bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1075: Conditionally active de novo IL-2 cytokine mimetics for targeted immunotherapy: de novo split technology
Authors: Quijano-Rubio Alfredo , Ali Lestat R. , Bhuiyan Md A. , Lajoie Marc J. , Dougan Michael , Baker David , Silva Daniel-Adriano
Published: September 2020

IL-2 is a highly potent, pleiomorphic cytokine approved for the treatment of melanoma and renal cell carcinoma. The therapeutic potential of this cytokine has been restricted by dose-limiting toxicities and off-tumor effects associated with systemic IL-2 administration. Scientists have long-pursued the development of targeted IL-2 variants to control biodistribution and improve the therapeutic index, e.g. IL-2-antibody fusions or immunocytokines. However, the potential benefit of targeting IL-2 is limited by the high affinity of the cytokine for its cellular receptor, which dominates biodistribution and results in activation of cells outside the tumor microenvironment. To address this challenge, we took advantage of the remarkable stability of Neoleukin-2/15 (Neo-2/15), a fully de novo cytokine mimetic that potently activates the beta and gamma subunits shared by the IL-2 and IL-15 receptors. We demonstrate that Neo-2/15 can be divided into two pieces that cannot signal individually, but that can recover their binding and cell-signaling potential when co-localized. Fusing the split molecules to independent targeting domains creates a modular and highly specific implementation of conditional activation. We demonstrate that if one split fragment is targeted to one tumor-associated antigen, and the complementary fragment is targeted to a second tumor-associated antigen, the ability to bind the IL-2 receptor and trigger signaling is only restored when these split fragments bind to the surface of cells expressing both targets. The split Neo-2/15 molecules have anti-tumor activity in syngeneic cancer models and have the potential to enhance the therapeutic index of cytokine mimetics in immunotherapy. To our knowledge, these are the first designed examples of strictly conditionally-active, targeted, cytokine-like molecules for immunotherapy.

*Current affiliation for D-A.S.: Neoleukin Therapeutics Inc, Seattle, WA, USA.

Citation Format: Alfredo Quijano-Rubio, Lestat R. Ali, Md A. Bhuiyan, Marc J. Lajoie, Michael Dougan, David Baker, Daniel-Adriano Silva. Conditionally active de novo IL-2 cytokine mimetics for targeted immunotherapy: de novo split technology [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 1075.
Abstract 1081: DNA methylation at the intersect of chromatin structure and transcriptome diversity
Authors: Ting Angela H. , Nanavaty Vishal , Abrash Elizabeth , Lee Byron , Fink Emily , Hwang Tae Hyun , Hong Changjin
Published: September 2020

Dysregulation of DNA methylation and mRNA alternative cleavage and polyadenylation (APA) are both prevalent in cancer, but they have been studied as independent processes. We discovered a DNA methylation regulated APA mechanism when we compared genome-wide DNA methylation and polyadenylation site usage between DNA methylation-competent and DNA methylation-deficient cells. Here we show that removal of DNA methylation enables CTCF binding and recruitment of the cohesin complex, which in turn, promotes proximal polyadenylation site usage. In this DNA de-methylated context, depletion of the RAD21 cohesin complex component can recover distal polyadenylation site usage. We also confirmed, using chromosomal conformation capture (3C), that CTCF and the cohesin complex mediate chromatin loops in the absence of DNA methylation to modulate polyadenylation site usage. Leveraging data from The Cancer Genome Atlas, we authenticated the relationship between DNA methylation and mRNA polyadenylation isoform expression in vivo. This DNA methylation regulated APA mechanism demonstrates how aberrant DNA methylation impacts transcriptome diversity and highlights the potential sequelae of global DNA methylation inhibition as a cancer treatment.

Citation Format: Angela H. Ting, Vishal Nanavaty, Elizabeth Abrash, Byron Lee, Emily Fink, Tae Hyun Hwang, Changjin Hong. DNA methylation at the intersect of chromatin structure and transcriptome diversity [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 1081.
Abstract 1082: Arid1a deletion results in enhanced osteosarcomagenesis and altered chromosome structure
Authors: Fatema Kaniz , Plyler Shawn , Pavek Adriene , Nartker Chris , Wang Yanliang , Jones Kevin , Barrott Jared
Published: September 2020

Osteosarcoma (OS) is a primary malignant bone tumor mainly affecting children and young adults. Early metastasis, chemoresistance and stagnation in the treatment options for the last 4 decades result in a less than 20% chance of long-term survival in metastatic OS. Investigating the molecular mechanisms underlying a more aggressive phenotype in osteosarcoma will certainly help develop novel treatment alternatives.

Through a transposon-mediated forward genetic screen, we were able to increase osteosarcomagenesis by random mutagenesis. Among the possible candidate genes on our list, we discovered a loss-of-function mutation in ARID1A. ARID1A is a member of the SWI/SNF chromatin-remodeling protein complex and has been implicated as a tumor suppressor in other cancers. We hypothesized that loss of ARID1A in osteosarcoma would increase the rate of tumor progression and metastasis by impacting the epigenetic configuration of the chromatin.

