bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3037: RSK4 inhibition promotes pseudo-hypoxic metabolic responses in lung adenocarcinoma
Authors: Thamlikitkul Lucksamon , Chai Peng , Nadal Cristina Balcells , Roy Rajat , Sun Xiao-Ming , MacFarlane Marion , Keun Hector , Seckl Michael , Pardo Olivier
Published: June 2022

Background: RSK4 is a member of the p90 Ribosomal S6 kinase family that promotes metastasis and chemoresistance in non-small cell lung cancer and its inhibition improves therapeutic outcome in in vivo and ex vivo models. Here, we show that RSK4 inhibition activates HIF-1alpha and also impacts glucose and glutamine metabolism in lung adenocarcinoma cells.

Methods: RSK4 was downregulated in lung adenocarcinoma cells by RNA interference or CRISPR/Cas9 technology. Glycolytic rate assay and mitochondrial stress test were performed using the Seahorse XFe96 analyzer. ATP levels were determined by the CellTiter Glo assay. Intracellular metabolites were profiled by untargeted mass spectrometry and U-13C glutamine labelling. 3D spheroids viability was assessed by CellTiter Glo 3D assay with or without glycolysis inhibition using 2-deoxy-D-glucose (2DG) or glutaminase inhibition using 6-Diazo-5-oxo-L-norleucine (DON). Protein expression was determined by Western blotting.

Results: RSK4 downregulation enhanced glycolysis, oxidative phosphorylation, and ATP production in A549 cells. The RSK4-silenced cells showed increased lactate production and glutamine consumption via reductive carboxylation. This sensitized RSK4-silenced cells to 2DG and DON treatments. Mechanistically, RSK4 knockdown upregulates HIF-1alpha in normoxia through decreased activation of PHD2, an enzymes that targets HIF-1alpha for degradation. Stabilization of HIF-1alpha resulted in increased transcription of HK2, the rate limiting enzyme of glycolysis, and GLS1, which hydrolyzes glutamine.

Conclusion: RSK4 knockdown promotes a pseudo-hypoxic phenotype in lung adenocarcinoma cells characterized by increased glycolysis and enhanced glutamine utilization via reductive carboxylation. Targeting these changes potentiates the effects of RSK4 inhibition and may represent new therapeutic opportunities for this tumor type.

Citation Format: Lucksamon Thamlikitkul, Peng Chai, Cristina Balcells Nadal, Rajat Roy, Xiao-Ming Sun, Marion MacFarlane, Hector Keun, Michael Seckl, Olivier Pardo. RSK4 inhibition promotes pseudo-hypoxic metabolic responses in lung adenocarcinoma [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 3037.
Abstract 3050: Equol as a radiosensitizer and therapeutic agent in prostate cancer
Authors: Jones Carli B. , Coulter Jonathan B. , Maynard Janielle P. , Mummert Luke A. , Phillips Ryan M. , Tran Phuoc T. , Sfanos Karen S.
Published: June 2022

The gastrointestinal microbiome and the metabolites it can produce have been linked to response to various cancer treatments, including in prostate cancer. Equol is a microbial metabolite that is exclusively produced by the conversion of soy isoflavones in the diet by specific intestinal bacteria. Interestingly, equol has previously been shown to exhibit anti-androgen effects on prostate cells and likewise may act as a radiosensitizer. We analyzed the microbial composition of fecal samples collected from men receiving stereotactic ablative radiation (SABR) versus observation to treat oligometastatic prostate cancer as part of the ORIOLE trial and found that patients harboring equol-producing bacteria had a lower risk of disease progression. We therefore aimed to study the effect of this compound on prostate cancer cell lines in vitro. MTT assay was used to assess the effects of equol treatment on cell viability in androgen receptor (AR) positive LNCaP cells and AR null PC3 cells over time. We assessed the ability of equol to reduce AR protein levels and/or subcellular localization in LNCaP cells using immunohistochemistry. Finally, western blotting was used to assess the effects of equol treatment on prostate specific antigen (PSA) levels to assess effects on the AR signaling pathway. Cell viability was significantly decreased over time in equol treated cells, but only in prostate cancer cell lines that were AR positive. The effects of equol on LNCaP viability was abrogated in an AR knockdown LNCaP cell line. Interestingly, AR protein levels were found to be markedly decreased upon equol treatment of LNCaP cells. We likewise observed a decrease in PSA protein levels in AR positive cells after equol treatment. Our results support the notion that equol can inhibit prostate cancer growth by inhibition of the androgen receptor. We are currently investigating whether combining equol exposure with radiation treatment produces a synergistic effect on DNA damage and reduction of prostate cancer cell viability and survival in vitro. Our future studies will investigate the effects of equol and equol-producing bacteria on tumor growth in in vivo models.

