Release Of BAK Research Articles

Combination with vorinostat overcomes ABT-263 (navitoclax) resistance of small cell lung cancer

ABSTRACTSmall cell lung cancer (SCLC) is an aggressive tumor type with high mortality. One promising approach for SCLC treatment would be to utilize agents targeting molecular abnormalities regulating resistance to apoptosis. BH3 mimetic antagonists, such as ABT-737 and its orally available derivative ABT-263 (navitoclax) have been developed to block the function of pro-survival BCL-2 family members. The sensitivity of SCLC to these drugs varies over a broad range in vitro and in clinical trials. We have previously shown that the expression of Noxa, a BH3-only pro-apoptotic BCL-2 family protein, is a critical determinant of sensitivity to ABT-737. Thus, pharmacological up-regulation of Noxa could enhance cell death induced by the BH3 mimetics. We find that the combination of ABT-263 and a HDAC inhibitor, vorinostat, efficiently induces apoptosis in a variety of SCLC cell lines, including ABT-263 resistant cell lines. Cell death induced by combined treatment is Noxa- and/or BIM-dependent in some cell lines but in others appears to be mediated by down-regulation of BCL-XL and release of BAK from BCL-XL and MCL-1. These results suggest that combination of HDAC inhibitors and BCL-2 inhibitors could be an alternative and effective regimen for SCLC treatment.

BIM-Mediated Membrane Insertion of the BAK Pore Domain Is an Essential Requirement for Apoptosis

SummaryBAK activation represents a key step during apoptosis, but how it converts into a mitochondria-permeabilizing pore remains unclear. By further delineating the structural rearrangements involved, we reveal that BAK activation progresses through a series of independent steps: BH3-domain exposure, N-terminal change, oligomerization, and membrane insertion. Employing a “BCL-XL-addiction” model, we show that neutralization of BCL-XL by the BH3 mimetic ABT-737 resulted in death only when cells were reconstituted with BCL-XL:BAK, but not BCL-2/ BCL-XL:BIM complexes. Although this resembles the indirect model, release of BAK from BCL-XL did not result in spontaneous adoption of the pore conformation. Commitment to apoptosis required association of the direct activator BIM with oligomeric BAK promoting its conversion to a membrane-inserted pore. The sequential nature of this cascade provides multiple opportunities for other BCL-2 proteins to interfere with or promote BAK activation and unites aspects of the indirect and direct activation models.

MEK inhibitors potentiate dexamethasone lethality in acute lymphoblastic leukemia cells through the pro-apoptotic molecule BIM

Glucocorticoids (GC) are common components of many chemotherapeutic regimens for lymphoid malignancies. GC-induced apoptosis involves an intrinsic mitochondria-dependent pathway. We and others have shown that BIM (BCL-2 interacting mediator of cell death), a BH3-only pro-apoptotic protein, is up-regulated by dexamethasone (Dex) treatment in acute lymphoblastic leukemia (ALL) cells and plays an essential role in Dex-induced apoptosis. Furthermore, BIM is inactivated by extracellular signal-regulated kinase (ERK)-mediated phosphorylation. We therefore hypothesized co-treatment with Dex and MEK/ERK inhibitors would promote apoptosis in ALL cells through BIM up-regulation and activation. We show here that MEK inhibitors (PD184352 and PD98059) synergistically enhance Dex lethality in a variety of ALL cells and in two primary ALL specimens. Co-treatment with Dex and PD184352 results in BIM accumulation, pro-apoptotic BAX/BAK activation, and cytochrome c release from mitochondria. Down-regulation of BIM by short-hairpin RNA in ALL cells suppressed BAX/BAK activation, cytochrome c release, and cell death by Dex/PD184352 co-treatment. BIM accumulated by this treatment sequesters anti-apoptotic BCL-XL/MCL-1, resulting in the release of BAK from these anti-apoptotic molecules. This study provides a rational foundation for future attempts to improve the activity of glucocorticoids with clinically relevant pharmacologic MEK inhibitors in the treatment of ALL and possibly other hematologic malignancies.

The Combination of a Histone Deacetylase (HDAC) Inhibitor with the BCL-2 Inhibitor GX15-070 Has Synergistic Antileukemia Effect by Inducing Both Apoptotic and Autophagic Pathways.

