PROJECT SUMMARY/ABSTRACT Juvenile myelomonocytic leukemia (JMML) is a common myeloproliferative neoplasm (MPN) in childhood. JMML is characterized as being Ras-driven due to mutations in NF1, CBL, KRAS, NRAS, or PTPN11, and cells from JMML patients show hypersensitivity to GM-CSF. Chemotherapeutic agents are mostly ineffective in JMML, and the only curative treatment is allogeneic hematopoietic stem cell transplantation (HSCT). A common clinical picture in JMML is that it presents as a hyperinflammatory syndrome, and is often difficult to distinguish from viral infections. Thus, a component of JMML is associated with hyperinflammatory state and hyperactive innate immune cells. Further, unlike other MPNs, JMML rarely progresses to blast crisis; rather, mortality is due to extramedullary myeloid cell expansion leading to organ failure. Importantly, following allogeneic HSCT, 50% of patients succumb to leukemia relapse, implicating a role for bone marrow microenvironment (BME) in JMML development and progression. The hyperinflammatory nature of JMML may damage the BME, altering the expansion of normal donor cells following transplant, permitting residual leukemia cells to outcompete the normal graft, and leading to relapse. Utilizing mouse models of JMML, we demonstrate relapse in mice bearing PTPN11 mutations, we show altered composition of the BME in PTPN11 bearing mice and provide evidence that JMML patients that have a higher neutrophil count at the time of HSCT are more likely to relapse. These data combined with previous studies demonstrating hyperactive and inflamed neutrophils due to PTPN11 mutations suggests that these cells may contribute to relapse. We will examine this in detail. We have been analyzing multiple RNA sequencing datasets for lncRNAs that are differentially expressed in JMML. In doing so, we identified several novel lncRNAs whose expression is differentially regulated. We will examine how one of these lncRNAs contributes to JMML pathogenesis. We have recently shown that PI3K catalytic subunit p110δ contributes to both Akt and Erk hyperactivation, and promotes PTPN11-induced GM-CSF hypersensitivity and hyperproliferation, thus partially contributing to the progression of JMML. Given the lack of complete rescue by loss of p110δ in PTPN11-induced JMML, we sought out putative tyrosine kinases that signal together with p110δ in the PI3K-Akt signaling pathway that must be targeted for optimal JMML therapy. We present preliminary data demonstrating that Bruton's Tyrosine Kinase (BTK) inhibition collaborates with PI3K p110δ inhibition to reduce the activation of Akt and Erk in PTPN11-expressing cells. We will study the mechanism behind this cooperation. Overall, the proposed Aims will shed novel insight into JMML development and pathogenesis as well as identification of novel therapeutic targets.

PROJECT NARRATIVE Few therapies are available for children with JMML. While allogenic hematopoietic stem cell transplant is curative, 50% of these patients relapse. The overall goal of this proposal is to find new innovative therapies for the treatment of JMML and to understand the mechanisms behind relapse.