Inhibition of the Intrinsic Apoptosis Pathway Downstream of Caspase-9 Activation Causes Chemotherapy Resistance in Diffuse Large B-Cell Lymphoma

Abstract

Purpose: Inhibition of the apoptosis cascade is an important cause of therapy resistance in diffuse large B-cell lymphomas (DLBCL). In this study, we investigated possible mechanisms and expression levels of apoptosis-related genes in the apoptosis pathway that may be responsible for differences in chemotherapy sensitivity between DLBCL patients. Experimental Design: Twenty-eight DLBCL patient samples were investigated for their expression levels of apoptosis-related genes using reverse transcription-multiplex ligation-dependent probe amplification analysis. Functional analysis of the intrinsic, caspase-9^mediated pathway was done using fluorescence-activated cell sorting analysis,Western blot analysis, and immunohistochemistry. Results:Two DLBCL groups were identified: one with low expression levels of both proapoptotic and antiapoptotic genes and one group with high expression levels of these genes. DLBCL with high expression levels of proapoptotic and antiapoptotic genes frequently seemed to be refractory to clinical chemotherapy. Functional analysis in these latter DLBCL samples and DLBCL cell lines with comparable expression profiles revealed high levels of spontaneous caspase-9 activity without induction of apoptosis, indicating disruption of the apoptosis pathway downstream of caspase-9 activation. This disruption of the apoptosis pathway could be restored using a smallmolecule XIAP antagonist. Conclusions: We conclude that the intrinsic, caspase-9^mediated apoptosis pathway is constitutively activated in part of chemotherapy-refractory DLBCL with concomitant downstream inhibition of the convergence apoptosis pathway and that inhibition of XIAP might be an alternative therapy for chemotherapy-refractory DLBCL.

Small-molecule XIAP antagonist restores caspase-9–mediated apoptosis in XIAP-positive diffuse large B-cell lymphoma cells

“…Furthermore, these findings explain why the XIAP antagonist did not sensitize DLBCL cells to etoposide-induced apoptosis, because the intrinsic pathway is already activated in XIAP-sensitive DLBCL cells, which cannot be further activated by etoposide. 9 In this study, we also show that the XIAP antagonist 1396-12 is active as a single agent and can directly induce apoptosis. Compared with conventional agents used in the treatment of DLBCL, the XIAP antagonist can induce apoptosis, even in cases with relatively low levels of etoposide-or rituximab-induced cell death.…”

“…9 The XIAP antagonist appeared to be especially effective in DLBCL samples with high expression levels of pro-and antiapoptotic genes (Table 1; P ϭ .02).…”

“…*Indicates expression levels of proapoptotic genes (including MCL1-short, Bax-Long, Noxa, Bad, Bak, Bim, Bid, Harakiri, PUMA, Bik, BNIP3, BNIP3L, MAP1, BMF, AIF, and antiapoptotic genes (including: BCL-W, BCL-XL, BCL-2, BCL2-A1, Appolon, BIRC7, NIAP, cIAP, cIAP2, XIAP, Survivin, as described previously. 9 †According to the previously described algorithm. 29 372 CILLESSEN et al BLOOD, 1 JANUARY 2008 ⅐ VOLUME 111, NUMBER 1…”

“…8 Subsequently, we revealed that high expression levels of proapoptotic genes are associated with constitutive activation of the intrinsic, caspase-9-mediated apoptosis pathway, and that apoptosis is inhibited downstream of caspase-9 activation. 9 Direct inhibitors of the downstream effector caspases of the intrinsic and extrinsic apoptosis pathways are the inhibitor of apoptosis proteins (IAPs). At present, 8 members of the IAP family have been identified in humans, including XIAP (X-linked inhibitor of apoptosis).…”

“…As a positive control for detection of apoptosis, Nalm6 cells were used. 9 Cell lines were cultured in RPMI 1640 medium (BioWhittaker) containing 10% fetal calf serum and 100 IU penicillin/100 g/mL streptomycin. …”