bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5230: Modulation of SF3B1 causes global intron retention and downregulation of the B-cell receptor pathway in chronic lymphocytic leukemia
Authors: Han Qimei , Wang Jianbo , Shull Austin Y. , Shi Fang , Deng Libin , Choi Jeong-Hyeon , Park Eun-Jeong , Tu Shuo , Pei Lirong , Awan Farrukh T. , Bollag Roni , Bryan Locke J. , Xin Hong-bo , Lagisetti Chandraiah , Webb Thomas R. , Jin Victor , Shi Huidong
Published: August 2019

Splicing factor SF3B1 is frequently mutated in chronic lymphocytic leukemia (CLL) patients and has been suggested as a potential therapeutic target. In this study, we demonstrated that SF3B1 modulator sudemycin D6 (SD6) effectively inhibits cell growth and induces apoptosis in CLL cells. RNA sequencing analysis revealed significant increases in global intron retention in SD6-treated CLL cells. Pathway analysis of the genes associated with increased intron retention suggested that B-cell receptor (BCR) and PI3K signaling pathways were among the most important pathways being affected by SD6. The increases in intron retention were inversely correlated with decreases in mRNA and protein levels of the affected BCR/PI3K pathway molecules including BLNK, BTK, AKT, PLCγ2, and PI3Kδ. SD6 treatment also induced a time-dependent exon-skipping event in MCL-1 mRNA and resulted in significant down-regulation of another anti-apoptotic gene TRAF1, thus collectively contributing to the SD6-induced apoptosis. Furthermore, SD6 treatment can overcome the pro-survival and pro-growth signals and synergize with established CLL therapies ibrutinib, idelalisib, and venetoclax to induce apoptosis in primary CLL cells co-cultured with bone marrow stromal cells and T-cell-derived cytokines. Finally, in vivo treatment with SD6 at 10mg/kg/day for 7 days significantly inhibited the growth of xenograft tumors that were established by subcutaneous inoculation of 5×106 MEC1 CLL cells into NOD mice. Collectively, these results provide a strong rationale for the future clinical development of spliceosome modulators and potential combination therapies for the treatment of CLL.

Citation Format: Qimei Han, Jianbo Wang, Austin Y. Shull, Fang Shi, Libin Deng, Jeong-Hyeon Choi, Eun-Jeong Park, Shuo Tu, Lirong Pei, Farrukh T. Awan, Roni Bollag, Locke J. Bryan, Hong-bo Xin, Chandraiah Lagisetti, Thomas R. Webb, Victor Jin, Huidong Shi. Modulation of SF3B1 causes global intron retention and downregulation of the B-cell receptor pathway in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5230.
Abstract 524: HMGB1-activated IRF3 and NF-κB contributes to UV radiation-induced immune suppression by upregulating PD-L1
Authors: Wang Wei , Chapman Nicole , Zhang Bo , Li Mingqi , Fan Meiyun , Laribee R. Nicholas , Zaidi M. Raza , Pfeffer Lawrence , Chi Hongbo , Wu Zhaohui
Published: August 2019

Solar ultraviolet radiation (UVR) suppresses skin immunity, which facilitates skin lesion initiation and establishment by promoting immune evasion. It is unclear whether immune checkpoints are involved in the modulation of skin immunity by UVR. Here we report that the expression of immune checkpoint molecule PD-L1 was significantly increased in melanocytes and melanoma cells upon UVR exposure. The damage-associated molecular patterns molecule HMGB1 was secreted by melanocytes and keratinocytes upon UVR, which subsequently activated the RAGE (receptor for advanced glycation endproducts) receptor to promote the NF-κB and IRF3-dependent transcription of PD-L1 in melanocytes. We also found that UVR exposure significantly reduced the susceptibility of melanoma cells to CD8+ T cell-dependent cytotoxicity, which was mitigated by inhibiting the HMGB1/TBK1/IRF3/NF-κB cascade or blocking the PD-1/PD-L1 checkpoint. Taken together, our findings demonstrate that UVR-induced PD-L1 upregulation contributes to the immune suppression in the skin microenvironment, which may promote immune evasion of oncogenic cells, and melanoma initiation and progression.

Citation Format: Wei Wang, Nicole Chapman, Bo Zhang, Mingqi Li, Meiyun Fan, R. Nicholas Laribee, M. Raza Zaidi, Lawrence Pfeffer, Hongbo Chi, Zhaohui Wu. HMGB1-activated IRF3 and NF-κB contributes to UV radiation-induced immune suppression by upregulating PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 524.
Abstract 5243: CRISPR knockout: How to get high biallelic knockout
Authors: Li Yongqi , Min Li , Liu Ellen , Wang Yiran
Published: August 2019

Although homology directed repair (HDR)-mediated gene knockout / knockin is well established, it cannot necessarily be applied in some cell types and organisms with low HDR frequencies. Herein we describe an improved technology for genome-wide gene knockout / knockin using CRISPR-Cas9 targeted genome editing: CRISPR KN2.0. CRISPR KN2.0 technology is specifically designed to provide an easy-to-use and get around 50% biallelic knockout. This gene knockout / knockin technology is designed to knock out any DNA locus and knock in a functional cassette containing Green Fluorescence Protein (GFP) and puromycin resistant gene to facilitate screening process. Both of these cassettes are expressed under EF1A promoter after genomic integration. CRISPR KN 2.0 has been used to successfully knock out many genes / knocked in functional cassette in HeLa, HEK293T and MIA PaCa-2 (a human pancreatic carcinoma cell line) cells. Studies carried out in house and by some colleagues show that CRISPR KN 2.0 is highly efficient and render improved knockout/knockin rate (over 50% positively integrated colonies after puromycin selection).

