bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3014: Gene signatures identify quinolate phosphoribosyltransferase as a key mediator of acquired resistance to Panobinostat and Bortezomib in glioma, and NAD+ biosynthesis as a targetable pathway to reverse treatment resistance
Authors: Jane Esther P. , Premkumar Daniel R. , Agnihotri Sameer , Myers Max , Chattopadhyay Ansuman , Taylor D. Lansing , Schurdak Mark , Stern Andrew , Pollack Ian F.
Published: August 2019

Resistance of tumor cells to the induction of apoptosis is an important reason for the failure of anticancer treatments in patients with gliomas. Several factors working in concert have been implicated as sources of this treatment resistance; therefore, innovative therapeutic approaches that conspire to attack key tumor vulnerabilities are needed. HDAC and proteasome inhibitors are two classes of agents that have shown some benefit in the clinic, but patients often rapidly manifest intrinsic or acquired resistance mechanisms that limits their individual efficacy. We demonstrate that the combination of the HDAC inhibitor panobinostat and the proteasome inhibitor bortezomib synergistically induces apoptosis of human glioma cell lines. However, acquired resistance is observed even with this initially effective combination. To examine the mechanism of this resistance, we performed RNA sequencing and pharmacological screening of resistant compared to sensitive cells. Based on these studies, we present evidence that quinolinic acid phosphoribosyltransferase (QPRT), an enzyme catalyzes a rate determining step in de novo NAD+ biosynthesis, provides a critical adaptive survival mechanism that allows cancer cells to evade an initially effective therapeutic combination. We identified 1004 genes that had a significant change between panobinostat and bortezomib- resistant cells versus inhibitor naïve control cells. Furthermore, pathway analysis revealed that the experimental regimen significantly altered metabolic pathways. By silencing QPRT (using siRNA) we demonstrated that we can overcome resistance, thus suggesting QPRT as a crucial crossroad for cancer cell survival and as a new anticancer target. We also showed that treatment of panobinostat and bortezomib resistant cells with FK866, niraparib, selisistat, epacadostat, gemcitabine, 5-fluoruracil, and methotrexate significantly increased cell death highlighting the importance of NAD+ and folate pathway inhibitors in resensitizing the resistant cells. Together, targeting QPRT or NAD+ consuming enzymes hold promise for eliminating recurrent disease in glioma.

Citation Format: Esther P. Jane, Daniel R. Premkumar, Sameer Agnihotri, Max Myers, Ansuman Chattopadhyay, D. Lansing Taylor, Mark Schurdak, Andrew Stern, Ian F. Pollack. Gene signatures identify quinolate phosphoribosyltransferase as a key mediator of acquired resistance to Panobinostat and Bortezomib in glioma, and NAD+ biosynthesis as a targetable pathway to reverse treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3014.
Abstract 3019: Dynamic kinome profiling of EGFRvIII-driven murine astrocyte models of glioblastoma reveals targets for dual kinase inhibitor therapy
Authors: Smithberger Erin , Shelton Abigail K. , Butler Madison K. , Flores Alex R. , Bash Ryan E. , Angus Steven P. , Sciaky Noah , Dhruv Harshil D. , Johnson Gary L. , Berens Michael E. , Furnari Frank B. , Miller C. Ryan
Published: August 2019

Glioblastoma (GBM) is an aggressive brain tumor with few effective treatments. Epidermal growth factor receptor (EGFR) is frequently amplified and mutated in GBM, leading to trials of several EGFR tyrosine kinase inhibitors, but none have proven successful. One potential reason for failure is acquired resistance, particularly acute, adaptive responses in the kinome. To study this adaptive resistance mechanism, we used RNA-seq and multiplex inhibitor bead/mass spectrometry (MIB-MS) to analyze transcriptomes and kinomes of genetically-engineered murine astrocytes with genotypes commonly seen in human GBM. We previously showed that 38% (86 of 228) of the expressed kinome varied among a panel of genetically diverse murine astrocytes harboring Cdkn2a deletion (C) plus Pten deletion (CP), wild-type human EGFR (CE) or EGFRvIII (CEv3) overexpression, or both overexpressed EGFRvIII and Pten deletion (CEv3P). Pairwise genotype comparisons revealed multiple differentially activated kinases, including Pdgfrb, Fgfr2, Lyn, Ddr1, and several Ephrin family members. We further investigated these potential targets for dual therapy with EGFR TKI by examining the transcriptional response of cultured astrocytes at 4, 24, and 48 hours after 3 μM afatinib. Afatinib induced no kinome changes in C and only 3 kinases (Fn3k, Prkg2, and Syk) were altered in CP astrocytes. Despite similar baseline gene expression profiles, CE astrocytes overexpressing wild-type EGFR responded significantly differently than C astrocytes without. Five kinases (Dclk1, Epha3, Epha7, Fgfr3, and Prkg1) were induced, while 14 were repressed. Six were similarly repressed in CEv3 (Bub1, Nek2, Pask, Plk4, Prkcb, and Vrk1). Whereas the kinase transcriptome response was blunted in C, CP, and CE astrocytes, afatinib induced altered expression of significantly more kinases in CEv3 (82) and CEv3P cells (49). One particularly attractive target in CEv3 astrocytes was Epha4, which afatinib induced >40-fold. Dual inhibition of EGFRvIII and Epha4 kinases may thus provide an opportunity for more effective targeted therapy.

