Resistance to Fas-mediated apoptosis manifested by hematopoietic cancers interferes with the efficacy of currently available chemotherapy regimens. We identified CD74, that is known to be overexpressed in hematological malignancies, as one of the factors interfering with Fas-mediated apoptosis.
Through stable suppression of CD74 expression in BJAB and Raji cells using shRNA technology, we have determined that cells overexpressing CD74 are more resistant to FasL-induced apoptosis than control non-target shRNA expressing cells (super FasL 10 ng/mL; 36 ± 3.3% vs.18 ± 0.1% and 35 ± 0.1% vs. 20 ± 1.5%; P< 0.01). Transfection of mice with plasmid encoding full-length CD74 protected mice from a lethal challenge with agonistic anti-Fas antibody Jo2. All 5 of vector-transfected mice died within 6 hours from challenge, while 5 out of 6 CD74-transfected mice survived the challenge (P< 0.05). Suppression of CD74 expression also sensitized BJAB cells to Fas-signaling dependent chemotherapies. Doxorubicin (125nM) killed 27 ± 4% of control and 48 ± 5% of CD74 knock-down cells (P< 0.001). Edelfosine (10 uM) induced apoptosis in 36 ± 7% of control and 64 ± 1% of CD74 knock-down cells (P< 0.001).
A detailed analysis of Fas signaling in cells lacking CD74 and control cells revealed increased cleavage/activation of pro-caspase-8 and corresponding enhancement of caspase-3 activation in the absence of CD74. This result suggested that CD74 affects the immediate early steps in Fas signaling at the plasma membrane. We next analyzed the levels of Fas receptor on the surface of cells by flow cytometry. Cells with suppressed CD74 expression had 30% higher staining of Fas receptor on their surface; the mean fluorescence intensity increased from 4017 ± 704 to 5122 ± 216 (P< 0.003).
We anticipate that specific targeting of the CD74 on the cell surface with milatuzumab will sensitize CD74-expressing cancer cells to Fas-mediated apoptosis and thus will increase effectiveness of chemotherapy regimens for hematological malignancies.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.