Citation Format: Yongqi Li, Li Min, Ellen Liu, Yiran Wang. CRISPR knockout: How to get high biallelic knockout [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5243.
Abstract 5249: The transcriptional regulation of CCND1 by miR-15a-5p in the nucleus of colorectal carcinoma cell lines
Authors: Liu Zhenzhen , Bai Xue , Li Chunyan
Published: August 2019

Although miRNAs are important post-transcriptional regulators of gene expression in cytoplasm, more and more evidences demonstrate that miRNAs also have nuclear functions. The majority of miRNAs are present in both the cytoplasm and the nucleus. The canonical function of translational repression in cytoplasm is well studied, but the functions of nuclear miRNAs are currently not well understood. From previous studies, we found that miR-15a-5p (miR-15a for short) can be detected in the nucleus of the colon cancer cell line. By transfected miR-15a labelled with biotin, we validated that miR-15a could bind to DNA (especially dsDNA) in the nucleus of colorectal carcinoma cell line HCT116. To find the targets of miR-15a in the nucleus, we scanned the promoter region of the potential targets which were predicted by Targetscan, miRWalk, and miRDB. The promoter of CCND1 has one region that is complementary to miR-15a. By integrating the RNA-seq data and miRNA-Seq data from NCI60 cell line panel and TCGA (The Cancer Genome Atlas), we found that CCND1 was negatively regulated by miR-15a in colorectal cancer. To confirm that miR-15a regulates CCND1’s transcription in the nucleus, we constructed the promoter of CCND1 and a luciferase reporter into one plasmid, then site-specific mutation was introduced into the potential binding site of miR-15a in the promoter of CCND1. The luciferase assay demonstrated that the mutation in the binding site decreased the expression of CCND1, which validate that miR-15a may regulate the expression of CCND1 by binding to the promoter of CCND1 in the nucleus. The next step, we will further study the function of nuclear miR-15a distinct from the canonical paradigm in carcinogenesis.

Note: This abstract was not presented at the meeting.

Citation Format: Zhenzhen Liu, Xue Bai, Chunyan Li. The transcriptional regulation of CCND1 by miR-15a-5p in the nucleus of colorectal carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5249.
Abstract 5254: CARMIL3 promotes tumor progression in triple negative breast cancer: A possible novel biomarker
Authors: Lee Jeeyeon , Baek Dong Won , Lee Soo Jung , Jeong Jae-Hwan , Lee In Hee , Jung Jin Hyang , Park Ho Yong , Chae Yee Soo , Kang Jieun
Published: August 2019

Purpose: CARMIL3 (LRRC16B, capping protein regulator and myosin 1 linker 3) was previously identified as a novel oncofetal-like gene. It influences cell proliferation, cell cycle control and transformation, possibly mediated by cyclin B1. This study aimed to investigate the function of CARMIL3 in triple negative breast cancer (TNBC) cells and tissues.

Methods: CARMIL3 mRNA was measured using a qRT-PCR in breast cancer cells (MDA-MB-231, MCF7, SK-BR3 and T-47D) and tissues from 20 patients with TNBC. The effects of CARMIL3 on cell proliferation, migration, and invasion were determined using MTT, wound healing, and Matrigel Transwell assays.

Results: Results showed that, the CARMIL3 mRNA expressions were significantly increased in breast cancer cell lines, especially in triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) when compared to normal breast epithelial cells (MCF10A). Downregulation of CARMIL3 inhibited the cell proliferation, migration and invasion of MDA-MB-231. Furthermore, high CARMIL3 mRNA expression was found in 20 TNBC specimens than normal controls (p<0.0001).

Conclusions: In conclusion, CARMIL3 expression in TNBC affects cancer progression, which suggests that CARMIL3 can be a new biomarker for patients with TNBC.

Citation Format: Jeeyeon Lee, Dong Won Baek, Soo Jung Lee, Jae-Hwan Jeong, In Hee Lee, Jin Hyang Jung, Ho Yong Park, Yee Soo Chae, Jieun Kang. CARMIL3 promotes tumor progression in triple negative breast cancer: A possible novel biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5254.
Abstract 5256: Molecular signatures associated with obesity-related tumor growth in breast cancer women
Authors: Prada Diddier , Arriaga-Canon Cristian , Diaz-Chavez Jose , Cortes Carlo , Andonegui-Elguera Marco , Perez-Collado Pedro , Muniz Rodolfo , Cantu-de-Leon David , Cabrera-Galeana Paula , Bargallo Enrique , Herrera Luis , Penaloza Fernando
Published: August 2019

