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Maritoclax and dinaciclib inhibit MCL-1 activity and induce apoptosis in both a MCL-1-dependent and -independent manner.

Varadarajan S, Poornima P, Milani M, Gowda K, Amin S, Wang HG, Cohen GM - Oncotarget (2015)

Bottom Line: Furthermore, maritoclax induced extensive mitochondrial fragmentation, and a Bax/Bak- but MCL-1-independent accumulation of mitochondrial reactive oxygen species (ROS), with an accompanying loss of complexes I and III of the electron transport chain.ROS scavengers, such as MitoQ, could not salvage maritoclax-mediated effects on mitochondrial structure and function.Although dinaciclib is clearly not a specific MCL-1 inhibitor, its ability to rapidly downregulate MCL-1 may be beneficial in many clinical settings, where it may reverse chemoresistance or sensitize to other chemotherapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Liverpool, UK.

ABSTRACT
The anti-apoptotic BCL-2 family proteins are important targets for cancer chemotherapy. Specific and potent inhibitors of the BCL-2 family, such as ABT-263 (navitoclax) and ABT-199, are only effective against some members of the BCL-2 family but do not target MCL-1, which is commonly amplified in tumors and associated with chemoresistance. In this report, the selectivity and potency of two putative MCL-1 inhibitors, dinaciclib and maritoclax, were assessed. Although both compounds induced Bax/Bak- and caspase-9-dependent apoptosis, dinaciclib was more potent than maritoclax in downregulating MCL-1 and also in inducing apoptosis. However, the compounds induced apoptosis, even in cells lacking MCL-1, suggesting multiple mechanisms of cell death. Furthermore, maritoclax induced extensive mitochondrial fragmentation, and a Bax/Bak- but MCL-1-independent accumulation of mitochondrial reactive oxygen species (ROS), with an accompanying loss of complexes I and III of the electron transport chain. ROS scavengers, such as MitoQ, could not salvage maritoclax-mediated effects on mitochondrial structure and function. Taken together, our data demonstrate that neither dinaciclib nor maritoclax exclusively target MCL-1. Although dinaciclib is clearly not a specific MCL-1 inhibitor, its ability to rapidly downregulate MCL-1 may be beneficial in many clinical settings, where it may reverse chemoresistance or sensitize to other chemotherapeutic agents.

No MeSH data available.


Related in: MedlinePlus

Dinaciclib and maritoclax diminish MCL-1 expression levels and induce MCL-1-dependent apoptosis in a cell-type specific manner(A) Whole cell lysates isolated from H460 cells, exposed to dinaciclib (30 nM) or maritoclax (3 μM) for the indicated times, were immunoblotted with the indicated antibodies. (B) H1299 cells were exposed to DMSO or ABT-263 (5 μM) for 30 min, followed for a further 24 h by the indicated concentrations of dinaciclib or maritoclax and cell death assessed by PS externalization. Western blots reveal changes in MCL-1 expression and PARP cleavage in H1299 cells, following 24 h of exposure to dinaciclib (30 nM) or maritoclax (3 μM), with or without a 30 min pretreatment of ABT-263 (5 μM). (C) Whole cell lysates from H1299 cells exposed to dinaciclib (30 nM) or maritoclax (3 μM) for the indicated times were immunoblotted with the indicated antibodies. (D) MEFs deficient in MCL-1 (dashed lines) along with their wild type counterparts (continuous bold lines) were exposed for 24 h to the indicated inhibitors and apoptosis assessed by PS externalization. (E) MCL-1f/fRosa-ERCreT2 MEFs were initially exposed to DMSO or 4-hydroxytamoxifen (4-HT) (100 nM) to delete endogenous MCL-1. The cells were then exposed for a further 24 h to the indicated inhibitors and apoptosis assessed. Western blots reveal changes in MCL-1 expression in different MEFs, following 4-HT exposure for 0, 24 or 48 h. Error bars represent the Mean ± SEM from three independent experiments. In all the graphs, the extent of apoptosis in untreated control cells matched the % apoptosis of the lowest concentration tested for both inhibitors.
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Figure 2: Dinaciclib and maritoclax diminish MCL-1 expression levels and induce MCL-1-dependent apoptosis in a cell-type specific manner(A) Whole cell lysates isolated from H460 cells, exposed to dinaciclib (30 nM) or maritoclax (3 μM) for the indicated times, were immunoblotted with the indicated antibodies. (B) H1299 cells were exposed to DMSO or ABT-263 (5 μM) for 30 min, followed for a further 24 h by the indicated concentrations of dinaciclib or maritoclax and cell death assessed by PS externalization. Western blots reveal changes in MCL-1 expression and PARP cleavage in H1299 cells, following 24 h of exposure to dinaciclib (30 nM) or maritoclax (3 μM), with or without a 30 min pretreatment of ABT-263 (5 μM). (C) Whole cell lysates from H1299 cells exposed to dinaciclib (30 nM) or maritoclax (3 μM) for the indicated times were immunoblotted with the indicated antibodies. (D) MEFs deficient in MCL-1 (dashed lines) along with their wild type counterparts (continuous bold lines) were exposed for 24 h to the indicated inhibitors and apoptosis assessed by PS externalization. (E) MCL-1f/fRosa-ERCreT2 MEFs were initially exposed to DMSO or 4-hydroxytamoxifen (4-HT) (100 nM) to delete endogenous MCL-1. The cells were then exposed for a further 24 h to the indicated inhibitors and apoptosis assessed. Western blots reveal changes in MCL-1 expression in different MEFs, following 4-HT exposure for 0, 24 or 48 h. Error bars represent the Mean ± SEM from three independent experiments. In all the graphs, the extent of apoptosis in untreated control cells matched the % apoptosis of the lowest concentration tested for both inhibitors.