In-vitro (CRISPR/Cas9) deletion in human OS cell lines and cell behavioral studies validated that ARID1A loss contributes to faster tumorigenesis. To understand its effect in-vivo, we have developed mice cohorts with or without ARID1A with a Cre-mediated, Osteoblast specific (Osx) conditional knockout system. We observed that Arid1a-mutant mice develop tumors and die on an average 13 weeks earlier than that of the wildtype cohort. By 3D micro CT scanning, we further confirmed a relatively higher number of primary and metastatic tumors in the Arid1a mutant mice, albeit the histology and morphology of the tumors did not change. The analysis of genome-wide chromatin accessibility by ATAC-seq confirmed an open chromatin organization in Arid1a mutant mouse samples.

We conclude that, acting as an epigenetic regulator, ARID1A plays a significant role in osteosarcoma progression and metastasis. Further investigation to strengthen this link would present the opportunity for alternatively targeting genetically unstable cancers with epigenetic drugs.

Citation Format: Kaniz Fatema, Shawn Plyler, Adriene Pavek, Chris Nartker, Yanliang Wang, Kevin Jones, Jared Barrott. Arid1a deletion results in enhanced osteosarcomagenesis and altered chromosome structure [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 1082.
Abstract 1086: Downstream kinase signaling is dictated by specific KRAS mutations
Authors: Budagyan Konstantin , Chernoff Jonathan
Published: September 2020

Oncogenic KRAS mutations are common in colorectal cancer (CRC), found in ~50% of tumors, and are associated with poor prognosis and resistance to therapy. There is substantial diversity of KRAS alleles observed in CRC. Importantly, emerging clinical and experimental analysis of relatively common KRAS mutations at amino acids G12, G13, A146, and Q61 suggest that each mutation differently influences the clinical properties of a disease and response to therapy. For example, KRAS G12 mutations confer resistance to EGFR-targeted therapy, while G13D mutations do not. Although there is clinical evidence to suggest biological differences between mutant KRAS alleles, it is not yet known what drives these differences and whether they can be exploited for allele-specific therapy. We hypothesized that different KRAS mutants elicit variable alterations in downstream signaling pathways. To investigate this hypothesis, we created a novel system by which we can model KRAS mutants in isogenic mouse colon epithelial cell lines. To generate the cell lines, we developed an assay using fluorescent co-selection for CRISPR-driven genome editing. This assay involves simultaneous introduction of single-guide RNAs (sgRNAs) to two different endogenous loci resulting in double-editing events. We first introduced Cas9 and blue fluorescent protein (BFP) into mouse colon epithelial cell line containing heterozygous KRAS G12D mutation. We then used sgRNAs targeting BFP and the mutant G12D KRAS allele along with homology-directed repair (HDR) templates for a GFP gene and a KRAS mutant allele of our choice. Cells that successfully undergo HDR are GFP-positive and contain the desired KRAS mutation. Therefore, selection for GFP-positive cells allows us to identify those with phenotypically silent KRAS edits. Ultimately, this method allows us to toggle between different mutant alleles while preserving the wild-type allele, all in an isogenic background. Using this method, we have generated cell lines with endogenous heterozygous KRAS mutations commonly seen in CRC (G12D, G12V, G12C, G12R, G13D). In order to elucidate cellular signaling pathway differences between the KRAS mutants, we screened the mutated cell lines using a small-molecule library of ~160 protein kinase inhibitors. We found that there are mutation-specific differences in drug sensitivity profiles. These observations suggest that KRAS mutants drive specific cellular signaling pathways, and that further exploration of these pathways may prove to be valuable for identification of novel therapeutic opportunities in CRC.

Citation Format: Konstantin Budagyan, Jonathan Chernoff. Downstream kinase signaling is dictated by specific KRAS mutations [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 1086.
Abstract 1090: Regulation of PTEN translation by PI3K signaling maintains pathway homeostasis
Authors: Mukherjee Radha , Vanaja Kiran Gireesan , Boyer Jacob A. , Qiu Juan , Chen Xiaoping , de Stanchina Elisa , Chandarlapaty Sarat , Levchenko Andre , Rosen Neal
Published: September 2020

The PI3K pathway is a key regulator of metabolism, cell proliferation and migration and some of its components (e.g. PIK3CA and PTEN) are frequently altered in cancer by genetic events that deregulate its output. However, PI3K signaling is not usually the primary driver of these tumors and inhibitors of components of the pathway have only modest antitumor effects. We now show that both physiologic and oncogenic activation of the PI3K signaling by growth factors and an activating hotspot PIK3CA mutation respectively, cause an increase in the expression of the lipid phosphatase PTEN, thus limiting the duration of the signal and the output of the pathway in tumors. Pharmacologic and physiologic inhibition of the pathway by HER2/PI3K/AKT/mTOR inhibitors and nutrient starvation respectively reduce PTEN, thus buffering the effects of inhibition and contributing to the rebound in pathway activity that occurs in tumors. This regulation is found to be a feature of multiple types of cancer, non-cancer cell line and PDX models thereby highlighting its role as a key conserved feedback loop within the PI3K signaling network, both in vitro and in vivo. Regulation of expression is due to mTOR/4EBP1 dependent control of PTEN translation and is lost when 4EBP1 is knocked out. Translational regulation of PTEN is therefore a major homeostatic regulator of physiologic PI3K signaling and plays a role in reducing the output of oncogenic mutants that deregulate the pathway and the antitumor activity of PI3K pathway inhibitors.

