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Characterization of a Dual CDC7/CDK9 Inhibitor in Multiple Myeloma Cellular Models.

Natoni A, Coyne MR, Jacobsen A, Rainey MD, O'Brien G, Healy S, Montagnoli A, Moll J, O'Dwyer M, Santocanale C - Cancers (Basel) (2013)

Bottom Line: These compounds also inhibit CDK9 which is relevant for MCL-1 expression.The activity and mechanism of action of the dual CDC7/CDK9 inhibitor PHA-767491 was assessed in a panel of multiple myeloma cell lines, in primary samples from patients, in the presence of stromal cells and in combination with drugs used in current chemotherapeutic regimens.We report that in all conditions myeloma cells undergo cell death upon PHA-767491 treatment and we report an overall additive effect with melphalan, bortezomib and doxorubicin, thus supporting further assessment of targeting CDC7 and CDK9 in multiple myeloma.

View Article: PubMed Central - PubMed

Affiliation: Centre for Chromosome Biology, School of Natural Sciences National University of Ireland Galway, Galway, Ireland. michael.odwyer@nuigalway.ie.

ABSTRACT
Two key features of myeloma cells are the deregulation of the cell cycle and the dependency on the expression of the BCL2 family of anti-apoptotic proteins. The cell division cycle 7 (CDC7) is an essential S-phase kinase and emerging CDC7 inhibitors are effective in a variety of preclinical cancer models. These compounds also inhibit CDK9 which is relevant for MCL-1 expression. The activity and mechanism of action of the dual CDC7/CDK9 inhibitor PHA-767491 was assessed in a panel of multiple myeloma cell lines, in primary samples from patients, in the presence of stromal cells and in combination with drugs used in current chemotherapeutic regimens. We report that in all conditions myeloma cells undergo cell death upon PHA-767491 treatment and we report an overall additive effect with melphalan, bortezomib and doxorubicin, thus supporting further assessment of targeting CDC7 and CDK9 in multiple myeloma.

No MeSH data available.


Related in: MedlinePlus

Analysis of pro- and anti-apoptotic proteins in response to PHA-767491. KMS-18 and MM1S myeloma cells were incubated with 5 μM PHA-767491 for the indicated time. Protein extracts were prepared and analyzed by immunoblotting using the indicated antibodies (A). In parallel samples, apoptosis (B) and DNA synthesis (C) were analysed by flow cytometry using AV staining and EdU incorporation assay respectively. Numbers in the gated regions represent the percentage of cells positive for either AV (B) or EdU (C) staining.
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cancers-05-00901-f003: Analysis of pro- and anti-apoptotic proteins in response to PHA-767491. KMS-18 and MM1S myeloma cells were incubated with 5 μM PHA-767491 for the indicated time. Protein extracts were prepared and analyzed by immunoblotting using the indicated antibodies (A). In parallel samples, apoptosis (B) and DNA synthesis (C) were analysed by flow cytometry using AV staining and EdU incorporation assay respectively. Numbers in the gated regions represent the percentage of cells positive for either AV (B) or EdU (C) staining.

