bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3463: Crosstalk between PKCalpha and TGF-beta signaling suppresses inhibitor of DNA binding 1 (Id1) expression in intestinal epithelial cells
Authors: Li Xinyue , Lum Michelle , Black Adrian , Black Jennifer
Published: August 2018

Inhibitor of DNA binding (Id) proteins (Id1-Id4) are dominant negative antagonists of basic helix-loop-helix (bHLH) transcription factors with recognized functions in development, stem cell self-renewal, and cell fate determination. Recent studies have identified Id proteins as master regulators of cancer-initiating cells and tumor aggressiveness, with critical roles in regulation of central hallmarks of cancer, including cell proliferation, survival, senescence, angiogenesis, migration, metastasis, and chemoresistance. In colorectal cancer (CRC), Id1 overexpression is associated with aggressive clinical behavior and poor prognosis, while decreasing sensitivity to chemotherapeutic agents. Protein kinase C α (PKCα) is a serine/threonine kinase that functions as a tumor suppressor in CRC. PKCα signaling induces cell cycle arrest in nontransformed IEC-18 intestinal epithelial cells via an ERK-dependent mechanism. A major target of PKCα negative control in these cells is Id1. To further characterize mechanisms underlying the tumor-suppressive effects of PKCα, we modulated PKCα expression and activation in CRC cells and nontransformed IEC-18 cells and performed RNA-seq analysis to identify changes in gene expression. This analysis identified transforming growth factor beta receptor 1 (TGFβR1) as an mRNA upregulated by PKCα signaling. Subsequent RT-PCR and Western blot analysis confirmed that PKCα upregulates TGFβR1 at both the mRNA and protein levels. The MEK inhibitors U0126 and PD98059 blocked PKCα upregulation of TGFβR1, pointing to an ERK-dependent mechanism. Notably, inhibition of TGFβR1 with GW788388 prevented PKCα-mediated Id1 downregulation, linking TGF-β signaling to the tumor-suppressive effects of PKCα. Further studies indicated that TGFβR1 activates a p38-mediated noncanonical pathway to downregulate Id1 in response to PKCα activity. Collectively, these findings identify a PKCα→Erk→TGFβR1→p38→Id1 signaling axis in intestinal epithelial cells. Both PKCα and TGF-β function as tumor suppressors in the intestinal epithelium and both pathways are inactivated during intestinal tumor progression. By providing a direct link between two tumor-suppressive signaling pathways with established effects on intestinal tumorigenesis, our findings broaden our understanding of CRC and open avenues for potential novel therapeutic approaches to this disease. (Supported by NIH grants DK60632, CA54807, CA036727 and CA191894.)

Citation Format: Xinyue Li, Michelle Lum, Adrian Black, Jennifer Black. Crosstalk between PKCalpha and TGF-beta signaling suppresses inhibitor of DNA binding 1 (Id1) expression in intestinal epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3463.
Abstract 3465: Biased signaling downstream of epidermal growth factor receptor regulates proliferative versus apoptotic response to ligand
Authors: Ali Remah , Brown Wells , Davisson Jo , Wendt Michael
Published: August 2018

Inhibition of EGFR signaling by small molecule kinase inhibitors and monocloncal antibodies has proven effective in the treatment of multiple metastatic cancers. In contrast, metastatic breast cancer (BC) derived from EGFR-driven mammary tumors is inherently resistant to EGFR-targeted therapies. Mechanisms that contribute to this inherent resistance remain poorly defined. Here we show that in contrast to primary tumors, ligand-mediated activation of EGFR is dominated by signaling transducer and activator of transcriptional 1 (STAT1) signaling in metastatic BC. This change in downstream signaling leads to apoptosis and growth inhibition in response to EGF in metastatic BC cells. Mechanistically, these changes in downstream signaling result from an increase in the internalized pool of EGFR in metastatic cells, increasing physical access to STAT1. Along these lines, an EGFR mutant that is defective in endocytosis is unable to elicit STAT1 phosphorylation and apoptosis. Additionally, inhibition of endosomal EGFR signaling using an EGFR inhibitor linked to a nuclear localization signal specifically prevents EGF-induced STAT1 phosphorylation and cell death without affecting EGFR:MAPK signaling. Pharmacologic blockade of MAPK signaling through the use of the MEK1/2 inhibitor trametinib led to a dramatic increase in EGF-mediated STAT1 phosphorylation and enhanced EGF-induced apoptosis in metastatic BC cells. Importantly, use of a trametinib:EGF combination also facilitated the apoptotic switch in EGFR function in primary tumor cells, but not normal mammary epithelial cells. These studies reveal a fundamental switch in EGFR in metastatic BC. Furthermore, they demonstrate that pharmacological biasing EGFR signaling toward STAT1 activation is capable of revealing the apoptotic function of this critical pathway.

