Emilio Cosimo

Dasatinib inhibits B cell receptor signalling in chronic lymphocytic leukaemia but novel combination approaches are required to overcome additional pro-survival microenvironmental signals

As antigenic stimulation of the B cell antigen receptor (BCR) is key to chronic lymphocytic leukaemia (CLL) pathogenesis, targeting dysregulated kinases involved in BCR signalling is an attractive therapeutic approach. We studied the effects of the Src/c‐Abl tyrosine kinase inhibitor dasatinib on BCR signal transduction in CLL cells. Treatment of CLL cells with 100 nmol/l dasatinib induced apoptosis by an average reduction in viability of 33·7% at 48 h, with dasatinib sensitivity correlating with inhibition of SykY348 phosphorylation. Dasatinib inhibited calcium flux, phosphatidylinositol‐3‐kinase and mitogen‐activated protein kinase activation following BCR crosslinking, and blocked the Mcl‐1‐dependent increase in CLL cell survival on prolonged BCR stimulation. However, the pro‐apoptotic effect of dasatinib was abrogated by stromal cell contact alone or in the presence of CD154 and interleukin (IL)‐4 (CD154L/IL‐4 system). Whilst dasatinib retained the ability to sensitize CLL cells in stromal co‐culture to both fludarabine and chlorambucil, the addition of CD154 and IL‐4 rendered cells resistant to these drug combinations. We demonstrate that the HSP90 inhibitor 17‐DMAG exhibited synergy with dasatinib in vitro, and moreover, induced apoptosis of CLL cells in the CD154L/IL‐4 system. Our data provide evidence that dasatinib would be most clinically effective in combination with agents able to target antigen‐independent microenvironmental signals.

[131I]meta-iodobenzylguanidine and topotecan combination treatment of tumors expressing the noradrenaline transporter.

Purpose: Both [131I]meta-iodobenzylguanidine ([131I]MIBG) and the topoisomerase I inhibitor topotecan are effective as single-agent treatments of neuroblastoma. The aim of this study was to investigate the efficacy of [131I]MIBG in combination with topotecan in vitro and in vivo. Experimental Design: The cell lines used were SK-N-BE(2c) (human neuroblastoma) and UVW/NAT (glioma cell line transfected with the noradrenaline transporter gene). Three different treatment schedules were assessed: topotecan given before (schedule 1), after (schedule 2), or simultaneously (schedule 3) with [131I]MIBG. DNA strand breakage was evaluated by comet assay, and cytotoxicity was determined by clonogenic survival. Efficacy was also measured by growth delay of tumor xenografts in nude mice. Results: Combination schedules 2 and 3 caused more cytotoxicity than schedule 1. Similarly, significant DNA damage was observed following treatment schedules 2 and 3 (P < 0.005) but not schedule 1. The mean number of days for a doubling in volume of SK-N-BE(2c) tumors and a 10-fold increase in volume of UVW/NAT tumors were 10.4 and 18.6 (untreated), 19.7 and 25.3 (topotecan alone), 22.8 and 31.9 ([131I]MIBG alone), 26.3 and 37.1 (combination schedule 1), 34.3 and 49.7 (combination schedule 2), and 53.2 and >71 (combination schedule 3), respectively. The highest rate of cure of both xenografts was observed following treatment with combination schedule 3. Conclusions: The combination of topotecan and [131I]MIBG compared with either treatment alone gave rise to greater than additive DNA damage, clonogenic cell kill, and tumor growth delay. These effects were dependent on the scheduling of the two agents.

Loss of Reelin Expression in Breast Cancer Is Epigenetically Controlled and Associated with Poor Prognosis

Reelin is a secreted, signaling protein associated with neuronal cell positioning and migration. Recently, reelin was found to be epigenetically silenced in gastric and pancreatic cancers in which down-regulation was associated with increased migratory ability and reduced survival. Here we analyzed reelin expression by immunohistochemistry in 17 normal breast tissue samples from reduction mammoplasties and in two independent tissue microarrays of 136 and more than 2000 breast cancer biopsy samples, respectively. Results were analyzed with regard to clinical parameters, including BRE (Bloom, Richardson, Elston) grade, nodal status, estrogen receptor and HER2 status, and overall survival. Reelin was expressed in the luminal epithelium and myoepithelium of the normal human breast but not in cancerous breasts. Loss of reelin protein expression correlated significantly with decreased survival (P=0.01) and positive lymph node status (P<0.001). By measuring reelin expression and promoter methylation status in 39 primary breast tumors, as well as in breast cancer-derived cell lines before and after decitabine treatment, we established that reelin expression levels correlated inversely with promoter methylation status, whereas demethylation increased reelin mRNA expression in vitro. Reelin overexpression in MDA-MB231 cells, as well as incubation with recombinant reelin, suppressed cell migration, invadopodia formation, and invasiveness in vitro. We conclude that reelin may play an important role in controlling invasiveness and metastatic potential of breast cancer cells and that its expression is controlled by promoter methylation.

