ALBERT

Description: Abstract 1652 Nuclear factor-κB (NF-κB) transcription factors are involved in cancer-relevant processes such as suppression of apoptosis, growth promotion, enhanced migration and invasiveness, although their actual role as oncogenic or tumor-suppressive activities remains controversial. Moreover, NF-κB-mediated suppression of apoptosis has been linked to chemoresistance. Interestingly, cellular senescence, a terminal cell-cycle arrest initiated via DNA-damaging chemotherapy as well, and known to improve long-term outcome, is associated with the massive induction of secretable NF-κB target genes, which, in turn, potentially reinforce the senescence phenotype. In this study, primary Eμ-myc transgenic mouse lymphomas as a well established model for human aggressive B-NHL, and information from human diffuse large B-cell lymphomas (DLBCL) were used in a cross-species approach to identify oncogenic networks in which chemotherapy-activated NF-κB signaling no longer mediates resistance but promotes therapy-induced senescence (TIS) and contributes to the outcome of anti-cancer treatment. Primary Myc-driven lymphoma cells, stably bcl2-transduced to block apoptosis, were exposed to the DNA-damaging chemotherapeutic agent adriamycin (ADR) to induce TIS. Gene set enrichment analysis of microarray-based gene expression profiles from drug-senescent vs. untreated cells found NF-κB target genes strongly skewed towards the TIS group, and multiplex ELISA-based analysis detected significantly higher DNA binding activities for the NF-κB family subunits p50, p52, p65 (RelA) and RelB in senescent cells. Inactivation of NF-κB by stable expression of the NF-κB super-repressor IκBα-δN (SR) lowered expression levels of NF-κB target genes in ADR-treated lymphomas. Matched pairs of individual primary lymphomas differing only in their SR status displayed compromised senescence induction in vivo when expressing the SR, indicating that TIS depends on intact NF-κB function. To assess the contribution of endogenous NF-κB signaling to long-term outcome, we grouped primary Myc-lymphomas by their NF-κB activity levels as “NF-κB low” (NL) or “NF-κB high” (NH). ADR-treated NH, but not NL lymphomas presented selective vulnerability to the SR moiety. Recapitulating the clinical outcome of patients suffering from DLBCL, around 60% of the mice harboring Eμ-myc lymphomas achieved long-term remissions, while the remaining 40% encountered a relapse after chemotherapy. Relapsing Myc-lymphomas exhibited substantially higher expression of the NF-κB targets IκBα and bcl2, reminiscent of activated B-cell-like (ABC) DLBCL, the clinically inferior subtype characterized by constitutively active NF-κB signaling. In contrast, germinal center B-cell-like (GCB) DLBCL rarely possess activating NF-κB mutations, but frequently develop in the context of a t(14;18) translocation that drives Bcl2 overexpression independent of NF-κB. Induced NF-κB target gene expression and increased TIS induction after overexpression of an NF-κB-activating CARD11 mutant suggested that higher NF-κB activity may contribute to treatment outcome via TIS promotion. Indeed, stratifying a large dataset of untreated GEP and corresponding clinical data after immunochemotherapy from 233 DLBCL patients by a 63-gene NF-κB expression signature (Shaffer-AL et al, Immunol Rev, 2006; Lenz-G et al, N Eng J Med, 2008), confirmed for the subset of GCB patients with above-median Bcl2 expression – the group whose genetic features we modeled before in the mouse – a significantly superior progression-free survival. In essence, our “cross-species” investigations demonstrate opposing roles of NF-κB in treatment outcome and have important ramifications for clinical trials that aim at inhibiting NF-κB signaling in DLBCL. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Premature senescence, a terminal cell-cycle arrest condition of viable cells in response to acute cellular stresses such as oncogenic activation or DNA-damaging anticancer therapy, is characterized by S-phase entry-controlling histone H3 lysine 9 trimethylation (H3K9me3). Previously, we reported an essential, tumor-suppressive role for the H3K9 histone methyltransferase Suv39h1 in oncogene-induced senescence (OIS) as a barrier to lymphoma development in vivo (Braig-M et al., Nature, 2005). In the current study, we focused, in addition to Suv39h1, on the H3K9-active demethylases LSD1 and JMJD2C in both OIS and therapy-induced senescence (TIS). Methods Human diploid fibroblasts (HDFs) and mouse embryo fibroblasts (MEFs) were stably transfected with H-RasG12V to induce OIS. S-phase-promoting E2F target gene control by H3K9me3 was analyzed by chromatin immunoprecipitation. Transformation was assessed by anchorage-independent colony formation in vitro and tumor development in nude mice. To establish TIS, primary Eµ-myc transgenic mouse lymphoma cells, retrovirally transduced with bcl2 to block apoptosis, were exposed to adriamycin in vitro or cyclophosphamide in vivo. Senescence was analyzed by staining for senescence-associated b-galactosidase activity (SA-b-gal), Ki67 and BrdU incorporation. Lymphoma formation and treatment in vivo were monitored by luciferase and GFP imaging, SA-b-gal/Ki67 staining in situ, and overall survival was assessed by Kaplan Meier analysis. Results H3K9-active demethylases – like overexpression of a dysfunctional H3R9 mutant – blocked cellular senescence, and permitted direct transformation under oncogenic Ras. In Myc-driven lymphomas, either loss of Suv39h1 or overexpression of LSD1 or JMJD2C cancelled TIS in vitro and in vivo. Notably, H3K9me3-impaired lymphomas resembled control lymphomas in their proliferation rate and sensitivity to drug-induced apoptosis, but displayed significantly shorter progression-free and overall survival after chemotherapy. Extended data sets on LSD1 and JMJD2C expression in human diffuse large B-cell lymphoma samples and their correlation to treatment outcome will be presented at the meeting. Conclusion The data underscore the essential albeit dynamic role of the H3K9me3 mark in OIS and TIS, and unveil the oncogenic potential of H3K9 demethylases, thereby providing a mechanistic basis for JMJD2C- or LSD1-targeting strategies in lymphoma therapy. Disclosures: No relevant conflicts of interest to declare.