bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1458: TRPM2 modulates neuroblastoma cell survival through Pyk2, CREB and MCU activation
Authors: Hirschler-Laszkiewicz Iwona Malgorzata , Chen Shu-jen , Bao Lei , Wang JuFang , Zhang Xue-Qian , Shanmughapriya Santhanam , Keefer Kerry , Madesh Muniswamy , Cheung Joseph Y. , Miller Barbara A.
Published: August 2018

Transient Receptor Potential Melastatin subfamily member 2 (TRPM2) is highly expressed in many cancers including neuroblastoma. Proline-rich tyrosine kinase 2 (Pyk2) is calcium-sensitive non-receptor tyrosine kinase which enhances cell survival and is activated by calcium influx through TRPM2. Pyk2 also modulates mitochondrial calcium uptake through phosphorylation of the mitochondrial calcium uniporter (MCU), impacting mitochondrial function, and through its effects on CREB, which regulates transcription of MCU. The purpose of this work was to investigate the mechanisms by which TRPM2 regulates cancer cell survival in order to develop new therapeutic strategies based on inhibition of TRPM2. Two models of TRPM2 inhibition were utilized: 1) reducing TRPM2 function in SH-SY5Y cells by expression of the dominant negative construct TRPM2-S, a short isoform of TRPM2, and 2) depletion of TRPM2 expression with CRISPR/Cas9 technology. Treatment with Doxorubicin (Doxo) was used as a model of oxidative stress. Doxorubicin reduced growth of cells in which TRPM2 was inhibited significantly more than control cells. Inhibition of TRPM2 also reduced Pyk2 and CREB expression and activation. Using shRNA technology we showed that Pyk2 mediated reduced expression and activation of CREB and activation of STAT3 in TRPM2 expressing cells. Fractionation experiments showed that depletion of TRPM2 reduced phosphorylation of whole cell and mitochondrial Src and Pyk2 and expression of mitochondrial CREB and MCU. Src has an important role in Pyk2 phosphorylation. In TRPM2 depleted cells, mitochondrial Ca uptake was significantly reduced since both peak mitochondrial calcium uniporter currents and amount of calcium transported (current-time integral) measured in mitoplasts were lower. Wt Pyk2 but not the phospho-deficient mutant Y402F and wt TRPM2 but not the TRPM2 Ca-impermeant mutant E960D, restored viability after Doxo treatment of SH-SY5Y cells in which TRPM2 was inhibited. In conclusion, these results suggest that TRPM2 inhibition leads to reduced viability in neuroblastoma through the Src-Pyk2-CREB-MCU pathway, which plays a key role in regulating mitochondrial function and cellular bioenergetics.

Citation Format: Iwona Malgorzata Hirschler-Laszkiewicz, Shu-jen Chen, Lei Bao, JuFang Wang, Xue-Qian Zhang, Santhanam Shanmughapriya, Kerry Keefer, Muniswamy Madesh, Joseph Y. Cheung, Barbara A. Miller. TRPM2 modulates neuroblastoma cell survival through Pyk2, CREB and MCU activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1458.
Abstract 1459: A mechanism counteracting micronucleation for maintenance of genomic integrity
Authors: Samwer Matthias , Schneider Maximilian W. , Hoefler Rudolf , Schmalhorst Philipp , Jude Julian , Zuber Johannes , Gerlich Daniel W.
Published: August 2018

A hallmark of eukaryotic cells is that they store all their chromosomes in a single nucleus. This is important for the maintenance of genomic integrity, as individual chromosomes packaged into separate micronuclei are prone to massive DNA damage. Micronuclei are characteristic for cancer cells and thought to drive the evolution of cancer genomes. However, the mechanisms normally promoting the packaging of all chromosomes into a single nucleus have remained largely elusive. Animal cells undergo an open mitosis, in which the cell disassembles its nucleus to release a set of individualized chromosomes. At the end of mitosis, cells reassemble a single nucleus around a complete set of chromosomes, utilizing endoplasmic reticulum-derived membranes. How cells restrict nuclear envelope assembly to the surface around the set of anaphase chromosomes is unclear. It has been proposed that the mitotic spindle may help anaphase chromosomes form a continuous surface by bringing them into proximity. Using live cell imaging, we found that the mitotic spindle is not required to form a single nucleus in human cells. We then performed an RNAi screen for spindle-independent factors that are required to shape a single nucleus and identified the protein barrier-to-autointegration factor (BAF). BAF has been previously suggested to contribute to nuclear assembly by linking chromatin to nuclear envelope membranes containing “LEM” domain (Lap2/Emerin/Man1) proteins. However, by RNAi and mutational analysis, we unexpectedly discovered that BAF's LEM-binding activity was dispensable for nuclear assembly in human cells. Instead, we found that BAF's ability to dimerize and cross-bridge distant DNA segments was essential to shape a single nucleus. By probing purified chromatin and recombinant BAF proteins with atomic force microscopy, we found that BAF forms a rigid shell at the chromatin surface, depending on its DNA-DNA cross-bridging activity. In cells, BAF restricted membranes to the surface formed by the set of anaphase chromosomes, thereby preventing the formation of micronuclei. Thus, BAF regulates chromosome mechanics to shape a single nucleus during mitotic exit.