Citation Format: Carli B. Jones, Jonathan B. Coulter, Janielle P. Maynard, Luke A. Mummert, Ryan M. Phillips, Phuoc T. Tran, Karen S. Sfanos. Equol as a radiosensitizer and therapeutic agent in prostate cancer [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 3050.
Abstract 3130: IL-27 signaling regulates anti-cancer immune response in hepatocellular carcinoma
Authors: Mazitova Aleksandra M. , Aghayev Turan , Fang Jennifer , Dzutsev Amiran , Trinchieri Giorgio , Campbell Kerry S. , Grivennikov Sergei I. , Koltsova Ekaterina K.
Published: June 2022

Hepatocellular carcinoma (HCC) is the most common form of liver cancer with poor survival and limited therapeutic options. HCC development is accompanied by underlying chronic inflammation, which represents a major unifying mechanism for tumor promotion. While some tumor-promoting inflammatory mechanisms had been proposed, the identity of immune mechanisms controlling anti-cancer immunity in HCC remain poorly understood. Interleukin (IL)-27 receptor signaling plays an anti-inflammatory role in a variety of infectious and chronic inflammatory diseases. Here, using genetic and pharmacological approaches we found that IL-27 receptor (IL-27R) signaling promotes HCC development in vivo. Genetic loss of IL-27R suppressed HCC in both carcinogen-induced and non-alcoholic steatohepatitis (NASH)-driven models. Mechanistically, the pro-tumorigenic effect was mediated by an immunoregulatory role of IL-27R within the tumor microenvironment, particularly the suppression of cytotoxic Natural killer (NK) cells. Single-cell RNA analysis further established the role of IL-27R signaling in restraining cytotoxic populations of NK cells. IL-27R ablation enhanced the accumulation and activation of cytotoxic NK cells during acute liver injury and in HCC tumors, while depletion or functional impairment of NK cells abrogated the effect of genetic IL-27R disruption. Taken together, our data suggest an unexpected role of IL-27R signaling as a novel immunological checkpoint regulating innate cytotoxic cell activity and promoting development of HCC of different etiologies.

Citation Format: Aleksandra M. Mazitova, Turan Aghayev, Jennifer Fang, Amiran Dzutsev, Giorgio Trinchieri, Kerry S. Campbell, Sergei I. Grivennikov, Ekaterina K. Koltsova. IL-27 signaling regulates anti-cancer immune response in hepatocellular 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 3130.
Abstract 3152: IFN-gamma stimulated cryopreserved mesenchymal stromal cells for treatment of radiation induced xerostomia
Authors: Paz Cristina , Blitzer Grace , Glassey Annemarie , Nickel Kwangok P. , Giri Jayeeta , Pennati Andrea , Robbins Daniel , Lunga Tadeas , Thibeault Susan , Galipeau Jacques , Kimple Randall
Published: June 2022

Purpose/Objectives: There is a critical need for a treatment that will safely and effectively alleviate radiation-induced xerostomia (RIX). Head and neck radiation results in hyposalivation and altered sialochemistry which patients experience as xerostomia. We performed a preclinical and a pilot clinical study to determine if IFN-ɣ stimulated marrow-derived mesenchymal stromal cells (MSC(M)) from HNC patients could function as a source of autologous cells for the treatment of RIX and as a tool in preclinical murine studies of RIX.

Materials/Methods: Bone marrow aspirates from HNC patients previously treated with chemoradiation were obtained (IRB #2019-0497, NCT 04007081). MSC(M)s were isolated from bone marrow. MSC(M) were expanded in culture, surface marker expression was confirmed via flow cytometry, stimulated with IFN-ɣ, and cryopreserved. Thawed MSC(M)s were profiled to evaluate their functionality after cryorecovery. Cells were tested for their ability to stimulate salivary production in a murine model of RIX by injecting 1x106 cells into the submandibular gland (SMG) one week after a single 15 Gy fraction of radiation. Efficacy was evaluated by saliva production and histology.

Results: MSC(M)s from six patients were expanded to >20x106 cells (median 15.5, range of 8-20 days). Post-thaw cultures demonstrated robust growth, with a median doubling time of 3.1 days. Cultured cells showed an MSC(M) phenotype, positive for CD73, CD90, and CD105 and negative for CD14, CD20, CD34, or CD45. IFN-ɣ stimulated MSC(M)s had immunomodulatory potential based on increased expression of IDO, ICAM-1, PD-L1, MHC I and MHC II expression compared with non-stimulated MSC(M)s (391%, 263%, 114%, 70% and 196% percent increase respectively). In a murine model of RIX, radiation resulted in structural changes to the gland as evidenced by increased fibrosis, decreased acinar size, and immune cell infiltration. Injection of MSC(M)s to the SMG following radiation resulted in improved saliva production, a reduction in fibrosis, and an increase in the size and density of acini within the tissue compared to control.

Conclusions: These data strongly support the feasibility of a first-in-human clinical trial of autologous IFN-ɣ stimulated MSC(M)s to treat RIX and the potential of using human-derived MSC(M)s in future murine studies of RIX.

Citation Format: Cristina Paz, Grace Blitzer, Annemarie Glassey, Kwangok P. Nickel, Jayeeta Giri, Andrea Pennati, Daniel Robbins, Tadeas Lunga, Susan Thibeault, Jacques Galipeau, Randall Kimple. IFN-gamma stimulated cryopreserved mesenchymal stromal cells for treatment of radiation induced xerostomia [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 3152.
Abstract 3154: Assessing hematopoietic stem cell fitness within a nanobioreactor in microgravity
Authors: Ladel Luisa , Pham Jessica , Oliver Isabelle , Balaian Larisa , Jamieson Catriona HM
Published: June 2022

Microgravity coupled with increased radiation exposure aboard the ISS provides a unique environment to simulate and study response to injury, inflammatory signaling, aging, and (pre-)malignant transformation of normal human hematopoietic stem cells (HSCs) in an accelerated timeframe. The NASA Twins study suggests that genomic, epigenomic, epitranscriptomic, and proteomic changes may detrimentally impact hematopoietic stem and immune cell fitness and induce stem cell exhaustion (Garrett-Bakelman et al., Science, 2019). Moreover, changes indicative of pre-cancer stem cell generation such as increased chromosome translocations and inversions occurred and persisted post-flight. Additionally, a recent publication entitled Multisystem Toxicity in Cancer: Lessons from NASA’s Countermeasures Program found significant similarities between the multisystem physiological toxicities in cancer patients and during spaceflight (Scott et al, Cell 2019). These reports highlight the benefits of studying injury response, changes in mutational profiles and cancer evolution in microgravity at the stem cell level.

