Chemogenomic screening Identifies the Hsp70 Co-chaperone HDJ2 as a Hub for Anticancer Drug Resistance

Abstract

24Heat shock protein 70 (Hsp70) is an important molecular chaperone that regulates 25 oncoprotein stability and tumorigenesis. However, attempts to develop anti-chaperone 26 drugs targeting molecules such as Hsp70 have been hampered by toxicity issues. Hsp70 27 is regulated by a suite of co-chaperone molecules that bring "clients" to the primary 28 chaperone for efficient folding. Therefore, rather than targeting Hsp70 itself, here we have 29 examined the feasibility of inhibiting the co-chaperone HDJ2, a member of the J domain 30 protein family, as a novel anticancer strategy. We found HDJ2 to be upregulated in a 31 variety of cancers, suggesting a role in malignancy. To confirm this role, we screened the 32 NIH Approved Oncology collection for chemical-genetic interactions with loss of HDJ2 in 33 cancer. 41 compounds showed strong synergy with HDJ2 loss, whereas 18 dramatically 34 lost potency. Several of these hits were validated using a HDJ2 inhibitor (116-9e) in 35 castration-resistant prostate cancer cell (CRPC) and spheroid models. Taken together, 36 these results confirmed that HDJ2 is a hub for anticancer drug resistance and that HDJ2 37 inhibition may be a potent strategy to sensitize cancer cells to current and future 38 therapeutics. 39 40 41 42 104 Materials and Methods 105 Cell culture. The HAP1 Chronic Myelogenous Leukemia cancer cell line and HDJ2 106 Knockout cell line was purchased from Horizon Discovery and were cultured in Iscove's 107 Modified Eagle Medium (Invitrogen) with 10% fetal bovine serum (Gibco), 100 units/ml 108 penicillin, and 100 μg/ml streptomycin at 5% CO2 and 37° C. The LNCaP cancer cell line 109 was purchased from ATCC and were cultured in RPMI-1640 medium (Invitrogen) with 110 10% fetal bovine serum (FBS, Clontech), 100 units/ml penicillin, and 100 μg/ml 111 streptomycin at 5% CO2 and 37° C. 112 6 Drug Screening. Approved Oncology Drug plates consisting of the most current FDA 113 approved anticancer drugs were obtained from National Cancer Institute (NCI). For 114 experiments delineating the synergy between the loss of HDJ2 and approved anticancer 115 drug, HAP1 cells and HAP1 (HDJ2 KO) cells were plated in growth media at 20% 116 confluency 1 day prior to drug treatment. On Day 1 of treatment, cells were treated with 117 DMSO (control), Approved oncology anticancer drugs at 50 µM for 72 hours. Following 118 drug treatments, Cell Titer-Glo reagent was added directly to the wells according to 119 manufacturer's instructions. The luminescence was measured on Bio-Tek Plate reader. 120 Luminescence reading was normalized to and expressed as a relative percentage of the 121 plate averaged DMSO control. The data shown are the mean and SEM of three 122 independent biological replicates. 123 Combination index (CI) calculations. For IC50 calculations, LNCaP cells were seeded 124 in triplicates in 96-well white bottom Nunc plates in growth media at 20% confluency 1 day 125 prior to initiation of drug treatment. On Day 1 of treatment, cells were treated with DMSO 126 (control) and ten ...

The C-terminal domain of Hsp70 is responsible for paralog-specific regulation of ribonucleotide reductase

“…The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.08.479504 doi: bioRxiv preprint 13 in mammalian cells, possibly in the hope of identifying novel ways to inhibit RNR in cancer, feasible given the role of DNAJA1 in anticancer drug resistance [59]…”

“…Although this study was carried out in yeast, Hsp70 and Hsp40 also bind RNR subunits in mammalian cells [34,35]. We envisage future studies that probe this complex interaction 13 in mammalian cells, possibly in the hope of identifying novel ways to inhibit RNR in cancer, feasible given the role of DNAJA1 in anticancer drug resistance [59] Materials and Methods…”