Therapeutic targets and novel anticancer agents for endocrine cancers

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

1. Identification of novel synergistic drug combination therapy for ACC 1.1 after publishing the MELK and CDK1-2 inhibitor, we were unable to identify a CDK1-2 inhibitor to replace RGB286638. Thus, a clinical trial development is on hold. 1.2 The novel synergistic combination of PI3K and HSP90 inhibitors in ACC. Status: The manuscript was revised and resubmitted. Aim: To identify and validate the effective novel synergistic drug combinations using PI3K inhibitor and Heat-shock protein 90 inhibitors and to study the molecular mechanisms of synergy identified by the computerized drug combination matrix screening in ACC cells. We analyzed publicly available databases and found that ACC overexpressed several isoforms of HSP90 and its clients involved in ACC initiation and progression. We validated the synergy in two ACC cell lines in a cell proliferation assay. ACC 3D tumor spheroid assays showed increased efficacy in the combination treatment group. Because PIK75 is not yet available for a clinical trial, we identified and validated the synergy between BGT226 and STA9090 or with HSP990 in multiple in vitro models at clinically achievable concentrations. We discovered that the synergistic mechanisms of cell death were completely different between PIK75 and BGT226. PIK75 combination induced G2M cell cycle arrest, followed by caspase-3/7 dependent apoptosis. We validated the treatment effects on PI3K/AKT/mTOR signaling pathway. RNA-seq data of ACC cells treated with these combinations showed BGT226 combination induced autophagy (not apoptosis), which we validated. We successfully validated the efficacy of BGT26 and STA9090 in patient-derived organoids (n=5) and in human ACC xenografts. 2. The identification of radiosensitizer and oxidative stress response in ACC. STATUS: The manuscript is in revision. Aim: To evaluate the effects and mechanism of action of auranofin in combination with radiotherapy (RT) in ACC, Summary: Most patients with ACC develop locoregional recurrence after surgery. After identifying auranofin from a quantitative high-throughput drug screening in two ACC cell lines as a novel radiosensitizer with a potent cytotoxic effect. The analyses of the independent databases of ACC samples showed the mRNAs of several genes involved in oxidative stress response, including the TXNRD pathway, targetable by auranofin, are differentially expressed in ACC. We showed downregulation of several anti-oxidative stress-related genes was inversely correlated with overexpression of MKi67 and CCNB1 (poor prognostic markers). We confirmed the synergistic activity of auranofin and RT in multiple in vitro models at clinically achievable concentrations, and in human ACC xenografts with inhibition of TXNRD activity. We observed that NCI-H295R cells (TP53 and CTNNb1 mutations) treated with RT and auranofin developed oxidative stress-induced ds-DNA break and repair, mitotic catastrophe, and PARP-induced apoptosis. However, SW13 cells (TP53 and SMARCA4 mutations) were more resistant to the combined treatment with ferroptosis as the key mechanism of cell death while inducing resistance via NRF2/KEAP1 nuclear translocation and TXNRD1 and HMOX1 overexpression. We excluded the role of corticosteroids in radiosensitivity in ACC using various treatments. We are in the process of knocking down SMARCA4 in NCI-H295R and two new ACC cell lines to test the effect on radiosensitivity for the revised manuscript. 2.2 Multi-Omic approach to identify genes and pathways involved in radioresistance in ACC. We completed bulk RNA-sequencing, total and phosphoproteomics studies of NCI-H295R receiving 2-week fractionated RT with a dosing scheme similar to patients to understand the genes and pathways. KPNA2 is the target gene that we pursue but the transient knockdown was too toxic to the cells and we are working on stable transfection. We have prepared CAS-9 stable-NCI-H295R for genome-wide CRISPR-KO screen. 3. Mechanism of TNF-a-related changes in the thyroid cancer microenvironment to enhance drug delivery Status: manuscript preparation is nearly completed but we discovered a cell line mixup and are repeating several experiments. Aim: to elucidate the mechanism of action of TNF-a in reducing tumor interstitial pressure leading to improved drug delivery efficiency. I. Summary: we found that TGF-b signaling played a central role in TNF-a-related improved drug delivery by downregulating LOX, decreasing hyaluronan (HA) synthesis, and increasing HA degradation. We confirmed the role of TGF-b and LOX in regulating vascular permeability via VEGF-R and VEGF-A secretion. To confirm the efficacy of the treatments using recombinant TNF-a and TGFb inhibition, we combined the TME treatments with paclitaxel in two iodine-resistant poorly differentiated (PDTC) (TPC1) and anaplastic thyroid cancer (ATC) (8505C) 3D tumor spheroids and found that the anti-tumor efficacy was higher in the combination treatment groups than single-treatment groups. Furthermore, we demonstrated the higher intratumoral penetration of fluorescent-labeled paclitaxel when thyroid cancer spheroids were treated with TGFb inhibitors and LOX inhibition (siLOX and BAPN which inhibits LOX enzymatic activity) and the intratumoral paclitaxel concentration was severalfold higher than the paclitaxel-only group. We validated the enhanced intratumoral paclitaxel delivery in vivo using HPLC by treating 8505C xenografts with nanogold particles carrying TNF-a and the TGFb inhibitor. Similar improvements in drug delivery efficiency and treatment efficacy can be seen in much denser co-cultured thyroid cancer spheroids with cancer-associated fibroblasts. 4. Characterization of a new ACC cell line: BD140A. We performed sc-RNA-seq, Whole genome sequencing, steroid profiling with ACTH and foskolin stimulation, and drug treatments. We are analyzing the results. 5. Characterization of ACC cancer stem cell (CSCs). We aim to overcome ACC treatment resistance by targeting ACC CSCs. Because ACC CSCs are not yet characterized and we hypothesize that ACC CSCs have different CSC markers depending on driver mutations, we first need to characterize ACC CSCs. Because cell lines have most enriched CSCs, we use 3 human ACC cell lines with different mutations and BD140A to characterize CSCs. We will use multiplexed mass cytometry with time of flight (CyToF) to characterize and isolate ACC CSCs to determine the oncogenic phenotypes and sensitivity to treatments in vitro and in vivo. We are validating 13 CyToF-ready antibodies in 6 non-ACC positive control cell lines.