Citation Format: Radha Mukherjee, Kiran Gireesan Vanaja, Jacob A. Boyer, Juan Qiu, Xiaoping Chen, Elisa de Stanchina, Sarat Chandarlapaty, Andre Levchenko, Neal Rosen. Regulation of PTEN translation by PI3K signaling maintains pathway homeostasis [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 1090.
Abstract 1131: Preventive effect of Celecoxib in Sorafenib-related hand-foot syndrome in hepatocellular carcinoma patients, a single-center, open-label, randomized, controlled clinical phase III trial
Authors: CHEN JIANCONG
Published: September 2020

Background: Skin toxicity, especially hand-foot syndrome (HFS), is one of the most common sorafenib-induced adverse events (AEs) in hepatocellular carcinoma (HCC) patients, leading to treatment interruption and failure. Mucocutaneous inflammation may cause HFS; therefore, we investigated whether celecoxib can alleviate HFS, improve patients' quality of life and increase survival when administered in conjunction with active therapy.

Methods: Our randomized, open-label study prospectively enrolled 116 advanced HCC patients receiving sorafenib as targeted therapy from July 2015 to July 2016. All patients were randomly assigned (1:1) via a computer-generated sequence to receive sorafenib with or without celecoxib. Sorafenib-related AEs were recorded, Survival was compared between the two groups.

Results: Compared to the Sorafenib group, the SoraCele group had lower incidence rates of ≥ grade 2 and grade 3 HFS (63.8% vs 29.3%, P < 0.001; 19.0% vs 3.4%, P = 0.008, respectively), hair loss, rash and abdominal pain. Kaplan-Meier analysis revealed a lower risk of ≥ grade 2 HFS (HR, 0.384; P = 0.002) and a lower dose reduction/interruption rate (46.6% to 15.5%, P < 0.001) in the SoraCele group. Cox proportional hazards regression analysis demonstrated that celecoxib was the only independent predictive factor of developing ≥ grade 2 HFS (HR, 0.414; P = 0.004). Longer progression-free survival (PFS) was also observed in the SoraCele group (P = 0.039), although overall survival was not prolonged (P = 0.305).

Conclusions: Sorafenib+Celecoxib administration alleviated sorafenib-related skin toxicity. Longer PFS was achieved in clinical practice, although overall survival was not prolonged (ClinicalTrials.gov: NCT02961998).

Keywords: hand-foot syndrome; celecoxib; sorafenib; hepatocellular carcinoma

Citation Format: JIANCONG CHEN. Preventive effect of Celecoxib in Sorafenib-related hand-foot syndrome in hepatocellular carcinoma patients, a single-center, open-label, randomized, controlled clinical phase III trial [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 1131.
Abstract 1133: Risk effect of primary prevention strategy with aspirin on lung cancer incidence depends on high-level use, bodyweight and age: A U.S. adults evidence
Authors: Dong Xuesi
Published: September 2020

Chemoprevention of cancer with aspirin is of particular interest as a primary prevention strategy, which was still in controversial. Using 101,462 individual participants' data from a U.S. adult's cohort, Cox regression model was used to detect the aspirin dosage effect on lung cancer risk. We calculated the effects of irregular use, low-dose use, and high-dose use of aspirin on lung cancer risk in population stratified by bodyweight and age. High-dose use of aspirin raised up 28% risk of lung cancer compared to no use (HR = 1.28, 95%CI = [1.14-1.45], P = 3.37×10-5). Meanwhile, the risk effect of high-dose aspirin significantly decreased with bodyweight increasing, indicating an antagonistic interaction (HRinteraction = 0.96, 95%CI = [0.94-0.99], P = 1.26×10-2). Furthermore, for these participants prone to lung cancer with bodyweight less than 80 kg, high-dose aspirin usage showed an elevated risk if their age less than 76 (HRage≤76 = 1.47, 95%CI = [1.25-1.73], P = 3.81×10-6), but did not exhibit risk effect in elders (HRage>76 = 1.13, 95%CI = [0.46-2.69], P = 0.80). The three-way interaction among dose of aspirin, bodyweight and age indicated that these vulnerable people (age ≤76 years and bodyweight <80 kg) with high lung cancer risk should be cautious to use high-dose aspirin for lung cancer chemoprevention.

Citation Format: Xuesi Dong. Risk effect of primary prevention strategy with aspirin on lung cancer incidence depends on high-level use, bodyweight and age: A U.S. adults evidence [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 1133.
Abstract 1150: Targeting PI3K inhibits ER-mammary tumor initiation and early dissemination
Authors: Zhang Yuan , Wang Jingyang , Xiao Yi , Seewaldt Victoria , Yu Dihua
Published: September 2020

