Cross-species genomic and functional analyses identify a combination therapy using a CHK1 inhibitor and a ribonucleotide reductase inhibitor to treat triple-negative breast cancer

INTRODUCTION: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is diagnosed in approximately 15% of all human breast cancer (BrCa) patients. Currently, no targeted therapies exist for this subtype of BrCa and prognosis remains poor. Our laboratory has previously identified a proliferation/DNA repair/cell cycle gene signature (Tag signature) that is characteristic of human TNBC. We hypothesize that targeting the dysregulated biological networks in the Tag gene signature will lead to the identification of improved combination therapies for TNBC. METHODS: Cross-species genomic analysis was used to identify human breast cancer cell lines that express the Tag signature. Knock-down of the up-regulated genes in the Tag signature by siRNA identified several genes that are critical for TNBC cell growth. Small molecule inhibitors to two of these genes were analyzed, alone and in combination, for their effects on cell proliferation, cell cycle, and apoptosis in vitro and tumor growth in vivo. Synergy between the two drugs was analyzed by the Chou-Talalay method. RESULTS: A custom siRNA screen was used to identify targets within the Tag signature that are critical for growth of TNBC cells. Ribonucleotide reductase 1 and 2 (RRM1 and 2) and checkpoint kinase 1 (CHK1) were found to be critical targets for TNBC cell survival. Combination therapy, to simultaneously attenuate cell cycle checkpoint control through inhibition of CHK1 while inducing DNA damage with gemcitabine, improved therapeutic efficacy in vitro and in xenograft models of TNBC. CONCLUSIONS: This combination therapy may have translational value for patients with TNBC and improve therapeutic response for this aggressive form of breast cancer

Measurement of psychological entitlement in 28 countries

This article presents the cross-cultural validation of the Entitlement Attitudes Questionnaire, a tool designed to measure three facets of psychological entitlement: active, passive, and revenge entitlement. Active entitlement was defined as the tendency to protect individual rights based on self-worthiness. Passive entitlement was defined as the belief in obligations to and expectations toward other people and institutions for the fulfillment of the individual’s needs. Revenge entitlement was defined as the tendency to protect one’s individual rights when violated by others and the tendency to reciprocate insults. The 15-item EAQ was validated in a series of three studies: the first one on a general Polish sample (N = 1,900), the second one on a sample of Polish students (N = 199), and the third one on student samples from 28 countries (N = 5,979). A three-factor solution was confirmed across all samples. Examination of measurement equivalence indicated partial metric invariance of EAQ for all national samples. Discriminant and convergent validity of the EAQ was also confirmed

A Gene Expression Signature of Acquired Chemoresistance to Cisplatin and Fluorouracil Combination Chemotherapy in Gastric Cancer Patients

We initiated a prospective trial to identify transcriptional alterations associated with acquired chemotherapy resistance from pre- and post-biopsy samples from the same patient and uncover potential molecular pathways involved in treatment failure to help guide therapeutic alternatives.A prospective, high-throughput transcriptional profiling study was performed using endoscopic biopsy samples from 123 metastatic gastric cancer patients prior to cisplatin and fluorouracil (CF) combination chemotherapy. 22 patients who initially responded to CF were re-biopsied after they developed resistance to CF. An acquired chemotherapy resistance signature was identified by analyzing the gene expression profiles from the matched pre- and post-CF treated samples. The acquired resistance signature was able to segregate a separate cohort of 101 newly-diagnosed gastric cancer patients according to the time to progression after CF. Hierarchical clustering using a 633-gene acquired resistance signature (feature selection at P<0.01) separated the 101 pretreatment patient samples into two groups with significantly different times to progression (2.5 vs. 4.7 months). This 633-gene signature included the upregulation of AKT1, EIF4B, and RPS6 (mTOR pathway), DNA repair and drug metabolism genes, and was enriched for genes overexpressed in embryonic stem cell signatures. A 72-gene acquired resistance signature (a subset of the 633 gene signature also identified in ES cell-related gene sets) was an independent predictor for time to progression (adjusted P = 0.011) and survival (adjusted P = 0.034) of these 101 patients.This signature may offer new insights into identifying new targets and therapies required to overcome the acquired resistance of gastric cancer to CF

Abstract 4734: Genes cooperatively downregulated by combined mTOR/histone deactylase (HDAC) inhibition are overexpressed in myeloma patients with lower survival

