Down-regulation of Mcl-1 potentiates HDACi-mediated apoptosis in leukemic cells.

Mcl-1 is an anti-apoptotic Bcl-2 family member, whose degradation is supposedly required for induction of apoptosis. However, histone deacetylase inhibitors (HDACi) induce apoptosis primarily through the Bak/Mcl-1/Noxa and Bim pathways without decreasing Mcl-1. To investigate this discrepancy, we examined the role of Mcl-1 on HDACi-mediated apoptosis. Inhibition of either Class I or Class II HDAC by selective HDACi caused an upregulation of Mcl-1 mRNA and protein. Down-regulation of Mcl-1 by three structurally unrelated cyclin dependent kinase inhibitors potentiated HDACi-mediated apoptosis in primary chronic lymphocytic leukemic (CLL) cells and K562 cells. Sensitivity to HDACi-induced apoptosis was increased ~10-fold by the cyclin dependent kinase inhibitors. Nanomolar concentrations of HDACi, ~300-fold lower than required to induce apoptosis alone, sensitized cells to TRAIL, emphasizing that the mechanism(s) whereby HDACi induce apoptosis is clearly distinct from those by which they sensitize to TRAIL. Furthermore knockdown of Mcl-1 potentiated HDACi-mediated apoptosis in K562 cells. Thus HDACi-mediated Mcl-1 upregulation plays an important anti-apoptotic regulatory role in limiting the efficacy of HDACi-induced apoptosis, which can be overcome by combination with an agent that down-regulates Mcl-1. Thus a clinical trial in some cancers is warranted using a combination of an HDACi with agents that down-regulate Mcl-1

Concurrent upregulation of BCL-XL and BCL2A1 induces ~1000-fold resistance to ABT-737 in chronic lymphocytic leukemia.

ABT-737 and its orally active analog, ABT-263, are rationally designed inhibitors of BCL2 and BCL-XL. ABT-263 shows promising activity in early phase 1 clinical trials in B-cell malignancies, particularly chronic lymphocytic leukemia (CLL). In vitro, peripheral blood CLL cells are extremely sensitive to ABT-737 (EC50 7 nM), with rapid induction of apoptosis in all 60 patients tested, independent of parameters associated with disease progression and chemotherapy resistance. In contrast to data from cell lines, ABT-737–induced apoptosis in CLL cells was largely MCL1-independent. Because CLL cells within lymph nodes are more resistant to apoptosis than those in peripheral blood, CLL cells were cultured on CD154-expressing fibroblasts in the presence of interleukin-4 (IL-4) to mimic the lymph node microenvironment. CLL cells thus cultured developed an approximately 1000-fold resistance to ABT-737 within 24 hours. Investigations of the underlying mechanism revealed that this resistance occurred upstream of mitochondrial perturbation and involved de novo synthesis of the antiapoptotic proteins BCL-XL and BCL2A1, which were responsible for resistance to low and high ABT-737 concentrations, respectively. Our data indicate that after therapy with ABT-737–related inhibitors, resistant CLL cells might develop in lymph nodes in vivo and that treatment strategies targeting multiple BCL2 antiapoptotic members simultaneously may have synergistic activity

TRAIL signals to apoptosis in CLL cells primarily through TRAIL R-1 whereas cross-linked agonistic TRAIL R-2 antibodies facilitate signalling via TRAIL R-2

TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF family, which is being developed as an anti-tumour agent due to its selective toxicity to tumour cells, induces apoptosis by binding to two membrane-bound receptors, TRAIL-R1 and TRAIL-R2. Clinical trials have been initiated with various preparations of TRAIL as well as agonistic mAbs to TRAIL-R1 and TRAILR2. Previously we reported that prior treatment of primary chronic lymphocytic leukaemia (CLL) cells with histone deacetylase inhibitors (HDACi) was required to sensitize CLL cells to TRAIL and using various receptor-selective TRAIL mutant ligands we demonstrated that CLL cells signalled to apoptosis primarily through TRAIL-R1. Some, but not all, agonistic TRAIL-receptor antibodies require cross-linking in order to induce apoptosis. We now demonstrate that CLL cells can signal to apoptosis through the TRAIL-R2 receptor, but only after cross-linking of the agonistic TRAIL-R2 antibodies, LBY135 and lexatumumab (HGS-ETR2). In contrast, signalling through TRAIL-R1 by receptor-selective ligands or certain agonistic antibodies, such as mapatumumab (HGS-ETR1) occurs in the absence of cross-linking. These results further highlight important differences in apoptotic signalling triggered through TRAIL-R1 and TRAIL-R2 in primary tumour cells. Such information is clearly important for the rational optimisation of TRAIL therapy in primary lymphoid malignancies, such as CLL