bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 575: Brain delivery of clinically suitable nanobioconjugates to inhibit glioblastoma growth through extracellular matrix-immune cell crosstalk
Authors: Ljubimov Alexander V. , Patil Rameshwar , Ding Hui , Israel Liron , Holler Eggehard , Ljubimova Julia Y. , Sun Tao , Black Keith L.
Published: April 2023

Introduction: Tumor growth, invasion, and escape from immune surveillance largely depend on cancer microenvironment. Laminins are trimeric proteins and essential components of glioblastoma (GBM) microenvironment/extracellular matrix (ECM). In brain glioma samples from 230 patients, we found a correlation between the overexpression of tumor ECM protein laminin-411 (α4β1γ1) and faster tumor recurrence with shorter patient survival. Laminin-411 is produced by endothelial cells, neutrophils, monocytes, platelets, lymphocytes, and glioma cells and can modulate the immune system. Novel nanotechnology approach to block trimeric laminin-411 and activate of brain local immune system with brain delivery of PD-1 checkpoint inhibitor was developed for future translational application.

Methods: Nanobioconjugates (NBC) based on poly (β-L-malic acid, P), were synthesized, characterized and used to intravenously treat mice with intracranial syngeneic GL261 or CT-2A GBM. The lead NBCs P/PEG/LLL(40%)/AON(α4β1)(2.0%)/AP-2(2%) and P/PEG/LLL(40%)/AP-2(2%)/αPD-1(0.2%) contained antisense to laminin-411 α4 and β1 chains, or αPD-1 antibody as well as trileucine (LLL) peptide for endosomal escape and AP-2 peptide for BBB crossing and tumor cell targeting. CRISPR/Cas9 constructs were used to knockdown α4 and β1 laminin chains in GBM ex vivo. Flow cytometry and RNA-seq analyses were performed to evaluate treatment.

Results: Laminin-411 depletion with CRISPR/Cas9 and multifunctional NBC in vivo treatment equally suppressed GBM growth and significantly prolonged animal survival. The brain privileged immune system was activated upon treatment with a significant increase of CD3+, CD8+ T cells, NK, IFNγ+ NK cells, and M1 macrophages. RNA-sec analyses after treatment with a combination of NBC suppressing laminin-411 and checkpoint PD-1 showed enhanced anti-tumor effect with upregulation of genes coding for apoptotic Caspase 3 and IFNγ, and reduction of proliferation markers EGFR, c-Myc and Ki-67.

Conclusion: Study describes novel GBM treatment strategy via NBCs crossing blood-brain barrier and targeting critical ECM and immune components of tumor microenvironment largely independent of heterogeneous genetic mutations in glioblastoma.

Citation Format: Alexander V. Ljubimov, Rameshwar Patil, Hui Ding, Liron Israel, Eggehard Holler, Julia Y. Ljubimova, Tao Sun, Keith L. Black. Brain delivery of clinically suitable nanobioconjugates to inhibit glioblastoma growth through extracellular matrix-immune cell crosstalk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 575.
Abstract 5754: Determining the regulatory logic of breast cancer cells using single-cell multi-omics
Authors: Regner Matthew J. , Thennavan Aatish , Garcia-Recio Susana , Wisniewska Kamila , Spanheimer Philip M. , Parker Joel S. , Perou Charles M. , Franco Hector L.
Published: April 2023

Cancer cells rewire regulatory elements scattered throughout the genome (such as enhancers) to drive aberrant gene expression. Thus, deconvoluting the regulatory mechanisms that contribute to oncogenic gene expression in cancer cells is key to understanding tumor biology. To this end, we have charted the transcriptional and epigenetic landscape of breast cancer at single-cell resolution to quantitatively link variation in chromatin accessibility to gene expression across malignant and non-malignant cell types. Our comprehensive dataset profiles the chromatin landscape (scATAC-seq) in concert with the transcriptional profiles (scRNA-seq) of 4 breast cancer cell lines, 12 primary breast tumors, and 4 normal mammary reduction tissue specimens collected and processed immediately after surgical resection. This dataset, encompassing over 250,000 individual cells, allowed us to define the regulatory logic of cancer cells by 1) revealing how the epigenome underlies cellular heterogeneity of these tumors in comparison to normal mammary tissue, 2) defining how malignant cells hijack enhancer elements to drive key transcriptional programs in a subtype-specific manner, and 3) annotating which cancer-specific enhancer-to-gene connections portend a worse outcome in patients. Notably, we discovered that cancer cells acquire de novo non-coding enhancer elements to modulate hallmark cancer pathways that were previously hidden using bulk genomics approaches. This highlights the potential for cancer-specific enhancers to serve as markers with diagnostic and prognostic potential, or even serve as tractable targets for therapeutic intervention. Together these data enable the annotation of the cellular composition, transcriptional, and epigenetic landscape of breast tumors to help pinpoint clinically relevant mechanisms of tumorigenesis.

