bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2974: Loss of TSC1 or TSC2 drives lineage infidelity and hamartoma formation in a renal organoid model of angiomyolipoma
Authors: Pietrobon Adam , Delaney Sean P. , Doré Carole , Yockell-Lelievre Julien , Julian Lisa , Stanford William L.
Published: July 2021

Renal angiomyolipomas (R-AMLs) are hamartomatous kidney tumors which stain positively for markers of adipocytic, vascular, smooth muscle, and melanocytic lineages. These lesions possess loss of function mutations in either TSC1 or TSC2, which are canonical negative regulators of mTORC1 signaling. To date, there exists no in vitro or in vivo model which faithfully recapitulates the architectural and molecular complexity of R-AMLs. Considering these lesions can be detected very early in life- even congenitally- we hypothesized they arise as a consequence of aberrant tissue development. To test this hypothesis, we generated TSC1-/- and TSC2-/- mutants in four human pluripotent stem cell (hPSC) backgrounds using CRISPR/Cas9 genome engineering. Wild type hPSCs differentiated into renal organoids express markers of the glomerulus, proximal and distal tubules, in a topology that resembles human nephron patterning. Remarkably, wild type renal organoids downregulate mTORC1 signaling compared to adjacent undifferentiated cells. In contrast, both TSC1-/- and TSC2-/- hPSCs exhibit substantial lineage infidelity upon differentiation, staining positively for adipocytic and melanocytic markers which are absent in matched wild type controls. Additionally, knockout lines formed nodular growths with disorganized architecture, resembling the hamartomatous organization of R-AML lesions. These lesions exhibit hyperactive mTORC1 signaling, consistent with human pathology. Together, these data suggest three primary findings: loss of TSC1/2 drives lineage infidelity; TSC1/2 may be required for architectural organization of the kidney parenchyma; and a developmental approach to R-AML modelling may best recapitulate the human disease.

Citation Format: Adam Pietrobon, Sean P. Delaney, Carole Doré, Julien Yockell-Lelievre, Lisa Julian, William L. Stanford. Loss of TSC1 or TSC2 drives lineage infidelity and hamartoma formation in a renal organoid model of angiomyolipoma [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 2974.
Abstract 3004: Establishment of KRAS G12C mutant patient-derived xenograft (PDX) models for pre-clinical evaluation of KRAS G12C targeted anticancer therapy
Authors: Shi Wenting , Tang Xuzhen , Zhang Jingying , Kuang Xinhong , Zhang Yan , Wang Jingjing , Gu Qingyang , Ji Qunsheng
Published: July 2021

KRAS is one of the most prevalent oncogenes in human cancers. The G12C mutation results in an amino acid substitution at position 12 in KRAS, from a glycine (G) to a cysteine (C), lead to accretion of GTP-bound activated state KRAS and activation of downstream signaling pathways. In non-small cell lung cancer (NSCLC), KRAS p.G12C is the most common mutation, comprising nearly half of all KRAS mutations. Recent breakthrough work led to the development of selective inhibitors targeting the KRAS G12C mutation, which have shown promise in early clinical trials. However, the therapeutic benefit of targeted therapies can be impaired by intrinsic or acquired resistance mechanisms, suggesting an urgent need of combinatorial strategies to overcome the resistance to drugs targeting KRAS G12C.Here we report on the establishment of a panel of KRAS G12C mutant PDX models, encompass non-small cell lung cancer, colorectal cancer and gastric cancer. The activity of AMG510, a selective KRAS G12C inhibitors in clinical trial, was characterized in these PDX models. Data showed that the validated KRAS G12C mutant models display a range of sensitivity to single treatment of AMG510. Given that in clinic only 50% of NSCLC patients with KRAS G12C mutation would benefit from KRAS G12C targeted inhibitors, in order to provide an insights for clinical therapeutic strategy, we investigated the combinatorial strategies in NSCLC PDX models: (1) AMG510 combined with Opdivo in KRAS G12C/CD274 amplification models in hPBMC humanized mice; (2) AMG510 combined with EGFR inhibitor in KRAS G12C/EGFR overexpression models. The possible mechanisms of combination effect were discussed. Altogether, the panel of KRAS G12C mutant PDX models are valuable for the pre-clinical evaluation of KRAS G12C targeted anti-cancer therapies.

Citation Format: Wenting Shi, Xuzhen Tang, Jingying Zhang, Xinhong Kuang, Yan Zhang, Jingjing Wang, Qingyang Gu, Qunsheng Ji. Establishment of KRAS G12C mutant patient-derived xenograft (PDX) models for pre-clinical evaluation of KRAS G12C targeted anticancer therapy [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 3004.
Abstract 3035: Identifying potential druggable targets for synovial sarcoma using comparative RNA-seq analysis
Authors: Vasquez Yvonne A. , Pfeil Jacob , Mueller Letitia , Beale Holly , Lyle Alfred G. , Sanders Lauren , Learned Katrina , Kephart Ellen , van den Bout Anouk , Cheney Allison , Hosseinzadeh Sahar , Bjork Isabel , Salama Sofie R. , Vaske Olena
Published: July 2021

