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Hippo Kinase Inactivation Contributes to Del(20q) Malignancies and Cooperates with JAK2-V617F to Promote Myelofibrosis in Mice


 Recurring chromosome abnormalities are frequent events in cancer and are especially prevalent in hematologic neoplasms. Somatic heterozygous deletions on chromosome 20q are detected in a variety of hematopoietic malignancies including myelodysplastic syndrome (MDS), classical myeloproliferative neoplasm (MPN), MDS/MPN overlap disorders such as chronic myelomonocytic leukemia (CMML), and acute leukemias. Del(20q) is especially prevalent in MPN patients (~10-15%), where it is the most commonly detected cytogenetic abnormality associated with primary myelofibrosis (PMF) and post-polycythemia vera myelofibrosis (MF). This suggests that heterozygous loss of genes in the del(20q) common deleted region (CDR) may contribute to adverse MPN progression. Despite these observations, relatively few genes located within the CDR have been unambiguously implicated, highlighting a significant need for further investigation. To identify genes that may play an important role in the biology of del(20q)-associated malignancies we utilized a published gene expression dataset of bone-marrow derived CD34+ cells from MDS patients and healthy controls (Gerstung et al, 2015). Comparison of the patients harboring del(20q) to healthy controls revealed STK4 (encoding Hippo kinase MST1) to be the most significantly downregulated gene (mean: 3.5-fold) among those located within the chromosome 20q CDR. We therefore set out to assess the role of Hippo kinase inactivation in hematologic malignancy using conditional gene inactivation in mice.
 We found that complete inactivation of both Hippo kinases (Stk4 and Stk3) within the hematopoietic system using Vav1-Cre (Stk4-/-Stk3-/-) resulted in a lethal bone marrow failure (median survival: 7 weeks) associated with myelodysplastic features and frequent extramedullary hematopoiesis in the spleen. A single copy of Stk4 rescued the lethality due to bone marrow failure, however sub-haploinsufficient mice displayed thrombocytopenia with a trend towards mild anemia; phenotypes that closely resemble those observed in MDS patients with isolated del(20q). Both a reduced number of mature megakaryocytes and the presence of dysplastic megakaryocytes were apparent in bone marrow sections. Inducible Hippo kinase inactivation in adult mice using the Mx1-Cre system similarly recapitulated several phenotypic features of both MDS and MPN. In competitive bone marrow transplant assays we found that Stk4-/-Stk3-/- hematopoietic stem cells (HSC) completely lacked engraftment potential and failed to reconstitute normal hematopoiesis, revealing a potential role for Hippo kinase function in HSC homing and retention in the bone marrow. Heterozygous HSCs maintained relatively normal steady-state hematopoiesis in peripheral blood and bone marrow for up to 48 weeks in primary and secondary transplantations, although upon aging these mice were prone to development of thrombocytopenia with increased mean platelet volume.
 Given the high frequency of del(20q) in MPN, especially PMF, we asked whether heterozygous Hippo kinase inactivation may cooperate with the common driver mutation JAK2-V617F to accelerate disease progression. Using an HSC-enriched retroviral transduction/transplantation model in C57BL/6 recipient mice, we monitored MPN progression for 36 weeks in heterozygous Stk4+/-Stk3+/-, or control (Vav1-Cre-), cells with or without expression of JAK2-V617F. While both JAK2-V617F groups initially displayed a similar degree of polycythemia relative to controls, we found heterozygous Hippo kinase inactivation to promote accelerated disease progression towards lethal bone marrow fibrosis during the course of observation. Recipients in this group showed significantly reduced overall survival, which was associated with higher grade fibrosis in bone marrow, elevated peripheral granulocyte counts, enhanced splenomegaly, and increased frequencies of hematopoietic stem and progenitor populations in the spleen. Together, these findings implicate aberrant Hippo kinase loss-of-function in the pathogenesis of del(20q)-associated hematologic malignancies, and shed new light on the molecular events that contribute to adverse MPN progression.
 
 
 Bejar: Genoptix: Consultancy; Modus Outcomes: Consultancy; Celgene: Consultancy, Honoraria; Takeda: Research Funding; Astex/Otsuka: Consultancy, Honoraria; AbbVie/Genentech: Consultancy, Honoraria; Foundation Medicine: Consultancy. Guan:Vivace: Equity Ownership.
