Therapeutically Targetable Recurrent PPP2R1A Mutations Drive Tumor Initiation and Dissemination in Uterine Serous Carcinoma

Abstract

Uterine serous carcinoma (USC) and uterine carcinosarcoma (UCS) tumors are uniquely aggressive suggesting that the primary tumor is intrinsically equipped to disseminate and metastasize. Previous work by our group and others identified mutational hotspots within PPP2R1A, which encodes the A-alpha scaffolding subunit of protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine phosphatase. Two recurrent heterozygous PPP2R1A mutations, P179R and S256F, occur exclusively within high-grade subtypes of uterine cancer. This dissertation aims to understand the functional role that the two recurrent PPP2R1A mutations play in the progression of high-grade endometrial cancer. Expression of these mutants in USC/UCS cell-lines enhanced the tumor-initiating capacity, drove a hybrid epithelial-to-mesenchymal (EM) plasticity profile, and enhanced the secretion of the tumorigenic cytokine IGFBP2. Therapeutic targeting of the IGFBP2/IGF1R signaling axis using small molecules and genetic approaches resulted in marked tumor growth inhibition. Next, we aimed to identify small molecular glues that could therapeutically correct trimer formation to the mutant scaffold. The lead molecule, PMG-2, promoted B56-alpha heterotrimerization to both the A-alpha P179R and S256F mutant A/C dimers. Importantly, PMG-2 did not affect the formation of other PP2A heterotrimers. Finally, PMG-2 caused tumor regressions and median survival extensions in multiple independent cell-line and patient-derived xenografts. Importantly, this activity was abrogated upon knockout of B56-alpha, highlighting the specificity of PMG-2 to its target. Mechanistically, IGFBP2 expression was determined to be regulated by PP2A through the transcription factor, NF-kappaB which harbors a B56 recognition motif. Stabilization of the B56alpha-PP2A heterotrimeric complex through PMG-2 induced the dephosphorylation of the serine 276 site of the p65 subunit of the NF-kappaB complex in vitro and in vivo. Analysis of EM plasticity and IGFP2 circulating levels were found to be significantly reduced following treatment with PMG-2 in vivo. Combined, these data demonstrate that PMGs are a promising novel therapeutic option for PPP2R1A mutant uterine cancer patients and represent the first small molecule approach targeting a driver of this aggressive disease. Collectively, these results identify a new role for PP2A in regulating paracrine cancer-cell signaling and highlight the potential therapeutic utility of PP2A molecular glues in driving tumor cell death in disease-relevant cellular and in vivo models of high-grade endometrial cancers. Furthermore, this body of work illustrates that the recurrent PPP2R1A mutants act as a molecular driver to promote the intrinsically aggressive nature observed in USC and UCS patients through a gain of tumor-initiating capacity, dissemination and metastatic potential, EM plasticity, and the secretion of tumorigenic cytokines. To conclude, this work is highly translatable as USC/UCS patients account for a disproportionate amount of endometrial cancer-related mortality, and the characterization of recurrent PPP2R1A mutants has led to the identification of novel therapeutic approaches for these highly vulnerable cancer patients.