Synovial sarcoma (SS) is an aggressive soft-tissue malignancy, accounting for 10% of all soft-tissue sarcomas. These tumors can occur at any age but most often affect young adults and adolescents, developing deep in the distal extremities. The prognosis of SS tumors is poor, with a 5-year survival rate of 36-76%, a high rate of metastasis and few treatments. The purpose of this study was to identify novel overexpressed oncogenes that could serve as druggable targets for treating synovial sarcoma patients. We compared the RNA-Seq expression profiles of a cohort of 36 synovial sarcomas to our compendium of RNA-Seq expression data from 12,236 tumor samples (treehousegenomics.ucsc.edu) from pediatric and adult cancer patients. In comparing gene expression in the synovial sarcoma cohort samples against the compendium samples, gene expression outliers were defined as having expression above the gene-specific outlier threshold as defined by the Tukey's outlier method. Among the overexpression outliers, pathway enrichment analysis was used to identify common and druggable pathways, with implications for potential therapeutics for patients with SS. Our analysis identified the overexpression of members of the Sonic Hedgehog pathway in the majority of synovial sarcoma samples. For example, GLI1 expression exceeded the outlier threshold in 35 out of 36 samples. This pathway can be targeted by available small molecule inhibitors. Ongoing work focuses on evaluating the role of Sonic Hedgehog signaling in the pathogenesis of SS using pharmacological inhibition, CRISPRi studies in cell line models of the disease and nanopore sequencing. We currently have 4 patient-derived synovial sarcoma cell lines (HSSY-II, SYO-1, YAMATO, and ASKA) that we can grow in both adherent conditions and in 3D cell culture as sarcospheres. We detected the expression of the SYT-SSX fusion transcript in each of the cell lines by RT-PCR to confirm the cell lines maintained expression of the pathogenic fusion. This work has implications for using comparative tumor RNA-seq derived gene expression data for nominating novel druggable targets specific to synovial sarcoma tumors.

Citation Format: Yvonne A. Vasquez, Jacob Pfeil, Letitia Mueller, Holly Beale, Alfred G. Lyle, Lauren Sanders, Katrina Learned, Ellen Kephart, Anouk van den Bout, Allison Cheney, Sahar Hosseinzadeh, Isabel Bjork, Sofie R. Salama, Olena Vaske. Identifying potential druggable targets for synovial sarcoma using comparative RNA-seq analysis [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 3035.
Abstract 3041: ASPSCR1-TFE3 gene fusion in transgenic mouse as a new pediatric kidney cancer model
Authors: Alasonyalilar-Demirer Aylin , Miller James L. , Miller Erin E. , Gleba Justyna J. , Tun Han W. , Copland John A.
Published: July 2021

A novel genetically engineered mouse (GEM) model for pediatric kidney cancer expressing proximal tubule specific ASPSCR1 :TFE3 fusion gene has been developed. The Rosa26 knock-in ASPSCR1 :TFE3 mouse was crossed with SGLT2-Cre mouse that expresses Cre recombinase in the proximal tubules of the kidney. The ASPSCR1 :TFE3 fusion gene retains the DNA binding and activation domain of TFE3, whereas the N-terminal region of TFE3 is replaced with ASPSCR1 sequences. ASPSCR1 :TFE3 is known to up-regulate the Met receptor tyrosine kinase gene and induce oncogenic phenotypes such as uncontrolled cell proliferation, invasion, and metastasis. However, the molecular roles of ASPSCR1 :TFE3 are poorly understood and no standard of care (SOC) exists for pediatric TFE3 fusion renal cell carcinoma (RCC). Ultrasound (US) and histological analyses of the kidneys of 5, 6 and 7 month old mice revealed bilateral cysts and tumors, while 6-week-old mouse kidney appears normal. At gross inspection, 5-month-old mouse had cysts with a diameter of 1-2 mm while 7-month-old mouse had a polycystic kidney and in the end stage disease. Histopathology revealed neoplastic changes in initial stages in the 5-month-old mouse whereas the neoplasia was predominantly a papillary cystic renal carcinoma; solid tumor formations were observed in the 7-month-old mouse. Neoplastic cells seen in within the blood vessel walls of the 7-month-old mouse indicated future metastasis. This new TFE3 fusion GEM will provide an avenue to understand molecular events of tumorigenesis and progression as well as a model for testing novel therapeutics with survival benefit for a cancer with no current SOC.

Citation Format: Aylin Alasonyalilar-Demirer, James L. Miller, Erin E. Miller, Justyna J. Gleba, Han W. Tun, John A. Copland. ASPSCR1-TFE3 gene fusion in transgenic mouse as a new pediatric kidney cancer model [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 3041.
Abstract 3051: On the origin of pediatric leukemia: studying somatic mutations in human blood
Authors: Brandsma Arianne
Published: July 2021

The acquisition of oncogenic mutations with age is believed to be rate limiting for carcinogenesis and aging is the biggest risk factor for cancer. However, children can also develop cancers even though their cells are less damaged by age. Here, we studied the cellular origin and time of onset during development of pediatric acute myeloid leukemia (pAML) to increase our understanding of why children can get cancer. We compared by whole genome sequencing individual hematopoietic stem and progenitor cells (HSPCs) and pAML blasts of the same leukemic bone marrow as well as nonmatching healthy HSPCs. We found that the number of clonal mutations in the majority of pAML cases was increased and mutational process contribution altered as compared to normal HSPCs, showing activity of specific processes that are either unique to fetal development or normally not observed in healthy HSPCs. These observations could be explained by altered selection dynamics early in life. Surprisingly, pAML cases with an overall mutation burden comparable to age matched healthy HSPCs showed worse overall and event-free survival. Identifying shared somatic mutations between individual HSPC clones and pAML using single-cell genotyping allowed us to pinpoint the developmental lineage that gave rise to the pAML. Our study provides new insights into the etiology of pAML and underscores the clinical potential of whole genome sequencing in pediatric leukemia.

