bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5761: Trends and regional distribution pattern of female lung cancer in China from 1992 to 2016
Authors: Ni Xue , Zhang Bei , Xu Ning , Zeng Xinying , Wang Qiang
Published: September 2020

Background: The risk factors of female lung cancer in China are still unclear. The growing trend of lung cancer in Chinese women has become a global concern of public health. This study aims to explore the secular trends and regional distribution of female lung cancer in China.

Method: The results of global burden of disease (GBD 2016) and the world standard population database (WHO 2000-2025) were used to extract age- sex- and province-specific incidence, mortality, and years of life lost due to premature death (YLL) for this report. The Joinpoint regression analysis was applied to fit the secular female lung cancer trends of age-standardized incidence rate (ASIR), age-standardized mortality rate (ASMR), and YLL rate for China and its 33 provincial administrative regions in 1992-2016.

Result: From 1992 to 2016, the secular trend of female lung cancer incidence in China rose upward and the trend of mortality or rate of YLL fell down. The average annual percent change (AAPC) of the ASIR, ASMR, and YLL rate were 0.7 (95%CI: 0.6,0.8%), -0.3% (95%CI: -0.5, -0.2%), and -0.7 (95%CI: -0.8, -0.6%) respectively. However, the secular trends among 33 provincial units are inconsistent. There were 5 regional patterns of joint trends in ASIR, ASMR, and YLL rate among 33 provinces, autonomous regions, and municipalities.

Conclusion: The regional pattern of the trends of female lung cancer in China suggested that the prevention and control of female lung cancer should be taken with targeted regional interventions.

Citation Format: Xue Ni, Bei Zhang, Ning Xu, Xinying Zeng, Qiang Wang. Trends and regional distribution pattern of female lung cancer in China from 1992 to 2016 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5761.
Abstract 5767: Multiple primary cancer from a retrospective population-based cancer registry, 1991 through 2015
Authors: Tanjak Pariyada , Thiengtrong Benjarat , Thamapala Kanokporn , Gerdsuriwong Tippawan , Songjang Cholticha , Rattanakumnerd Aem-on , Acharayothin Onchira , Karakate Supatee , Thamnongdee Nongnard , Therasakvichya Suwanit , Laiteerapong Somsri , Chinswangwatanakul Vitoon
Published: September 2020

Background Multiple primary cancer refers to the synchronous or metachronous appearances in the same individual. The accurate approach, which distinguishes between multiple primary cancer and metastasis, is important to make an appropriate management. This study aims to systematically classify the synchronous and metachronous multiple primary cancers, and to identify multiple primary cancer from cancer metastasis.

Methods Cancer registry was compiled at the Faculty of Medicine Siriraj Hospital, Mahidol University based on guidelines from the Surveillance Epidemiology and End Results (SEER) project. Data was validated through clinical records, coded, and verified for multiple primary cancer according to International Classification of Diseases for Oncology 3rdEdition (ICD-O-3). The cancer registry for the year 1991-2015 was queried for numbers of the multiple primary cancer.

Results A total of 1,913 patients presented multiple primary cancer, accounting for 1.48% of all patients (129,216). Median age at diagnosis of the first cancer was 60. The most multiple primary cancer in female is synchronous breast cancers, diagnosed two or more histologically distinct cancer within two months. In male, the most combination is prostate, urinary tract, and gastrointestinal cancer. In addition to understanding of the connections between primary and subsequent cancer, we plan to perform panel sequencing in each primary site of multiple gastrointestinal primary cancer to access for the causal driver mutation.

Conclusion Cancer registry is a valuable approach to punctually identify and record multiple primary cancer. Further study in genetic profiles of each primary cancer may improve our understanding of multiple primary cancer for enhancing cancer management.

Citation Format: Pariyada Tanjak, Benjarat Thiengtrong, Kanokporn Thamapala, Tippawan Gerdsuriwong, Cholticha Songjang, Aem-on Rattanakumnerd, Onchira Acharayothin, Supatee Karakate, Nongnard Thamnongdee, Suwanit Therasakvichya, Somsri Laiteerapong, Vitoon Chinswangwatanakul. Multiple primary cancer from a retrospective population-based cancer registry, 1991 through 2015 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5767.
Abstract 5808: Examination of clinically relevant inhibitor selectivity in live cells across the CDK family
Authors: Vasta James , Wells Carrow , Corona Cesear , Wilkinson Jennifer , Zimprich Chad , Ingold Morgan , Ogno Domenic , Schwinn Marie , Machleidt Thomas , Cong Mei , Fan Frank , Huwiler Kristin , Landreman Amy , Pickett Julie E. , Huber Kilian , Robers Matthew
Published: September 2020