Mentions: We have previously shown that in CLL primary cells, PHA-767491 induces apoptosis through the intrinsic pathway most likely by downregulating MCL-1 protein levels and that upregulation of BCL-XL upon CD40 stimulation can limit apoptotic cell death [26]. In order to gain a better understanding of the molecular determinants of PHA-767491-induced apoptosis in myeloma cells, we analysed the levels and dynamics of several pro- and anti-apoptotic proteins in both KMS-18 and MM1S cells in a time course experiment after exposure to 5 µM PHA-767491. Samples were also taken to assess DNA synthesis and levels of apoptosis by measuring the percentage of cells incorporating the thymidine analogue EdU in their DNA and exposing phosphatidylserine on the outer membrane respectively. The levels of pSer2 RNA Pol II decreased very rapidly following PHA-767491 treatment in both cell lines (Figure 3A). PHA-767491 treatment also diminished the levels of pSer40 albeit with slower kinetics compared to pSer2 RNA Pol II (Figure 3A). PHA-767491 induced a marked and rapid downregulation of MCL-1 proteins levels in KMS-18 which was rather slow and less pronounced in MM1S at these early time points (Figure 3A). Downregulation of MCL-1 levels in KMS-18 correlated with a robust caspase-3 activation, PARP cleavage and loss of X-IAP. Again, these events were less evident in MM1S, supporting a direct correlation between MCL-1 downregulation and caspase activation. Moreover, the appearance of AV+ cells in both cell lines and a complete chase of DNA synthesis were observed and these effects occurred faster and were more pronounced in KMS-18 than in MMS1 cells (Figure 3B,C), although at later time points the number of apoptotic cells was equivalent for both cell lines. The percentage of AV+ cells directly correlated with the relative amount of cleaved PARP and cleaved caspase 3 detected in the extracts and inversely correlated with the levels of anti-apoptotic protein MCL-1. The relative levels of the anti-apoptotic protein BCL2 were higher in KMS-18 than MM1S and did not change significantly during the time of treatment (Figure 3A). Intriguingly the pro-apoptotic protein NOXA, that normally binds and counteracts MCL-1 [36], was undetectable in MM1S cells and was downregulated in KMS-18 following PHA-767491 treatment arguing against a role of NOXA in PHA-767491-induced apoptosis in myeloma cells. Instead, the levels of BCL-XL were higher in MM1S than KMS-18 and were not affected by the PHA-767491 treatment (Figure 3A). To better understand the mechanism(s) of action of PHA-767491, the protein levels of BIM which together with NOXA represents the major binding partner of MCL-1 [36], were analysed in both KMS-18 and MM1S cells in a time course experiment after exposure to 5 µM PHA-767491. Again, PHA-767491 induced a marked downregulation of MCL-1 which was accompanied by an increase in AV+ cells, however the protein levels of BIM were stable with a slight decrease in KMS-18 at 9 h post-treatment probably due to the high levels of apoptosis (Figure 4A). Importantly, the decrease in the MCL-1 protein levels were not a consequence of caspase activation as inhibition of caspases by the broad spectrum caspase inhibitor QVD-OPH did not prevent downregulation of MCL-1 although completely prevented phosphatidylserine exposure and PARP cleavage (Figure 4B). Altogether these results suggest that the downregulation of MCL-1 contributes to PHA-767491-induced apoptosis.


Characterization of a Dual CDC7/CDK9 Inhibitor in Multiple Myeloma Cellular Models.

Natoni A, Coyne MR, Jacobsen A, Rainey MD, O'Brien G, Healy S, Montagnoli A, Moll J, O'Dwyer M, Santocanale C - Cancers (Basel) (2013)

Analysis of pro- and anti-apoptotic proteins in response to PHA-767491. KMS-18 and MM1S myeloma cells were incubated with 5 μM PHA-767491 for the indicated time. Protein extracts were prepared and analyzed by immunoblotting using the indicated antibodies (A). In parallel samples, apoptosis (B) and DNA synthesis (C) were analysed by flow cytometry using AV staining and EdU incorporation assay respectively. Numbers in the gated regions represent the percentage of cells positive for either AV (B) or EdU (C) staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3795371&req=5

