In the last decade taxane-based therapy has emerged as a standard of care for hormone-refractory prostate cancer. Nevertheless, a significant fraction of tumors show no appreciable response to the treatment, while the others develop resistance and recur. Despite years of intense research, the mechanisms of taxane resistance in prostate cancer and other malignancies are poorly understood and remain a topic of intense investigation. We have used improved mutagenesis via random insertion of a strong promoter to search for events, which enable survival of prostate cancer cells after Taxol exposure. High-throughput mapping of the integration sites pointed to the PRKAR2A gene, which codes for a type II-α regulatory subunit of protein kinase A, as a candidate modulator of drug response. Both full-length and N-terminally truncated forms of the PRKAR2A gene product markedly increased survival of prostate cancer cells lines treated with Taxol and Taxotere. Suppression of protein kinase A enzymatic activity is the likely mechanism of action of the overexpressed proteins. Accordingly, protein kinase A inhibitor PKI (6-22) amide reduced toxicity of Taxol to prostate cancer cells. Our findings support the role of protein kinase A and its constituent proteins in cell response to chemotherapy.
BackgroundT-cell receptor (TCR) and B-cell receptor (BCR) repertoire profiling holds great potential for understanding disease mechanisms and for the development of new therapeutics in infectious diseases, autoimmunity and in immuno-oncology. However, this potential could be greatly improved by combining information about receptor clonotypes with immuno-phenotypes of T and B cells.MethodsTo facilitate these studies, we developed a novel technology for combined profiling of all human TCR and BCR variable regions and phenotypic characterization of immune cells in bulk and at the single-cell level in PBMC and immune cell fraction samples. The developed TCR/BCR Immunophenotyping method involves multiplex RT-PCR amplification and sequencing of CDR3 regions of TCR and BCR genes and a set of the most informative T- and B-cell phenotyping genes. Bioinformatics analysis of NGS data allows profiling of TCR/BCR clonotypes, and identification of major immune cell subtypes and their activation status.ResultsData will be presented showing how combined TCR/BCR clonotype analysis combined with targeted expression profiling of immune cells can be applied for large-scale discovery of novel cell typing and activation biomarkers in several immune-responsive model systems.ConclusionsPreliminary studies demonstrate the assay has unparalleled throughput, sensitivity, and improved cost-effectiveness for high-throughput immunity biomarker discovery applications.
Introduction: Selecting patients predisposed to respond to existing and novel immunotherapy treatments requires the development of novel prognostic and predictive biomarkers. Recent reports have identified several gene expression signatures specific for immunity status and immune contexture in solid tumor microenvironments, and enable predictions of efficacy of a number of chemo- and immunotherapeutics. We used these expression signatures to develop a robust standardized assay—the Cancer Immunotherapy 2000 (CI2000) panel—that would dissect immunosurveillance mechanisms and discover novel prognostic and predictive immune response biomarkers. Material and Method: The CI2000 panel provides signatures for approximately 400 immunity-related genes from 16 predictive and prognostic core genes that have been validated in recent chemo- and immunotherapy clinical trials across several tumor types, including melanoma, colorectal, breast, and lung cancers. In addition, using computation analysis of immunotherapy network model, we predicted key nodes in pathways specific for antigen presentation and recognition, inhibition, activation and motility of immune cells, adhesion, and apoptosis of cancer cells. This computational analysis produced approximately 400 more targets beyond the core panel. The CI2000 panel also includes a comprehensive set of 1,200 genes specific for detection and quantitative profiling in the tumor microenvironment of different types of activated immune cells of adaptive and innate immunity. Further, an additional set of housekeeping genes with constant expression between different cancer types was included in the assay for data normalization. Results and Discussion: The CI2000 assay quantitatively profiles the expression levels of approximately 2,000 key cancer genes from 10-100ng of total RNA using multiplex RT-PCR amplification followed by next-generation HT sequencing (NGS). Built-in standards for each gene target enable sample-to-sample calibration of the NGS data and provide a reference to adjust for background noise which often depends on the quality of samples. Control studies have shown that the CI2000 assay quantifiably measures 4 orders of magnitude variation in the expression levels of 2,000 key cancer immune-related genes from as few as 100 cells in whole lysate, and profiles from frozen biopsies, surgically-removed tumor samples, PBMCs, and FFPE clinical samples show a comparable range and sensitivity. We will present profiling data from infiltrating immune cells and key intact and deficient immune mechanisms in the tumor microenvironment of breast cancer samples to demonstrate the performance and utility of the CI2000 assay. Conclusions: Comprehensive profiling of tumor-associated immune cells with the CI2000 gene panel provides a sensitive, standardized, and quantitative method to investigate cancer immunoediting mechanisms. Robust methods for molecular characterizations of the immune mechanism in the tumor microenvironment are essential to meet the imminent need for diagnostic approaches that identify patient population responsive to the growing number of immunotherapeutic treatments. Toward this goal, the described CI2000 assay provides a platform for the discovery prognostic and predictive immune response biomarker signatures. Citation Format: Alex Chenchik, Leonid Iakoubov, Michael Makhanov, Costa Frangou. Cancer immunotherapy biomarker profiling assay. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A010.
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