bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1324: Profile of common prostate cancer risk variants in an unscreened Romanian population
Authors: Iordache Paul D. , Halldórsson Bjarni , Manolescu Andrei , Mates Dana , Ursu Radu , Jinga Viorel
Published: July 2017

In the present study we investigated for the first time the profile of common prostate cancer risk variants in an unscreened Romanian population. The study population consisted of 990 unrelated histopathologically confirmed prostate cancer (PCa) cases and 1,034 male controls consisting of patients admitted for urological and surgical conditions, excluding cancer. DNA was extracted from whole blood at deCODE Genetics (Reykjavik, Iceland) and genotyped using Illumina SNP arrays, 24.295.558 variants were imputed using the 1000 Genomes dataset in the 2,024 Romanian subjects. A systematic literature review for variants associated with prostate cancer identified in previous GWAS‘ was done using the NHGRI catalog as a starting point, identifying 238 unique variants from 28 studies. None of the tested variants in a Romanian only GWAS reached a genome wide significance (p-value lower than 5x10-8) but 807 markers reached p-values of 1x10-6. Thirty-one of the previously-reported SNPs replicated in the Romanian cohort, with the strongest associations seen at: 8q24.21, 11q13.3, 6q25.3, 5p15.33, 22q13.2, 17q12 and 3q13.2. The most signficantly replicated variants in Romania are rs1016343 at 8q24.21 (P = 2.2x10-4), rs7929962 at 11q13.3 (P = 2.7x10-4) and rs9364554 at 6q25.2 (P = 4.7x10-4). Our present study is the first GWAS on prostate cancer performed on a Romanian cohort. The high proportion of clinically significant disease in the Romanian prostate cancer cases will allow the dissection of the association between genetic variation and severe disease. Acknowledgements: This study was funded in part by the European Union FP7 Program (ProMark project 202059) and by the EEA grant (ROMCAN project RO14-0017; EEA-JRP-RO-NO-20131-10191).

Citation Format: Paul D. Iordache, Bjarni Halldórsson, Andrei Manolescu, Dana Mates, Radu Ursu, Viorel Jinga. Profile of common prostate cancer risk variants in an unscreened Romanian population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1324. doi:10.1158/1538-7445.AM2017-1324
Abstract 134: A dual mTOR-PI3K inhibitor DCB-HDG2-57 with Hedgehog signaling pathway antagonist activity
Authors: Lee Ying-Shuan , Kuo Mann-Yan , Liu Chia Wei , Tseng Yu-Wen , Wei Win Yin , Chiou Shian-Yi , Lin Her Sheng , Lu Y.-Y. , Liao Chu-Bin , Pen Shao-Zheng
Published: July 2017

The Hedgehog (Hh) signaling pathway is a critical regulator of embryonic patterning, and aberrant Hh pathway activation has been implicated in a diverse spectrum of cancers. Therefore, components of the Shh pathway (such as Shh, SMO, and GLI1/2) are viable therapeutic targets for anti-tumor strategy. Smo antagonists such as GDC-0449 and NVP-LDE225 have received FDA approval for treating basal cell carcinoma. However, acquired resistance has emerged as a challenge to targeted therapeutics and may limit their anti-cancer efficacy. Studies have linked the hyperactive phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR signaling pathway in a variety of human cancers and drug resistance condition. The synergistic role of PI3K/AKT/mTOR in Hh signaling in embryonic development and Hh-dependent tumors has been reported. Both the PI3K/Akt/mTOR and Hh pathway also play important role in maintaining the stem cell-like properties of cancer stem cell (CSCs). We report here the development of a potent dual mTOR-PI3K inhibitor designated DCB-HDG2-57 with Hh signaling pathway antagonist activity. In vitro biochemical assays showed that DCB-HDG2-57 inhibited recombinant pI3Kα and mTOR kinase with IC50 of 27.2 and 133 nM, respectively. DCB-HDG2-57 is also highly active in cellular assays, as evidenced by inhibition of phosphorylation of cellular downstream targets of PI3K-mTOR kinases ribosomal protein S6 kinase (S6K1, Ser240/24) in the colon (LS-180) and pancreatic cancer (MiaPaCa2 and PANC1) cell lines. Interestingly, DCB-HDG2-57 demonstrated Hh signaling pathway antagonist activity in a 293 cell-based Gli-luciferase inhibition assay upon agonist treatment, and retains inhibition activity against the Smo wild-type and D473H mutant co-transfection with IC50 of 261.8 and 327.7 nM, respectively. In addition, DCB-HDG2-57 can significantly decrease the cancer stem cell liked side population of PANC1 pancreatic cell, and inhibited the migration of E3LZ10.7 pancreatic cancer cell. DCB-HDG2-57 demonstrated in vivo tumor growth inhibition activity in LS-180 colon cancer xenograft model and reduced the Gli-1 RNA abundance in the tumor. Taken together, our data demonstrate that combined pharmacological blockade of mTOR-PI3K and Hh pathway of DCB-HDG2-57 can provide a therapeutic strategy for targeting ligand-dependent Hh cancer.