Citation Format: Erin Smithberger, Abigail K. Shelton, Madison K. Butler, Alex R. Flores, Ryan E. Bash, Steven P. Angus, Noah Sciaky, Harshil D. Dhruv, Gary L. Johnson, Michael E. Berens, Frank B. Furnari, C. Ryan Miller. Dynamic kinome profiling of EGFRvIII-driven murine astrocyte models of glioblastoma reveals targets for dual kinase inhibitor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3019.
Abstract 3023: Clonal expansion of TP53 mutated cells is associated with chemoresistance in acute myeloid leukemia
Authors: Yan Bowen , Qiu Yi
Published: August 2019

Chemoresistance is a major burden for the treatment of many cancers, including acute myeloid leukemia (AML). AML is a clonal disease and chemoresistant cells may either evolved from the expansion of a subclone of the primary clone or from the clone gained additional mutations during therapy. We have generated chemoresistant cell lines from AML patient sample derived cell lines, OCI-AML2 and MV4-11, to study the chemoresistant mechanism. Interestingly, we found that both established resistant lines have TP53 mutations (Y220C or R248W) but not in the parental cell lines by Sanger sequencing. However, high resolution sequencing result shows that residual TP53 mutation is present in both parental cell lines. These small population of cells with TP53 mutation was expanded under the selection by chemotherapy and eventually became a dominant clone in chemoresistant lines. TCGA AML patient sample sequencing data also shows, there’re around 3% of patients have residual TP53 mutation reads. However, since the reads are low, these patients are considered TP53 wild type conventionally. Importantly, these patients have worse outcome of survival as compared to the TP53 wild type patients, indicating the residual TP53 mutation may play an important role on chemoresistance in patients. Consistent with TP53 mutation in chemoresistant cells, Gene Set Enrichment Analysis (GSEA) from RNA-seq data shows p53 target genes are repressed in resistant cells. The cyclin-dependent kinase inhibitor p21 is one of p53 target genes and is down regulated in chemoresistant cells. Knocking down of p21 in parental cells increased cell chemoresistance, indicating p21 gene repression contributes to chemoresistance. Data from TCGA also shows patients with lower p21 expression have worse outcome of survival. In chemoresistant cell line, there is reduced p53 recruitment at p21 promoter, leading to failure of p21 activation. The epigenetic drug Romidepsin can elevate histone acetylation at p21 promoter region, reactivates p21 gene expression and induces cell death. Furthermore, GSEA from RNA-seq data shows Romidepsin can reactivate genes that repressed by p53 mutation. Our data indicate that TP53 mutation is directly linked to chemoresistance. Residual TP53 mutation in patients was neglected previously, however, it can be an important driver for clonal evolution and chemoresistance in AML. In addition, epigenetic drug Romidepsin can be a potential therapeutic drug to prevent chemoresistance development especially for patients with TP53 mutation or residual TP53 mutation.

Citation Format: Bowen Yan, Yi Qiu. Clonal expansion of TP53 mutated cells is associated with chemoresistance in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3023.
Abstract 3025: Investigation of AKT3 in the resistance of non-small cell lung cancer to EGFR-TKI
Authors: Huang Shih-Hsiang , Shih Jin-Yuan , Chen Ching-Chow
Published: August 2019

Lung cancer is the leading cause of cancer deaths worldwide, and EGFR-TKI is the first-line treatment for non-small cell lung cancer (NSCLC) harboring EGFR activation mutation. However, most patients develop acquired resistance around 12 months. Two resistant cells, HCC827/IR (IRESSA resistance) and H1975/AR (AZD9291 resistance) exhibiting EMT phenotypes were generated to investigate the molecular and cellular characteristics of the EGFR-TKI acquired resistance.

The expression and activation of EGFR were reduced in both resistant cells. Upregulation of AXL and FGFR1 were found in HCC827/IR but not H1975/AR cells. HCC827/IR (without T790M) and H1975/AR (without C797S) showed “off target resistance”. Activation of AKT (p-AKT) in both parental cells was more sensitive to EGFR-TKI than that in resistant cells. RNA-seq revealed that AKT3 was upregulated in both resistant cells. High expression of AKT3 was predicted to correlate with the poor survival of lung adenocarcinoma patients. Knockdown of AKT3 in HCC827/IR cells inhibited cell migration and increased the protein expression of E-cadherin, as well as inhibited S phase population to reduce cell proliferation. Knockdown of AKT3 in H1975/AR cells enhanced AZD9291-induced inhibition on AKT activation (p-AKT). Immunoprecipitation of AKT3 in both resistant cells demonstrated its involvement in AKT activation, and AKT3 activation (p-AKT3) was not sensitive to inhibition by gefitinib and AZD9291 in resistant cells. We also found that protein but not mRNA of AKT3 was upregulated in gefitinib-treated HCC827/IR and AZD9291-treated H1975/AR cells. Therefore, AKT3 might serve as a predictive biomarker for EGFR-TKIs therapy, and its upregulation might be one of the mechanisms for EGFR-TKIs acquired resistance. In addition, combination of AZD9291 with AKT inhibitors elicited synergistic inhibition on cell viability of H1975/AR cells.

This work was supported by the National Health Research Institutes (NHRI)- NHRI-EX107-10707BI.