Despite recent advances in cancer treatment, including targeted therapies and immunotherapies, many women still die of breast cancer. To ultimately reduce breast cancer death, it is of urgent need to develop more effective prevention and early intervention strategies. Although tamoxifen and aromatase inhibitors decrease incidences of estrogen receptor positive (ER+) breast cancer, there is no effective agent for prevention of ER negative (ER-) breast cancer. In this study, we identified that PI3K pathway is significantly activated in premalignant ER- breast lesions compared to paired normal tissue from breast cancer patients. We therefore tested whether blocking PI3K pathway by genetic knockdown or using a clinically applicable PI3K inhibitor (GDC0941) can inhibit ER- tumor initiation and progression. Indeed, both genetic knockdown of PI3K and low dose GDC0941 intervention reversed the disorganized three-dimensional growth of semi-transformed human ER- mammary epithelial cells in vitro. More importantly, low dose GDC0941 treatment significantly delayed the tumor initiation in MMTV-neu mouse model which develops ER-HER2+ mammary tumors. Notably, low dose GDC0941 treatment did not show discernable adverse effects, including no significant changes in body weight and liver functions, no immuno-toxicity. Since PI3K activation is crucial for early disseminated tumor cells, we collected bone marrow from treated mice versus vehicle control group, and examined the impact of GDC0941 treatment on early dissemination of tumor cells into the bone marrow. Excitingly, we found that administration of low dose GDC0941 significantly decreased CD45-ErBb2+/CK8/18+ early disseminated mammary tumor cells into the bone marrow, indicating that low dose PI3K inhibitor not only delayed primary tumor initiation but also prevented tumor cell early dissemination in the bone marrow. Taken together, these data support the notion that using low dose targeting drug to block PI3K pathway is safe and promising for ER- breast cancer prevention.

Citation Format: Yuan Zhang, Jingyang Wang, Yi Xiao, Victoria Seewaldt, Dihua Yu. Targeting PI3K inhibits ER-mammary tumor initiation and early dissemination [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 1150.
Abstract 1170: Socioeconomic disparities in patients with esophageal cancer at time of diagnosis: A national cancer data base analysis
Authors: Sadaps Meena , Mehta Neal , Wei Wei , Tullio Katherine , Bhatt Amit , Khorana Alok
Published: September 2020

Introduction: There have been noted to be significant gender and socioeconomic disparities in the incidence, prognosis, and clinical outcomes of patients with esophageal cancer. The purpose of this study was to characterize disparities amongst patients with esophageal cancer at time of diagnosis, as stratified by their stage, as there is a paucity of published data in this realm.

Methods: We identified patients in the National Cancer Data Base diagnosed with esophageal cancer, from 2004 to 2016 and grouped them by stage: early stage (stage I-III) and advanced stage (stage IV). These groupings were then compared based on various patient demographics and socioeconomic factors, including age, gender, insurance status, education, and income. p-values were determined by chi-square test.

Results: 102,540 patients (79.5% male; 84.0% Caucasian) were included in our analysis. There was a statistically significant increase in the diagnosis of de novo metastatic disease seen with younger age (⇐59 years), male gender, lack of insurance coverage, residential communities with lower education, and lower income (<$38,000 annual income) as shown in Table 1.

Summary Table of Stage at Diagnosis by Patient CharacteristicsTotal NTotal %Stage 0-IIINStage 0-III%Stage IVNStage IV %p-valueAge⇐59298301001840361.691142738.31<0.000160-74475031003244668.31505731.7>=75252071001789971.01730828.99GenderFemale210701001510671.69596428.31<0.0001Male814701005364265.842782834.16InsuranceUnknown2902100194967.1695332.84Government612231004204368.671918031.33<0.0001Not Insured3450100184053.33161046.67Private349651002291665.541204934.46Community Median IncomeUnknown52610035567.4917132.51<38K185611001221065.78635134.22<0.000138-63K526941003531767.021737732.98>=63K307591002086667.84989332.16Percent No High School Degree Quartiles 2012-2016Unknown142710091664.1951135.81>=17.6%197021001290265.49680034.51<0.000110.9-17.5%274621001845067.18901232.826.3-10.8%300291002013867.06989132.94<6.3%239201001634267.053379232.95

Conclusion: Data from the National Cancer Data Base supports the association of younger age and lower socioeconomic status with higher stage at diagnosis in patients with esophageal cancer, highlighting the need for increased awareness and education in these at-risk subgroups.

Citation Format: Meena Sadaps, Neal Mehta, Wei Wei, Katherine Tullio, Amit Bhatt, Alok Khorana. Socioeconomic disparities in patients with esophageal cancer at time of diagnosis: A national cancer data base analysis [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 1170.
Abstract 1232: Synergistic roles played by autophagy and Hsp70 in cancer cell survival
Authors: Sannino Sara , Brodsky Jeffrey L.
Published: September 2020

To avoid cell death, cancer cells adapt to extreme conditions. Chaperone upregulation, modulation of the unfolded protein response (UPR), and induction of protein degradation pathways are critical for cancer cell survival and metastasis in some cancers. Thus, it is not surprising that the molecular chaperone Hsp70 is upregulated in many cancers. For example, increased Hsp70 abundance correlates with metastasis formation and poor prognosis in breast cancer patients. We previously demonstrated that inhibition of Hsp70, by treating cells with a specific inhibitor known as MAL3-101, induces rhabdomyosarcoma cancer cell death due to induction of a UPR-mediated apoptotic response (Sabinis et al., 2016). Moreover, by taking advantage of a MAL3-101-resistant cell line, we established that protein degradation pathways act as a compensatory mechanism in MAL3-101 resistant rhabdomyosarcoma cells. In particular, we found that autophagy inhibition re-sensitized resistant cells to Hsp70 inhibition, suggesting that autophagy is a key compensatory mechanism for Hsp70 inhibition. Autophagy was further induced by MAL3-101 treatment in these cells, as evidenced by both autophagy related gene mRNA and protein accumulation as well as by autophagic-like structures detected by electron microscopy (Sannino et al., 2018). These data highlight a pro-survival role for autophagy induction upon exposure to an Hsp70 inhibitor in cancer, and provide a link between Hsp70, proteasome activity, the UPR, and autophagy in rhabdomyosarcoma. We next asked if other cancers might be dependent on Hsp70 activity and investigated the benefit of combined treatment with autophagy and/or proteasome inhibitors together with MAL3-101 in breast cancer cells. Our recent studies suggest that diverse breast cancer cell lines are either sensitive or resistant to Hsp70 inhibition. MAL3-101 resistant breast cancer cells have higher autophagy levels compared to MAL3-101 sensitive lines. Moreover, autophagy is upregulated upon Hsp70 inhibition in resistant breast cancer cells, again highlighting the potential benefit of combined treatment with autophagy and Hsp70 inhibitors. Overall, our work positions autophagy as a critical compensatory mechanism when molecular chaperone function is overwhelmed and misfolded proteins accumulate in cancer cells. Further investigations will reveal the potential for Hsp70 inhibitors and the role of protein degradation pathways in vivo in breast cancer.

