Abstract 2825: Stromal cell derived IL6 inhibits the extrinsic apoptotic pathway in multiple myeloma cell lines
Authors: Ackley James C. , McCachren Samuel , Lonial Sagar , Green Damien J. , Riddell Stanley R. , Hill Geoffrey R. , Dhodapkar Madhav V. , Boise Lawrence H. 
Published: June 2022

Abstract
Multiple myeloma (MM) is a disease of malignant plasma cells that resides in the bone marrow microenvironment (BMM). While advances in therapy have improved patient outcomes, the majority of patients relapse. Clinical trials have shown that BCMA targeted CAR-T therapy induces MM remission in most patients with heavily pretreated relapsed refractory MM; however, the duration of the response has been disappointing. CAR-T cells induce target cell death through two primary mechanisms, granzyme B and FASL. To define the role of FAS-induced cell death in CAR-T therapy in MM, we first evaluated if FAS-induced cell death was important for CAR-T activity in MM. We utilized CRISPR-Cas9 to generate FAS KOs in three MM cell lines: RPMI8226, KMS18, and OCIMY5. These lines were utilized in vitro in cytotoxic CAR-T assays where MM cells were cultured with BMCA CAR-T cells at five effector to target (E:T) ratios (range: 0:1 to 1:1). The loss of FAS protected KMS18 and OCIMY5 from CAR-T induced cell death, changing the E:T ratio needed to induce 50% MM cell death 3.2 and 3.4 fold respectively, but did not protect RPMI8226. We determined the expression of FAS negatively correlated with the EC50 of rFASL, suggesting that FAS expression levels are important for this response. To determine the role of the intrinsic apoptotic pathway in FASL-induced death, we knocked out BAK and BAX in KMS12 PE and OCIMY5. This did not protect KMS12 PE or OCIMY5 from rFASL-induced cell death, suggesting that FAS uses the type I mitochondria-independent pathway in these cell lines. The BMM supports MM progression and promotes drug resistance in part by altering the apoptotic threshold. Therefore, we examined the impact of the BMM on FASL-induced cell death. Coculture of MM cells with the HS5 stromal cell line protected KSM12 PE and KMS18 from rFASL, with the EC50 of rFASL changing 2.72 and 3.1 fold respectively, but did not protect OCIMY5. To determine if the protection observed was cell contact dependent, or only required soluble factors, we utilized HS5 conditioned media (CM). HS5 CM protected KMS12 PE, KMS18, and surprisingly OCIMY5 from rFASL, causing a change in EC50 from control of 1.65 - 2.23 fold. CM lacking IL6 did not protect MM cells from rFASL. Therefore, we tested the effects of IL6 addition IL6(10 ng/ml) and observed protection from rFASL in KMS12 PE and KMS18 cells with a change in EC50 of 2.03 and 3.84 fold respectively; however, we did not see protection in OCIMY5. To identify the mechanism of IL6-induced resistance to rFASL, we cultured BAK/BAX KO cells in HS5 CM and observed protection of these apoptosis-deficient KMS12 PE and OCIMY5 cells from rFASL. This suggests that soluble factors in the BMM can convert cells from type I to type II (mitochondrial-dependent) death receptor signaling and that inhibiting BMM signals such as IL6 may enhance FASL-induced cell death in MM. However, the data also demonstrate that the role of FAS in CAR-T killing is cell context dependent.
Citation Format: James C. Ackley, Samuel McCachren, Sagar Lonial, Damien J. Green, Stanley R. Riddell, Geoffrey R. Hill, Madhav V. Dhodapkar, Lawrence H. Boise. Stromal cell derived IL6 inhibits the extrinsic apoptotic pathway in multiple myeloma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2825.