tbuckley/cabot-search huggingface dataset:
Abstract A015: Multiplexed live-cell imaging for drug responses in patient-derived organoid models of endometrial cancer
Authors: Kaitriana E. Colling, Emily L. Symons, Hiruni K. Sumanasiri, Kristina W. Thiel
Journal: Clinical Cancer Research (2024)

Abstract Patient-derived organoid (PDO) models of endometrial cancer are rapidly emerging as a multifunctional research model to recapitulate in vivo cancer progression and therapeutic response. PDO models are generated by reconstituting patient tumor cells in extracellular basement membrane extract (BME) and culturing as three-dimensional domes supplemented with a specialized niche of growth factors. Despite the rise in popularity of PDO models to assess drug response in vitro, there are limited commercially available reagents that are compatible with BME. In addition, most reagents are endpoint assays that require cell lysis, which is problematic because PDOs are a limited resource. Reducing drug response assessment to a single timepoint misses out on valuable kinetic response data, and samples cannot be used for subsequent downstream analyses, such as RNA sequencing or protein assays. Herein we describe a method to assess drug effects in PDO models using kinetic live-cell imaging. Live-cell imaging is a sophisticated form of automated microscopy that, when combined with fluorescent dyes, has the capacity to quantify multiple cellular responses simultaneously. Importantly, live-cell imaging reagents do not perturb cell viability or induce DNA damage, thereby allowing for accurate repeated assessment of PDO response to treatment over time. In proof-of-concept studies, we first established the utility of this approach to assess the effect of cytotoxic and cytostatic agents in PDO models of endometrial cancer. Specific parameters of interest include PDO number, area, cell health and induction of apoptosis. Agents that produced a cytotoxic effect promoted a decrease in organoid area and number over time, concomitant with an increase in apoptosis as measured by Annexin V staining. Ongoing studies are examining the dose-dependent phenotypic effects of cytostatic agents such as progestins in endometrial cancer PDO models. To evaluate the safety of the compounds, we have also conducted experiments using patient-derived organoids of normal endometrium, which was collected from uninvolved regions of the uterus in cytoreductive surgery. We envision that this automated kinetic imaging platform can be expanded to other phenotypic readouts to better understand diverse therapeutic effects of different classes of drugs under investigation for endometrial cancer. Citation Format: Kaitriana E. Colling, Emily L. Symons, Hiruni K. Sumanasiri, Kristina W. Thiel. Multiplexed live-cell imaging for drug responses in patient-derived organoid models of endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A015.
Abstract A009: Addressing obesity’s impact on the PTEN mutant endometrium
Authors: Hilary J. Skalski, Shannon K. Harkins, Amelia R. Arendt, Madison MacLachlan, Katelyn Becker, Marc Wegener, Marie Adams, Galen Hostetter, Ronald L. Chandler
Journal: Clinical Cancer Research (2024)

Abstract Obesity is a rising epidemic in the United States, with approximately 50% of adult women predicted to be obese by 2030. Obesity is a risk factor for endometrial hyperplasia (EH), which is characterized by the benign overgrowth of the endometrial epithelium and is considered a precursor lesion for endometrial cancer (EC). Somatic PTEN mutations are commonly observed in EH and EC, with PTEN loss being one of the earliest mutations observed in the progression to EC. Previous obesity work in our lab utilized high-fat diet (HFD)-induced obese WT mice and found that obesity affected different cell types within the endometrium, including non-tumor prone cells. Our preliminary findings suggest that 60% HFD prolongs the estrus stage of the estrus cycle in WT mice. The aims of this study are to determine the cell-specific effects of obesity on the endometrial epithelia carrying PTEN mutations. Female PTENfl/fl; Pax8Cre/+ mutant and Pax8Cre+/+ control mice on a C57BL/6J background were fed either a 60% HFD or it’s sucrose-matched control diet (CD) for a minimum of 22 