1155 - Loss of AID exacerbates the malignant progression of CLL

Introduction
One of the major prognostic indicators for chronic lymphocytic leukemia (CLL) is the mutational status of the immunoglobulin heavy chain locus (IgHV). Patients with mutated IgHV have longer survival and a better response to chemotherapy than those with unmutated IgHV. This immunoglobulin mutation status is mediated by activation-induced cytidine deaminase (AID), an enzyme primarily expressed in B cells and during the germinal reaction when two gene-modifying processes critical for antibody function occur: class switch recombination and somatic hypermutation. In human patients, it has been shown that there is a correlation between higher levels of AID mRNA and a worse prognosis, although circulating CLL cells do not express AID protein. In contrast, the expression of AID has been shown to be targetable to kill CLL cells and may be important in the development of the relatively indolent mutated CLL. The function of AID in the progression of mutated versus unmutated CLL is not well understood, and the molecular mechanisms behind the development of CLL in an AID-deficient background have not been thoroughly studied.

Hypothesis, Methods, and Results
We generated an AID knockout CLL mouse model, AID-/-/Eμ-TCL1, and found that these mice die significantly earlier than their AID-proficient counterparts. As shown by immunoblots, AID-deficient Eμ-TCL1 CLL cells exhibited higher B cell receptor (BCR) signaling and a higher ER stress response compared to Eμ-TCL1 controls, particularly through activation of the IRE1/XBP1s pathway. Analysis of mRNA expression by qPCR in AID deficient B cells showed higher levels of spliced and unspliced XBP1 mRNA; knockout of secretory IgM from AID deficient B cells caused downregulation of XBP1s expression. Further, flow cytometry showed that AID-/-/Eμ-TCL1 CLL cells downregulate the tumor suppressive SMAD1/S1PR2 pathway and have altered migration. AID-/-/Eμ-TCL1 mice exhibited increased CLL cells in the livers, lungs, and peritoneal cavities by immunohistochemistry. Translationally, CLL cells from patients with IgHV-unmutated disease express higher levels of XBP1s mRNA compared to those from patients with IgHV-mutated CLL. Treatment of CLL cells from mice and human patients with combinations of the S1PR2 agonist CYM-5520 and the IRE1/XBP1s pathway inhibitor B-I09 delivered enhanced cytotoxicity.

Conclusions
We found that AID deficiency leads to upregulation of the IRE1/XBP1s pathway in both B cells and CLL cells. This upregulation of XBP1 is transcriptionally augmented by the increased secretory IgM found in AID-deficient B cells. The higher levels of XBP1s found in AID-/-/Eμ-TCL1 CLL cells support BCR signaling, contributing to CLL growth and survival. Conversely, downregulation of the SMAD1/S1PR2 pathway in AID-deficient CLL cells results in altered CLL migration, with leukemia cells homing to the non-lymphoid organs of the AID-/-/Eμ-TCL1 mice, potentially explaining their shorter survival. Finally, combined targeting of S1PR2 and XBP1s could be a useful strategy for CLL therapy. Our studies thus reveal novel mechanisms by which the loss of AID leads to worsened CLL and may explain why IgHV-unmutated CLL is more aggressive than IgHV-mutated CLL.