Indiana University-Purdue University Indianapolis (IUPUI) / Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm that lacks effective chemotherapies. Most commonly, patients have gain-of-function (GOF) oncogenic mutations in SHP2, leading to hyperactivation of ERK and AKT and hyperproliferation of cells in response to granulocyte macrophage-colony stimulating factor (GM-CSF). Our lab previously showed that p110δ, the hematopoietic-specific catalytic subunit of phosphoinositide 3-kinase, is a crucial mediator of mutant Shp2-induced GM-CSF hypersensitivity in vitro.
We treated oncogenic Shp2-expressing mice with a p110δ inhibitor and showed that the strong effect our lab observed in vitro translated into reduced splenomegaly and prolonged survival in vivo. We investigated molecules potentially cooperating with p110δ signaling and discovered that Bruton’s tyrosine kinase (BTK) is hyperphosphorylated in GOF Shp2 myeloid cells. We used specific BTK and p110δ inhibitors to demonstrate that BTK cooperates with p110δ to hyperactivate Akt/Erk and to promote hyperproliferation. GOF Shp2-expressing mice treated in vivo with the drug combination targeting p110δ and BTK have significantly decreased splenomegaly and WBC counts. We also explored the mechanism of BTK signaling and hypothesized that B cell adaptor for PI3K (BCAP) mediated BTK upregulation of PI3K activity. In mutant Shp2 macrophages, we observed BCAP phosphorylation specifically in the larger isoforms needed for PI3K activation, and BTK inhibition led to a dose-dependent reduction in this phosphorylation. We also demonstrated reduced interaction between BCAP and the PI3K regulatory p85α subunit bearing mutated SH2 domains.
Finally, we investigated the effects of mutated DNA methyltransferase 3A (Dnmt3a) in conjunction with GOF Shp2. Double mutant mice quickly became moribund with pronounced splenomegaly and leukocytosis. There was an expansion of mature myeloid cells in the periphery and myeloid progenitors in the bone marrow, plus anemia with evidence of compensatory erythropoiesis in the spleen.
Our findings show that the myeloproliferative neoplasm caused by GOF Shp2 is due to hyperactive p110δ, and this is further promoted by BTK, which forms a positive feedback loop with PI3K and BCAP, thus leading to more Akt/Erk hyperphosphorylation and more hyperproliferation in response to GM-CSF. The dual inhibition of p110δ and BTK represents a novel effective treatment strategy for JMML and other diseases induced by oncogenic Shp2.
| Identifer | oai:union.ndltd.org:IUPUI/oai:scholarworks.iupui.edu:1805/15832 |
| Date | January 2018 |
| Creators | Deng, Lisa |
| Contributors | Chan, Rebecca, J., Kapur, Reuben, Herbert, Brittney-Shea, Ware, Stephanie M., Yoder, Mervin |
| Source Sets | Indiana University-Purdue University Indianapolis |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation |
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