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Somatic mutation of the protein phosphatase 2A (PP2A) Aα-subunit gene PPP2R1A is highly prevalent in high-grade endometrial carcinoma. The structural, molecular, and biological basis by which the most recurrent endometrial carcinoma–specific mutation site P179 facilitates features of endometrial carcinoma malignancy has yet to be fully determined. Here, we used a series of structural, biochemical, and biological approaches to investigate the impact of the P179R missense mutation on PP2A function. Enhanced sampling molecular dynamics simulations showed that arginine-to-proline substitution at the P179 residue changes the protein's stable conformation profile. A crystal structure of the tumor-derived PP2A mutant revealed marked changes in A-subunit conformation. Binding to the PP2A catalytic subunit was significantly impaired, disrupting holoenzyme formation and enzymatic activity. Cancer cells were dependent on PP2A disruption for sustained tumorigenic potential, and restoration of wild-type Aα in a patient-derived P179R-mutant cell line restored enzyme function and significantly attenuated tumorigenesis and metastasis in vivo. Furthermore, small molecule–mediated therapeutic reactivation of PP2A significantly inhibited tumorigenicity in vivo. These outcomes implicate PP2A functional inactivation as a critical component of high-grade endometrial carcinoma disease pathogenesis. Moreover, they highlight PP2A reactivation as a potential therapeutic strategy for patients who harbor P179R PPP2R1A mutations. Significance: This study characterizes a highly recurrent, disease-specific PP2A PPP2R1A mutation as a driver of endometrial carcinoma and a target for novel therapeutic development. See related commentary by Haines and Huang, p. 4009

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by red cell aplasia, congenital anomalies and a predisposition to cancer. The erythroid defects of DBA include macrocytosis, reticulocytopenia, and a selective decrease or absence of erythroid precursors in the bone marrow. It was the first disease linked to ribosomal dysfunction and >70% of patients have since been shown to have a haploinsufficiency of a ribosomal protein (RP) gene. Among these RP mutations, RPS19 mutations were the first to be identified and are the most common, found in up to 25% of DBA patients. Current therapies for DBA include corticosteroids, chronic transfusions, and/or hematopoietic stem cell transplantation. However, each of these therapies have potential for significant side effects and as such there is an urgent need to develop new therapies for the management of these patients. In addition there is a need for improved understanding of the mechanistic basis for the wide range of phenotypic expression and responses to therapy between patients with the same mutation, which implies a key role for modifier genes in this disease. To identify potential modifiers in the phenotype of patients and for developing potential novel therapeutic targets, we performed a genome wide CRISPR screen using A549 cells with RPS19 haploinsufficiency as a cellular model for DBA, which enabled large-scale screening. As the p21 pathway is known to be central to the pathophysiology of DBA, we identified hits that resulted in higher and lower levels of p21 expression in the DBA model vs. control, respectively. Among these hits, we consistently identified Calbindin 1 (CALB1), a member of the calcium-binding superfamily that includes calmodulin and buffers entry of calcium into cells upon stimulation of glutamate receptors. Given the recent findings that calmodulin inhibition can rescue anemia in DBA models by blocking the p90 ribosomal S6 kinase (RSK)-p70 (p70S6K) pathway and the role for glucose and glutamine metabolism in the regulation of human HSC lineage specification, we decided to pursue CALB1 as a potential target of interest. We used peripheral blood derived-CD34+ cells placed in erythroid stimulating media following knockdown of RPS19, and investigated the effects of CALB1 on cell cycle progression and erythroid differentiation by flow cytometry. We observed that knockdown of CALB1 significantly improved cell viability, increased G0/G1 population and promoted erythroid maturation with increased expression of CD36 and glycophorin A in the context of RPS19 deficiency. Together, these data suggest that the knockdown of CALB1 rescues human erythropoiesis in a model of DBA. We are investigating the effects of CALB1 knockdown on signaling pathways and genes involved in DBA due to RPS19 mutations notably GATA-1/HSP70 and TNF-alpha. We anticipate that the identification of a role for CALB1 in the pathophysiology of DBA will provide a new therapeutic target for DBA either alone or in combination with steroids or calmodulin inhibition. Disclosures Glader: Agios Pharmaceuticals, Inc.: Consultancy.