bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2498: MCL1 binds and negatively regulates the transcriptional function of tumor suppressor p73
Authors: Widden Hayley , Kaczmarczyk Aneta , Whitaker Robert H. , Placzek William J.
Published: August 2019

MCL1 is an anti-apoptotic member of the Bcl-2 family, which has become a popular therapeutic target in cancer research over the past decade due to its propensity to promote cancer cell survival and facilitate chemoresistance. Through a phage display screen, we identified a novel MCL1 binding motif that is the reverse of the canonical BH3-binding helix that mediates interactions amongst Bcl-2 family members. Upon a BLAST sequence analysis of native proteins, we identified a putative reverse BH3 (rBH3) motif in the tumor suppressor protein, p73. Here, we show that MCL1 and p73 bind through a direct protein-protein interaction. Using fluorescence polarization assay, we characterize the strength of this interaction and reduce binding down to the rBH3-containing alpha helix in the tetramerization domain of p73. To elucidate biological function, we employ electrophoretic mobility shift assay to show that excess MCL1 negatively impacts p73 binding to DNA. Finally, using two p53-/- cancer cell lines, we demonstrate that MCL1 inhibits the transcriptional function of p73 through analysis of p73 target gene expression. In summary, we characterize a novel protein-protein interaction between MCL1 and p73 and show that MCL1 negatively impacts p73 target gene activation. This work establishes a novel function of MCL1 outside of apoptotic regulation at the mitochondria and provides new evidence of cross talk between the Bcl-2 family and the DNA damage response pathway.

Citation Format: Hayley Widden, Aneta Kaczmarczyk, Robert H. Whitaker, William J. Placzek. MCL1 binds and negatively regulates the transcriptional function of tumor suppressor p73 [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 2498.
Abstract 2499: In vitro and in vivo efficacy of MCL1 inhibitor S63845 in small cell lung cancer
Authors: Yasuda Yuto , Ozasa Hiroaki , Tsuji Takahiro , Nomizo Takashi , Yamamoto Tomoko , Ajimizu Hitomi , Yoshida Hironori , Sakamori Yuichi , Hirai Toyohiro , Kim Young Hak
Published: August 2019

MCL1 is a member of the BCL-2 family, which regulates apoptosis. Targeting MCL1 represents a potential breakthrough of cancer treatment. We tested S63845, a MCL1 inhibitor, in four SCLC cell lines (DMS114, DMS53, SW1271, and NCI-H69) and, in addition, one patient derived SCLC cell line (KTOR201). S63845 had greater efficacy in two of five SCLC cell lines (DMS114 and KTOR201). These two SCLC cell lines had higher expression of MCL1 and lower expression of BCL-XL, which is another member of the BCL-2 family. The other three SCLC cell lines (DMS53, SW1271 and NCI-H69) were resistant to S63845 and had a higher expression of BCL-XL or lower expression of MCL1. In vivo activity was evaluated in xenograft models. DMS114 was subcutaneously implanted into immunocompromised mice. When the tumor volume was reached 150-200 mm3, mice were administered with S63845 (25 mg/kg) or vehicle intravenously twice a week. S63845 showed significant antitumor efficacy in vivo. Finally, immunohistochemical evaluation of MCL1 and BCL-XL was assessed in SCLC obtained from 33 patients at Kyoto University Hospital. High MCL1 expression with low BCL-XL expression was observed in 16 specimens (48.5%).These data suggested that S63845 might be a powerful treatment of SCLC as a new therapeutic strategy. It is possible that the expressions of MCL1 and other members of the BCL-2 family predict the sensitivity of S63845.

Citation Format: Yuto Yasuda, Hiroaki Ozasa, Takahiro Tsuji, Takashi Nomizo, Tomoko Yamamoto, Hitomi Ajimizu, Hironori Yoshida, Yuichi Sakamori, Toyohiro Hirai, Young Hak Kim. In vitro and in vivo efficacy of MCL1 inhibitor S63845 in small cell lung 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 2499.
Abstract 2511: Analysis of circulating tumor DNA by targeted ultra-deep sequencing across various non-Hodgkin lymphoma subtypes
Authors: Kim Yeon Jeong , Shin Seung-Ho , Lee Danbi , Park Woong-Yang , Kim Seok Jin , Kim Won Seog , Park Donghyun
Published: August 2019

Although targeted deep sequencing of cell-free DNA (cfDNA) was recently used to investigate tumor somatic mutations in particular subtypes of non-Hodgkin lymphomas (NHLs), the immense genetic heterogeneity across subtypes poses a hurdle to design a universal gene panel applicable for diverse subtypes of NHLs. We designed a panel targeting 66 genes associated with NHLs and performed targeted deep sequencing to analyze plasma cfDNA from patients with various subtypes of NHLs. Genetic profiling in plasma cfDNA using the method resulted in 88.0% sensitivity and >99% specificity in detecting mutations present at a frequency greater than 20% in the tumor biopsies. Furthermore, the level of ctDNA significantly decreased and increased depending on designated clinical responses to therapy and disease progression. These results demonstrated that ctDNA sensitively indicated the presence of cancer and reliably correlated with tumor burden, suggesting potential utility of the method for patients with various subtypes of NHLs.

