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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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SNS-032 and TRAIL co-treatment eradicates established lung tumors in vivo. (a) Experimental treatment schedule is shown. (b) In week three after treatment tumor burden was quantified by bioluminescence imaging (Photon Flux). Values are means ±S.E.M. Dots represent individual mice (n=8 per group). Three representative mice from each group are shown. (c) Paraffin sections of lungs from all mice were stained with H&E and subjected to microscopical analysis quantifying the percentage of total lung area occupied by tumour tissue. Values are means ±S.E.M. Dots represent lungs from individual mice, (n=8 per group). Representative histological images are shown (arrows indicate tumor tissue). *P<0.05; **P<0.01, ***P<0.001; Student's t-test
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fig7: SNS-032 and TRAIL co-treatment eradicates established lung tumors in vivo. (a) Experimental treatment schedule is shown. (b) In week three after treatment tumor burden was quantified by bioluminescence imaging (Photon Flux). Values are means ±S.E.M. Dots represent individual mice (n=8 per group). Three representative mice from each group are shown. (c) Paraffin sections of lungs from all mice were stained with H&E and subjected to microscopical analysis quantifying the percentage of total lung area occupied by tumour tissue. Values are means ±S.E.M. Dots represent lungs from individual mice, (n=8 per group). Representative histological images are shown (arrows indicate tumor tissue). *P<0.05; **P<0.01, ***P<0.001; Student's t-test

Mentions: Having established an applicable therapeutic window for our newly identified combination of TRAIL with SNS-032 in vitro, we next assessed this combination's potency in an orthotopic model of lung cancer in vivo. To this end, we induced lung tumors via tail vein injection of A549 cells stably expressing luciferase (A549-luc). After 7 days, mice were randomized to create treatment groups of mice with comparable tumor burden in each group (Supplementary Figure S7). Subsequently, a 4-day treatment regime was started with either vehicle, TRAIL, SNS-032 or the combination of SNS-032 and TRAIL (Figure 7a). Whereas TRAIL treatment alone had a slight growth inhibitory effect, and SNS-032 only marginally affected lung tumor burden, combined treatment with TRAIL and SNS-032 induced a drastic antitumor effect. TRAIL/SNS-032 treatment completely eradicated established lung tumors in most mice, as determined by in vivo bioluminescence imaging (Figure 7b) and subsequent histopathological inspection of lung sections (Figure 7c). Strikingly, and in line with the bioluminescence data, seven out of eight mice that had received TRAIL combined with SNS-032 were histologically tumor free after a 4-day treatment cycle.


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)

SNS-032 and TRAIL co-treatment eradicates established lung tumors in vivo. (a) Experimental treatment schedule is shown. (b) In week three after treatment tumor burden was quantified by bioluminescence imaging (Photon Flux). Values are means ±S.E.M. Dots represent individual mice (n=8 per group). Three representative mice from each group are shown. (c) Paraffin sections of lungs from all mice were stained with H&E and subjected to microscopical analysis quantifying the percentage of total lung area occupied by tumour tissue. Values are means ±S.E.M. Dots represent lungs from individual mice, (n=8 per group). Representative histological images are shown (arrows indicate tumor tissue). *P<0.05; **P<0.01, ***P<0.001; Student's t-test
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921597&req=5

fig7: SNS-032 and TRAIL co-treatment eradicates established lung tumors in vivo. (a) Experimental treatment schedule is shown. (b) In week three after treatment tumor burden was quantified by bioluminescence imaging (Photon Flux). Values are means ±S.E.M. Dots represent individual mice (n=8 per group). Three representative mice from each group are shown. (c) Paraffin sections of lungs from all mice were stained with H&E and subjected to microscopical analysis quantifying the percentage of total lung area occupied by tumour tissue. Values are means ±S.E.M. Dots represent lungs from individual mice, (n=8 per group). Representative histological images are shown (arrows indicate tumor tissue). *P<0.05; **P<0.01, ***P<0.001; Student's t-test
Mentions: Having established an applicable therapeutic window for our newly identified combination of TRAIL with SNS-032 in vitro, we next assessed this combination's potency in an orthotopic model of lung cancer in vivo. To this end, we induced lung tumors via tail vein injection of A549 cells stably expressing luciferase (A549-luc). After 7 days, mice were randomized to create treatment groups of mice with comparable tumor burden in each group (Supplementary Figure S7). Subsequently, a 4-day treatment regime was started with either vehicle, TRAIL, SNS-032 or the combination of SNS-032 and TRAIL (Figure 7a). Whereas TRAIL treatment alone had a slight growth inhibitory effect, and SNS-032 only marginally affected lung tumor burden, combined treatment with TRAIL and SNS-032 induced a drastic antitumor effect. TRAIL/SNS-032 treatment completely eradicated established lung tumors in most mice, as determined by in vivo bioluminescence imaging (Figure 7b) and subsequent histopathological inspection of lung sections (Figure 7c). Strikingly, and in line with the bioluminescence data, seven out of eight mice that had received TRAIL combined with SNS-032 were histologically tumor free after a 4-day treatment cycle.

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