Citation Format: Matthias Samwer, Maximilian W. Schneider, Rudolf Hoefler, Philipp Schmalhorst, Julian Jude, Johannes Zuber, Daniel W. Gerlich. A mechanism counteracting micronucleation for maintenance of genomic integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1459.
Abstract 1462: The oncogene MYC induces chromosomal instability through dysregulating mitotic progression evoking a dependency on TPX2
Authors: Rohrberg Julia , Corella Alexandra , Taileb Moufida , Jokisch Marie-Lena , Camarda Roman , Balakrishnan Sanjeev , Zhou Alicia , Goga Andrei
Published: August 2018

More than 70% of common solid tumors are aneuploid. Aneuploidy has been implicated in tumorigenesis for decades. A direct cause of aneuploidy is chromosomal instability (CIN) that fuels tumor evolution by providing a heterogeneous subpopulation of cells that contributes to the lethal outcome of cancer, metastasis, therapeutic failure, and drug resistance. The oncogene c-MYC is overexpressed in many cancers and has been shown to induce structural and numerical chromosomal aberrations and aneuploidy. While the role of MYC in overcoming the G1/S checkpoint is well established, the role of MYC in altering mitotic dynamics and eliciting CIN remains poorly understood. Here, we identify a direct influence of increased MYC expression on mitotic progression and spindle assembly. We observe MYC-dependent microtubule nucleation and polymerization defects in mitosis. MYC overexpression induces altered spindle morphology promoting chromosome segregation defects resulting in micronuclei formation, CIN and aneuploidy. Importantly, attenuation of MYC expression reverses the mitotic defects observed, even in established tumor cell lines, implicating an ongoing role for MYC in the persistence of a CIN phenotype in tumors. Gene expression data reveal that MYC regulates numerous genes involved in mitosis, kinetochore function and microtubule behavior. We performed a screen to identify microtubule regulators required for the survival of MYC overexpressing cells. MYC overexpressing cells are reliant on high TPX2 expression, a master microtubule regulator. High TPX2 expression is permissive for mitotic spindle assembly and chromosome segregation in MYC high cells, while TPX2 depletion attenuates cell viability and tumor growth in vivo. Our data suggest that TPX2 is a transcriptional target of MYC. We postulate that high levels of TPX2 are required to tolerate mitotic stress induced by MYC. TPX2 compensates for impaired mitotic progression and prevents the development of physiologically intolerable levels of CIN. High levels of TPX2 might be required for tumors with oncogene-induced mitotic stress to faithfully proceed through mitosis providing an explanation for the abundant upregulation of TPX2 in cancer. Thus, we describe a conditional role for MYC in altering spindle assembly and dynamics, resulting in a dependency on TPX2 for tumor survival.

Citation Format: Julia Rohrberg, Alexandra Corella, Moufida Taileb, Marie-Lena Jokisch, Roman Camarda, Sanjeev Balakrishnan, Alicia Zhou, Andrei Goga. The oncogene MYC induces chromosomal instability through dysregulating mitotic progression evoking a dependency on TPX2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1462.
Abstract 1481: Selective protein expression in tumor cells controlled by progression elevated gene-3 promoter
Authors: Pando Ruben , Shrestha Tej , Pyle Marla , Troyer Deryl
Published: August 2018

The progression elevated gene-3 (PEG-3) promoter is a rodent gene that has shown strong activity in tumor progression. Demonstrating tumor specificity and activity in at least five different human cancers, PEG-3 is considered a novel promoter for tumor-specific gene expression. Understanding how PEG-3 controls expression of reporter proteins is of biomedical significance because of the role it could play in new therapeutic strategies against certain types of cancer. In this study, we aim to use the PEG-3 promoter to observe the expression of firefly luciferase 2 (luc 2), a bioluminescent reporter protein, in the transfection of mammalian cells. Ultimately, we plan to use PEG-3 to express a therapeutic gene, cytosine deaminase, in cancer specific areas of breast, lung, and ovary.

Citation Format: Ruben Pando, Tej Shrestha, Marla Pyle, Deryl Troyer. Selective protein expression in tumor cells controlled by progression elevated gene-3 promoter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1481.
Abstract 1486: Cxcr4/Sdf-1 expression in Her2/Neu breast cancer biopsies and its relation to adjuvant treatment
Authors: Ayala Natalia C. , Campos Melina N. Lorenzini , Pescie Mariana M. Gomez , Alsina Angel E. , Zimerman Jorge R. , Zimmermann Maria C.
Published: August 2018