Citation Format: Ying-Shuan Lee, Mann-Yan Kuo, Chia Wei Liu, Yu-Wen Tseng, Win Yin Wei, Shian-Yi Chiou, Her Sheng Lin, Y.-Y. Lu, Chu-Bin Liao, Shao-Zheng Pen. A dual mTOR-PI3K inhibitor DCB-HDG2-57 with Hedgehog signaling pathway antagonist activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 134. doi:10.1158/1538-7445.AM2017-134
Abstract 1341: Talin plays an important role in cell-cell interactions
Authors: Patel Devang M. , Kumar Akhilesh , Morgan William , Birnbaum Michael D. , Jiang Chaunhua , Zhang Fangliang
Published: July 2017

Appropriate cell adhesion is necessary for numerous physiological processes and can be deranged in many diseases, including cancer. It has long been recognized that the cadherins mediated cell-cell adhesion receptor is an important determinant of tumor progression, serving as a suppressor of invasion and metastasis. Recently a role for the C-terminal region of talin (the VAD fragment) in cadherin mediated cell-cell attachment has been suggested, but its molecular mechanism remained unclear. Here, we identified a small region of the VAD fragment required for its localization to cadherin-mediated cell-cell junctions and also found that its localization to cell-cell junctions is independent of its actin binding properties. To identify the binding partner of the VAD fragment at cell-cell junctions we utilized immune-pulldown and found that it interacts with catenins, an important component of cadherin-mediated cell-cell junction. Based on our data, we proposed that the VAD-fragment of talin stabilized and/or stimulated the cell-cell junction by interacting with catenins.

Citation Format: Devang M. Patel, Akhilesh Kumar, William Morgan, Michael D. Birnbaum, Chaunhua Jiang, Fangliang Zhang. Talin plays an important role in cell-cell interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1341. doi:10.1158/1538-7445.AM2017-1341
Abstract 1347: In vivo efficacy of eflapegrastim in rats with chemotherapy-induced neutropenia
Authors: Kim Young Hoon , Choi InYoung , Kolli Prasad , Reddy Guru
Published: July 2017

Introduction: Eflapegrastim (SPI-2012, HM10460A) is a novel, long-acting recombinant human granulocyte colony-stimulating factor (rhG-CSF). Eflapegrastim consists of an rhG-CSF conjugated to a recombinant E. coli derived Fc fragment of IgG4 via a polyethylene glycol linker. Eflapegrastim is in clinical development for the treatment of chemotherapy-induced neutropenia in cancer patients. The purpose of this study was to evaluate and compare the efficacy of eflapegrastim, pegfilgrastim and filgrastim in rats with chemotherapy-induced neutropenia.

Methods: Rats were treated with 50 mg/kg of cyclophosphamide (CPA) intraperitoneally to induce neutropenia. Pegfilgrastim was administered subcutaneously as a single dose of 100 µg/kg on Day 1 and filgrastim was administered subcutaneously at a dose of 20 µg/kg daily for five days on days 1 to 5. Eflapegrastim was administered subcutaneously as a single dose on day 1, at doses ranging from 32 µg /kg to 322 µg/kg (or 8.8 µg/kg to 88 µg/kg as G-CSF equivalent). Blood samples were collected for 8 days after drug administration for the measurement of neutrophil counts and the Duration of Severe Neutropenia (DSN).

Results: The DSN in neutropenic rats treated with eflapegrastim was compared with the DSN in neutropenic rats treated with pegfilgrastim or filgrastim. The DSN was 0.2 days when eflapegrastim was administered as a single dose at 88 µg/kg (as G-CSF equivalent) 24 hours after administering CPA. In contrast, the DSN was 3.04 days with filgrastim administered at a dose of 20 µg/kg for 5 days from Day 1 to Day 5 and 2.8 days with pegfilgrastim administered as a single dose of 100 µg/kg 24 hours after administering CPA. At the lowest eflapegrastim dose of 8.8 µg/kg that was about 1/10 of G-CSF equivalent dose for pegfilgrastim, the DSN in eflapegrastim treated rats was 2.94 days. Thus, eflapegrastim was found to be more potent in shortening the DSN with a lower G-CSF equivalent dose when compared to either pegfilgrastim or filgrastim.

Conclusion: Eflapegrastim was about 10 times more potent than pegfilgrastim at G-CSF equivalent doses in this neutropenic rat model

Citation Format: Young Hoon Kim, InYoung Choi, Prasad Kolli, Guru Reddy. In vivo efficacy of eflapegrastim in rats with chemotherapy-induced neutropenia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1347. doi:10.1158/1538-7445.AM2017-1347
Abstract 1351: Alternative splicing of neurofibromin 1 is associated with elevated MAPK activity and poor prognosis in glioma
Authors: Siddaway Robert , Ramani Arun , Yu Man , Brudno Michael , Hawkins Cynthia
Published: July 2017