Cyclin-dependent kinases (CDK) play key roles in diverse cellular functions including cell cycle control, cell proliferation, and transcriptional regulation. Due to these important roles, CDKs have been targeted for cancer therapeutic development, which has resulted in hundreds of small molecule inhibitors. However, conflicting data of CDK inhibitor potency has been reported and a rigorous comparison of inhibitor affinity and selectivity for intracellular CDKs is lacking. To address this need, we have developed a panel of cell-permeable energy transfer probes or tracers to enable comprehensive quantitation of inhibitor target occupancy and affinity for CDKs in live cells via a bioluminescent energy transfer (BRET) method. Specifically, the quantitative BRET-based capability is achieved via energy transfer from cell-permeable fluorescent tracers reversibly engaged to NanoLuc luciferase-tagged CDK proteins expressed in live cells. An untagged cyclin can be introduced into the cells at the same time as the NanoLuc-CDK, allowing for the interrogation of a specific CDK-cyclin pair. The BRET-based method can be used in equilibrium binding analysis. In addition, time-dependent target-compound occupancy (or residence time) can also be obtained with this method. We will present a comprehensive evaluation of intracellular isozyme potency and selectivity for a collection of clinically-advanced CDK inhibitors and chemical probes across the CDK family. We observed unexpected intracellular activity profiles for some clinically-advanced CDK inhibitors. We further evaluated mechanisms for achieving target selectivity through target residence time under non-equilibrium cell culture conditions. This BRET-based method is broadly applicable for evaluating the occupancy, affinity, and selectivity of chemical matter for CDKs in live cells.

Citation Format: James Vasta, Carrow Wells, Cesear Corona, Jennifer Wilkinson, Chad Zimprich, Morgan Ingold, Domenic Ogno, Marie Schwinn, Thomas Machleidt, Mei Cong, Frank Fan, Kristin Huwiler, Amy Landreman, Julie E. Pickett, Kilian Huber, Matthew Robers. Examination of clinically relevant inhibitor selectivity in live cells across the CDK family [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5808.
Abstract 5810: Fyn kinase negatively regulates telomere length in stem cells and cancer cells
Authors: Paul Souren , Le Thi My Le , Ryu Joohyun , Bae Ki Beom , Bode Ann M. , Dong Zigang
Published: September 2020

Telomere maintenance is on the critical biological process in stem cell, aging, and cancer. Cells maintain telomere either by Telomerase (TERT) dependent pathway or by alternative lengthening of telomere (ALT) pathway. Here we report a link between Fyn kinase expression and telomere length. Fyn deficient mouse and mouse embryonic stem cells (mESC) maintain a stable telomere length through activation of ALT pathway. Fyn deficient mice are also resistant to the aging process as compared to wild type mice. We uncover a novel molecular mechanism of telomere maintenance. Menin, an essential component of an MLL/SET1 histone methyltransferase (HMT) complex that specifically methylates 'Lys-4' of histone H3 (H3K4), is a potential phosphorylation target of Fyn. Phosphorylation of Menin facilitates SUMOylation and binding to Telomerase RNA Component (TERC). The telomere dysfunction is one pivotal reason for many genetic disorders, including telomere related diseases like dyskeratosis congenita (DC). Currently, there are no curative therapies for these diseases of telomere dysfunction. We knockout Fyn gene from DC mice model and also used 4-Amino-5-(4-chlorophenyl)-7-(dimethyl ethyl) pyrazolo[3,4-d]pyrimidine (PP2), a selective inhibitor of Fyn kinase. Our results demonstrate that Fyn could be a therapeutic target in telomere related diseases. Knockout of Fyn or inhibition of Fyn by using inhibitor improves DC phenotype in mice like bone marrow failure. Human iPSC generated from DC patient's skin fibroblast shows higher expression of p-Menin and maintains a stable telomere length when we inhibited Fyn.

Citation Format: Souren Paul, Thi My Le Le, Joohyun Ryu, Ki Beom Bae, Ann M. Bode, Zigang Dong. Fyn kinase negatively regulates telomere length in stem cells and cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5810.
Abstract 5812: FOXD1 mediates G2/M transition in clear cell renal cell carcinoma
Authors: Bond Kyle Henry , Jaikaran Ryan , Qiu Suizhen , Oxburgh Leif
Published: September 2020

The forkhead family of transcription factors are highly conserved and essential during embryonic development. There have been reports showing increased expression of forkheads in various cancers, but little is known about the role of these proteins in the disease context. Analysis of 500 clear cell renal cell carcinoma (ccRCC) transcriptomes in The Cancer Genome Database revealed that FOXD1 is overexpressed in ccRCC and correlates with poor patient outcomes (median survival FOXD1-low=2830 days; FOXD1-high=1913 days). Immunohistochemistry of 46 patient tumors showed that FOXD1 is expressed the majority of tumor cells (95% CI [50.6%, 79.4%]). To understand the biological role of FOXD1, we generated FOXD1-null variants of the 786-O ccRCC cell line. Using an in vivo xenograft model, FOXD1-null cells had smaller initiating tumor sizes (parent=71.05mm3; FOXD1-null=36.39mm3) and stunted tumor growth. In vitro, FOXD1-null lines had significantly reduced growth rates compared to the parental line, and showed increased cell size. Cell cycle analysis revealed that FOXD1-null cells had an increased number of cells in G2 compared to the parental line (parent=21.1%; FOXD1-null=37.1%). After G1 synchronization, all lines progressed through G1, S, and G2 at similar rates (parent=10 hours; FOXD1-null=10 hours). However, FOXD1-null clones spent increased time in G2 compared to the parental line (parent= 4 hours; FOXD1-null= 8 hours). Western blot analysis revealed decreased levels of Cyclin B1 and p-H3, as well as increased levels of p27 10 hours post-synchronization in FOXD1-null cells compared to the parent line. Staining with yH2AX did not indicate any significant change in the number of double strand breaks at any phase. These findings suggest that FOXD1 is a mediator of the transition between G2 and M, and that its loss initializes the G2/M checkpoint. Extrapolating back to our clinical observation, we propose that increased FOXD1 expression in tumor cells accelerates the transition between G2 and M, a point that is normally critical for survey and repair of DNA damage. This could result in greater tolerance for DNA damage, accelerating tumor evolution.

