Perforin-Induced Killing in Leukemic Cells Is Driven By NFAT2-Mediated Cytoskeletal Remodeling

Cytoskeletal remodeling is critical for the migration, infiltration, metastasis, and overall aggressiveness of malignant cells. In healthy cells, cytoskeletal remodeling also protects cytotoxic lymphocytes from the contents of their own cytotoxic granules. In immunotherapeutic settings, such as administration of the anti-CD20 antibody rituximab in chronic lymphocytic leukemia (CLL) therapy, cytoskeletal remodeling prevents perforin/granzyme-induced lysis in antibody-dependent cellular cytotoxicity (ADCC). We have previously shown that loss of the transcription factor NFAT2 (NFATc1) in CLL cells correlates with an aggressive disease course. Since NFAT2 is also involved in the regulation of cytoskeletal reorganization of immune cells, we here investigated its involvement in the susceptibility of CLL cells to perforin-induced lysis.

To this end, we generated a CRISPR/Cas9-based NFAT2 knockout (KO) in MEC-1 cells. Cytotoxicity assays revealed a profoundly increased resistance of NFAT2 KO cells to NK cell killing compared to scrambled control (SCR) cells, without affecting the level of recognition as revealed by analysis of NK cell activation, degranulation and IFNy release. Treatment of MEC-1 cells with NK cell isolates containing cytotoxic granule content revealed increased resistance of NFAT2 KO cells to membrane permeabilization as compared to SCR controls, with perforin blockade offsetting the susceptibility to membrane permeabilization, confirming the involvement of NFAT2 in maintaining membrane integrity.

Next, we investigated the role of NFAT2 in the Eµ-TCL1 mouse model of CLL by inducing a conditional B cell-specific NFAT2 knockout. We have previously shown that TCL1 NFAT2 KO mice have significantly reduced overall survival compared to wildtype TCL1 mice, confirming the role of NFAT2 in poorer CLL outcome. Using this model, we found that CLL cells from TCL1 NFAT2 KO mice were less susceptible to perforin-mediated lysis compared to those from TCL1 mice. In line, leukemic cells from CLL patients classified according to their NFAT2 transcript levels showed that low NFAT2 levels correlated with higher resistance to perforin-mediated lysis and reduced perforin binding in the cell membrane, further confirming our findings obtained in MEC-1 and murine CLL cells.

In turn, inhibition of CDC42, a key regulator of actin cytoskeleton remodeling, with ZCL278 sensitized MEC-1 NFAT2 KO cells to perforin-mediated lysis. Similar effects were observed with the tubulin cytoskeletal stabilizer paclitaxel, providing further evidence that cytoskeletal remodeling is critical for perforin resistance.

Taken together, our results demonstrate that loss of NFAT2 allows CLL cells to evade NK cell effector function. This is true for both constitutive cytotoxicity and therapeutically induced ADCC and is due to a reduced susceptibility to perforin-mediated membrane permeabilization that can be overcome by inhibition of cytoskeletal rearrangement. Thus, the loss of NFAT2 facilitates the resistance of CLL cells to immunotherapeutic treatment, which could be exploited in the future for therapeutic modulation to enhance efficacy.

Heitmann:Bicony Therapeutics GmbH: Current Employment. Salih:TWYCE GmbB: Current equity holder in private company; Philogen S.p.A.: Membership on an entity's Board of Directors or advisory committees; BiConY GmbH: Consultancy; Synimmune GmbH: Research Funding.