High-grade gliomas (HGG) are invasive with poor prognosis regardless of age: diffuse intrinsic pontine gliomas (DIPG), arising in the brainstem, are almost universally fatal and the leading cause of brain-tumor death in children; while adult anaplastic astrocytoma and glioblastoma multiforme (GBM) have median survivals of 1-3 years. The mutational spectra of adult and pediatric HGG differ, with pediatric tumours containing recurrent mutations of H3F3A and HIST1H3B. However, alterations leading to RAS/MAPK/PI3K pathway activation, including PDGFRA amplification, EGFRvIII, BRAF-V600E, NF1 deletion, are frequently found, although not all tumours will have mutations in this pathway. The neurofibromin 1 (NF1) gene negatively regulates RAS signalling by stimulating RAS-GTP turnover, thereby leading to RAS-inactivation. The two major isoforms, NF1-I and NF1-II, differ only by inclusion of the 21 aa exon23a in the GAP-related domain of NF1-II. Exon23a-inclusion has been shown to render NF1 10 times less active towards RAS, leading to elevated MAPK signalling. The brain expresses predominantly NF1-I, while the major isoform elsewhere is NF1-II. Here we used RNA-Seq to identify genes alternatively spliced between DIPG and normal brain, identifying an isoform switch from NF1-I in normal brain to NF1-II; we additionally found the same isoform switch in the TCGA adult GBM and LGG cohorts. For both GBM and LGG, RAS/MAPK/PI3K wild-type tumors with elevated NF1-II conferred significantly reduced patient survival compared to RAS/MAPK/PI3K mutant tumors. NF1-exon23a inclusion is known to be repressed in cell model systems by the CELF and ELAV-like families of splice regulators. We further show that members of these gene families are downregulated in HGG. Together, our results indicate a novel mechanism by which gliomas can activate signaling downstream from RAS independent of mutations and tumor grade, which promotes tumorigenesis by regulating pathways such as proliferation and invasion.

Citation Format: Robert Siddaway, Arun Ramani, Man Yu, Michael Brudno, Cynthia Hawkins. Alternative splicing of neurofibromin 1 is associated with elevated MAPK activity and poor prognosis in glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1351. doi:10.1158/1538-7445.AM2017-1351
Abstract 1355: Linking AP/MS-derived protein-protein interaction networks and pairwise sgRNA screens defines new KRAS effectors in non-small cell lung cancer
Authors: Kelly Marcus R.
Published: July 2017

The KRAS oncogene is mutated in over one fourth of human cancers. Despite decades of work studying known effector pathways, no drugs in clinical use specifically target KRAS-mutant tumors. Uncharacterized feedback mechanisms and parallel pathways have stymied the treatment of KRAS-mutant tumors in patients, and the KRas protein does not easily accommodate the binding of inhibitory small molecules. These challenges demand better characterization of the physical and genetic relationships between Ras regulators and effectors. To that end, we used tandem affinity purification of Kras, Hras and Nras and twelve proteins with known regulatory or effector roles to generate a high-confidence protein-protein interaction (PPI). Interactions were cross-referenced with public PPI, genetic susceptibility, and patient data to assemble a physical PPI map annotated with important functional determinants. The final map contains 287 genes and 990 physical interactions among them. This map was used to construct a library of >1000 sgRNAs covering >100 genes of probable relevance to the Ras pathway. This library was screened for pairwise genetic effects in a dual sgRNA vector system to identify quantitative changes in the growth of the A549 NSCLC line. We will present the quantitative results of this screen and additional validation studies on several previously undescribed regulators and effectors of the Ras proteins. In particular, we highlight unknown interactions between KRAS and the cell adhesion regulator RADIL, and the specificity of Ras-family protein interaction with the Raf and Ral GEF family proteins. These data underscore the limitations of our current understanding of Kras-driven cancers and reveal their genetic vulnerabilities.

Citation Format: Marcus R. Kelly. Linking AP/MS-derived protein-protein interaction networks and pairwise sgRNA screens defines new KRAS effectors in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1355. doi:10.1158/1538-7445.AM2017-1355
Abstract 1357: The small GTPase Arf6 potentiates melanoma metastasis by activating Akt
Authors: Acosta Lehi , Rogers Aaron , Yoo Jae H. , Odelberg Shannon J. , Li Dean Y. , Holmen Sheri , Grossmann Allie H.
Published: July 2017

Arf6 is a member of the Ras-superfamily of small GTPases and controls membrane trafficking and cytoskeletal remodeling, functioning mainly in endocytosis pathways at the cell periphery. Arf6 is activated by various extracellular signals and oncogenic events and has been shown to promote cell migration and pro-invasive phenotype in human cancer cells. Small molecule inhibition of ARF6 reduces spontaneous metastasis in xenograft models of human cutaneous melanoma, suggesting that ARF6 is necessary for disease progression. Using a genetically-engineered mouse model of BRAF-mutant melanoma, we determined whether activation of Arf6 is sufficient to induce spontaneous metastasis in vivo. For melanocyte-specific primary tumor induction, Cre recombinase was delivered via local injection of RCAS virus into DCT-TVA;Cdkn2alox/lox;BRAFV600E mice. In the experimental mice, a constitutively active mutant form of Arf6 (Q67L) was virally delivered with Cre. In this study, we observed a significant increase in spontaneous metastatic disease burden in Arf6 Q67L mice. Likewise, tail vein injection of melanoma cell lines derived from Arf6 Q67L tumors consistently show a diffuse pattern of pulmonary metastasis compared to controls that show rare, microscopic metastasis. Recently, it has been demonstrated that activation of Akt, but not Pten loss, leads to an aggressive phenotype in melanoma that includes the acquisition of brain metastases. Immunohistochemical analysis of phospho-Akt revealed that Arf6 Q67L is sufficient to induce Akt activation in tumors. In addition, ARF6 is necessary for AKT activation in human melanoma lines. We did not observe brain metastasis in DCT-TVA;Cdkn2alox/lox;BRAFV600E + Arf6 Q67L mice. When we added Ptenlox/lox allele to this genetic background, however, we observed microscopic brain metastases at a low frequency, suggesting that the combination of Pten loss and Arf6 activation reaches a threshold level of Akt activation that is sufficient to cause brain metastasis. Taken together our data indicate that activation of Arf6 is sufficient to potentiate melanoma metastasis, consistent with the proinvasive cellular phenotype attributed to Arf6. In addition, our data suggests a novel signaling mechanism by which Arf6, a small GTPase involved in trafficking, is somehow involved in Akt activation and that this step may be important for the acquisition of metastatic capacity.

