The rituximab-PKCζ/Raf-1/mTOR connection

Rituximab (RTX; chimeric anti-CD20 mAb), the first antibody approved by the Food and Drug Administration (FDA) for cancer treatment, is used in the treatment of B-cell non-Hodgkin lymphoma (B-NHL) alone, or in combination with chemotherapy, with significant clinical responses. While antibody-dependent cellular toxicity, complement-dependent toxicity, and apoptosis have been proposed as underlying mechanisms of RTX activity in vivo, the exact mechanisms of responsiveness and unresponsiveness of tumor cells remain unknown. Several reports have investigated the potential ways by which RTX mediates its inhibitory effect on cell proliferation, induction of apoptosis, and chemo-immunosensitization.^1,2  These studies demonstrated RTX's ability to induce mobilization of CD20 into lipid rafts, activate the sphingomyelin pathway, and inhibit Src kinases and multiple cell survival signaling pathways (such as the Raf-1/NF-κB/p38MAPK/AKT). However, these studies did not identify the proximal targets involved following RTX triggering.

The study by Leseux and colleagues in this issue of Blood identifies a key player in cell signaling by RTX, PKCζ, which leads to inhibition of the Raf-1/mTOR pathways. PKCζ's involvement was suspected based on previous findings that RTX activates the sphingomyelin cycle and augments ceramide, which was reported to target PKCζ. The authors demonstrate that RTX inhibits the activity of the atypical PKC subfamily isoform, PKCζ. In addition, while RTX triggers the sphingomyelin cascade in FL, diffuse large B-cell lymphoma (DLBCL), and mantle cell lymphoma (MCL), there was no inhibition of PKCζ activity in DLBCL and MCL; there was only selective inhibition in FL, for reasons not yet understood. Another novel finding in Leseux et al's study is that RTX-mediated inhibition of PKCζ results in inhibition of the MAPK pathway and leads to inhibition of the mTOR pathway that is, in part, regulated by MAPK. Hence, the authors have established the link between RTX and-PKCζ/Raf-1/mTOR dysregulation, resulting in inhibition of cell growth. Leseux et al's novel findings identify a pivotal proximal kinase in the mechanism by which RTX inhibits survival pathways by inhibiting PKCζ activity and establish PKCζ and mTOR as targets for therapeutic intervention.

The study by Leseux et al raises several intriguing questions that deserve attention. For instance, the selective activity in FL, and not in other lymphomas, supports the phenotypic and genetic heterogeneity of these diseases and their response to rituximab. Hence, different therapeutic approaches are needed to treat these various malignancies. PKCζ is highly activated in FL, but it is not clear what regulates its hyperactivation, whether con-stitutively or by autocrine/paracrine loops. PKCζ activation depends on the phosphatidylinositol (PI)-3, 4, 5-triphosphate (PIP³ ), which is mainly induced by PI-3 kinase. 3′-PI–dependent protein kinase 1, which binds with high affinity to PIP,³  phosphorylates and activates PKCζ.³  Leseux et al show that the role of PKCζ in the inhibition of Raf-1 signaling does not involve Raf-1 kinase inhibitor protein (RKIP). They find that, unlike in Burkitt lymphoma,²  RTX does not induce RKIP overexpression in FL. RKIP has been identified as a member of the phosphatidylethanolamine binding protein (PEBP) family and shown to bind Raf-1 and inhibit MEK binding to Raf-1⁴  and inhibit downstream Raf-1–induced transformation and AP-1–dependent transcription. Corbit et al⁵  reported that PKCζ phosphorylates RKIP at Ser153, resulting in the dissociation of RKIP from Raf-1. Thus, inhibition of PKCζ by RTX should result in the inhibition of phosphorylation of RKIP and prevent RKIP dissociation from Raf-1, thereby inhibiting Raf-1 signaling. The failure of RTX to up-regulate RKIP expression in FL in Leseux et al's findings, however, does not rule out the possibility that RTX inhibited phosphoRKIP via inhibition of PKCζ activity. The findings on the role of mTOR inhibition by RTX and the demonstration of synergy achieved by combination of RTX and rapamycine is of paramount clinical significance.

The continued high fatality rates in NHL patients, despite recent advances, illustrate the need for innovative approaches. mTOR inhibitors are good candidates for intervention, and several inhibitors that show promise are currently in clinical trials. However, the effects of mTOR inhibitors in cancer cells are largely unknown, and better understanding of the mTOR pathway can lead to more rational therapies. Since patients can develop resistance to RTX treatment and also resist mTOR inhibitors, Leseux et al's examination of FL cell lines that have developed resistance to these agents will be useful.