Citation Format: Matthew J. Regner, Aatish Thennavan, Susana Garcia-Recio, Kamila Wisniewska, Philip M. Spanheimer, Joel S. Parker, Charles M. Perou, Hector L. Franco. Determining the regulatory logic of breast cancer cells using single-cell multi-omics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5754.
Abstract 5777: Race as a predictive confounder in the rate of transdifferentiation and drug response in human pancreatic acinar ductal metaplasia
Authors: Perkins Corey Melissa , Jiang Jinmai , Hakimjavadi Hesam , Brant Jason , Zhang Zhongyue , Lee Ji-Hyun , Quashie David , Mao Yating , Ali Jamel , Kim Sarah , Campbell-Thompson Martha , Schmittgen Thomas
Published: April 2023

Pancreatic diseases (chronic pancreatitis, type 2 diabetes, pancreatic cancer) disproportionately affect the Black/African American (AA) community in comparison to non-White Hispanics and Whites. Acinar to ductal metaplasia (ADM), the process by which pancreatic acinar cells transdifferentiate into ductal epithelial cells, is believed to be an initiating event of pancreatic ductal adenocarcinoma. Our lab has developed a 3D organoid assay to display ADM using primary, human pancreatic acinar cells to study the rate of transdifferentiation amongst these three different races. Preliminary data shows that the rate of ADM is occurring significantly faster (p < 0.05) in Blacks/AAs (White=16, Hispanic=11, Black/AA=5), which may explain the disproportionately behind the incidence and mortality rates for this race in pancreatic cancer. A sigmoid Emax function best captured the longitudinal changes in the percentage of ADM in nonlinear mixed effects modeling analysis performed in Monolix. Covariate analysis on top of the selected model demonstrated that race was the only covariate affecting the individual trajectories in the percentage of ADM. We additionally use nanoparticles to study the biomechanical properties (I.e., viscoelasticity, storage modulus) of the ADM microenvironment which shows a stiffer microenvironment in Blacks/AAs than for the other races (White=4,Hispanic=4, Black/AA=1). Furthermore, we have used a histone deacetylase (HDAC) inhibitor in reversing the process of ADM, which has consequently shown race-related outcomes, with Blacks/AAs displaying a significant chemoresistance (p < 0.05) to HDAC treatment (White=6,Hispanic=6, Black/AA=3) by utilizing an ADM reversal index (ADMRI). Through further analysis, the plan is to continue procuring human samples from these three races to isolate an ADM-specific biomarker in relation race and drug reversal by studying the expression/activity of pancreatic associated genes by bulk-RNA and single-cell sequencing.

Citation Format: Corey Melissa Perkins, Jinmai Jiang, Hesam Hakimjavadi, Jason Brant, Zhongyue Zhang, Ji-Hyun Lee, David Quashie, Yating Mao, Jamel Ali, Sarah Kim, Martha Campbell-Thompson, Thomas Schmittgen. Race as a predictive confounder in the rate of transdifferentiation and drug response in human pancreatic acinar ductal metaplasia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5777.
Abstract 5852: Vimentin content associated with shear wave speed in hepatocellular carcinoma: Non-invasive identification of more aggressive tumor phenotypes
Authors: Cortes Andrea C. , Minamiguchi Kiyoyuki , Da Costa Lopez Patricia L. , Anfossi Simone , Malea L Williams , Stenkamp Maria S. , Avritscher Rony , Fowlkes Natalie W.
Published: April 2023

Tumor stromal heterogeneity can negatively impact immune surveillance and treatment outcomes. Current cross-sectional imaging tools rely solely on tumor size and perfusion to monitor therapy and fail to characterize tumor biomechanics. Shear-wave elastography has emerged as a non-invasive technique that can quantify stromal elasticity. The goal of the present study is to characterize the molecular underpinnings associated with shear wave elastography measurements in liver cancer. To this end, rat hepatoma cell line McA-RH7777 stably expressing green fluorescent protein genes (fluc/GFP) were implanted in the left liver lobe of Buffalo rats. Three weeks after tumor implantation, adequate tumor size was verified by B-mode ultrasound. A 7-color panel was developed to assess the percentage of vimentin, α-smooth muscle actin, IBA-1, S100A4, TGF-β, and glial fibrillary acidic protein (GFAP) in tumor sections. Shear wave elastography data was used to stratify tumor nodules as stiff or soft according to the mean value of the shear wave (3.05 versus 2,26 m/s). Our results showed two distinct tumor phenotypes. Stiffer tumors, as determined by higher shear wave speed measurements on ultrasound, were characterized by higher percentages of vimentin-positive cells, low S100A4 and IBA-1 macrophages expressing TGF-b, and intermediate expression of IBA-1 macrophages and GFAP cells. Slow shear wave speed, characteristic of softer tumors, displayed the opposite phenotype (two-factor ANOVA; P≤0.001). The most remarkable difference between stiffness phenotypes was the percentage of cells expressing vimentin, with a mean percentage in stiffer tumors of 26.54 compared with 13.68% in softer tumors (P<0.003). Our results indicate that high expression of vimentin is highly correlated with increased shear wave speeds. Given the association between vimentin expression and epithelial-mesenchymal transition a marker of aggressive characteristics of tumor cells (increased migration and invasion), our findings suggest that longitudinal quantification of shear wave speed can be useful to assess functional changes in the stromal component and identify more aggressive tumor phenotypes with a higher likelihood of metastatic potential.

