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Uterine serous carcinoma (USC) is a highly aggressive endometrial cancer subtype with limited therapeutic options and a lack of targeted therapies. While mutations to PPP2R1A, which encodes the predominant protein phosphatase 2A (PP2A) scaffolding protein Aα, occur in 30% to 40% of USC cases, the clinical actionability of these mutations has not been studied. Using a high-throughput screening approach, we showed that mutations in Aα results in synthetic lethality following treatment with inhibitors of ribonucleotide reductase (RNR). In vivo, multiple models of Aα mutant uterine serous tumors were sensitive to clofarabine, an RNR inhibitor (RNRi). Aα-mutant cells displayed impaired checkpoint signaling upon RNRi treatment and subsequently accumulated more DNA damage than wild-type (WT) cells. Consistently, inhibition of PP2A activity using LB-100, a catalytic inhibitor, sensitized WT USC cells to RNRi. Analysis of The Cancer Genome Atlas data indicated that inactivation of PP2A, through loss of PP2A subunit expression, was prevalent in USC, with 88% of patients with USC harboring loss of at least one PP2A gene. In contrast, loss of PP2A subunit expression was rare in uterine endometrioid carcinomas. While RNRi are not routinely used for uterine cancers, a retrospective analysis of patients treated with gemcitabine as a second- or later-line therapy revealed a trend for improved outcomes in patients with USC treated with RNRi gemcitabine compared with patients with endometrioid histology. Overall, our data provide experimental evidence to support the use of ribonucleotide reductase inhibitors for the treatment of USC.Significance:A drug repurposing screen identifies synthetic lethal interactions in PP2A-deficient uterine serous carcinoma, providing potential therapeutic avenues for treating this deadly endometrial cancer.

Supplementary Figure from Targeting Ribonucleotide Reductase Induces Synthetic Lethality in PP2A-Deficient Uterine Serous Carcinoma

Supplementary Data from Targeting Ribonucleotide Reductase Induces Synthetic Lethality in PP2A-Deficient Uterine Serous Carcinoma

Uterine serous carcinoma (USC) is a highly aggressive endometrial cancer subtype with limited therapeutic options and a lack of targeted therapies. While mutations to PPP2R1A, which encodes the predominant protein phosphatase 2A (PP2A) scaffolding protein Aα, occur in 30% to 40% of USC cases, the clinical actionability of these mutations has not been studied. Using a high-throughput screening approach, we showed that mutations in Aα results in synthetic lethality following treatment with inhibitors of ribonucleotide reductase (RNR). In vivo, multiple models of Aα mutant uterine serous tumors were sensitive to clofarabine, an RNR inhibitor (RNRi). Aα-mutant cells displayed impaired checkpoint signaling upon RNRi treatment and subsequently accumulated more DNA damage than wild-type (WT) cells. Consistently, inhibition of PP2A activity using LB-100, a catalytic inhibitor, sensitized WT USC cells to RNRi. Analysis of The Cancer Genome Atlas data indicated that inactivation of PP2A, through loss of PP2A subunit expression, was prevalent in USC, with 88% of patients with USC harboring loss of at least one PP2A gene. In contrast, loss of PP2A subunit expression was rare in uterine endometrioid carcinomas. While RNRi are not routinely used for uterine cancers, a retrospective analysis of patients treated with gemcitabine as a second- or later-line therapy revealed a trend for improved outcomes in patients with USC treated with RNRi gemcitabine compared with patients with endometrioid histology. Overall, our data provide experimental evidence to support the use of ribonucleotide reductase inhibitors for the treatment of USC. Significance: A drug repurposing screen identifies synthetic lethal interactions in PP2A-deficient uterine serous carcinoma, providing potential therapeutic avenues for treating this deadly endometrial cancer.

Uterine serous carcinoma (USC) is a highly aggressive endometrial cancer subtype with limited therapeutic options and a lack of targeted therapies. While mutations to PPP2R1A, encoding the predominant protein phosphatase 2A (PP2A) scaffolding protein Aα, occur in 30-40% of cases, the clinical actionability of these mutations has not been studied. Here, we show that mutation to Aα results in synthetic lethality to treatment with inhibitors of ribonucleotide reductase (RNR), and multiple models of Aα mutant uterine serous tumors were sensitive to Clofarabine, an RNR inhibitor in vivo. Aα mutant cells displayed impaired checkpoint signaling upon RNRi treatment, and subsequently accumulated more DNA damage than wild type cells. This was PP2A dependent as complete inhibition of PP2A activity using LB-100, a catalytic site inhibitor, sensitized wild type USC cells to RNRi. Analysis of TCGA data indicated that inactivation of PP2A, through loss of PP2A subunit expression, was prevalent in USC, with 88% of USC patients harboring loss of at least one PP2A gene. In contrast, loss of PP2A subunit expression was rare in uterine endometrioid carcinomas. While RNR inhibitors are not routinely used for uterine cancers, we identified a cohort of patients with recurrent disease treated with gemcitabine at MSKCC as a second or later line therapy. In a retrospective analysis of this cohort there was a trend for improved outcomes in USC patients treated with RNRi gemcitabine compared to patients with endometrioid histology. Overall, our data provide experimental evidence to support the use of ribonucleotide reductase inhibitors for the treatment of USC. Citation Format: Caitlin M. O'Connor, Sarah E. Taylor, Kathryn M. Miller, Lauren Hurst, Terrance J. Haanen, Tahra K. Suhan, Kaitlin P. Zawacki, Fallon K. Noto, Jonida Trako, Arathi Mohan, Jaya Sangodkar, Dmitriy Zamarin, Analisa DiFeo, Goutham Narla. Synthetic lethality by targeting ribonucleotide reductase in PP2A deficient uterine serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3341.