Obesity has been widely associated with chronic diseases, including cancer. Obesity increases the risk of developing several types of cancer, both in women and men, especially breast cancer in postmenopausal women. However, the role of obesity after tumors is established and its potential mechanisms are unknown. Our study aims to determine the molecular signatures associated with obesity-related tumor growth in a cohort of breast cancer patients from a country with an obesity epidemic. We evaluated a cohort of patients with breast cancer (n=151) at diagnosis, including patients in all clinical stages and 78% of them showed overweight or obesity. We determined the role of body mass index (BMI) on clinical characteristics and, using RNA-seq analyses in biopsy samples at diagnosis, we determined potential genes associated. We found a positive correlation between BMI and tumor size at diagnosis (r = 0.147, p-value = 0.006) and age (r=0.116, p-value=0.03). Lower BMI was observed when the number of positive nodes increased (p-value = 0.015). In a random subset from these patients (n=12) and using robust linear models, we determined that 10 genes (GPRC5B, SLITRK6, KRT23, VTCN1, TNFRSF12, PTP4A2, BACE2, EPH8, CYP21A1P, and TMEM52) were positively associated with BMI. CSTA and IGKV1-16 showed a negative association with BMI. Gene ontology analyses revealed that VTCN1 and PTP4A3 were linked to increased proliferation and tumor progression. CSTA, a protease inhibitor, was also associated with poor prognosis. Our study suggested a deleterious effect of obesity on breast cancer patients, which could be caused by changes in cell expression on key genes in tumor cells.

Citation Format: Diddier Prada, Cristian Arriaga-Canon, Jose Diaz-Chavez, Carlo Cortes, Marco Andonegui-Elguera, Pedro Perez-Collado, Rodolfo Muniz, David Cantu-de-Leon, Paula Cabrera-Galeana, Enrique Bargallo, Luis Herrera, Fernando Penaloza. Molecular signatures associated with obesity-related tumor growth in breast cancer women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5256.
Abstract 5259: Fatty acid oxidation represents a metabolic vulnerability in glioblastoma in nutrient-deprived conditions
Authors: Kant Shiva , Dayalan Antony , Kesarwani Pravin , Chinnaiyan Prakash
Published: August 2019

Glioblastoma represents an aggressive, primary brain tumor with limited treatment options. We performed an integrative, cross-platform analysis coupling global metabolomics and gene expression profiling in >100 patient-derived gliomas to begin to understand the diverse metabolic programs driving the aggressive phenotype of this malignancy. Alterations in fatty acid β-oxidation (FAO) emerged as a key metabolic node differentiating glioblastoma from low-grade astrocytoma, as demonstrated by an accumulation of acylcarnitines. Metabolic heterogeneity was observed within glioblastoma that could further define tumors as FAO ‘high’ and ‘low’. Integrative analyses identified these metabolic subtypes to be enriched with mesenchymal (MES) and proneural (PN) glioblastoma subtypes, respectively. These findings were metabolomically and functionally recapitulated in molecular subtype-specific preclinical models. Analysis of gene expression profiles from these lines uncovered an orchestrated transcriptional program designed to promote fatty acid uptake, activation, and mitochondrial oxidation. Studies designed to determine the biologic consequence of enhanced FAO in glioblastoma only identified a role in glucose-deprived conditions, where it served as a vital, alternate source for ATP synthesis. Based on these findings, we tested the hypothesis that dual targeting of glycolysis and FAO would elicit energetic stress-mediated cell death in glioblastoma. Accordingly, the glycolysis inhibitor 2DG and the FAO inhibitor etomoxir only demonstrated anti-proliferative activity in MES glioblastoma cell lines when used as single agents in vitro, while the combination resulted in robust, necroptosis-mediated cell death. Synergistic anti-tumor activity was observed following combined glycolysis and FAO inhibition when extended in vivo in an orthotopic model. Collectively, our findings suggest FAO provides metabolic plasticity in MES glioblastoma, allowing these cells to adapt to nutrient-deprived microenvironments. Combinatorial strategies designed to inhibit glycolysis and FAO represents an attractive therapeutic approach in glioblastoma.

Citation Format: Shiva Kant, Antony Dayalan, Pravin Kesarwani, Prakash Chinnaiyan. Fatty acid oxidation represents a metabolic vulnerability in glioblastoma in nutrient-deprived conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5259.
Abstract 5262: Lipidomic profiling of neuroblastoma cells after treatment with novel withalongolide A 4, 19, 27-triacetate (WGA-TA)
Authors: Subramanian Chitra , Thekkelnaycke Rajendiran , Soni Tanu , Opipari Valerie P. , Timmermann Barbara N. , Cohen Mark S.
Published: August 2019

Introduction: Neuroblastoma (NB) is a pediatric solid tumor that affects approximately 700 children every year in the United States. Despite advances in treatment, the cure rate for high risk advanced NB having N-myc amplification has a poor response of 40-50%. Standard of care treatment for high risk NB patient consists of surgery, intensive chemotherapy and radiotherapy. Recently, we and others have shown that withanolide treatment of NB induce apoptosis of NB cells, through several mechanism including lipid oxidation, down regulation of N-myc and suppression of Akt/mTOR/NF-κB activation. Therefore, we hypothesized that mass spectrometric analysis of untargeted lipidomic profiling after treatment of NB cells with withanolide WGA-TA will identify novel prognostic and diagnostic biomarkers of NB.

Methods: Validated neuroblastoma cell lines IMR32 (N-myc amplified) and SH-EP (N-myc non-amplified) were grown in 2D culture in appropriate growth medium. Lipids were extracted from cells using modified Bligh and Dyer method. Lipidomic profiling was carried out using LC MS/MS. Principal component analysis (PCA) was used to identify differentially regulated metabolites.

