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Selective CDK9 inhibition overcomes TRAIL resistance by concomitant suppression of cFlip and Mcl-1.

Lemke J, von Karstedt S, Abd El Hay M, Conti A, Arce F, Montinaro A, Papenfuss K, El-Bahrawy MA, Walczak H - Cell Death Differ. (2013)

Bottom Line: Mechanistically, CDK9 inhibition resulted in downregulation of cellular FLICE-like inhibitory protein (cFlip) and Mcl-1 at both the mRNA and protein levels.Primary human hepatocytes did not succumb to the same treatment regime, defining a therapeutic window.Importantly, TRAIL in combination with SNS-032 eradicated established, orthotopic lung cancer xenografts in vivo.

View Article: PubMed Central - PubMed

Affiliation: 1] Centre for Cell Death, Cancer and Inflammation, UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6DD, UK [2] Clinic of General and Visceral Surgery, University of Ulm, Albert-Einstein-Allee 23, 89081 Ulm, Germany.

ABSTRACT
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in many cancer cells without causing toxicity in vivo. However, to date, TRAIL-receptor agonists have only shown limited therapeutic benefit in clinical trials. This can, most likely, be attributed to the fact that 50% of all cancer cell lines and most primary human cancers are TRAIL resistant. Consequently, future TRAIL-based therapies will require the addition of sensitizing agents that remove crucial blocks in the TRAIL apoptosis pathway. Here, we identify PIK-75, a small molecule inhibitor of the p110α isoform of phosphoinositide-3 kinase (PI3K), as an exceptionally potent TRAIL apoptosis sensitizer. Surprisingly, PI3K inhibition was not responsible for this activity. A kinome-wide in vitro screen revealed that PIK-75 strongly inhibits a panel of 27 kinases in addition to p110α. Within this panel, we identified cyclin-dependent kinase 9 (CDK9) as responsible for TRAIL resistance of cancer cells. Combination of CDK9 inhibition with TRAIL effectively induced apoptosis even in highly TRAIL-resistant cancer cells. Mechanistically, CDK9 inhibition resulted in downregulation of cellular FLICE-like inhibitory protein (cFlip) and Mcl-1 at both the mRNA and protein levels. Concomitant cFlip and Mcl-1 downregulation was required and sufficient for TRAIL sensitization by CDK9 inhibition. When evaluating cancer selectivity of TRAIL combined with SNS-032, the most selective and clinically used inhibitor of CDK9, we found that a panel of mostly TRAIL-resistant non-small cell lung cancer cell lines was readily killed, even at low concentrations of TRAIL. Primary human hepatocytes did not succumb to the same treatment regime, defining a therapeutic window. Importantly, TRAIL in combination with SNS-032 eradicated established, orthotopic lung cancer xenografts in vivo. Based on the high potency of CDK9 inhibition as a cancer cell-selective TRAIL-sensitizing strategy, we envisage the development of new, highly effective cancer therapies.

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CDK9 mediates TRAIL resistance by promoting concomitant transcription of cFlip and Mcl-1. (a) A549 or HeLa cells were incubated with SNS-032 (300 nM) or PIK-75 (100 nM) for 6 h and subsequently stained for surface expression of TRAIL-R1 and TRAIL-R2. One representative of two independent experiments is shown. (b) A549 cells were treated with PIK-75 (100 nM) or SNS-032 (300 nM) for the indicated times. Cells were lysed and subjected to western blotting. One representative of two independent experiments is shown. (c) HeLa cells were subjected to the indicated knockdowns for 48 or 72 h. zVAD was added at 20 μM 24 h after transfection where indicated. Cells were lysed and subjected to western blotting. One representative of two independent experiments is shown. (d) A549 and HeLa cells were incubated with SNS-032 (300 nM) for different times. cFlipL, cFlipS and Mcl-1 mRNA expression was quantified by RT-PCR. Values are means±S.E.M. of three independent experiments. Z, zVAD
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fig4: CDK9 mediates TRAIL resistance by promoting concomitant transcription of cFlip and Mcl-1. (a) A549 or HeLa cells were incubated with SNS-032 (300 nM) or PIK-75 (100 nM) for 6 h and subsequently stained for surface expression of TRAIL-R1 and TRAIL-R2. One representative of two independent experiments is shown. (b) A549 cells were treated with PIK-75 (100 nM) or SNS-032 (300 nM) for the indicated times. Cells were lysed and subjected to western blotting. One representative of two independent experiments is shown. (c) HeLa cells were subjected to the indicated knockdowns for 48 or 72 h. zVAD was added at 20 μM 24 h after transfection where indicated. Cells were lysed and subjected to western blotting. One representative of two independent experiments is shown. (d) A549 and HeLa cells were incubated with SNS-032 (300 nM) for different times. cFlipL, cFlipS and Mcl-1 mRNA expression was quantified by RT-PCR. Values are means±S.E.M. of three independent experiments. Z, zVAD