Citation Format: Arianne Brandsma. On the origin of pediatric leukemia: studying somatic mutations in human blood [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 3051.
Abstract 3089: Study of HOX genes in the stem cell origin of CRC
Authors: Osmond Brian , Facey Caroline O. , Opdenaker Lynn M. , Boman Bruce M.
Published: July 2021

Our goal is to ascertain how HOX-regulatory mechanisms play a role in the SC origin of colorectal cancer (CRC). While HOX genes encode master regulatory transcription factors that regulate stem cells (SCs), their role in cancer etiology is under-studied. We and others find that HOX genes are aberrantly expressed in CRC. We previously reported that HOXA4 and HOXD10 along with aldehyde dehydrogenase (ALDH) and the other components of the retinoic acid (RA) signaling pathway are selectively expressed in human colon SCs. That RA signaling regulates HOX expression indicates that RA regulation of HOX gene expression mainly occurs through ALDH+ SCs in the colon. We hypothesize that specific HOX genes regulate growth and differentiation of SCs in human colon and HOX gene dysregulation leads to decreased differentiation and SC overpopulation. Indeed, our bioinformatics analysis shows that many HOX genes are overexpressed in CRCs, which parallels the overpopulation of ALDH+ SC that occurs during CRC tumorigenesis. We are using immunohistochemical mapping to measure proportions of SCs and HOX-positive cells in normal and malignant human colon tissues. We find that specific HOX proteins have increased expression levels in malignant colon tissues. In addition, immunofluorescence co-staining of human colon tissues is used to validate HOX gene expression in ALDH+ SCs. We are also evaluating the effects of all-trans-RA (ATRA) on HOX gene expression and on cellular proliferation and differentiation using CRC cell lines. Our results show that ATRA treatment of HT29 CRC cells leads to reduced proliferation and decreased expression of select HOX genes. These results indicate that HOX genes like HOXA4, HOXA9, and HOXD10, are expressed colonic SCs, and are regulated by RA signaling. Overall, the overexpression of HOX genes in cancer SCs appears to contribute to the SC origin of CRC.

Citation Format: Brian Osmond, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. Study of HOX genes in the stem cell origin of CRC [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 3089.
Abstract 3090: Targeting WNT and retinoid signaling pathways to promote differentiation of cancer stem cells along the neuroendocrine lineage in CRC
Authors: Facey Caroline O. , Hunsu Victoria O. , Osmond Brian T. , Opdenaker Lynn M. , Boman Bruce M.
Published: July 2021

Since APC mutations lead to overpopulation of ALDH+ stem cells (SCs) in colorectal cancers (CRCs) and that ALDH is a key component of the retinoic acid (RA) signaling pathway, the hypothesis was developed that human CRC evolves due to an imbalance between WNT and RA signaling. Indeed, we previously discovered that progressive overpopulation of ALDH+ SCs occurs in normal, mutant, adenomatous, and malignant colon crypt tissues, which coincided with the change in the APC mutation zygosity state in familial adenomatous polyposis (FAP) patients. Therefore, the SC overpopulation that drives development of CRC appears to result from dysregulated WNT signaling, of which APC is a negative regulator. Normal colonic SC differentiation is feedback regulated by neuroendocrine cells (NECs) that reside adjacent to SCs in the bottom of the colon crypt. Our goal was to determine if APC mutation and subsequent dysregulated WNT signaling contributes to the inability of SCs to differentiate into NECs, leading to overpopulation of cancer SCs (CSCs) during tumor development. Although RA signaling is altered in CRC, we still found that all- trans-retinoic acid (ATRA) could reduce the number of ALDH+ CSCs in several CRC cell lines. We also found that administering ATRA promotes differentiation of CSCs along the neuroendocrine lineage. Moreover, induction of wild-type (wt)-APC expression in HT29 CRC cells led to increased expression of several NEC markers including CGA, GLP2R, NSE, SSTR1; and decreased WNT/β-catenin signaling when tested via TCF reporter assay. Inducing wt-APC also led to reduced β-catenin and change in protein expression of several proteins known to be targeted by WNT signaling including c-MYC, c-JUN, MET, and CD44. Our preliminary results also show that inducing wt-APC expression increases the number of GLPR2+ NECs. Thus, a link between WNT and RA signaling provides a possible mechanism that might explain how mutant APC leads to decreased maturation of ALDH+ SCs along the NEC lineage and an increased the number of ALDH+ SCs. Therefore, discovering ways to increase differentiation of SCs along the NEC lineage in vivo might lead to new more effective treatment strategies for CRC patients.