Citation Format: Kyle Henry Bond, Ryan Jaikaran, Suizhen Qiu, Leif Oxburgh. FOXD1 mediates G2/M transition in clear cell renal cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5812.
Abstract 5814: Emergent functions of cyclin-dependent kinase 4 regulating aurora b and cenpp transcription
Authors: Lee Sung Hyun , Rodriguez Liliana R. , de Marval Paula L. Miliani , Rodriguez-Puebla Marcelo L.
Published: September 2020

A better understanding of the precise molecular mechanisms that control cell proliferation is crucial for the identification of novel cancer therapeutic targets. A critical molecule in this process is the Cyclin-Dependent Kinase 4 (CDK4), which regulates key aspects of the G1 to S phase transition through phosphorylation of the retinoblastoma (Rb) family of proteins. Since dysregulation of this cell cycle mechanism is a hallmark of most cancers, drugs have been developed to inhibit the kinase activity of CDK4. However, such drugs have been met with variable degrees of success and, in some cases, little efficacy. Research performed in the last few years has suggested that CDK4 have alternative targets other than the Rb proteins. Consistent with this idea, we found that CDK4 plays additional roles not related to the canonical function in the CDK-Rb axis. Here, we show that CDK4 remains attached to the chromatin fraction of Balb/MK2 keratinocytes upon the extraction of the soluble components (nucleoplasm and cytoplasm). Chromatin-immunoprecipitation (ChIP) analysis shows that CDK4 occupies the regulatory sites of genes associated with chromosomal segregation, such as Aurkb (Aurora B) and Cenpp (Centromere Protein P). Moreover, gain- and loss-of-function experiments showed that the AurkB and Cenpp promoters are regulated through CDK4 binding. In line with these findings, studies in skin carcinogenesis revealed that transgenic expression of CDK4 in mouse epidermis induces chromosome instability (CIN) and centrosome amplification (CA), two events linked to overexpression of Aurora B. Moreover, Aurora-B has a crucial role in chromosome bi-orientation and the spindle-assembly checkpoint, whereas Cenpp is required for proper kinetochore function. Remarkably, a persistent dysregulation of Aurora B has been reported in numerous human cancers. Thus, our results suggest that CDK4 plays a role in the maintenance of chromosomal stability, implying that additional functions could be inhibited by therapeutically targeting CDK4. Our conclusions are supported by previous reports showing that pharmacological inhibition of CDK4 led to delayed progression from G2 to mitosis in Rb-positive and as well as Rb-negative cells.

Citation Format: Sung Hyun Lee, Liliana R. Rodriguez, Paula L. Miliani de Marval, Marcelo L. Rodriguez-Puebla. Emergent functions of cyclin-dependent kinase 4 regulating aurora b and cenpp transcription [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5814.
Abstract 5826: Molecular mechanism of OLIG2-CDK2 interaction in glioma cells
Authors: Saito Norihiko , Hirai Nozomi , Sato Sho , Hiramoto Yu , Fujita Satoshi , Nakayama Haruo , Hayashi Morito , Sakurai Takatoshi , Iwabuchi Satoshi
Published: September 2020

Oligodendrocyte transcription factor 2 (Olig2) promotes proliferation of normal neural stem/progenitor cells and glioma cells. However, the mechanisms underlying the regulation of Olig2 remain largely unknown. Here we identified Olig2 as a critical phosphorylation target for cyclin-dependent kinase 2 (CDK2). CDK2-mediated Olig2 phosphorylation stabilizes Olig2 protein from proteasomal degradation. Phosphorylated Olig2 binds to the E-Box regions of p27 promoter and represses p27 transcription, which in turn activates CDK2 in positive feedback manner. CDK2-mediated Olig2 phosphorylation promotes cell cycle progression, cell proliferation, and tumorigenesis. Olig2 inhibition disrupted cell cycle control mechanism by decreasing CDK2 and elevating apoptosis-related molecules. Inhibition of CDK2 activity disrupted Olig2-CDK2 interactions and attenuated Olig2 protein stability. In addition, Olig2-high glioma initiating cells are highly sensitive to CDK2 inhibitor treatment, indicating that Olig2 can be a biomarker for personalized treatment for glioblastoma patients with CDK2 inhibitors. Further investigation on these mechanisms may lead to novel targeted therapy on GBMs with high Olig2 expression.