Citation Format: Remah Ali, Wells Brown, Jo Davisson, Michael Wendt. Biased signaling downstream of epidermal growth factor receptor regulates proliferative versus apoptotic response to ligand [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3465.
Abstract 3470: Ibrutinib and sorafenib synergistically inhibit HCC growth in preclinical models
Authors: Ghoshal Kalpana , Lin Cho-hao , Elkholy Khadija , Wani Nissar A. , Li Ding , Barajas Juan M. , Hu Peng , Zhang Xiaoli , Yu Lianbo , Motiwala Tasneem
Published: August 2018

Hepatocellular carcinoma (HCC) is the second major cause of cancer-related death worldwide with limit therapeutic options. Thus, there is an urgent need to develop novel alternative therapies for HCC. In this study, we report that ibrutinib, recently approved for the treatment of B cell malignancies, and a covalent inhibitor of TEC (BTK, ITK etc.) and ERBB (EGFR, Her2 etc.) family of tyrosine kinases, inhibits tumorigenic functions of human HCC cells in vitro and in xenografts. More importantly, co-treatment with ibrutinib and sorafenib, an approved targeted therapy for advanced HCCs that marginally improve patients' survival, synergistically inhibited proliferation and clonogenic survival of HCC cells including those with acquired sorafenib resistance. Mechanistically, ibrutinib inhibits Akt and ERK signaling pathways through inactivating EGFR, its irreversible substrate in HCC cells. Besides, tumor sphere formation and expression of cancer stem cell markers were suppressed by ibrutinib and sorafenib co-treatment in HCC cells. Ectopic expression of the constitutively active Akt mutant abrogated the synergism of these two kinase inhibitors on HCC cell survival. Ibrutinib and sorafenib combination therapy significantly suppressed tumor growth of highly aggressive HCCLM3 subcutaneous xenografts in NSG mice. Collectively, these results demonstrate that ibrutinib could be a re-purposed anti-HCC drug, and our data provides the evidence for the therapeutic potential of ibrutinib and sorafenib combination as an effective and attractive strategy for treating HCCs including those with sorafenib resistance.

Citation Format: Kalpana Ghoshal, Cho-hao Lin, Khadija Elkholy, Nissar A. Wani, Ding Li, Juan M. Barajas, Peng Hu, Xiaoli Zhang, Lianbo Yu, Tasneem Motiwala. Ibrutinib and sorafenib synergistically inhibit HCC growth in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3470.
Abstract 3473: Investigating RAS and RTK signaling in MPNST progression and drug resistance
Authors: King Candace N. , Graveel Carrie , Steensma Matthew
Published: August 2018

Neurofibromatosis, type 1 (NF1) is a tumor predisposition syndrome affecting about 1 in 2,500 live births in the United States. The protein product of the NF1 gene, neurofibromin, loses its function as a negative regulator of the RAS protein, leading to the development of painful and disfiguring benign tumors known as a neurofibromas. Eight to thirteen percent of these neurofibromas will transform into malignant peripheral nerve sheath tumors (MPNSTs). MPNSTs are aggressive soft tissue sarcomas that do not respond to the standard sarcoma treatment of chemotherapy and surgical resection. There is a critical need for efficacious targeted therapy to increase the average life span of these patients. Current targeted therapy efforts have shown modest efficacy clinically due to the establishment of compensatory feedback mechanisms that propagate tumor progression and recurrence. These feedback mechanisms are not completely understood in MPNSTs. In lung cancer, inhibition of MEK in the RAS pathway has been shown to activate upstream receptor tyrosine kinases (RTK). Genomic studies of MPNST samples have shown that 30% of tumors have amplification of the RTKs MET and EGFR. Furthermore, in a study of the genomic progression of a NF1 patient pre- and post-treatment, we found amplification of MET and EGFR that increased following treatment. In a study on triple-negative breast cancer cells, it has been shown that MET and EGFR crosstalk may be responsible for the reactivation of the inhibited cancer pathways and drug resistance. Therefore, we hypothesize that coordinated deregulated RAS signaling and MEK inhibition leads to MET and EGFR amplification, expression, activation, and crosstalk, which drives MPNST progression, drug resistance, and tumor recurrence. To test our hypothesis, we will use both in vitro and in vivo experimental methods to pursue the following specific aims: (1) determine the effect of MEK inhibition on establishing compensatory feedback loops that increase RTK activation and downstream signaling, (2) determine the role of MET-EGFR crosstalk signaling in MPNST tumor progression and resistance to kinase inhibition, and (3) determine the efficacy of combination RTK and MEK inhibition in vivo.

Citation Format: Candace N. King, Carrie Graveel, Matthew Steensma. Investigating RAS and RTK signaling in MPNST progression and drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3473.
Abstract 3488: Elucidation of Warburg effect and cell cycle regulations through analysis of cancer tissue omics data and kinetic modeling
Authors: Sheng Tao
Published: August 2018

Cancer cells tend to depend more on anerobic glycolysis to generate adenosine 5'- triphosphate (ATP), which is an inefficient energy producing process termed as “The Warburg effect”. The causes or functions of Warburg effect have been extensively studied, however, the growth advantage it confers to cancer cells still remains unclear. In this study, we propose that the metabolic shift in cancer cells would facilitate the accumulation of nutrients and biomass (e.g., nucleotides, amino acids and lipids), which would promote the cell cycle initiation and progression as unicellular organisms do. Specifically, by comprehensively analyze the gene expression and mutational profile in 16 cancer types, we identified (1) the gene expression profile of cancer cells is evolutionarily more unicellular like when compared to normal; and (2) certain cell cycle associated mutations are associated with the metabolic alternations in cancer cells, such as nucleotide synthesis pathways and lipids metabolism. Moreover, we developed a system of kinetic equations to capture the metabolic relations between nutrient metabolism and cell cycle initiation and progression. The kinetic model shows that the rapid nucleotide synthesis as well as high influx of glutamate would affect the activities of cell cycle signaling and CDKs genes. Our study provides a novel mechanistic view in linking the Warburg effect and cancer cell cycle initiation and progression.