Citation Format: Caroline O. Facey, Victoria O. Hunsu, Brian T. Osmond, Lynn M. Opdenaker, Bruce M. Boman. Targeting WNT and retinoid signaling pathways to promote differentiation of cancer stem cells along the neuroendocrine lineage in CRC [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 3090.
Abstract 3092: Effect of cancer stem cell-specific progerin expression on metastatic potential
Authors: Morgenroth Philipp , Söhnchen Christoph , Spitschak Alf , Puetzer Brigitte M. , Logotheti Stella
Published: July 2021

Metastasis signals the final and treatment-refractory stage of cancer disease. Formation of metastases is fueled by a small cell subpopulation within the tumor, the so-called cancer stem cells (CSCs), which have the ability to rejuvenate the heterogeneous tumor population and support self-renewal, survival and tumor cell heterogeneity. Stemness is a cell property rather than a distinct subpopulation of the tumor structure. In this respect, even if CSCs are specifically targeted to be killed, their surrounding cells dedifferentiate to replace assaulted CSCs. Herein, we sought to target CSCs with progerin, a truncated form of lamin A, which causes the Hutchinson-Gilford childhood progeria syndrome, a rare genetic disease of accelerated aging. We combined a promoter responsive to two stemness factors (Yamanaka factors) with the progerin gene and produced a lentiviral construct, where progerin is set under the transcriptional control of stemness cascades. Stable transduction of cancer cells with this construct establishes a dynamic gene modification of the tumor cell population, where all tumor cells carry the progerin gene, but only the ones where the levels of stemness factors are initially high or secondarily upregulated are able to express it. This way, stemness programs are transcriptionally linked with a gene that, when expressed in normal stem cells, causes premature exhaustion of adult stem cells, inhibits the regenerative capacity of tissues and accelerates senescence. We monitor the effects of CSC-specific progerin expression on the CSC reservoir and the tumor cell population dynamics over time, in comparison with CSC-specific expression of its wild-type laminA counterpart, as well as non CSC-specific progerin expression. We further examine how targeting the CSC turnover within the tumor population projects into metastasis-initiating processes, such as cell invasion, migration, epithelial-mesenchymal transition, and tumor metabolism. Our approach provides a model for addressing the potential crosstalk between stemness and senescence in the context of CSCs and holds promise to translate the findings into anti-metastatic therapeutic solutions.

Funding: This project is funded by FORUN (889040) and P.M. has a scholarship from the German Cancer Aid (70114135).

Citation Format: Philipp Morgenroth, Christoph Söhnchen, Alf Spitschak, Brigitte M. Puetzer, Stella Logotheti. Effect of cancer stem cell-specific progerin expression on metastatic potential [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 3092.
Abstract 3095: Targeting ALDH1A1 and regulatory networks that support stemness in ovarian cancer cells
Authors: Muralikrishnan Vaishnavi , Nephew Kenneth P. , Hurley Thomas D.
Published: July 2021

Epithelial ovarian cancer (OC) is the deadliest amongst gynecologic cancers with the majority of deaths caused by cancer recurrence. A new paradigm explaining tumor relapse involves the persistence of cancer stem cells after chemotherapy. OC stem cells (OCSCs) are hypothesized to be largely responsible for the emergence of chemoresistant tumors, and we have previously shown that OCSCs contribute to recurrent, drug resistant high-grade serous OC (HGSOC) using ALDH (aldehyde dehydrogenase) activity as a robust functional marker to identify OCSCs. Of the ALDH isoforms, ALDH1A1 is an intracellular enzyme that oxidizes toxic aldehydes to carboxylic acids and plays a role in controlling cell differentiation pathways. High levels of ALDH1A1 expression have been associated with poor outcome in OC patients. Our group and others have demonstrated that OCSCs with high expression of ALDH1A1 have a greater ability to initiate ovarian tumors in vivo. However, the mechanism by which ALDH1A1 maintains stemness phenotype remains poorly understood. In the current study, we hypothesized that ALDH1A1 upregulation in OCSCs was associated with genetic changes that mediate the cellular signals needed for survival. To test this hypothesis, we used a novel ALDH1A1-specific small molecule inhibitor named compound 974. Treatment of HGSOC cell lines with compound 974 reduced ALDH enzyme activity (p<0.01) and inhibited stem-like properties including spheroid formation (p<0.01) and clonogenic survival (p<0.05). To further examine the effect of compound 974 to inhibit ALDH1A1 and consequently tumor initiation, mice were injected with 106, 105 and 104 OVCAR3 cells treated in vitro with compound 974 (5µM for 48h). Tumor initiation was delayed by 974 treatment compared to vehicle-treated group. Extreme limiting dilution analysis revealed that compound 974 reduced CSC frequency compared to control. Transcriptomic sequencing of ovarian cancer cells treated with compound 974 revealed significant inhibition of genes and pathways associated with stemness (NF-κB, KLF4, FZD7), chemoresistance (ABCB1) and senescence pathway genes (p15, p21). Furthermore, key genes associated with senescence associated secretory phenotype (SASP) such as IL6, IL8 and IL1α were significantly inhibited. Taken together, these data warrant further investigation into the mechanism of ALDH1A1 inhibition and loss of stemness in HGSOC.