Mentions: Although both dinaciclib and maritoclax induce apoptosis in MCL-1-addictive cell lines, the mechanisms by which the compounds inhibit MCL-1 are quite distinct. In the MCL-1-dependent H460 cells, dinaciclib caused a significant reduction of MCL-1 protein levels, as early as 2 h and was almost complete at 8 h (Fig. 2A). Dinaciclib also resulted in a time-dependent, near-complete loss of BIM and NOXA, but the loss of BAK was more modest (Fig. 2A). The loss of MCL-1 and its interacting partners preceded the cleavage of the canonical caspase substrate, poly (ADP-ribose) polymerase (PARP) (Fig. 2A). Maritoclax resulted in a much more modest loss of MCL-1, which, at later time points, was not just restored but resulted in protein levels higher than control cells (Fig. 2A). However, maritoclax also resulted in PARP cleavage at 16-24 h (Fig. 2A), suggesting that the early loss of MCL-1 was sufficient to trigger apoptotic events in these cells. Next, we wished to assess if this early loss of MCL-1 could synergize with BCL-2 family inhibitors, such as navitoclax, which inhibit BCL-2, BCL-XL and BCL-w, but not MCL-1, in enhancing apoptosis. For this experiment, we used H1299 cells, which depend on BCL-XL and MCL-1 for survival [16]. Pretreatment of H1299 cells for 30 min with navitoclax greatly enhanced the ability of dinaciclib to induce apoptosis (Fig. 2B). This potentiation of apoptosis was accompanied by the total loss of MCL-1 and significant PARP cleavage (Figs. 2B and 2C). In marked contrast, the extent of apoptosis induction, as evidenced by PARP processing and PS externalization, in cells exposed to navitoclax and maritoclax, was more modest (Figs. 2B and 2C). These observations suggest that a complete loss of MCL-1, as observed with dinaciclib, may be required to observe synergy with navitoclax.


Maritoclax and dinaciclib inhibit MCL-1 activity and induce apoptosis in both a MCL-1-dependent and -independent manner.

Varadarajan S, Poornima P, Milani M, Gowda K, Amin S, Wang HG, Cohen GM - Oncotarget (2015)

