CD40 FasL and CTLA-4 FasL Fusion Proteins Induce Apoptosis in Malignant Cell Lines by Dual Signaling

Evolution of apoptosis resistance in both lymphoma and leukemia cells is well documented, and induction of apoptosis in malignant cells is a major goal of cancer therapy. Up-regulation of anti-apoptotic signals is one of the mechanisms whereby resistance to apoptosis emerges. We have previously described the fusion proteins CD40 FasL and CTLA-4 FasL, which are formed from two functional membrane proteins and induce apoptosis of activated T cells. The present study explores the potential use of CD40 FasL and CTLA-4 FasL for the killing of malignant cells of lymphatic origin. Using malignant B and T cell lines that differ in surface expression of costimulatory molecules, we found that CTLA-4 FasL induces effective apoptosis of cells expressing CD95 and activates caspases 3, 8, and 9. Only B7-expressing B cells responded to CTLA-4 FasL with rapid abrogation of cFLIP expression. CD40 FasL effectively killed only the T cells that express high levels of CD40L in addition to CD95. In these cells, CD40 FasL significantly diminished cFLIP expression. Importantly, each of the fusion proteins is more potent than its respective components parts, alone or in combination. Thus, the proteins with their two functional ends deliver a pro-apoptotic signal and, in parallel, inhibit an antiapoptotic signal, thus optimizing the wanted, deathinducing effect. Therefore, these proteins emerge as promising agents to be used for targeted and specific tumor cell killing. (Am J Pathol 2010, 177:3159–3168; DOI: 10.2353/ajpath.2010.100301) Induction of effective and selective apoptotic death in malignant cells is a major goal for cancer therapy. The evolution of apoptotic resistance in malignant tumor cell populations is by now well documented. Apoptosis resistance can result from several mechanisms: loss of cell surface Fas receptor expression, interference with death complex (Death inducing signaling complex; DISC) formation, interference with the initiation of the caspase cascade at the DISC level, down-regulation of caspases downstream to the DISC, and up-regulation of anti-apoptotic signals at multiple levels of the death cascade. Lymphoma and leukemia cells are resistant to apoptosis induced by chemotherapy and/or Fas ligand, presumably because of the up-regulation of anti-apoptotic or pro-surviving signals such as Bcl-xL, Mcl-1, nuclear factor B (NFB), survivin, cFLIP, and even viral FLIP. The up-regulation of anti-apoptotic signals, which occurs normally during the activation of lymphocytes, is thought to be triggered, at least partially, by co-stimulatory signaling. When CD40 ligand (CD40L) triggers the CD40 receptor on B cells, a strong survival signal is generated in both normal and malignant B cells, which in turn leads to cell proliferation and enhanced survival in vitro. Triggering of CD40 on malignant B cells is followed by up-regulation of anti-apoptotic signals. CD40L can be presented to malignant B cells by infiltrating T cells. Interestingly, however, CD40L and CD40 are co-expressed by several types of B-cell malignancies, for example, large B-cell lymphoma. Therefore, an autocrine/paracrine CD40L/CD40 survival loop has been suggested as a mechanism that could explain the resistance to apoptosis of these B-cell neoplasms. A similar survival loop was recently proposed for cutaneous T-cell lymphoma (mycosis fungoides), where the neoplastic