Abstract The molecular pathogenesis of many cancer types, including multiple myeloma (MM) and mantle cell lymphoma (MCL), involves alterations in the PI3K/Akt/mTOR and cyclin/CDK/CDKI/Rb (Rb) pathways. Previously, we showed that the combination of an HDAC inhibitor (HDACi) with rapamycin synergistically inhibited proliferation in 88% of human MM cell lines tested, and effectively controlled tumor growth in preclinical studies. To gain an initial understanding of the molecular mechanism of the synergistic action of the drug combination, we used an unbiased systems-level approach to analyze our gene expression profile (GEP) data with weighted gene co-expression network analysis (WGCNA). This analysis delineated the contribution of HDACi and rapamycin, singly and in combination, to the overall gene expression change of the combination by considering not only measures of fold-change and significance testing, but also the degree of gene expression inter-connectedness. WGCNA identified five gene modules, each representing a particular gene expression effect of the combination. Each gene module was individually tested for functional and clinical enrichment using gene set enrichment analysis (GSEA) and survival analyses with Cox regression. Of particular interest, the module containing genes cooperatively affected by both compounds was highly enriched (p<0.001) for genes involved in cell cycle (especially mitotic processes), immune recognition, and DNA damage/repair, which we investigated further. Genes down-regulated by the drug combination were most significantly correlated with genes over-expressed in MM patients. Furthermore, analysis of the cooperative drug signature in publicly available patient GEP datasets with survival annotation found it predictive of increased survival (p<0.01), thus linking the drug combination-induced transcriptional changes to predictions for enhanced survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4734. doi:1538-7445.AM2012-473

Investigation into diagnostic agreement using automated computer-assisted histopathology pattern recognition image analysis

The extent to which histopathology pattern recognition image analysis (PRIA) agrees with microscopic assessment has not been established. Thus, a commercial PRIA platform was evaluated in two applications using whole-slide images. Substantial agreement, lacking significant constant or proportional errors, between PRIA and manual morphometric image segmentation was obtained for pulmonary metastatic cancer areas (Passing/Bablok regression). Bland-Altman analysis indicated heteroscedastic measurements and tendency toward increasing variance with increasing tumor burden, but no significant trend in mean bias. The average between-methods percent tumor content difference was -0.64. Analysis of between-methods measurement differences relative to the percent tumor magnitude revealed that method disagreement had an impact primarily in the smallest measurements (tumor burden <3%). Regression-based 95% limits of agreement indicated substantial agreement for method interchangeability. Repeated measures revealed concordance correlation of >0.988, indicating high reproducibility for both methods, yet PRIA reproducibility was superior (C.V.: PRIA = 7.4, manual = 17.1). Evaluation of PRIA on morphologically complex teratomas led to diagnostic agreement with pathologist assessments of pluripotency on subsets of teratomas. Accommodation of the diversity of teratoma histologic features frequently resulted in detrimental trade-offs, increasing PRIA error elsewhere in images. PRIA error was nonrandom and influenced by variations in histomorphology. File-size limitations encountered while training algorithms and consequences of spectral image processing dominance contributed to diagnostic inaccuracies experienced for some teratomas. PRIA appeared better suited for tissues with limited phenotypic diversity. Technical improvements may enhance diagnostic agreement, and consistent pathologist input will benefit further development and application of PRIA

Abstract 2217: A systems pharmacogenomic approach to identify synergistic molecular mechanisms of combined mTOR/HDAC inhibition

Abstract The necessity of combining targeted therapeutics to achieve optimal, lasting clinical benefit is clear, but standardized approaches for identifying the interactive effects of these combinations are not yet established. Discerning drug synergy at the molecular level has proven particularly challenging, yet identification of cooperatively responding, biologically-relevant targets could be useful for defining patient subsets for which the combination would be active. Here we used a transcriptional co-expression systems-level analysis to define the cooperative molecular response to the synergistic combination of mTOR/HDAC inhibitors in multiple myeloma (MM), and in other tumor types including triple negative breast cancer. Co-expression analysis of cells treated individually and in combination defined the contribution of each drug to the combination, and identified a distinct network of 126 genes cooperatively targeted by both drugs. We interrogated the cooperative network genes for differential expression between normal and malignant cells, as well as for correlation with survival in a large patient dataset. 37 of the cooperatively affected genes were both differentially expressed in MM and predictive of survival (p<0.01). Analysis of additional tumor types showed similar results. The pharmacodynamic response of the survival-linked signature to the drug combination was evaluated using the NanoString gene expression platform in a large number of cell lines from multiple tumor types and in ex vivo-treated primary patient samples before and after treatment. We found the expression change of signature genes to be highly specific for biological response to the drug combination across tumor types. Additionally, to link the response signature to a central molecular effect of combination treatment, Ingenuity transcription factor enrichment testing was performed. Based on these predictions, subsequent analysis of CHIP-Seq datasets was performed, and two oncogenic transcription factors (TFs) were found to bind nearly all genes of this signature. We then experimentally linked drug combination response to diminished expression of these TFs at the protein level ahead of cell cycle and apoptotic changes. Further experiments have been performed to establish a direct link between these TFs, our gene signature, and drug response. Thus, a systems-level genomic approach has identified a gene signature indicative of drug combination activity, mechanism, disease specificity, and clinical potential. Citation Format: John K. Simmons, Aleksandra M. Michalowski, Ben Gamache, Jyoti Patel, Adriana Zingone, Ke Zhang, Michael Kuehl, Jing Huang, Ola Landgren, Beverly A. Mock. A systems pharmacogenomic approach to identify synergistic molecular mechanisms of combined mTOR/HDAC inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2217. doi:10.1158/1538-7445.AM2013-221

