Reinhild Brinker

Disruption of T cell suppression in chronic lymphocytic leukemia by CD200 blockade

CD200 plays a key role in regulating the immune system and has been shown to be upregulated on the surface of different tumors including chronic lymphocytic leukemia. In this study we addressed the effects of CD200 over-expression in CLL cells on autologous T cells in a mixed lymphocyte reaction system. We used native and CD40 ligand (CD40L)-stimulated CLL cells as antigen-presenting cells (APCs) to expand autologous T cells of 14 patients. T cell proliferation over 3 weeks of in vitro culture was significantly enhanced compared to control cells when using CD40L-stimulated APCs and the anti-CD200 antibody 1B9 (p=0.0004). CD200 blockade was further shown to stimulate antigen-specific T cell responses towards the CLL-associated antigen fibromodulin (p=0.04). Finally, the number of CD4+/CD25high/FOXP3+ T cells (T(reg)) was significantly decreased adding anti-CD200 antibody (p=0.04). In summary, CD200 blockade may provide therapeutic benefits in CLL by augmenting an antigen-specific T cell response with suppression of regulatory T cells.

B-cell receptor triggers drug sensitivity of primary CLL cells by controlling glucosylation of ceramides

Survival of chronic lymphocytic leukemia (CLL) cells is triggered by several stimuli, such as the B-cell receptor (BCR), CD40 ligand (CD40L), or interleukin-4 (IL-4). We identified that these stimuli regulate apoptosis resistance by modulating sphingolipid metabolism. Applying liquid chromatography electrospray ionization tandem mass spectrometry, we revealed a significant decrease of proapoptotic ceramide in BCR/IL-4/CD40L-stimulated primary CLL cells compared with untreated controls. Antiapoptotic glucosylceramide levels were significantly increased after BCR cross-linking. We identified BCR engagement to catalyze the crucial modification of ceramide to glucosylceramide via UDP-glucose ceramide glucosyltransferase (UGCG). Besides specific UGCG inhibitors, our data demonstrate that IgM-mediated UGCG expression was inhibited by the novel and highly effective PI3Kδ and BTK inhibitors CAL-101 and PCI-32765, which reverted IgM-induced resistance toward apoptosis of CLL cells. Sphingolipids were recently shown to be crucial for mediation of apoptosis via mitochondria. Our data reveal ABT-737, a mitochondria-targeting drug, as interesting candidate partner for PI3Kδ and BTK inhibition, resulting in synergistic apoptosis, even under protection by the BCR. In summary, we identified the mode of action of novel kinase inhibitors CAL-101 and PCI-32765 by controlling the UGCG-mediated ceramide/glucosylceramide equilibrium as a downstream molecular switch of BCR signaling, also providing novel targeted treatment options beyond current chemotherapy-based regimens.

Novel X‐linked inhibitor of apoptosis inhibiting compound as sensitizer for TRAIL‐mediated apoptosis in chronic lymphocytic leukaemia with poor prognosis

Given that aggressive DNA damaging chemotherapy shows suboptimal efficacy in chronic lymphocytic leukaemia (CLL), alternative therapeutic approaches are needed. Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is able to induce tumour‐specific apoptosis. However, apoptosis might be inhibited by elevated levels of X‐linked inhibitor of apoptosis (XIAP). Use of XIAP‐inhibiting compounds might sensitize primary CLL cells towards TRAIL‐mediated apoptosis. A novel small molecule, compound A (CA), an inhibitor of XIAP, was used in combination with TRAIL to induce apoptosis in primary CLL cells (n = 48). XIAP was significantly more highly expressed in primary CLL cells (n = 28) compared to healthy B cells (n = 16) (P = 0·02). Our data obtained by specific knock‐down of XIAP by siRNA identified XIAP as the key factor conferring resistance to TRAIL in CLL. Combined treatment with CA/TRAIL significantly increased apoptosis compared to untreated (P = 8·5 × 10−10), solely CA (P = 4·1 × 10−12) or TRAIL treated (P = 4·8 × 10−10) CLL cells. CA rendered 40 of 48 (83·3%) primary CLL samples susceptible to TRAIL‐mediated apoptosis. In particular, cells derived from patients with poor prognosis CLL (ZAP‐70+, IGHV unmutated, 17p‐) were highly responsive to this drug combination. Our highly‐effective XIAP inhibitor CA, in concert with TRAIL, shows potential for the treatment of CLL cases with poor prognosis and therefore warrants further clinical investigation.

Sustained NF-kappaB activity in chronic lymphocytic leukemia is independent of genetic and epigenetic alterations in the TNFAIP3 (A20) locus

Inappropriate nuclear factor (NF) κB activity is one major hallmark of B‐cell malignancies and chronic lymphocytic leukemia (CLL). NFκB‐dependent genes are involved in antiapoptosis, cell proliferation and metastasis and are responsible for survival and proliferation of tumors. However, the mechanisms of NFκB activity in CLL still need to be elucidated. Previously, we identified translocations in a region on chromosome 6q that encodes tumor necrosis factor alpha‐induced protein 3, which is a key player in negative feedback loop regulation of NFκB. Inactivation of this ubiquitin‐editing enzyme is involved in immunopathologies and in tumorigenesis. Frequent mutations in the A20 locus—leading to sustained NFκB activity—could be shown to play a dominant role in development of different B‐cell malignancies. To check if A20 is involved in upregulation of NFκB activity in CLL, we sequenced Exons 2–9 of the A20 gene in 55 CLL DNA samples. Furthermore, we determined the methylation status of the promoter region in 63 CLL DNA samples and compared to 10 control DNAs of B cells from healthy donors. Contrary to reports from other B‐cell malignancies, the A20 region showed neither mutations nor aberrant DNA methylation. Moreover, its expression could be confirmed by immunoblotting and showing comparable results to healthy B cells. These results indicate that malignant development in CLL differs from most of other B‐cell malignancies, which show frequent inactivation of A20.

Erufosine, a novel alkylphosphocholine, induces apoptosis in CLL through a caspase-dependent pathway.

The alkylphosphocholine (APC) erufosine is a synthetic phospholipid analogue with antineoplastic activity. APC are known to interact with lipid metabolism and modulate cellular signaling pathways, particularly the phosphorylation of Akt. Here, in primary CLL cells induction of apoptosis was detected with an IC50 of 22muM whereas healthy donor PBMC were less sensitive towards erufosine. Treatment with erufosine caused dose-dependent cleavage of PARP, co-incubation with caspase inhibitor z-VAD almost completely abrogated the cytotoxic effect of erufosine indicating a caspase-dependent mechanism of erufosine. Erufosine was shown to induce apoptosis in primary CLL cells and merits further investigation regarding therapeutic options in CLL.