Citation Format: Lehi Acosta, Aaron Rogers, Jae H. Yoo, Shannon J. Odelberg, Dean Y. Li, Sheri Holmen, Allie H. Grossmann. The small GTPase Arf6 potentiates melanoma metastasis by activating Akt [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1357. doi:10.1158/1538-7445.AM2017-1357
Abstract 1361: Characterization of TpeL, a RAS specific clostridial toxin
Authors: Abreu-Blanco Maria T.
Published: July 2017

Clostridium perfringes lethal toxin (TpeL) belongs to the family of large clostridial glycosylating cytotoxins. Clostridial toxins are glycosyltransferases that modify and deactivate small GTPases of the RHO and RAS subfamily. TpeL mono-glycosylates in the switch I domain (Thr35) of RAC and RAS small GTPases. Glycosylation of RAS at Thr35 prevents binding to the primary effector, RAF kinase and results in a blockade RAS signal transduction. RAS family proteins function as key regulators of cell proliferation, differentiation, survival and gene expression. Moreover, mutations in RAS proteins are highly prevalent in human cancers. Considering the specificity of TpeL for RAS, we decided to investigate the biochemical interaction between TpeL and RAS, and potentially develop tools to disrupt the RAS signaling pathway. In order to assess the effect of TpeL in vivo, we use RAS-dependent MEFs expressing different KRAS mutations and BRAF V600E. We expect that proliferation should be inhibited in MEF cells expressing RAS isoforms, but MEF cells expressing BRAF V600E should not be affected by any toxin. We found that TpeL treatment did not affect the viability of the BRAF V600E cells and only induced toxicity in RAS expressing cells. Consistent with this result, TpeL treatment inhibited MAPK signal transduction in the RAS expressing cells, but not in the BRAF V600E cells. Surprisingly, both cell viability as well as pERK levels remained unaffected in the KRAS Q61R MEF cells treated with TpeL, suggesting that KRAS Q61R is resistant to glycosylation. This may be due in part to the relatively slow intrinsic rate of GTP hydrolysis and very high levels of KRAS-GTP in KRAS Q61R cells. We also developed and adapted biochemical assays to study TpeL-KRAS interaction in vitro. Based on Alpha Assay and UDP Glow, TpeL activity does not appear to be nucleotide specific, in contrast to previously reported findings. We are currently testing if TpeL RAS glycosylation is competitive with or is inhibited by other molecules that bind to switch I, such as the RBD domain of RAF1 or GEF/GAP proteins. These data may indicate that the lack of activity in KRAS Q61R expressing MEF cells is due not to the high levels of RAS-GTP, but to the constitutive binding to effector molecules, which prevent TpeL from accessing KRAS. We also hope to identify the critical residues required for TpeL binding and catalysis. By elucidating the mechanism of substrate binding and substrate specificity we may gain insight into novel binding pockets that could be exploited therapeutically.

Citation Format: Maria T. Abreu-Blanco. Characterization of TpeL, a RAS specific clostridial toxin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1361. doi:10.1158/1538-7445.AM2017-1361
Abstract 1362: Argonaute 2 controls RAS activation in mouse embryonic fibroblasts
Authors: Siebenaler Ronald F. , Shankar Sunita , Dommeti Vijaya L. , Mody Malay , Chinnaiyan Arul
Published: July 2017

The RAS gene family is among the most commonly mutated genes within cancer, and while much research has elucidated the major downstream pathways, including MAPK and PI3K, little progress has been made in successfully targeting mutant RAS in cancer. We recently identified an interaction between the N terminal domain of Argonaute 2 (AGO2), a core component of RNA-induced silencing complex (RISC), and the Switch II domain of KRAS. Furthermore, this interaction was found in all cell lines tested, expressing either wild-type (WT) or mutant KRAS. We found that stable knockdown of AGO2 in KRAS dependent cell lines lead to a decrease in KRAS protein expression with a subsequent decrease in cellular proliferation. Conversely, the overexpression of AGO2 in these cells lead to both an increase in KRAS expression and oncogenesis. In addition, this interaction inhibits the RNAi function of AGO2 by preventing microRNA unwinding in the presence of oncogenic KRAS compared to WT-KRAS. Despite a clear association between mutant KRAS and AGO2 mediating increased KRAS mediated oncogenesis, the precise function of this interaction remains unclear in normal physiology. In order to better assess the endogenous function of the KRAS-AGO2 interaction, we analyzed two mouse embryonic fibroblast cell lines (NIH 3T3 and MEF) with complete knockout of AGO2. Utilizing a Raf-1 RAS binding domain (RBD) pulldown method, we assessed activated WT-RAS levels in AGO2 null NIH 3T3 and MEF cells. We found that knockout of AGO2 lead to an increase in WT RAS-GTP activation compared to normal control cells. Immunoblot analysis also indicates that AGO2 null fibroblasts lead to increase in RAS downstream signaling through the MAPK/ERK and PI3K/AKT pathways. Furthermore, rescue of AGO2 knockout using full length mouse AGO2 decreased wild type RAS activation and its downstream signaling. Taken together, these observations suggest that the AGO2 interaction may suppress WT-KRAS activation, leading to maintenance of RAS-GDP levels. Using RNA-seq, proteome and microRNA analysis, we have begun to identify the pathways that may be involved in RAS activation in AGO2 null cells. Early analyses indicate that AGO2 controls WT-KRAS levels and activity through multiple mechanisms, laying the foundation for a better understanding of the RAS-AGO2 interaction in normal physiology.