Citation Format: Andrea C. Cortes, Kiyoyuki Minamiguchi, Patricia L. Da Costa Lopez, Simone Anfossi, Williams Malea L, Maria S. Stenkamp, Rony Avritscher, Natalie W. Fowlkes. Vimentin content associated with shear wave speed in hepatocellular carcinoma: Non-invasive identification of more aggressive tumor phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5852.
Abstract 5863: Blockade of tumor glucocorticoid production inhibits Treg function and tumor growth
Authors: Otsuka Shizuka , Taves Matthew D. , Donahue Kaitlynn M. , Ashwell Jonathan D.
Published: April 2023

Glucocorticoids (GCs) are steroid hormones with potent immunosuppressive properties. GCs are primary produced from cholesterol in the adrenals via the de novo synthetic pathway. In some tissues, however, there is also a recycling pathway in which the enzyme 11β-HSD1 generates GCs from the inactive metabolites dehydrocorticosterone (rodent) or cortisone (human). Here we find that multiple tumor types produce GCs via 11β-HSD1 in vitro and in vivo. We used CRISPR/Cas9 technology to knock out Hsd11b1 in GC-producing tumor cell lines and found that lack of 11β-HSD1 in B16 melanoma, Panc02 pancreatic adenocarcinoma, and MC38 colon carcinoma lines reduced their growth in vivo. Reduced tumor growth corresponded with increased expression of the activation marker CD44 and production of the effector cytokines IFNγ and TNFα by tumor-infiltrating CD8+ T cells (TILs). Rather than directly suppressing effector cell function, recent findings have suggested that GCs instead act primarily by activating Treg cells, and that these Treg are responsible for inhibition of effector cell function. Consistent with this, we found that tumor-derived GCs upregulate Treg expression of miRNA-342, which downregulates the mTOR component Rictor and activates Treg activity. A role of tumor-derived GCs in enhancing Treg function was demonstrated by inoculating tumor cells into mice with Treg-specific glucocorticoid receptor deficiency (GRFoxp3Cre mice). Notably, growth of tumors was reduced in GRFoxp3Cre mice, along with increased CD8+ TIL activation. Importantly, pharmacologic inhibition of 11β-HSD1 reduced WT tumor growth to the same degree as knockout of 11β-HSD1, and rendered immunotherapy-resistant tumors susceptible to inhibition with anti-PD-1 antibodies. Given that 11β-HSD1 expression is upregulated in many human tumors, these data suggest that its inhibition, which has been accomplished in a number of phase 3 clinical trials for other indications and is well-tolerated, may be beneficial in their treatment.

Citation Format: Shizuka Otsuka, Matthew D. Taves, Kaitlynn M. Donahue, Jonathan D. Ashwell. Blockade of tumor glucocorticoid production inhibits Treg function and tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5863.
Abstract 5868: Uncovering key microenvironmental features for immunotherapy response at the subclonal level in pancreatic cancer
Authors: Yen Er-Yen , Ho I-Lin , Li Chieh-Yuan , Dyke Charles , Jiang Shan , Citron Francesca , Attanasio Sergio , Shah Rutvi , Chen Ko-Chien , Draetta Giulio , Viale Andrea
Published: April 2023

The lack of suitable experimental approaches to investigate and model functional heterogeneity in vivo has had a profound negative impact on our understanding of how heterogeneity affects the response to immunotherapy. To bridge this technological gap, we leverage a new platform that visualizes the spatial architecture of subclones and microenvironments. To study heterogeneous populations of cells and their clonal dynamics in vivo, we performed barcode lineage tracing with next-generation sequencing analysis. It allows a quantitative evaluation of the spatial distribution and temporal clonal dynamic in vivo with multiple pharmacological perturbations, such as immune checkpoint blockade (ICB). We found that treatment with a PD1 inhibitor, despite a limited effect on tumor volume, induced dramatic changes in tumor clonal architecture. Furthermore, we revealed that subclones from well-defined geographical domains in vivo share differential behavior upon immune checkpoint blockade (ICB). By visualizing 40 different markers with multiplexed staining technology, we further identified that the microenvironment also exhibits a high level of spatial heterogeneity. Therefore, we coupled spatial barcode sequencing with high-content imaging and revealed the differential microenvironment feature of sensitive and resistant clones. We will further explore the mechanisms of immune evasion at the subclonal level. Through isolation and deep characterization of clonal lineages endowed with a distinct ability to engage the immune response, we hope to identify new vulnerabilities to overcome resistance to ICB.

Citation Format: Er-Yen Yen, I-Lin Ho, Chieh-Yuan Li, Charles Dyke, Shan Jiang, Francesca Citron, Sergio Attanasio, Rutvi Shah, Ko-Chien Chen, Giulio Draetta, Andrea Viale. Uncovering key microenvironmental features for immunotherapy response at the subclonal level in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5868.
Abstract 5872: Spatial immunophenotypes contribute to predict survival of triple-negative breast cancer assessed as Miller-Payne grade 4 and N0 after neoadjuvant therapy
Authors: Ma Jianli , Deng Yuwei , Chen Dawei , Li Xiaomei , Yu Zhiyong , Wang Haibo , Zhong Lei , Li Yingjie , Wang Chengqin , Zhou Xiaoping , Li Xiang , Zhang Qingyuan , Yu Jinming
Published: April 2023

Objectives Some Miller-Payne 4 and N0 patients with triple-negative breast cancer (TNBC) after neoadjuvant chemotherapy (NACT) who had better survival should avoid treatment escalation. We aimed to identify these patients based on spatial distributions of immunophenotypes at initial diagnosis.