Complement deficiencies limit CD20 monoclonal antibody treatment efficacy in CLL

Monoclonal antibodies (MAbs) form a central part of chronic lymphocytic leukaemia (CLL) treatment. We therefore evaluated whether complement defects in CLL patients reduced the induction of complement-dependent cytotoxicity (CDC) by using anti-CD20 MAbs rituximab (RTX) and ofatumumab (OFA). Ofatumumab elicited higher CDC levels than RTX in all CLL samples examined, particularly in poor prognosis cohorts (11q− and 17p−). Serum sample analyses revealed that 38.1% of patients were deficient in one or more complement components, correlating with reduced CDC responses. Although a proportion of patients with deficient complement levels initially induced high levels of CDC, on secondary challenge CDC activity in sera was significantly reduced, compared with that in normal human serum (NHS; P<0.01; n=52). In addition, a high CLL cell number contributed to rapid complement exhaustion. Supplementing CLL serum with NHS or individual complement components, particularly C2, restored CDC on secondary challenge to NHS levels (P<0.0001; n=9). In vivo studies revealed that complement components were exhausted in CLL patient sera post RTX treatment, correlating with an inability to elicit CDC. Supplementing MAb treatment with fresh-frozen plasma may therefore maintain CDC levels in CLL patients with a complement deficiency or high white blood cell count. This study has important implications for CLL patients receiving anti-CD20 MAb therapy.

Inhibition of NF-κB–Mediated Signaling by the Cyclin-Dependent Kinase Inhibitor CR8 Overcomes Prosurvival Stimuli to Induce Apoptosis in Chronic Lymphocytic Leukemia Cells

Purpose: Chronic lymphocytic leukemia (CLL) is currently incurable with standard chemotherapeutic agents, highlighting the need for novel therapies. Overcoming proliferative and cytoprotective signals generated within the microenvironment of lymphoid organs is essential for limiting CLL progression and ultimately developing a cure. Experimental Design: We assessed the potency of cyclin-dependent kinase (CDK) inhibitor CR8, a roscovitine analog, to induce apoptosis in primary CLL from distinct prognostic subsets using flow cytometry–based assays. CLL cells were cultured in in vitro prosurvival and proproliferative conditions to mimic microenvironmental signals in the lymphoid organs, to elucidate the mechanism of action of CR8 in quiescent and proliferating CLL cells using flow cytometry, Western blotting, and quantitative real-time PCR. Results: CR8 was 100-fold more potent at inducing apoptosis in primary CLL cells than roscovitine, both in isolated culture and stromal-coculture conditions. Importantly, CR8 induced apoptosis in CD40-ligated CLL cells and preferentially targeted actively proliferating cells within these cultures. CR8 treatment induced downregulation of the antiapoptotic proteins Mcl-1 and XIAP, through inhibition of RNA polymerase II, and inhibition of NF-κB signaling at the transcriptional level and through inhibition of the inhibitor of IκB kinase (IKK) complex, resulting in stabilization of IκBα expression. Conclusions: CR8 is a potent CDK inhibitor that subverts pivotal prosurvival and proproliferative signals present in the tumor microenvironment of CLL patient lymphoid organs. Our data support the clinical development of selective CDK inhibitors as novel therapies for CLL. Clin Cancer Res; 19(9); 2393–405. ©2013 AACR.

Modulation of PKC-α promotes lineage reprogramming of committed B lymphocytes.

During hematopoietic lineage development, hematopoietic stem cells sequentially commit toward myeloid or lymphoid lineages in a tightly regulated manner, which under normal circumstances is irreversible. However, studies have established that targeted deletion of the B‐lineage specific transcription factor, paired box gene 5 (Pax5), enables B cells to differentiate toward other hematopoietic lineages, in addition to generating progenitor B‐cell lymphomas. Our previous studies showed that subversion of protein kinase C (PKC)‐α in developing B cells transformed B‐lineage cells. Here, we demonstrate that PKC‐α modulation in committed CD19+ B lymphocytes also promoted lineage conversion toward myeloid, NK‐, and T‐cell lineages upon Notch ligation. This occurred via a reduction in Pax5 expression resulting from a downregulation of E47, a product of the E2A gene. T‐cell lineage commitment was indicated by the expression of T‐cell associated genes Ptcra, Cd3e, and gene rearrangement at the Tcrb gene locus. Importantly, the lineage‐converted T cells carried Igh gene rearrangements reminiscent of their B‐cell origin. Our findings suggest that modulation of PKC‐α induces hematopoietic‐lineage plasticity in committed B‐lineage cells by perturbing expression of critical B‐lineage transcription factors, and deregulation of PKC‐α activity/expression represents a potential mechanism for lineage trans‐differentiation during malignancies.

Generation of a poor prognostic chronic lymphocytic leukemia-like disease model: PKCα subversion induces up-regulation of PKCβII expression in B lymphocytes.

Overwhelming evidence identifies the microenvironment as a critical factor in the development and progression of chronic lymphocytic leukemia, underlining the importance of developing suitable translational models to study the pathogenesis of the disease. We previously established that stable expression of kinase dead protein kinase C alpha in hematopoietic progenitor cells resulted in the development of a chronic lymphocytic leukemia-like disease in mice. Here we demonstrate that this chronic lymphocytic leukemia model resembles the more aggressive subset of chronic lymphocytic leukemia, expressing predominantly unmutated immunoglobulin heavy chain genes, with upregulated tyrosine kinase ZAP-70 expression and elevated ERK-MAPK-mTor signaling, resulting in enhanced proliferation and increased tumor load in lymphoid organs. Reduced function of PKCα leads to an up-regulation of PKCβII expression, which is also associated with a poor prognostic subset of human chronic lymphocytic leukemia samples. Treatment of chronic lymphocytic leukemia-like cells with the selective PKCβ inhibitor enzastaurin caused cell cycle arrest and apoptosis both in vitro and in vivo, and a reduction in the leukemic burden in vivo. These results demonstrate the importance of PKCβII in chronic lymphocytic leukemia-like disease progression and suggest a role for PKCα subversion in creating permissive conditions for leukemogenesis.