Citation Format: Yeon Jeong Kim, Seung-Ho Shin, Danbi Lee, Woong-Yang Park, Seok Jin Kim, Won Seog Kim, Donghyun Park. Analysis of circulating tumor DNA by targeted ultra-deep sequencing across various non-Hodgkin lymphoma subtypes [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 2511.
Abstract 2515: Comprehensive analysis of the histologically distinct components of an adenosquamous carcinoma of the lung
Authors: Krause Arthur , Filippo Maria R. De , Lorber Thomas , Savic Spasenija , Piscuoglio Salvatore , Ng Charlotte K. , Bubendorf Lukas
Published: August 2019

Background: Adenosquamous carcinoma (ASC) is a rare subtype of non-small-cell lung cancer, consisting of an adenocarcinoma (AC) and a squamous cell carcinoma (SCC) component. Little is known about the molecular makeup of the two components in ASC. In this study, we applied a comprehensive analysis including whole exome sequencing (WES) and RNA sequencing to understand the evolutionary relationship between the AC and SCC components of an ASC of the lung.

Methods:FFPE tumor tissue of three ASC patients was used in this study. Samples were stained with FastRed to visualize the nuclei. AC and SCC components were scratched separately from histological slides for DNA and RNA extraction. WES analysis was performed to assess the mutational profile and copy number alterations (CNAs). The transcriptome was investigated in each of the components using RNA sequencing.

Results:WES analysis of the AC and SCC components of one patient revealed a common trunk of 118 mutations, including driver mutations EGFR exon 19 deletion and TP53 p.192*, indicating a common clonal origin of the components and the presence of bona fide drivers in the common ancestor. AC shows more private mutations than SCC (234 vs 134). Potential drivers are private to AC included TP53 and AKT1 mutations, while TLX1 and TRBV5-1 mutations are restricted to SCC. AC and SCC have highly similar CNA profiles, suggesting that all CNAs are early evolutionary events of the ASC. Mutational signature of the truncal mutations is similar to that of the mutations private to AC, while mutations private to SCC showed a distinct pattern enriched in T>G, suggesting that SCC may have derived from an AC ancestor cell. Transcriptomic profiling shows that genes related to oxidative phosphorylation, fatty acid metabolism and peroxisome pathways are highly expressed in AC, in contrast to genes related to epithelial-mesenchymal-transition, hedgehog signaling and IL6-JAK-STAT3-signaling pathways in SCC.

Conclusion:Macroscopic dissection and separate analysis of the histologically distinct components of an ASC of the lung demonstrated the clonal relatedness of the AC and SCC. The diverse phenotype of the components is associated with distinct genetic profiles and mutational signatures. Analysis of additional ASCs of the lung may reveal genetic and/or transcriptomic underpinnings of the phenotypes.

Citation Format: Arthur Krause, Maria R. De Filippo, Thomas Lorber, Spasenija Savic, Salvatore Piscuoglio, Charlotte K. Ng, Lukas Bubendorf. Comprehensive analysis of the histologically distinct components of an adenosquamous carcinoma of the lung [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 2515.
Abstract 2526: Measuring DNA quantity, FFPE degradation, and genomic DNA contamination of ccfDNA in a single assay
Authors: Schagat Trista L. , Mook Jennifer , Steffen Leta , Wieczorek Doug , Knox Curtis
Published: August 2019

FFPE tumor biopsies are being increasingly used as a source of DNA for translational research in pursuit of personalized medicine. In addition, circulating cell-free DNA in liquid biopsy fractions shows promise as a means of tracking tumor burden, tumor regression, and/or cancer relapse. However, both sources come with significant DNA quality risks related to sample preparation that can result in downstream sequencing failures or false negatives. For FFPE samples, chemical fixation of the tissue crosslinks DNA, resulting in DNA fragmentation and damage that cannot be completely reversed. For liquid biopsy samples, contamination with genomic DNA can reduce sensitivity for detecting the tumor-specific fraction of ccfDNA. The ProNex® DNA QC Assay is a multiplex qPCR assay that provides both quantification and quality control for small or fragmented DNA samples. ProNex® DNA QC Assay includes a 75bp, 150bp, and 300bp target as well as an internal positive control. Unlike absorption, fluorescent DNA binding dyes, and electrophoresis-based methods, qPCR can be used to quantify specifically amplifiable DNA able to participate in downstream PCR or sequencing applications. The ratio of amplification at 75bp and 300bp sizes can be used to assess fragmentation of FFPE samples, which is correlated with improved coverage uniformity in NGS applications. With ccfDNA, the ratio can also be used to determine the degree of genomic DNA contamination and indicate when further clean-up is necessary prior to sequencing.