cancers-05-00901-f003: Analysis of pro- and anti-apoptotic proteins in response to PHA-767491. KMS-18 and MM1S myeloma cells were incubated with 5 μM PHA-767491 for the indicated time. Protein extracts were prepared and analyzed by immunoblotting using the indicated antibodies (A). In parallel samples, apoptosis (B) and DNA synthesis (C) were analysed by flow cytometry using AV staining and EdU incorporation assay respectively. Numbers in the gated regions represent the percentage of cells positive for either AV (B) or EdU (C) staining.
Mentions: We have previously shown that in CLL primary cells, PHA-767491 induces apoptosis through the intrinsic pathway most likely by downregulating MCL-1 protein levels and that upregulation of BCL-XL upon CD40 stimulation can limit apoptotic cell death [26]. In order to gain a better understanding of the molecular determinants of PHA-767491-induced apoptosis in myeloma cells, we analysed the levels and dynamics of several pro- and anti-apoptotic proteins in both KMS-18 and MM1S cells in a time course experiment after exposure to 5 µM PHA-767491. Samples were also taken to assess DNA synthesis and levels of apoptosis by measuring the percentage of cells incorporating the thymidine analogue EdU in their DNA and exposing phosphatidylserine on the outer membrane respectively. The levels of pSer2 RNA Pol II decreased very rapidly following PHA-767491 treatment in both cell lines (Figure 3A). PHA-767491 treatment also diminished the levels of pSer40 albeit with slower kinetics compared to pSer2 RNA Pol II (Figure 3A). PHA-767491 induced a marked and rapid downregulation of MCL-1 proteins levels in KMS-18 which was rather slow and less pronounced in MM1S at these early time points (Figure 3A). Downregulation of MCL-1 levels in KMS-18 correlated with a robust caspase-3 activation, PARP cleavage and loss of X-IAP. Again, these events were less evident in MM1S, supporting a direct correlation between MCL-1 downregulation and caspase activation. Moreover, the appearance of AV+ cells in both cell lines and a complete chase of DNA synthesis were observed and these effects occurred faster and were more pronounced in KMS-18 than in MMS1 cells (Figure 3B,C), although at later time points the number of apoptotic cells was equivalent for both cell lines. The percentage of AV+ cells directly correlated with the relative amount of cleaved PARP and cleaved caspase 3 detected in the extracts and inversely correlated with the levels of anti-apoptotic protein MCL-1. The relative levels of the anti-apoptotic protein BCL2 were higher in KMS-18 than MM1S and did not change significantly during the time of treatment (Figure 3A). Intriguingly the pro-apoptotic protein NOXA, that normally binds and counteracts MCL-1 [36], was undetectable in MM1S cells and was downregulated in KMS-18 following PHA-767491 treatment arguing against a role of NOXA in PHA-767491-induced apoptosis in myeloma cells. Instead, the levels of BCL-XL were higher in MM1S than KMS-18 and were not affected by the PHA-767491 treatment (Figure 3A). To better understand the mechanism(s) of action of PHA-767491, the protein levels of BIM which together with NOXA represents the major binding partner of MCL-1 [36], were analysed in both KMS-18 and MM1S cells in a time course experiment after exposure to 5 µM PHA-767491. Again, PHA-767491 induced a marked downregulation of MCL-1 which was accompanied by an increase in AV+ cells, however the protein levels of BIM were stable with a slight decrease in KMS-18 at 9 h post-treatment probably due to the high levels of apoptosis (Figure 4A). Importantly, the decrease in the MCL-1 protein levels were not a consequence of caspase activation as inhibition of caspases by the broad spectrum caspase inhibitor QVD-OPH did not prevent downregulation of MCL-1 although completely prevented phosphatidylserine exposure and PARP cleavage (Figure 4B). Altogether these results suggest that the downregulation of MCL-1 contributes to PHA-767491-induced apoptosis.

Bottom Line: These compounds also inhibit CDK9 which is relevant for MCL-1 expression.The activity and mechanism of action of the dual CDC7/CDK9 inhibitor PHA-767491 was assessed in a panel of multiple myeloma cell lines, in primary samples from patients, in the presence of stromal cells and in combination with drugs used in current chemotherapeutic regimens.We report that in all conditions myeloma cells undergo cell death upon PHA-767491 treatment and we report an overall additive effect with melphalan, bortezomib and doxorubicin, thus supporting further assessment of targeting CDC7 and CDK9 in multiple myeloma.

View Article: PubMed Central - PubMed

Affiliation: Centre for Chromosome Biology, School of Natural Sciences National University of Ireland Galway, Galway, Ireland. michael.odwyer@nuigalway.ie.

ABSTRACT
Two key features of myeloma cells are the deregulation of the cell cycle and the dependency on the expression of the BCL2 family of anti-apoptotic proteins. The cell division cycle 7 (CDC7) is an essential S-phase kinase and emerging CDC7 inhibitors are effective in a variety of preclinical cancer models. These compounds also inhibit CDK9 which is relevant for MCL-1 expression. The activity and mechanism of action of the dual CDC7/CDK9 inhibitor PHA-767491 was assessed in a panel of multiple myeloma cell lines, in primary samples from patients, in the presence of stromal cells and in combination with drugs used in current chemotherapeutic regimens. We report that in all conditions myeloma cells undergo cell death upon PHA-767491 treatment and we report an overall additive effect with melphalan, bortezomib and doxorubicin, thus supporting further assessment of targeting CDC7 and CDK9 in multiple myeloma.

No MeSH data available.


Related in: MedlinePlus