Citation Format: Shih-Hsiang Huang, Jin-Yuan Shih, Ching-Chow Chen. Investigation of AKT3 in the resistance of non-small cell lung cancer to EGFR-TKI [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3025.
Abstract 3027: Pharmacological regulation of the ABCG2 multidrug transporter in A549 non-small cell lung cancer cells
Authors: Kovacsics Daniella , Mészáros Nikolett , Tihanyi Borbála , Matula Zsolt , Borsy Adrienn , Szabó Edit , Várady György , Orbán Tamás , Apáti Ágota , Fóthi Ábel , Brózik Anna , Sarkadi Balázs
Published: August 2019

The notoriously drug resistant non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths and the ABCG2 multidrug transporter was shown to be involved in NSCLC drug resistance. Direct drug interactions with this transporter have been extensively studied, while a potential modulation of ABCG2 expression by drugs or hormones is less explored. To study the drug-dependent regulation of ABCG2 expression, we used CRISPR-Cas9 and homologous recombination-mediated DNA repair to generate fluorescent lung cancer reporter cells with preserved endogenous regulatory regions. In A549 lung adenocarcinoma cells, which have two wild type copies of the ABCG2 gene, we replaced the initial coding region of ABCG2 with a green fluorescent protein (eGFP)-coding sequence. In addition, since our previous studies demonstrated that an N-terminally eGFP-tagged ABCG2 protein is properly localized and functional, we have generated A549 cells in which the native ABCG2 coding region was modified to express an eGFP-tagged ABCG2 protein. The eGFP knock-in cells sensitively reported ABCG2 transcriptional regulation, while the cells expressing an eGFP-ABCG2 fusion protein allowed the assessment of the full, functional transporter. Using the engineered A549 cells, we found that several drugs, including anti-cancer medications, glucocorticoids, HDAC inhibitors and hypoxia-mimicking agents, caused a significant induction of ABCG2 expression. In these experiments, an intronic regulatory element was found to play a key role in the specific glucocorticoid induction of ABCG2. We suggest that our transgenic reporter cell lines, amenable to high-throughput studies, can be used to investigate the induction of ABCG2-based drug resistance in cancer and predict potential drug-drug interactions. Understanding how the generation of drug-tolerant persister cells occurs via ABCG2 expression may help the selective elimination of these multidrug resistant cancer cells. Supported by the NKFIH-OTKA grant K115375.

Citation Format: Daniella Kovacsics, Nikolett Mészáros, Borbála Tihanyi, Zsolt Matula, Adrienn Borsy, Edit Szabó, György Várady, Tamás Orbán, Ágota Apáti, Ábel Fóthi, Anna Brózik, Balázs Sarkadi. Pharmacological regulation of the ABCG2 multidrug transporter in A549 non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3027.
Abstract 3034: TP53 mutant cell lines selected for resistance to MDM2 inhibitors retain growth inhibition by MAPK pathway inhibitors but a reduced apoptotic response
Authors: Wu Chiao-En , Koay Tsin Shue , Ho Yi-Hsuan , Lovat Penny , Lunec John
Published: August 2019

Emergence of resistance to molecular targeted therapy constitutes a potential limitation to clinical benefits in cancer treatment. Cross-resistance commonly happens with chemotherapeutic agents but might not with targeted agents. In the current study, TP53 wild -type cell lines with druggable MAPK pathway mutations (BRAFV600E (WM35) or NRAS Q61K (SJSA-1)) were compared with their TP53 mutant sublines (WM35-R, SN40R2) derived by selection for resistance to MDM2/p53 binding antagonists. The continued presence of the druggable MAPK pathway targets in the TP53 mutant (TP53MUT) WM35-R and SN40R2 cells was confirmed. Trametinib and vemurafenib were tested on the paired WM35/WM35-R and SJSA-1/SN40R2 cells and similar growth inhibitory effects on the paired cell lines was observed. However, apoptotic responses to trametinib and vemurafenib were greater in WM35 than WM35-R, evidenced by FACS analysis and caspase 3/7 activity, indicating that these MAPK inhibitors acted on the cells partially through p53-regulated pathways. SiRNA mediated knockdown of p53 expression in WM35 replicated the same pattern of response to trametinib and vemurafenib as seen in WM35-R, confirming that p53 plays a role in trametinib and vemurafenib induced apoptosis. In contrast, these differences in apoptotic response between WM35 and WM35-R were not seen with the SJSA-1/SN40R2 cell line pair. This is likely due to p53 suppression by overexpressed MDM2 in SJSA-1. In conclusion, the TP53MUT cells selected by resistance to MDM2 inhibitors nevertheless retained growth inhibitory but not apoptotic response to MAPK pathway inhibitors.

Citation Format: Chiao-En Wu, Tsin Shue Koay, Yi-Hsuan Ho, Penny Lovat, John Lunec. TP53 mutant cell lines selected for resistance to MDM2 inhibitors retain growth inhibition by MAPK pathway inhibitors but a reduced apoptotic response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3034.
Abstract 3035: Collagen hydroxylation promotes TNBC chemoresistance through stabilizing HIF1á
Authors: Xiong Gaofeng , Stewart Rachel , Chen Jie , Gao Tianyan , Scott Timothy , Samayoa Luis , O’Connor Kathleen , Lane Andrew , Xu Ren
Published: August 2019

Collagen prolyl 4-hydroxylase (P4H) expression and collagen hydroxylation in cancer cells are necessary for breast cancer progression. Here, we show that P4H alpha 1 subunit (P4HA1) protein expression is induced in triple-negative breast cancer (TNBC). By modulating alpha ketoglutarate (α-KG) and succinate levels P4HA1 expression reduces proline hydroxylation on hypoxia-inducible factor (HIF) 1α, enhancing its stability in cancer cells. Activation of the P4HA/HIF-1 axis enhances cancer cell stemness. Inhibition of P4HA1 sensitizes TNBC to the chemotherapeutic agent in xenografts and patient-derived models. We also show that increased P4HA1 expression correlates with short relapse-free survival in TNBC patients who received chemotherapy. These results suggest that P4HA1 promotes chemoresistance by modulating HIF-1-dependent cancer cell stemness. Targeting collagen P4H is a promising strategy to inhibit tumor progression and sensitize TNBC to chemotherapeutic agents.