For this study, we designed a novel bioreactor system to support the culture of donor-derived human HSCs in low Earth orbit (LEO). A sponge matrix and stromal cells model the microenvironment HSCs reside in within the bone marrow niche. Testing on Earth confirmed our system’s ability to maintain stem cell fitness over several weeks. To assess stem cell physiology in LEO over time, we lentivirally transduce a reporter into the HSCs pre-flight (Pineda et al., Scientific Reports 2016), which allows for cell cycle tracking via fluorescence imaging. This will provide data for assessment of stem cell health, maintenance and functionality. Furthermore, we will be analyzing mutational status post-flight, with a focus on signatures we have previously connected to (pre-)malignant transformation via RNA sequencing analysis (Jiang, Cancer Cell 2019). Our bioreactors are scheduled to launch as part of the SpaceX CRS-24 mission on Dec 21, 2021. This investigation may provide valuable insights into the maintenance of hematopoietic stem cell health and functionality, response to injury through accumulation of mutations and, eventually, the mechanisms fueling long-term (pre-)malignant transformation into leukemia stem cells.

Citation Format: Luisa Ladel, Jessica Pham, Isabelle Oliver, Larisa Balaian, Catriona HM Jamieson. Assessing hematopoietic stem cell fitness within a nanobioreactor in microgravity [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 3154.
Abstract 3163: PR/STAT3 nuclear transcriptional complexes mediate aurora-A kinase-induced stemness plasticity in ER+ breast cancer
Authors: D'Assoro Antonino B.
Published: June 2022

Purpose: Emergence of de novo or acquired resistance to standard of care endocrine therapies represents a major challenge in the clinical management of estrogen receptor alpha positive (ER+) breast tumors. This process is characterized by high tumor plasticity that confers to cancer cells the ability to shift from a differentiated state, with limited tumorigenic potential, to an undifferentiated state which is responsible for drug resistance and tumor relapse. In ER+ breast tumors, this plasticity is manifest by the enrichment of breast tumor initiating cells (BTICs) with stemness features, such as self-renewal capacity, CD44 expression and high ALDH activity, and ER down-regulation. Development of clinically relevant ER+ breast tumor models to test novel therapeutic strategies that inhibit the enhanced tumor cell plasticity and restore or extend endocrine sensitivity are urgently needed.

Experimental Design: Unique endocrine resistant and metastatic breast cancer models were utilized to define in vitro and in vivo the pivotal role of Aurora-A mitotic kinase (AURKA) in the induction of stemness plasticity, endocrine therapy resistance and tumor progression. Unbiased phospho-proteomic analysis was performed in the absence or presence of alisertib, a clinically relevant AURKA inhibitor, that was used to identify novel down-stream targets of AURKA signaling pathway.

Results: Phospho-proteomic analysis showed that AURKA targeting reduced the phosphorylation of STAT3 transcription factor at the serine-727 site. Significantly, herein we define a novel mechanism by which AURKA-induced self-renewal capacity is mediated through the activation of a phosphorylated serine727-STAT3/serine294-progesterone receptor (PR) transcriptional complex that regulates the expression of KLF4 stemness reprogramming gene. Remarkable, pharmacologic and genetic targeting of AURKA/STAT3/PR oncogenic axis impairs self-renewal capacity of endocrine therapy resistant breast cancer cells.

Conclusions: In this study we demonstrate the pivotal role of a novel AURKA/STAT3/PR oncogenic axis in promoting stemness plasticity and endocrine therapy resistance. These findings provide the strong preclinical rationale for the development of novel combinatorial therapeutic strategies targeting cooperative AURKA/serine727-STAT3 and serine294-PR pathways predicted to result in the selective eradication of CD44High/ALDHHigh BTICs from the bulk tumor with consequent benefits to the clinical outcomes of patients with advanced ER+ breast cancer refractory to standard of care endocrine therapy.

Citation Format: Antonino B. D'Assoro. PR/STAT3 nuclear transcriptional complexes mediate aurora-A kinase-induced stemness plasticity in ER+ breast cancer [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 3163.
Abstract 3187: Understanding the role of cancer-associated fibroblasts (CAFs) on pathogenesis of KRAS mutated cancers by defining differences in the basal state proteome and secretome
Authors: Lau Rachel , Yu Lu , Roumeliotis Theodoros , Pickard Lisa , Stewart Adam , Choudhary Jyoti , Banerji Udai
Published: June 2022

Background: KRAS is commonly mutated in solid tumors. Cancer-associated fibroblasts (CAFs) are a key tumor stroma component, which can affect cancer progression and drug response. We hypothesized that defining the differential secreted proteins between CAFs and KRAS mutant-cancer cells could reveal potential mechanisms by which CAFs influence cancer progression and drug resistance.

