Janine Schwamb

Sensitizing Protective Tumor Microenvironments to Antibody-Mediated Therapy

Therapy-resistant microenvironments represent a major barrier toward effective elimination of disseminated malignancies. Here, we show that select microenvironments can underlie resistance to antibody-based therapy. Using a humanized model of treatment refractory B cell leukemia, we find that infiltration of leukemia cells into the bone marrow rewires the tumor microenvironment to inhibit engulfment of antibody-targeted tumor cells. Resistance to macrophage-mediated killing can be overcome by combination regimens involving therapeutic antibodies and chemotherapy. Specifically, the nitrogen mustard cyclophosphamide induces an acute secretory activating phenotype (ASAP), releasing CCL4, IL8, VEGF, and TNFα from treated tumor cells. These factors induce macrophage infiltration and phagocytic activity in the bone marrow. Thus, the acute induction of stress-related cytokines can effectively target cancer cells for removal by the innate immune system. This synergistic chemoimmunotherapeutic regimen represents a potent strategy for using conventional anticancer agents to alter the tumor microenvironment and promote the efficacy of targeted therapeutics.

Targeting lipid metabolism by the lipoprotein lipase inhibitor orlistat results in apoptosis of B-cell chronic lymphocytic leukemia cells

Constitutively activated pathways contribute to apoptosis resistance in chronic lymphocytic leukemia (CLL). Little is known about the metabolism of lipids and function of lipases in CLL cells. Performing gene expression profiling including B-cell receptor (BCR) stimulation of CLL cells in comparison to healthy donor CD5+ B cells, we found significant overexpression of lipases and phospholipases in CLL cells. In addition, we observed that the recently defined prognostic factor lipoprotein lipase (LPL) is induced by stimulation of BCR in CLL cells but not in CD5+ normal B cells. CLL cellular lysates exhibited significantly higher lipase activity compared to healthy donor controls. Incubation of primary CLL cells (n=26) with the lipase inhibitor orlistat resulted in induction of apoptosis, with a half-maximal dose (IC50) of 2.35 μM. In healthy B cells a significantly higher mean IC50 of 148.5 μM of orlistat was observed, while no apoptosis was induced in healthy peripheral blood mononuclear cells (PBMCs; P<0.001). Orlistat-mediated cytotoxicity was decreased by BCR stimulation. Finally, the cytotoxic effects of orlistat on primary CLL cells were enhanced by the simultaneous incubation with fludarabine (P=0.003). In summary, alterations of lipid metabolism are involved in CLL pathogenesis and might represent a novel therapeutic target in CLL.

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.

Overexpression of TOSO in CLL is triggered by B-cell receptor signaling and associated with progressive disease

Resistance toward apoptotic stimuli mediated by overexpression of antiapoptotic factors or extracellular survival signals is considered to be responsible for accumulation of malignant B cells in chronic lymphocytic leukemia (CLL). TOSO was identified as overexpressed candidate gene in CLL, applying unit-transformation assays of publicly available microarray datasets. Based on CLL samples from 106 patients, TOSO was identified to exhibit elevated relative expression (RE) of 6.8 compared with healthy donor B cells using quantitative real-time polymerase chain reaction (PCR; P = .004). High levels of TOSO expression in CLL correlated with high leukocyte count, advanced Binet stage, previous need for chemotherapy, and unmutated IgV(H) status. CD38(+) CLL subsets harboring proliferative activity showed enhanced TOSO expression. We evaluated functional mechanisms of aberrant TOSO expression and identified TOSO expression significantly induced by B-cell receptor (BCR) stimulation compared with control cells (RE; 8.25 vs 4.86; P = .01). In contrast, CD40L signaling significantly reduced TOSO expression (RE, 2.60; P = .01). In summary, we show that the antiapoptotic factor TOSO is associated with progressive disease and enhanced in the proliferative CD38(+) CLL subset. Both association with unmutated IgV(H) and the specific induction of TOSO via the BCR suggest autoreactive BCR signaling as a key mediator of apoptosis resistance in CLL.

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.