Citation Format: Gaofeng Xiong, Rachel Stewart, Jie Chen, Tianyan Gao, Timothy Scott, Luis Samayoa, Kathleen O’Connor, Andrew Lane, Ren Xu. Collagen hydroxylation promotes TNBC chemoresistance through stabilizing HIF1á [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3035.
Abstract 3043: Examination of NMT1 and NMT2 as independent prognostic markers and novel drug targets in adult acute myeloid leukemia
Authors: Mackey John R. , Iyer Aishwarya , Yap Megan C. , Zak Zoulika , Vincent Krista , Beauchamp Erwan , Postovit Lynne M. , Brandwein Joseph , Berthiaume Luc G.
Published: August 2019

Myristoylation is required for biological activity of >200 intracellular proteins. N-myristoyltransferases (NMTs) transfer the fatty acid myristate to N-terminal glycine residue; there are two isoforms, NMT1 and 2. In acute myeloid leukemia (AML), upregulation of Lyn and Src, important myristoylated proteins, contribute to cell survival and proliferation. The specific roles of NMT1 and NMT2 are unknown in this context. The relationships among NMT1/NMT2 expression and acute AML patient outcomes were studied using RNA-sequencing and microarray cohorts with over 350 patients. We found that high NMT1 and low NMT2 expression were associated with reduced overall and event-free survival in adult AML, which was independent of other prognostic markers on multivariate analysis. High NMT1, but not NMT2, expression was associated with proliferative gene sets in AML cell lines, indicating potential for distinct isozyme substrates. Given these results, we examined NMT1 and NMT2 levels in AML cell lines and AML patient blast cells using Western blotting and flow cytometry. We determined that NMT2 expression varied greatly among patients, but was markedly reduced in most cases, while NMT1 expression was relatively preserved. A potent small molecule NMT inhibitor, PCLX-001, preferentially induced apoptosis and reduced viability in NMT2-deficient AML cell lines cultured in vitro compared with normal lymphocytes and peripheral blood mononuclear cells. PCLX-001 also killed freshly isolated human AML blasts ex vivo with an IC50 of ~170nM regardless of their mutational background. In a murine AML xenograft model, subcutaneously delivered PCLX-001 monotherapy demonstrated dose-dependent anticancer activity and produced complete remissions after five daily 50 mg/kg doses. NMT expression provides independent prognostic information to refine existing clinical stratification, and NMT inhibition is a promising novel therapeutic strategy for AML.

Citation Format: John R. Mackey, Aishwarya Iyer, Megan C. Yap, Zoulika Zak, Krista Vincent, Erwan Beauchamp, Lynne M. Postovit, Joseph Brandwein, Luc G. Berthiaume. Examination of NMT1 and NMT2 as independent prognostic markers and novel drug targets in adult acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3043.
Abstract 3046: Targeting N-myristoylation in B cell lymphomas as a therapeutic strategy
Authors: Mackey John R. , Beauchamp Erwan , Yap Megan C. , Iyer Aishwarya , Perinpanayagam Maneka A. , Vincent Krista M. , Al-Momany Abass M. , Heit Ryan J. , Sim Jacky Y. , Lai Raymond , Dong Wei-feng , Lakshmanan Manikandan , Raju Anandhkumar , Tergaonkar Vinay , Tan Soo Yong , Lim Soon Thye , Postovit Lynne M. , Read Kevin D. , Gray David W. , Wyatt Paul G. , Berthiaume Luc G.
Published: August 2019

Treatment of aggressive lymphoma is toxic, expensive, and a substantial proportion of patients relapse and die. There is an urgent need for more effective treatments. Myristoylation is required for biological activity of >200 intracellular proteins. N-myristoyltransferases (NMTs) transfer the fatty acid myristate to N-terminal glycine residue; there are two isoforms, NMT1 and 2. Since they are critical to intracellular signaling, NMTs are potential anti-cancer targets. We tested a novel potent pan-NMT inhibitor, PCLX-001, in B cell lymphoma cell lines. In vitro assays included cell viability, immunoblotting, and metabolic labeling of lymphoma cell lines. Immunohistochemistry was performed on formalin fixed paraffin embedded lymphoma specimens from patients. In vivo experiments included cell line derived murine xenografts and a patient derived mouse xenograft treated with increasing concentrations of PCLX-001. PCLX-001 selectively killed lymphoma cells, while sparing normal cells in vitro and in 3 mouse xenograft models, eradicating tumors in two of these models including a patient-derived xenograft from a R-CHOP refractory lymphoma patient. While NMT2 is overexpressed in some cancers, loss of NMT2 expression is common in numerous cancers and occurs at the highest prevalence in lymphomas, where it is independently linked to a worse prognosis. This NMT2 suppression occurred through epigenetic mechanisms and may account for lymphoma sensitivity to NMT inhibition. The global myristoylation of lymphoma cell proteins, including that of the protein tyrosine kinase oncogene Src, is profoundly inhibited by PCLX-001. Loss of Src myristoylation is accompanied by loss of Src activity and may account for loss of prosurvival signals causing lymphoma cell death. Targeting NMT2 deficient B cell lymphoma with a pan-NMT inhibitor suppresses the residual NMT1 function provides a novel, selective, and effective therapeutic strategy.