Background: Chemokines have a fundamental role in inflammation and in cancer progression. Chemokine receptor 4 (CXCR4) is a transmembrane receptor that belongs to the CXC chemokine receptor family and is the only known receptor for the Stromal Cell derived Factor-1(SDF-1). Many retrospective studies have documented that the expression of various chemokine receptors, particularly CXCR4, was associated with a poor prognosis in patients with breast cancer. In the present study, we proposed an SDF-1/CXCR4 induced expression in HER2/neu positive breast cancer biopsies. We, additionally, study its relation to patient's treatment, prior and post adjuvant therapy. Methods: Immunohistochemical staining of her2/neu and ki64 were analyzed in mammary tumor biopsies. Reduction mammoplasty of normal patients were also studied as normal controls. Stained hematoxilyn and eosin slides where previously revised to confirm histological diagnostic and tumoral classification based on established criteria. Additional information considering biological markers such as estrogen receptor (ER), progesterone receptor (PR) expression were also observed in histopathological reports. Quantitative PCR was performed by amplifying a conserved region of the CXCR-4 and SDF-1 genes, and their correlation to Her2/neu expression and adjuvant therapy were analyzed. Statistical analysis was assess using appropriate parametric and nonparametric tests. Results: Her2/neu breast tumor specimens expressed SDF-1 at varying levels and differed upon tumor classification. Expression of the receptor correlates mainly with the tumor type and degree. Quantitative PCR showed low constitutive CXCR4 expression in normal breast tissue that increased in breast tumor tissue, particularly in those expressing her2/neu. Variations upon treatment correspond to those of tumor progression to malignancies and recurrence. Conclusions: We established a correlation between the expression of SDF-1/CXCR4 and the tumoral characteristics prior and post adjuvant treatment. A more careful examination of experimental/clinical data may help to explain the mechanism(s) involving the SDF-1/CXCR4 signaling axis in the breast cancer pathophysiology.

Citation Format: Natalia C. Ayala, Melina N. Lorenzini Campos, Mariana M. Gomez Pescie, Angel E. Alsina, Jorge R. Zimerman, Maria C. Zimmermann. Cxcr4/Sdf-1 expression in Her2/Neu breast cancer biopsies and its relation to adjuvant treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1486.
Abstract 1498: The role of tissues specific super-enhancers in mediating the genetic risk of ovarian cancer
Authors: Vavra Kevin C. , Corona Rosario I. , Seo Ji-Heui , Coetzee Simon G. , Lee Janet M. , Freedman Matthew L. , Pharoah Paul D. , Hazelett Dennis J. , Gayther Simon A. , Lawrenson Kate
Published: August 2018

Super-enhancers demarcate regions of master regulators involved in cellular differentiation and tumorigenesis. While many trait-associated variants discovered through genome wide association studies are enriched in regulatory elements, the role of super-enhancers in disease susceptibility is less well understood. We evaluated the role of super-enhancers, defined by H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) analysis of epithelial ovarian cancers (EOC) and precursor cells, in mediating the genetic risk of EOC. We characterized super-enhancer-risk SNP intersections to identify a series of risk-associated loci where multiple candidate causal alleles overlap with ovarian super-enhancers (approximately 33% of candidate causal loci). In particular, a risk locus at 3q25 marked by SNP rs7651446, (odds ratio = 1.59, p = 1.5x10-34), is tagged by 79 candidate causal risk SNPs, more than half of which coincide with super-enhancers detected in EOC precursor cells (ovarian and fallopian tube epithelia) but not in ovarian cancer cell lines or primary tumors suggesting that loss of activity of this super-enhancer is associated with EOC development.

We hypothesized that SNPs at the 3q25 locus modulate activity of super-enhancers that regulate in cis the expression of genes involved in the identity of normal tissue precursors, and that these genes are downregulated during tumorigenesis. We tested the effect of decreasing the binding affinity of BRD4, a protein that binds super-enhancer regions, using a small molecule inhibitor (JQ1). We measured the expression of genes at this locus following JQ1 treatment in EOC precursor cells, identifying three candidate genes, LEKR1, SSR3, and TiPARP, that show decreased expression due to BRD4 binding inhibition. For SSR3 and TIPARP, these expression level changes mirrored the differential expression observed between normal precursor cells and primary high grade serous ovarian cancer. We then established stable shRNA-mediated knockdown models for SSR3 and TIPARP in normal EOC precursor cell lines to establish a role for these genes in ovarian cancer development. In summary, we have identified a potential role for super-enhancers at a subset of EOC risk loci, and present a functional pipeline for identification of the target gene/genes and evaluation of their role in neoplastic transformation.

Citation Format: Kevin C. Vavra, Rosario I. Corona, Ji-Heui Seo, Simon G. Coetzee, Janet M. Lee, Matthew L. Freedman, Paul D. Pharoah, Dennis J. Hazelett, Simon A. Gayther, Kate Lawrenson. The role of tissues specific super-enhancers in mediating the genetic risk of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1498.
Abstract 1499: Vitamin D promotes differentiation in patient-derived organoids in benign and cancer prostate models
Authors: McCray Tara N. , Lugli Giovani , Nonn Larisa
Published: August 2018