Citation Format: Vaishnavi Muralikrishnan, Kenneth P. Nephew, Thomas D. Hurley. Targeting ALDH1A1 and regulatory networks that support stemness in ovarian cancer cells [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 3095.
Abstract 3104: EMT and CSC Enrichment Induced by ERK Inhibition in NSCLC
Authors: Cai Shurui , Bai Xuetao , Li Na , Banerjee Ananya , Lavudi Kousalya , Pal Tejinder
Published: July 2021

The activation of Mitogen-activated protein kinase pathway (MAPK/ERK pathway) has been found to play an important role in many cancers including lung cancer, and multiple inhibitors targeting this pathway have been developed. Several ERK inhibitors (ERKi, e.g., Ulixertinib, SCH772984) have been shown to be active against different cancers harboring RAS, BRAF, or MEK mutations, as well as cancers with developed BRAF/MEK inhibitor resistance. Nevertheless, sustained inhibition of ERK can lead to resistance and is likely to cause tumor relapse as well. Studies over the years have shown that a small portion of cells within tumors, which can reproduce themselves and sustain cancer, being called “cancer stem cells (CSCs)”, is the cause of tumor initiation and progression. Many reports have implicated the association of Epithelial-to-Mesenchymal Transition (EMT) program with CSCs and identified EMT-transcription factors (TFs) as CSC regulators. Here, we demonstrated that ERKi treatment induces EMT in non-small cell lung cancer cells (NSCLC), probably by inducing Slug expression. We also found that ERKi treatment can enrich the cancer stem cell (CSC) like population in NSCLC cells, evidenced by increased ALDH+ cells, enhanced in vitro sphere formation capability, and enhanced tumorigenicity in immunodeficient mice. Moreover, in NSCLC cells, we proved ERK knockdown or ERKi treatment can reduce C/EBPα expression, which is believed to be a master epithelial “gatekeeper” whose expression is required to prevent unwarranted mesenchymal transition. Our findings reveal a novel mechanism underlying the tumor relapse after ERKi treatment in NSCLC, and ultimately will help us improve current NSCLC therapeutic methods.

Citation Format: Shurui Cai, Xuetao Bai, Na Li, Ananya Banerjee, Kousalya Lavudi, Tejinder Pal. EMT and CSC Enrichment Induced by ERK Inhibition in NSCLC [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 3104.
Abstract 3108: Human telomerase reverse transcriptase regulates stemness of pancreatic cancer
Authors: Ye Juanjuan , Yamakawa Keiko , Narusawa Yuko , Matsuda Yoko
Published: July 2021

Backgrounds: Human telomerase reverse transcriptase (hTERT) plays a key role on immortalization of cancer cells. In the present study, we clarified the effect of small interfering RNA (siRNA) targeting hTERT in pancreatic cancer cell lines.

Methods: Two sequences of siRNA targeting hTERT and negative control siRNA were transfected into PANC-1 and MIA PaCa-2 human pancreatic cancer cell lines. Cell proliferation (WST-8 cell counting assay), stemness (sphere formation) and migration (scratch assay) were analyzed using hTERT-siRNA transfected cells and control-siRNA transfected cells. The expression levels of CDK1, SOX9 and nestin were analyzed by real time PCR.

Results: As compared to control-siRNA, hTERT-siRNA suppressed cell proliferation and stemness of pancreatic cancer cell lines. Cell migration did not show any difference between hTERT-siRNA and control-siRNA transfected cells. The expression levels of CDK1, SOX9 and nestin in hTERT-siRNA transfected cells were lower than those in control-siRNA transfected cells.

Conclusion: The present study indicates that inhibition of hTERT in pancreatic cancer cells suppressed stemness via alteration of the expression levels of CDK1, SOX9 and nestin.

Citation Format: Juanjuan Ye, Keiko Yamakawa, Yuko Narusawa, Yoko Matsuda. Human telomerase reverse transcriptase regulates stemness of pancreatic cancer [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 3108.
Abstract 314: Highly polymeric grape seed proanthocyanidins: A call for establishing the safe dose
Authors: Thilakarathna Wasitha , Rupasinghe H.P. Vasantha
Published: July 2021

Introduction: Proanthocyanidins (PC) are the oligomers and polymers of flavan-3-ols, especially catechin and epicatechin. PC in general are proven to be beneficial against cancers and widely available as over the counter (OTC) supplements. However, role of highly polymeric PC (HP-PC) on human cancer prevention remain unknown. Therefore, the effects of HP-PC supplementation on hepatic pathology in the context of hepatocellular carcinoma (HCC) were evaluated. HP-PC supplementation on mice gut microbiota was also evaluated considering the association of dysbiosis-mediated inflammation with HCC.

Methodology: Crude PC was extracted from grape seeds (GS) using 70% acetone (in water) as the extraction solvent. PC mixture was purified and fractionated by flash chromatography. Monomeric and oligomeric PC was eluted with 100% ethanol and the HP-PC was collected by eluting with 70% acetone. C57BL/6 mice (49 - 56 days old) were supplemented with HP-PC (200 mg/kg body weight per day) for 14 days and sacrificed to collect livers and blood serum. Feces samples were collected from mice colon to evaluate the composition of gut microbiota using the 16S rRNA sequencing technology. Mice liver histopathology was examined by staining with hematoxylin and eosin. The level of alanine aminotransferase (ALT) in mice blood serum was measured as a marker of liver damage.