Methods: We studied 12 cell lines: 3 KRAS mutant-colorectal cancer cell lines (H747, LIM2099 and SW620), 3 KRAS mutant-lung cancer cell lines (H1792, H2030 and H23), 3 KRAS mutant-pancreatic cancer cell lines (CAPAN1, DANG, MIAPACA2) and 3 CAF cell line models (colorectal and lung CAF and pancreatic stellate cells). We quantified proteins in cell lines and serum-free media after 24 hour incubation in cells using mass spectrometry in a multiplexed manner (12plex) in triplicates to characterize the proteome and the profile of secreted proteins (secretome), respectively. Prediction software (signalP, secretomeP, TMHMM) and databases (surfaceome and cell surface protein atlas) were used to annotate secreted or transmembrane proteins.

Results: Our proteome analysis quantified 9307 proteins. T-test between the cell types (CAF vs cancer) identified 1770 differentially expressed proteins, where 712 proteins were CAF-enriched. This included known CAF markers (e.g α smooth muscle actin) but also proteins not known to be CAF markers such as heat shock protein beta-5/6 (HSPB5/6). The secretome analysis identified 2330 secreted or transmembrane proteins where 202 were differentially expressed between cancer cells and CAFs. 190 proteins were CAF-enriched with the highest ranking being syndecan-2 (SDC2), collagen α-28(I) chain (COL28A1), decorin (DCN) and wnt family member 5B (WNT5B). Interestingly, basal proteome analysis of KRAS mutant-cancer cells showed expression of receptors corresponding to the CAF-enriched ligands, such as receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2) which respond to WNT5B. This suggests that CAF-enriched ligands may activate certain downstream pathways, including non-canonical WNT signaling, in KRAS mutant-cancer cells.

Conclusion: We undertook deep quantitative proteomics where we identified potential novel CAF markers HSPB5/6. We have also found proteins in the CAF secretome, such as WNT5B, which may drive drug resistance in KRAS mutant-cancer cells.

Citation Format: Rachel Lau, Lu Yu, Theodoros Roumeliotis, Lisa Pickard, Adam Stewart, Jyoti Choudhary, Udai Banerji. Understanding the role of cancer-associated fibroblasts (CAFs) on pathogenesis of KRAS mutated cancers by defining differences in the basal state proteome and secretome [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 3187.
Abstract 3195: Tumor inhibitory CAFs in CNS metastases
Authors: Simon Thomas , Buckley David , Salhia Bodour
Published: June 2022

The 2-year survival rate for patients with central nervous system (CNS) metastases (CMs) remains below 2%. With the aim to define new therapeutic options, researchers have investigated how interactions between CM cells and the surrounding tumor microenvironment (TME) support metastatic progression. As an important component of the TME, cancer associated fibroblasts (CAFs) have largely been reported as tumor supportive. But new evidence for the high heterogeneity of the CAF compartment, including in vivo studies from our lab, showed that certain CAF subpopulations could exhibit tumor inhibitory capabilities. Interestingly, recent reports revealed that CAFs showing high expression of Immunoglobulin Superfamily containing Leucin Rich Repeat (ISLR) could show tumor inhibitory capabilities in both pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC). Yet, despite the invaluable clinical potential of tumor inhibitory CAFs, the CAF compartment in the TME of CMs is still largely under described. Using CM patient tissue and CM patient-derived cell lines established in our lab, we performed single cell RNA sequencing (scRNA-seq) to define CAF subpopulations present in the TME of CMs. We identified distinct clusters characterized by differential expression of ISLR and tumor supportive CAF marker Actin Alpha 2, Smooth Muscle (ACTA2/α-SMA). CAF subpopulations were thus defined as follows: ISLRhigh/α-SMAlow, ISLRlow/α-SMAhigh, ISLRlow/α-SMAlow. Altogether, we hypothesized that ISLRhigh/α-SMAlow CAFs could act to restrain tumor progression in the TME of CMs, as opposed to potentially tumor supportive ISLRlow/α-SMAhighCAFs. ISLRhigh and ISLRlow CAFs were obtained through fluorescence-activated cell sorting (FACS) performed on one of our patient-derived CAF cell lines represented in all defined scRNA-seq clusters. ISLRhigh CAFs were validated to express lower levels of α-SMA (ISLRhigh/α-SMAlow CAFs) compared to ISLRlow cells (ISLRlow/α-SMAhigh CAFs). This mutually exclusive expression of ISLR and α-SMA in different CAF subpopulations is in accordance with recent data in PDAC and CRC and could indicate common underlying mechanisms driving the phenotype of each subpopulation. Further in vitro and in vivo experiments are currently underway in our lab to determine the tumor supportive capabilities of both ISLRhigh/α-SMAlow and ISLRlow/α-SMAhigh CAFs. Ultimately, we expect the current study to unravel the diverse and dynamic nature of CAFs in the TME of CMs, which is still largely unknown.

Citation Format: Thomas Simon, David Buckley, Bodour Salhia. Tumor inhibitory CAFs in CNS metastases [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 3195.
Abstract 3219: Changes and intra-individual reliability in computed tomography image-assessed adiposity and skeletal muscles areas after breast cancer surgery
Authors: Aduse-Poku Livingstone , Gopireddy Dheeraj , Henandez Mauricio , Lall Chandana , Divaker Joel , Falzarano Sara , Masood Shahla , Zhang Weizhou , Datta Susmita , Bian Jiang , Cheng Ting-Yuan David
Published: June 2022

Background: Levels of adiposity and skeletal muscles are prognostic indicators in breast cancer patients; however, data are limited on temporal changes in body composition, particularly in different densities of muscles, before and after mastectomy.