Triple-negative breast cancer (TNBC) is an aggressive subtype most prevalent among women of Western Sub-Saharan African ancestry. It accounts for 15-25% of African American (AA) breast cancers (BC) and up to 80% of Ghanaian breast cancers, thus contributing to outcome disparities in BC for black women. The aggressive biology of TNBC has been shown to be regulated partially by breast cancer stem cells (BCSC) which mediate tumor recurrence and metastasis and are more abundant in African breast tumors.We studied the biological differences between TNBC in women with African ancestry and those of Caucasian women by comparing the gene expression of the BCSC. From low-passage patient derived xenografts (PDX) from Ghanaian (GH), AA, and Caucasian American (CA) TNBCs, we sorted for and sequenced the stem cell populations and analyzed for differential gene enrichment.In our cohort of TNBC tumors, we observed that the ALDH expressing stem cells display distinct ethnic specific gene expression patterns, with the largest difference existing between the GH and AA ALDH+ cells. Furthermore, the tumors from the women of African ancestry [GH/AA] had ALDH stem cell (SC) enrichment for expression of immune related genes and processes. Among the significantly upregulated genes were CD274 (PD-L1), CXCR9, CXCR10 and IFI27, which could serve as potential drug targets.Further exploration of the role of immune regulated genes and biological processes in BCSC may offer insight into developing novel approaches to treating TNBC to help ameliorate survival disparities in women with African ancestry.

To design interventions to reduce the burden of breast cancer (BC) in women with African ancestry, there is the need to understand the factors - heritable or non-heritable, modifiable or non-modifiable - that are associated with the disease. Patients and tumors from black women have been underrepresented in clinical research which are the main route to identifying new and improved treatments. Triple negative breast cancer (TNBC) is a clinically aggressive form of BC with propensity to metastasize and is enriched in patients with hereditary predisposition to BC. TNBC is most prevalent among black women despite lacking strong links with the known BC. This phenotype prevalence contributes to disparities in BC outcomes between blacks and other populations. There is an urgent need to represent black tumors in clinical research. Here we describe the process we used to transport fresh breast tumors from Ghana, west Africa to the USA to develop PDXs. Considering the shared ancestry between west Africans and diasporan African American, the results of this work will benefit both populations. Through an international collaboration between investigators at University of Michigan (UM) and Komfo Anokye Teaching Hospital (KATH) in Ghana, regular team site visits were initiated to generate surgical specimens for transport to the United States of America (USA). These specimens were used to create in vivo models. With informed consent, excess patient tumor tissue was taken from the operating room, at KATH. The tumors were cut into small pieces, and slow- frozen to -80°C in 10% DMSO/90%FBS. The samples were then transported overnight on dry ice in a commercial flight to UM. At UM, the tumor pieces were rapidly thawed and rinsed off with HBSS or PBS. The tumor sample was cut into smaller fragments (approximately 1-2 mm in diameter), mixed with matrigel (BD), and 25 μl of matrigel per injection site was injected into the mammary fat pad of female NOD/scid/IL2Rgnull (NSG) mice with an 18-gauge needle. From 2012 to 2017, we obtained fresh tumor samples from 49 Ghanaian patients. Of these, 23 (47%) were TNBC and 26 (53%) were other subtypes. Of the 23 TNBC samples, we implanted 18 into NSG mice and developed PDXs from 12. This gave us a success rate of 67%. We have fully characterized these tumors and their corresponding primary tumors. Our results demonstrate a maintenance of the histology and marker expression in the corresponding PDXs. Additionally, we obtained samples and developed PDXs from 17 Caucasian Americans, 13 African Americans, and 2 Asian patients from UM. 20 samples were TNBCs and 13 were non-TNBC. 18 were implanted in mice (12 TNBC, 4 ER+, and 2 Her2+). Of these, 11 developed into PDXs (9 TNBC, 2 ER+). Transporting African tumors to the USA is expensive, challenging, and limits large scale studies. These challenges can be overcome by enhancing African based research resources in order to conduct research in Africa. Citation Format: Evelyn M. Jiagge, Tahra K. Suhan, Jessica M. Bensenhaver, Michele Dziubinski, Joseph K. Oppong, Francis Aitpillah, Awuah Baffour, John D. Carpten, Lisa A. Newman, Sofia D. Merajver, Max S. Wicha. Creating PDXs from continental African breast tumors: addressing racial inequity through science [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2996.