Citation Format: Norihiko Saito, Nozomi Hirai, Sho Sato, Yu Hiramoto, Satoshi Fujita, Haruo Nakayama, Morito Hayashi, Takatoshi Sakurai, Satoshi Iwabuchi. Molecular mechanism of OLIG2-CDK2 interaction in glioma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5826.
Abstract 5827: Alterations of telomere and SOX9 play key roles on pancreatic carcinogenesis
Authors: Matsuda Yoko , Yamakawa Keiko , Narusawa Yuko
Published: September 2020

The morbidity of pancreatic cancer is predicted to increase because of the growing elderly population. In this study, we analyzed alterations of telomere length and SOX9 expression to clarify the relationship between precancerous lesions (pancreatic intraepithelial neoplasia; PanIN), pancreatic cancer and aging. The incidence of PanIN in the elderly patients was 60%. The telomere, which can protect the chromosome ends, was shortened in the following order; pancreatic duct>PanIN-1>PanIN-2>PanIN-3>pancreatic cancer. The telomeres in normal duct epithelium became shorter with aging, and those in cancers were shorter than in controls, suggesting that telomere shortening occurs when histological changes are absent. SOX9, which regulates pancreatic progenitor cells, was the highest in carcinomas, followed by PanIN-3, PanIN-2, PanIN-1, and duct epithelium. SOX9 expression was positively correlated with telomere length in cancer cells. Inhibition of SOX9 using siRNA showed decrease of human telomerase reverse transcriptase (hTERT) expression and decrease of sphere formation in human pancreatic cancer cell lines. In conclusion, upregulation of SOX9 in the duct epithelium, which harbors shortened telomeres, may give rise to PanINs and pancreatic cancers.

Citation Format: Yoko Matsuda, Keiko Yamakawa, Yuko Narusawa. Alterations of telomere and SOX9 play key roles on pancreatic carcinogenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5827.
Abstract 5836: Autophagy inhibition as a new therapeutic strategy for cancers with transcription elongation defects
Authors: Muhammad Belal , Vinnedge Lisa , Komurov KAkajan
Published: September 2020

Increasing evidence suggests that the tumorigenic process is driven by various forms of transcriptional and epigenetic defects beyond somatic mutations. We recently described a novel non-genomic phenomenon associated with tumorigenesis that we call Transcription Elongation Defects (TEdeff), which is characterized by high levels of truncated transcripts, intron retention, and loss of gene body exon expression. The TEdeff phenotype exists in more than 20% of all human tumors and is linked to immunotherapy resistance (Modur et al, 2018). The aim of this study was to find a new therapeutic strategy for these types of tumors by targeting cellular pathways that are essential for TEdeff tumor survival. Computational analyses of RNA-sequencing data indicate that TEdeff tumors have extensive overexpression of the cytoplasmic protein homeostasis machinery, including several components of autophagy and proteasome degradation pathways. This strongly suggests that these cells may have increased dependency on cellular protein quality control due to elevated proteotoxic stress. Accordingly, we experimentally show that TEdeff cancer cell lines have a profound accumulation of protein aggregates (aggresomes) that appear to be associated with autophagy for clearance. Importantly, TEdeff cells, but not control cells with proficient transcription elongation (TEprof), are highly sensitive to autophagy inhibitors like chloroquine (CQ), but not proteasome inhibitors, which indicates their increased dependency on autophagy for survival. We also show evidence that the proteasomal degradation system may be defective in TEdeff cells, further supporting the hypothesis that these cells are dependent on alternative protein quality control mechanisms. Mechanistically, we propose that blocking the autophagy pathway maximizes proteotoxic stress, due to the accumulation of toxic aggresomes and proteins generated from defective transcripts, leading to a strong synthetic lethality in TEdeff cells. Therefore, autophagy inhibition may be a potent treatment strategy for TEdeff tumors, which are inherently resistant to current immunotherapies.

Citation Format: Belal Muhammad, Lisa Vinnedge, KAkajan Komurov. Autophagy inhibition as a new therapeutic strategy for cancers with transcription elongation defects [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5836.
Abstract 5839: The unfolded protein response gene Ire1α is required for tissue renewal and normal differentiation in the mouse tongue and esophagus
Authors: Chalmers Fiona E. , Stairs Douglas B. , Glick Adam B.
Published: September 2020

IRE1α is a well-characterized stress response signaling protein that adapts the cell to conditions of endoplasmic reticulum (ER) stress. In addition to well-established sources of ER stress such as viral infection, hypoxia and nutrient deprivation, it has recently been proposed that the morphological and epigenetic changes associated with routine cell differentiation are also a source of ER stress for the cell, and that IRE1α has an important role in adapting the cell to its newly differentiated state. Here, we provide evidence that IRE1α is essential for the differentiation of stratified squamous epithelium. We utilized mice expressing K14CreERT2 and floxed IRE1α to enable tamoxifen-inducible deletion of IRE1α targeted to the basal layer and stem cells of stratified squamous epithelium. After tamoxifen injection, IRE1α knockout mice displayed severe weight loss resulting in death within 10 days of treatment. Histological analysis revealed that epithelial homeostasis in the tongue and esophagus was severely disrupted leading to significantly decreased food intake and, as a result, death. The tongue epithelium and esophageal mucosal layer were found to be significantly thinner, and filiform papillae were lost from the tongue. Immunohistochemical staining showed significant downregulation of the proliferative marker Ki67, as well as a marker of differentiation, Sox2, suggesting an almost complete loss of tissue renewal as early as four days after IRE1α deletion. This work highlights the complexity of IRE1α signaling beyond adaptation to external sources of stress and proposes a role of IRE1α in the normal physiology of tissue homeostasis. As inhibition of the IRE1α signaling pathway has been proposed as a therapeutic target in certain cancers, an increased understanding of how broad the scope of IRE1α activity is may provide insight into potential side effects resulting from these treatments.