Results: Principal component analysis of NB cell lines and WGA-TA treatment showed complete separation of samples as well as internal controls. Initial analysis of lipid profiling of IMR32 and SH-EP cells showed differential regulation of several lipids. N-myc non-amplified SH-EP cells have upregulation of phospholipids such as phosphocholine (PC), phosphor ethanol amine (PE), sphingomyelin (SM) and free fatty acid, whereas glycerol lipid diacyl glycerol (DG) is down regulated in SH-EP cells compared to IMR32 cells. Treatment of IMR32 cells with WGA-TA led to up regulation of phospho lipids such as PC, phosphoserine (PS), SM, Lyso PE, free fatty acids just like N-myc non-amplified cells. But, triglycerides (TG) were up regulated after treatment with WGA-TA in both IMR32 and SH-EP cells.

Conclusions: WGA-TA treatment of N-myc amplified cells led to upregulation of phospholipids similar to that of N-myc non-amplified cells indicating that CDP-Choline pathway lipids might play an important role in NB. Further analysis is needed to unravel the link between N-myc and lipid metabolism in NB.

Citation Format: Chitra Subramanian, Rajendiran Thekkelnaycke, Tanu Soni, Valerie P. Opipari, Barbara N. Timmermann, Mark S. Cohen. Lipidomic profiling of neuroblastoma cells after treatment with novel withalongolide A 4, 19, 27-triacetate (WGA-TA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5262.
Abstract 5275: Cancer specific caloric restriction using novel small molecule improves the therapeutic regime for triple negative breast cancer
Authors: Tailor Dhanir , Resendez Angel , Kumar Vineet , Going Catherine , Pitteri Sharon , Malhotra Sanjay
Published: August 2019

Hyperglycemic and hyper insulin condition are the signature symptoms for type two diabetes which is the most favorable condition for the development of cancer. Women having a type two diabetes has 20-27% high risk to develop breast cancer including triple negative breast cancer (TNBC). Microenvironment including hyperglycemic, hyper insulin condition leads to the therapy resistance. According to ‘Warburg’s effect’ cancer cells consume a high amount of glucose in comparison to normal cells via aerobic glycolysis process. To take care of this condition currently, several clinical studies are going on for the combination therapy of type 2 diabetes and chemotherapy drugs together including erlotinib and metformin. The major drawback of this kind of type 2 diabetes drugs is that they won't affect cancer cells under hyperglycemic and hyper insulin conditions. These drugs including metformin work via AMP-activated protein kinase (AMPK) activation to switch aerobic glycolysis to oxidative phosphorylation. We have developed a novel AMPK activator (SU-18) who selectively induces an oxidative phosphorylation in TNBC cells and glycolysis in normal cells. We found that SU-18 induces G2/M phase arrest, mitochondrial stress and apoptosis in TNBC cells as same as metformin but at 1000 fold less concentration. It selectively reduces the glucose consumption, lactate and ATP production in TNBC cells where normal cells act opposite to this. SU-18 activity is through AMPK activation which got abolished in presence of AMPK inhibitor (Compound c). During mouse xenograft studies, it inhibits the tumor growth and progression. It also improves the blood glucose level in hyperglycemic mice. Comparative studies with metformin suggest that SU-18 works irrespective of hyperglycemia and insulin conditions, whereas metformin, is ineffective in this condition. FDG-PET scan analysis also supported to our in vitro glucose consumption data that treatment with SU-18 reduces the FDG accumulation in tumor cells. This study advocates the clinical candidature of SU-18 for TNBC patients.

Citation Format: Dhanir Tailor, Angel Resendez, Vineet Kumar, Catherine Going, Sharon Pitteri, Sanjay Malhotra. Cancer specific caloric restriction using novel small molecule improves the therapeutic regime for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5275.
Abstract 5284: Hypoxia promote self-renewal in pancreatic cancer by modulating L-2-Hydroxyglutarate level
Authors: Gupta Vineet K. , Sharma Nikita , Kesh Kousik , Hadad Roey , Dudeja Vikas , Saluja Ashok , Banerjee Sulagna
Published: August 2019

Pancreatic cancer cells undergo extensive metabolic reprogramming to fulfil the demands of metabolic nutrients to fuel their rapid proliferation. This results in abnormal accumulation of various metabolites which causes both metabolic and nonmetabolic dysregulation and potential transformation to malignancy and therefore termed as “oncometabolites”. Hypoxia is a major hallmark of pancreatic tumors which associated with increase in self-renewal. The present study investigates the role of Hypoxia mediated altered oncometabolite in regulating self-renewal in pancreatic cancer. GC-mass spectrometric analysis of pancreatic cancer cells under hypoxia showed significant accumulation of 2-hydroxyglutarate (2-HG) both in cells and culture supernatant. Further analysis of hypoxic cells showed selective accumulation L- enantiomer form. Self-renewal requires critical balance between stemness and differentiation. Present study showed for the first time that L-2-HG regulates self-renewal by increasing expression of genes associated with stemness (Sox-2, CD133) and by decreasing expression of differentiation genes (PdX-1, HB9, NKX6.1). Further analysis showed that L-2-HG mediated epigenetic changes regulates these self-renewal gens in pancreatic cancer. Our results indicated that hypoxia mediated metabolic changes results in accumulation of L-2-HG which upregulate self-renewal by shifting critical balance of gene expression towards stemness in pancreatic cancer which contribute to chemoresistance and metastasis in Pancreatic cancer.