Citation Format: Tao Sheng. Elucidation of Warburg effect and cell cycle regulations through analysis of cancer tissue omics data and kinetic modeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3488.
Abstract 35: Triple-negative breast cancer cells alter the pulmonary landscape to favor metastasis via exosome-mediated release of NM23
Authors: Duan Suzann , Nordmeier Senny , Byrnes Aidan E. , Brown Miles A. , Buxton Iain L.
Published: August 2018

There is strong evidence implicating exosomes, cell-derived nanoscale vesicles, and their protein cargo in facilitating dialogue between primary tumor cells and the premetastatic niche. Identified by our lab as one such protein, secreted nucleoside diphosphate kinase (NM23/NDPK) has been shown to activate endothelial remodeling in support of proangiogenic and prometastatic events. We have previously demonstrated that triple-negative breast cancer (MDA-MB-231) cells elaborate exosomes enriched with NDPK transphosphorylase activity and target the lung as a future site of metastasis. Using in vitro and in vivo approaches, we interrogate the functional role of NDPK in exosome-mediated lung remodeling. In cell-based assays, we demonstrate that MDA-MB-231 exosomes stimulate migration of pulmonary vascular endothelial cells and enhance monolayer permeability. These effects are reversed following treatment with an inhibitor of NDPK activity or an antagonist to the purinergic P2Y1 receptor. Further, we show that immune-compromised mice receiving intravenous injections of MDA-MB-231 exosomes exhibit enhanced pulmonary vascular leakage, whereas treatment with an inhibitor of NDPK attenuates this effect. Using tandem mass tag labeling and mass spectrometry, we profiled proteomic changes to the lung and identified the purinergic signaling pathway as among those significantly affected by exosome treatment. These results elucidate the functional role of exosomal NDPK in compromising vascular integrity and transforming the pulmonary landscape to support colonization by circulating breast cancer cells.

Citation Format: Suzann Duan, Senny Nordmeier, Aidan E. Byrnes, Miles A. Brown, Iain L. Buxton. Triple-negative breast cancer cells alter the pulmonary landscape to favor metastasis via exosome-mediated release of NM23 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 35.
Abstract 3500: A pipeline to identify driver mutations
Authors: Chakroborty Deepankar , Kurppa Kari , Paatero Ilkka , Elo Laura , Elenius Klaus
Published: August 2018

Introduction: Cancer tissues harbor thousands of mutations while the current list of clinically validated actionable variants contains only about a dozen genetic markers. Most of the somatic mutations in cancer are expected to be inconsequential passenger mutations that reflect the general instability of the tumors. The discovery of most of the currently known driver mutations has been facilitated by their accumulation in mutation hot-spots within their respective genes. However, a great majority of mutations in cancer tissues are rare and no information is currently available about their functional significance. Several lines of in vitro and in vivo clinical evidence also indicate that there is a significant number of, as yet unidentified, activating driver mutations that could serve as predictive markers for oncology.

Method: To identify functional driver mutations, we have established a functional genetics screen based on expression of random gene variants and the ability of driver mutations to promote a growth-advantage in vitro, as compared to passenger mutations. The screen was set up using a randomly mutated expression library encoding thousands of variants of epidermal growth factor receptor (EGFR), a well-known oncogene, as a model. The library was retrovirally introduced into murine lymphoid Ba/F3 cells, that normally require interleukin-3 (IL-3) for survival but can exploit ectopic expression of activated variants of oncogenic kinases to compensate for the deficiency of exogenous IL-3. While expression of wild-type EGFR did not promote IL3-independent survival of the Ba/F3 cells, as expected, transduction of the mutant EGFR library did, indicating that the surviving Ba/F3 pool included EGFR mutations with ligand-independent activity. To identify and quantify the frequency of these mutations, targeted next-generation sequencing of the EGFR inserts was carried out.

Results: Using the method we were able to identify approximately 20 candidate activating mutations out of the 7000 random EGFR mutations in the original library. The 20 candidates included the well-known activating EGFR mutation, L858R, validating the pipeline. A previously unidentified activating mutation was also found, and the growth promoted by the mutant was susceptible to inhibition by clinically available EGFR inhibitors.

