Sorafenib improves rituximab and ofatumumab efficacy by decreasing the expression of complement regulatory proteins

Despite major therapeutic advances in chronic lymphocytic leukaemia (CLL), there is still no curative strategy and new treatment approaches with improved efficacy are urgently needed. Although showing clinical activity in CLL patients, anti-CD20 monoclonal antibodies in chemoimmunotherapeutic regimens display rather limited efficacy. Thus combinations of targeted therapies with immunotherapeutic approaches might constitute a promising strategy to enhance antitumour effects. To date, several preclinical studies have reported the sensitivity of CLL cells to sorafenib, an oral compound targeting the activity of numerous membrane-bound and cytosolic kinases (BRAF, c-Raf, vascular endothelial growth factor receptor, platelet-derived growth factor receptor, fms-related tyrosine kinase 3, c-Kit), suggesting that it could represent a viable option as a therapy for CLL. Sorafenib has been reported to induce proliferation arrest and apoptosis in B-cell tumours. Antitumour effects of sorafenib in CLL are not abrogated by pro-survival signals from stromal cells indicating that treatment with sorafenib may overcome drug resistance. At present, sorafenib is being investigated in 4500 clinical trials, mostly in solid tumours and also in B-cell malignancies. Recently, the combination of perifosine (AKT/phosphoinositide 3-kinase inhibitor) and sorafenib has demonstrated promising activity in Hodgkin’s lymphoma patients. Phase II studies in diffuse-large B-cell lymphoma and refractory B-cell lymphoma patients revealed good tolerance to sorafenib. Moreover, a phase I/II clinical trial is currently initiated to assess the effects of sorafenib in CLL patients. Considering the accumulating number of preclinical and clinical studies confirming that combined regimens including sorafenib might be beneficial in CLL therapy, in the present study we assess the influence of sorafenib on antitumour activity of anti-CD20 monoclonal antibodies (mAbs) in lymphoma/leukaemia cell lines and primary CLL samples. We observe that sorafenib potentiates antitumour effects of anti-CD20 mAbs, an effect likely mediated by downregulation of membrane-bound complement regulatory proteins (mCRPs). In our primary experiments using a standard MTT (3-(4,5)dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) cytotoxicity assay, we observe that a 48-h preincubation of Raji, Ramos or Daudi cells (Burkitt’s lymphomas), as well as DoHH2 cells (follicular lymphoma), with sorafenib significantly potentiates the ability of rituximab to induce complement-dependent cytotoxicity (CDC; Figure 1a). Also, flow cytometry experiments with antibodies recognizing neo-epitope within C5b-9 reveal strong augmentation in incorporated membrane attack complexes (MAC) in Raji cells preincubated with sorafenib (Figure 1b, Supplementary Figure 1A). In rituximab-resistant CLL cells (MEC-1), sorafenib fails to increase rituximab-mediated CDC but effectively potentiates CDC triggered by ofatumumab (Figure 1c), another anti-CD20 mAb reported to strongly induce CDC. As anti-CD20 mAbs also activate antibody-dependent cellmediated cytotoxicity (ADCC), we sought to determine the influence of sorafenib on natural killer (NK) cells. The results of preclinical studies reporting immunoregulatory effects of sorafenib on NK cells activity are controversial and not conclusive. For example, sorafenib was shown to alter the microenvironment of hepatocellular carcinomas and trigger activation of NK cells by restoring classical macrophage polarization. Moreover, sorafenib sensitized hepatocellular carcinoma cells to NK cell-mediated functions by