Citation Format: John R. Mackey, Erwan Beauchamp, Megan C. Yap, Aishwarya Iyer, Maneka A. Perinpanayagam, Krista M. Vincent, Abass M. Al-Momany, Ryan J. Heit, Jacky Y. Sim, Raymond Lai, Wei-feng Dong, Manikandan Lakshmanan, Anandhkumar Raju, Vinay Tergaonkar, Soo Yong Tan, Soon Thye Lim, Lynne M. Postovit, Kevin D. Read, David W. Gray, Paul G. Wyatt, Luc G. Berthiaume. Targeting N-myristoylation in B cell lymphomas as a therapeutic strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3046.
Abstract 3047: Connective tissue growth factor: Novel therapeutic target for non-small cell lung cancer
Authors: Chaudhary Priyanka , Labat Marie-Liesse , Wang Guangfang , Onaitis Mark
Published: August 2019

Lung cancer is the leading cause of cancer deaths worldwide. Non-small cell lung cancer (NSCLC), which accounts for 85% of all lung cancer cases, includes adenocarcinoma, squamous cell lung carcinoma and large-cell lung carcinoma. Despite recent advances in targeted therapies for adenocarcinoma, they are effective in only a small percentage of patients while there are no targeted therapies available for squamous cell lung cancer. Therefore, it is critical to identify novel intervention strategies against NSCLC. In the present study, we have identified connective tissue growth factor (CTGF) as a possible therapeutic target for NSCLC. CTGF is an extracellular-matrix (ECM) associated protein, which is involved in cellular processes like cell adhesion, migration, proliferation, differentiation, survival, apoptosis and senescence. CTGF has been associated with both oncogenic as well as tumor suppressive activities in context of different cancer types. The function of CTGF in lung cancer pathogenesis remains controversial. We performed immunohistochemistry on tissue microarray containing 60 cases, which clearly suggested that CTGF expression increases in lung carcinoma. More specifically, among other subtypes, squamous cell lung cancer cases expressed more CTGF compared to their matched normal tissues. To determine the functional role of CTGF in NSCLC, we employed both genetic and pharmacologic approaches to inhibit CTGF activity. Our results clearly demonstrated that CTGF inhibition by both lentiviral knockdown as well as monoclonal antibody against CTGF (FG-3019) caused marked inhibition in cell proliferation, colony formation and xenograft growth in immunodeficient mice. We have also found that CTGF knockdown causes cell cycle arrest and apoptosis. Furthermore, we tested the efficacy of FG-3019 along with drugs like cisplatin and gemcitabine in patient-derived xenograft (PDX) model of squamous cell lung cancer. Our results demonstrated that CTGF inhibition enhances the effects of gemcitabine chemotherapy in PDX model of squamous cell lung carcinoma. These results clearly suggested that CTGF has a profound effect on lung tumorigenesis, as its inhibition not only causes attenuation in tumor growth but also potentiates the chemotherapeutic sensitivity of commonly used drugs for NSCLC. We have also elucidated that tumor hypoxia and Yap signaling are responsible for CTGF up-regulation in lung carcinoma. Overall, our study underscores the significance of CTGF inhibition in non-small cell lung carcinoma and offers the potential use of FG-3019 for its treatment.

Citation Format: Priyanka Chaudhary, Marie-Liesse Labat, Guangfang Wang, Mark Onaitis. Connective tissue growth factor: Novel therapeutic target for non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3047.
Abstract 3051: Targeting cdk12/13 re-sensitizes trastuzumab resistant her2+ breast cancers
Authors: Vena Francesca , Aceti Max , Bayle Simon , Quereda Victor , Monastyrskyi Andrii , Roush William , Duckett Derek
Published: August 2019

Overexpression or amplification of the human epidermal growth factor receptor 2 (erbB2), which encodes for a receptor tyrosine kinase often referred to as HER2, occurs in about 20% breast cancer patients and is associated with aggressive disease and poor clinical outcome. The anti-HER2 therapy trastuzumab (a monoclonal antibody targeting HER2) has proven highly effective, increasing relapse free and overall survival for HER2 positive breast cancer patients. Nevertheless, acquisition of therapeutic resistance occurs frequently in advance disease. Recent studies have demonstrated that HER2 co-amplified genes may play a role in mechanisms of disease resistance. Cyclin dependent kinase 12 (CDK12) is among the genes that have been reported to be amplified in addition to HER2, and thus we aimed to investigate whether targeting CDK12 could be exploited as a therapeutic target and enhance and/or restore sensitivity in HER2 positive inhibitor resistant models of breast cancer.

We have developed an in-house, highly selective, potent, and orally bioavailable CDK12/13 inhibitor, which effectively blocks proliferation and induces apoptosis in a panel of breast cancer cell lines and acts in synergy with trastuzumab in HER2 treatment resistant cell lines. Furthermore, our lead compound significantly slows tumor growth in trastuzumab resistant patient derived xenograft (PDX) models (as a single agent) and importantly, provokes greater efficacy in combination with trastuzumab. We are beginning to identify the signaling pathways underlying HER2 positive treatment resistance and how inhibition of CDK12/13 thwarts these resistance mechanisms. Accordingly, we anticipate that the development of CDK12/13 inhibitors holds promise as an anti-breast cancer strategy and future treatment option for HER2 positive breast cancer patients with recurrent disease.