Deficiency in the prohormone vitamin D is associated with increased risk of lethal prostate cancer (PCa) and less-differentiated, more aggressive tumors. As cancer progresses there is aberrant expression of vitamin D metabolic genes causing dysregulation of the vitamin D pathway, indicating its role in disease. Vitamin D has many chemopreventive actions in vitro, including promotion of differentiation and apoptosis. The ability for vitamin D to drive differentiation has been explored in various cell types and cancer lines, but its mechanism of action in the prostate is not well defined. Our lab found that vitamin D treatment globally upregulates microRNAs (miRs) in prostate cell lines, primary cells and patient samples. Temporal regulation of miRs is critical for differentiation and levels of mature miRs are globally lower in PCa. Given the relationship between vitamin D and microRNAs, and their influence over differentiation, we hypothesize that vitamin D promotes differentiation in the prostate in a microRNA-dependent manner and that this process is disrupted during cancer. Here we examined organoid differentiation in the presence of 1,25D, the active metabolite of vitamin D. Organoids were grown in matrigel from human primary prostate epithelial progenitor cells. Differentiation was assessed by organoid size and branching, and by expression of epithelial markers. Organoids grown in the presence of 1,25D were strikingly larger and formed more complex branching structures than those treated with control. Whole mount immunofluorescence revealed expression of basal and luminal epithelial cells in the organoids. Flow-sorted organoids had a greater composition of basal cells when grown in the presence of 1,25D. RT-qPCR analysis of the flow-sorted epithelial populations revealed an enhancement in the WNT pathway with 1,25D, this pathway is critical in prostate development and dysregulated in cancer. The role of miRNAs was determined by reducing global miRNA levels via knockdown of Drosha, an essential miRNA-processing protein. Knockdown abrogated the effect of 1,25D, as evidenced by reduced size, round shape, and protein expression. Together our findings indicate a role of 1,25D in normal prostate development and branching, a phenotype that is lost when mature miR levels are reduced. Ongoing studies examine these phenotypes in PCa organoids.

Citation Format: Tara N. McCray, Giovani Lugli, Larisa Nonn. Vitamin D promotes differentiation in patient-derived organoids in benign and cancer prostate models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1499.
Abstract 1512: Regulation of the CDC42 signaling pathway by IKZF1 in T-cell acute lymphoblastic leukemia
Authors: Soliman Mario , Song Chunhua , Payne Jonathon L. , Ge Zheng , Gowda Chandrika , Ding Yali , Payne Kimberly J. , Dovat Sinisa
Published: August 2018

The IKZF1 gene encodes Ikaros—a DNA-binding, Kruppel-like zinc finger protein that functions as a tumor suppressor in T-cell acute lymphoblastic leukemia (T-ALL). Ikaros regulates transcription of a large number of genes that are involved in control of cellular proliferation. However, the molecular mechanisms through which Ikaros regulates proliferation of T-ALL cells are still unknown. The use of quantitative chromatin immunoprecipitation (qChIP) showed that Ikaros binds to the promoter regions of CDC42 in vivo in primary T-ALL patient samples as well as in T-ALL cell lines. Ikaros overexpression in B-ALL cells via retroviral transduction results in decreased transcription of CDC42, as evidenced by qRT-PCR and Western blot. The luciferase reporter assay showed that Ikaros represses CDC42 transcription by directly binding to its promoter. The shRNA-mediated knock-down of Ikaros in T-ALL resulted in increased expression of CDC42. These results suggest that Ikaros functions as a transcriptional repressor of CDC42 in T-ALL. Next, we studied the upstream signaling pathways that regulate Ikaros-mediated control of CDC42 transcription in T-ALL. Because Ikaros activity in leukemia is regulated via direct phosphorylation by Casein Kinase II (CK2), we tested the effect of CK2 inhibition on Ikaros-mediated regulation of CDC42 expression. Treatment of T-ALL cells with a specific CK2 inhibitor, CX-4945, resulted in reduced expression of CDC42 in T-ALL cells. This was associated with an increase in DNA-binding of Ikaros to the promoter of the CDC42 gene. Ikaros knock-down restored high expression of CDC42 in T-ALL cells that were treated with CK2 inhibitors. These data suggest that transcriptional repression of the CDC42 gene by Ikaros is impaired in T-ALL due to increased CK2 activity and reduced Ikaros DNA-binding affinity toward the CDC42 gene promoter. Inhibition of CK2 restores Ikaros function and results in transcriptional repression of the CDC42 gene. In conclusion, our results suggest that Ikaros regulates CDC42 signaling pathway via transcriptional repression of the small GTPase, CDC42, in T-ALL. Ikaros-mediated repression of CDC42 in T-ALL is negatively regulated by CK2. These results suggest that regulation of CDC42 expression is one of the mechanisms through which Ikaros regulates cellular proliferation in T-ALL.