HDAC inhibitors have limited single agent clinical activity in leukemia, a probable effect of dysregulated proapoptotic pathways. We hypothesized that the combination of a HDAC inhibitor with a Bcl-2 inhibitor may result in synergistic antileukemia activity. To test this concept, we modeled in vitro the combination of MGCD0103, a class 1 specific HDAC inhibitor, with GX15-070 (Obatoclax), a novel Bcl-2 homology domain-3 (BH3) mimetic The cytotoxic potential of the combination was first assessed measuring cell viability using trypan blue extraction assays in leukemia cell lines HL60, U937, THP1, and Molt4. Mathematical modeling analysis indicated a strong synergistic effect of the combination of MGCD0103 and GX15-070 in terms of growth inhibition. Inhibitory effect of the combination at concentrations of MGCD0103 600 nM and GX15-070 200 nM was 64 fold the effect of single agent MGCD0103 and 16 fold that of GX15-070. Analysis of sequence of treatment indicated that the optimal sequence was addition of GX15-070 first followed by MGCD0103. We then analyzed the effect of the combination on the induction of apoptosis using Annexin V flow assays. The combination also showed a marked synergistic effect in terms of apoptosis induction (the combination was 217% more potent than single agent MGCD0103 and 79% more than GX15-070). Western blot analysis indicated that the use of the combination substantially enhanced the cleavage/activation of caspase-3 and the subsequent cleavage of caspase-3 substrate PARP. As demonstrated in AML and other cell types previously, we showed that GX15-070 dissociated the pro-apoptotic protein BAK from MCL-1. The release of BAK has been demonstrated to play an important role in caspase activation. Western blot analysis also demonstrated that while anti-apoptotic protein XIAP, the major inhibitor of caspase 3, was reduced in MGCD0103 treated cells, the reduction was enhanced with the combination. These results suggest that GX15-070 may sensitize XIAP to the down-regulation effect of MGCD0103, which in turn further activates caspase 3. The combinatory effect of BAK release by GX-15-070, with the suppression of XIAP by MGCD0103, that is potentiated by GX15-070, may play a critical role in the synergistic effect of these two agents on caspase activation, which then results in increased induction of apoptosis. Because in initial experiments we evidenced a discrepancy between cell viability and apoptosis, we also studied the effect of the combination on the induction of autophagy using electron microscopy: non-apoptotic cells after MGCD0103 treatment did not show increase autophagic characteristics compared to control cells, while more non-apoptotic cells under GX15-070 evidenced swollen endoreticulum, a feature of early autophagy. Most non-apoptotic cells treated with MGCD0103 and GX15-070 combination showed accumulation of macro-autophagosomes, a marker of late autophagy, indicating that more severe autophagy was induced by the combination. Furthermore, induction of LC3-II, a marker of autophagy, was observed after combination. We confirmed these results (both in terms of apoptosis and autophagy) ex vivo in primary cells (n=8) obtained from patients with relapsed/refractory AML. In conclusion, the combination of an HDAC inhibitor with GX15-070 has synergistic antileukemia activity and should be studied in human clinical trials.

Targetting a Pro-Apoptotic Molecule BIM by Dexamethasone and MEK Inhibitors Synergistically Induces Apoptosis in Acute Lymphoblastic Leukemia Cells.

Glucocorticoids (GC) are common components in many chemotherapeutic protocols for lymphoid/myeloid malignancies, including ALL, multiple myeloma, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. GC-induced apoptosis is essentially divided three stages:an initiation stage, which involves glucocorticoid receptor (GR) activation and GR-mediated gene regulation;a decision stage, which engages pro- and anti-apoptotic BCL-2 family proteins at the mitochondrial level; andexecution stage, which implicates caspases and endonucleases activation.Although the effects of GC on lymphocytes have been scrutinized for many years, the molecular mechanisms of sensitivity and resistance are still elusive. Since GC-resistant leukemia and myeloma are often associated with GR defects, bypassing the receptor by targeting downstream molecules may develop new therapeutic strategies. Numerous novel drug combinations are currently being tested to prevent resistance and improve GC efficacy in the therapy of lymphoid malignancies. However, it is still not entirely clear how the signaling pathways and their downstream target molecules, including the BCL-2 family members, participate in GC-induced cell death. We and others have shown that BIM (BCL-2 interacting mediator of cell death), a BH3-only pro-apoptotic BCL-2 family protein, is up-regulated by dexamethasone (Dex) treatment in acute lymphoblastic leukemia (ALL) cells and plays an essential role in Dex-induced apoptosis. Furthermore, BIM is inactivated by extracellular signal-regulated kinase (ERK)-mediated phosphorylation. We therefore hypothesized co-treatment with Dex and MEK/ERK inhibitors would promote apoptosis in ALL cells through BIM upregulation and activation. We show here that MEK inhibitors (PD184352 and PD98059) synergistically enhance Dex lethality in both T-ALL and B-ALL cells. Co-treatment with Dex and PD184352 results in BIM accumulation, pro-apoptotic BAX/BAK activation, and cytochrome c release from mitochondria. Down-regulation of BIM by short-hairpin RNA in CCRF-CEM (T-ALL) and RS4;11 (B-ALL) cells almost completely suppressed BAX/BAK activation, cytochrome c release, and cell death by Dex/PD184352 co-treatment. BIM accumulated by this co-treatment sequesters anti-apoptotic BCL-XL/MCL-1, resulting in the release of BAK from these anti-apoptotic molecules. The main concept is that we have a novel and potentially effective way to increase GC activity against ALL cells, which may reflect the fact thatGCs up-regulate BIM; andpharmacologic MEK inhibitors further potentiate BIM activation by blocking BIM phosphorylation and degradation.Our study provides a rational foundation for future attempts to improve the effect of glucocorticoids with clinically relevant pharmacologic MEK inhibitors in the treatment of ALL and possibly other hematological malignancies. BIM also plays an important role in cell death induced by other chemotherapeutic drugs such as STI571 (imatinib mesylate) and histone deacetylase inhibitors. Thus, targeting BIM by drugs combination could be effective on other tumors.