Results: Evaluation of liver histopathology revealed signs of steatosis in the mice supplemented with HP-PC. Interestingly, rare foci of monocytic lobular inflammation was visible in the hepatic tissues of the mice supplemented with HP-PC. The alpha diversity of the mice gut microbiome was evaluated in terms of Shannon Index, Pielou's evenness, Faith's phylogenic diversity, and observed operational taxonomic units. A significant difference was not observed for the alpha diversity between the HP-PC supplemented and control group mice gut microbiota. The levels of ALT in the mice blood serum were not significantly different between HP-PC supplemented and control group mice.

Conclusions: The preliminary experimental data suggests the potential of HP-PC to promote steatosis and hepatic inflammation. However, further studies must be conducted to understand potential harmful effect of HP-PC (if any) in hepatic toxicity and safer dose to use for potential health benefits. Furthermore, the results of this study emphasize the critical importance of establishing the safe dose of HP-PC or any OTC supplements.

Citation Format: Wasitha Thilakarathna, H.P. Vasantha Rupasinghe. Highly polymeric grape seed proanthocyanidins: A call for establishing the safe dose [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 314.
Abstract 3144: Intratumor heterogeneity quantification using 3D segmentation of organoids co-culture systems
Authors: Mukashyaka Patience , Noorbakhsh Javad , Kumar Pooja , Courtois Elise , Anczukow Olga , Robson Paul , Liu Edson , Chuang Jeffrey
Published: July 2021

Intratumoral heterogeneity is an essential aspect of cancer biology, as the diversity of cancer cells can increase challenges of designing effective treatment. Cancer cells within the same tumor evolve and compete for shared resources and may also respond differently to drug treatments. In previous work, by closely analyzing triple negative breast cancer xenografts, we have identified a system of closely-related subclonal populations within a tumor that respond differentially to chemotherapy. To explore the dynamics of competition and cooperation between these subclones, we derived cell lines from each and tagged them with fluorescent markers. We then co-cultured these cell lines in 3D organoids and tracked them using Opera Phenix high content screening system (3D confocal fluorescence microscopy). To determine the number of cells and the location of each subclones within organoids, we developed a 3D cell segmentation pipeline. Our pipeline relies on a two-step process where organoids are segmented first and then individual cells are detected within each organoid. Preliminary analysis of hundreds of organoids suggests that the two cell types preserve their relative ratio and sensitivity to treatment regardless of organoid size, suggesting that the system is in a state of ecological coexistence. We will present results on cellular mixing during growth and treatment and the implications for adaptive therapy ecologies in tumors.

Citation Format: Patience Mukashyaka, Javad Noorbakhsh, Pooja Kumar, Elise Courtois, Olga Anczukow, Paul Robson, Edson Liu, Jeffrey Chuang. Intratumor heterogeneity quantification using 3D segmentation of organoids co-culture systems [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 3144.
Abstract 3149: Investigation of the potential therapeutic value of CAF-derived milk fat globule-EGF factor 8 protein (MFG-E8) in esophageal squamous cell carcinoma
Authors: Liu Beilei
Published: July 2021

The physiological state of the tumor microenvironment (TME) is closely linked with tumorigenesis. RNA sequencing data from our lab showed a broad range of dysregulated genes between ESCC isolated CAFs and paired peripheral blood isolated Fbcs. Among the 2347 up-regulated genes, we found the GO analysis is many focused in extracellular matrix and the top 4 GO terms enriched in cell development and adhesion. Due to this, enriched CAF-secreted extarcellular proteins attracts our attention. MFG-E8 is highly expressed secreted protein in CAFs but low expressed in tumor cells. TCGA database revealed high expression of MFG-E8 is associated with poor overall survival and disease-free survival. In addition, ESCC cell line k410 and K180 with MFG-E8 protein showed a strong ability in cell migration, cell invasion and apoptosis inhibition compared with the PBS group. The further western blot demonstrated MFG-E8 promoted ESCC cell migration and invasion by triggering EMT, and activated an altered akt/stat3 signaling pathway. Moreover, Anti-MFG-E8 antibody effectively reduced tumor size, especially combined with cisplatin. Therefore, MFG-E8 may act as a potential target effective therapeutic strategies.

Citation Format: Beilei Liu. Investigation of the potential therapeutic value of CAF-derived milk fat globule-EGF factor 8 protein (MFG-E8) in esophageal squamous cell carcinoma [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 3149.
Abstract 3188: β-catenin in metastatic ovarian cancer cells mediates polyploidy induced by tumor-associated macrophages
Authors: To Sally Kit Yan , Shi Jue , Wong Alice Sze Tsai
Published: July 2021

Tumor-associated macrophages (TAMs) closely associate with tumor progression and could promote metastatic spread. However, how TAMs interact with individual cancer cell in the heterogeneous cancer cell population is unknown. Here, using an isogenic cell line pair with different metastatic potentials, we showed that highly metastatic (HM) cells, but not non-metastatic (NM) cells, could skew the co-cultured macrophages to TAM-like phenotypes. Single-cell tracking by time lapse imaging revealed that during co-culture, macrophages could induce failed cytokinesis in a subset of HM cells. Subsequent analyses suggested that this induction could be facilitated by direct cell-cell contact. We further identified that the cell-surface metadherin expression induced by β-catenin in HM cells is involved in this induction, while surface CEACAM1 expressed on macrophages could be responsible for the interaction with metadherin. HM-macrophage interaction promotes macrophage secretion of the cytokine CCL3, a strong chemoattractant for monocytes, which may further amplify the interaction. In an orthotopic humanized mice model, knockdown of β-catenin in HM cells reduced tumor burden as well as the number of tumor-infiltrating CD163+ TAMs. These findings demonstrate a positive feedback loop between macrophages and a subpopulation of cancer cells mediated by β-catenin to enhance the metastatic cascade (This work is supported by RGC GRF grant 17141216).