Citation Format: Trista L. Schagat, Jennifer Mook, Leta Steffen, Doug Wieczorek, Curtis Knox. Measuring DNA quantity, FFPE degradation, and genomic DNA contamination of ccfDNA in a single assay [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 2526.
Abstract 2528: Mapping tumoral and immune heterogeneity in PD-1 responsive glioblastoma
Authors: Restrepo Paula , Yong Raymund , Laface Ilaria , Tsankova Nadejda , Gnjatic Sacha , Hormigo Adilia , Losic Bojan
Published: August 2019

Clonal evolution and the immune microenvironment in glioblastoma (GBM) ultimately determine the potential benefit of checkpoint inhibition. We present a case study of a 67- year-old patient who underwent a resection of a large right temporal GBM. The patient received standard-of-care treatment consisting of tumor resection, radiation therapy and concurrent temozolomide. Her tumor recurred as a gliosarcoma after 2 cycles of adjuvant Temozolomide. Subsequently, she received 26 cycles of a PD-1 checkpoint inhibitor and 20 cycles of bevacizumab, a VEGF-A inhibitor. She survived 25 months after diagnosis including six after immunotherapy and VEGF-A blockade was discontinued. We integrated RNA-seq and whole exome sequencing data from the primary tumor and several recurrent biopsies to characterize the genomic and molecular landscape within the context of clonal evolution. Specifically, we surveyed tumor-immune interactions by computing the mutational and neo-epitope tumoral burden, along with the magnitude and clonality of infiltrating lymphocytes using VDJ-sequencing. Furthermore, we intersected genomic instability measures with the regional gene expression profiles among the sectors. Finally, we integrated TCGA-GBM RNA-seq data to examine the expression of our patient sectors among the spectrum of known GBM variation. We found significant regional heterogeneity in the predicted neoantigen and infiltrating lymphocyte landscape, with a pronounced shift towards lower predicted immunogenicity and lower immune infiltration in all recurrent biopsies. We also observed profound large-scale immune variance among the regional sectors when examined in the context of TCGA. Overall, this case study quantifies intra-tumoral clonal and immune heterogeneity with multiregional glioblastoma omics data, providing reasonable priors for further assessing the efficacy of checkpoint inhibition for GBM.

Citation Format: Paula Restrepo, Raymund Yong, Ilaria Laface, Nadejda Tsankova, Sacha Gnjatic, Adilia Hormigo, Bojan Losic. Mapping tumoral and immune heterogeneity in PD-1 responsive glioblastoma [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 2528.
Abstract 2530: Understanding cancer heterogeneity in relation to minor sub-clones at single cell profiling level in renal cell carcinoma
Authors: Villarreal Enrique I. Velazquez , Carpten John D. , Craig David W.
Published: August 2019

Renal cell carcinoma (RCC) is one of the most lethal urological malignancies that would benefit from understanding cancer heterogeneity to identify molecular signatures providing potential innovative therapeutic targets. Renal cell carcinoma is characterized by extensive cancer heterogeneity. Single cell sequencing has the potential to improve our understanding of cancer heterogeneity by providing clonotype detection. We examined cancer heterogeneity using single-cell copy-number analysis and clonotype detection in four renal cancer tumors. The technology used for processing single cell was 10x ChromiumTM Technology that provides detection of 100 Kb CNV events, calling clonotypes down to 10 of 1000 cell inputs. By processing each of the four samples it resulted 469 total cells from sample one where 268 were tumor and 171 were normal; sample two resulted with 888 cells where 51 were tumor and 820 were normal; sample three resulted with 673 total cells where 167 were tumor and 485 normal; finally, sample four resulted with 1298 cells where 511 were tumor and 758 normal. Genome-wide ploidy analysis of the four tumor cells showed variable median ploidies. The regional copy number was estimated by processing our data in 20 Kb cases of reads. Multiple sub-clones were identified in sample number one where four clusters of sub-clones characterized cancer heterogeneity. Fast maximum-likelihood (ML) genetic clustering and Bayesian Information Criterion (BIC) were implemented on sample one to select the optimal clustering solution and provide a better insight of sub-clonal evolution. These sub-clones reported events such as copy number changes on entire chromosomes arms. The single cell resolution provided the ability of variant detection where we identified VHL gene mutations that are consistent with recent reports that describe its role as a molecular signature of disease outcome and drug efficacy in RCC. In conclusion, our findings describe sub-clones with large-scale events that provide new insights in understanding cancer heterogeneity in RCC.

Citation Format: Enrique I. Velazquez Villarreal, John D. Carpten, David W. Craig. Understanding cancer heterogeneity in relation to minor sub-clones at single cell profiling level in renal cell carcinoma [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 2530.
Abstract 2537: Targeting chromosomal passenger complex by disruption of INCENP function inhibits tumor progression in neuroblastoma
Authors: Sun Ming , Veschi Veronica , Lam Norris , Bagchi Sukriti , Xu Man , Mendoza Arnulfo , Liu Zhihui , Thiele Carol J.
Published: August 2019

Chromosomal passenger complex (CPC), which is composed of Aurora B and Survivin, INCENP and Borealin, regulates crucial mitotic events including chromosome alignment, segregation and cytokinesis during the mitosis. Disruption of CPC function by Aurora B and Survivin inhibition has been demonstrated to be effective at killing tumor cells and show promising results in many different cancer types including neuroblastoma (NB). However, chemical inhibition of either Aurora B or Survivin is unable to target CPC specifically due to off-target effects of Aurora B inhibitors on the other kinases and antiapoptotic activities of Survivin independent of this complex. In a chromatin-focused siRNA screen, we found that NB cells were particularly vulnerable to the silencing of INCENP, a gene encoding a key scaffolding and regulatory component of the CPC. In this study, we found that INCENP was highly expressed in NB cells and its expression levels were decreased upon Retinoic Acid (RA)-induced NB cell differentiation. Genetic silencing of INCENP reduced the growth of both MYCN single copy and MYCN amplified NB cell lines in vitro and led to significant decreases in NB xenograft growth and increases in murine survival in vivo. Elevated levels of INCENP were significantly associated with poor prognosis in primary NB tumors whereas low INCENP expression levels were predictive of better outcomes. Mechanistically, we found that INCENP depletion suppressed NB cell growth by inducing massive polyploidization, mitotic arrest, senescence and cell death (apoptosis). We also observed that in the majority of NB cell lines tested in vitro, cell death represented the primary cell fate after INCENP silencing due to a strong induction of DNA damage response and activation of the p53-p21 axis. Therefore, targeting INCENP phenocopies treatment with Aurora B inhibitor, providing a novel strategy to disrupt the activity of CPC and inhibit tumor progression in NB.

