Combined Pharmacological Inhibition of Bruton's Tyrosine Kinase (BTK) and Phosphoinositide 3-Kinase (PI3K) p110δ Rescues Monocytosis, Thrombocytopenia, and Splenomegaly in a Genetic Mouse Model for JMML
Authors: Patel Roshni , Ramdas Baskar , Deng Lisa , Jideonwo-Auman Victoria , Chan Rebecca J , Kapur Reuben 
Published: October 2019

Juvenile myelomonocytic leukemia (JMML) is an aggressive childhood myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN). It has no effective treatments and the only treatment that prolongs survival is allogeneic hematopoietic stem cell transplant (HSCT). However, even with this aggressive intervention, approximately 50% of patients relapse with JMML within five years. The most commonly mutated gene in JMML patients is PTPN11, which encodes for the non-receptor protein tyrosine phosphatase SHP2. Gain-of-function (GOF) mutations in SHP2 lead to hyperactive RAS signaling and PTPN11 is recognized as a well-established oncogene in various leukemia's. Our lab has shown that p110δ, the hematopoietic-specific catalytic subunit of phosphoinositide 3-kinase (PI3K), plays an important role downstream of SHP2-signaling. Recently, we have shown that in vivo treatment with a specific pharmacologic p110δ inhibitor significantly prolongs the overall survival and reduces the splenomegaly seen in GOF Shp2-expressing mice. Having observed the effectiveness of PI3K p110δ inhibition in correcting the mutant Shp2-induced leukemia phenotype in vitro and in prolonging survival of mice in vivo, we next explored signaling molecules with which p110δ may be interacting to promote the aberrant Shp2 signaling in myeloid cells. In recent years, a key player in B cell receptor (BCR) signaling, Bruton's tyrosine kinase (BTK), has come under intense study in the field of lymphocytic leukemia and lymphoma research. Ibrutinib, a small molecule inhibitor targeting BTK, has proven to be very effective and has received FDA-approval for the treatment of a variety of B cell malignancies. Although research on BTK up to this point has focused on B cell malignancies, BTK is also highly expressed in myeloid cells and mice lacking BTK are defective in many myeloid cell functions. Thus, it is possible that BTK signaling may also play an important role in myeloid malignancies, such as JMML. Given the collaboration of BTK and p110δ in BCR signaling, the key role of p110δ in GOF Shp2-induced leukemia, and the high expression of BTK in myeloid cells, we hypothesized that BTK and p110δ function cooperatively in GOF Shp2-expressing myeloid cells to promote MPN. To test this hypothesis, we examined the potential collaboration of GM-CSF-stimulated BTK and p110δ in GOF Shp2-expressing myeloid cells and tested the inhibitor combination in vivo. We show that BTK cooperates with p110δ to promote GOF Shp2-induced leukemia. We show a role for B cell adaptor for PI3K (BCAP) in BTK upregulation of PI3K activity. In mutant Shp2 macrophages, BCAP phosphorylation is increased specifically in the larger isoforms regulating PI3K activation, and BTK inhibition results in a dose-dependent reduction in this phosphorylation. Our findings show that the MPN caused by GOF Shp2 is due to cooperative signaling between p110δ and BTK, which forms a positive feedback loop with BCAP, thus leading to more Akt/Erk hyperphosphorylation and hyperproliferation in response to GM-CSF.

Given these in vitro observations, we treated GOF Shp2 bearing mice with a combination of a PI3K p110δ-specific inhibitor and, a BTK-specific inhibitor, or single agents alone and performed hematopoietic analysis. The combination treatment scheme completely rescued monocytosis and uniquely ameliorated thrombocytopenia compared to single agent treatment of diseased mice. In addition, splenomegaly was also completely rescued in all three treatment groups compared to vehicle. Detailed flow cytometric analysis of bone marrow hematopoietic stem and progenitor cell populations revealed significant repression of LSK, HPC1 and granulocyte macrophage progenitors (GMPs) with a concomitant increase in the megakaryocyte precursors (MKPs) in drug combination treated mice vs. other groups. Consistent with these observations, a significant reduction in mature myeloid cells was noted in the spleen and a complete rescue in the production of peripheral blood platelets was observed in the mutant mice treated with a combination of the two drugs. Thus, combination therapy with PI3K p110δ- and BTK-specific inhibitors profoundly rescues disease state hallmarks of JMML, and may warrant further clinical investigation.

Disclosures

No relevant conflicts of interest to declare.