Abstract (Project 3) Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile myelomonocytic leukemia(JMML), an aggressive myeloproliferative neoplasm (MPN) for which the standard of care is hematopoietic stem cell transplant (HSCT). Unfortunately, relapse rates are high after HSCT, particularly in NF1 patients. We discovered that NF1 functions as a tumor suppressor gene in hematopoietic cells and showed that NF1 inactivation results in deregulated Ras/Raf/MEK/ERK signaling. This work suggested a central role of hyperactive Ras signaling in JMML pathogenesis, and our group and others subsequently identified mutations in other Ras pathway genes including NRAS, KRAS, PTPN11, and CBL. Ras pathway mutations are invariably present at high allelic frequency at both diagnosis and relapse. Progression to acute myeloid leukemia (AML) occurs in 20-30% of JMML patients and is frequently associated with outgrowth of subclones that harbor secondary mutations, most commonly in SETBP1 or SH2B3. Consistent with the molecular genetics of JMML, using the Mx1-Cre transgene to inactivate the conditional mutant Nf1flox allele generated or to express oncogenic KrasG12D or NrasG12D in hematopoietic cells induces a JMML-like MPN in mice. We utilized these genetically engineered mouse (GEM) models to perform preclinical trials and observed remarkable efficacy of MEK inhibitors in Kras and Nf1 mutant mice with MPN. During this initial cycle of SPORE funding, we opened a national phase 2 clinical trial of the MEK inhibitor trametinib for patients with relapsed/refractory JMML (ADVL1521) and have observed objective responses in patients treated to date. Enrollment is ongoing and we are collaborating with Cores B (Omics) and C (Pathology) to perform correlative molecular and pathologic analyses. We also developed sensitive and reproducible assays for monitoring molecular responses during the current funding period that we deployed to address additional translational research questions. Finally, we collaborated with colleagues in Europe and Japan to identify global DNA methylation as a novel biomarker of outcome with the most hypermethylated samples portending the worst prognosis independent of genotype. We will extend these novel observations through the following aims: Aim 1. To conduct innovative clinical trials for patients with JMML that emanate from our laboratory and preclinical studies. We will complete the ongoing ADVL1521 trial and have developed the first interventional risk-stratified clinical trial in newly diagnosed JMML; and, Aim 2. To perform biologic and preclinical studies of promising therapies in JMML patient specimens and GEM models to inform clinical translation. Our overall goal is to develop more effective and less toxic therapies for infants and children with JMML by targeting the underlying molecular pathogenesis. These studies also have implications for the fundamental problem of improving the treatment of other cancers driven by hyp...