Direct Inhibition of Fas Activation by CD74.

Abstract 3770

Poster Board III-706

Hematopoietic cancers are commonly resistant to Fas-mediated apoptosis that contributes to their resistance to chemotherapy and reversing Fas resistance has become a primary interest in treating chemotherapy-resistant hematopoietic cancers. Wide expression of Fas receptor yet the limited extent of Fas-mediated apoptosis in tissues suggest a strict regulation of Fas signaling. A recently identified tissue-specific modulator of Fas-mediated apoptosis is hepatocyte growth factor (HGF) receptor HGFR/Met. HGFR/Met binds Fas and inhibits Fas signaling at an immediate early step. However, upon binding of ligand HGF to HGFR/Met, Fas is released and Fas signaling is restored. We hypothesized that hematopoietic cancers may express similar inhibitors of Fas, that can be targeted to resensitize cancer cells to Fas and thus also to therapy. We screen lymphoma cells for for such inhibitors. B cell lymphoma-derived BJAB cells and primary patient samples were incubated with Fas agonistic antibody. Upon removal of antibody excess, cell lysates were immuno-depleted of CH-11 antibody-bound and thus activated Fas. The remaining supernatant was then precipitated a second time with anti-Fas antibody B-10 that binds to the intracellular region of Fas. Protein bands present in B-10 precipitates and absent in CH-11 precipitates were excised from silver-stained gel and analyzed by mass-spectroscopy. CD74, also termed the invariant chain of MHC II and known to mediate pro-survival signaling as a receptor for macrophage migration inhibitory factor (MIF), was found to exclusively associate with activation-resistant Fas. Intriguingly, CD74 is known to be overexpressed in 80% to 95% of hematopoietic cancers and also in some solid tumors.

Using RNA interference we showed that CD74 negative cells are more sensitive to Fas-mediated apoptosis and that CD74 overexpression confers resistance to Fas both in vitro and in mouse tissues. Incubation of cells with peptides or anti-CD74 antibody LL1 disrupts the CD74-Fas inhibitory complex and sensitizes CD74-positive cells to Fas-mediated apoptosis. Analysis at the molecular level revealed that CD74 interferes with the immediate early steps in Fas signaling – binding of agonistic CH-11 antibody and subsequent clustering of Fas receptor.

We anticipate that specific disruption of the CD74-Fas interaction by CD74-derived peptides or anti-CD74 antibodies will sensitize cancer cells to Fas-mediated apoptosis. This approach represents a novel strategy for modulation of cell surface death receptors to produce effective treatments for CD74-positive cancers.