Citation Format: Mario Soliman, Chunhua Song, Jonathon L. Payne, Zheng Ge, Chandrika Gowda, Yali Ding, Kimberly J. Payne, Sinisa Dovat. Regulation of the CDC42 signaling pathway by IKZF1 in T-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1512.
Abstract 1516: BAT-1 up-regulation decreases cell invasion and migration in vitro
Authors: Garcia-Vargas Aileen M. , Sanchez Maria , Martinez-Ferrer Magaly
Published: August 2018

Prostate cancer is the most common cancer in men in the United States and is the third cause of cancer related deaths in men. In Puerto Rico, prostate cancer is the most common type of cancer and the leading cause of cancer death in men. Approximately one out of seven men will be diagnosed with prostate cancer in his lifetime. The current available biomarkers are unable to predict malignant outcomes such as recurrence. Thus, there is a critical demand for the development of innovative diagnostic and prognostic tools for the management of prostate cancer. In the current study, we evaluated the biological role of BAT-1 in prostate cancer. Preliminary data from patients who had prostate cancer recurrence identified that HLA-B associated transcript 1 (BAT-1) was down-regulated in patients with prostate cancer recurrence when compared with non-recurrent patients. We up-regulated BAT-1 in androgen independent PC3 and androgen dependent 22RV1 prostate cancer cell lines using a BAT-1 recombinant protein. In vitro assays were performed to measure proliferation, migration and invasion. Proliferation assays using MTS showed no significant change in BAT-1 recombinant protein concentrations ranging from 0.25-2.0μg/mL at 12, 24 and 48hrs for PC3 and 22RV1 cells. Wound healing assay showed that cells treated with BAT-1 recombinant protein at 0.25μg/mL and 2.0μg/mL significantly decreased migration at 6, 12hrs and 24hrs in PC3 cells when compared to control. Boyden assay showed that cells treated with BAT-1 recombinant protein at 0.25μg/mL and 2.0μg/mL significantly decreased invasion in PC3 and 22RV1 cells at 24hrs when compared to control. Our results showed that BAT-1 up-regulation decreased migration and cell motility. Cells did not show a significant change in proliferation. These results were expected due to previous data showing that BAT-1 down-regulation increased cell migration and invasion, suggesting that BAT-1 expression promotes aggressiveness in prostate cancer recurrence. A possible mechanism of action for BAT1 is the modulation of cell migration and invasion.

Citation Format: Aileen M. Garcia-Vargas, Maria Sanchez, Magaly Martinez-Ferrer. BAT-1 up-regulation decreases cell invasion and migration in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1516.
Abstract 1527: Splicing factors determine breast cancer cell mitosis through control of sister chromatid cohesion
Authors: Koedoot Esmee , Martens John , Dévédec Sylvia E. Le , Water Bob van de
Published: August 2018

Breast cancer is the mostly diagnosed cancer in women worldwide and is also the leading cause of death from cancer in women, mainly due to metastasis formation. Accumulating evidence suggests that RNA splicing is critical in breast cancer progression. To investigate the role of every single component of the splicing machinery (the spliceosome) in this process, we systematically evaluated the effect of siRNA-mediated knockdown of 244 splicing factors on proliferation in two highly proliferative breast cancer cell lines MDA-MB-231 and Hs578T. Hits of the primary screen were validated with single siRNAs and separated based on their effects on migration and proliferation respectively. Knockdown of nine validated proliferation hits, amongst which was SF3B1, a known breast cancer driver gene, resulted for all nine hits in a distinct poly-lobed nuclear phenotype in both cell types, accompanied by increased DNA content and higher number of cells in G1-S transition. Increased levels of phospho-Histone H3 and lack of metaphase alignment suggested a defect in mitosis, which was confirmed by decreased RNA expression of sister chromatid cohesion factors MAU2, ESPL1 and SMC1A and interestingly, increased CDCA5 RNA levels. Upon splicing factor knockdown, CDCA5 intron 1 was retained resulting in decreased CDCA5 protein levels, inaccurate metaphase alignment and finally cell death. To identify the direct partners of the splicing factors that control mitosis, we coupled pull-down of GFP fused splicing factors with mass spectrometry. Seven out of the nine splicing factors that were identified in our screen causing poly-lobed nuclear phenotypes, resided in the same protein complex together with proteins involved in snRNP assembly and some mitosis-related factors. In conclusion, we have identified several splicing factors that are critically determining breast cancer cell proliferation through modulating the expression of chromatid cohesion factors and thereby mediating the successful metaphase alignment and ultimate mitosis.

Citation Format: Esmee Koedoot, John Martens, Sylvia E. Le Dévédec, Bob van de Water. Splicing factors determine breast cancer cell mitosis through control of sister chromatid cohesion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1527.
Abstract 153: A genome-wide CRISPR screen in hESC uncovers JNK pathway mediated inhibition of human definitive endoderm formation
Authors: Li Qing , Huangfu Danwei
Published: August 2018

Human embryonic stem cells (hESCs) are uniquely suitable for interrogating human development and birth defects with high-throughput genetic manipulation. Using CRISPR/Cas, we have conducted a forward genetic screen for genes that regulate the formation of human definitive endoderm (DE), the first step towards the generation of cells in respiratory and gastrointestinal organs. Our screen identified both known and unknown regulators of endoderm differentiation, including a previously unrecognized inhibitory mechanism mediated by the JNK/JUN pathway. JNK inhibitor treatment significantly improves the efficiency of hESC differentiation to endoderm, leading to more efficient creation of endoderm-derived pancreatic and lung lineage cells. The AP1 transcription factor JUN co-occupies ESC enhancers with SMAD2/3 and OCT4, and impedes the re-configuration of the SMAD2/3 chromatin-binding landscape during the ESC-to-DE transition. Thus the hESC-based CRISPR screen enables unbiased interrogation of the functional genome during human development, which can inform hESC directed differentiation for regenerative medicine and disease modeling.