Citation Format: Ronald F. Siebenaler, Sunita Shankar, Vijaya L. Dommeti, Malay Mody, Arul Chinnaiyan. Argonaute 2 controls RAS activation in mouse embryonic fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1362. doi:10.1158/1538-7445.AM2017-1362
Abstract 1365: Sensitivity of oncogenic KRAS to adenosine triphosphate suppression
Authors: Holderfield Matthew , Sharma Kanika
Published: July 2017

KRAS proteins regulate many cellular processes including initiation of cell division in response to mitogens. Gain of function KRAS mutations appear in approximately 30% of all human cancers and activate effector signal transduction pathways independent of mitogenic stimuli. KRAS driven cancers also frequently have high metabolic rates and may have increased sensitivity to metabolic inhibitors but the underlying mechanism is not fully understood. In this study, we characterized the cellular mechanism of a small molecule inhibitor identified using a transgenic Drosophila melanogaster model that expresses human KRAS-G12V in the fly wing. The compound partially restored wing development in the KRAS-G12V flies and inhibited growth and signal transduction in multiple KRAS driven cancer cells and KRAS-G12V dependent MEF cells but not in MEF cells expressing BRAF-V600E. The compound also had a profound impact on mitochondrial function by inhibiting electron transport (ETC) complex I. Multiple other ETC or ATP synthase inhibitors similarly impaired oncogenic KRAS signal transduction. ATP production through glucose metabolism was sufficient to rescue KRAS function and cellular growth after ETC inhibition, suggesting that inhibition of MAPK signaling is due to low ATP levels rather than a direct consequence of ETC or complex I inhibition. These data indicate that oncogenic KRAS signal transduction requires high cellular ATP levels and suggests a synthetic lethal interaction between KRAS and multiple metabolic targets that could be exploited to target KRAS driven cancers.

Citation Format: Matthew Holderfield, Kanika Sharma. Sensitivity of oncogenic KRAS to adenosine triphosphate suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1365. doi:10.1158/1538-7445.AM2017-1365
Abstract 1369: DNA, protein, and cell line reagents to facilitate RAS pathway drug discovery efforts
Authors: Esposito Dominic
Published: July 2017

The RAS Reagents Core within the NCI RAS Initiative produces and distributes qualified DNA vectors, cell lines, and protein production reagents to the external community to facilitate research into RAS biology and development of RAS cancer therapeutics. DNA vectors representing a variety of RAS mutations, as well as a complete collection of 180 genes in the RAS pathway are available as Gateway-compatible Entry clones. These clones represent the most commonly expressed transcript forms of the 180 genes, many of which were not previously available in full-length, wildtype forms. The vectors can be used with the FNLCR combinatorial cloning library to easily generate a large number of different types of expression clones with various promoters, fusion tags, and plasmid backbones. Cell line reagents include a variety of Ras-dependent mouse embryonic fibroblasts (MEFs) which have a defined mutant allele of KRAS and which serve as isogenic cell lines for investigating allele and mutant specific RAS biology, as well as for valuable screening tools. Protein reagents include E. coli and insect cell constructs and cell lines for production of a variety of Ras-related proteins. In particular, we have materials for the high yield production of properly processed, prenylated KRAS protein using a unique engineered insect cell expression system. This system can also be used to produce other prenylated small GTPases at levels not previously attainable. Many of the resources of the RAS Reference Core are readily available to the academic community through repositories or material transfer agreements, and can be licensed to industry scientists as well. The program is also interested in identifying the needs of the community for additional RAS-related resources, and is developing methods for vetting requests from the outside to provide additional reagents.

Citation Format: Dominic Esposito. DNA, protein, and cell line reagents to facilitate RAS pathway drug discovery efforts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1369. doi:10.1158/1538-7445.AM2017-1369
Abstract 137: FT-1518, a new generation selective and potent mTORC1 and mTORC2 inhibitor: an in vitro and in vivo profile
Authors: Mita Alain C. , Mita Monica M. , William Anthony D. , Pasha Khalid , Siddamadappa Chandra , Zikaras Kevin , Garzon Felix T.
Published: July 2017

Mammalian target of rapamycin (mTOR) is a clinically validated target in the treatment of cancer. mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2 which regulate cell growth, metabolism, proliferation, and survival. Rapamycin analogues target only the mTORC1 complex but do not affect the mTORC2 complex, which is an important driver for cancer cell growth and survival. The new generation of “Selective” mTOR inhibitors, blocking both mTORC1 and mTORC2 signaling might increase the efficacy and safety while expanding the therapeutic potential of these anticancer agents. Herein we describe FT-1518, a low nanomolar potent, kinase and PI3K sub family selective mTOR inhibitor. FT-1518 not only exhibited high oral bioavailability in preclinical species but has demonstrated excellent microsomal stability with no inhibitory activity towards undesired CYPs. FT-1518 showed high sustained tumor exposure and target Inhibition in a single oral dose xenograft model. FT-1518 depicted very good growth inhibitory activity across a large panel of hematologic and solid tumor cell lines with most activities falling into low nanomolar range. mTOR kinase inhibition in cells, by FT-1518, resulted in more potent inhibition of the mTOR pathway biomarkers (mTORC 1 & 2 biomarkers [pAkt(S473) and pS6(S240/244) or p70 S6K), no inhibition of PI3K biomarker [pAkt(T308)], and improved anti-proliferative activity as compared with rapamycin. FT-1518 exhibited dose-dependent and higher tumor growth inhibition (TGI) in multiple solid tumor xenografts compared with rapalogs and is poised to enter the clinic with a favorable toxicology profile.