Citation Format: Deepankar Chakroborty, Kari Kurppa, Ilkka Paatero, Laura Elo, Klaus Elenius. A pipeline to identify driver mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3500.
Abstract 3501: Adipocytes sustain pancreatic cancer progression through a non-canonical WNT paracrine network inducing ROR2 nuclear shuttling
Authors: Carbone Carmine , Piro Geny , Santoro Raffaela , Simionato Francesca , Merz Valeria , Zecchetto Camilla , Scarpa Aldo , Tortora Giampaolo , Melisi Davide
Published: August 2018

Background: Solid epidemiological evidences connect obesity with incidence, stage, and survival in pancreatic cancer. However, the underlying mechanistic basis linking adipocytes to pancreatic cancer progression remain largely elusive. We hypothesized that factors secreted by adipocytes could be responsible for epithelial-to-mesenchymal transition (EMT) induction and, in turn, a more aggressive phenotype in models of pancreatic preneoplastic lesions.

Methods: We studied the role of factors secreted by two adipogenic model systems from primary human Bone Marrow Stromal Cells (hBMSC) in an in vitro experimental cell transformation model system of human pancreatic ductal epithelial (HPDE) cell stably expressing activated KRAS (HPDE/KRAS).

Results: We measured a significant induction of EMT and aggressiveness in HPDE and HPDE/KRAS cell lines when cultured with medium conditioned by fully differentiated adipocytes (ADIPOCM) if compared with the same cells cultured with medium conditioned by hBMSC (hBMSCCM) from two different healthy donors. Several genes coding for soluble modulators of the non-canonical WNT signaling pathway, including FRZB, SFRP2, RSPO1, WNT5A and 5B were significantly overexpressed in fully differentiated adipocytes than in their respective in hBMSC. ADIPOCM induced the overexpression and the nuclear translocation of the Frizzled family member Receptor tyrosine kinase-like orphan receptor (Ror) 2 in HPDE and HPDE/KRAS cells. Vantictumab, an anti-Frizzled monoclonal antibody, reduced ROR2 nuclear translocation and in turn the EMT and aggressiveness in HPDE and HPDE/KRAS cells.

Conclusions: We demonstrated that adipocytes could induce EMT and aggressiveness in models of pancreatic preneoplastic lesions by orchestrating a complex paracrine signaling of soluble modulators of the non-canonical WNT signaling pathway that determine, in turn, the activation and nuclear translocation of ROR2. This signaling pathway could represent a novel target for pancreatic cancer chemoprevention. Most importantly, these factors could serve as novel biomarkers to

select a risk population among obese subjects for screening and, thus, early diagnosis of pancreatic cancer.

Citation Format: Carmine Carbone, Geny Piro, Raffaela Santoro, Francesca Simionato, Valeria Merz, Camilla Zecchetto, Aldo Scarpa, Giampaolo Tortora, Davide Melisi. Adipocytes sustain pancreatic cancer progression through a non-canonical WNT paracrine network inducing ROR2 nuclear shuttling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3501.
Abstract 3502: Chemoproteomics provide insights into cell surface reprogramming during KRAS-mediated transformation
Authors: Nandagopal Neethi , Aubert Léo , Nourreddine Sami , Lavoie Geneviève , Roux Philippe
Published: August 2018

KRAS is among the most frequently mutated genes driving human cancers, including ~45% of colorectal cancers (CRC). Despite decades of research, mutant KRAS is considered undruggable, and is frequently associated with resistance to chemotherapy and other targeted therapies. While the effect of mutant KRAS on regulating intracellular signaling events is well studied, much less is known about its impact on the cell surface proteome (surfaceome), which constitutes the predominant therapeutic target for FDA-approved drugs. To study the cell surfaceome, we optimized a chemoproteomics approach that selectively isolates cell surface proteins and allows their relative quantification using mass spectrometry. When comparing normal intestinal cells to their counterparts expressing oncogenic KRAS (G12V), we identified 104 (~13%) and 170 (~22%) cell surface proteins that were significantly upregulated or downregulated, respectively, in KRAS-transformed cells. Overall, a myriad of proteins, including receptors, solute carriers, adhesion molecules, etc., were modulated by mutant KRAS, and their potential roles in KRAS-mediated transformation are currently being studied. We also found that most of the cell surface proteins were altered in response to transcriptional changes induced by KRAS signaling, and due to increased global endocytosis in KRAS-mutated cells. Our study suggests potential cell surface proteins that could be targeted in KRAS-mutant cells, which will be verified in subsequent studies using human CRC models. Our work helps assemble a global view of how oncogenic KRAS alters the spatial distribution of several cell surface proteins, and thereby potentially impacts tumor functions and therapeutic response.

Citation Format: Neethi Nandagopal, Léo Aubert, Sami Nourreddine, Geneviève Lavoie, Philippe Roux. Chemoproteomics provide insights into cell surface reprogramming during KRAS-mediated transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3502.
Abstract 351: Epigenetic mechanisms regulate DNA amplification and drug-resistant gene selection
Authors: Mishra Sweta , Rechem Capucine Van , Chakraborty Damayanti , Whetstine Johnathan R.
Published: August 2018

DNA copy gains and amplifications are often associated with tumorigenesis, poor prognosis and drug resistance. Little is known about how these regions of the genome are selected for amplification. Our group discovered that modulation of epigenetic factors and their associated chromatin states control DNA copy gains of drug-resistant regions. In fact, transient site-specific copy number gains (TSSGs) occur in both normal and cancer cells upon manipulation of a histone H3 lysine 9/36 (H3K9/36) tri-demethylase KDM4A. However, the mechanism by which KDM4A is targeted to specific genomic regions and whether additional chromatin regulators are involved in this process were unknown. To begin addressing these questions, we performed a series of unbiased genetic screens against all the histone lysine-modifying enzymes and followed their ability to generate copy gains of regions within chromosome 1. Our screen uncovered insights into both KDM4A targeting and the identification of other epigenetic factors modulating DNA amplification of drug-resistant regions of the genome. We have uncovered KDM4A-independent DNA amplifications, which established that additional regions are undergoing epigenetic control for amplification. These currently unpublished data will be discussed at the meeting.