Methods We retrospectively analyzed 272 TNBC patients with Miller-Payne grade 4/5 and N0 for tumor-infiltrating lymphocytes (TILs) in relation to clinical survival. The spatial immunophenotypes were analyzed by multiplexed ion beam imaging by time of flight combined with proteomic. A prognostic classifier was established by the random forest algorithm.

Results The 5-year disease-free survival (DFS) was 63.8% for Miller-Payne 4 and 83.0% for Miller-Payne 5 (p=0.003), and overall survival (OS) was 71.0% and 85.5% respectively (p=0.007). High TILs were significantly associated with better DFS and OS in the Miller-Payne 4 patients (both p=0.016). Spatially, Miller-Payne 4 with good prognosis exhibited inflamed phenotype, with dominant CD8+ T cells on tumor center, few scattered CD68+ myeloid-derived cells far away from T cells, and deposit of increased lymphocyte activate molecules. Miller-Payne 4 with poor prognosis presented excluded phenotype, with few CD8+ T cells restricted to invasive margin, high density of CD14+CD68+CD11c+ myeloid cells with related molecules. These spatial immunophenotypes provided a good classifier model (AUC=0.975) to identify Miller-Payne 4 patients with different prognosis. We also observed similar signatures in Miller-Payne 5.

Conclusion Spatial immunophenotypes may indicate the prognosis in TNBC assessed as Miller-Payne 4 and N0.

Citation Format: Jianli Ma, Yuwei Deng, Dawei Chen, Xiaomei Li, Zhiyong Yu, Haibo Wang, Lei Zhong, Yingjie Li, Chengqin Wang, Xiaoping Zhou, Xiang Li, Qingyuan Zhang, Jinming Yu. Spatial immunophenotypes contribute to predict survival of triple-negative breast cancer assessed as Miller-Payne grade 4 and N0 after neoadjuvant therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5872.
Abstract 5893: Suppression of anti-tumor immunity by the Fusobacterium nucleatum protease fusolisin
Authors: Shhadeh Amjad , Dassa Liat , Fahoum Jamal , Haj Nicole , Wiener Reuven , Mandelboim Ofer , Bachrach Gilad
Published: April 2023

Fusobacterium nucleatum is an oral pathogen associated with periodontal disease, preterm births and the exacerbation of colorectal, esophageal, pancreatic, and breast cancers. F. nucleatum was previously found to inhibit killing of cancer cells by natural killer (NK) cells and tumor infiltrating T cells by inducing the NK cells killing-suppressing receptors TIGIT and CEACAM1. In this study, we show that the incubation of F. nucleatum with primary human natural killer (NK) cells causes the cleavage of the activating receptors CD16, NKp44 and NKp46 by fusolisin. Fusolisin is a fusobacterial outer-membrane, auto-transporter, serine protease and to date the only functional protease found in F. nucleatum. We previously showed that fusolisin is essential for fusobacterial growth in culture. High expression of a functional recombinant fusolisin in E. coli was enabled using codon optimization and replacement of the fusolisin's signal peptide with that of the E. coli OmpA. Recombinant fusolisin was found to cleave the same NK-activating receptors degraded by F. nucleatum. Cleavage of these activating receptors by fusolisin inhibited NK cells activity including killing of tumor cells in-vitro and in-vivo. Our results provide a new bacterial protease-dependent mechanism in which tumors colonized by F. nucleatum are protected from NK cells attack by exploiting fusolisin proteolytic activity. Importantly, our previous and current results suggest that fusolisin might serve to target for tumor-colonized fusobacteria.

Citation Format: Amjad Shhadeh, Liat Dassa, Jamal Fahoum, Nicole Haj, Reuven Wiener, Ofer Mandelboim, Gilad Bachrach. Suppression of anti-tumor immunity by the Fusobacterium nucleatum protease fusolisin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5893.
Abstract 5994: INX-315, a potent and selective CDK2 inhibitor, demonstrates robust antitumor activity in CCNE1-amplified cancers
Authors: Trub Alec G. , Bisi John E. , Dietrich Catherine , Taylor Michael , Strum Jay C. , Goel Shom , Roberts Patrick J.
Published: April 2023