Citation Format: Francesca Vena, Max Aceti, Simon Bayle, Victor Quereda, Andrii Monastyrskyi, William Roush, Derek Duckett. Targeting cdk12/13 re-sensitizes trastuzumab resistant her2+ breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3051.
Abstract 3052: MGMT inhibition is associated with MAPK pathway inhibition and enhances Raf, MEK, ERK inhibitors and restores meaningful Temozolomide activity in melanoma
Authors: Bobustuc George C. , Kassam Amin B. , Bosenko Dmitry , Donohoe Deborah L. , Rovin Richard A. , Konduri Santhi D.
Published: August 2019

Braf and MEK inhibition leads to limited survival gains in melanoma. In this research we show that, in vitro, MGMT controls all MAPK pathway effectors. We show that triple lock - upstream and downstream, along the MAPK pathway - effectively restores durable Braf and MEK inhibitor activity and significantly sensitizes melanoma to Temozolomide. The advantage of a multiple lock approach on the MAPK pathway is substantiated by the lack of signaling cross talk. We briefly discuss how this simple MGMT based regulatory paradigm could be immediately exploited in the care of melanoma patients. We also show how this strategy was exploited in two, surviving, metastatic (to include CNS disease) melanoma patients who had failed Braf and MEK inhibition (2 and 5 years ago) who now show stable or negligible residual disease burden while continuing on an intermittent, low treatment density, combination regimen

Citation Format: George C. Bobustuc, Amin B. Kassam, Dmitry Bosenko, Deborah L. Donohoe, Richard A. Rovin, Santhi D. Konduri. MGMT inhibition is associated with MAPK pathway inhibition and enhances Raf, MEK, ERK inhibitors and restores meaningful Temozolomide activity in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3052.
Abstract 3056: Development of an integrated CRISPR interference system targeting ΔNp63 to treat lung and esophageal squamous cell carcinoma
Authors: Yoshida Masakazu , Yokota Etsuko , Sakuma Tetsushi , Takigawa Nagio , Ushijima Toshikazu , Yamamoto Takashi , Naomoto Yoshio , Fukazawa Takuya , Yamatsuji Tomoki
Published: August 2019

TP63 encodes two different transcripts that have opposite functions in transcriptional control. One transcript encodes TAp63 that is functionally similar to the tumor suppressor TP53. The other transcript, ΔNp63, lacks the transcription-activating domain of TAp63 and has been proposed to function as a potent oncogene in squamous cell carcinomas. In this study, we have developed an integrated CRISPR interference system to selectively suppress ΔNp63 and we have termed this system CRISPRiΔNp63. We engineered the CRISPRi using tandemized guide RNA expression cassettes to target the 50 to 100 bp downstream region of the transcription start site of ΔNp63 and we used inactivated Cas9 linked to the transcription repression module Krüppel-associated box repressor domain. The plasmid vector harboring CRISPRiΔNp63 repressed ΔNp63 transcriptional activity in lung and esophageal squamous cell carcinoma cells that express ΔNp63. Moreover, an all-in-one adenoviral vector containing tandemized gRNAs and the dCas9/KRAB expression cassette, Ad-CRISPRiΔNp63, suppressed ΔNp63 expression in squamous cell carcinomas cells. Ad-CRISPRiΔNp63 effectively decreased cell proliferation and colony formation and induced apoptosis in lung and esophageal squamous cell carcinoma cells in vitro. Moreover, the all-in-one vector significantly inhibited tumor growth in a lung squamous cell carcinoma xenograft mouse model in vivo. These results indicate that ΔNp63 suppression by CRISPRiΔNp63 might be an effective strategy for the treatment of lung and esophageal squamous cell carcinoma.

Citation Format: Masakazu Yoshida, Etsuko Yokota, Tetsushi Sakuma, Nagio Takigawa, Toshikazu Ushijima, Takashi Yamamoto, Yoshio Naomoto, Takuya Fukazawa, Tomoki Yamatsuji. Development of an integrated CRISPR interference system targeting ΔNp63 to treat lung and esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3056.
Abstract 3060: YAP1 induces STAT3 phosphorylation & combined blockade enhances chemotherapeutic efficacy
Authors: Shibata Masahiro , Ooki Akira , Inokawa Yoshikuni , Izumchenko Evgeny , Munari Enrico , Bogina Giuseppe , Gabrielson Edward , Singh Anju , Hoque Mohammad Obaidul
Published: August 2019

Recent studies have identified transcriptions factors YAP1 and STAT3, as therapeutic targets in cancer, but efforts targeting either YAP1 or STAT3 pathway have met with limited success. Our study is focused on understanding the cross-talk between YAP1 and STAT3 signaling, impact of this cross-talk on lung tumorigenesis, drug resistance and blocking this pathway as a potential therapeutic strategy in lung adenocarcinoma (LUAD). Mechanistically, we demonstrate that YAP1 promotes the phosphorylation of STAT3 through IL-6 upregulation. In clinical LUAD samples, a positive correlation between YAP1 and phosphorylated STAT3 protein as well as an association between YAP1 and IL-6 mRNA was observed. Pre-clinically, genetic and pharmacologic dual inhibitions of YAP1 and STAT3 showed decreased cell growth and increased sensitivity to chemotherapy through the attenuation of cancer-stemness like features. Although verteporfin and S3I-201 were employed as YAP1 and STAT3 inhibitors respectively, verteporfin was found to suppress not only YAP1 but also STAT3, and their combination induced synergistic cytotoxic effects. Furthermore, addition of verteporfin and S3I-201 to standard-of-care chemotherapy regimen significantly inhibited tumor growth in patient-derived xenograft mouse models and attenuated in vivo expansion of malignant stemness. These findings provide the conceptual framework for combined targeting of YAP1 and STAT3 in LUAD.