Background: Ewing sarcoma is the second most common pediatric bone cancer. Metastatic disease is almost always fatal, and there is currently no method to predict which patients are at risk for metastasis. Better therapies are needed to prevent and treat metastatic disease, which means the mechanisms that drive Ewing sarcoma metastasis must be better elucidated. It is becoming increasingly clear that interactions between tumor cells and the tumor microenvironment (TME) play an essential role in metastasis, but the specific mechanisms through which the TME contributes to Ewing sarcoma progression remain largely unknown. Our previous work has demonstrated that activation of canonical Wnt and TGF-β pathways in a subset of Ewing sarcoma cells induces changes in gene expression and protein secretion that are associated with enhanced metastatic engraftment, changes in the extracellular matrix (ECM), and increased angiogenesis. As bone, the primary site for Ewing sarcoma, is an excellent source of ligands for both pathways, we hypothesize that crosstalk between Wnt/TGF-β-activated tumor cells and the local bone microenvironment contributes to osteolysis and metastatic progression. Methods: Ewing sarcoma cells were treated with control or Wnt3a media +/- recombinant TGF-β1, and then the mRNA levels of target genes were measured by Q-RT-PCR. ELISA and Luminex assays were also performed to determine the levels of secreted proteins. Conditioned media collected from stimulated cells were used to treat osteoblast precursor cells, which were then assayed for effects on differentiation and function. Subcutaneous, femur, and vossicle transplant xenograft models were established in mice and are being validated. Results: We have demonstrated that in response to Wnt3a and TGF-β1, Ewing sarcoma cells upregulate expression of canonical Wnt targets (e.g., LEF1), ECM-associated genes (e.g., TNC, COL1A1, and MMP2), and the pro-osteolytic factor PTHrP. We have also shown that osteoblast precursor cells can be induced to differentiate in the presence of conditioned media from Ewing sarcoma cell lines. Immunohistochemical staining is being used to evaluate Wnt and TGF-β pathway activation, angiogenesis, osteolysis, and the ECM in our bone tumor xenograft models. Conclusions: Wnt3a/TGF-β1-stimulated Ewing sarcoma cells upregulate expression of genes associated with the ECM and osteolysis. We are in the process of demonstrating the functional consequences of those changes using in vitro conditioned media experiments and in vivo transplant models. Understanding the mechanisms by which these pathways contribute to Ewing sarcoma phenotypes is important for the development of new therapeutics for patients with metastatic disease. Citation Format: Kelsey Temprine, Sydney Treichel, Allegra Hawkins, Tahra Suhan, Wei Jiang, Parker Acevedo, Amy Koh, Kurt Hankenson, Laurie K. McCauley, Elizabeth R. Lawlor. Investigating the role of tumor:bone microenvironment crosstalk in Ewing sarcoma progression [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B74.

Ewing sarcomas predominantly arise in pelvic and stylopod bones (i.e., femur and humerus), likely as a consequence of EWS-FLI1 oncogene-induced transformation of mesenchymal stem/progenitor cells (MSCs). MSCs located in the embryonic superficial zone cells (eSZ) of limbs express anatomically distinct posterior Hox genes. Significantly, high expression of posterior HOXD genes, especially HOXD13, is a hallmark of Ewing sarcoma. These data drove our hypothesis that Hox genes in posterior skeleton MSCs contribute to Ewing sarcoma tumorigenesis. We isolated eSZ cells from stylopod and zeugopod (i.e., tibia/fibula, radius/ulna) bones, from wild-type and Hoxd13 mutant embryos, and tested the impact of EWS-FLI1 transduction on cell proliferation, gene expression, and tumorigenicity. Our data demonstrate that both stylopod and zeugopod eSZ cells tolerate EWS-FLI1 but that stylopod eSZ cells are relatively more susceptible, demonstrating changes in proliferation and gene expression consistent with initiation of malignant transformation. Significantly, loss of Hoxd13 had no impact, showing that it is dispensable for the initiation of EWS-FLI1-induced transformation in mouse MSCs. These findings show that MSCs from anatomically distinct sites are differentially susceptible to EWS-FLI1-induced transformation, supporting the premise that the dominant presentation of Ewing sarcoma in pelvic and stylopod bones is attributable to anatomically-defined differences in MSCs.