Citation Format: Qing Li, Danwei Huangfu. A genome-wide CRISPR screen in hESC uncovers JNK pathway mediated inhibition of human definitive endoderm formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 153.
Abstract 1531: A bispecific chimeric-antigen receptor T-cell factory for triple-negative breast cancer
Authors: Kuiper Emily G. , Chan Leo L. , Chang Matthew R. , Zhu Quan K. , Marasco Wayne A.
Published: August 2018

Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer with poor clinical prognosis. While treated with chemotherapies, the high incidence of relapse signifies the need for novel, targeted therapies. Immune therapies offer an exciting therapeutic option for TNBC. Our goal is to engineer a chimeric-antigen receptor (CAR) T-cell factory, a CAR T-cell that secretes immune-modulating antibodies, for TNBC.

Chimeric-antigen receptor (CAR) T-cell therapies redirect a patient's T-cells to kill tumor cells by the exogenous expression of a CAR. A CAR is a membrane spanning fusion protein that links the antigen recognition domain of an antibody to the intracellular signaling domains of the T-cell receptor and co-receptor. Solid tumors offer unique challenges for CAR-T therapies. Unlike blood cancers, tumor-associated target proteins are overexpressed between the tumor and healthy tissue resulting in on-target/off-tumor T-cell killing of healthy tissues. Furthermore, immune repression in the tumor microenvironment (TME) limits the activation of CAR-T cells towards killing the tumor.

We hypothesize that a bispecific CAR targeting two antigens on TNBC will mitigate on-target/off-tumor T-cell killing and that the secretion of a checkpoint blockade antibody will remove repression in the tumor microenvironment. Following local immune restoration, the CAR-T cells and other cells in the TME will work synergistically to shrink and clear tumors. Our current work evaluates human single-chain variable fragments (scFvs) to serve as CAR-targeting moieties. We are evaluating the efficacy of scFvs to specifically kill target cells in vitro. This work will define lead scFvs used for engineering of our bispecific CAR. Future work will evaluate this therapy in humanized mouse models of TNBC.

Citation Format: Emily G. Kuiper, Leo L. Chan, Matthew R. Chang, Quan K. Zhu, Wayne A. Marasco. A bispecific chimeric-antigen receptor T-cell factory for triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1531.
Abstract 1540: Allogeneic chimeric antigen receptor T cells targeting B cell maturation antigen
Authors: Dar Henia , Henderson Daniel , Padalia Zinkal , Porras Ashley , Mu Dakai , Maeng Kyungah , Police Seshi , Kalaitzidis Demetrios , Terrett Jonathan , Sagert Jason
Published: August 2018

B cell maturation antigen (BCMA) is a tumor necrosis factor family cell surface receptor that binds B cell activating factor (BAFF) and a proliferation inducing ligand (APRIL) and is involved in the long-term survival of B cells. BCMA has been shown to be expressed broadly on monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) cells and for this reason has been pursued as a potential antigen for the treatment of MM. Indeed, clinical studies evaluating autologous chimeric antigen receptor T cells (CAR-T cells) targeting BCMA for the treatment of MM have shown promising efficacy with >80% response rates. However, logistical challenges will potentially limit the number of patients that have access to autologous therapies. Using the CRISPR/CAS9 system, we generated allogeneic CAR-T cells targeting BCMA by disrupting the beta-2-microglobulin (B2M) and T cell receptor alpha constant (TRAC) genes and inserting an anti-BCMA CAR into the TRAC locus. This results in allogeneic CAR-T cells lacking the major histocompatibility complex (MHC) and endogenous T cell receptor (TCR) expression while being potently cytotoxic towards cells expressing BCMA. These data show proof-of-concept for an “off the shelf” CAR-T therapy targeting BCMA.

Citation Format: Henia Dar, Daniel Henderson, Zinkal Padalia, Ashley Porras, Dakai Mu, Kyungah Maeng, Seshi Police, Demetrios Kalaitzidis, Jonathan Terrett, Jason Sagert. Allogeneic chimeric antigen receptor T cells targeting B cell maturation antigen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1540.
Abstract 1549: LAPTM5 in clear cell renal cell carcinoma
Authors: Ryu Hyewon , Kim Solbi , Kim Nayoung , Kim Jin-Man , Lee Hyo Jin
Published: August 2018