weeks. Weekly mouse and food weights were collected. Mice were tested for glucose tolerance after being on diet for at least 4.5 months. Uterus and blood were collected at estrus stage. Uteri were processed for single-cell RNA seq. PTENfl/fl; Pax8Cre/+ mice on both the HFD and CD had a diminished weight gain compared to Pax8Cre+/+ mice on the same diet. HFD-treated mice exhibited hyperphagia, weighed significantly more than the CD mice, and demonstrated glucose intolerance. H&amp;E staining of the adipose tissue and liver revealed adipocyte hypertrophy and fatty liver infiltration in HFD mice. IHC of PTENfl/fl; Pax8Cre/+ uteri confirmed epithelial-specific loss of PTEN and displayed endometrial hyperplasia. PTENfl/fl; Pax8Cre/+ uteri had an influx of Ly6G+ neutrophils invading the endometrial epithelium compared to the Pax8Cre+/+ mice. Strikingly in PTENfl/+; Pax8Cre/+ we observe clonal loss of PTEN immunoreactivity in a subset of endometrial glands with similar phenotypic features. We propose that obesity’s impact on non-tumor cell types within the endometrium will be shaped by early genetic mutations in tumor-prone epithelia. Citation Format: Hilary J. Skalski, Shannon K. Harkins, Amelia R. Arendt, Madison MacLachlan, Katelyn Becker, Marc Wegener, Marie Adams, Galen Hostetter, Ronald L. Chandler. Addressing obesity’s impact on the PTEN mutant endometrium [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A009.
Abstract PR005: Patients with endometrial cancer and benign gynecologic conditions exhibit unique vaginal and rectal microbiomes
Authors: Nicole R. Jimenez, Chloe Herman, Pawel Laniewski, Emily K. Cope, Jamal Mourad, Nichole Mahnert, Dana M. Chase, J. G. Caporaso, Melissa M. Herbst-Kralovetz
Journal: Clinical Cancer Research (2024)

Abstract Endometrial cancer (EC) rates are increasing in the United States, with 66,200 women diagnosed this year. The microbiome may be a key target for understanding EC etiology and progression and hold vital information for prevention strategies. With the largest cross-sectional cohort to date, a rich set of patient-related meta-data, and samples from multiple mucosal sites, we explored the gut-vagina axis in EC compared to benign gynecologic conditions. Vaginal and rectal swabs were collected from 192 patients diagnosed with benign conditions (n=108), hyperplasia (n= 18), and EC (n= 66). 16S rRNA amplicon sequencing was conducted on DNA samples, and data analysis was performed with QIIME 2 and R software. The clinical and demographic analysis identified risk factors associated with EC, such as BMI and menopausal status, that differed between disease groups. Global microbial analyses identified a distinct microbial composition between benign and EC, as well as higher diversity in rectal samples and lower diversity in vaginal samples in benign patients compared to cancer patients. Differential abundance analysis, even after adjustment for known risk factors, revealed unique microbial features such as depletion of Lactobacillus and increase of Anaerococcus, Porphyromonas, and Peptoniphilus in vaginal samples when EC is compared to benign conditions. While Rectal samples indicated a depletion of Peptoniphilus and Prevotella and an increase in Buttiaxella in EC compared to benign conditions. Additionally, community networks were different between cancer patients compared to benign, where taxa in Peptoniphilaceae had the most microbial network connections in EC compared to benign conditions. Notably, putative metabolic pathway analysis identified dysregulation of amino acid metabolism, complex carbohydrate metabolism, and hormone metabolism between cancer patients and benign, which provide key insight into the potential pathophysiological mechanisms employed by the microbiome in EC. This study provides key findings on unique singular and community microbial signatures that may directly affect EC development and be important targets for prevention. Citation Format: Nicole R. Jimenez, Chloe Herman, Pawel Laniewski, Emily K. Cope, Jamal Mourad, Nichole Mahnert, Dana M. Chase, J. G. Caporaso, Melissa M. Herbst-Kralovetz. Patients with endometrial cancer and benign gynecologic conditions exhibit unique vaginal and rectal microbiomes [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr PR005.