Citation Format: Masahiro Shibata, Akira Ooki, Yoshikuni Inokawa, Evgeny Izumchenko, Enrico Munari, Giuseppe Bogina, Edward Gabrielson, Anju Singh, Mohammad Obaidul Hoque. YAP1 induces STAT3 phosphorylation & combined blockade enhances chemotherapeutic efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3060.
Abstract 3061: Role of ID4 (inhibitor of DNA binding protein 4) in FKBP52-AR pathway
Authors: Komaragiri Shravan Kumar
Published: August 2019

Introduction: Inhibitor of DNA binding/differentiation protein 4 (ID4), acts as a dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors. In normal prostate epithelial cells, ID4 collaborates with androgen receptor (AR) and p53 to exert its tumor suppressor activity. Previous studies have shown that ID4 promotes tumor suppressive function of AR whereas loss of ID4 results in tumor promoter activity of AR through selective interaction with FKBP52. Pharmacological inhibition of FKBP52, by MJC13, attenuated the tumor growth, weight, and volume of LNCaP cells lacking ID4(L(-)ID4) xenografts. The L(-)ID4 cells also gains CRPC phenotype through FKBP52-mediated AR signaling. In this study we investigated the role of ID4 in pharmacological inhibition of FKBP52 with GMC1 (Initial hit molecule by in-silico screening with the most potent inhibition of FKBP52) in regulating the AR pathway.

Experimental procedures: GMC1 drug treatments in LNCaP(-)ID4 and CW22RV1 generated xenograft tumors were further analyzed for tumor volume and tumor weight. We also performed immunohistochemistry on GMC1 treated xenografts to determine AR, PSA, FKBP51 and FKBP52 protein expression. Our in vivo studies with GMC1 drug treatments in castrated male SCID mice injected with LNCaP-ID4 and CW22RV1 cells showed decreased tumor size and volume when compared to untreated mice tumors.

Results: IHC results on GMC1 treated LNCaP(-)ID4 and CW22RV1 generated xenograft tumors showed no change in ID4 expression with decrease expression of AR, PSA, FKBP51, FKBP52 and KI67 when compared to untreated xenograft tumors. These results suggest that inhibition of FKBP52-regulated AR activity (via GMC1) demonstrated a significant reduction in the tumor volume of subcutaneous xenografts in vivo. Overall, ID4 appears as a potential tumor suppressor gene that selectively regulates AR activity through direct interaction with FKBP52, and its loss, promotes CRPC through FKBP52-mediated AR signaling.

Conclusions: In conclusion, we demonstrate that loss of ID4 in LNCaP cells elevates FKBP52 levels, transcriptionally potentiates AR activity which in turn then leads to progression of androgen-independent prostate cancer. GMC1 is a novel inhibitor that inhibits the binding of FKBP52 to AR thereby prevents the AR transcriptional hyperactivity in PCa

Acknowledgement: These studies were supported by DOD, Grant # W81XWH-17-1-0437

Citation Format: Shravan Kumar Komaragiri. Role of ID4 (inhibitor of DNA binding protein 4) in FKBP52-AR pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3061.
Abstract 3062: PLK1: a new therapeutic target in mucinous ovarian carcinoma
Authors: Affatato Roberta , Chilà Rosaria , Lupi Monica , Restelli Valentina , Erlander Mark , Carrassa Laura , Damia Giovanna
Published: August 2019

Epithelial ovarian cancer (EOC) is the most lethal gynaecological cancer. Amongst the five histotypes (high-grade serous, clear-cell, endometrioid, mucinous, and low-grade serous), mucinous epithelial ovarian cancer (mEOC) is a rare subset and represents approximately 3% of EOC. Beside these different histotypes are unique clinical entities with a different prognosis and response to therapy, they are still treated as a homogeneous group with a similar chemotherapeutic approach (platinum-paclitaxel combination), to which however mEOC is generally poor responsive. New specific and active therapeutic strategies are needed for mEOC.

To find new therapeutic options for mEOC, we have performed a high-throughput screening using a siRNA library directed against 719 human protein kinases in the mEOC MCAS cell line. After two independent screenings and validation experiments with specific esiRNAs, Polo-like kinase1 (PLK1), a mitotic serine/threonine kinase, was identified as the most significant kinase whose downregulation interfered with both cell proliferation and survival. Both PLK1 siRNA and nanomolar concentrations of commercially available inhibitors against PLK1 (onvansertib (NMS-P937) and volasertib) inhibited cell growth in other two additional mEOC cell lines (EFO-27 and JHOM-1). In addition, in the mEOC cell lines PLK1 inhibition was able to induce apoptosis and to block cells in the G2-M phase of the cell cycle. We then evaluated in vitro combinations between the PLK1 inhibitors and different chemotherapeutic drugs used in mEOC, including cisplatin and paclitaxel used in front line therapy in all the available mEOC cell lines. No synergism could be found in any of the cell lines, when both PLK1 inhibitors were combined with cisplatin. On the contrary, strong synergism could be observed in two out the three mEOC cell lines investigated when onvansertib and volasertib were combined with both paclitaxel and eribulin. On the basis of the strong in vitro synergistic effect between onvansertib and paclitaxel, this combination was tested in vivo in nude mice transplanted with MCAS cells. Onvansertib as single agent was inactive, while paclitaxel at the dose used was active. Interestingly enough, the combination of the two drugs was well tolerated and was much more active than paclitaxel single agent, with both a stronger tumor regression and more sustained tumor growth inhibition that translated in a longer survival time. These data suggest that onvansertib and paclitaxel could represent a new active therapeutic option in mEOC.