Citation Format: Sweta Mishra, Capucine Van Rechem, Damayanti Chakraborty, Johnathan R. Whetstine. Epigenetic mechanisms regulate DNA amplification and drug-resistant gene selection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 351.
Abstract 3516: Discovery of novel SHMT small molecule inhibitors for cancer treatment
Authors: Bartosik Anna , Guzik Pawel , Sowinska Marta , Gluza Karolina , Krol Marcin , Wrobel Anna , Dreas Agnieszka , Iwanska Faustyna , Zastawna Magdalena , Kulesza Urszula , Boutard Nicolas , Schultz David , Wujkowska Justyna , Pyziak Karolina , Sroka-Porada Agnieszka , Przybylowicz Agnieszka , Adamus Agnieszka , Sieprawska-Lupa Magdalena , Golik Przemyslaw , Kowalczyk Piotr , Brzozka Krzysztof , Rzymski Tomasz , Nowak Mateusz
Published: August 2018

Over-activation of the serine synthesis pathway, upregulation of SHMT2 has been described in over 20% of solid tumors (e.g. breast, lung, colorectal cancers). Such cancer cells are highly dependent on serine. Serine hydroxymethyltransferase (SHMT) plays a key role in a so-called one-carbon pathway, a group of biochemical reactions involved in amino acid metabolism. SHMT catalyzes the conversion of serine to glycine and also plays a role in the folate (vitamin B9) cycle. Antagonists of folate metabolism or antifolates are an established chemotherapy in certain cancers. Folate antagonism disrupts cell division, DNA/RNA synthesis and protein synthesis. Pemetrexed (for non-small cell lung carcinoma, mesothelioma) and methotreaxate (for autoimmune conditions like rheumatoid arthritis and certain cancers) are two well established and effective antifolates. The main drawback with antifolates in cancer treatment, however, is the development of resistance. In this study we report development of a series of small molecule SHMT1/2 inhibitors. Synthetized compounds exert potency in SHMT1/2 biochemical assay as well as in cellular assay (measured by the C13 serine to glycine conversion) with the low nanomolar range. Therapeutic effect of the compounds was investigated in the panel of cancer cell lines with different genetic background as well as with different SHMT2 levels. We identified several cell lines in which tested compounds inhibited cancer cell grow with nM GI50 values. Taken together, presented data supports our rationale for using SHMT1/2 inhibitors as a novel and interesting approach for the cancer therapy.

Citation Format: Anna Bartosik, Pawel Guzik, Marta Sowinska, Karolina Gluza, Marcin Krol, Anna Wrobel, Agnieszka Dreas, Faustyna Iwanska, Magdalena Zastawna, Urszula Kulesza, Nicolas Boutard, David Schultz, Justyna Wujkowska, Karolina Pyziak, Agnieszka Sroka-Porada, Agnieszka Przybylowicz, Agnieszka Adamus, Magdalena Sieprawska-Lupa, Przemyslaw Golik, Piotr Kowalczyk, Krzysztof Brzozka, Tomasz Rzymski, Mateusz Nowak. Discovery of novel SHMT small molecule inhibitors for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3516.
Abstract 352: DNA replication licensing factor MCM5 is a target for SETD8 mediated methylation
Authors: Pittella Fabio , Blum Gil , Zhu Yongxia , Senevirathne Chamara , Luo Minkui
Published: August 2018

SETD8 methyltransferase activity is implicated in several fundamental cellular processes such as transcriptional regulation and heterochromatin formation as well as processes that ensures genomic stability including DNA replication and the DNA damage response. It has been proposed that SETD8 affects DNA replication as a positive regulator of origin licensing through H4K20 methylation and by supporting Okazaki fragment processing through PCNA methylation. However, there is no evidence whether other key element in the replication machinery is directly modified by SETD8. To address this question, we used a combination of SILAC-based BPPM (Bioorthogonal Profiling of Protein Methylation) to profile new substrates for SETD8. We genetically engineered SETD8 and identified mutants amenable to accommodate non-native SAM analogues containing a terminal alkyne moiety for click chemistry. The engineered SETD8 can transfer this unique chemical moiety into target proteins for subsequent identification of the modified substrates. Stable cell lines expressing catalytically active or catalytically dead SETD8 mutants were systematically cultured with distinct stable isotope labeled amino acids allowing quantitative proteomic analysis of the identified substrates. With this method we could validate previously known substrates of SETD8 and identify novel targets, including MCM5, a subunit of the hexameric minichromosome maintenance (MCM) DNA helicase complex. MCM5 directly interacts with MCM2 and 3 to form the MCM2-7 hexamer which associates with the origins of DNA replication to form part of the pre-replicative complex (preRC), playing a major role during replication initiation and elongation. We found that SETD8 mediated methylation of MCM5 directly affects its binding affinity to MCM2 and 3. Amino acid substitutions at the methylated lysine further evidenced a stronger binding to its interacting partners contributing to the formation of the MCM hexamer. In addition, CRISPR based modifications on MCM5 methylated lysine affected the replication process resulting in defective cell cycle progression. Taken together our results indicate that MCM5 methylation contributes to the assembly of the MCM complex revealing a novel role for SETD8 in replication initiation. Our findings brings new perspectives on the biological importance of SETD8 during DNA replication.

