Combination Therapy Using CDK9 and BRD4 Inhibitors to Combat Lung Adenocarcinoma
Abstract
Methods: Specific Aim 1. To evaluate the molecular mechanisms by which CDK9 and BRD4 regulate lung cancer progression: A panel of K-Ras (A549 and H460) and EGFR (H1650 and PC9) mutant, as well as Osimertinib-resistant lung adenocarcinoma cells and four different patient-derived lung organoids will be used for these studies. Cells will be treated with inhibitors of CDK9 (SNS032, AZD4573 or LY2857785) Sand BRD4 (JQ1), alone or in combination, and analyzed for biological changes such as changes in viability, proliferation, adherence-independent growth (soft agar colony formation), self-renewal in stem cell selective medium, migration, and invasion. To assess the viability of lung organoids we will use the live/dead cell viability assay using DiOC18, following the manufacturer’s protocol. Organoids will be stained with DiOC18, treated with the inhibitors as single agents or in combination, and stained with propidium iodide prior to analysis on a fluorescent microscope. Additionally, the cells and organoids will be analyzed by immunofluorescence and confocal microscopy for changes in expression and distribution of CDK9; c-Myc; BRD4; markers of EMT (Ecadherin, fibronectin, vimentin, Slug, Snail); stemness (Sox2, Sox9, Oct4), etc. Proximity Ligation Assays (PLA) will be conducted on organoids and lung TMAs to examine the interaction between CDK9, BRD4, Myc, and its targets. An unbiased RNA-Seq (in triplicates) and metabolome analysis (in six replicates) will be conducted to examine for global changes in gene expression and metabolome, and the data will be analyzed with help from the Institutional Core Facilities and Shared Resources. The top hits from the RNA-Seq will be validated by RT-PCR. The data from metabolic studies will be analyzed on MZmine 2 to identify altered metabolites and the major metabolic pathways affected will be identified by GeneGo MetaCore pathway analysis. A ChIP-qPCR will be conducted to determine the occupancy of CDK9 and BRD4 at the proximal promoters and distal enhancer elements of c-Myc and its target genes as well as other identified relevant genes from the RNA-Seq analysis, which will be confirmed by RT-PCR analysis. Since both BRD4 and CDK9 are known to regulate chromatin remodeling, we will analyze for global changes in occupancy of H3K27Ac at the gene promoters by ChIP-Seq and identified genes confirmed by ChIP-qPCR. This study will be repeated with cells that are depleted of CDK9 and BRD4, alone or in combination, to establish specificity. Furthermore, multispectral imaging using Vectra Polaris System will be conducted on TMAs to enable analysis of multiple proteins and establish that the proteins identified under this study are indeed altered in lung cancer patients, thus validating that targeting CDK9 and BRD4 simultaneously is a viable approach to combat lung adenocarcinoma. Specific Aim 2. To determine the efficacy of CDK9 and BRD4 inhibitors, alone and in combination, in vivo. Patient-derived organoids stably expressing a luciferase gene will be orthotopically implanted into the left lung of athymic nude mice. Mice will be divided into groups of 10 each for treatment with vehicle, CDK9 inhibitor, BRD4 inhibitor or a combination of both. Tumors will be monitored once a week by in vivo imaging using IVIS 200 system. Once the mice show signs of discomfort, tumors and any visible metastasized organs will be excised and analyzed for changes in expression of CDK9, BRD4, Myc, and its targets, as well as other relevant proteins identified under Specific Aim 1. Apoptosis and proliferation will be assessed by TUNEL and Ki67 analysis. These studies will also be conducted in 129 syngeneic mice, subcutaneously implanted with 344SQ cells. Flow cytometry as well as IHC will be conducted to assess how the inhibitors, alone or in combination, affect T-cell infiltration of the tumors. Further, spleen and bone marrow will be harvested and the proportion of Tregs and active T-cells ass
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Dec 05, 2021
- Source ID
- W81XWH2110627
Entities
People
- Jaya Padmanabhan
Organizations
- H. Lee Moffitt Cancer Center & Research Institute
- United States Army