Citation Format: Ming Sun, Veronica Veschi, Norris Lam, Sukriti Bagchi, Man Xu, Arnulfo Mendoza, Zhihui Liu, Carol J. Thiele. Targeting chromosomal passenger complex by disruption of INCENP function inhibits tumor progression in neuroblastoma [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 2537.
Abstract 2551: Inhibition of mTOR abrogates mitosis progression
Authors: Kuo Hsiao-Hui , Fang Chieh-Ting , Yih Ling-Huei
Published: August 2019

The mammalian target of rapamycin (mTOR) mediates the coordination of cell growth and cell cycle progression in response to nutrient supply. It is frequently activated in cancers and confers resistance to anti-cancer drugs. Accumulated studies have demonstrated that inhibition of mitotic kinases (PLK1 or aurora kinase A) enhances mTOR activities and the mTOR activation is associated with cell survival to anti-mitotic drugs. In this study, whether mTOR signaling is involved in mitosis progression is explored. Treatment of cells with mTOR inhibitors for 24 h resulted in the accumulation of G2 and mitotic cells and increased the percentage of mitotic cells with spindle abnormalities and chromosome missegregation. In addition, mTOR inhibitors sensitized cancer cells to taxol by enhancing apoptosis. Our results indicate that mTOR signaling pathway may be required for faithful mitosis progression and represent a potential resistance mechanism to the use of anti-mitotic drugs.

Citation Format: Hsiao-Hui Kuo, Chieh-Ting Fang, Ling-Huei Yih. Inhibition of mTOR abrogates mitosis progression [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 2551.
Abstract 2552: Senescence induced by CDK4/6 inhibition facilitates anti-tumor T cell responses
Authors: Blevins Ashlyn , Mont Stacey , Lawrence Hunter , Saleh Nabil , Chen Sheau-Chiann , Richmond Ann , Vilgelm Anna
Published: August 2019

Cancer immunotherapies have shown remarkable clinical activity across several tumor types but responses have been modest in breast cancers. This is attributed to low levels of neo-antigens in breast tumors and immunosuppressive nature of mammary tumor microenvironment. Here we investigated if we can counteract these obstacles to an effective immunotherapy by inducing senescence in breast cancer cells. Senescence is one of the physiological stress response programs characterized by loss of proliferative potential and secretion of a variety of pro-inflammatory markers which promotes identification and removal of senescent cells by the immune cells. Here we hypothesized that senescence will stimulate anti-tumor immune response by establishing immune-permissive microenvironment. We utilized immunocompetent and humanized PDX-bearing mice to investigate senescence-induced modulation of tumor secretome proteome, transcriptome and immune cell recruitment and activity. An FDA-approved small molecule CDK4/6 inhibitor was used to induce senescence. We detected induction of multiple markers of senescence after CDK4/6 inhibitor treatment of cells and tumors. Senescent cells and tumors had increased secretion of chemokines CCL4, CCL5, CXCL9, CXCL10 and CXCL11 which facilitated T cell homing into the tumor. This was a result of their transcriptional induction by NF-κB. Consequently, we observed the dynamic enrichment of tumor immune microenvironment with T cells in tumors treated with CDK4/6 inhibitor. Furthermore, the recruitment of adoptively transferred ex-vivo activated anti-tumor T cells was significantly enhanced in tumors that were treated with CDK4/6 inhibitor. Finally, CDK4/6 inhibitor-treated tumors became responsive to T cell-activating immunotherapy with antibody agonists of T cell co-stimulatory receptors OX40 and 4-1BB. In summary, our findings suggest that CDK4/6 inhibition can facilitate T cell recruitment into the mammary tumors which can lead to an improved immunotherapy response. This pre-clinical study provides rationale for clinical development of CDK4/6 inhibitors and T cell agonists and uncovers the potential of senescence-inducing approach for sensitizing tumors to immune therapy.

Citation Format: Ashlyn Blevins, Stacey Mont, Hunter Lawrence, Nabil Saleh, Sheau-Chiann Chen, Ann Richmond, Anna Vilgelm. Senescence induced by CDK4/6 inhibition facilitates anti-tumor T cell responses [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 2552.
Abstract 2554: Branched chain amino acid transaminase 1 in claudin-low breast cancer
Authors: Reynen Lisa C. , Jones Robert , Moorehead Roger
Published: August 2019