Citation Format: Sara Sannino, Jeffrey L. Brodsky. Synergistic roles played by autophagy and Hsp70 in cancer cell survival [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 1232.
Abstract 1251: Epigenetics in early onset colorectal cancer (EOCRC)
Authors: Riggins Karen , Musher Benjamin , Scheurer Michael , Yang Li , Castro Patricia , Zarrin-Khameh Neda , Shen Lanlan
Published: September 2020

Colorectal cancer (CRC) incidence rates have increased among young adults while the rates have decreased among older adults over 50 years of age. While the etiology of the growing prevalence of young adults with newly diagnosed CRC is unknown, there has been growing interest in the roles implicated for diabetes, obesity, environmental changes, and epigenetics. Among the epigenetic marks implicated in EOCRC, the most widely studied are the global DNA hypomethylation. Using mouse studies, our group has demonstrated that DNA methylation as an epigenetic mechanism to control gene expression is essential to postnatal development. We have discovered a key set of genes that undergo epigenetic changes in colonic epithelial stem cells, and these changes are stable over a lifetime. We have shown that the gut microbiome influences this process. Importantly, perturbation of established intestinal DNA methylation leads to accelerated intestinal tumor development. To translate our mechanistic studies in the animal model to humans, we have confirmed these CpG loci that are concordant between the two species (i.e. homologous regions and/or conserved CpG islands). Here, we hypothesize that the dysregulation of developmentally programmed intestinal DNA methylation is associated with enhanced susceptibility to colon cancer in young adult. We have identified a population of patients within the Harris Health and Baylor College of Medicine system under the age of 50 with mismatch repair stable colorectal adenocarcinoma. Patient characteristics including age, sex, race, and primary tumor profile (stage at diagnosis, histology, and RAS status) have been defined. Using a panel of 6 genes and bisulfite pyrosequencing for quantitative assessment of DNA methylation, we compared the level of methylation in EOCRC tumors to paired normal adjacent tissues as well as normal adult tissue (age 50 or greater) to traditional CRC tumors. Our preliminary data suggest hypermethylation patterns are distinct in patients of EOCRC relative to traditional CRC. Our ultimate goal is to better understand the etiology of EOCRC in order to devise better prevention and treatment strategies for EOCRC.

Citation Format: Karen Riggins, Benjamin Musher, Michael Scheurer, Li Yang, Patricia Castro, Neda Zarrin-Khameh, Lanlan Shen. Epigenetics in early onset colorectal cancer (EOCRC) [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 1251.
Abstract 1274: Metformin, not γ-tocotrienol, increases gene methylation and decreases expression of genes responsible for cell signal transduction in prostate cancer cells
Authors: Moore Christine Ann , Lightner Janet , Duffourc Michelle , Krishnan Koyamangalath
Published: September 2020

Metformin and γ-tocotrienol (GT3) are promising chemopreventative agents with studies showing improved patient outcomes with metformin use in several cancer types, including prostate cancer. However, the cellular mechanisms underlying the improved survival rates in cancer patients treated with these drugs are incompletely understood. Inhibition of mammalian target of rapamycin (mTOR), alteration of p-c-JUN and p-ERK signaling pathways and inhibition of mitochondrial complex 1 have all been implicated as part of metformin's anti-tumorigenesis activities. More recently, metformin was shown to influence the activity of epigenetic modifying enzymes resulting in genome-wide alterations in DNA methylation patterns in breast cancer cell lines. In this study, we investigated whether GT3 and metformin treatment would alter methylation pathways in prostate cancer cells.The prostate cancer cell line PC-3 (androgen independent) and normal prostate control cell line RWPE-1 were treated with varying concentrations of either GT3, metformin, the methylation inhibitor azacitidine, or metformin plus azacitidine for 6 hours. Genomic DNA was isolated, and methylation assessed using the Illumina Infinium methylation EPIC BeadChip array. Analysis revealed treatment of PC-3 cells with metformin promoted hypermethylation of CC2D2B, GHDC, LINC01377, PLPP2, and PTK2 and decreased methylation of TSPY4 as compared to normal prostate controls. qRT-PCR experiments confirmed decreased expression of PTK2 mRNA, consistent with hypermethylation of PTK2. In contrast, GT3 did not affect methylation of any identifiable recognizable genes. This study suggests metformin, but not GT3, may inhibit prostate cancer proliferation partially through epigenetic changes which disrupt downstream focal adhesion kinase (FAK) signaling pathways. Metformin is a promising chemopreventive agent; studies investigating the impact of metformin on cellular adhesion and FAK pathways are ongoing in our laboratory.