Objectives: We examined the change in CT-assessed adipose tissue and different density of skeletal muscle areas using a high-resolution segmentation method in breast cancer patients before and after surgery.

Methods: This study included 50 newly-diagnosed breast cancer patients who had taken CT scans before and after surgery. We analyzed their third lumbar (L3) CT images to measure the areas of adipose tissue and five-level (from very-high-density to very-low-density) skeletal muscle components. We assessed mean and percent changes in areas using paired t-test and calculated the intra-class correlation (ICC).

Results: Patients’ mean age at diagnosis was 58.3 (SD=12.5) years. The mean duration between scans was 591 (SD=537) days. There was a significant decrease in the areas of subcutaneous adipose tissue (mean change= -36.17 cm2, 11.72% decrease, p=0.029), total adipose tissue (mean change= -45.64 cm2, 10.18% decrease, p=0.032), and very-high-density muscle (mean change= -0.37 cm2, 11.08% decrease, p=0.010). The ratio of total adipose tissue and total skeletal muscle decreased from 3.67 to 3.32 after surgery (mean change= -0.36, 9.81% decrease, p=0.061). There was a strong correlation in adipose tissues and skeletal muscle areas of pre-and post-surgery scans taken within 3 years apart (ICC=0.802 to 0.999, all p<0.05), but the correlations were attenuated in scans taken >3 years apart (ICC = -0.055 to 0.738, all p>0.05).

Conclusion: In this sample of breast cancer patients who have undergone surgery, there was a significant decrease in subcutaneous adipose tissues, total adipose tissues, and very-high-density muscle areas over time. Body composition assessed by CT images is reliable for a 3- year period involving breast cancer surgery. These findings serve as a baseline for prospective studies examining the impact of these changes on breast cancer outcomes.

Keywords: Adipose tissue, skeletal muscle, computed tomography scans, breast cancer, surgery

Citation Format: Livingstone Aduse-Poku, Dheeraj Gopireddy, Mauricio Henandez, Chandana Lall, Joel Divaker, Sara Falzarano, Shahla Masood, Weizhou Zhang, Susmita Datta, Jiang Bian, Ting-Yuan David Cheng. Changes and intra-individual reliability in computed tomography image-assessed adiposity and skeletal muscles areas after breast cancer surgery [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 3219.
Abstract 3249: Targeting polyploid giant cancer cells to overcome resistance to PARP inhibitors in ovarian cancer
Authors: Zhang Xudong , Yao Jun , Li Xiaoran , Niu Na , Liu Yan , Sood Anil K. , Liu Jinsong
Published: June 2022

PARP inhibitors (PARPi) offer major clinical benefit for patients with ovarian or other cancers, especially those with deficiencies in the homologous recombination DNA repair pathway. However, most patients with advanced cancer eventually acquire resistance to PARPi, and the mechanisms by which tumors escape PARPi therapy are still not fully understood. Here, we report that treatment with the PARPi olaparib induces the formation of polyploid giant cancer cells (PGCCs) in ovarian and breast cancer cell lines (Hey, SKOV3, MCF-7), three human high-grade serous cancer (HGSC)-derived organoids, and HGSC patient-derived xenografts. Live-cell fluorescence time-lapse tracking of ovarian cancer cells labeled with FUCCI (fluorescent ubiquitination cell-cycle indicator) or histone H2B-mCherry/α-tubulin-EGFP revealed that PGCCs primarily developed from the endoreplication of diploid cancer cells after exposure to sublethal concentrations of olaparib. PGCCs exhibited markers of senescent cells; however, they were able to escape senescence to generate mitotically competent daughter cells via budding, multipolar mitosis, and acytokinetic mitosis following olaparib withdrawal. PGCCs and derived daughter cells conferred resistance to olaparib-induced cytotoxicity, which could be blocked by the anti-progesterone drug mifepristone. Whole transcriptome analysis by RNA sequencing of ovarian cancer cell lines and ovarian cancer-derived organoids showed activation of a senescence-associated secretory phenotype with upregulated cytokines and chemokines and downregulation of MYC signaling in PGCCs. Mifepristone/olaparib combination therapy significantly mitigated tumor growth in patient-derived xenograft models with acquired resistance to olaparib. Thus, targeting of PGCCs may represent a promising approach to overcome PARPi resistance in recurrent ovarian cancer.

Citation Format: Xudong Zhang, Jun Yao, Xiaoran Li, Na Niu, Yan Liu, Anil K. Sood, Jinsong Liu. Targeting polyploid giant cancer cells to overcome resistance to PARP inhibitors in ovarian cancer [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 3249.
Abstract 3250: Microbial metabolite, Urolithin A acts as chemo-sensitizing adjuvant in conventional drug therapies via modulation of drug transporters and EMT markers
Authors: Ghosh Sweta , Singh Rajbir , Vanwinkle Zachary M. , Guo Haixun , Vemula Praveen K. , Goel Ajay , Haribabu Bodduluri , Jala Venkatakrishna Rao
Published: June 2022