Citation Format: Roberta Affatato, Rosaria Chilà, Monica Lupi, Valentina Restelli, Mark Erlander, Laura Carrassa, Giovanna Damia. PLK1: a new therapeutic target in mucinous ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3062.
Abstract 3063: Over expression of protein kinase Nek1 and its modulation by Thioridazine in prostate cancer
Authors: Singh Vibha , Jaiswal Praveen K. , Koul Hari K. , Yu Xiuping , Benedetti Arrigo
Published: August 2019

Introduction: Prostate Cancer (PCa) is one of the most common urological malignancies in men in the United States. We have identified NIMA kinase NEK1 through a proteomic approach, that interacts and co-localizes with Tousled Like kinases (TLKs) and has a role in the DDR, upstream of ATR and Chk1. In the present study we have checked the NEK1 activity in PCa patient samples and tested Thioridazine (THD), an anti-psychotic drug and inhibitor of TLKs in inhibiting the activity of NEK1 in human and mice derived prostate cancer cell lines and xenografts derived from LNCaP cells.

Material & Methods: We have used tissue microarray (TMA) derived from patient samples and respective benign control. We have used a human prostate cancer cell lines: LNCaP, C4-2, C4-2b, 22Rv1, DU145 and PC3 and normal prostate cell line RWPE-1 as well as TRAMP-C2 cell lines derived from TRAMP mice. Clonogenic activity was measured by colony formation assay. Immunoblotting and immunohistochemistry was done for TLKs, NEK1, p-ATR, p-Chk1, cell cycle and apoptosis related protein in PCa cell lines and tumor tissues from xenografts. Xenografts derived from LNCaP and LNCaP-NEK1 mutant were used in present study. We have used THD (alone) or in combination with the anti-androgen drug bicalutamide in PCa cell lines as well as in xenografts mice model.

Results: We found a significant over expression of NEK1 activity as measured by p-NEK1 with PCa samples as compare to benign control (p<0.001) and also correlated with high Gleason score patients. Treatment with THD significantly impaired the colony formation efficiency in human PCa cells in vitro (even of androgen independent lines) as well in TRAMP-C2 cells. THD treatment halts the PCa cells in subG0 phase and affects the cyclin D1, p27, p21 levels and leads to apoptosis with induction of cleaved PARP and Caspase-3 activity. Further p-ATR and p-Chk1 axis was inhibited by treatment with THD and in combination with bicalutamide. Combination therapy of THD and Bicalutamide remarkably inhibited the tumor growth in LNCaP xenografts model while tumor growth in LNCaP xenografts with NEK1 mutant were inhibited by Bicalutamide treatment alone. Following DNA damage, addition of the THD impaired ATR and Chk1 activation, that leads to TLK1>NEK1>ATR>Chk1 pathway activation in PCa.

Conclusions: Our data indicated that NEK1 is over-expressed in PCa patients and its activity can be suppressed by treatment with THD (that inhibits its activating kinase, TLK1) in vitro and in vivo.

Citation Format: Vibha Singh, Praveen K. Jaiswal, Hari K. Koul, Xiuping Yu, Arrigo Benedetti. Over expression of protein kinase Nek1 and its modulation by Thioridazine in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3063.
Abstract 3064: Epigenetic manipulation can sensitize AML cells to differentiate with ATRA
Authors: Heimdal Kalsi , Ghimire Bikalpa , Hernandez Edjay Ralph , Super Heidi J. Gill
Published: August 2019

Human acute myeloid leukemia (AML), the most common type of acute leukemia, has approximately eight subtypes, many of which have poor prognosis. Most of these subtypes are associated with specific, recurrent chromosome translocations. These translocations result in fusion genes, which encode oncoproteins that block differentiation and promote proliferation of immature cells. The Myeloid Lymphoid Leukemia gene (MLL) is frequently involved in these translocations, and is considered a driver of the AML. Differentiation promoting drugs, such as all-trans-retinoic acid (ATRA) are an attractive alternative to cytotoxic chemotherapy, but few types of AML, other than acute promyelocytic leukemia (APL), respond to ATRA. We hypothesize that specific genes must be activated or inhibited in AML for drugs like ATRA to induce differentiation, and that gene activation or inhibition may be the result of specific epigenetic modification. We also hypothesize that AML with different genetic alterations may respond differently to specific epigenetic inhibitors. Our initial studies have focused on two MLL-driven AML cell lines, MV4;11 and THP-1, showing translocations t(4;11) and t(9;11), respectively, and one non-MLL related AML cell line, U937. MV4;11, THP-1, and U937 were treated with specific epigenetic modifiers, including tranylcypromine (TCP), an inhibitor of histone demethylase KMD1A/LSD1, N-acetyl-dinaline (CI-994), a general histone deacetylase inhibitor, and 3-deazaneplanocin A (DZNep), a S-adenosylhomocystein hydrolase inhibitor that depletes EZH2 and thus its associated H3K27me3 activity. Evidence for differentiation was noted in a variety of assays, including reduced cell proliferation as measured directly and by MTT assay, upregulation of myeloid-specific cell surface markers such as CD11b as measured by fluorescence-activated cell sorting (FACS), myeloid-related nuclear morphological changes noted with cytospin analysis of cells, and decreased AML-associated gene expression with qPCR. All three drugs seemed to sensitize U937 and THP-1 cells to differentiate when treated with ATRA. MV4;11 cells were sensitized to differentiate by CI-994 and DZNep with or without ATRA, although there was no indication of CD11b upregulation. However, MV4;11 was not sensitized by TCP to differentiate with or without ATRA. These experiments suggest epigenetic inhibitors may increase sensitivity to ATRA differentiation therapy, but since the two MLL cell lines responded differently the response may still be dependent on the specific MLL partner gene driving the AML.

Citation Format: Kalsi Heimdal, Bikalpa Ghimire, Edjay Ralph Hernandez, Heidi J. Gill Super. Epigenetic manipulation can sensitize AML cells to differentiate with ATRA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3064.