ALBERT

Description: Abstract 2382 Poster Board II-359 Although recent advances in treatment-strategies for chronic lymphocytic leukemia (CLL) have resulted in increased remission rates and response duration, the disease eventually relapses, which necessitates repeated cycles of therapy. Eventually most patients develop chemo-resistant disease which infers a very poor prognosis. The activity of purine-analogs and alkylating agents, the backbone of current treatment regimens, depends on functional p53 and chemo-resistance is highly associated with a dysfunctional p53-response. P53-independent sensitization of CLL cells to these compounds could represent a novel strategy to overcome chemo-resistance. Platinum-based compounds have been successfully applied in relapsed lymphoma and recently also in high-risk CLL. In various cancer-types, the activity of such compounds has been found to be p53-independent and in part mediated by p73. In this study we investigated the efficacy and mechanism of action of platinum-based compounds in chemo-refractory CLL. Neither cisplatinum nor oxaliplatin as a single agent induced cell death in clinically relevant doses. However, independent of p53-functional status, platinum-based compounds acted synergistically with fludarabine, which was found to be caspase-dependent. Combination-treatment resulted in strong upregulation of the pro-apoptotic BH3-only protein Noxa. We did not find evidence for a role of p73; however, the observed synergy was found to involve generation of reactive oxygen species (ROS). Co-treatment with ROS-scavengers completely abrogated Noxa-upregulation and cell-death upon combination treatment in p53-dysfunctional CLL. Noxa RNA-interference markedly decreased sensitivity to combination treatment, supporting a key role for Noxa as mediator between ROS signaling and apoptosis induction. In addition to these findings, we tested the effects of platinum-based compounds and fludarabine on drug-resistance resulting from CD40-ligand stimulation of CLL cells, which represents a model for CLL cells in the protective micro-environment of the secondary lymph node-tissue (Hallaert et al Blood 2008 112(13):5141). Combination treatment could overcome CD40-ligand induced chemo-resistance and was, at least in part, mediated by the generation of ROS and marked induction of expression of Noxa. Our data indicate that interference with the cellular redox-balance represents an interesting target to overcome drug resistance due to both p53-dysfunction as well as micro-environmental protective stimuli in CLL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1825 Chronic lymphocytic leukemia (CLL) is characterized by relentless relapses. It is postulated that this might be due to microenvironment-induced resistance to cytotoxic agents. Previously we have shown that in vitro CD40 stimulation of peripheral blood derived from CLL patients can to a certain extent mimic the lymph node (LN) microenvironment and result in resistance to cytotoxic drugs. This is correlated with CD40-induced changes in apoptosis regulating Bcl-2 family members. The BH3 mimetic drug ABT-737 antagonizes the anti-apoptotic molecules Bcl-XL and Bcl-2, but not Mcl-1 or Bfl-1. At present it is unknown whether patient-specific variations in expression of apoptosis regulating proteins occur and to what extent they correlate with sensitivity or resistance to ABT-737. In the present investigation we assessed the variability in response to ABT-737 in CD40 stimulated CLL cells. Although in all tested CD40-stimulated CLL patients Mcl-1 and Bfl-1 levels increased, there was a heterogeneous response in cell death to ABT-737 when used as a single agent. Notably, time course analyses showed variations in Noxa levels upon CD40 triggering, which differed between CLL patients. High Noxa/Mcl-1 ratios correlated with sensitivity to ABT-737. This was confirmed by Noxa knockdown as well as Mcl-1 and Bfl-1 overexpression, which resulted in resistance to ABT-737. To determine the signaling pathways involved in Noxa regulation, CLL cells were stimulated with CD40L in the presence of various kinase inhibitors. We observed that Noxa levels were specifically inhibited by the p38 MAP kinase inhibitor SB203580. Preliminary data indicate that after CD40 stimulation, phosphorylation of p38 decreased over time, correlating with a decrease in Noxa levels. Importantly however, a minority of CD40 stimulated CLL samples were sensitive to ABT-737 and showed high levels of phosphorylated p38 and increasing Noxa levels over time. In conclusion, we demonstrate that following CD40 triggering of CLL cells the p38 MAPK signaling pathway regulates Noxa levels and that patient-specific variations in this pathway contribute to the responsiveness to ABT-737. These findings raise the question whether assessment of p38 MAPK activation status and Noxa levels in vivo could predict the response of CLL patients to ABT-737. Moreover our findings offer possible new targets for the treatment of CLL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 711 Mutations or deletions of the tumor suppressor p53 or its upstream kinase ATM are well-known determinants of poor prognosis in Chronic Lymphocytic Leukemia (CLL). In recent years, genome wide sequencing has uncovered novel gene mutations that correspond with poor prognosis. Specifically, recurrent mutations in the splicing factor SF3B1 and the Notch and NRAS/KRAS oncogenes have been found. These mutations were (in part) mutually exclusive with p53 and/or ATM mutations, which suggested overlap in biological function. Here, we report results of a comparative analysis of p53 target genes and in vitro drug responses in CLL samples with either p53 (n=9), ATM (n=10), SF3B1 (n=11), Notch (n=6), or NRAS/KRAS (n=4) gene deletions/mutations. We found that upon irradiation, mRNA induction of all tested p53 targets genes (p21, Puma, CD95, Bax, PCNA, FXDR) was clearly decreased in all SF3B1 mutated CLL samples (overall p