Citation Format: Christine Ann Moore, Janet Lightner, Michelle Duffourc, Koyamangalath Krishnan. Metformin, not γ-tocotrienol, increases gene methylation and decreases expression of genes responsible for cell signal transduction in prostate 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 1274.
Abstract 1324: Genomic and clinical characteristics of MET exon14 alterations in 26,391 Chinese cancer patients
Authors: Liu Sisi , Song Danni , Tong Xiaoling , Wu Xue , Wang Xiaonan , Shao Yang Washington
Published: September 2020

Background: Alterations in MET exon 14 (METex14) and its flanking intronic regions have been identified in a variety of cancers, and suggested to cause enhanced MET activity and oncogenesis. Clinical evidences suggested that patients with METex14 alterations often benefit from MET inhibitors such as crizotinib. Given the unique mutation profiles of Chinese lung cancer patients, it is necessary to investigate the prevalence of METex14 alterations in a large cohort of cancer patients.

Patients and methods: Cases carrying METex14 alterations were screened from 26,391 Chinese cancer patients, and the clinicopathologic and molecular characteristics of the cohort were reviewed. PD-L1 immunohistochemistry and the treatment response to MET inhibitors were also analyzed for cases with such information available.

Results: Compared to Western cancer population (3~7%), the frequency of METex14 alterations is much lower in Chinese cancer patients (0.7%, n=184) and in Chinese lung cancer patients (1.1%, n=175). Seventy-eight distinct METex14 alterations were detected, with majority of alterations are base substitutions at splice donor site (42%) and insertion/deletions (indels) in intron 13 (28%). Concurrent MET copy gain and non-exon14 MET mutations were found in 4% and 11% of cases, respectively. Other driver alterations, such as EGFR copy gain (11%) and mutations (8%), KRAS (5%) and PIK3CA (5%), appeared in a mutually exclusive pattern. In 71 patients sequenced by 425-gene panel, gene copy gains were prevalent in MDM2 (24%), CDK4 (14%), MCL1 (13%), TERT (13%), MYC (11%), CD274 (7%) and CDK6 (6%). Female patients contain much less TP53 mutations than male patients (65% vs. 24%, FDR = 0.01). Of 9 tissue specimens having PD-L1 immunohistochemistry (IHC) results, 5 of them (55.5%) were found PD-L1 positive, indicating the option for immunotherapy. In 14 patients who received crizotinib-treatment, the median progression free survival (mPFS) was 7 months with the longest PFS of 17 months. Upon resistance to crizotinib, two patients acquired secondary mutations in MET and one patient acquired BRAF p.K601E that can be a novel resistance mechanism to crizotinib treatment.

Conclusion: Chinese cancer patients have a relatively lower frequency of METex14 alterations compared to Western patients, and these patients can be potentially treated by immunotherapy and crizotinib.

Citation Format: Sisi Liu, Danni Song, Xiaoling Tong, Xue Wu, Xiaonan Wang, Yang Washington Shao. Genomic and clinical characteristics of MET exon14 alterations in 26,391 Chinese cancer patients [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 1324.
Abstract 133: Micro-environment on demand (MOD): A versatile technology for cognate T-cell receptor - neoantigen discovery
Authors: Srinivasan Maithreyan , Madrigal Justin , Schoepp Nathan , Cole Russell
Published: September 2020

NCI estimates that cancer will be the leading cause of death in 2030, worldwide. Checkpoint inhibitors and adoptive cell therapies (ACTs) cost up to ~$2 million/patient and have shown durable responses in a few patients while many patients experience life-threatening side-effects. So, there is a critical need to identify cost-effective therapies to treat cancer with minimal to no side-effects. Deep mapping of cancer antigens and their cognate immune cells is one approach that has the potential for a more universal/targeted treatment. However, systematic methods do not exist to identify cancer antigens and their cognate immune cells functionally and link phenotypic readouts to antigen and immune cell DNA sequences. Here we present a single cell handling and analysis platform based on Printed Droplet Microfluidics, a new droplet microfluidic technology that enables the precise construction of cell-cell interaction experiments with single cell resolution, followed by functional and genomic analysis. At the core of the technology is a fluorescence-activated flow sorter repurposed to deterministically sort and merge droplets containing single cells, beads, reagents, and drugs, collect and assay merged droplets and resort assay-positive droplets for downstream genomic analyses. The selectivity of the sorting/merging technology enables both the construction of experiments with up to one million precise cell combinations and the performance of flexible multi-step molecular biology workflows. We will show proof-of-concept for the MOD platform that integrates three technologies after fluorescently labeled single-cells are encapsulated in droplets - droplet-based cell-sorting and merging, single-cell-based cytokine assays and selective sequencing of T-cell receptor variable regions based on cytokine signals. The technology has the potential to rapidly accelerate the rate of immunotherapy discovery by enabling a larger number of experiments to be performed with higher precision, more modes of analysis, and less starting material than competing bulk technologies.