Abstract B006: Mutant <i>PPP2R1A</i> induces the expression and secretion of IGFBP2 to promote uterine serous carcinoma metastasis
Authors: Terrance James Haanen, Sophie Boock, Sarah Taylor, Caitlin O'Connor, Analisa DiFeo, Goutham Narla
Journal: Clinical Cancer Research (2024)

Abstract Uterine serous carcinoma (USC) tumors demonstrate a unique aggressiveness suggesting that the primary tumor is intrinsically equipped to disseminate, metastasize and initiate tumor formation at secondary sites. Yet the drivers and pathways regulating dissemination and metastasis in USC remain incompletely understood. Previous work by our group and others identified a mutational hotspot within PPP2R1A, which encodes the Aa scaffolding subunit of protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine phosphatase and known tumor suppressor. Two recurrent mutations, P179R and S256F, occur exclusively within high-grade subtypes of uterine cancer at a rate of 30-40%. We previously published that the P179R mutant drives USC tumorigenesis through the inactivation of PP2A, and genetic correction of this mutant resulted in decreased primary and metastatic tumor formation. The underlying mechanisms for these observations, however, remain unclear. To study the mechanistic basis for these observations, we generated isogenic patient-derived USC cell lines expressing the Aa – P179R and S256F mutants. Expression of these mutants enhanced the tumor-initiating capacity (TIC), a hybrid epithelial to mesenchymal (EM) plasticity profile, and the secretion of tumor-promoting factors in vitro and in vivo. Molecular analysis of these cell lines and tumors demonstrated that these mutants drove increased NF-kb phosphorylation resulting in the upregulation and secretion of the EM cytokine, IGFBP2. We found that the Aa mutant phenotype was dependent on the IGFBP2 cytokine function in vitro and in vivo. Future experiments will focus on identifying which PP2A B regulatory subunits are responsible for regulating the onset of the NF-kb transcriptional program. Cellular reprogramming through the IGFBP2-induced NF-kb signaling pathway mediated by recurrent Aa mutants may explain the intrinsically aggressive nature of USC, through enhanced dissemination and metastasis. Citation Format: Terrance James Haanen, Sophie Boock, Sarah Taylor, Caitlin O'Connor, Analisa DiFeo, Goutham Narla. Mutant PPP2R1A induces the expression and secretion of IGFBP2 to promote uterine serous carcinoma metastasis [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B006.
Abstract IA021: Novel genomic features of POLE-mutant tumors: the utility for tumor classification and identification of new driver alleles
Authors: Daria Ostroverkhova, Kathryn Tyryshkin, Annette K. Beach, Elizabeth A. Moore, Yosef Masoudi-Sobhanzadeh, Stephanie R. Barbari, Igor B Rogozin, Konstantin V Shaitan, Anna R Panchenko, Polina V. Shcherbakova
Journal: Clinical Cancer Research (2024)

Abstract Alterations in the exonuclease domain of POLE drive the development of ultramutated endometrial tumors with better prognosis. A hallmark of these tumors is the accumulation of G&amp;gt;T mutations in AGA sequences; however, understanding of their genomic features beyond the trinucleotide motifs is limited. We applied a novel computational framework to the whole-exome sequencing data of 524 endometrial tumors reported by The Cancer Genome Atlas network to analyze the extended DNA sequence context of ultramutation. We found that the presence of POLE driver alleles is associated with an increased frequency of G&amp;gt;T mutations in polypurine tracts. Sequences containing three consecutive purines, including the classic AGA context, are only moderately mutable, but the mutability increased abruptly as the tract length reached six or more purines. Moreover, mutations showed a strong preference for certain positions within the tracts, creating a characteristic quantifiable pattern. Using this signature, we developed a machine learning classifier to identify tumors with hitherto unknown POLE drivers and validated two new drivers, POLE-E978G and POLE-S461L, by functional assays in yeast. Unlike previously known pathogenic variants, the E978G substitution affects the DNA polymerase rather than exonuclease domain of POLE. We further show that the extended genomic signature of POLE ultramutation is shared by tumors with POLD1 drivers. These findings uncover new mechanisms of ultramutation and highlight the limitations of the current focus on POLE exonuclease domain variants when stratifying patients. Citation Format: Daria Ostroverkhova, Kathryn Tyryshkin, Annette K. Beach, Elizabeth A. Moore, Yosef Masoudi-Sobhanzadeh, Stephanie R. Barbari, Igor B Rogozin, Konstantin V Shaitan, Anna R Panchenko, Polina V. Shcherbakova. Novel genomic features of POLE-mutant tumors: the utility for tumor classification and identification of new driver alleles [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA021.