Citation Format: Alain C. Mita, Monica M. Mita, Anthony D. William, Khalid Pasha, Chandra Siddamadappa, Kevin Zikaras, Felix T. Garzon. FT-1518, a new generation selective and potent mTORC1 and mTORC2 inhibitor: an in vitro and in vivo profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 137. doi:10.1158/1538-7445.AM2017-137
Abstract 1371: Isoprenylcysteine carboxylmethyltransferase (ICMT) function is critical for epithelial malignant transformation by mutant-Ras
Authors: Lau Hiu Yeung , Tang Jingyi , Casey Patrick J. , Wang Mei
Published: July 2017

Purpose Limited progress has been made in the development of direct inhibitors of Ras. Therefore, targeting isoprenylcysteine carboxylmethyltransferase (ICMT), the final enzyme in the prenylation pathway for Ras and other proteins containing the so-called CaaX-motif, remains an important strategy to inhibit Ras function in cancers. In this study, we investigate the role ICMT plays in the initiation of human epithelial cancers involving constitutively-active mutant Ras using genetic manipulation strategies.

Methods Small T antigen and a shRNA against p53 (shp53) were sequentially expressed in human mammary epithelial cells immortalized by hTERT (HME1-hTERT) to create pre-malignant HME1-shp53 cells. All 4 isoforms of wild-type or mutant Ras, namely H-Ras, N-Ras, K-Ras-4A and K-Ras-4B, were independently introduced into HME1-shp53 cells to determine their transforming ability. Using CRISPR-Cas9 genome editing technology, ICMT-null HME1-shp53 cells were generated and mutant Ras isoforms were subsequently introduced; tumor formation abilities of thus generated cells were determined using soft-agar anchorage-independent colony formation assay and subcutaneous xenografts in mice. ICMT knock-out was also performed in MiaPaCa-2 and MDA-MB-231 cells, both expressing mutant K-Ras, and subcutaneous xenograft tumor formation in mice was measured.

Results Pre-malignant HME1-shp53 cells can be transformed by all mutant Ras isoforms but not by any wild-type Ras. ICMT loss-of-function dramatically reduced anchorage-independent colony formation caused by all mutant Ras isoforms in HME1-shp53 cells, and in vivo xenograft formation was completely abolished. ICMT loss-of-function also dramatically reduced in vivo xenograft formation for mutant K-Ras-expressing MiaPaCa-2 and MDA-MB-231 naturally-occurring cancer cells.

Conclusion ICMT function is essential for the malignant transformation of HME1 cells by all isoforms of Ras. Furthermore, ICMT function is also crucial for the maintenance of the malignant phenotype in mutant K-Ras-expresing cells such as MiaPaCa-2 and MDA-MB-231 cells.

Citation Format: Hiu Yeung Lau, Jingyi Tang, Patrick J. Casey, Mei Wang. Isoprenylcysteine carboxylmethyltransferase (ICMT) function is critical for epithelial malignant transformation by mutant-Ras [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1371. doi:10.1158/1538-7445.AM2017-1371
Abstract 1373: The role of the ASH1L SET domain in leukemogenesis
Authors: Rogawski David , Miao Hongzhi , Kempinska Katarzyna , Chen Lili , Chase Jen , Friedman Ann , Yan Xiaomei , Nishioka Kenichi , Huang Gang , Ernst Patricia , Muntean Andy , Maillard Ivan , Cierpicki Tomasz , Grembecka Jolanta
Published: July 2017

ASH1L (absent, small, or homeotic-like 1) is a histone lysine methyltransferase (KMTase) specific for histone 3, lysine 36 that has an emerging role in multiple types of cancer. During development, ASH1L activates HOX genes and regulates self-renewal potential of long-term hematopoietic stem cells. Interestingly, HOX genes are oncogenic drivers in a variety of cancer types. For example, overexpression of HOXA9 is highly associated with a poor prognosis in acute myeloid leukemia (AML). In addition, ASH1L is overexpressed in a variety of cancers, including breast and thyroid cancer, but its role in cancer remains poorly characterized. Recent data suggest that ASH1L promotes leukemogenesis in mixed-lineage leukemia by recruiting the MLL epigenetic regulator to target genes. However, ASH1L is a large protein with multiple chromatin-interacting domains, and which regions of the ASH1L protein are important in leukemia has not been reported. Specifically, the role of ASH1L’s KMTase activity in leukemogenesis remains unknown.