Citation Format: Fabio Pittella, Gil Blum, Yongxia Zhu, Chamara Senevirathne, Minkui Luo. DNA replication licensing factor MCM5 is a target for SETD8 mediated methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 352.
Abstract 353: Using cancer-associated variants to dissect functions of SETD8
Authors: Zhu Yongxia , Wang Lihui , Senevirathne Chamara , Chen Shi , Silva Fabio Pittella , Luo Minkui
Published: August 2018

Protein lysine methyltransferases (PKMTs) play important roles in carcinogenesis through methylation of histone and non-histone substrates. SETD8 is the sole human methyltransferase for the monomethylation of histone H4 Lys20 (H4K20me1). Besides histone substrates, SETD8 modifies multiple non-histone substrates including proliferating cell nuclear antigen (PCNA), Numb, and p53. The biological functions related to the methyltransferase activity of SETD8 have been implicated in many essential biological processes including DNA replication, DNA damage response, cell cycle regulation, and transcription modulation. The SET domain of SETD8 is required to catalyze the methylation in substrates using the cofactor S-adenosyl-L-methionine (SAM). The C-terminal region post the SET domain (cSET) contains both the cofactor and protein substrate-binding sites. Recent genomics data from samples of cancer patients have revealed distinct variants in the SET domain of SETD8. Missense mutations, such as, E289Q, R292L, V315I, and H347Q were found in multiple types of cancers. To help dissect the roles of SETD8, we proposed the identification and characterization of SETD8 variants with partial loss-of-function phenotypes. Our data showed that overexpression of the SETD8 mutants increased the migration and tumor sphere formation of breast cancer cells without affecting H4K20me1. In vitro biochemical assay also confirmed that the SETD8 mutants lose the methyltransferase activity on H4K20 substrate. The RNA-seq data in the context of SETD8 mutant indicated that the c-Myc pathway was upregulated without affecting c-Myc expression. With the technology of Bioorthogonal Profiling of Protein Methylation (BPPM), we have found that SETD8 can methylate cancer-associated transcription factors in a cellular context. Collectively, these findings suggest that native SETD8 can act on cancer driving-associated transcription factors and its mutant maintains this methyltransferase activity but spare H4K20 methylation for cancer malignancy.

Acknowledgements: This project was supported by 1R01GM120570.

Citation Format: Yongxia Zhu, Lihui Wang, Chamara Senevirathne, Shi Chen, Fabio Pittella Silva, Minkui Luo. Using cancer-associated variants to dissect functions of SETD8 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 353.
Abstract 3533: Elevated expression of heme related proteins in viable tumor cells treated with vascular disrupting agent, combretastatin A-4 phosphate in xenograft models of NSCLC
Authors: Dey Sanchareeka , Kumari Sharda , Kalainayakan Sarada Preeta , Ghosh Poorva , Zhang Li , Liu Li
Published: August 2018

Tumors cannot grow without a supply of oxygen and nutrients. Hence, targeting tumor vasculature with Vascular Disrupting Agents (VDAs) represents a promising therapeutic strategy, especially for the treatment of solid tumors. However, VDAs cause central necrosis, leaving a rim of viable tumor cells that often eventually lead to tumor recurrence and development of resistance. Here, we aim to better understand the underlying molecular mechanisms leading to the acquisition of VDA-resistance and its correlation with the extent of hypoxia and respiratory functions.

A well-known VDA, Combretastatin A-4 phosphate (CA4P), was used for the study. First, a NSCLC cell line expressing luciferase was implanted into the right flanks of female SCID mice. Next, CA4P was administered intraperitoneally. The effect was monitored 3 and 24 hours post injection of CA4P through BLI (Perkin Elmer's IVIS Lumina III) after subcutaneous injection of luciferin. After sacrificing the mice, the tumors were harvested, formalin-fixed, and paraffin-embedded for Immunohistochemistry (IHC).

Our BLI data indicated significant decrease in radiance 3 hour post treatment (total flux in photons per second) which remained so at 24 hours, when luciferin was re-administered. Our IHC data indicated enhanced levels of enzymes involved in heme biosynthesis, uptake and degradation, and hemoproteins involved in oxidative phosphorylation. Furthermore, we examined the effect of a mitochondrial targeting agent (MTA) that undermined the expression of these proteins, on the progression of subcutaneous tumor xenografts.