Citation Format: Vineet K. Gupta, Nikita Sharma, Kousik Kesh, Roey Hadad, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee. Hypoxia promote self-renewal in pancreatic cancer by modulating L-2-Hydroxyglutarate level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5284.
Abstract 5289: Gain-of-function mutant p53 predisposes head and neck keratinocytes and squamous cell carcinoma cells to replicative stress and genomic instability through minichromosome maintenance complex component 5
Authors: Zhao Mei , Wang Tianxiao , Zhen Chen , Wang Jing , Pickering Curtis R. , Chen Junjie , Myers Jeffrey N. , Zhou Ge
Published: August 2019

As the guardian of genome, p53 plays important roles in maintaining the genome integrity, and TP53 mutations thereby often lead to genomic instability in cancers. In addition to the loss-of-function, many TP53 mutations can lead to the gain-of-functions (GOF) that often promote tumor development and tumor progression. As an effort to identify the mechanisms involved in mutant p53 GOF activities in head and neck squamous cell carcinoma (HNSCC), we performed an unbiased proteomic screen to identify the mutant p53 G245D interactome in HNSCC cells by immunoprecipitation, and quantitative proteomics using stable isotope labeling with amino acids in cell culture and liquid chromatography coupled with tandem mass spectrometry. Our results identified that MCM5, a member of replication licensing heterohexameric origin recognition complex minichromosome maintenance 2-7 (MCM2-7), interacts with mutant p53s, which suggests a possible role of mutant p53 in regulation of DNA replication. Consistent with this, we show that overexpression of mutant p53 in p53-depelted oral keratinocytes and HNSCC cells predisposes cells susceptibility to replication stress by inhibiting the dormant origin firing, leading to increased chromosomal instability (CIN) under replication stress. Moreover, by overexpressing and downregulating MCM5, we demonstrate that MCM5 modulates GOF mutant p53-mediated susceptibility to replication stress and CIN. Given the importance of MCM2-7 in replication licensing, initiation, and elongation, our discovery of the relationship between mutant p53 and MCM5 suggests that the MCM2-7 complex is one of the key downstream effectors, through which GOF mutant p53s regulate DNA replication and genomic instability in HNSCC.

Citation Format: Mei Zhao, Tianxiao Wang, Chen Zhen, Jing Wang, Curtis R. Pickering, Junjie Chen, Jeffrey N. Myers, Ge Zhou. Gain-of-function mutant p53 predisposes head and neck keratinocytes and squamous cell carcinoma cells to replicative stress and genomic instability through minichromosome maintenance complex component 5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5289.
Abstract 5293: Celf2 suppresses non-small cell lung carcinoma growth by inhibiting the prex2-pten interaction
Authors: Yeung Yiu To , Fan Suyu , Lu Bingbing , Li Xiang , Bode Ann M. , Dong Zigang
Published: August 2019

The phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway is important in the regulation of cell proliferation through its production of phosphatidylinositol 3,4,5-triphosphate (PIP3). Activation of this pathway is frequently observed in human cancers, including non-small cell lung cancer. The PI3- K/Akt pathway is negatively regulated by the dual-specificity phosphatase and tensin homolog (PTEN) protein. PTEN acts as a direct antagonist of PI3-K by dephosphorylating PIP3. Studies showed that PTEN phosphatase activity is inhibited by PREX2, a guanine nucleotide exchanger factor (GEF). Multiple studies revealed that CELF2, an RNA binding protein, cooperates synergistically with PTEN as a tumor suppressor in many cancers. However, the underlying mechanism as to how CELF2 enhances PTEN activity remains unclear. Here, we report that CELF2 interacts with PREX2 and reduces the association of PREX2 with PTEN. Consistent with this observation, PTEN phosphatase activity is upregulated with CELF2 overexpression and overexpression of CELF2 represses both Akt phosphorylation and cell proliferation only in the presence of PTEN. CELF2 gene delivery significantly inhibited PDX tumor growth. We analyzed 87 paired clinical lung adenocarcinoma samples and results showed that CELF2 protein expression is down-regulated in tumor tissues and associated with poor prognosis. Analysis of TCGA datasets showed that CELF2 expression is also associated with shorter patient survival time in many cancers. Overall, our work suggests that CELF2 plays a novel role in PI3-K signaling by antagonizing the oncogenic effect of PREX2.