Cyclin-dependent kinases (CDK) are a family of serine/threonine kinases that heterodimerize with regulatory subunits called cyclins to drive cell cycle progression. Uncontrolled cellular proliferation is a hallmark of cancer commonly driven by dysregulated kinase activity of specific CDK family members, including cyclin-dependent kinase 2 (CDK2). Aberrant CDK2 activity most frequently occurs through amplification of CCNE1 and/or overexpression of its protein product cyclin E1, which is a canonical binding partner of CDK2. Overexpression of cyclin E1 is observed in many solid tumors including in patients with high grade serous ovarian cancer (HGSOC), gastric cancer, and ER-positive breast cancer patients whose tumors have progressed on a prior CDK4/6 inhibitor regimen. Selective inhibition of CDK2 is thus a compelling therapeutic approach to regain cell cycle control. Here, we report preclinical data supporting the development of INX-315 for patients with cancers in which proliferation is CDK2-dependent. INX-315 is a potent inhibitor of CDK2/cyclin E (0.6 nM biochemical IC50) with high selectivity over other CDK family members in both biochemical and intracellular NanoBRET assays. In CCNE1-amplified human ovarian and gastric cancer cell lines, INX-315 potently inhibited Rb phosphorylation, induced a G1 cell cycle arrest, and inhibited proliferation. INX-315 also showed potent anti-proliferative activity in luminal breast cancer cell lines that had been cultured in CDK4/6 inhibitor (+/- anti-estrogen therapy) for prolonged periods to the point of developing drug resistance. While these cell lines did not readily respond to either CDK4/6 or CDK2 inhibition alone, combination treatment again suppressed Rb phosphorylation, accompanied by G1 arrest and a senescent-like phenotype. Lastly, in CCNE1-amplified xenograft models of ovarian and gastric carcinomas, INX-315 inhibited Rb-phosphorylation and induced tumor regression. These data demonstrate INX-315 to be a potent and selective CDK2 inhibitor that may benefit patients with CDK2/cyclin E driven cancers.

Citation Format: Alec G. Trub, John E. Bisi, Catherine Dietrich, Michael Taylor, Jay C. Strum, Shom Goel, Patrick J. Roberts. INX-315, a potent and selective CDK2 inhibitor, demonstrates robust antitumor activity in CCNE1-amplified cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5994.
Abstract 6041: Investigating metabolic dependencies of group 3 medulloblastoma
Authors: Dang Derek , Smith Kyle S. , Panwalkar Pooja , McKolay John , Animasahun Olamide , Achreja Abhinav , Nagrath Deepak , Northcott Paul A. , Venneti Sriram
Published: April 2023

Medulloblastomas (MB) are the most common pediatric brain malignancy. Transcriptomic, genomic, and epigenomic insights have stratified these tumors into four distinct subtypes: SHH, WNT, Group 3 and Group 4. Of the four subtypes, Group 3 tumors bear the worst prognosis. Each MB subtype is distinguished by their unique transcriptome profile and epigenetic landscape. Metabolic reprogramming is a hallmark of cancer and allows cells to actively promote the utilization of nutrients to support their uncontrolled proliferation. We identified distinct transcriptional metabolic profiles in medulloblastomas. We found that Group 3 MB show upregulation of key anabolic pathways. We then cross-referenced these data from comprehensive single-cell RNA sequencing profiles of cerebellar developmental niches. We found that Group 3 MB exhibits metabolic signature similar that of early progenitors, rhombic lip progenitors, and cerebellar ventricular zone derivatives. Finally, we identify DLAT, the E2 subunit of the pyruvate dehydrogenase complex, as a uniquely upregulated gene in Group 3 MB. Knockdown of DLAT inhibited tumor growth in-vitro and in-vivo suggesting a potential targets for future therapeutic regimens.

Citation Format: Derek Dang, Kyle S. Smith, Pooja Panwalkar, John McKolay, Olamide Animasahun, Abhinav Achreja, Deepak Nagrath, Paul A. Northcott, Sriram Venneti. Investigating metabolic dependencies of group 3 medulloblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6041.
Abstract 6044: Identification and molecular characterization of cancer cells survived under glucose starvation
Authors: Nesher Elimelech , Levi Bar , Levy Hadas , Cohen-Harazi Raichel , Koman Igor
Published: April 2023

Tumors are heterogeneous, consisting of different cell subpopulations to allow tumor survival in variable conditions. We observed the survival of a small percentage of HCT116 cells under glucose starvation. We hypothesized that these cells represent a previously undescribed subpopulation able to adjust their metabolism in the absence of glucose. Here we aimed to characterize the metabolic and molecular properties allowing these cells to survive. We propagated individual clones from HCT116 cells and further analyzed a single clone descendants were able to survive glucose starvation. Biochemical and molecular analyses were performed under standard glucose conditions, under glucose starvation during adaptation period, and under glucose starvation after resumption of proliferation. Metabolic analysis by Seahorse revealed that surviving cells completely shifted their metabolism from glycolysis to oxidative phosphorylation. LC-MS metabolomics analysis revealed that during senescence-like adaptation the cells accumulate metabolites required for NAD synthesis. FAD, a co-factor involved in the TCA cycle, was not found in the dormant cells, while higher levels of carnosine, essential for ROS elimination, were observed. The metabolic shift toward enhanced oxidative phosphorylation was accompanied by increased production of ROS leading to DNA damage, reflected in an upregulation of the genes NUPR, ST6GAL1, and EGRF as observed by RNAseq. Cells that resumed proliferation were also characterized by enhanced expression of DNA damage repair genes and upregulated subunits of ATP-synthase, a key enzyme in oxidative phosphorylation. We suggest that cells predisposed to effective DNA repair have more time and capabilities to adjust their metabolism and to survive.