To further investigate the function of ASH1L in leukemia, we knocked down expression of ASH1L in leukemia cell lines. We found that ASH1L knockdown slowed cell growth and caused a decrease in expression of HOXA genes and their collaborator MEIS1. To specifically investigate the role of ASH1L KMTase activity in leukemia, we used a mouse model with a targeted in-frame deletion of the ASH1L SET domain (ΔSET). ΔSET cells express a shorter version of ASH1L that does not contain the SET domain. We transduced bone marrow cells (BMCs) isolated from ΔSET and WT mice with several oncogenes relevant to AML to determine the role of the ASH1L SET domain in leukemic transformation. We found that ΔSET BMCs were significantly impaired in leukemic transformation compared to WT BMCs, as manifested by decreased colony-forming ability and reduced expression of HOXA genes. These findings suggest that the ASH1L SET domain plays an important role in leukemogenesis mediated by multiple different oncogenes. Future studies will further characterize ASH1L’s role in leukemia by identifying the complete set of genetic loci that it methylates and regulates, and will determine whether the ASH1L SET domain represents a viable therapeutic target.

Citation Format: David Rogawski, Hongzhi Miao, Katarzyna Kempinska, Lili Chen, Jen Chase, Ann Friedman, Xiaomei Yan, Kenichi Nishioka, Gang Huang, Patricia Ernst, Andy Muntean, Ivan Maillard, Tomasz Cierpicki, Jolanta Grembecka. The role of the ASH1L SET domain in leukemogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1373. doi:10.1158/1538-7445.AM2017-1373
Abstract 1375: Loss of C-MYC and chromatin acetylation induce epigenetic reprogramming in acute lymphoblastic leukemia
Authors: Costa Elodie M. Da , Armaos Gregory , Jacques-Ricard Simon , Beaudry Annie , St-Onge Pascal , Caron Maxime , Sinnett Daniel , McGraw Serge , Raynal Noël J.
Published: July 2017

Epigenetic modifications play a key role in establishing and maintaining gene expression. In cancer, they are highly altered and responsable of gene expression deregulation. Epigenetic drugs have the ability to reset cancer cell epigenome producing cancer cell differentiation and apoptosis. In a drug screening initiative, we recently reported a series of FDA-approved drugs with unsuspected epigenetic and anticancer activities. Here, we tested in a secondary screen the activity of these drugs against acute lymphoblastic leukemia (ALL) cell lines (MOLT-4 and NALM-6). We found that Proscillaridin A, a cardiac glycoside used for heart failure treatment, was the most active with IC50 values in the low nanomolar range, suggesting drug repositioning potential. Proscillaridin treatments induced a significant decrease in RNA and protein levels of C-MYC, a master oncogenic driver in ALL. Shortly after proscillaridin A treatment, C-MYC exhibited a 75% reduction in lysine acetylation, a post-translational modification known to prevent its degradation. Loss of acetylation was associated with down-regulation of lysine acetyltransferases CBP, P300 and Tip60, which also correlated with a reduction in histone 3 and 4 acetylation levels (H3K14ac, H3K9ac, H3K27ac and H4K5ac). Preliminary analysis with si-RNA experiments reveal that independent HAT activities are not responsible of C-MYC downregulation. RNA sequencing and gene set enrichment analysis in proscillaridin-treated ALL cells (5 nM for 48h) showed that genes associated with cell differentiation and apoptosis pathways were up-regulated whereas down-regulated genes were associated with C-MYC target genes. Altogether, our findings show that acetylation through lysine acetyltransferase down-regulation simultaneously induces loss of C-MYC and H3 acetylation leading to epigenetic reprogramming in ALL cells. This drug repositioning strategy, using proscillaridin A, has the potential to reprogram cancer cells that are driven by MYC overexpression or hyperactivation.

Citation Format: Elodie M. Da Costa, Gregory Armaos, Simon Jacques-Ricard, Annie Beaudry, Pascal St-Onge, Maxime Caron, Daniel Sinnett, Serge McGraw, Noël J. Raynal. Loss of C-MYC and chromatin acetylation induce epigenetic reprogramming in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1375. doi:10.1158/1538-7445.AM2017-1375
Abstract 1377: Set7 is a novel histone methyltransferase in Schizosaccharomyces Pombe
Authors: Luccio Eric Di , Shen Yunpeng , Mevius Damiaan E.H.F , Noh Yeon Jeong , Kim Jihyeon , Morishita Masayo
Published: July 2017

Dynamics and plasticity of chromatin regulation is mediated by a molecular ballet of writers, readers and erasers of epigenetic marks on both DNA and histones. The pattern of histone modifications may define a histone code, which is part of intricate networks that ultimately regulate transcriptional events. Dysfunction of histone methylation affects chromatin regulation and is involved in an increasing number of pathologies from cellular transformation to tumor progression and other diseases. However, histone methyltransferase (HMTase) pathways remain to be further explored and better understood.

Fission yeast (Schizosaccharomyces pombe) is an ideal model organism to investigate the fundamental mechanisms of chromatin dynamics. Fission yeast possesses 13 SET-containing proteins (Set1 to 13), several of which methylate histones or ribosomes. The catalytic SET domain is highly conserved across eukaryotes (e.g. Set2, a homologue of the oncoprotein NSD2/MMSET/WHSC1 in human). Structure-function studies of HMTases are essential from basic science to translational research. However, full-length structures of fission yeast methyltransferases are still scarce.