Citation Format: Fiona E. Chalmers, Douglas B. Stairs, Adam B. Glick. The unfolded protein response gene Ire1α is required for tissue renewal and normal differentiation in the mouse tongue and esophagus [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5839.
Abstract 584: TGN1412 induces dose and time dependent human cytokines in a humanized PBMC mouse model for cytokine release syndrome
Authors: Yang Hongyuan , Jiao Jing , Cai Danying , Cheng Mingshan , Brehm Michael , Grainer Dale , Shultz Leonard , Keck James
Published: September 2020

There have been remarkable progresses in the development of immune therapies for cancer. However, the promise of immune therapy comes with significant challenges of a variety of adverse effects. Due to the lack of predictive preclinical model to evaluate antibody mediated adverse effects, TGN1412, a humanized anti-CD28 monoclonal antibody which acts as a superagonist of CD28 to activate T cells, unfortunately caused life-threatening cytokine release syndrome (CRS) during the first-in-man trial. Therefore there is a critical need for translational protocols that could predict immune toxicity more accurately.

We have developed a sensitive and reproducible PBMC-humanized mouse model that can measure cytokine release induced by bispecifics or checkpoint inhibitors in vivo. In this study the utility of this platform was demonstrated using TGN1412 as a model. NSG™ mice or derivative strains such as SGM3 mice were engrafted with human PBMCs for 6 days before being treated with TGN1412 at different concentrations (0.2, 1.0 and 2 mg/kg). Mouse blood was collected at 1, 2, 4, 6, 24 and 48 hours post dose for flow cytometry and cytokine analysis. We observed dose and time dependent increase of human IFN-γ, TNFα, IL-2, IL-4, IL-6 and IL-10 levels in the serum following TGN1412 treatment with TNFα peaking at 1 h and the others peaking at 2-4 h post dose. A significant decrease in hCD45+ cells was observed in the peripheral blood collected at 6h post treatment. TGN1412 was tested in the humanized NSG™ mice with PBMCs from 7 different donors, and a significant decrease in hCD45+ cell population in the peripheral blood was observed in all donors. We have demonstrated that the novel mouse model using PBMC-humanized NSG™ can successfully detect CRS induced by TGN1412 in a dose and time dependent manner, suggesting that indeed CRS could be a strong adverse effect associated with TGN1412 treatment in human. Our data supports that our newly developed mouse model for CRS assessment may be a valuable tool to assess in vivo safety of experimental human therapeutic antibodies including immune checkpoint inhibitors and bispecifics with better reliability than an in vitro assay.

Citation Format: Hongyuan Yang, Jing Jiao, Danying Cai, Mingshan Cheng, Michael Brehm, Dale Grainer, Leonard Shultz, James Keck. TGN1412 induces dose and time dependent human cytokines in a humanized PBMC mouse model for cytokine release syndrome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 584.
Abstract 5840: IRE1a is required for ROS homeostasis and PERK protein stability in response to UVB irradiation
Authors: Son Jeongin , Worrel Stephen , Glick Adam
Published: September 2020

IRE1α is required for ROS homeostasis and PERK protein stability in response to UVB irradiation

UV induced stress is one of the primary driving factors of human skin cancer. UV irradiation can activate Endoplasmic Reticulum (ER) stress and the Unfolded Protein Response (UPR). Inositol-requiring enzyme 1a (IRE1α) is one of the key players in the UPR. Previous studies have shown that UVB irradiation can activate IRE1α in human and mouse skin cells, but the function of IRE1α in the UV response has not yet been characterized. Here we show that IRE1α expression is transiently downregulated after UVB irradiation and that in the absence of IRE1α the stability and activation of PKR-like endoplasmic reticulum kinase (PERK) is compromised. Basal levels of PERK are reduced in the absecence of IRE1α. Following UV irradiation, loss of IRE1α not only causes rapid degradation of PERK protein but also suppresses phosphorylation of the PERK target, Nuclear factor erythroid-2-related factor 2 (NRF2), and NRF2 dependent antioxidant gene expression. Similarly, PERK protein is rapidly degraded following thapsigargin treatment in the absence of IRE1α, suggesting a general regulation of PERK stability by IRE1α. Additionally, keratinocytes with either knockout or knockdown of IRE1α have increased intracellular ROS and cytosolic calcium. Overexpression of both wildtype and RNase defective human IRE1α can rescue the increased ROS levels. Additionally, the increased ROS is also suppressed by treatment with an inhibitor of the ER localized Inositol triphosphate receptor (InsP3R) which regulates calcium efflux from the ER. Because PERK regulates cytosolic calcium through inhibiting InsP3R, our result suggests that IRE1α is critical for suppressing intracellular ROS through stabilization of PERK. We conclude that crosstalk between IRE1α and PERK is essential for the basal and UVB- induced oxidative stress response, and this may have important implications for UV related damage and skin cancer development.