Breast cancer, the most commonly diagnosed cancer in women, can be classified into five distinct subtypes. One subtype, claudin-low breast cancer, accounts for approximately 7% of the breast cancer cases and these tumors are aggressive and highly metastatic. RNA sequencing of human claudin-low breast cancers by other groups and RNA sequencing of a murine claudin-low mammary tumor cell line by our group has revealed that Bcat1 is significantly up-regulated in this breast cancer subtype. Bcat1 regulates the metabolism of branched chain amino acids and has been linked to numerous pathologies including heart disease, diabetes, and cancer. Based on this data we hypothesized that the expression of Bcat1 in claudin-low mammary tumors is driving the aggressive and metastatic nature of this cancer subtype and disrupting Bcat1 will deter these features. Elevated expression of Bcat1 in the murine claudin-low cell line RJ423, compared to the murine luminal mammary tumor cell line RJ345, has been confirmed at the mRNA and protein level. Bcat1 has been transiently down-regulated ~70% in RJ423 cells using siRNA and this suppression of Bcat1, contrary to the anticipated result, showed no effect on proliferation based on phospho-histone H3 immunofluorescence. However, when the cell population doubling time was investigated, a significant difference was seen between the RJ423 cell line and the cell lines with suppressed Bcat1 expression. To elucidate these contradicting results, cell cycle analysis using Bromodeoxyuridine and 7-AAD through flow cytometry is currently underway. RJ423 stably expressing Bcat1 shRNA and RJ345 stably expressing a Bcat1 expression vector are currently being selected. Further in vitro and in vivo studies will utilize these stably transfected cell lines. This study will determine whether further investigation into the effects of Bcat1 on claudin-low human breast cancer is prudent and if Bcat1 may be used as a therapeutic target.

Citation Format: Lisa C. Reynen, Robert Jones, Roger Moorehead. Branched chain amino acid transaminase 1 in claudin-low 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 2554.
Abstract 256: Repurposing neuroleptic drug for brain tumor therapy
Authors: Kaushik Iti S. , Ranjan Alok , Schwettmann Blake , Srivastava Sanjay
Published: August 2019

Brain tumor is considered as one of the most aggressive and incurable form of cancer. Majority of the brain tumors have a median survival rate of only 12%. Even though advanced treatments such as surgical removal, chemotherapy and radiotherapy are available, brain tumor persists to be lethal. Temozolomide (TMZ) is the main therapy for brain tumor but resistance to TMZ is the major cause of treatment failure. This resistance in tumor cells has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT). Pimozide (PMZ) is a neuroleptic drug used for the treatment of schizophrenia and chronic psychosis. In our study, we observed that pimozide significantly reduced the viability of U87MG, U251MG, DAOY and GBM28 cell lines with an IC50 ranging from 11µM to 20µM after 24 h of treatment. Pimozide induced apoptosis in these cell lines as evaluated by FITC/Annexin assay and further validated by the cleavage of caspase 3 as well as PARP. Western blot analysis resulted in the concentration dependent decrease in the expression of STAT3 signaling mainly targeting the anti-apoptotic markers BCl-XL, BCl-2 and Survivin etc in U87MG, U251MG, DAOY and GBM28 brain cancer cell lines. PMZ alone suppressed the growth of U87MG cells with an IC50 of 10µM, after 96 hours of treatment. On the other hand, treatment of U87MG cells with 750 µM of TMZ in combination with 10µM PMZ for 96 hours increased the killing of U87MG cells by 50% as compared to 10% when treated with TMZ. In addition, PMZ treatment reduced the survival of TMZ resistant U251R cells with an IC50 ranging from 15-20 µM after 24 h of treatment. Our results further demonstrated that oral administration of PMZ (25mg/kg) significantly inhibits the growth of brain tumors in an intracranial tumor model without any apparent signs of toxicity. It is important to note that PMZ is an FDA approved drug with no considerable toxicity. Overall, this study depicts that PMZ is a potential candidate to target brain tumors.

Citation Format: Iti S. Kaushik, Alok Ranjan, Blake Schwettmann, Sanjay Srivastava. Repurposing neuroleptic drug for brain tumor therapy [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 256.
Abstract 2560: Werner syndrome helicase is a selective vulnerability of microsatellite instability-high tumor cells
Authors: Lieb Simone , Blaha-Ostermann Silvia , Kamper Elisabeth , Ehrenhöfer-Wölfer Katharina , Schlattl Andreas , Wernitznig Andreas , Lipp Jesse , Nagasaka Kota , Bader Gerd , Neumueller Ralph , Kraut Norbert , Pearson Mark , Woehrle Simon , Petronczki Mark
Published: August 2019

Targeted cancer therapy is based on exploiting selective dependencies of tumor cells. By leveraging recent large-scale genomic profiling and functional screening of cancer cell lines we identified Werner syndrome helicase (WRN) as a novel specific vulnerability of microsatellite instability-high (MSI-H) cancer cells. MSI, caused by defective mismatch repair is frequently detected in human malignancies, in particular in colorectal, endometrial and gastric cancers. We demonstrate that WRN inactivation selectively impairs the viability of MSI-H but not microsatellite stable (MSS) colorectal and endometrial cancer cell lines. In MSI-H cells, WRN loss results in the emergence of chromosome breaks, chromatin bridges and micronuclei highlighting defective genome integrity. WRN variants harboring mutations abrogating the ATPase function of WRN helicase fail to rescue the viability phenotype of WRN-depleted MSI-H colorectal cells. Our study suggests that pharmacological inhibition of WRN helicase function might represent a novel opportunity to develop a targeted therapy for MSI-H cancers.