Citation Format: Sanchareeka Dey, Sharda Kumari, Sarada Preeta Kalainayakan, Poorva Ghosh, Li Zhang, Li Liu. Elevated expression of heme related proteins in viable tumor cells treated with vascular disrupting agent, combretastatin A-4 phosphate in xenograft models of NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3533.
Abstract 3545: Identifying the effects of lung cancer exosomes on non-tumorigenic human bronchial epithelial cells
Authors: Kubo Hana , Humphrey Sean E. , Kitdumrongthum Sarunya , Tian Feng , Allen Sarah , Kasinski Andrea L.
Published: August 2018

Lung cancer is the leading cause of death from cancer in most industrialized countries and many lung cancer patients remain undiagnosed until the development of locally advanced or metastatic lung cancer. Despite extensive research, universal biomarkers for the early diagnosis of lung cancer remain elusive, and therapies for treating lung cancer patients remain ineffective and unfocused. This makes the identification of new diagnostic biomarkers and therapeutic targets for the treatment of lung cancer a critical need for patients. Exosomes, nanovesicles secreted by a variety of cell types, are known to transfer bioactive molecules such as proteins and nucleic acids that induce physiological changes in recipient cells. Exosomes secreted by cancer cells have been shown to promote tumor initiation, progression, and metastasis in various cancer types, but have been understudied in lung cancer. Our study aims to identify the effects lung cancer secreted exosomes may have on non-cancerous cells of the tumor microenvironment by assessing their tumorigenic effects on normal lung epithelial cells. To that end, we treated normal lung epithelial cells with exosomes derived from a panel of human non-small lung cancer cell lines. Treated cells acquired the ability to invade and migrate. As a proxy for metastasis, cancer exosomes are also being assayed for their ability to permeabilize a monolayer of normal lung epithelial cells. Further assessing the molecules within cancer exosomes critical for promoting these oncogenic behaviors in non-tumorigenic cells could lead to the discovery of potential diagnostic biomarkers and therapeutic targets for inhibiting tumor growth in affected patients.

Citation Format: Hana Kubo, Sean E. Humphrey, Sarunya Kitdumrongthum, Feng Tian, Sarah Allen, Andrea L. Kasinski. Identifying the effects of lung cancer exosomes on non-tumorigenic human bronchial epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3545.
Abstract 3547: Properties of lipid rafts in two epigenetically distinct subtypes of the oncogenic cell-line SW13
Authors: Espejo Luis , Leyva Kathryn J. , Hull Elizabeth E.
Published: August 2018

The epithelial SW13 cancer cell line has two naturally occurring well-defined cell subtypes. The SW13(Vim-) has a phenotype that resembles typical epithelial tumors, displaying increased growth and proliferation with a low level of invasiveness. The SW13(Vim+) subtype has a mesenchymal-like phenotype, commonly associated with more invasive and metastatic states of epithelial cancers. SW13(Vim-) can be converted to a pseudo SW13(Vim+) phenotype when treated with histone deacetylase inhibitors (HDACi). This model is used to study the epigenetic phenotypic differences between tumor-like cancer cells and cells that display a more metastatic phenotype. Gene chip data obtained from the SW13 treated and untreated cells displays differential expression in many genes, specifically genes for cellular membrane proteins and genes involved in cholesterol, sphingolipid, and GPI-anchored protein metabolism; all of which are essential components of lipid rafts. Additionally, recent improved outcomes noted in patients using statin drugs further implicates that cholesterol plays a role in cancer progression, although the mechanism for this improvement is not well understood. This suggests lipid rafts are a candidate for a physical structure that manifests the differential effect of cholesterol perturbations in cancer phenotypes. We hypothesize that lipid raft protein composition is distinctly different between the two SW13 cell subtypes, and that the differences in the composition of lipid rafts may play a role in the unique oncogenic phenotypes of the two subtypes. Utilizing low-temperature detergent-resistant lipid raft isolation techniques, western blotting, and mass spectrometry we investigate protein composition differences between the two SW13 cell subtypes. Cholesterol assays are applied to assess the distribution and total cholesterol content of each SW13 subtype, this also provides translational confirmation of the gene chip data obtained from the subtypes. Cholesterol depletion techniques utilizing cyclodextrin for acute disruption and simvastatin for chronic disruption are being implemented to perturb lipid rafts and observe their role in the oncogenic phenotypes of the SW13 cell subtypes. Specifically, metastatic qualities are measured after lipid raft and cholesterol perturbations. Initial findings support differential cholesterol representation in the two SW13 cell subtypes. Potential findings may explain a mechanism in which cholesterol perturbations are capable of decreasing metastasis and improving the outcome of patients with epithelial cancers.