Citation Format: Elimelech Nesher, Bar Levi, Hadas Levy, Raichel Cohen-Harazi, Igor Koman. Identification and molecular characterization of cancer cells survived under glucose starvation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6044.
Abstract 6050: Analysis of diverse metabolic pathways using bioluminescent cell-based assays
Authors: Leippe Donna , Karassina Natasha , Valley Mike , Sylvester Kayla , Vidugiriene Jolanta
Published: April 2023

Cellular energy metabolism is a complex system of many interconnected metabolic pathways that govern both cell health and function. Cancer cells are adept at quickly switching between metabolic pathways to adapt to nutrient limitations in the environment, enhancing their growth advantages while negatively impacting other cells in the tumor microenvironment such as immune cells. To facilitate metabolic characterization of cancer cells, we developed assays for detection of metabolites involved in key pathways, such as glycolysis and glutaminolysis, using a bioluminescent NADH detection technology coupled to specific dehydrogenases. We have now extended these assays to include metabolites involved in glycogen, lipid, ketone body and amino acid metabolism, as well as intermediates in the TCA cycle such as malate, isocitrate, and 2-hydroxyglutarate. A second core bioluminescent hydrogen peroxide detection system was also used to develop an assay for pyruvate. Here we describe the use of these assays to measure metabolite levels in cell lysates and cell culture media collected from cancer cell lines plated in 96- or 384-well plates and in 2D or 3D cultures. Changes in metabolite levels were monitored in response to altered media composition and drug treatments. Levels were also compared between different cell lines. In comparative studies of glioblastoma cell lines, 2-hydroxyglutarate production was higher in cells expressing mutant isocitrate dehydrogenase. By reconfiguring the NADH detection technology to measure cellular enzyme activity rather than metabolite levels, we were able to detect activity of dehydrogenases that participate in the glycolysis pathway, the TCA cycle, and pentose phosphate pathway. Assays for cellular malate dehydrogenase, isocitrate dehydrogenase and other dehydrogenase enzymes were sensitive, requiring ≤ 400 lysed cells per assay. The expanded collection of bioluminescent assays provides a way to quickly characterize the metabolic states and behaviors of cancer cells to help identify pathways that may present therapeutic targets.

Citation Format: Donna Leippe, Natasha Karassina, Mike Valley, Kayla Sylvester, Jolanta Vidugiriene. Analysis of diverse metabolic pathways using bioluminescent cell-based assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6050.
Abstract 6098: Genome wide CRISPR screen reveals genetic vulnerabilities of next generation PARP1 inhibitor AZD5305
Authors: Yin Ling , Chen Junjie
Published: April 2023

The first-generation PARP inhibitors (PARPi) olaparib, niraparib, talazoparib and rucaparib have been clinically approved for several cancers, like breast, ovarian and prostate, especially in BRCA-mutant tumors which are of homologous recombination repair (HRR)-deficiency. All PARPi both target PARP1 and PARP2, causing cancer cell deaths deficient in HRR. However, only inhibition of PARP1 is required for synthetic lethality in HRR-deficient cells. AZD5305 is a highly selective PARP1 inhibitor which has been used in clinical trials. In this study, we used the Toronto Knock Out Library version 3 (TKOv3), which contains 70948 gRNAs targeting 18,053 protein-coding genes to perform whole-genome CRISPR-Cas9 screens with three isogenic cell lines 293A, MCF10A, and Hela to uncover known and new high-confidence genes synthetic lethal interacted with AZD5305. MAGeCK and Drug Z were used to analyze the results and genes were ranked according to their drugZ scores. We identify that in all three cell lines, PARP1 was in the top hit in positive selection which proves the AZD5305 indeed a PARP1 inhibitor. Moreover, through a comprehensive and comparative analysis with the screen results of first generation PARPi olaparib, we reveal known and new essential genes which are proved to be a common mechanism for synthetic lethality. We identify the gene AUNIP (Aurora Kinase A and Ninein Interacting Protein), in the top hit from AZD5305 screen but not in olaparib screen, suggesting that AUNIP may be an additional target for synthetic lethality with PARP1 loss. Taken together, this screen reveals the potential molecular genes related with PARP1 inhibitor that can be further explored targeted and combined cancer therapy.

Citation Format: Ling Yin, Junjie Chen. Genome wide CRISPR screen reveals genetic vulnerabilities of next generation PARP1 inhibitor AZD5305 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6098.
Abstract 612: Control of the Basal Molecular Subtype of Murine Bladder Cancer by Adaptive Immunity
Authors: Johnson Burles Avner , Feng Mingxiao , Choi Woonyoung , Colocho Gabriela , Arbuiso Alyssa , Jin Samuel , Aragaki Adam K. , Ruland Charles , Rapiey Stanley , Hahn Noah M. , McConkey David J.
Published: April 2023