Abstract B033: Functional characterization of <i>ALK5</i> (<i>TGFBR1</i>) mutations in endometrial cancer
Authors: Eun-Jeong Yu, Daphne W. Bell
Journal: Clinical Cancer Research (2024)

Abstract ALK5 (TGFBR1) is a transmembrane receptor serine/threonine kinase that transduces TGF-b (Transforming Growth Factor b) signaling to activate SMAD2/3-dependent and -independent pathways. The purpose of this study is to determine the functional effects of ALK5 mutations in endometrial cancer (EC). Using nine in silico algorithms, we found that 79% (23 of 39) of ALK5 kinase domain mutations in EC are predicted to impact protein function. To test these predictions experimentally, constructs expressing wildtype-, constitutively active-, kinase-dead, or mutant-ALK5, including ALK5-A230V, were transfected into NIH3T3 cells, which have low endogenous ALK5 levels. Following TGF-β stimulation, we observed that transient exogenous expression of ALK5-A230V, located in the ATP-binding pocket, delayed SMAD2/3 signal transduction and altered SMAD-independent signaling. We further showed that the ALK5-A230V mutant has reduced protein stability via a ubiquitin-dependent protein degradation mechanism. Our structural modeling predicts that SB431542, a small molecule ATP-competitive inhibitor of ALK5 will bind to the ALK5-A230V mutant with less affinity than to wildtype ALK5. We, therefore, examined the inhibitory effect of SB431542 on wildtype- and mutant-ALK5 activity using a Smad-binding element (SBE) luciferase reporter assay in combination with TGF-β stimulation. SBE luciferase activity in ALK5-A230V-transfected cells was inhibited less by SB431542 than in wildtype-ALK5 transfected cells indicating that ALK5-A230V is less sensitive to SB431542 than wildtype ALK5, potentially due to the changes in affinity. Our findings are novel and show that the ALK5-A230V mutant is a partial loss-of-function mutant that attenuates TGF-b signal transduction and has reduced sensitivity to an ALK5 small molecule inhibitor. Citation Format: Eun-Jeong Yu, Daphne W. Bell. Functional characterization of ALK5 (TGFBR1) mutations in endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B033.
Abstract B020: A clinically relevant orthotopic endometrial carcinosarcoma mouse model using human patient-derived organoids
Authors: Megan Gorman, Divya Gowthaman, Arielle Katcher, Charlie Chung, Brian Yueh, Mali Barbi, Libia Garcia, Marina Frimer, Gary L. Goldberg, Semir Beyaz
Journal: Clinical Cancer Research (2024)

Abstract Uterine carcinosarcoma (CS) is a rare but aggressive endometrial malignancy. The incidence of CS has been increasing and is projected to continue to rise. African American women in particular have an increased incidence of CS and overall increased mortality related to endometrial cancer, underscoring known disparities in cancer care across racial and ethnic groups. Compared to other histologic subtypes, CS remains rare and understudied on a molecular and cellular basis, and under-represented in clinical trials. Therefore, current treatment therapies available for CS have been extrapolated from treatments used in other histologies of endometrial cancer, highlighting a significant need for effective treatment targeted at this subtype. Due to this paucity of data surrounding targeted therapies for CS, patients who are diagnosed with this aggressive and lethal cancer tend to have worse outcomes. We have developed a clinically relevant CS murine model by injecting human CS patient-derived organoids (PDO) from diverse ancestries into the endometrium of immunocompromised mice. Tumor progression in the mice was monitored with physical examination, PET/CT scan, contrast enhanced CT scan, ultrasound, and bioluciferase imaging. The tumor was established after three months, and the mice were sacrificed following the experiment. The diagnosis of invasive CS was confirmed by macroscopic and histologic analyses in the orthotopic model. Our orthotopic CS PDO transplantation model has many potential applications for studying tumor biology of CS in the pre-clinical setting. Future directions include trialing the efficacy of standard of care chemotherapy in the orthotopic model. Using this model, we aim to identify targeted therapy for CS and potentially advance to Phase I human clinical trials. Citation Format: Megan Gorman, Divya Gowthaman, Arielle Katcher, Charlie Chung, Brian Yueh, Mali Barbi, Libia Garcia, Marina Frimer, Gary L. Goldberg, Semir Beyaz. A clinically relevant orthotopic endometrial carcinosarcoma mouse model using human patient-derived organoids [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B020.