Dinaciclib and maritoclax diminish MCL-1 expression levels and induce MCL-1-dependent apoptosis in a cell-type specific manner(A) Whole cell lysates isolated from H460 cells, exposed to dinaciclib (30 nM) or maritoclax (3 μM) for the indicated times, were immunoblotted with the indicated antibodies. (B) H1299 cells were exposed to DMSO or ABT-263 (5 μM) for 30 min, followed for a further 24 h by the indicated concentrations of dinaciclib or maritoclax and cell death assessed by PS externalization. Western blots reveal changes in MCL-1 expression and PARP cleavage in H1299 cells, following 24 h of exposure to dinaciclib (30 nM) or maritoclax (3 μM), with or without a 30 min pretreatment of ABT-263 (5 μM). (C) Whole cell lysates from H1299 cells exposed to dinaciclib (30 nM) or maritoclax (3 μM) for the indicated times were immunoblotted with the indicated antibodies. (D) MEFs deficient in MCL-1 (dashed lines) along with their wild type counterparts (continuous bold lines) were exposed for 24 h to the indicated inhibitors and apoptosis assessed by PS externalization. (E) MCL-1f/fRosa-ERCreT2 MEFs were initially exposed to DMSO or 4-hydroxytamoxifen (4-HT) (100 nM) to delete endogenous MCL-1. The cells were then exposed for a further 24 h to the indicated inhibitors and apoptosis assessed. Western blots reveal changes in MCL-1 expression in different MEFs, following 4-HT exposure for 0, 24 or 48 h. Error bars represent the Mean ± SEM from three independent experiments. In all the graphs, the extent of apoptosis in untreated control cells matched the % apoptosis of the lowest concentration tested for both inhibitors.
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Figure 2: Dinaciclib and maritoclax diminish MCL-1 expression levels and induce MCL-1-dependent apoptosis in a cell-type specific manner(A) Whole cell lysates isolated from H460 cells, exposed to dinaciclib (30 nM) or maritoclax (3 μM) for the indicated times, were immunoblotted with the indicated antibodies. (B) H1299 cells were exposed to DMSO or ABT-263 (5 μM) for 30 min, followed for a further 24 h by the indicated concentrations of dinaciclib or maritoclax and cell death assessed by PS externalization. Western blots reveal changes in MCL-1 expression and PARP cleavage in H1299 cells, following 24 h of exposure to dinaciclib (30 nM) or maritoclax (3 μM), with or without a 30 min pretreatment of ABT-263 (5 μM). (C) Whole cell lysates from H1299 cells exposed to dinaciclib (30 nM) or maritoclax (3 μM) for the indicated times were immunoblotted with the indicated antibodies. (D) MEFs deficient in MCL-1 (dashed lines) along with their wild type counterparts (continuous bold lines) were exposed for 24 h to the indicated inhibitors and apoptosis assessed by PS externalization. (E) MCL-1f/fRosa-ERCreT2 MEFs were initially exposed to DMSO or 4-hydroxytamoxifen (4-HT) (100 nM) to delete endogenous MCL-1. The cells were then exposed for a further 24 h to the indicated inhibitors and apoptosis assessed. Western blots reveal changes in MCL-1 expression in different MEFs, following 4-HT exposure for 0, 24 or 48 h. Error bars represent the Mean ± SEM from three independent experiments. In all the graphs, the extent of apoptosis in untreated control cells matched the % apoptosis of the lowest concentration tested for both inhibitors.
Mentions: Although both dinaciclib and maritoclax induce apoptosis in MCL-1-addictive cell lines, the mechanisms by which the compounds inhibit MCL-1 are quite distinct. In the MCL-1-dependent H460 cells, dinaciclib caused a significant reduction of MCL-1 protein levels, as early as 2 h and was almost complete at 8 h (Fig. 2A). Dinaciclib also resulted in a time-dependent, near-complete loss of BIM and NOXA, but the loss of BAK was more modest (Fig. 2A). The loss of MCL-1 and its interacting partners preceded the cleavage of the canonical caspase substrate, poly (ADP-ribose) polymerase (PARP) (Fig. 2A). Maritoclax resulted in a much more modest loss of MCL-1, which, at later time points, was not just restored but resulted in protein levels higher than control cells (Fig. 2A). However, maritoclax also resulted in PARP cleavage at 16-24 h (Fig. 2A), suggesting that the early loss of MCL-1 was sufficient to trigger apoptotic events in these cells. Next, we wished to assess if this early loss of MCL-1 could synergize with BCL-2 family inhibitors, such as navitoclax, which inhibit BCL-2, BCL-XL and BCL-w, but not MCL-1, in enhancing apoptosis. For this experiment, we used H1299 cells, which depend on BCL-XL and MCL-1 for survival [16]. Pretreatment of H1299 cells for 30 min with navitoclax greatly enhanced the ability of dinaciclib to induce apoptosis (Fig. 2B). This potentiation of apoptosis was accompanied by the total loss of MCL-1 and significant PARP cleavage (Figs. 2B and 2C). In marked contrast, the extent of apoptosis induction, as evidenced by PARP processing and PS externalization, in cells exposed to navitoclax and maritoclax, was more modest (Figs. 2B and 2C). These observations suggest that a complete loss of MCL-1, as observed with dinaciclib, may be required to observe synergy with navitoclax.

Bottom Line: Furthermore, maritoclax induced extensive mitochondrial fragmentation, and a Bax/Bak- but MCL-1-independent accumulation of mitochondrial reactive oxygen species (ROS), with an accompanying loss of complexes I and III of the electron transport chain.ROS scavengers, such as MitoQ, could not salvage maritoclax-mediated effects on mitochondrial structure and function.Although dinaciclib is clearly not a specific MCL-1 inhibitor, its ability to rapidly downregulate MCL-1 may be beneficial in many clinical settings, where it may reverse chemoresistance or sensitize to other chemotherapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Liverpool, UK.

ABSTRACT
The anti-apoptotic BCL-2 family proteins are important targets for cancer chemotherapy. Specific and potent inhibitors of the BCL-2 family, such as ABT-263 (navitoclax) and ABT-199, are only effective against some members of the BCL-2 family but do not target MCL-1, which is commonly amplified in tumors and associated with chemoresistance. In this report, the selectivity and potency of two putative MCL-1 inhibitors, dinaciclib and maritoclax, were assessed. Although both compounds induced Bax/Bak- and caspase-9-dependent apoptosis, dinaciclib was more potent than maritoclax in downregulating MCL-1 and also in inducing apoptosis. However, the compounds induced apoptosis, even in cells lacking MCL-1, suggesting multiple mechanisms of cell death. Furthermore, maritoclax induced extensive mitochondrial fragmentation, and a Bax/Bak- but MCL-1-independent accumulation of mitochondrial reactive oxygen species (ROS), with an accompanying loss of complexes I and III of the electron transport chain. ROS scavengers, such as MitoQ, could not salvage maritoclax-mediated effects on mitochondrial structure and function. Taken together, our data demonstrate that neither dinaciclib nor maritoclax exclusively target MCL-1. Although dinaciclib is clearly not a specific MCL-1 inhibitor, its ability to rapidly downregulate MCL-1 may be beneficial in many clinical settings, where it may reverse chemoresistance or sensitize to other chemotherapeutic agents.

No MeSH data available.


Related in: MedlinePlus