Lysosomal-associated multispanning membrane protein 5 (LAPTM5) is membrane protein that mostly expressed in immune cells and hematopoietic cells. Recently, it has been demonstrated that LAPTM5 expression levels are decreased in several cancer cells by inducing regression of Hela cells and neuroblastomas. However, its exact role in cancer has yet to be defined. Furthermore, the clinical effect of LAPTM5 expression in cancer has not been reported so far. Thus, we investigated the association between LAPTM5 expression and its clinicopathological characteristics in 112 cases of clear cell renal cell carcinoma using tissue microarrays by immunohistochemical staining. Tumors were classified into four grades based on the staining intensity: 0 = no staining, +1 = weak, +2 = intermediate, and +3 = strong. The staining intensity was classified into LAPTM5-negative group or LAPTM5-positive groups. (0; negative, +1 ~ +3; positive). Of all 112 cases, 9 cases showed LAPTM5-negative, while 93 cases showed LAPTM5-positive: +1 (35 cases), +2 (28 cases), and +3 (30 cases). LAPTM5-positivity was significantly associated with decreased tumor recurrence. In addition, recurrence-free survival (RFS) (p = 0.010), cancer-specific survival (CSS) (p = 0.022), and overall survival (OS) (p = 0.022) were significantly decreased in the LAPTM5-negative group. Univariate analysis indicated that tumor size (HR = 3.44; 95% CI, 1.000-11.836), Fuhrman nuclear grade (HR = 1.863; 95% CI, 1.169-2.971), and negative LAPTM5 expression (HR = 3.173; 95% CI, 1.249-8.064) significantly affected the RFS of clear cell renal cell carcinoma patients. Moreover, CSS and OS were significantly affected by Fuhrman nuclear grade (HR = 2.177; 95% CI, 1.261-3.757 and HR = 1.673, 95% CI, 1.062-2.635, respectively) and negative LAPTM5 expression (HR = 3.398; 95% CI, 1.110-10.405 and HR = 2.826; 95% CI, 1.136-7.031, respectively). Multivariate analysis showed that a high Fuhrman nuclear grade and negative LAPTM5 expression were significant independent risk factors. Based on these results, loss of LAPTM5 was associated with poor oncological outcomes; this suggests that LAPTM5 might be a useful prognostic marker in patients with clear cell renal cell carcinoma.

Citation Format: Hyewon Ryu, Solbi Kim, Nayoung Kim, Jin-Man Kim, Hyo Jin Lee. LAPTM5 in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1549.
Abstract 155: KDM4 inhibition targets breast cancer stem-like cells
Authors: Maurer Jochen
Published: August 2018

Traditional treatments for breast cancer fail to address therapy-resistant cancer stem-like cells that have been characterized by changes in epigenetic regulators such as the lysine demethylase KDM4. Here we describe an orally available, selective and potent KDM4 inhibitor (QC6352) with unique preclinical characteristics. To assess the antitumor properties of QC6352, we established a method to isolate and propagate breast cancer stem-like cells (BCSC) from individual triple-negative tumors resected from patients after neoadjuvant chemotherapy. Limiting-dilution orthotopic xenografts of these BCSC regenerated original patient tumor histology and gene expression. QC6352 blocked BCSC proliferation, sphere formation and xenograft tumor formation. QC6352 also abrogated expression of EGFR, which drives the growth of therapy-resistant triple-negative breast cancer cells. Our findings validate a unique BCSC culture system for drug screening and offer preclinical proof of concept for KDM4 inhibition as a new strategy to treat triple-negative breast cancer.

Citation Format: Jochen Maurer. KDM4 inhibition targets breast cancer stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 155.
Abstract 1551: Determination of the origin of oral squamous cell carcinoma by microarray analysis
Authors: Kinouchi Makoto , Izumi Sayaka , Uchida Daisuke , Kuribayashi Nobuyuki , Komiyama Yuske , Tsuchida Shuji , Fujita Atsushi , Kawamata Hitoshi
Published: August 2018

More than 90% of oral cancer is histopathologically squamous cell carcinoma (SCC). According to its clinical behavior and histopathological features, we hypothesize that oral SCC can originate from either oral squamous epithelium or the minor salivary glands. In this study, we attempted to clarify the possibility of some oral SCC originating from the minor salivary glands, and to investigate whether these tumors showed particularly aggressive biological behavior. The mRNA expression profile of samples obtained from 6 patients with SCC of the oral floor (5 men, 1 woman; mean age 62.7 years) was analyzed by microarray containing 32,878 genes. Clustering analysis of the expression level of the 845 genes that were differentially expressed in normal oral squamous epithelium and normal salivary glands revealed that the 6 samples can be divided into 2 groups. The expression profile in four cases was similar to that of normal oral squamous epithelium, and in two cases was similar to that of normal salivary glands. Furthermore, we identified nine genes that reveal the origin of the oral SCC. Subsequently, we examined the expression levels of these 9 marker genes by reverse transcriptase-polymerase chain reaction to determine the origin of 66 oral SCCs. Twelve of the 66 oral SCCs were considered to originate from minor salivary glands, and these tumors showed high metastatic potential (p<0.05) and poor event free survival rate (p<0.05). In conclusion, determination of the origin of the oral SCC is helpful when making a treatment plan for patients with oral SCC.

Citation Format: Makoto Kinouchi, Sayaka Izumi, Daisuke Uchida, Nobuyuki Kuribayashi, Yuske Komiyama, Shuji Tsuchida, Atsushi Fujita, Hitoshi Kawamata. Determination of the origin of oral squamous cell carcinoma by microarray analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1551.
Abstract 1559: Detection and retrieval of multiple myeloma cells with single-cell molecular confirmation from peripheral blood by multiparameter immunofluorescence, automated scanning, and image analysis
Authors: U'Ren Lance , Ericson Nolan , Campton Daniel , Ramirez Arturo , Blau C. Anthony , Kaldljian Eric
Published: August 2018

Background: The detection of multiple myeloma (MM) cells is currently performed by analysis of bone marrow aspirates. Detection of MM cells from the peripheral blood would allow for less invasive and more frequent assessments to monitor residual disease. Using the RareCyte platform, we developed a 5-parameter assay to visualize and retrieve MM cells from peripheral blood and applied it to MM patient samples under an IRB-approved protocol. We also used this to identify plasma cells from the peripheral blood of healthy volunteer donors.

