Indiana University-Purdue University Indianapolis (IUPUI) / Juvenile Myelomonocytic Leukemia (JMML) is rare, fatal myeloproliferative disease (MPD) affecting young children, and is characterized by expansion of monocyte lineage cells and hypersensitivity to Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) stimulation. JMML is frequently associated with gain-of-function mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase, Shp2. Activating Shp2 mutations are known to promote hyperactivation of the Ras-Erk signaling pathway, but Akt is also observed to have enhanced phosphorylation, suggesting a potential role for Phosphatidylinositol-3-Kinase (PI3K)-Akt signaling in mutant Shp2-induced GM-CSF hypersensitivity and leukemogenesis. Having demonstrated that Class IA PI3K is hyperactivated in the presence of mutant Shp2 and contributes to GM-CSF hypersensitivity, I hypothesized the hematopoietic-specific Class IA PI3K catalytic subunit p110δ is a crucial mediator of mutant Shp2-induced PI3K hyperactivation and GM-CSF hypersensitivity in vitro and MPD development in vivo. I crossed gain-of-function mutant Shp2 D61Y inducible knockin mice, which develop fatal MPD, with mice expressing kinase-dead mutant p110δ D910A to evaluate p110δ’s role in mutant Shp2-induced GM-CSF hypersensitivity in vitro and MPD development in vivo. As a comparison, I also crossed Shp2 D61Y inducible knockin mice with mice bearing inducible knockout of the ubiquitously expressed Class IA PI3K catalytic subunit, p110α. I found that genetic interruption of p110δ, but not p110α, significantly reduced GM-CSF-stimulated hyperactivation of both the Ras-Erk and PI3K-Akt signaling pathways, and as a consequence, resulted in reduced GM-CSF-stimulated hyper-proliferation in vitro. Furthermore, I found that mice bearing genetic disruption of p110δ, but not p110α, in the presence of gain-of-function mutant Shp2 D61Y, had on average, smaller spleen sizes, suggesting that loss of p110δ activity reduced MPD severity in vivo. I also investigated the effects of three PI3K inhibitors with high specificity for p110δ, IC87114, GDC-0941, and GS-9820 (formerly known as CAL-120), on mutant Shp2-induced GM-CSF hypersensitivity. These inhibitors with high specificity for p110δ significantly reduced GM-CSF-stimulated hyperactivation of PI3K-Akt and Ras-Erk signaling and reduced GM-CSF-stimulated hyperproliferation in cells expressing gain-of-function Shp2 mutants. Collectively, these findings show that p110δ-dependent PI3K hyperactivation contributes to mutant Shp2-induced GM-CSF hypersensitivity and MPD development, and that p110δ represents a potential novel therapeutic target for JMML.

Indiana University-Purdue University Indianapolis (IUPUI) / Phosphatases of regenerating liver, consisting of PRL-1, PRL-2 and PRL-3, belong to a novel protein tyrosine phosphatases subfamily, whose overexpression promotes cell proliferation, migration and invasion and contributes to tumorigenesis and metastasis. However, although great efforts have been made to uncover the biological function of PRLs, limited knowledge is available on the underlying mechanism of PRLs’ actions, therapeutic value by targeting PRLs, as well as the physiological function of PRLs in vivo. To answer these questions, we first screened a phage display library and identified p115 RhoGAP as a novel PRL-1 binding partner. Mechanistically, we demonstrated that PRL-1 activates RhoA and ERK1/2 by decreasing the association between active RhoA with GAP domain of p115 RhoGAP, and displacing MEKK1 from the SH3 domain of p115 RhoGAP, respectively, leading to enhanced cell proliferation and migration. Secondly, structure-based virtual screening was employed to discover small molecule inhibitors blocking PRL-1 trimer formation which has been suggested to play an important role for PRL-1 mediated oncogenesis. We identified Cmpd-43 as a novel PRL-1 trimer disruptor. Structural study demonstrated the binding mode of PRL-1 with the trimer disruptor. Most importantly, cellular data revealed that Cmpd-43 inhibited PRL-1 induced cell proliferation and migration in breast cancer cell line MDA-MB-231 and lung cancer cell line H1299. Finally, in order to investigate the physiological function of PRLs, we generated mouse knockout models for Prl-1, Prl-2 and Prl-3. Although mice deficient for Prl-1 and Prl-3 were normally developed, Prl-2-null mice displayed growth retardation, impaired male reproductive ability and insufficient hematopoiesis. To further investigate the in vivo function of Prl-1, we generated Prl-1-/-/Prl-2+/- and Prl-1+/-/Prl-2-/- mice. Similar to Prl-2 deficient male mice, Prl-1-/-/Prl-2+/- males also have impaired spermatogenesis and reproductivity. More strikingly, Prl-1+/-/Prl-2-/- mice are completely infertile, suggesting that, in addition to PRL-2, PRL-1 also plays an important role in maintaining normal testis function. In summary, these studies demonstrated for the first time that PRL-1 activates ERK1/2 and RhoA through the novel interaction with p115 RhoGAP, targeting PRL-1 trimer interface is a novel anti-cancer therapeutic treatment and both PRL-1 and PRL-2 contribute to spermatogenesis and male mice reproductivity.