Citation Format: Jeongin Son, Stephen Worrel, Adam Glick. IRE1a is required for ROS homeostasis and PERK protein stability in response to UVB irradiation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5840.
Abstract 585: Colitis in tumor-bearing mice undergoing checkpoint inhibitor therapy
Authors: Parello Caitlin S. , Cuiffo Benjamin G. , Reardon Kasey , Fusco Alexandria , Van Dam Brett , Lyng Gregory D. , Sonis Stephen T.
Published: September 2020

Checkpoint inhibitor (CPI) therapy represents an exciting new treatment option for patients with advanced malignancies. However, several adverse events (AEs) that affect multiple body systems are associated with CPI treatment, including colitis. The mechanisms mediating these AEs remain to be fully elucidated. That mice treated with CPI fail to develop the spectrum of AEs observed in patients hampers the field's ability to develop supportive care treatment options. Here we sought to examine colitis in tumor bearing mice undergoing CPI therapy. Briefly, naïve TH cells and Treg cells were harvested from C57Bl/6 donor mice and were adoptively transferred into RAG2−/- recipients. Animals were inoculated with B16F10 cells into the left flank seven days later. T cell engraftment was assessed after 13 days, and animals were randomized into treatment groups by rate of T cell engraftment; animals were treated Q3D with either vehicle or anti-CTLA4 for an additional four weeks. Animals were monitored for body weight loss and systemic AEs (gastrointestinal, pulmonary, neurological) daily and tumor volume was measured by digital caliper three times per week. Extent of colitis was evaluated by video endoscopy at several timepoints and colitis severity was scored using a 0-4 scale. Animals developed body weight loss approximately two weeks after T cell transfer and CPI treatment increased the severity of body weight loss observed. Colitis was seen by video endoscopy within a similar timeframe. Mean tumor volume was decreased in animals treated with CPI as compared to those treated with vehicle. Diarrhea, pulmonary symptoms, and neurological symptoms were observed in a subset of animals over the study period; anti-CTLA4 treatment increased the overall incidence of pulmonary symptoms. These data demonstrate the utility of this system for assessment of colitis or other AE supportive care treatment options in the context of CPI-treated, tumor-bearing mice.

Citation Format: Caitlin S. Parello, Benjamin G. Cuiffo, Kasey Reardon, Alexandria Fusco, Brett Van Dam, Gregory D. Lyng, Stephen T. Sonis. Colitis in tumor-bearing mice undergoing checkpoint inhibitor therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 585.
Abstract 5854: New therapeutic targets of ER positive breast cancer are revealed through nascent RNA transcription time series
Authors: Azofeifa Joseph , Basken Joel , Lai Maria , Langendorf Ryan , Norris Laura , Read Tim , Robbins-Pianka Adam
Published: September 2020

Mechanistic understanding of Estrogen Receptor (ER) signaling will lead to new therapeutic targets for ER positive breast cancer. To this end, Arpeggio performed nascent RNA sequencing following estradiol (E2) treatment in MCF7 breast cancer cells every 15 minutes for 6 hours. To date, our study represents the densest time series of estrogen signaling ever reported. Arpeggio noted three transcriptional waves following E2 treatment: peaks at 30 minutes, 2.5 hours and 5 hours, respectively. We noted that the early response was enriched for canonical ER signaling, however, mid and late response were enriched for metabolic processes. Because our study was statistically powered with 24 time points, we could use delay-coordinate embedding to look for causal gene interactions. We implicated NME2 as the causal driver of the 5-hour transcriptional wave. Indeed, NME2 and Estrogen Receptor (ER) have been shown to co-localize where NME2 blocks ER effect. Our study represents a new methodology for target discovery in hormone-driven cancers.

Citation Format: Joseph Azofeifa, Joel Basken, Maria Lai, Ryan Langendorf, Laura Norris, Tim Read, Adam Robbins-Pianka. New therapeutic targets of ER positive breast cancer are revealed through nascent RNA transcription time series [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5854.
Abstract 5861: BOP1 expression contributes to the proliferative/invasive phenotype in melanoma
Authors: Stark Mitchell S. , Hammerlindl Sabrina , Tom Lisa , Jagirdar Kasturee , Tan Jean-Marie , Schaider Helmut , Sturm Richard A. , O'Brien Blake , Walsh Michael , Collins Angus , Soyer H.Peter
Published: September 2020

We recently performed whole exome sequencing of 30 melanocytic nevi which surprisingly revealed numerous regions of copy number loss. It was found that common reticular nevi (mostly with a junctional component) have a high incidence of copy number aberrations (CNA). Importantly, concurrent with the loss of tumor suppressor genes (e.g. CDKN2A, TP53, and NF1) was the loss of potent oncogenes (e.g. NRAS, MITF, and MDM2). We postulated that the balanced nature of these CNAs confers protection from transformation, thus keeping the lesion in the benign state. Along with copy-number loss of these well-known genes, further interrogation of these data revealed a loss on chr8q24.3 which was frequently observed in 7/30 (23%) nevi. In the smallest region of overlap, the block of proliferation 1 (BOP1) gene was identified as a candidate for functional validation. BOP1 is part of the trimeric “PeBoW” complex, essential for ribosome biogenesis and cell cycle progression. In melanoma cell lines (MM96L, HT144, and MM253), we used siRNA mediated gene targeting to achieve differing BOP1 expression levels (high, low, absent), which resulted in altered cell states. For example, high BOP1 expression caused an increase in proliferation and colony formation, whereas low BOP1 expression conferred reduced proliferation, colony formation, and a senescent like phenotype. Interestingly, low BOP1 expression also resulted in increased migration and invasive capacity. BOP1 immunofluorescence assessment of benign nevi revealed limited expression whereas in early invasive melanomas, BOP1 expression was clearly evident in proliferative melanoma nests. These data support the role of BOP1 playing a role in melanoma progression by contributing to phenotype switching.

