Exploiting ER Stress to Eliminate Inhibitor Antibodies in Hemophilia

Hemophilia is the most common severe bleeding disorder, affecting up to 1 in 5,000 males. The most feared complication for patients with hemophilia A is the development of inhibitor antibodies to infused FVIII, occurring in 30% of patients with hemophilia A. Inhibitor antibodies are produced in large numbers by the professional secretory cells of the immune system, the plasma cells, in response to exposures to FVIII. The development of these professional secretory cells has been shown to cause stress in the endoplasmic reticulum (ER) and mechanisms of the unfolded protein response (UPR) are integral to the adaptation of these cells. Proteasome inhibitors, such as bortezomib (Velcade), have been shown to increase ER stress and kill plasma cells by inhibiting the destruction of misfolded proteins and inducing proteotoxicity. Current therapeutic strategies for hemophilia patients with inhibitors revolve around inducing tolerance to FVIII by treating patients with large doses of FVIII daily. We believe this therapy induces antibody production, stressing the plasma cells that manufacture the inhibitor antibodies.

We have tested the hypothesis that the one-two punch of ER stress directed at the anti-FVIII plasma cells and proteasome inhibiting or CHOP inducing agents will induce apoptosis. To this end we have tested bortezomib in vitro and determined that it does potently induce ER stress and the UPR. Further, we have shown that this induces apoptosis and cell death and that cells lacking ATF6α, an upstream mediator of the UPR, are more sensitive to bortezomib while those lacking CHOP, a pro-apoptotic downstream mediator of UPR, are slightly less sensitive. Additionally we have found that the super-oxide scavenger, tiron, protects cells from bortezomib, suggesting a role for reactive oxygen species upstream of UPR activation by bortezomib. We have studied bortezomib in a mouse model of hemophilia A inhibitors and have found that it can effectively eradicate inhibitor antibody producing cells in this model. In contrast when bortezomib was injected into CHOP knock-out inhibitor mice the inhibitor antibodies were not eradicated, in contrast to the wild type controls in which the inhibitor antibodies were eliminated. This is strong evidence in support of CHOP as a primary mediator of plasma cell death induced by bortezomib and suggests that other CHOP inducing agents may also be promising therapeutics for hemophilia inhibitors. To that end we are investigating agents identified through a cell based, luciferase reporter, high throughput screen of 300,000 compounds for CHOP inhibitors. We hope that this work will identify agents that can be used to eradicate inhibitor antibodies in patients with Hemophilia.