Colon cancer is the third leading cause in cancer related deaths in United States of America. Chemoresistance (drug resistance) of tumors is the primary reason for the failure of chemotherapy and a major cause of mortality in colon cancer. The molecular mechanisms involved in chemoresistance or methods to chemosensitization of cancer cells to chemotherapy remain elusive. Recent studies suggest that gut microbiota and microbial metabolites play a crucial role in the development and progression of colon cancer. Urolithin A (UroA) is a gut microbial metabolite derived from ellagitannin/ellagic acid rich-diets such as pomegranate and berries. Here, we identified that UroA and its novel structural analogue (UAS03) chemosensitize 5FU resistant (5FUR) colon cancer cells to 5FU treatment. Our data suggested that combination of 5FU with UroA or UAS03 significantly reduced cell viability of 5FUR colon cancer cell lines with combination index (CI) less than 1 suggesting their synergism. Moreover, UroA or UAS03 in combination with 5FU significantly inhibited 5FUR colon cancer cell proliferation, migration as well as induced apoptosis. Mechanistically, UroA or UAS03 treatment down regulated drug transporters (MDR1, BCRP and MRP2) leading to decreased efflux of drug (e.g., 5FU) and potentially responsible for chemosensitization. Furthermore, treatment with these compounds also modulated EMT makers (e.g., downregulated Snail and β-catenin; upregulated ZO-1 and E-cadherin) to reduce EMT transition. Treatment of UroA or UAS03 in combination with 5FU significantly reduced the growth of 5FUR-tumor xenografts in NRGS mice. Taken together, utilization of UroA or UAS03 in combination with 5FU treatment may provide better therapeutic options for 5FUR colon cancer alleviation.

Citation Format: Sweta Ghosh, Rajbir Singh, Zachary M. Vanwinkle, Haixun Guo, Praveen K. Vemula, Ajay Goel, Bodduluri Haribabu, Venkatakrishna Rao Jala. Microbial metabolite, Urolithin A acts as chemo-sensitizing adjuvant in conventional drug therapies via modulation of drug transporters and EMT markers [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 3250.
Abstract 3288: The use of in vitro assays to assess and measure gene therapy immunogenicity
Authors: Schockaert Jana , Janss Thibaut J. , Lamy Juliette , Mazy Aurélie , Boelen Ellen , Lefevre Simon , Pattijn Sofie
Published: June 2022

Introducing the gene therapy product into human cells requires a vector that will deliver the gene into the cells and incorporate those genes into the gene expression mechanism in those cells. One of the issues is the induction of an unwanted immune response that can have an influence on the efficacy and potency of the treatment. Additionally, pre-existing immunity towards AAV and CRISPR can also neutralize the therapeutic effect. Comprehensive assessment of human immune responses to gene therapy candidates includes characterization of humoral and cellular immunogenicity specific for both the viral vector and the expressed transgene (protein) product before and after dose administration. In vitro assays such as dendritic cell activation and T cell proliferation assays can be used to assess this unwanted immunogenicity in an early phase. Additionally, innate assays such as dendritic cell activation assays can be used for the evaluation of potential impurities and innate response inducing contaminants. Sensitive Fluorospot assays can be used to monitor patient’ specific immunogenicity both at the T and B cell level. The advantage of the Fluorospot technology is the sensitivity to detect responses on a single cell level and measure up to 4 analytes simultaneously. For all in vitro assays, the quality and functionality of the primary cells is important to assure reproducibility and solid assay performance.

Citation Format: Jana Schockaert, Thibaut J. Janss, Juliette Lamy, Aurélie Mazy, Ellen Boelen, Simon Lefevre, Sofie Pattijn. The use of in vitro assays to assess and measure gene therapy immunogenicity [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 3288.
Abstract 331: Pre-clinical evaluation in endocrine resistant breast cancer of a novel antibody-drug conjugate targeting cell surface cleaved Amphiregulin
Authors: Lofgren Kristopher A. , Sreekumar Sreeja , Jenkins E. Charles , Ernzen Kyle J. , Kenny Paraic A.
Published: June 2022

The EGFR ligand, Amphiregulin, is a key proliferative effector of estrogen receptor signaling in breast cancer and also plays a role in other malignancies. Amphiregulin is a single-pass transmembrane protein proteolytically processed by TACE/ADAM17 to release the soluble EGFR ligand, leaving a residual transmembrane stalk that is subsequently internalized. Here, we report the development of an antibody drug conjugate, GMF-1A3-MMAE, targeting an AREG neo-epitope revealed following ADAM17-mediated cleavage. The antibody does not interact with uncleaved Amphiregulin, providing a novel means of targeting cells with high rates of Amphiregulin shedding. Using fluorescent dye conjugation, we demonstrated that the antibody is internalized by cancer cells in a manner dependent on the presence of cell surface cleaved Amphiregulin. Antibodies conjugated with monomethyl auristatin E (MMAE) were cytotoxic in vitro and induced rapid regression of established breast tumor xenografts in immunocompromised mice. We also show that these antibodies recognize the Amphiregulin neo-epitope in formalin fixed paraffin embedded tumor tissue, suggesting their utility as a companion diagnostic for patient selection. Among ER-positive (n = 88) and ER-negative (n = 50) tumors evaluated, the proportions of tumors exhibiting medium/high intensity staining with 1A3 were essentially equivalent (69.3% v 70%, respectively). These data provide proof-of-principle that cell surface proteolytic cleavage products can represent good targets for antibody drug conjugate development and highlight the potential of using this antibody drug conjugate against tumors shedding high levels of Amphiregulin.