Abstract B028: Epithelial-specific loss of <i>PIK3R1</i> in a murine model result in endometrial hyperplasia
Authors: Shannon K. Harkins, Hilary J. Skalski, Amelia Arendt, Laura Pavliscak, Mike R. Wilson, Ronald L. Chandler
Journal: Clinical Cancer Research (2024)

Abstract Somatic mutations in the phosphatidylinositol 3-kinase (PI3K) pathway occur in 84% of endometrial carcinomas (EC) and up to 68% of endometrial hyperplasia (EH). Regulation of PI3K/Akt/mTOR signaling is integral for normal endometrial function and is governed by tumor suppressor genes PTEN and PIK3R1 and proto-oncogene PIK3CA. While the actions of PTEN and PIK3CA are in direct opposition as a phosphatase and kinase, respectively, PIK3R1 regulates PI3K/Akt/mTOR signaling in two distinct ways. As the regulatory subunit of the PI3K enzyme, PIK3R1 controls the catalytic activity of PIK3CA and augments PTEN activity. Mutations or alterations in PIK3R1 are present in 33% of EC and 9% of EH, the majority of which lead to loss of PIK3R1 protein (p85a). Both loss of PTEN and PIK3R1 lead to increased proliferation through PI3K/Akt/mTOR signaling, but the structural, functional, and mechanistic implications of PIK3R1 loss in the endometrial epithelium (EE) in vivo are largely unexplored. To further interrogate the changes in the endometrium resulting from PIK3R1 loss, we have created a murine model of endometrial epithelial-specific PIK3R1 loss driven by Pax8-Cre. Mice were estrus cycle staged and sacrificed in the diestrus phase to reduce confounding variables when assessing phenotypes between genotypes. PIK3R1 loss was confirmed by immunohistochemistry (IHC). Histology and immunostaining of control and PIK3R1 knockout (KO) mice were assessed by IHC and immunofluorescence. We found that PIK3R1 loss in the EE leads to EH distinct from what is observed with PTEN loss. In contrast to the EH with atypia seen In PTEN KO mice, the EH resulting from PIKR1 loss more closely resembles EH without atypia. The endometrium of PIK3R1 KO mice is characterized by thickened luminal epithelia with jagged edges and irregularities, mild glandular crowding, and occasional nuclear atypia. PIK3R1 KO mice also showed an increase in proliferation (Ki-67) and downstream PI3K signaling (phospho-S6) in the EE. Gene expression changes occurring within the EE from PIK3R1 KO and Cre-negative control mice were also assessed by RNA-seq. In vivo genomic analyses were compared to in vitro knockdown of PIK3R1 and publicly available patient data. We found that PIK3R1 loss leads to an increase in signaling through the PI3K/Akt/mTOR and MAPK pathways. Our data show that the effect of PIK3R1 loss is sufficient to cause EH and may have shared and distinct actions from PTEN. Citation Format: Shannon K. Harkins, Hilary J. Skalski, Amelia Arendt, Laura Pavliscak, Mike R. Wilson, Ronald L. Chandler. Epithelial-specific loss of PIK3R1 in a murine model result in endometrial hyperplasia [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B028.