-dervied growth factor β receptor (PDGFβR) family have been described in patients with chronic myelomonocytic leukemia (CMML). Like other tyrosine kinase driven myeloproliferative syndromes, CMML is characterized by progression leading to highly aggressive acute leukemia. In order to understand the progression of these malignancies we are studying tyrosine kinase pathway regulatory genes. In this study we focus on the functional role of the sprouty gene family in the regulation of PDGFβR fusion oncogenes. Sprouty (Spry) has recently been identified as a repressor of receptor tyrosine kinases signaling in vertebrates and invertebrates. The studies of sprouty in the mammalian system have thus far mostly focused on the regulation of the epidermal and fibroblast growth factor receptor, while nothing is known about the possible regulation of PDGF receptors by sprouty proteins and nothing is known about regulation of mutationally activated tyrosine kinases. Expression plasmids containing human sprouty wildtype genes (Spry1-3 WT) were constructed, along with a series of plasmids containing dominant negative variants by site-direct mutagenesis in critically conserved domains [Spry1(Y53F), Spry2(Y55F), Spry3(Y27F)]. Stable cell lines containing these plasmids have been generated in the BaF3 background with or without the constitutively activated Rabaptin-5/PDGFβR (R/P) fusion oncoprotein. Effects on cell growth and downstream signaling events were studied. Spry1 WT and Spry3 WT signifcantly inhibit growth of R/P transformed BaF3 cell lines. This inhibition was much more pronounced in IL3 depleted media indicating that the inhibition is mediated through PDGFβR tyrosine kinase inhibition. The dominant negative forms, Spry1(Y53F) and Spry3(Y27F) stimulated growth of the the same BaF3 cell lines. Results from [³H]thymidine uptake studies in these cell lines showed decreased uptake in Spry1 WT and Spry3 WT transduced cells and increased uptake in the dominant negative forms, indicating that the effects are through increased proliferation rather than decreased apoptosis. Interestingly, R/P transformed BaF3 cell lines transfected with plasmid containing Spry2 WT and Spry2(Y55F) showed inverse results, Spry2 WT stimulated growth while Spry2(Y55F) inhibited growth. A possible explanation for stimulatory effects of Spry2 is that this Spry variant contains a Cbl binding domain previously shown to prevent Cbl mediated ubiquitylation and degradation of RTKs. We are currently studying the downstream targets of the Spry regulation of PDGFβR focusing on Ras and MAPkinase pathways.

In conclusion, we have shown that Spry1 and Spry3 inhibits growth of PDGFβR transformed BaF3 cell lines, while Spry2 stimulates growth. This is the first evidence for regulatory role of Sprouty genes in activated fusion tyrosine kinase. This conserved family of tyrosine kinase regulatory genes is an ideal target for studies of disease progression in tyrosine kinase driven malignancies.