Citation Format: Kristopher A. Lofgren, Sreeja Sreekumar, E. Charles Jenkins, Kyle J. Ernzen, Paraic A. Kenny. Pre-clinical evaluation in endocrine resistant breast cancer of a novel antibody-drug conjugate targeting cell surface cleaved Amphiregulin [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 331.
Abstract 3331: Effective multitargeted kinase inhibitors for metastatic castration-resistant prostate cancer predicted by integrative systems-based modeling
Authors: Bello Thomas , Paindelli Claudia , Diaz-Gomez Luis , Mikos Antonios , Nelson Peter , Gujral Taranjit , Dondossola Eleonora
Published: June 2022

Metastatic castration-resistant prostate cancer (CRPC) is an advanced subtype of prostate cancer (PCa) with limited therapeutic opportunities. Here, we applied an unbiased systems-based modeling approach called Kinome Regularization (KiR) to identify candidate kinase inhibitors (KIs) for the treatment of CRPC. KiR is a method of target deconvolution that harnesses the polypharmacology of KIs to identify critical kinases involved in a specific cellular activity (i.e., cell proliferation). By computationally integrating the results of a functional drug screen with prior biochemical knowledge, we generated KiR models for a panel of PCa cell lines that predict KIs effective at abrogating CRPC cell growth and proliferation. Two predicted KIs (PP121 and SC-1) showed significant inhibition of PCa cell growth and kinase-mediated growth factor signaling in vitro and in primary subcutaneous tumors. However, therapy resistance emerged in bone metastases, as identified by analysis in a 3D in vitro engineered bone mimetic environment and in tumors implanted in mouse bones. A combination of PP121 and SC-1 with standard-of-care chemotherapy overcame the resistance, enhancing both tumor response and mouse survival. These results suggest that combining multi-targeted KIs with chemotherapy may represent an effective strategy for CRPC treatment.

Overall, our findings demonstrate the application of a multidisciplinary strategy that blends bench science with machine-learning approaches for rapidly identifying KIs that result in desired phenotypic effects.

Citation Format: Thomas Bello, Claudia Paindelli, Luis Diaz-Gomez, Antonios Mikos, Peter Nelson, Taranjit Gujral, Eleonora Dondossola. Effective multitargeted kinase inhibitors for metastatic castration-resistant prostate cancer predicted by integrative systems-based modeling [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 3331.
Abstract 3340: Small molecule mediated stabilization of PP2A modulates the homologous recombination pathway and potentiates DNA damage-induced cell death
Authors: Avelar Rita A. , Armstrong Amy , Narla Goutham , DiFeo Analisa
Published: June 2022

High Grade Serous Carcinoma (HGSC) is the most lethal ovarian cancer subtype and accounts for approximately 60% of all ovarian tumors. Despite recent advances in drug development and increased understanding of genetic alterations that drive HGSC progression, mortality has not declined, highlighting the need for novel therapies. PARP inhibitors (PARPi) have become the mainstay of HGSC targeted therapy research given that these tumors are driven by a high degree of genomic instability resulting from the combination of fast DNA replication rates and numerous defects in the DNA-damage response (DDR) pathway. Nonetheless, only ~25% of these patients initially respond to treatment and a significant percentage eventually relapses with resistant disease. Here, we discovered that a Small Molecule Activator of Protein Phosphatase 2A (PP2A) (SMAP-061) induces apoptosis in both established and patient-derived HGSC cell lines as well as in genetically distinct Patient-Derived Xenograft (PDX) mouse models. Interestingly, we also uncovered that several genes that make-up the heterotrimer PP2A tumor suppressor protein are heterozygously lost in more than 95% of HGSC tumors, second only to p53. Mechanistically, we show that stabilization of PP2A protein by SMAP-061 inhibits the Homologous Recombination (HR) pathway via the direct inhibition of RAD51, ultimately leading to chronic accumulation of DNA damage and thus programmed cell death. Furthermore, we found that SMAP-061’s ability to inhibit HR potentiated the effects of PARP inhibition and resulted in synergistic cell death in both HR proficient and deficient models. These studies emphasize the potential of PP2A activators to expand the patient population that can benefit from PARPi therapies and possibly overcome PARPi resistance. In sum, our data highlights a new role of PP2A in regulating the DDR pathway in HGSC and supports the use of SMAPs in both HR proficient and deficient HGSC tumors.

Citation Format: Rita A. Avelar, Amy Armstrong, Goutham Narla, Analisa DiFeo. Small molecule mediated stabilization of PP2A modulates the homologous recombination pathway and potentiates DNA damage-induced cell death [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 3340.
Abstract 342: Development and characterization of a novel anti-CLDN6 antibody drug conjugate for the treatment of CLDN6 positive cancers
Authors: McDermott Martina S. , Gong Ke Wei , O'Brien Neil A. , Conklin Dylan , Hoffstrom Benjamin , Lu Ming , Zhang Jun , Luo Tong , Jia Weiping , Hong Jenny J. , Chau Kevin , Davenport Simon , Press Michael F. , Handly-Santana Abram , Brugge Joan S. , Drapkin Ronny , Glaspy John A. , Presta Leonard , Slamon Dennis J.
Published: June 2022

Background: Claudin 6 (CLDN6), a member of the claudin family of tight junction proteins, is expressed at high levels in multiple human malignancies including ovarian and endometrial cancers. Conversely it has little or no expression in normal tissues. This expression profile makes CLDN6 an ideal target for development of potential therapeutic antibody-drug conjugates (ADCs). This study describes the generation and preclinical characterization of an anti-CLDN6 ADC consisting of a humanized anti-CLDN6 monoclonal antibody coupled to MMAE via a cleavable linker.