Citation Format: Simone Lieb, Silvia Blaha-Ostermann, Elisabeth Kamper, Katharina Ehrenhöfer-Wölfer, Andreas Schlattl, Andreas Wernitznig, Jesse Lipp, Kota Nagasaka, Gerd Bader, Ralph Neumueller, Norbert Kraut, Mark Pearson, Simon Woehrle, Mark Petronczki. Werner syndrome helicase is a selective vulnerability of microsatellite instability-high tumor cells [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 2560.
Abstract 2565: Type I interferons enhance chemosensitivity of breast cancer by inhibiting DNA repair
Authors: Ka Na-Lee , Lim Ga Young , Lee Mi-Ock
Published: August 2019

Type I interferons (IFNs) are known to have antineoplastic effects against several tumors by stimulating anticancer immune response. In this study, we identified a novel function of type I IFNs in DNA repair, which enhanced chemosensitivity in breast cancer cells. We investigated that IFNα and IFNβ inhibited both non-homologous recombination and homologous recombination double strand breaks repair. Moreover, IFNs accelerated the release of DNA into the cytoplasm in response to DNA damaging agent, doxorubicin. In agreement, IFNs synergistically activated DNA damage-induced STING pathway and increased the production of chemokines, CCL5 and CXCL11. In agreement, IFNβ greatly increased the sensitivity of cells to doxorubicin-induced suppression of cell survival. This sensitizing effect of IFNβ was also supported by in vivo xenograft experiments. Thus, combination therapy with chemotherapeutic agents and IFNs may be effective against breast cancer, particularly in patients with defective in DNA damage response pathways. The downstream effectors that modulate the effect of IFNs should be investigated as predictive biomarker in the treatment of breast cancer.

Citation Format: Na-Lee Ka, Ga Young Lim, Mi-Ock Lee. Type I interferons enhance chemosensitivity of breast cancer by inhibiting DNA repair [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 2565.
Abstract 2566: CYT01B suppresses RAD51 focus formation and induces replication catastrophe following AID induced DNA damage
Authors: Day Melinda , Cyr Amber , Maclay Tyler , Mills Kevin
Published: August 2019

Activation Induced Cytidine Deaminase (AID) is a DNA-directed cytidine deaminase that plays a critical role in somatic hypermutation and immunoglobulin class switching in activated B- lymphocytes. AID is often ectopically expressed and promotes genomic hypermutation in a range of lymphoid malignancies. Deamination of genomic cytidines results in point mutations, single strand DNA breaks (SSB), and double strand DNA breaks (DSB). The resulting DNA damage stress leads to an obligate dependency on homologous recombination for cellular survival. RAD51 is a critical component of the homologous recombination pathway, forming nucleoprotein filaments at sites of DNA damage or replication fork stalls. RAD51 mediates homologous DNA strand exchange to promote recombinational repair of breaks and damaged replication forks. We have previously shown that RAD51 depletion leads to accumulation of DNA breaks, and ultimately cell death, in AID expressing cells. We now describe the mechanism of action for a novel small molecule, CYT01B, that targets RAD51 mediated recombination. Treatment of AID-expressing cells with CYT01B, attenuates RAD51 focus formation, reduces the nuclear concentration of RAD51, and promotes RAD51 protein degradation. CYT01B reduces homologous recombination activity in cells, culminating in cytologically visible chromosome fragmentation following DNA damage. Finally, CYT01B treated cells undergo replication catastrophe associated with caspase 3/7 activation and persistent PCNA nuclear signal. We conclude that CYT01B is a novel homologous recombination inhibitor, and propose a new synthetic lethality mechanism, induced by CYT01B, mediated by DNA replication stress and replication catastrophe.

Citation Format: Melinda Day, Amber Cyr, Tyler Maclay, Kevin Mills. CYT01B suppresses RAD51 focus formation and induces replication catastrophe following AID induced DNA damage [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 2566.
Abstract 2574: Werner syndrome helicase is required for the survival of cancer cells with microsatellite instability
Authors: Kategaya Lorn , Perumal Senthil K. , Hager Jeffrey H. , Belmont Lisa D.
Published: August 2019

Werner Syndrome protein (WRN) is a RecQ enzyme involved in the maintenance of genome integrity. Germline loss of function mutations in WRN lead to premature aging and pre-disposition to cancer. We evaluated synthetic lethality (SL) between not only of WRN, but also of another human RecQ helicase, BLM, with DNA damage response genes in cancer cell lines. We found that WRN exhibited SL with a DNA mismatch repair protein MutL homolog 1 (MLH1), loss of which is associated with high microsatellite instability (MSI-H). We then demonstrated that MSI-H cells exhibited increased double-stranded DNA breaks, altered cell cycles and decreased viability in response to WRN knockdown. Although WRN is the only human RecQ enzyme with a distinct exonuclease domain, only loss of helicase activity drives the MSI SL interaction. This SL interaction in MSI cancer cells positions WRN as a relevant therapeutic target in patients with MSI-H tumors.

