Abstract 6059: Targeting the anthracycline metabolizing enzyme AKR1C3 in adipocytes to improve cytotoxicity

Abstract

Abstract Introduction: We have reported that mouse and human adipocytes take up and metabolize the anthracycline, daunorubicin (DNR), reducing its concentration in the adipocyte microenvironments. This may contribute to anthracycline resistance for cancers, which reside in adipocyte rich environments such as omentum and bone marrow. Adipocytes express several carbonyl- and aldo-keto reductases (CBRs and AKRs) which metabolize and inactivate anthracyclines, and it is unclear which of these might be important targets to improve treatment outcome. Experimental Procedures: We knocked out AKR1C3 in the human preadipocyte cell line Chub S7 using CRISPR/Cas9 gene editing technology. We chose AKR1C3 first as it is one of the overexpressed enzymes in adipocytes with the highest anthracycline-metabolizing activity. We delivered ribonucleoprotein complexes of CRISPR-Cas9 enzyme plus guide RNAs by nucleofection. Then we established single-cell derived clones and tested for successful KO by Western blot. Finally, we quantified adipocyte lysate AKR activity using a colorimetric assay based on NADPH-dependent reduction of phenanthrenequinone. Data Summary: We chose three Chub S7 preadipocyte clones that demonstrated successful AKR1C3 knockout based on almost undetectable protein expression by western blot. AKR activity was significantly reduced in all three clones; control preadipocytes had 44±4.7, while clones had activity of 28±7.3, 33±3.4, and 35±5.7 pmol/min/µg (p<0.05 for all comparisons, n=3 for each assay). Conclusions: These findings demonstrate that AKR1C3 knockout can be successfully done in human preadipocytes using a CRISPR-Cas9 system. Knockout of AKR1C3 significantly reduces overall aldoketoreductase activity in preadipocyte cells. This implies that a substantial portion of preadipocyte AKR activity is dependent on the isoenzyme, AKR1C3. Future testing is needed to determine whether AKR1C3 KO will reduce the clearance of anthracyclines from the cancer microenvironment, and may represent a treatment target to enhance anthracycline cytotoxicity. Citation Format: Vladislava Paharkova, Etan Orgel, Michael Neely, Stan. Louie, Steven Mittelman. Targeting the anthracycline metabolizing enzyme AKR1C3 in adipocytes to improve cytotoxicity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6059.