Citation Format: Maithreyan Srinivasan, Justin Madrigal, Nathan Schoepp, Russell Cole. Micro-environment on demand (MOD): A versatile technology for cognate T-cell receptor - neoantigen discovery [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 133.
Abstract 1374: Preclinical investigation of ATR inhibition alone and in combination with PARP inhibition in high risk neuroblastoma
Authors: Southgate Harriet E. , Chen Lindi , Curtin Nicola J. , Tweddle Deborah A.
Published: September 2020

Background:Neuroblastoma (NB) is the commonest extra-cranial malignant solid tumour of childhood and one of the most difficult to cure. DNA damage response (DDR) defects are frequently observed in high risk NB including allelic loss and loss of function mutations in key DDR genes, oncogene induced replication stress (RS) and cell cycle checkpoint dysfunction. Cancer cells with defective cell cycle checkpoint signalling and/or increased oncogene-driven RS are acutely dependent on the DNA damage sensor kinase ATR. This study aims to identify features of NB cell lines leading to ATR inhibitor sensitivity. As PARP inhibition causes RS through unrepaired single strand DNA breaks progressing to replication, we hypothesise that ATR inhibition will increase PARP inhibitor cytotoxicity.

Aims: 1) To determine which molecular features lead to sensitivity to VE-821 (ATR inhibitor) in cell lines derived from high-risk NB tumours. 2) To assess synergism between PARP inhibition with olaparib and ATR inhibition in high risk NB cell lines and to measure RS.

Materials and Methods: Cell proliferation in response to 72 hours treatment with VE-821 was assessed by XTT assay (Roche) in a panel of 11 NB cell lines and the effect of ATR inhibition on olaparib growth inhibition in 4 cell lines: SHSY5Y, SKNAS, NGP and N20_R1. CHK1S345 and H2AXS129 phosphorylation was assessed using Western blotting to determine ATR activity and RS respectively. RS was also measured by γH2AX foci formation using immunofluorescent microscopy.

Results: VE-821 caused significantly more growth inhibition in MYCN amplified cell lines and cell lines with low ATM protein expression by XTT assay (p<0.05 Mann-Whitney U test). Olaparib (5 µM) treatment increased CHK1S345 and H2AXS129 phosphorylation after 24 hours treatment in all cell lines. H2AXS129 phosphorylation and foci number was further increased with the addition of VE-821 (1 µM). ATR inhibition prevented CHK1S345 phosphorylation. In cell proliferation assays, combination index analysis (Calcusyn) showed that ATR inhibition by VE-821 is synergistic with olaparib at sub lethal concentrations (<1 µM) (CI value 0.04-0.89).

Conclusion: MCYN amplification and low ATM protein expression are determinants of ATRi sensitivity in NB cell lines. ATR inhibition by VE-821 is synergistic with olaparib at sub lethal concentrations (<1 µM) and further increases the replication stress caused by PARP inhibition.

Citation Format: Harriet E. Southgate, Lindi Chen, Nicola J. Curtin, Deborah A. Tweddle. Preclinical investigation of ATR inhibition alone and in combination with PARP inhibition in high risk neuroblastoma [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 1374.
Abstract 1378: Targeting the metabolic-epigenetic axis to sensitize HR-proficient ovarian cancer to PARP inhibitors
Authors: Dahl Erika S. , Leon Kelly E. , Chen Chi-Wei , Jia Qingyuan , Buj Raquel , Snyder Nathaniel W. , Aird Katherine M.
Published: September 2020

Overexpression of the oncogene cyclin E1 is considered one of the initiating factors in fallopian tube transformation and ovarian tumorigenesis. Patients with high cyclin E1 expression have worse overall survival than patients with low expression. Cyclin E1 overexpression increases expression of DNA damage response (DDR) genes in order to tolerate DNA damage and bypass senescence, a state of cell cycle arrest. However, the mechanism by which cyclin E1-high cells transcriptionally increase DDR gene expression to bypass senescence remains unclear. We found that cyclin E1 overexpression alters the metabolic-epigenetic axis through wildtype isocitrate dehydrogenase I (wtIDH1), a TCA cycle enzyme. Upregulation of wtIDH1 in cyclin E1-high cells increased the transcription of multiple DDR genes related to homologous recombination (HR), including BRCA1, BRCA2, and RAD51. Published data from our lab demonstrates that wtIDH1 primarily converts isocitrate to alpha-ketoglutarate (αKG) in cyclin E1-high ovarian cancer cells, and suppression of wtIDH1 and αKG pools induces senescence through increased repressive histone marks. Our current data suggest that wtIDH1 is both necessary and sufficient for HR gene expression in cyclin E1-high cells in part through modulation of histone methylation. Functionally, inhibition of wtIDH1 in cyclin E1-high fallopian tube cells induced senescence via decreased HR and marked accumulation of DNA damage. Cyclin E1-high ovarian cancer tumors are HR-proficient and resistant to emerging poly(ADP-ribose) polymerase (PARP) inhibitors. Interestingly, inhibition of both wtIDH1 and PARP in combination increased apoptosis of cyclin E1-high ovarian cancer cells in vitro, suggesting this may be a novel therapy for HR-proficient ovarian cancers. Together, our data suggest that wtIDH1-mediated metabolism affects the epigenome in cyclin E1-high cells, which contributes to both fallopian tube transformation and HR-proficiency. Targeting wtIDH1 with current FDA-approved inhibitors may therefore be a rational therapeutic strategy for cyclin E1-high ovarian cancer patients.

