Computationally Validation Of Transcriptional Drivers And Therapeutic Combinations In CRPC

Superior Essays
Aim 3. Validation of transcriptional drivers and therapeutic combinations in CRPC.
Rationale and strategy: To experimentally validate computationally predicted drivers of CRPC, we will use loss- and gain-of-function approaches for in vitro (i.e., cell lines) and in vivo (i.e., xenograft models) experimental validation to determine whether these genes are essential for drug-resistance. Computationally inferred drug combinations will be validated for their effect on MRs’ activity and ability to govern drug resistance and tumorigenicity. Furthermore, inferred drug combinations will be analyzed using RNA sequencing of treated tumors for their synergistic action and potential benefits for patients with castration-resistant prostate cancer.
Approach
…show more content…
For this, we will utilize mouse (NKP) as well as human castration-resistant (e.g., CWR-R1, C4-2, C4-2AT6, PC3, DU145) (citation)cell lines. We will perform shRNA-mediated silencing of the activated MRs by lentiviral infection(citation), which will be confirmed by quantitative RT-PCR at 48 hours. To analyze ability of MRs to abrogate castration-resistance, we will perform analyses of cellular proliferation, using BrdU incorporation and Ki67 staining, colony formation essays and invasion essays after silencing. As a control, we will silence 3-5 genes that are neither differentially expressed nor differentially active in castration-resistant tumors and are not regulated by any MR. Conversely, MRs whose activity decreases in castration-resistant state will be tested in over-expression essays using lentiviral cDNA expression …show more content…
To overcome this limitation, we will over-express MRs of CRPC (that are not sufficiently expressed/active) in cell lines used for functional in vitro and in vivo validation in Aim 3.1 (Validation of drug combinations). Secondly, since therapeutic response is dose-dependent, we will perform a short pilot study to determine optimal drug dosages for single and combination drugs. Thirdly, we understand that survival analysis might depend on multiple factors, including age, Gleason score, pre-treatment etc. We will consider these factors in multivariate statistical analysis to evaluate independent predictive value for the transcriptional drivers of CRPC.

H. Projected Timeline
We anticipate to conduct the proposed R01 grant within a 5-year period, from July 1st 2017 to June 30th 2022 (Fig 9). We will begin with Aim 1, which will uncover transcriptional drivers of treatment failure in castration-resistant prostate cancer. While conducting Aim 1, we will start working on (i) Aim 2, where we will gather and analyze datasets with available single-agent treatment data to identify drugs and drug combinations that could be used to preclude or overcome resistance; and (ii) Aim 3.1, which will carry experimental validation of identified Figure 9. Time-line for

Related Documents

  • Superior Essays

    To examine whether these candidates are also altered in pancreatic ASC, I will analyze the RNA levels of top targets in pancreatic ASC samples that contain UPF1 mutations. To determine the function of the identified candidates from high-throughput sequencing, I will perform overexpression and knockdown experiments in primary pancreatic cell lines to examine for corresponding gain or loss in proliferation. In addition, I will determine whether overexpression of the candidates transforms primary pancreatic cells. In summary, results from this aim will elucidate the mechanism by which UPF1 mutations promote pancreatic…

    • 1047 Words
    • 4 Pages
    Superior Essays
  • Great Essays

    These steps include dendritic cells promoting tumor antigens obtained from tumor microenvironments or dead tumor cells, recruitment of activated T-cells to cause responses and circumventing immunosuppression in the tumor microenvironment (Kershaw, Devaud et. 2013). Dendritic cells can present antigens using MHC class I and II molecules by moving to the lymph nodes to expose antigens to T cells upon receiving a stimulus (Vegh , Wang et. 1993). The activated naïve T cells can migrate to region containing antigens and target cancer cells displaying these antigens.…

    • 1172 Words
    • 5 Pages
    Great Essays
  • Great Essays

    Deliverables for Specific Aim 2: Any candidate gene identified in AA PCa will be subjected to screening EA PCa cohort. Depending on the nature of the candidate gene we will choose PCR, FISH, IHC and RNA-ISH approach to study the recurrent nature. Our systematic analysis of sequencing data we will address the following questions: Among the many fusion genes in a sample, how many of them are causal aberrations (driver)? How many of them are responsible for tumor growth (passenger)? How do so many abnormal gene fusion products cooperate in a tumor environment?…

