Abstract 4990: Genotoxic chemotherapy impedes antibody-mediated complement dependent cytotoxicity (CDC), via a Chk1-CD59 axis
Authors: Chan Allison S. , Jaynes Patrick W. , Mustafa Nurulhuda B. , Anbuselvan Akshaya A. , Ong Charmaine Z. , Jeyasekharan Anand D. 
Published: March 2024

Monoclonal antibodies such as rituximab exert targeted tumor cell lysis via complement dependent cytotoxicity (CDC). These antibodies are often combined with chemotherapy in clinical treatment protocols. However, the impact of chemotherapy on cell-intrinsic pathways that regulate antibody-mediated CDC is not known. Through a phage display screen, we identified that genotoxic stress caused upregulation of a membrane complement regulatory protein (mCRP), CD46, prompting further investigation into its implications and mechanism.

Using cell lines from diffuse large B cell lymphoma (DLBCL) as our model, we confirmed that genotoxic chemotherapy upregulates CD46, as well as other mCRPs such as CD55 and CD59. We hypothesized that upregulated mCRPs could impede Rituximab-mediated CDC. We assessed this in-vitro using rituximab and complement human serum. Aligned with our hypothesis, chemotherapy treatment of DLBCL cells significantly impeded rituximab-induced CDC. Using blocking antibodies to each mCRP, reversal of the effect of chemotherapy on CDC only occurred after co-treatment with a CD59 blocking antibody, suggesting that CD59 is a key player for chemotherapy-invoked resistance to CDC.

To further elucidate the underlying cellular pathway, we performed a high-throughput small molecule screen using rituximab-induced CDC as a readout, with or without cotreatment of a genotoxic chemotherapeutic (etoposide). We identified Chk1 inhibitors as consistent top rescuers. qPCR and flow cytometry revealed that Chk1 inhibition prevented the upregulation of mCRPs transcriptionally. Sp1 is a known transcription factor driving constitutive CD59 expression. Inhibiting Sp1 similarly prevented chemotherapy-induced upregulation of CD59 and resistance to CDC. However, CHIP-qPCR showed no increased binding of total Sp1 protein to the CD59 promoter after chemotherapy. Instead, positive co-immunoprecipitation of pATM and p300 with Sp1 only with etoposide and not with co-treatment of Chk1 inhibitor suggests Chk1-mediated activation of Sp1. Mechanistically, we have found that chemotherapy-activated Chk1 modulates Sp1-mediated CD59 transcription and that the induced upregulation of CD59 is able to prevent antibody-mediated CDC.

Overall, these results indicate novel immune regulation by DNA damage response, particularly in the context of complement regulatory proteins. Our results advocate consideration when using simultaneous administration of chemotherapy with CDC-inducing monoclonal antibodies, and suggest Chk1 blockade as a possible therapeutic addition.

Citation Format: Allison S. Chan, Patrick W. Jaynes, Nurulhuda B. Mustafa, Akshaya A. Anbuselvan, Charmaine Z. Ong, Anand D. Jeyasekharan. Genotoxic chemotherapy impedes antibody-mediated complement dependent cytotoxicity (CDC), via a Chk1-CD59 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4990.