Project Summary Juvenile myelomonocytic leukemia (JMML), a clonal hematological malignancy of early childhood, has limited therapeutic options. While the current standard of care for patients with JMML relies on allogeneic stem cell transplant, relapse remains the main cause of treatment failure, most likely due to the persistence of leukemic stem cells (LSCs), a small population of self-renewing precursor cells that gives rise to the bulk of tumor cells. This reservoir of tumor cells is responsible for the long-term maintenance of leukemia growth and is also a major source of drug resistance. It remains a critical challenge to develop effective therapeutics to eradicate these tumor initiating cells. A novel treatment approach focused on the unique characteristics and vulnerabilities of LSCs is needed in order to address this problem. JMML is associated with genetic mutations in the signaling proteins involved in the Ras pathway, among which the protein tyrosine phosphatase Ptpn11 (Shp2), a positive regulator of Ras signaling, is most frequently mutated (heterozygous). These mutations cause greatly increased catalytic activity of Shp2, and JMML patients with Ptpn11 mutations have the worst prognosis in all subtypes of JMML. We created a line of conditional knock-in mice with the Ptpn11E76K mutation, the most common mutation found in JMML. Induction of the Ptpn11E76K/+ mutation in the hematopoietic system (Ptpn11E76K/+/Mx1-Cre+ mice) resulted in JMML-like myeloproliferative neoplasm (MPN) with full penetrance, suggesting a causative role of this mutation in the pathogenesis of JMML. With this unique mouse model, we have recently discovered that while the Ras/Erk signaling pathway was aberrantly enhanced, Stat3 activity as reflected by tyrosine phosphorylation (Tyr705) decreased by ~70% in Ptpn11E76K/+ mutant stem cells (referred to as LSCs since they could reproduce the same hematological malignancy in transplants) compared to that in wild-type (Ptpn11+/+/Mx1-Cre+) control stem cells. The decrease in Stat3 activity was apparently caused by the accelerated dephosphorylation of Stat3 by the hyperactive Shp2 E76K mutant because Stat3 is one of Shp2 substrate proteins. Importantly, Ptpn11E76K/+ LSCs appear to rely on Stat3 for self-renewal and maintenance - the deletion of Stat3 dramatically decreased the LSC pool in Ptpn11E76K/+/Mx1-Cre+/Stat3fl/fl double mutant mice. Consequently, these double mutant mice died rapidly, while none of Ptpn11E76K/+/Mx1-Cre+/Stat3+/+ or Ptpn11+/+/Mx1-Cre+/Stat3fl/fl single mutant mice did. The synthetic lethality induced by Stat3 deletion in Ptpn11 E76K mutant stem cells raises an intriguing possibility, that is, diminished Stat3 activity is an Achilles' heel of Ptpn11 mutated (Shp2 hyperactivated) LSCs, making them vulnerable to pharmacological inhibition of Stat3. We plan to test this hypothesis and accomplish the objective of this proposal by pursuing the following two aims. 1) To define the role of Stat3 in maintena...