Citation Format: Lorn Kategaya, Senthil K. Perumal, Jeffrey H. Hager, Lisa D. Belmont. Werner syndrome helicase is required for the survival of cancer cells with microsatellite instability [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 2574.
Abstract 2585: The development of a novel microRNA replacement therapy for ovarian cancer - a potential of patient derived-exosomes as a carrier
Authors: Kobayashi Masaki , Sawada Kenjiro , Shimizu Aasa , Miyamoto Mayuko , Nakatsuka Erika , Kawano Mahiru , Matsumoto Yuri , Kodama Michiko , Hashimoto Kae , Mabuchi Seiji , Kimura Tadashi
Published: August 2019

Objective: microRNAs (miRNAs) affect key features of cancer cells and some are identified as tumor suppressor (TS) miRNAs. In spite of many preclinical studies with promising results, all translational clinical trials with TS miRNAs have failed due to the difficulties of miRNA systemic delivery. Therefore, the development of a novel carrier is urgently needed. Exosomes, non-sized vesicles secreted from cells, are stable in body fluid and therefore can serve as a promising candidate for miRNA carrier. In ovarian cancer treatment, omentum should be taken at surgery and normal fibroblasts can be collected from omentum. The aim of this study is to pursue the possibility of fibroblast derived-exosomes as a carrier for miRNA replacement therapy to treat ovarian cancer.

Methods: At surgery, omentum was collected and fibroblasts were primary cultured. Thereafter, exosomes were collected from culture media. miR-199a-3p was selected as a TS miRNA. Synthesized miR-199a-3p was corporated into exosomes by electroporation. miR-199a-3p-loaded exosomes (M199-exosomes) were treated with ovarian cancer cells and the expression levels of miR-199a-3p and target genes were assessed. The treatment effect of M199-exosomes was analyzed focusing on peritoneal dissemination.

Results: The treatment of M199-exosomes drastically increased miR-199a-3p expression in ovarian cancer cells (CaOV3; 8592-fold, SKOV3; 67188-fold, OVCAR3; 2280-fold). Treatment with M199-exosomes suppressed c-Met expression and significantly inhibited the proliferation and invasion of ovarian cancer cells in vitro (P<0.01). In a xenograft study, M199-exosomes significantly inhibited peritoneal dissemination and extended the survival of mice.

Citation Format: Masaki Kobayashi, Kenjiro Sawada, Aasa Shimizu, Mayuko Miyamoto, Erika Nakatsuka, Mahiru Kawano, Yuri Matsumoto, Michiko Kodama, Kae Hashimoto, Seiji Mabuchi, Tadashi Kimura. The development of a novel microRNA replacement therapy for ovarian cancer - a potential of patient derived-exosomes as a carrier [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 2585.
Abstract 2586: Therapeutic potential of the topical treatment of miR-634 ointment for cutaneous squamous cell carcinoma
Authors: Inoue Jun , Gokita Kentaro , Kishikawa Masahiro , Hamamoto Hidetoshi , Kobayashi Katsunori , Fujiwara Kyoko , Asakage Takahiro , Kojima Kazuyuki , Inazawa Johji
Published: August 2019

Cutaneous squamous cell carcinoma (CSCC) is the second most common nonmelanoma skin cancers. Surgery represents the gold standard for CSCC treatment, however it is needed to develop the noninvasive and effective treatments as the nonsurgical options for CSCC patients whose conditions are not suitable for or who refuse operation. On the other hand, microRNAs (miRs) are endogenous small non-coding RNAs negatively regulate gene expression by interfering with the translation or stability of target transcripts. Some tumor-suppressive miRs can target multiple cancer-promoting genes concurrently and may be useful as a therapeutic agent for cancer therapy. We have previously found that overexpression of miR-634 activated the mitochondrial apoptotic pathway by directly targeting genes associated with cytoprotective processes, including mitochondrial homeostasis, anti-apoptosis, antioxidant ability, and autophagy, in various cancer cells. The development of drug delivery system (DDS) is critical for the implementation of miR-based therapeutics. While topical pharmacotherapy represents a nonsurgical option to consider in CSCC, we validated the therapeutic potential of topical treatment of miR-634 ointment in mice model of CSCC. In xenograft tumors of A431 cells, a human epidermoid carcinoma cell line, in mice the topical treatment of ointment incorporated double-strand miR-634 mimics significantly reduced tumor growth. Furthermore, the topical treatment of miR-634-ointment also inhibited tumor growth in carcinogen-induced mouse skin papilloma model. Taken together, these findings suggest that topical treatment of miR-634-ointment may be useful as a noninvasive and effective treatments for CSCC.

Citation Format: Jun Inoue, Kentaro Gokita, Masahiro Kishikawa, Hidetoshi Hamamoto, Katsunori Kobayashi, Kyoko Fujiwara, Takahiro Asakage, Kazuyuki Kojima, Johji Inazawa. Therapeutic potential of the topical treatment of miR-634 ointment for cutaneous squamous cell carcinoma [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 2586.
Abstract 26: Development of potent lead compounds in multiple tumor types
Authors: Raj Ganesh V. , Liu Xihui , Ekoue Dede , Ahn Jung-Mo , Vadlamudi Ratna
Published: August 2019

Background: We had earlier reported a novel therapeutic agent, ERX-11, that modulates estrogen receptor coregulator interactions. For lead optimization, we designed, synthesized and tested over 500 analogs of ERX-11 in multiple models of BC. We also tested this library of compounds for activity against other cancer cell lines both in vitro and in vivo and against primary tumors ex vivo.