Introduction: Developing optimized bladder cancer mouse models that can be used in the evaluation of immune based therapeutics remains a high priority in ongoing research.Experimental Procedures: In previous work by our collaborators, murine cell lines were generated that recapitulate the basal (BBN964, BBN966, and BBN975) molecular subtype of human bladder cancer (Saito et al. Cancer Res. 2018;78(14):3954-3968). Here we compared their tumorigenicity and growth rates in immunocompetent C57BL/6 mice and in immunodeficient Rag1-KO mice lacking B and T cells. Changes in the tumor and stromal compartments were correlated with the differences in growth rates observed. Observations and New Data: Tumors derived from all three BBN basal cell lines grew robustly in Rag1-KO mice (10/10 BBN964 tumors, 19/19 BBN966 tumors and 19/19 BBN975 tumors >100 mm3 by 10 weeks post-injection). Conversely, very few tumors grew in immunocompetent (C57BL/6, e.g. B6) mice (0/9 BBN964 at 8 weeks, 0/17 BBN966 at 16 weeks, 4/15 BBN975 at 9 weeks). BBN966 tumors conditioned to grow in Rag1-KO mice were also highly tumorigenic when they were subsequently transferred into immunocompetent mice (Rag1-KO ◊ B6, 16/20 mice with tumors >100 mm3 at 4 weeks), and they displayed increased tumorigenicity when they were reintroduced into immunodeficient mice (Rag1-KO ◊ Rag1-KO, 20/20 mice with tumors >100 mm3 at 3 weeks). BBN966 tumors harvested from B6 mice and passaged in vitro for 5 weeks maintained tumorigenicity when reimplanted into immunocompetent mice, and BBN966 tumors grown in female B6 mice grew robustly when transferred into male B6 mice. Bulk RNA sequencing of tumors comparing the BBN966 Rag1-KO (1st passage) to BBN966 Rag1-KO (2nd passage) demonstrated decreased expression of genes associated with tumor infiltrating macrophages in the 2nd passage tumors. Conclusions: Our results demonstrate that basal BBN syngeneic bladder tumors display markedly different growth rates in wild-type and T- and B-cell-deficient mice. The results suggest that adaptive immunity controls basal bladder cancer tumorigenicity, which is consistent with the immune infiltrated landscape observed in basal human bladder cancers. Further results described in this poster will include deeper molecular characterizations of the immune microenvironments of these tumors.

Citation Format: Burles Avner Johnson, Mingxiao Feng, Woonyoung Choi, Gabriela Colocho, Alyssa Arbuiso, Samuel Jin, Adam K. Aragaki, Charles Ruland, Stanley Rapiey, Noah M. Hahn, David J. McConkey. Control of the Basal Molecular Subtype of Murine Bladder Cancer by Adaptive Immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 612.
Abstract 6154: Imipramine induces apoptosis and inhibits metastasis via suppression of EGFR signaling pathway in bladder cancer
Authors: Lo Tsai Lin , Tsai Jai-Jen , Hsu Fei-Ting , Chang Yuan
Published: April 2023

Bladder cancer (BC) is one of the most common malignant diseases of the urinary system, with poor prognosis and high recurrence and metastasis rate. Recent literatures suggested that BC patients have proportionately worse depression and mental health, as well as being at increased risk of suicidal death when compared to the general population, while relatively sane BC patients have a 2.2 times higher mortality rate, which means that depression can make BC worse. Imipramine is a tricyclic antidepressant, was used to treat neuropathic pain, nocturnal enuresis, and depression. Recently, imipramine has been reported to owns anti-tumor efficacy in various type of cancer. However, the effects and underlying mechanisms of imipramine on BC is remaining unclear. First, we indicated that imipramine may induce cytotoxicity of BC by MTT assay. Flow cytometry also showed that imipramine may trigger the loss of mitochondrial membrane potential, the accumulation of oxidative stress (ROS), and the activation of cleaved-caspase-3, -8 and -9. Our western blotting assay and immunofluorescence translocation staining also verified that imipramine markedly inhibited BC progression by inhibiting both EGFR/AKT/NFΚB and EGFR/ERK/NΚB signaling pathways. Furthermore, transwell and wound healing assay indicated that imipramine effectively reduced the metastatic ability of BC. In addition, the anti-tumor effect of imipramine was also validated by MB49 bearing animal model. Most importantly biochemistry level and pathology was not affected by imipramine. In conclusion, imipramine may not only suppressed BC progression by inactivation of EGFR/AKT/NFΚB and EGFR/ERK/NFΚB signaling pathways and induction of apoptosis pathways. These results suggested that imipramine has the opportunity to be a new therapeutic strategy for BC patients.

Citation Format: Tsai Lin Lo, Jai-Jen Tsai, Fei-Ting Hsu, Yuan Chang. Imipramine induces apoptosis and inhibits metastasis via suppression of EGFR signaling pathway in bladder cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6154.
Abstract 6189: Targeting DNA damage responsive pathways in cancer therapy
Authors: Chen Junjie
Published: April 2023

DNA double strand breaks (DSBs) result in activation of several key DNA damage response (DDR) kinases including ATM, ATR, and DNA-PK. These protein kinases not only promote DNA damage-induced checkpoint control, but also facilitate DSB repair in humans. Thus, these DDR kinases have become promising drug targets for cancer therapy. However, the benefits of targeting DDR kinases remain to be realized, in part due to the lack of predictive biomarkers. By undertaking CRISPR screens with inhibitors targeting key DDR kinases, we obtained a global and unbiased view of genetic interactions with DDR inhibition. Additionally, we compared the synergistic effects of combining different DDR inhibitors and found that an ATM inhibitor plus a PARP inhibitor induced dramatic levels of cell death, probably through promoting apoptosis. Our results provide a better understanding of DDR pathways, which may facilitate the use of these DDR-targeting agents in cancer therapy. Moreover, To provide comprehensive and unbiased perspective of DDR signaling pathways, we performed 30 fluorescence-activated cell sorting-based genome-wide CRISPR screens with antibodies recognizing distinct endogenous DNA damage-signaling proteins to identify new regulators involved in DNA damage response (DDR). We discovered that proteasome-mediated processing is an early and prerequisite event for cells to trigger camptothecin- and etoposide-induced DDR signaling. Furthermore, we identified PRMT1 and PRMT5 as new modulators that regulate ATM protein level. Moreover, we discovered that GNB1L is a master regulator of DDR signaling via its role as a co-chaperone for PIKK proteins. Collectively, these screens offer a rich resource for further investigation of DDR, which may provide insight into strategies of targeting these DDR pathways to improve therapeutic outcomes.