    • 1479 Words
    • 6 Pages
    Great Essays
  • Improved Essays

    Provide Statistics. Explain how microarray technology can be used to learn how genetic risks can be identified. ○ What is the purpose of microarray? The microarray technology helps scientists identify the change in cells by comparing gene expression and repression within a normal and cancer cell. ○ Explain the process of microarray In a microarray the mRNA is removed from both the infected and uninfected cell.…

    • 1004 Words
    • 5 Pages
    Improved Essays
  • Improved Essays

    The aCGH assay will be an essential tool to assess the genomic changes. Based on the results of SKY and aCGH analyses, representative CTC cultures will be subjected to mutation screening in CTC, CTD-PDX and biopsy materials by whole genome sequencing using NGS for comprehensive molecular profiling of abnormalities at the nucleotide level. NGS data will be transferred to Palanisamy, who will carry out biostatistics and bioinformatics analysis. Given the unbiased nature of NGS analysis, we anticipate identifying new druggable genomic mutations on a personalized…

    • 1048 Words
    • 4 Pages
    Improved Essays
  • Improved Essays

    The aim of gene therapy is to introduce the transgenes or gene into appropriate target cells to restore normal function, therefore, treating and preventing the disease .Similar technologies are being used in gene immunotherapy and DNA vaccines against cancer and other infectious diseases. For a long period of time, current focus with gene therapy is the need for high level of gene expression and to minimize safety issues such as immune reaction against transgene product or transfer vectors and associated cancer…

    • 994 Words
    • 4 Pages
    Improved Essays
  • Improved Essays

    T Cells Essay

    • 1316 Words
    • 6 Pages

    Scientists have demonstrated that adaptive immunity intervened by T-cell lymphocytes gives important information and support for implementing and maintaining antitumor response. (citation). a broad tumor ingression by cytotoxic CD8+ T cells was heavily correlated with patient survival and react to therapy. Hence, additional studies have observed that cancer reversion and autoimmunity caused by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma. Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is an essential immune-regulatory molecule (showed on triggered T cells and a portion of regulatory T cells) able to down-regulate T cell activation.…

    • 1316 Words
    • 6 Pages
    Improved Essays
  • Improved Essays

    To maximize the likelihood our findings are relevant to pancreatic cancer, I will collaborate with Dr. William Jarnagin’s group in the Pancreatic Center at MSKCC to examine whether the identified genes are deregulated in human patients with pancreatic ASC. To elucidate whether the shortlisted candidates have oncogenic potential, I will overexpress candidates in pancreatic cell lines and examine for increased cell proliferation and ability to transform primary cells. As a proof-of-concept that inhibiting these candidates can block pancreatic ASC progression, I will knockout selected candidates using CRISPR-Cas9 system and analyze for either increased apoptosis or reduced cellular proliferation in patient cell lines generated from pancreatic…

    • 1024 Words
    • 4 Pages
    Improved Essays
  • Superior Essays

    Detailed focus question: What is the process of self-renewal and duplication in breast cancer stem cells through E113, the Wnt-beta catenin signaling pathways and the tumor regulator p53? I. Introduction: To explain the cellular and molecular features of my focus question I will provide background on cancer stem cells, some of the various signaling pathways and breast cancer specifically. A. Cancer Stem Cells (Tumor-initiating cells) 1.…

    • 1133 Words
    • 5 Pages
    Superior Essays
  • Superior Essays

    Analysis Of HCV DNA

    • 975 Words
    • 4 Pages

    Several PNA probe combinations will be evaluated experimentally to determine the most efficient combination. For each target RNA, 2 PNA probe pairs [G-probe with C-terminus FITC and A-probe (antigen, or biotin or antibody)] will be designed and evaluated. Probe design will be performed via Primer Express version two software. The software will create probe sets designed. Probe hybridization sequences will be evaluated by BLAST to select regions conserved across multiple transcript variants.…

    • 975 Words
    • 4 Pages
    Superior Essays