Citation Format: Erika S. Dahl, Kelly E. Leon, Chi-Wei Chen, Qingyuan Jia, Raquel Buj, Nathaniel W. Snyder, Katherine M. Aird. Targeting the metabolic-epigenetic axis to sensitize HR-proficient ovarian cancer to PARP inhibitors [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 1378.
Abstract 1389: Exploiting inherent DNA damage repair defects in IDH1/2 mutated gliomas with the CNS penetrant PARP inhibitor, pamiparib
Authors: Hong Christopher , Sule Amrita , Beckta Jason , Sundaram Ranjini , Bindra Ranjit
Published: September 2020

Our group recently demonstrated that IDH1/2 mutated gliomas exhibit intrinsic homologous recombination (HR) defects mediated by the oncometabolite, 2HG. These defects render IDH1/2 mutated cells exquisitely sensitive to PARP inhibitors (PARPi's), which suggests a novel therapeutic strategy. Here, we studied the potential in vitro and in vivo efficacy of a CNS penetrant PARPi, pamiparib, in combination with radiation therapy (RT) and temozolomide (TMZ) against IDH1/2 mutated gliomas. Our central hypothesis is that multi-modality therapy using agents that are active against 2HG producing cells will increase efficacy, while allowing lower doses of RT and TMZ as a means to mitigate normal tissue toxicity. We performed a comprehensive series of DNA repair functional studies, short and long term viability assays, as well as in vivo studies, using engineered and patient-derived IDH1/2 mutant glioma models. We tested unique combinations of pamiparib, TMZ and RT in these experiments. In vitro short term viability assays and long-term clonogenic survival assays in model IDH1 wild-type (WT) and R132H mutated glioma cells demonstrated that the mutation confers enhanced TMZ sensitivity (IC50 602 vs. 9.5 uM, respectively). A similar differential sensitivity was observed with RT. We also demonstrated significant, mutant IDH1 selective pamiparib sensitivity in vitro (IC50 0.67 vs. 141 uM). Western blot analysis of PARylation, a functional readout of PARPi activity, confirmed PARylation inhibition by pamiparib in a dose dependent manner. Subsequently, we demonstrated synergistic interactions between TMZ and RT combined with pamiparib, particularly at low doses of both DNA damaging agents, in an IDH1 selective manner. Finally, we demonstrated the activity of PARPi's against IDH1 mutant tumors in vivo, including pharmacodynamic and pharmacokinetic properties of pamiparib on target activity within the brain. Pamiparib is a promising, CNS penetrant PARP inhibitor that may selectively target IDH1/2 mutated gliomas, based on intrinsic HR defects associated with these tumors. Pamiparib appears to act synergistically with TMZ and RT, which are the backbone of GBM therapies. These data lay the groundwork for a future trial testing “trimodality therapy” (i.e., pamiparib, RT and TMZ) against IDH1/2 mutant gliomas.

Citation Format: Christopher Hong, Amrita Sule, Jason Beckta, Ranjini Sundaram, Ranjit Bindra. Exploiting inherent DNA damage repair defects in IDH1/2 mutated gliomas with the CNS penetrant PARP inhibitor, pamiparib [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 1389.
Abstract 1390: Inhibition of the catalytic activity of hUNG2 sensitizes multiple cancer cell lines to 5-FdUrd killing
Authors: Christenson Eric S. , Cui Junru , Gizzi Anthony , Meshesha Mesfin , Stivers James T.
Published: September 2020

Previously reported shRNA knockdown studies have established that expression of the DNA repair enzyme human uracil DNA glycosylase (hUNG2) is protective against 5-flourodeoxyuridine (5FdUrd) cell killing in p53 mutated cancer cell lines, but not in cell lines with functional p53. Here we take a different approach to understanding the role of hUNG2 in 5FdUrd chemotherapy by selectively inhibiting the catalytic activity of hUNG2 using the small bacterial inhibitor protein UGI. UGI binds extremely tightly to the DNA binding and catalytic site of hUNG2, effectively mimicking the impact of a small molecule inhibitor and allowing us to explore the potential therapeutic vulnerabilities that loss of hUNG2 activity produces in p53 mutated and wild-type cancer cells. Accordingly, we used lentiviral transduction to introduce UGI, under control of a doxycycline-inducible promoter, into a large panel of cancer cell lines. In vitro hUNG2 activity measurements using cell extracts established that all lines had robust activity before induction of UGI and undetectable activity after induction. We find that some wild-type and mutant p53 cancer lines from different tissues show significant increases in drug sensitivity. For instance, the p53 wild-type MCF7 breast cancer line showed a 10,000-fold increase in sensitivity, while the p53 mutant HT29 colorectal cancer line showed a 50-fold increased sensitivity. Overall, the results do not support the proposal that p53 status itself determines whether a cancer cell will be sensitized to 5FdUrd in the presence of hUNG2 inhibition. Ongoing studies are focused on identifying the cellular factors that contribute to drug sensitivity under conditions of hUNG2 inhibition. Realizing the full therapeutic benefit of 5FdUrd requires identification of the cancer cell phenotypes that are sensitized upon hUNG2 inhibition.

Citation Format: Eric S. Christenson, Junru Cui, Anthony Gizzi, Mesfin Meshesha, James T. Stivers. Inhibition of the catalytic activity of hUNG2 sensitizes multiple cancer cell lines to 5-FdUrd killing [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 1390.