Abstract 5850: Nrf2-mediated oxidative stress response is altered during acquired resistance to the proteasome inhibitor, oprozomib, in multiple myeloma

Abstract Multiple myeloma (MM) is a hematologic neoplasm characterized by malignant proliferation of plasma cells in the bone marrow. Proteasome inhibitors are widely used in treatment regimens for MM. Although initial responses to PI (e.g., bortezomib, carfilzomib) treatments have been promising, patients often develop resistance and become refractory to disease. Understanding molecular alterations in signaling cascades influenced by proteasome inhibitors and mechanisms underlying acquired resistance is needed. In this study, we have established a clinically relevant oproxomib-resistant subline (KMS28BMONYX) of the MM cell line KMS28BM. The KMS28BMONYX cell line is pan-resistant to PIs with a 10-fold increase in IC50 for oprozomib as compared to the parental line. To identify genes involved in modulating drug resistance, we analyzed gene expression profiles of both parental and resistant cell lines using the Affymetrix GeneChip Human Genome U133 Plus 2.0 array. Ingenuity Pathway Analysis of microarray data comparing the parental and resistant cells revealed an acute dependence on stress response proteins to maintain PI-resistance. Activation of nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2; gene symbol NFE2L2) coupled with elevated levels of sequestosome 1/p62 (SQSTM1/p62) were prominent features of the KMS28BMONYX cell line. Altered levels of SQSTM1 correlated with resistance to oprozomib in several MM cell lines. Simultaneously, the KMS28BMONYX cell line showed increased expression of MYC and MCL1. Oprozomib treatment stabilized c-Myc expression in the KMS28BMONYX line. The Champion ChiP Transcription Factor Search Portal database DECODE predicted two c-Myc transcription factor binding sites in the SQSTM1 promoter. CHIP-seq data for MYC in MM1s cells also indicates strong binding in the promoter region of SQSTM1. Our data suggest that therapies targeting the SQSTM1/p62-Nrf2 pathway may help overcome proteasome inhibitor resistance in refractory MM patients. Citation Format: Snehal M. Gaikwad, Adriana Zingone, Aleksandra Michalowski, Susana Najera, Anaisa Quintanilla-Artega, Sayeh Gorjifard, John Simmons, Nick Watson, Ola Landgren, Jing Huang, Beverly Mock. Nrf2-mediated oxidative stress response is altered during acquired resistance to the proteasome inhibitor, oprozomib, in multiple myeloma [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 5850

RasGRP3 Contributes to Formation and Maintenance of the Prostate Cancer Phenotype

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A Small Molecule Stabilizer of the MYC G4-Quadruplex Induces Endoplasmic Reticulum Stress, Senescence and Pyroptosis in Multiple Myeloma

New approaches to target MYC include the stabilization of a guanine-rich, G-quadruplex (G4) tertiary DNA structure in the NHE III region of its promoter. Recent screening of a small molecule microarray platform identified a benzofuran, D089, that can stabilize the MYC G4 and inhibit its transcription. D089 induced both dose- and time-dependent multiple myeloma cell death mediated by endoplasmic reticulum induced stress. Unexpectedly, we uncovered two mechanisms of cell death: cellular senescence, as evidenced by increased levels of p16, p21 and &gamma;-H2AX proteins and a caspase 3-independent mechanism consistent with pyroptosis. Cells treated with D089 exhibited high levels of the cleaved form of initiator caspase 8; but failed to show cleavage of executioner caspase 3, a classical apoptotic marker. Cotreatment with the a pan-caspase inhibitor Q-VD-OPh did not affect the cytotoxic effect of D089. In contrast, cleaved caspase 1, an inflammatory caspase downstream of caspases 8/9, was increased by D089 treatment. Cells treated with D089 in addition to either a caspase 1 inhibitor or siRNA-caspase 1 showed increased IC50 values, indicating a contribution of cleaved caspase 1 to cell death. Downstream effects of caspase 1 activation after drug treatment included increases in IL1B, gasdermin D cleavage, and HMGB1 translocation from the nucleus to the cytoplasm. Drug treated cells underwent a &lsquo;ballooning&rsquo; morphology characteristic of pyroptosis, rather than &lsquo;blebbing&rsquo; typically associated with apoptosis. ASC specks colocalized with NLRP3 in proximity ligation assays after drug treatment, indicating inflammasome activation and further confirming pyroptosis as a contributor to cell death. Thus, the small molecule MYC G4 stabilizer, D089, provides a new tool compound for studying pyroptosis. These studies suggest that inducing both tumor senescence and pyroptosis may have therapeutic potential for cancer treatment