Materials and Methods: A fully humanized anti-CLDN6 antibody was initially characterized for binding affinity, selectivity/specificity, internalization characteristics and in vivo efficacy. It was then conjugated to MMAE resulting in the potential therapeutic anti-CLDN6 ADC. The anti-tumor efficacy of the ADC was next assessed for anti-tumor efficacy in CLDN6 positive (CLDN6+) and negative (CLDN6-) xenografts and patient-derived xenograft (PDX) models of specific cancers including ovarian and endometrial cancer.

Results: Selective binding of the ADC to CLDN6, without cross reactivity to other CLDN family members CLDN3, CLDN4 and CLDN9, was confirmed in human cancer cell lines and cells engineered to overexpress each protein. The ADC was also shown to rapidly internalize in CLDN6+ cells. Robust tumor regressions following treatment with the ADC were observed in CLDN6+ xenografts that were sustained beyond the treatment window. Conversely, there was limited to no activity of the ADC in CLDN6- xenografts models. In addition, the prevalence of CLDN6 expression in human ovarian and endometrial cancers was assessed by IHC in tissue microarrays and found to be 28% (ovarian epithelial carcinomas) and 11% (endometrial carcinomas), respectively.

Discussion: Overall, these data suggest that our anti-CLDN6 ADC may be a promising treatment for patients with CLDN6+ tumors and it is currently in Phase I clinical testing.

Citation Format: Martina S. McDermott, Ke Wei Gong, Neil A. O'Brien, Dylan Conklin, Benjamin Hoffstrom, Ming Lu, Jun Zhang, Tong Luo, Weiping Jia, Jenny J. Hong, Kevin Chau, Simon Davenport, Michael F. Press, Abram Handly-Santana, Joan S. Brugge, Ronny Drapkin, John A. Glaspy, Leonard Presta, Dennis J. Slamon. Development and characterization of a novel anti-CLDN6 antibody drug conjugate for the treatment of CLDN6 positive cancers [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 342.
Abstract 345: Development of a novel and direct peptide Helicon࣪ inhibitor of β-catenin-TCF interaction with in vivo validation of transcriptional modulation and anti-tumor activity
Authors: Si Yaguang , White Brian , Cappucci Sarah , Ramirez Jessica , Ponthier Charles , Visness Erica , Ling Kevin , Du Peicheng , Choi Minjung , Thomson Ty , Alfaro-Lopez Josue , Beerepoot Pieter , Agarwal Sorabh , Ortet Paula , Lech Miroslaw , Li Zhi , Olokpa Voké , Jewett Ivan , La Daniel , Yu Lihua , McGee John , Tremblay Martin , Hurov Jonathan , Verdine Greg
Published: June 2022

Wnt pathway mutations can be found in nearly all colorectal cancers and a significant number of cancers of the liver, breast, prostate, endometrium and lung, among others. β-catenin is a key signaling hub in the Wnt pathway. Activated nuclear β-catenin forms a complex with TCF/LEF (T-cell factor/lymphoid enhancer binding factor) and drives the transcription of genes essential for cancer cell proliferation, survival, and metabolism. Blocking the β-catenin-TCF/LEF interaction offers an attractive therapeutic strategy to treat a large population of patients with WNT pathway mutations. However, β-catenin is considered as an “undruggable” target because it lacks tractable hydrophobic pockets for small-molecule binding. To address this challenge, we have successfully discovered and developed Helicon࣪ peptides targeting the β-catenin-TCF/LEF interaction. Using this novel modality, linear peptides are locked in a helical structure via a proprietary tethering technology to yield macrocyclic stapled peptides.

Our helicons exhibit picomolar β-catenin binding affinity and nanomolar anti-proliferative cell-based activity. Cells treated with lead helicons followed by unbiased RNAseq GSEA indicate that the WNT/β-catenin pathways represent the top downregulated transcriptional signatures after treatment. Furthermore, PRISM cell line screening of more than 900 cell lines reveals that the most sensitive lines are enriched with APC and CTNNB1 mutations. Mechanistically, helicon treatment reduces nuclear β-catenin and alters the levels of cyclin D2/D3 and p27 in the sensitive lines. In vivo, our helicons display favorable pharmacokinetic properties, broad tissue distribution, and potent anti-tumor effects. Taken together, our data demonstrate β-catenin-targeting Helicon࣪ peptides have the potential to become the next-generation class of therapeutics to treat cancers with defined genetic mutations.

Citation Format: Yaguang Si, Brian White, Sarah Cappucci, Jessica Ramirez, Charles Ponthier, Erica Visness, Kevin Ling, Peicheng Du, Minjung Choi, Ty Thomson, Josue Alfaro-Lopez, Pieter Beerepoot, Sorabh Agarwal, Paula Ortet, Miroslaw Lech, Zhi Li, Voké Olokpa, Ivan Jewett, Daniel La, Lihua Yu, John McGee, Martin Tremblay, Jonathan Hurov, Greg Verdine. Development of a novel and direct peptide Helicon࣪ inhibitor of β-catenin-TCF interaction with in vivo validation of transcriptional modulation and anti-tumor activity [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 345.