Constitutive activation of the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3), via co-expression of its ligand or by genetic mutation, is common in acute myeloid leukemia (AML). In this study we show that FLT3 activation inhibits the activity of the tumor suppressor, protein phosphatase 2A (PP2A). Using BaF3 cells transduced with wildtype or mutant FLT3, we show that FLT3-induced PP2A inhibition sensitizes cells to the pharmacological PP2A activators, FTY720 and AAL(S). FTY720 and AAL(S) induced cell death and inhibited colony formation of FLT3 activated cells. Furthermore, PP2A activators reduced the phosphorylation of ERK and AKT, downstream targets shared by both FLT3 and PP2A, in FLT3/ITD+ BaF3 and MV4-11 cell lines. PP2A activity was lower in primary human bone marrow derived AML blasts compared to normal bone marrow, with blasts from FLT3-ITD patients displaying lower PP2A activity than WT-FLT3 blasts. Reduced PP2A activity was associated with hyperphosphorylation of the PP2A catalytic subunit, and reduced expression of PP2A structural and regulatory subunits. AML patient blasts were also sensitive to cell death induced by FTY720 and AAL(S), but these compounds had minimal effect on normal CD34+ bone marrow derived monocytes. Finally, PP2A activating compounds displayed synergistic effects when used in combination with tyrosine kinase inhibitors in FLT3-ITD+ cells. A combination of Sorafenib and FTY720 was also synergistic in the presence of a protective stromal microenvironment. Thus combining a PP2A activating compound and a FLT3 inhibitor may be a novel therapeutic approach for treating AML.

The serine/threonine protein phosphatase 2A (PP2A) is a master regulator of the complex cellular signaling that occurs during all stages of mammalian development. PP2A is composed of a catalytic, a structural, and regulatory subunit, for which there are multiple isoforms. The association of specific regulatory subunits determines substrate specificity and localization of phosphatase activity, however, the precise role of each regulatory subunit in development is not known. Here we report the generation of the first knockout mouse for the Ppp2r2a gene, encoding the PP2A-B55α regulatory subunit, using CRISPR/Cas9. Heterozygous animals developed and grew as normal, however, homozygous knockout mice were not viable. Analysis of embryos at different developmental stages found a normal Mendelian ratio of Ppp2r2a −/− embryos at embryonic day (E) 10.5 (25%), but reduced Ppp2r2a −/− embryos at E14.5 (18%), and further reduced at E18.5 (10%). No live Ppp2r2a −/− pups were observed at birth. Ppp2r2a −/− embryos were significantly smaller than wild-type or heterozygous littermates and displayed a variety of neural defects such as exencephaly, spina bifida, and cranial vault collapse, as well as syndactyly and severe epidermal defects; all processes driven by growth and differentiation of the ectoderm. Ppp2r2a −/− embryos had incomplete epidermal barrier acquisition, associated with thin, poorly differentiated stratified epithelium with weak attachment to the underlying dermis. The basal keratinocytes in Ppp2r2a −/− embryos were highly disorganized, with reduced immunolabeling of integrins and basement membrane proteins, suggesting impaired focal adhesion and hemidesmosome assembly. The spinous and granular layers were thinner in the Ppp2r2a −/− embryos, with aberrant expression of adherens and tight junction associated proteins. The overlying stratum corneum was either absent or incomplete. Thus PP2A-B55α is an essential regulator of epidermal stratification, and is essential for ectodermal development during embryogenesis.

These results suggest that loss of specific PP2A regulatory subunits is functionally important in breast tumourigenesis, and support strategies to enhance PP2A activity as a therapeutic approach in breast cancer.

Breast cancer is the most common cancer in women and a leading cause of death. Dysregulation of cellular signalling pathways controlling proliferation, survival and migration, such as the PI3K/Akt and Ras/MAPK pathways, are key features of breast cancer. Protein phosphatase 2A (PP2A) negatively regulates many components of these pathways. PP2A is a family of trimeric serine/threonine phosphatases, each consisting of a structural, a catalytic and a regulatory subunit of which there are multiple isoforms. The addition of specific regulatory subunits provides subcellular targeting and substrate specificity to the enzyme. While PP2A is generally considered a tumor suppressor, a specific role for individual PP2A subunits in breast cancer has not been described. To address this, we first examined PP2A subunit expression in human breast tumors. Immunohistochemical analysis revealed significantly lower expression of the structural subunit, PP2A-A, and regulatory subunits PP2A-B55α and PP2A-B56α, in primary tumors and metastases, compared to adjacent normal mammary tissue. We further found an association of low PP2A-B55α with aggressive breast cancer subtypes, and with worse disease-free and overall survival. Functionally, shRNA-mediated knockdown of PP2A-B55α in normal mammary epithelial 3D cultures induced a tumorigenic phenotype, characterised by increased proliferation and enlarged multi-lobular acini. In contrast, overexpression of PP2A-B55α in breast cancer cells inhibited proliferation. Thus PP2A inactivation, in particular loss of B55α, is functionally important in breast tumorigenesis. PP2A-B55α complexes play an important role in DNA damage repair, and we found B55α knockdown impaired DNA damage repair. Thus low PP2A-B55α may contribute to genomic instability. To examine the functional role of PP2A-B55α in vivo, we have generated the first PP2A-B55α (Ppp2r2a) knockout mouse. Constitutive knockout of Ppp2r2a is embryonic lethal, with embryos dying during late development, post 14.5 days p.c. Heterozygous Pppr2ra mice (Ppp2r2a+/-) are viable, despite expressing only ~10% of PP2A-B55α protein levels compared to WT mice. Interestingly, Ppp2r2a+/- mice have significantly reduced branching in the developing mammary gland, similar to that observed in mice with mammary-specific loss of the breast tumor susceptibility gene, Brca1. Analysis of breast tumor formation in these mice, either alone or when crossed with MMTV-Neu animals, is underway. Finally, we show that pharmacological activation of PP2A, using FTY720, or the non-phosphorylatable analogue, AAL(S), inhibits tumor growth and metastases in an orthotopic xenograft model of aggressive, triple negative breast cancer (MDA-MB-231). Together this work demonstrates the importance of PP2A as a tumor suppressor in breast cancer, and suggests that targeting PP2A is a potential therapeutic strategy for poor outcome patients. Citation Format: Nikita Panicker, Abdul Mannan, Lauren F. Watt, Ben Copeland, Matt D. Dun, Simon King, Megan Clarke, Kathryn Skelding, Severine Roselli, Nicole M. Verrills. Functional role of the tumor suppressor protein phosphatase, PP2A-B55α, in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2375. doi:10.1158/1538-7445.AM2017-2375

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