Citation Format: Yiu To Yeung, Suyu Fan, Bingbing Lu, Xiang Li, Ann M. Bode, Zigang Dong. Celf2 suppresses non-small cell lung carcinoma growth by inhibiting the prex2-pten interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5293.
Abstract 5295: Targeting Rb phosphatase activity to circumvent CDK4/6 inhibitor resistance
Authors: DiSalvo Kathleen H. , Velez Brian C. , Kravtsov Rebecca L. , Krucher Nancy A.
Published: August 2019

The Retinoblastoma (Rb) tumor suppressor protein plays a role in the control of proliferation, apoptosis, differentiation, invasion and the maintenance of genome stability. Its activity is regulated by phosphorylation on several serine/threonine sites, which is thought to inactivate its tumor suppressive function. Cyclin dependent kinases (CDKs) are responsible for most of the Rb phosphorylation that occurs in cells. It is well established that alterations in the Rb pathway (consisting of CDK4/6, D-type cyclins, the CDK inhibitor p16INK4a, and Rb itself) that lead to excessive phosphorylation of Rb have been observed in almost all cancer types. Alterations that lead to increased CDK activity toward Rb are more common than mutations to the Rb gene itself, a finding that prompted investigations into the development of clinical treatments that inhibit the phosphorylation of Rb by CDKs. The CDK4/6 inhibitors palbociclib and abemaciclib were recently clinically approved for use in breast cancer. However, acquired resistance to CDK4/6 inhibition has been shown to limit clinical efficiency. Targeting Rb phosphorylation by CDK4/6 inhibition has been shown to activate alternate pro-survival pathways such as the AKT pathway. In our studies we developed a method of siRNA-mediated activation of Rb phosphatase that targets Rb phosphorylation in attempt to circumvent the development of resistance. Phosphatase Nuclear Targeting Subunit (PNUTS) associates with and regulates PP1 activity toward Rb. In several cancer cell types, such as HCT116 colon cancer, MIA PaCa-2 pancreatic cancer and MCF7 breast cancer, PNUTS depletion causes dephosphorylation of Rb without AKT activation. Furthermore, in Panc1 pancreatic cancer cells, activation of AKT is observed after 1-hour palbociclib (500nM) treatment, whereas PNUTS depletion does not induce AKT. In MD-MBA-231 breast cancer cells, 1-hour treatment of palbociclib or abemaciclib (20nM) induces the AKT pathway as well as the STAT pathways, however PNUTS depletion in these cells does not induce AKT activation. It has been shown that CDK4/6 inhibitors activate AKT by causing loss of Rb interaction with SIN1, a component the mTORC2 complex. Our studies reveal that PNUTS depletion causes dephosphorylation of Rb, without disrupting Rb-mediated inhibition of mTORC2. Thus, the stimulation of Rb phosphatase activity may target Rb phosphorylation without the development of resistance.

Citation Format: Kathleen H. DiSalvo, Brian C. Velez, Rebecca L. Kravtsov, Nancy A. Krucher. Targeting Rb phosphatase activity to circumvent CDK4/6 inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5295.
Abstract 5296: DNAJA1 promotes cancer progression by stabilizing mutant p53
Authors: Iwakuma Tomoo
Published: August 2019

The tumor suppressor p53 is mutated in approximately 50% of human cancers. Majority of them are missense mutations, which results in accumulation of dysfunctional p53 proteins in tumors. Increasing evidence suggests that accumulation of mutant p53 is crucial for its oncogenic gain-of-function activities, such as proliferation, metastasis, and chemotherapy resistance. Previous work from our lab has revealed that DNAJA1, a HSP40 family member, specifically binds to and stabilizes conformational/misfolded mutant p53. DNAJA1 downregulation induces ubiquitination and proteasomal degradation of conformational p53 mutants, but does not affect the levels of wild-type p53 or DNA contact mutants. Based on these observations, we hypothesize that DNAJA1 promotes cancer progression in a manner dependent on conformational/misfolded mutant p53. To test this hypothesis, we examined the effects of DNAJA1 deletion/knockdown on malignant properties of head and neck squamous cell carcinoma (HNSCC) cells. Genetic deletion/knockdown of DNAJA1 resulted in reduced proliferation, colony formation, migration, and orthotopic tumor growth of HNSCC cells expressing conformational mutant p53, but not DNA contact mutant p53. These cellular phenotypes induced by DNAJA1 knockdown/knockout were substantially rescued by overexpression of DNAJA1 or mutant p53. We also conducted RNA sequencing analyses using HN31 cells (control, DNAJA1 knockdown or mutant p53 knockdown). Notably, 77.2% of downregulated genes by p53 knockdown were overlapped with genes downregulated by DNAJA1 knockdown. Moreover, genes involved in cancer-related pathways, including adhesion and actin cytoskeleton organization, were commonly downregulated. These results strongly suggest that DNAJA1 promotes HNSCC progression mostly dependent on conformational mutant p53. Our results may also suggest that DNAJA1 could be a novel therapeutic target for cancers carrying conformational/misfolded mutant p53.

Citation Format: Tomoo Iwakuma. DNAJA1 promotes cancer progression by stabilizing mutant p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5296.
Abstract 5301: The role of ID4 in regulating SAT1 gene expression in prostate cancer phenotype
Authors: Al-Zahrani Majid
Published: August 2019

Introduction: Inhibitor of DNA binding 4 (ID4), a bHLH protein and a transcriptional regulator, is a tumor suppressor gene in prostate cancer. ID4 is expressed in prostate cancer cells (LNCaP), which are androgen sensitive, but epigenetically silenced in a more aggressive DU145 cells. The epigenetic silencing of ID4 is associated with increasing tumor grade and castration resistant prostate cancer (CRPC). ID4 regulates the expression of some other genes such as SAT1, which is a rate limiting enzyme found in the X chromosome involved in the catabolic pathway of polyamine metabolism by catalyzing the N(1)-acetylation of spermidine and spermine. Recently, SAT1 was also shown to mediate histone H3 acetylation suggesting its role in regulating gene expression. Next generation sequencing (NGS) data showed a significant low expression of SAT1 when ID4 is up regulated and vise-versa, which led to a hypothesis that ID4 might regulate the expression of SAT1 gene in prostate cancer cells.