Citation Format: Junjie Chen. Targeting DNA damage responsive pathways in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6189.
Abstract 6211: Development and validation of a novel sSTRIDE-MMR functional assay to study the efficiency of MMR inhibitors
Authors: Solarczyk Kamil , Uznańska Karolina , Wójcikowska Olga , Białecka Maja , Waligórska Agnieszka , Koman Szymon , Kordon-Kiszala Magdalena
Published: April 2023

Mismatch repair (MMR) is considered to be one of the fundamental pathways that guards genome stability and mutations in genes encoding for crucial MMR proteins have been shown to promote cancer initiation. On the other hand, inactivation of MMR components in cancer cells leads to increased neoantigen formation and in consequence, exposes the tumor to the immune system. Therapeutic strategies aiming at inhibiting MMR are thus under development, yet methods reporting on the status of MMR are lacking. We report here the development, optimization and validation of a first MMR-specific functional assay. The assay, which is based on the STRIDE platform technology, detects DNA nicks localized in close proximity to PMS2 protein and can thus be utilized as a direct reporter of inhibition of PSM2 itself or upstream acting proteins. The optimization phase of assay development was performed in U2OS cells. First, in untreated cells we have shown that the sSTRIDE-MMR signals constitute ca. 10-15% of total single-strand DNA breaks foci detected by the classic sSTRIDE assay variant. In a series of technical negative controls we have then shown that the number of false-positive foci never exceeds 10% of the total number of signals, with as little as 2% for the isotype controls. The assay was further validated in HAP1 wild-type and PMS2-KO isogenic cell line pair. As expected, the number of sSTRIDE-MMR foci in the PMS2-KO cell line was significantly lower than in wild-type cells and additionally, no response to the treatment with 6TG, a known inducer of MMR, was detected in PMS2 null cells. Finally, the assay was used to verify the efficiency of first-in-class small-molecule MMR inhibitors. After treatment with the compounds, the number of detected sSTRIDE-MMR foci was significantly lower when compared to the number of foci in untreated cells. In conclusion, we show here that sSTRIDE-MMR is an assay characterized by high specificity, low false positives rate and a high-dynamic range. We believe that sSTRIDE-MMR can prove to be a very useful cell-based solution to study the efficiency of MMR inhibitors.

Citation Format: Kamil Solarczyk, Karolina Uznańska, Olga Wójcikowska, Maja Białecka, Agnieszka Waligórska, Szymon Koman, Magdalena Kordon-Kiszala. Development and validation of a novel sSTRIDE-MMR functional assay to study the efficiency of MMR inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6211.
Abstract 6212: Inhibition of ATM-dependent checkpoint control and DNA double-strand break repair enhances the efficacy of ATR inhibitors
Authors: Turchick Audrey , Zimmermann Astrid , Chiu Li-Ya , Dahmen Heike , Elenbaas Brian , Zenke Frank T. , Blaukat Andree , Vassilev Lyubomir T.
Published: April 2023

Ataxia telangiectasia and Rad3 related (ATR) and ataxia telangiectasia mutated (ATM) protein kinases play key roles in the DNA damage response (DDR) by responding to replication stress and double-strand DNA breaks respectively to pause the cell cycle and promote DNA repair. ATR kinase inhibition in cancer cells disrupts cell cycle control and causes unrepaired DNA lesions and cytotoxicity. Accordingly, several ATR inhibitors (ATRi) are in clinical development as monotherapies and in combination with DNA damaging chemotherapies and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that while treatment of cancer cells with an ATRi inhibits the ATR pathway, it simultaneously activates the ATM signaling pathway as shown by increased levels of p-ATM, p-CHK2, p-KAP1 and p-p53. In p53 wild-type cancer cells, ATR inhibition causes an ATM-mediated G1 cell cycle arrest which diminishes the DNA lesions and cytotoxic effects of ATR inhibition. Combination of an ATRi with a selective ATM inhibitor (ATMi) synergistically potentiated efficacy in cancer cells in vitro and increased efficacy in vivo at doses that did not show overt toxicities. In a panel of patient-derived xenograft (PDX) models of triple-negative breast cancer treated with an ATRi/ATMi combination, substantial improvement in efficacy was observed. Thus, activation of the ATM pathway by an ATRi acts as a compensatory resistance pathway for ATR inhibition. These results suggest a novel and efficacious combination approach for cancer therapy by dual inhibition of these two key DDR kinases.

Citation Format: Audrey Turchick, Astrid Zimmermann, Li-Ya Chiu, Heike Dahmen, Brian Elenbaas, Frank T. Zenke, Andree Blaukat, Lyubomir T. Vassilev. Inhibition of ATM-dependent checkpoint control and DNA double-strand break repair enhances the efficacy of ATR inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6212.