Citation Format: Mitchell S. Stark, Sabrina Hammerlindl, Lisa Tom, Kasturee Jagirdar, Jean-Marie Tan, Helmut Schaider, Richard A. Sturm, Blake O'Brien, Michael Walsh, Angus Collins, H.Peter Soyer. BOP1 expression contributes to the proliferative/invasive phenotype in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5861.
Abstract 5863: Improved conversion in extraction, library construction, and capture improve sensitivity for variants in liquid biopsy samples
Authors: Roseman Nicole , Parakh Shilpa , Huang Hsiao-Yun , Lai Kevin , Barnes Timothy , Lewis Lyn , Chakravarty Ushati Das , Potts Anastasia , Jackson Alisa , Yoder Amy , Sheu Jessica , Chen Tzu-Chun
Published: September 2020

Demonstrate performance of a complete automation and reagent workflow for analysis of cfDNA from bodily fluids. The efficient extraction of cfDNA from bodily fluids is a unique challenge due to the very low concentrations of nucleic acid. The extraction process along with library preparation is a laborious workflow, where human variability can lead to increased variability in the downstream analysis. Integrated DNA Technology (IDT) and Beckman Coulter (BC) have teamed up to provide a complete automation and reagent workflow for analysis of low frequency variants in cfDNA. The Apostle MiniMax™ High Efficiency Isolation Kit from BC provides complex, utilized magnetic nanoparticles to effectively capture cfDNA. IDT's library prep kit utilizes novel chemistry to maximize conversion, suppress adapter-dimer formation, reduce chimera rates, and facilitate double strand consensus analysis to call ultra-low frequency variants. Finally, IDT's xGen™ hybrid capture products maintain high library diversity and on-target rates to enable low frequency variant calling regardless of panel size. The Biomek i5 and i7 Hybrid workstations bring out the best performance from these reagents. The Biomek NGS workstations protocol is written with a modular design with safe stop points, making it customizable for each lab. The automated protocol uses Beckman's Demonstrated Method Interface tools which include: Biomek Method Launcher to run the method without going into Biomek software, Method Options Selector to choose the run parameters with a user friendly interface, Guided labware Setup to set the deck with labware based on the run parameters, DeckOptix Final Check software to help reduce deck setup errors. We demonstrate the performance of this complete workflow with a range of plasma inputs (4-8 mL). Using control samples with known variant frequencies, the workflow yields high library complexity, 100% positive predictive value, and reliable detection of <0.5% mutant allele frequency variants. With real cfDNA, the workflow demonstrates both high cfDNA and sequencing library yields along with high library complexity. The combination of these reagents on the Biomek workstations provides a robust and reproducible solution for the analysis of cfDNA.

Citation Format: Nicole Roseman, Shilpa Parakh, Hsiao-Yun Huang, Kevin Lai, Timothy Barnes, Lyn Lewis, Ushati Das Chakravarty, Anastasia Potts, Alisa Jackson, Amy Yoder, Jessica Sheu, Tzu-Chun Chen. Improved conversion in extraction, library construction, and capture improve sensitivity for variants in liquid biopsy samples [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5863.
Abstract 5869: Characterization and therapeutic implications of SMARCA4 mutations in cancer
Authors: Fernando Tharu M. , Piskol Robert , Bainer Russell , Sokol Ethan , Trabucco Sally , Zhang Qing , Trinh Huong , Maund Sophia , Kschonsak Marc , Chaudhuri Subhra , Januario Tom , Modrusan Zora , Yauch Robert L.
Published: September 2020

Genomic studies performed in cancer patients have identified a high frequency of alterations in the core catalytic subunit of the mammalian switch/sucrose non-fermentable (mSWI/SNF) chromatin remodeling complex, SMARCA4. Cells with inactivating SMARCA4 mutations rely on its paralog, SMARCA2, making it an attractive therapeutic target. Here we report the results of genomic profiling of 131,668 cases from patients with solid tumors and identified 8,588 cases with one or more SMARCA4 alterations. We observed a high prevalence of homozygous SMARCA4 truncating and non-truncating mutations in certain tumor types, notably non-small cell lung cancer (NSCLC) and cancers of unknown primary. We found that homozygous SMARCA4 mutations in NSCLC were mutually exclusive with other mutations in mSWI/SNF and oncogenic drivers, including KRAS and EGFR. A retrospective study of NSCLC patients who were treated in the Flatiron Health network (>265 oncology practices in the U.S.) and underwent FoundationOne® or FoundationOne CDx® tumor sequencing as part of routine care revealed that real-world patients with homozygous truncating SMARCA4 mutations had significantly reduced overall survival. The large sample size of our cohort also elucidated novel hotspot missense mutations within the helicase domain of SMARCA4. Functional modeling of these mutations using biochemical and cell-based (ATAC-seq) assays demonstrated that all evaluated mutations have significantly reduced nucleosome remodeling activity upon in vitro reconstitution in SMARCA4-deficient cells. We also addressed whether SMARCA4 missense mutations could rescue the cell growth defects and loss in chromatin accessibility induced upon SMARCA2 loss. Interestingly, we observe that a few missense mutants were able to either partially or fully rescue growth, suggesting hypomorphic activity under the selective pressure of SMARCA2 loss. Overall, these studies highlight NSCLC patients with homozygous truncating SMARCA4 mutations as a novel population with an unmet need who may benefit from SMARCA2-targeted therapy and underscore that careful selection criteria must be employed to identify patients with inactivating, homozygous SMARCA4 missense mutations.