Experimental procedures: The NGS (exon sequencing) was performed on prostate cancer cells either over-expressing ID4 (DU145(+)ID4) or cells in which ID4 was genetically silenced (LNCaP(-)ID4). Moreover, immunohistochemistry (IHC) analysis was performed on mice prostates tissues (WT and ID4-/- mice) and DU145(+)ID4 tumor xenografts tissues to analyze SAT1 protein expression in the presence and the absence of ID4.

Results: The NGS and IHC data analysis suggested that Spermidine/spermine N(1)-acetyltransferase (SAT1) is up-regulated in cells lacking ID4 such as (LNCaP(-)ID4) but down-regulated in cells which express ID4 (DU145(+)ID4). These results prompted us to investigate the effect of silencing SAT1 on ID4 expression and on cancer phenotype.

Conclusion: The expression of ID4 gene in prostate cancer cell lines LNCaP, and DU145(+)ID4 cells showed low expression of SAT1 gene compared with cells lacking ID4 gene as in LNCaP(-)ID4 and DU145 cells.

Acknowledgment: These studies were supported by the NIH/NIMHD/P20 Grant

#P20MD002285.

Citation Format: Majid Al-Zahrani. The role of ID4 in regulating SAT1 gene expression in prostate cancer phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5301.
Abstract 5308: BRMS1L, a metastatic suppressor gene, is transcriptionally regulated by p53
Authors: Tokino Takashi , Koyama Ryota , Idogawa Masashi , Sasaki Yasushi
Published: August 2019

The tumor suppressor p53 plays a pivotal role in the cell fate determination in response to diverse upstream signals. p53 regulates a number of genes that are involved in cell cycle arrest, apoptosis, senescence, and maintenance of genomic stability. Recent studies revealed that p53 is also important for the regulation of cell invasion and migration. Our group has previously revealed that the p53 transactivates anti-invasion genes such as CLCA2, ICAM2, and LIMA1. However, the role of the p53 downstream pathway in cancer invasion, migration, and metastasis remains unclear. In this study, microarray analysis showed that breast cancer metastasis-suppressor 1- like (BRMS1L) is upregulated by p53. We identified two responsive elements of p53 family proteins in the first intron and upstream of BRMS1L. These response elements are well conserved among mammals. Functional analysis showed that ectopic expression of BRMS1L inhibited cancer cell invasion and migration; knockdown of BRMS1L by siRNA induced the opposite effect. Importantly, clinical databases revealed that reduced BRMS1L expression correlated with poor prognosis in patients with breast and brain cancer. Together, these results strongly demonstrate that BRMS1L is one of the mediators downstream of the p53 pathway, and that BRMS1L inhibits cancer cell invasion and migration, which are essential steps in cancer metastasis. Collectively, our results suggest that BRMS1L is involved in cancer cell invasion and migration, and could be a therapeutic target for cancer.

Citation Format: Takashi Tokino, Ryota Koyama, Masashi Idogawa, Yasushi Sasaki. BRMS1L, a metastatic suppressor gene, is transcriptionally regulated by p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5308.
Abstract 548: Evaluation of novel SIRP antibodies as potential cancer therapeutics
Authors: Hiebsch Ronald R. , Bouchlaka Myriam N. , Capoccia Benjamin J. , Donio Michael J. , Chakraborty Prabir , Wilson W. Casey , Puro Robyn J. , Pereira Daniel S.
Published: August 2019

Targeting immune checkpoints of adaptive immunity has shown great therapeutic efficacy in oncology, but in a limited fraction of patients. Innate immune cells represent the most abundant immune cell types in many solid tumors and are often linked to a poor prognosis.

SIRPα is expressed by innate immune cells and its interaction with CD47, expressed by most tumor cells, is an important immune checkpoint of the innate response, involved in the regulation of phagocytosis by macrophages, dendritic cells and neutrophils.

Recently, first generation agents targeting CD47 (CD47 antibodies and SIRPα-Fc fusion proteins) have shown promise in clinical trials, but they have also experienced hematological toxicities such as anemia or thrombocytopenia. Consequently, we have previously reported on the development of AO-176, a next generation anti-CD47 antibody that not only blocks the CD47/SIRPα interaction and induces phagocytosis, but also preferentially binds tumor versus normal cells (including RBCs where it binds negligibly) and directly kills tumor cells via a programmed cell death type III and an immunogenic cell death process.

Here we report the discovery of novel anti-SIRP antibodies that recognize either SIRPα selectively or SIRPα/γ. These antibodies are being evaluated for their ability to induce phagocytosis of tumor cells - we have identified antibodies that induce phagocytosis of tumor cells alone and in combination with Rituxan. The ability of our anti-SIRP antibodies to induce immunomodulatory activities in a variety of ex vivo cultured immune cells expressing either SIRPα or SIRPα/γ is also under investigation and will be presented.