Materials and Methods: Healthy volunteer or MM patient peripheral blood was collected into RareCyte blood collection tubes. RPMI8226 cells spiked into normal donor blood samples were used as a model for MM cell assay development. Buffy coats isolated from 7.5mL of blood by AccuCyte® separation were spread onto slides. Slides were stained with a 5-marker panel that included antibodies to CD138, CD38, CD56, CD45, and a nuclear dye on the Leica Bond Rx auto-stainer. Slides were scanned with CyteFinder® and MM cells identified by positive CD138 and CD56 staining and negative CD45 staining. Identified MM cells and WBC control cells were mechanically retrieved with the integrated CytePicker module, whole genome amplified, and Sanger sequencing was performed for specific mutations in four genes (IDH1, NRAS, PHLDA1, RPH3AL) identified by genomic analysis of a bone marrow sample. Plasma cells from normal donors were identified by co-expression of CD138 and CD38.

Results: In a pretreatment MM sample, the 5-parameter assay identified 55 MM cells per mL of whole blood. In isolated single cells with at least one successful sequencing reaction, 0 of 13 WBCs contained mutations in at least one of the genes. Mutations were found in 9 of 15 MM cells (defined as CD138+, CD56+, CD38-, and CD45-). In healthy volunteer samples, 5 plasma cells (defined as CD138+, CD38+, and CD45 dim/negative) were identified per mL of whole blood.

Conclusions: The 5-parameter assay successfully detected rare cells with an MM phenotype from the peripheral blood of an MM patient. Individually retrieved MM cells were demonstrated to contain mutations specific to the patient's tumor, confirming that the cells identified were MM cells. This approach may allow for less invasive assessment of MM and monitoring of residual disease.

Citation Format: Lance U'Ren, Nolan Ericson, Daniel Campton, Arturo Ramirez, C. Anthony Blau, Eric Kaldljian. Detection and retrieval of multiple myeloma cells with single-cell molecular confirmation from peripheral blood by multiparameter immunofluorescence, automated scanning, and image analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1559.
Abstract 156: Identifying novel therapeutics to inhibit the WNT self-renewal pathway in leukemia stem cells
Authors: Haney Meghan Green , Liu Chunming , Blackburn Jessica
Published: August 2018

The relapse rate of pediatric Acute Lymphoblastic Leukemia (ALL) is 15-20%, and these patients have limited treatment options and a poor prognosis. Relapse rate is likely due to a small population of cells known as leukemia stem cells (LSCs), which have the ability to self-renew and can re-form a leukemia from a single cell. Additionally, a major clinical concern is whether these LSCs are effectively killed by conventional cytotoxic chemotherapies. Current efforts to study LSCs have faced serious limitations, which have impeded our understanding of this important population of cells.

Prior work in our lab has established a zebrafish Myc-induced T-cell acute lymphoblastic leukemia (T-ALL) model that mimics the most aggressive and treatment resistant form of human T-ALL. Using this system, we isolated single LSCs through a novel transplantation strategy. Limiting dilution analysis showed significant differences in the rate of self-renewal between different LSCs, suggesting heterogeneity in LSC relapse potential. We generated a library of zebrafish T-ALL with high self-renewal rates (about 1 in 10 leukemia cells is a LSC) for use in bulk RNAseq, as well as single cell qPCR and single cell RNAseq to identify a self-renewal signature unique to LSCs. Single cell analyses showed a population of cells that expressed known self-renewal genes and clustered separately from the rest of the leukemia cells in the population. Ongoing work in our laboratory is focused on functionally testing whether this gene expression profile is linked with self-renewal. Additionally from this analysis, the Wnt pathway, more specifically ß-catenin, was enriched in the putative LSC population. These data support previous findings in mammalian models of ALL.

We hypothesize that inhibitors of the Wnt pathway will inhibit self-renewal of LSCs and force them to terminally differentiate. However, there are no WNT inhibitors clinically available due to undesirable side-effects. We are currently using a 6xTCF/LEF:GFP zebrafish Wnt reporter model for high-throughput screens to identify new, less toxic Wnt pathway inhibitors. Thus far, several compounds have showed significantly decreased TCF/LEF activity after drug treatment, with no effects on animal health. Future studies will test these inhibitors in our zebrafish high LSC leukemia samples and patient derived xenograft models. Ultimately, these inhibitors may represent a potential therapeutic strategy for targeting treatment-resistant LSCs and preventing ALL relapse.

Citation Format: Meghan Green Haney, Chunming Liu, Jessica Blackburn. Identifying novel therapeutics to inhibit the WNT self-renewal pathway in leukemia stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 156.