Methods: In vitro activity was tested using Cell titer glo, MTT, and apoptosis assays.The utility of the ERX analogs in treating therapy resistant ER-positive BC was evaluated using models with acquired resistance (Tamoxifen, Letrozole), and engineered models that express ER mutations. Xenografts were used for testing the utility of ERX analogs in vivo. Primary patient tumor derived explants were used for ex vivo testing of ERX analogs.

Results: Our screening studies identified several ERX analogs with potent activity against BC cells. Subtle changes in the ERX analogs appear to have significant ramifications on both their potency against ER+ BC cell lines and against other tumors types. Some analogs like ERX-41 were more potent than ERX-11 in their ability to block the proliferation of multiple ER-positive BC cell lines (IC50 ranging from 20-200nM). Other analogs like ERX-208 showed similar activity as ERX-11 against ER-positive BC cell lines (IC50 ranging from 100-500nM) but had potent activity against ovarian cancer cell lines. Through iterative changes, we have identified leads compounds with significant activity against other cancers, including in gliomas, ovarian and pancreatic cancers. Although all these compounds were designed to better target the ligand binding pocket of ER, these active compounds do not all target ER and appear to target other proteins, including other nuclear receptors. Some compounds for example have activity in ER-negative breast cancers. In several xenograft models, including pancreatic cancer and ER-negative, the activity of the compounds have been confirmed by oral administration of the ERX analog in vivo. We have also validated the activity of these analogs in patient-derived explants cultured ex vivoin multiple tumor types.

Conclusions: From our studies to develop a more potent ERX-11 lead analog, we have identified multiple analogs with distinct activities against multiple cancers. While the intended target of these analogs was ER, our library of analogs has potent activity against both ER-positive and ER-negative tumors. In collaboration, we are pursuing further leads in multiple cancers to further delineate the mechanism of action of these various analogs.

Citation Format: Ganesh V. Raj, Xihui Liu, Dede Ekoue, Jung-Mo Ahn, Ratna Vadlamudi. Development of potent lead compounds in multiple tumor types [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 26.
Abstract 2600: Interaction of host cell factor-1 with the oncoprotein transcription factor MYC
Authors: Popay Tessa M. , Thomas Lance R. , Wang Jing , Liu Qi , Tansey William P.
Published: August 2019

Host cell factor (HCF)-1 is a highly-conserved epigenetic scaffolding protein that is essential for development, cell cycle progression, and cellular proliferation. Through integration into several chromatin modifying complexes, HCF-1 plays a critical role in regulating the activity of key transcription factors, such as the E2F family of proteins. HCF-1 is also linked to leukemogenesis, through its regulation by the BAP1 tumor suppressor and its incorporation into the oncogenic MLL complex. Despite its role in the cell cycle, presence at chromatin, and well-defined interactions, the function of HCF-1 in cancer remains under-studied and undetermined. Here we describe the role of HCF-1 in the context of its interaction with the transcription factor and oncoprotein MYC. MYC is overexpressed in most cancers and drives tumorigenesis by regulating the expression of genes involved in ribosome biogenesis, cell growth, and metabolism. The region of MYC responsible for interaction with HCF-1 is evolutionarily conserved in all vertebrate MYC family members, and we have previously demonstrated that mutations in MYC that selectively disable interaction with HCF-1 attenuate the tumorigenicity of MYC in vivo. This demonstrates that HCF-1 is an important co-factor for MYC-driven cancer. Using a battery of contemporary genomic approaches, including ChIP-seq, SLAM-seq, RNA-seq, and PRO-seq, coupled to a decisive CRISPR/Cas9 editing approach, we reveal the relationship of MYC and HCF-1 at chromatin and how this modulates the function of MYC as a transcription factor and oncoprotein.

Citation Format: Tessa M. Popay, Lance R. Thomas, Jing Wang, Qi Liu, William P. Tansey. Interaction of host cell factor-1 with the oncoprotein transcription factor MYC [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 2600.
Abstract 2603: Herbal extract SH003 suppresses tumor growth and metastasis of MDA-MB-231 breast cancer cells by inhibiting STAT3-IL-6 signaling
Authors: Choi Yu-Jeong , Kim Myeong-Sun , Kang SooYeon , Lee Seo Yeon , Kim Ji Hye , Choi Youn Kyung , Oh Sukjoong , Ko Seong-Gyu
Published: August 2019

Cancer inflammation promotes cancer progression, resulting in a high risk of cancer. Here, we demonstrate that our new herbal extract, SH003, suppresses both tumor growth and metastasis of MDA-MB-231 breast cancer cells via inhibiting STAT3-IL-6 signaling path. Our new herbal formula, SH003, mixed extract from Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii Maximowicz, suppressed MDA-MB-231 tumor growth and lung metastasis in vivo and reduced the viability and metastatic abilities of MDA-MB-231 cells in vitro. Furthermore, SH003 inhibited STAT3 activation, which resulted in a reduction of IL-6 production. Therefore, we conclude that SH003 suppresses highly metastatic breast cancer growth and metastasis by inhibiting STAT3-IL-6 signaling path.

Citation Format: Yu-Jeong Choi, Myeong-Sun Kim, SooYeon Kang, Seo Yeon Lee, Ji Hye Kim, Youn Kyung Choi, Sukjoong Oh, Seong-Gyu Ko. Herbal extract SH003 suppresses tumor growth and metastasis of MDA-MB-231 breast cancer cells by inhibiting STAT3-IL-6 signaling [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 2603.