Mentions: Having established that CDK9 inhibition efficiently sensitizes cancer cell lines to TRAIL-induced apoptosis, we next addressed which molecular changes are responsible for this effect. Upregulation of TRAIL-R1 and/or TRAIL-R2 often correlates with, and sometimes also contributes to, TRAIL apoptosis sensitization.36 However, treatment of HeLa or A549 cells with PIK-75 or SNS-032 did not alter TRAIL-R1/R2 surface expression (Figure 4a), in line with similar recruitment of TRAIL-R1/2 in the DISC analysis (Figure 3e). Consequently, TRAIL sensitization by CDK9 inhibition is likely to require changes in intracellular modulators of the TRAIL apoptosis pathway that should enhance DISC activity and possibly additional downstream steps in the pathway. We, therefore, next investigated whether known components of the TRAIL–DISC and the downstream apoptosis pathway it activates are regulated by PIK-75 or SNS-032 treatment. Whereas the majority of the DISC components and downstream pro- and anti-apoptotic proteins remained unchanged, cFlip and Mcl-1 protein levels were rapidly suppressed by pharmacological CDK9 inhibition by SNS-032 or PIK-75 (Figure 4b and Supplementary Figure S4a). Because siRNA-mediated suppression of CDK9, performed in the presence or absence of pan-caspase inhibition to exclude a possible impact of CDK9-silencing-induced apoptosis, also resulted in downregulation of cFlip and Mcl-1, we can conclude that CDK9 is required to maintain high expression of these anti-apoptotic proteins in cancer cells (Figure 4c).


Selective CDK9 inhibition overcomes TRAIL resistance by concomitant suppression of cFlip and Mcl-1.

Lemke J, von Karstedt S, Abd El Hay M, Conti A, Arce F, Montinaro A, Papenfuss K, El-Bahrawy MA, Walczak H - Cell Death Differ. (2013)

CDK9 mediates TRAIL resistance by promoting concomitant transcription of cFlip and Mcl-1. (a) A549 or HeLa cells were incubated with SNS-032 (300 nM) or PIK-75 (100 nM) for 6 h and subsequently stained for surface expression of TRAIL-R1 and TRAIL-R2. One representative of two independent experiments is shown. (b) A549 cells were treated with PIK-75 (100 nM) or SNS-032 (300 nM) for the indicated times. Cells were lysed and subjected to western blotting. One representative of two independent experiments is shown. (c) HeLa cells were subjected to the indicated knockdowns for 48 or 72 h. zVAD was added at 20 μM 24 h after transfection where indicated. Cells were lysed and subjected to western blotting. One representative of two independent experiments is shown. (d) A549 and HeLa cells were incubated with SNS-032 (300 nM) for different times. cFlipL, cFlipS and Mcl-1 mRNA expression was quantified by RT-PCR. Values are means±S.E.M. of three independent experiments. Z, zVAD
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fig4: CDK9 mediates TRAIL resistance by promoting concomitant transcription of cFlip and Mcl-1. (a) A549 or HeLa cells were incubated with SNS-032 (300 nM) or PIK-75 (100 nM) for 6 h and subsequently stained for surface expression of TRAIL-R1 and TRAIL-R2. One representative of two independent experiments is shown. (b) A549 cells were treated with PIK-75 (100 nM) or SNS-032 (300 nM) for the indicated times. Cells were lysed and subjected to western blotting. One representative of two independent experiments is shown. (c) HeLa cells were subjected to the indicated knockdowns for 48 or 72 h. zVAD was added at 20 μM 24 h after transfection where indicated. Cells were lysed and subjected to western blotting. One representative of two independent experiments is shown. (d) A549 and HeLa cells were incubated with SNS-032 (300 nM) for different times. cFlipL, cFlipS and Mcl-1 mRNA expression was quantified by RT-PCR. Values are means±S.E.M. of three independent experiments. Z, zVAD
Mentions: Having established that CDK9 inhibition efficiently sensitizes cancer cell lines to TRAIL-induced apoptosis, we next addressed which molecular changes are responsible for this effect. Upregulation of TRAIL-R1 and/or TRAIL-R2 often correlates with, and sometimes also contributes to, TRAIL apoptosis sensitization.36 However, treatment of HeLa or A549 cells with PIK-75 or SNS-032 did not alter TRAIL-R1/R2 surface expression (Figure 4a), in line with similar recruitment of TRAIL-R1/2 in the DISC analysis (Figure 3e). Consequently, TRAIL sensitization by CDK9 inhibition is likely to require changes in intracellular modulators of the TRAIL apoptosis pathway that should enhance DISC activity and possibly additional downstream steps in the pathway. We, therefore, next investigated whether known components of the TRAIL–DISC and the downstream apoptosis pathway it activates are regulated by PIK-75 or SNS-032 treatment. Whereas the majority of the DISC components and downstream pro- and anti-apoptotic proteins remained unchanged, cFlip and Mcl-1 protein levels were rapidly suppressed by pharmacological CDK9 inhibition by SNS-032 or PIK-75 (Figure 4b and Supplementary Figure S4a). Because siRNA-mediated suppression of CDK9, performed in the presence or absence of pan-caspase inhibition to exclude a possible impact of CDK9-silencing-induced apoptosis, also resulted in downregulation of cFlip and Mcl-1, we can conclude that CDK9 is required to maintain high expression of these anti-apoptotic proteins in cancer cells (Figure 4c).