Citation Format: Ronald R. Hiebsch, Myriam N. Bouchlaka, Benjamin J. Capoccia, Michael J. Donio, Prabir Chakraborty, W. Casey Wilson, Robyn J. Puro, Daniel S. Pereira. Evaluation of novel SIRP antibodies as potential cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 548.
Abstract 555: A novel human TNFR2 antibody (MM-401) modulates T cell responses in anti-cancer immunity
Authors: Sampson James F. , Kurella Vinodh B. , Paragas Violette , Kumar Sandeep , Lulo James E. , Qiu James A. , Razlog Maja , Fulton Ross B. , Camblin Adam J. , Richards Jennifer M. , Wong Christina S. , Koshkaryev Alexander , Suchy James J. , Grabow Stephanie , Muda Marco , Raue Andreas , Drummond Daryl C. , Tam Eric M.
Published: August 2019

TNFR2 is an emerging therapeutic target in immuno-oncology. We have previously shown that an agonistic mouse TNFR2 antibody was able to activate CD8+ T cells in vitro and has anti-tumor activity in multiple syngeneic mouse tumor models. This activity required CD8+ T cell and NK cell response and involves Fc γ receptor engagement. Based on this compelling evidence, we have developed MM-401, a human antibody targeting the TNFR2 receptor. MM-401 was humanized from a mouse hybridoma antibody by CDR grafting and includes additional mutations for improved affinity and biophysical properties. Consequently, MM-401 binds with low nanomolar affinity to a region in human TNFR2 that corresponds to the mouse TNFR2 epitope of our mouse surrogate antibody. Although the antibody competes with TNF α in binding the receptor, MM-401 has agonistic activity and induces TNFR2 signaling as observed using a NFκB reporter cell assay. Upon incubation of MM-401 with CD4+ and CD8+ T cells from healthy human blood, we observed upregulation of activation markers and cytokine production comparable to utomilumab (anti-4-1BB), MEDI6469 (anti-OX40), and TRX518 (anti-GITR). We also observed that MM-401 promotes antibody-dependent cellular cytotoxicity (ADCC) in an NK cell-mediated in vitro assay and a reduction in the number regulatory T (Treg) cells in ovarian cancer ascites samples. These data suggest that MM-401 could also promote anti-tumor immunity by mediating ADCC, as well as by direct co-stimulation of T cell responses. Currently, we are evaluating MM-401 in patient derived xenograft (PDX) models in humanized mice generated using NSG-SGM3 mice with cord blood CD34+ hematopoietic stem cells. These results justify the continued development of MM-401 as a modulator of anti-tumor immunity for the treatment of cancer.

Citation Format: James F. Sampson, Vinodh B. Kurella, Violette Paragas, Sandeep Kumar, James E. Lulo, James A. Qiu, Maja Razlog, Ross B. Fulton, Adam J. Camblin, Jennifer M. Richards, Christina S. Wong, Alexander Koshkaryev, James J. Suchy, Stephanie Grabow, Marco Muda, Andreas Raue, Daryl C. Drummond, Eric M. Tam. A novel human TNFR2 antibody (MM-401) modulates T cell responses in anti-cancer immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 555.
Abstract 609: Vitamin D binding protein and risk of renal cell carcinoma in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial
Authors: Kratzer Tyler B. , Mondul Alison M. , Weinstein Stephanie J. , Albanes Demetrius
Published: August 2019

Introduction/Study purpose: Vitamin D binding protein (DBP) is the major carrier of vitamin D in circulation. In addition to its role in vitamin D transport, DBP has several other biologic mechanisms that may be relevant to cancer risk including macrophage activation, apoptosis, and angiogenesis. Although previous studies have demonstrated no association between vitamin D and RCC, our group has conducted two previous studies which found strong inverse associations between DBP and RCC. We undertook the current analysis to replicate our findings in a different study population.

Methods: We conducted a nested case-control study in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO). Cases (n=323) were matched 1:1 to controls on age (+/- 1 year), race, date of blood collection (+/- 30 days) and sex. We performed conditional logistic regression to estimate the odds ratios and 95% confidence intervals for the association between quartiles of circulating DBP and risk of RCC. In addition to conditioning on the matching factors, multivariable models were further adjusted for history of diabetes or hypertension, family history of renal cancer, body mass index (BMI), and smoking status. Additionally, we performed stratified analyses by sex, family history of renal cancer, BMI, smoking status, and tumor stage at diagnosis.

Results: We observed a statistically significant positive association between DBP and RCC that persisted after multivariable adjustment (mv-adj Q4 vs. Q1 OR=3.1, 95% CI=1.8-5.6; p-trend <0.0001). Sensitivity analyses were performed removing matched pairs where the case was diagnosed within 2 years of blood collection or experienced a large weight loss (>40 lbs), with little effect on the overall trend (Q4 vs. Q1 OR=2.5 CI=1.4-4.6 p-trend=<0.0001).

Conclusions: In the present study, those with higher serum concentrations of DBP were at increased risk of RCC, which differs from previously-published findings. Further research is necessary to determine the true

association between DBP and risk of RCC, and whether different DBP phenotypes via GC variation may have different associations with risk of RCC.

Note: This abstract was not presented at the meeting.

Citation Format: Tyler B. Kratzer, Alison M. Mondul, Stephanie J. Weinstein, Demetrius Albanes. Vitamin D binding protein and risk of renal cell carcinoma in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 609.