Citation Format: Sally Kit Yan To, Jue Shi, Alice Sze Tsai Wong. β-catenin in metastatic ovarian cancer cells mediates polyploidy induced by tumor-associated macrophages [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 3188.
Abstract 383: Efficacy of immune checkpoint inhibitors in lung cancer patients with EGFR P719A mutation: A case report
Authors: Gong Fuyu , Zheng Kai
Published: July 2021

Background: Results from previous studies indicated that lung cancer patients harboring EGFR sensitizing mutations may receive unfavorable outcomes from immunotherapy. The present study aims to investigate the efficacy of inhibitors of programmed cell death protein-1in lung cancer patients with driver oncogene EGFR P719A mutations and strong positive PD-L1 expression in one special case.

Methods: Tissue samples were subjected to NGS in our College of American Pathologists-certified and Clinical Laboratory Improvement Amendments-accredited lab for driver oncogene mutations and PD-L1 expression.

Results: A 52-year-old male never-smoker who complained of a persistent cough was examined by computed tomography (CT), which revealed a 39 mm wide tumor in the upper region of the right lobe of the lung in May 2019. Tumor biopsy pathology revealed that the patient had a stage IIIB (T2N3M0) adenocarcinoma. NGS performed on a formalin-fixed, paraffin-embedded tumor specimen to identify genomic aberrations. The tumor was positive for EGFR, EGFR P719A mutation. The PD-L1 expression have been tested by FDA-approved 22C3 antibodies, and PD-L1 expression level of 95%. The patients received anti-PD-1 combination treatment. He has reached partial response (PR) after 4 cycle treatment. After ten months, he has the opportunity to receive the operation. The following anti-PD-1/L1 monotherapy is still ongoing until now.

Conclusion: Our real-world study supported that NSCLC patients harboring sensitizing oncogenic mutations may potentially benefit from anti-PD-1/L1 especially for those with positive indicators of immunotherapy with strong positive PD-L1 expression.

Citation Format: Fuyu Gong, Kai Zheng. Efficacy of immune checkpoint inhibitors in lung cancer patients with EGFR P719A mutation: A case report [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 383.
Abstract 406: Development of a gene signature assessing ER modulation by SERMs and SERDs as a target engagement biomarker for endocrine therapy in breast cancer
Authors: Lee Joon Sang , Shomali Maysoun , Bouaboula Monsif , Wang Emma , Dos-Santos-Bele Wilson , Dib Colette , Boitier Eric , Pelekanou Vasiliki , Ternes Nils , Gosselin Alice , Cohen Patrick , Celanovic Marina , Soria Christopher , Protopopov Alexei , Pollard Jack
Published: July 2021

Hormone or endocrine therapy is the primary treatment for estrogen receptor-positive (ER+) breast cancer. Selective Estrogen Receptor Modulators (SERMs) have been used for women with ER+ invasive cancer in the adjuvant setting. However, they do not fully inhibit ER transcriptional activity. Therefore, selective estrogen receptor degraders (SERDs) such as fulvestrant have been developed to overcome the partial modulation of ER transcriptional activity by SERMs. However, the clinical benefit of fulvestrant is limited by its pharmaceutical properties, and burden of intramuscular administration. We developed amcenestrant (SAR439859), a novel, orally bioavailable SERD to improve drug properties and is under clinical investigation.To assess the relative ER modulating activity, we developed a gene signature by transcriptional profiling of multiple ER+ cell lines. We identified 87 genes that were either up- or down-regulated by estradiol and reversed by SERM and SERD compounds or differentially expressed between a SERM/SERD compound and estradiol. We assessed ER activity scores by applying Gene Set Variation Analysis (GSVA) method to the ER gene signature and evaluated the effect of 6 SERD compounds. Amcenestrant and fulvestrant illuminated a deeper inhibition of ER activity compared to the other compounds. In addition, these ER activity scores could potentially be utilized in clinics as pharmacodynamic (PD) biomarker to assess target engagement. We applied this ER signature to RNA-seq data from paired pre- and post-treatment tumor biopsies from patients of NCT03284957 study (AMEERA-1), an open-label, phase 1/2 study of amcenestrant in postmenopausal women with ER+/HER2- metastatic breast cancer. The results of our analysis showed that ER activity is down-regulated by amcenestrant (oral SERD) for 3 out of 5 patients, in parallel with clinical benefit. Validation in a larger patient cohort is needed.