Citation Format: Luis Espejo, Kathryn J. Leyva, Elizabeth E. Hull. Properties of lipid rafts in two epigenetically distinct subtypes of the oncogenic cell-line SW13 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3547.
Abstract 3556: Sam68 is required for the growth and survival of non-melanoma skin cancer
Authors: Fu Kai , Wan Fengyi
Published: August 2018

Skin cancer is the most commonly diagnosed malignancy in the United States, and the incidence of skin cancer has increased dramatically over the last few decades. Non-melanoma skin cancer (NMSC), which includes squamous cell carcinomas (SCC) and basal cell carcinomas (BCC), is the most common type of skin cancer with substantial associated morbidity and mortality. Whereas targeting DNA repair signaling pathways has emerged as a promising therapeutic for skin cancer, the relevance of DNA damage responses (DDR) in the development and survival of non-melanoma skin cancer (NMSC) remains obscure. Here we report that Src-associated substrate during mitosis of 68 kDa (Sam68), an early signaling molecule in DDR, is elevated in skin tumor tissues derived from NMSC patients and skin lesions from Gli2-transgenic mice. Downregulation of Sam68 impacts the growth and survival of human tumor keratinocytes and genetic ablation of Sam68 delays the onset of basal cell carcinomas (BCC) in Gli2-transgenic mice. Moreover, Sam68 plays a critical role in DNA damage-induced DNA repair and nuclear factor kappa B (NF-κB) signaling pathways in keratinocytes, hence conferring keratinocyte sensitivity to DNA damaging agents. Together our data reveal a novel function of Sam68 in regulating DDR in keratinocytes that is crucial for the growth and survival of NMSC.

Citation Format: Kai Fu, Fengyi Wan. Sam68 is required for the growth and survival of non-melanoma skin cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3556.
Abstract 3557: Beta-hydroxybutyrate inhibits oncogenic signaling and cellular motility in pancreatic cancer cells
Authors: Shukla Surendra K. , Chaika Nina V. , Singh Pankaj K.
Published: August 2018

Pancreatic ductal adenocarcinoma is a devastating malignancy with a five-year survival rate less than 7%. It is characterized by a plethora of metabolic and signaling pathways, which plays a critical role in the pathogenesis of the disease. Caloric restriction and ketogenic diets have been shown to be beneficial in the management of several kinds of malignancies including pancreatic cancer. During caloric restriction or ketogenic diet supplementation, blood level of ketone bodies (beta-hydroxy butyrate or BHB, acetoacetate, acetone) gets elevated in the body. Among, these ketone bodies, BHB, the most abundant ketone body in mammals has been shown to act as the modulator of several signaling and inflammatory pathways. We have evaluated the effect of BHB on oncogenic signaling pathways and motility of pancreatic cancer cells. We observed reduced activation of key oncogenic signaling pathways, including mTOR. We also observed inhibition of cellular motility in a dose-dependent manner. Furthermore, we observed reduced expression of c-Myc, a key oncogene and a downstream effector molecule of mTOR. Correspondingly, we also observed reduced expression of several c-Myc target genes. We have also evaluated the effect of BHB on epithelial-mesenchymal transition (EMT) markers and observed altered expression of EMT markers like N-cadherin and E-cadherin. We also evaluated the effect of BHB on the expression of inflammatory cytokines in the pancreatic cancer cells and observed that BHB treatment leads to the reduction of expression of several inflammatory cytokines including IL6 and IL1-beta. Overall, our findings suggest that BHB inhibits inflammatory signaling in pancreatic cancer cells and reduces the cellular motility by modulating the expression of EMT regulators.

Citation Format: Surendra K. Shukla, Nina V. Chaika, Pankaj K. Singh. Beta-hydroxybutyrate inhibits oncogenic signaling and cellular motility in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3557.
Abstract 3564: A combination of chimeric switch-receptor T cells targeting both PD1 and CTLA4 suppresses tumor growth
Authors: Li Peng , Qin Le , Wei Xinru , Zhao Ruocong , Lai Yunxin
Published: August 2018

Chimeric antigen receptor (CAR) T cells have been successfully used to treat B cell malignancies, but faces many obstacles in solid tumors. A major challenge is the immune-suppressive effects induced by PD-L1 expression in tumors and CD80/86 that ligate with CTLA-4 and are expressed in myeloid-derived immune suppressive cells. Dual blockade of PD-1 and CTLA-4 with monoclonal antibodies has achieved promising anti-tumor effects in clinics. Here, we designed two switch receptor constructs. One contains the truncated extracellular domain of human PD1, costimulatory domains of 4-1BB and TLR2, and the CD3z signaling domain (cPD-1). cPD-1 T cells specifically lysed PD-L1+ tumor cell lines and augmented cytokine secretion in vitro. In addition, cPD-1 T cells were able to suppress the growth of lung cancer, gastric cancer, and osteosarcoma in xenografts. The other construct, similarly, is composed of the truncated extracellular domain of murine Ctla4, human CD28, TLR2, and CD3z signaling domains (cmCtla-4). Because CD80/CD86 are expressed in murine myeloid cells rather than human tumor cells in PDX models, cmCtla4 T cells targeted mouse myeloid cells that were abundant in tumors but not human tumor cells. We found that cmCtla4 T cells hindered tumor growth in gastric cancer PDX. In combination, cPD-1 and cmCtla4 T cells synergistically induced superior anti-tumor effects in lung cancer PDX. Taken together, our findings demonstrated the anti-tumor function of switched immune checkpoint receptors and revealed a strategy of combining T cells targeting both tumor cells and tumor microenvironment, promoting clinical investigation of these chimeric switch-receptor T cells for cancer treatment.

Citation Format: Peng Li, Le Qin, Xinru Wei, Ruocong Zhao, Yunxin Lai. A combination of chimeric switch-receptor T cells targeting both PD1 and CTLA4 suppresses tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3564.