Bottom Line: Mechanistically, CDK9 inhibition resulted in downregulation of cellular FLICE-like inhibitory protein (cFlip) and Mcl-1 at both the mRNA and protein levels.Primary human hepatocytes did not succumb to the same treatment regime, defining a therapeutic window.Importantly, TRAIL in combination with SNS-032 eradicated established, orthotopic lung cancer xenografts in vivo.

View Article: PubMed Central - PubMed

Affiliation: 1] Centre for Cell Death, Cancer and Inflammation, UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6DD, UK [2] Clinic of General and Visceral Surgery, University of Ulm, Albert-Einstein-Allee 23, 89081 Ulm, Germany.

ABSTRACT
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in many cancer cells without causing toxicity in vivo. However, to date, TRAIL-receptor agonists have only shown limited therapeutic benefit in clinical trials. This can, most likely, be attributed to the fact that 50% of all cancer cell lines and most primary human cancers are TRAIL resistant. Consequently, future TRAIL-based therapies will require the addition of sensitizing agents that remove crucial blocks in the TRAIL apoptosis pathway. Here, we identify PIK-75, a small molecule inhibitor of the p110α isoform of phosphoinositide-3 kinase (PI3K), as an exceptionally potent TRAIL apoptosis sensitizer. Surprisingly, PI3K inhibition was not responsible for this activity. A kinome-wide in vitro screen revealed that PIK-75 strongly inhibits a panel of 27 kinases in addition to p110α. Within this panel, we identified cyclin-dependent kinase 9 (CDK9) as responsible for TRAIL resistance of cancer cells. Combination of CDK9 inhibition with TRAIL effectively induced apoptosis even in highly TRAIL-resistant cancer cells. Mechanistically, CDK9 inhibition resulted in downregulation of cellular FLICE-like inhibitory protein (cFlip) and Mcl-1 at both the mRNA and protein levels. Concomitant cFlip and Mcl-1 downregulation was required and sufficient for TRAIL sensitization by CDK9 inhibition. When evaluating cancer selectivity of TRAIL combined with SNS-032, the most selective and clinically used inhibitor of CDK9, we found that a panel of mostly TRAIL-resistant non-small cell lung cancer cell lines was readily killed, even at low concentrations of TRAIL. Primary human hepatocytes did not succumb to the same treatment regime, defining a therapeutic window. Importantly, TRAIL in combination with SNS-032 eradicated established, orthotopic lung cancer xenografts in vivo. Based on the high potency of CDK9 inhibition as a cancer cell-selective TRAIL-sensitizing strategy, we envisage the development of new, highly effective cancer therapies.

Show MeSH
Related in: MedlinePlus