Citation Format: Joon Sang Lee, Maysoun Shomali, Monsif Bouaboula, Emma Wang, Wilson Dos-Santos-Bele, Colette Dib, Eric Boitier, Vasiliki Pelekanou, Nils Ternes, Alice Gosselin, Patrick Cohen, Marina Celanovic, Christopher Soria, Alexei Protopopov, Jack Pollard. Development of a gene signature assessing ER modulation by SERMs and SERDs as a target engagement biomarker for endocrine therapy in breast cancer [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 406.
Abstract 44: The discovery of ARV-471, an orally bioavailable estrogen receptor degrading PROTAC for the treatment of patients with breast cancer
Authors: Snyder Lawrence B. , Flanagan John J. , Qian Yimin , Gough Sheryl M. , Andreoli Monica , Bookbinder Mark , Cadelina Gregory , Bradley John , Rousseau Emma , Chandler Julian , Willard Ryan , Pizzano Jennifer , Crews Craig M. , Crew Andrew P. , Houston John , Moore Marcia Dougan , Peck Ron , Taylor Ian
Published: July 2021

ARV-471, an estrogen receptor (ER) alpha PROTAC® protein degrader, is a hetero-bifunctional molecule that facilitates the interactions between estrogen receptor alpha and an intracellular E3 ligase complex, leading to the ubiquitylation and subsequent degradation of estrogen receptors via the proteasome. ARV-471 robustly degrades ER in ER-positive breast cancer cell lines with a half-maximal degradation concentration (DC50) of ~ 1 nM. PROTAC® mediated ER degradation decreases the expression of classically regulated ER-target genes and inhibits cell proliferation of ER-dependent cell lines (MCF7, T47D). Additionally, ARV-471 degrades clinically relevant ESR1 variants (Y537S and D538G) and inhibits growth of cell lines expressing those variants. In an immature rat uterotrophic model, ARV-471 degrades rat uterine ER and demonstrates no agonist activity. Daily, oral-administration of single agent ARV-471 (3, 10, and 30 mpk) leads to significant anti-tumor activity of estradiol-dependent MCF7 xenografts and concomitant tumor ER protein reductions of >90% at study termination. Moreover, when a CDK4/6 inhibitor is combined with ARV-471 in the MCF7 model, even more pronounced tumor growth inhibition is observed (131% TGI), accompanied by significant reductions in ER protein levels. In an ESR1 Y537S, hormone-independent patient-derived xenograft model, ARV-471 at 10 mpk completely inhibited growth and also significantly reduced mutant ER protein levels. Taken together, the preclinical data of ARV-471 supports its continued development as a best-in-class oral ER PROTAC® protein degrader. These preclinical data supported the clinical development of ARV-471 for the treatment of patients with breast cancer. The discovery, chemical structure and initial clinical data of ARV-471 will be presented.

Citation Format: Lawrence B. Snyder, John J. Flanagan, Yimin Qian, Sheryl M. Gough, Monica Andreoli, Mark Bookbinder, Gregory Cadelina, John Bradley, Emma Rousseau, Julian Chandler, Ryan Willard, Jennifer Pizzano, Craig M. Crews, Andrew P. Crew, John Houston, Marcia Dougan Moore, Ron Peck, Ian Taylor. The discovery of ARV-471, an orally bioavailable estrogen receptor degrading PROTAC for the treatment of patients with breast cancer [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 44.
Abstract 443: Bintrafusp alfa enhances the antitumor efficacy of anti-VEGF and VEGF receptor kinase inhibitors in preclinical solid tumor models
Authors: Yeung Tsz-Lun , Lazorchak Adam , Yu Huakui , Qi Jin , Qin Guozhong , Marelli Bo , Fu Kai , Lan Yan
Published: July 2021

The targeting of pathways that regulate tumor angiogenesis is an effective strategy for the treatment of solid tumors. Transforming growth factor (TGF)-β and vascular endothelial growth factor (VEGF) are master regulators of tumor angiogenesis that limit the efficacy of cancer therapies and suppress the anti-tumor immune response. Combination therapies consisting of immune checkpoint inhibitors and tumor angiogenesis inhibitors are emerging as effective treatments for solid cancers. Bintrafusp alfa (M7824) is a bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 antibody blocking PD-L1. Here, we tested the antitumor efficacy of a combination therapy consisting of bintrafusp alfa plus a VEGF receptor kinase inhibitor (pazopanib, lenvatinib, regorafenib, axitinib, or nintedanib), or a VEGF neutralizing antibody (B20) in multiple preclinical tumor models. The combination of bintrafusp alfa with lenvatinib, regorafenib, axitinib, or nintedanib showed significantly greater antitumor efficacy than the respective monotherapies in the CT26 mouse syngeneic colon carcinoma model. In the 4T1 mouse breast carcinoma model, the combination of bintrafusp alfa with anti-VEGF, lenvatinib, or regorafenib was significantly more efficacious than the respective monotherapies. Additionally, the combination of bintrafusp alfa with pazopanib also showed superior antitumor efficacy in an orthotopic Renca mouse tumor model. These data indicate that bintrafusp alfa may be a viable combination partner with multiple angiogenesis inhibitors and provide a rational for the continued development of these combination therapies to treat solid cancers.

Citation Format: Tsz-Lun Yeung, Adam Lazorchak, Huakui Yu, Jin Qi, Guozhong Qin, Bo Marelli, Kai Fu, Yan Lan. Bintrafusp alfa enhances the antitumor efficacy of anti-VEGF and VEGF receptor kinase inhibitors in preclinical solid tumor models [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 443.