Citation Format: Tharu M. Fernando, Robert Piskol, Russell Bainer, Ethan Sokol, Sally Trabucco, Qing Zhang, Huong Trinh, Sophia Maund, Marc Kschonsak, Subhra Chaudhuri, Tom Januario, Zora Modrusan, Robert L. Yauch. Characterization and therapeutic implications of SMARCA4 mutations in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5869.
Abstract 5879: Spatially-segmented single-cell transcriptomics by diffusional accessibility to a small-molecule dye
Authors: Morse David B. , Ceribelli Michele , De Jonghe Joachim , Michalowski Aleksandra , Muus Christoph , Vias Maria , Boyle Samantha , Weitz David A. , Brenton James , Buenrostro Jason D. , Thomas Craig , Knowles Tuomas P.
Published: September 2020

High-throughput single-cell RNA-seq (scRNA-seq) is used to describe complex tissues by characterizing transcriptional states of individual cells. Defining a cell's position, both in regard to tissue margins and its social context, is essential for understanding the intrinsic and extrinsic variables that effect the transcriptional identity of individual cells. Conventional high-throughput scRNA-seq assays, however, decouple cells from their original locations within tissues. In situ hybridization readouts of gene expression and in situ sequencing preserve spatial information in tissues, but currently have a lower total read threshold than NextGen sequencing – imposing a restriction on either cell throughput or transcriptional breadth. Combining the above methods or using regional barcodes to define 2D positions have spatially-reconstructed tissue regions but are seldom employed in unspecialized laboratories. We describe SEgmentation by Exogenous Perfusion (SEEP), a rapid, integrated, method for providing 3D spatial-segmentation to scRNA-seq data. Tissues are divided into layers based on accessibility of a fluorescent dye allowing sorted cells to be characterized by transcriptomic and regional identity. We use SEEP to explore how the transcriptional states of cells in high-grade serous ovarian cancer vary with respect to intratumoral position. We describe an integrated method for correlating 3D radial-spatial cell positions with scRNA-seq data. By employing a basic stain-and-sort method using an off-the-shelf live-dead stain, we are able to correlate transcriptional profiles with radial-spatial positions in both radially symmetric tissues (e.g., spheroids, organoids, spherical tumor masses) and linear tissue samples (e.g., punch biopsies). SEEP uses an imaging-based calibration step to inform the parameters of a FACS and sequencing based measurement step. Across spheroid, organoid, and PDX models of high-grade serous ovarian cancer, we find common positional transcriptomic profiles, ranging from single-gene proclivities to multi-gene-set enrichments across tissue layers.

Citation Format: David B. Morse, Michele Ceribelli, Joachim De Jonghe, Aleksandra Michalowski, Christoph Muus, Maria Vias, Samantha Boyle, David A. Weitz, James Brenton, Jason D. Buenrostro, Craig Thomas, Tuomas P. Knowles. Spatially-segmented single-cell transcriptomics by diffusional accessibility to a small-molecule dye [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5879.
Abstract 589: Immune biomarker profiling and safety evaluation response to 4-1BB antibody (Urelumab) in humanized 4-1BB mice
Authors: Xiang Jie , Li Dirui , Yang Hao , Ng Gary , Shen Yuelei
Published: September 2020

4-1BB (CD137) belongs to the TNFR superfamily and is expressed by activated CD4 helper T cells, B cells, natural killer cells, natural killer T cells, dendritic cells and Treg cells. 4-1BB is a potential therapeutic target as it enhances tumor rejection by upregulating T cells following activation and its engagement increases T cell proliferation and pro-inflammatory cytokine production. However, the liver toxicity associated with over immune activation has obstructed the clinical development of 4-1BB targeting therapies. To address the issue, Biocytogen has developed humanized B-h4-1BB mice as an efficient platform of both in vivo MOA and safety studies of 4-1BB antibody.

In B-h4-1BB mice, murine colon cancer MC38 cells were subcutaneously implanted. Biomarkers, such as, mCD45, mCD3, mCD4, mCD8, mNK, mFoxp3, mCD11b, and etc., were profiled after the treatment with an 4-1BB antibody (Urelumab). FACS, IHC, and IF data showed that Urelumab have increased the ratio of CD8+ T cell, but on the other hand decreased Treg cell, the PD-1 and Tim3 (T cell exhausted marker) upon treatment. Toxicity have also been with the treatment of Urelumab using this model. The blood chemistry assays showed that the high dose Urelumab had toxicity effects compared with the control group and WT C57BL/6 mice. Based on these findings, we conclude that the B-h4-1BB humanized mouse model is a powerful platform for both MOA (biomarker) discovery and safety evaluation of 4-1BB antibody.

Citation Format: Jie Xiang, Dirui Li, Hao Yang, Gary Ng, Yuelei Shen. Immune biomarker profiling and safety evaluation response to 4-1BB antibody (Urelumab) in humanized 4-1BB mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 589.