Here, in a bid to better understand histone methylation and to gain insight for drug-design, we report the identification and structural characterization of a novel histone methyltransferase Set7 (SPCC297.04c), in Schizosaccharomyces Pombe. In this study, we investigated Sp.Set7’s cellular localization and the effect of Sp.Set7 knock-out on the cell cycle and sporulation. Next, we elucidated Sp.Set7’s Lysine substrate specificity on histone H3 and H4 by biochemical assays and mass spectrometry. Finally, we solved the X-ray structure of the apo Sp.Set7 at 2.0 Å resolution.

In summary, we report the biochemical and structural characterization of a novel histone methyltransferase in fission yeast, which has implications for better understanding the fundamental mechanism of HMTases.

Citation Format: Eric Di Luccio, Yunpeng Shen, Damiaan E.H.F Mevius, Yeon Jeong Noh, Jihyeon Kim, Masayo Morishita. Set7 is a novel histone methyltransferase in Schizosaccharomyces Pombe [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1377. doi:10.1158/1538-7445.AM2017-1377
Abstract 1381: Human CRL4DDB2 E3 ubiquitin ligase regulates post-repair chromatin restoration of epigenetic marker H3K56Ac through recruitment of histone chaperon CAF-1
Authors: Zhu Qianzheng , Sharma Nidhi , He Jinshan , Wani Gulzar , Wani Altaf A.
Published: July 2017

Epigenetic marker acetylated histone H3 lysine 56 (H3K56Ac) diminishes as an early DNA damage response but is restored in chromatin after DNA repair. Here, we report that human CRL4DDB2 ubiquitin ligase regulates the DNA repair-driven chromatin restoration of H3K56Ac by monitoring the recruitment of histone chaperon CAF-1. We show that the H3K56Ac accumulates at DNA damage sites. The post-repair H3K56Ac restoration depends on CAF-1 and CBP/p300 function. The CRL4DDB2 components DDB1, DDB2 and CUL4A are required not only for maintaining the H3K56Ac level in chromatin, but also for restoring H3K56Ac following the repair of DNA photolesions and strand breaks. Depletion of CUL4A decreases the recruitment of CAF-1 p60 and p150 to ultraviolet radiation (UVR)- and phleomycin-induced DNA damage. Inhibition of neddylation renders CRL4DDB2 inactive and concomitantly diminishes the recruitment of CAF-1 and prevents post-repair H3K56Ac restoration. Additionally, a PCNA-interacting protein (PIP) motif is shown to exist within DDB2. The mutation in the PIP box of DDB2 compromises its capability to maintain the H3K56Ac level but does not affect CRL4DDB2-mediated XPC ubiquitination. These results suggest a novel role of CRL4DDB2 in regulating post-repair chromatin restoration, which differs from its canonical role in the process of nucleotide excision repair.

Citation Format: Qianzheng Zhu, Nidhi Sharma, Jinshan He, Gulzar Wani, Altaf A. Wani. Human CRL4DDB2 E3 ubiquitin ligase regulates post-repair chromatin restoration of epigenetic marker H3K56Ac through recruitment of histone chaperon CAF-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1381. doi:10.1158/1538-7445.AM2017-1381
Abstract 1386: Effects of histone deacetylase inhibitors on differentiation markers and growth of colon and bladder cancer cells
Authors: Lea Michael A. , Cué Lauren , Batista Elizabeth , Lew Erik , desBordes Charles
Published: July 2017

Alkaline phosphatase and dipeptidyl peptidase are markers of differentiation in colon cancer cells. In colon cancer cells differentiation can often be induced by treatment with inhibitors of histone deacetylase (HDAC) activity. In some colon cancer cells butyrate causes a several-fold induction of alkaline phosphatase but only modest effects on dipeptidyl peptidase. There is relatively little information on changes in activity of cell surface hydrolases in bladder cancer. However, there is some evidence for decreased alkaline phosphatase activity in bladder cancer. We tested the hypothesis that there may be retention of activity in more slowly growing cancer cells and the activity will be regulated by HDAC inhibitors in a manner similar to that in colon cancer. In our initial studies we screened seven human bladder cancer cell lines for alkaline phosphatase activity. We identified three cell lines (5637, HT1197 and HT1376) with activity comparable to that in more differentiated colon cancer cell lines. The response to incubation with the HDAC inhibitors butyrate and valproate was compared with effects in three colon cancer cell lines Caco-2, HT29 and SW1116. In all six cell lines there were growth inhibitory effects that tended to be a little greater with butyrate than with valproate. Induction of alkaline phosphatase activity was generally greater with butyrate than with valproate and was apparent even without normalizing the activity on a protein basis. After determining the activity per unit protein there were increases in dipeptidyl peptidase activity after treatment with the HDAC inhibitors that were smaller than for alkaline phosphatase. Growth inhibition was observed with all the HDAC inhibitors examined including LMK235, nicotinamide, RGFP966, romidepsin and tubacin but increased activity of alkaline phosphatase was not always seen. Activities of the enzymes were very low in CRL1790 human colonocytes of fetal origin and there was little or no induction with HDAC inhibitors. Although pyruvate and carnitine have been reported to be HDAC inhibitors, incubation with colon and bladder cancer cells did not cause induction of alkaline phosphatase activity. It was concluded that regulation of alkaline phosphatase and dipeptidyl peptidase by HDAC inhibitors can be similar in bladder and colon cancer cells.

Citation Format: Michael A. Lea, Lauren Cué, Elizabeth Batista, Erik Lew, Charles desBordes. Effects of histone deacetylase inhibitors on differentiation markers and growth of colon and bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1386. doi:10.1158/1538-7445.AM2017-1386