Limits...
Small-molecule multi-targeted kinase inhibitor RGB-286638 triggers P53-dependent and -independent anti-multiple myeloma activity through inhibition of transcriptional CDKs.

Cirstea D, Hideshima T, Santo L, Eda H, Mishima Y, Nemani N, Hu Y, Mimura N, Cottini F, Gorgun G, Ohguchi H, Suzuki R, Loferer H, Munshi NC, Anderson KC, Raje N - Leukemia (2013)

Bottom Line: RGB-286638 displayed caspase-dependent apoptosis in both wt-p53 and mutant-p53 cells that was closely associated with the downregulation of RNA polymerase II phosphorylation and inhibition of transcription.We also demonstrated downregulation of oncogenic miR-19, miR-92a-1 and miR-21.Our data provide the rationale for the development of transcriptional CDKIs as therapeutic agents, which activate p53 in competent cells, while circumventing p53 deficiency through alternative p53-independent cell death mechanisms in p53-mutant/deleted cells.

View Article: PubMed Central - PubMed

Affiliation: 1] MGH Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA [2] Leebow Institute of Myeloma Therapeutics and Jerome Lipper Multiple Myeloma Disease Center, Dana-Farber Cancer Institute, Boston, MA, USA.

ABSTRACT
Small-molecule multi-targeted cyclin-dependent kinase (CDK) inhibitors (CDKIs) are of particular interest due to their potent antitumor activity independent of p53 gene alterations. P53 deletion is associated with a very poor prognosis in multiple myeloma (MM). In this regard, we tested the anti-MM activity of RGB-286638, an indenopyrazole-derived CDKI with Ki-nanomolar activity against transcriptional CDKs. We examined RGB-286638's mode-of-action in MM cell lines with wild-type (wt)-p53 and those expressing mutant p53. RGB-286638 treatment resulted in MM cytotoxicity in vitro associated with inhibition of MM tumor growth and prolonged survival in vivo. RGB-286638 displayed caspase-dependent apoptosis in both wt-p53 and mutant-p53 cells that was closely associated with the downregulation of RNA polymerase II phosphorylation and inhibition of transcription. RGB-286638 triggered p53 accumulation via nucleolar stress and loss of Mdm2, accompanied by induction of p53 DNA-binding activity. In addition, RGB-286638 mediated p53-independent activity, which was confirmed by cytotoxicity in p53-knockdown and p53-mutant cells. We also demonstrated downregulation of oncogenic miR-19, miR-92a-1 and miR-21. Our data provide the rationale for the development of transcriptional CDKIs as therapeutic agents, which activate p53 in competent cells, while circumventing p53 deficiency through alternative p53-independent cell death mechanisms in p53-mutant/deleted cells.

Show MeSH

Related in: MedlinePlus

RGB-286638 Mediated p53-Independent Apoptosis in MM Cells(A) RGB-286638 induced cytotoxicity in p53 knockdown MM.1S. P53 shRNA- or empty vector-transduced MM.1S were cultured with RGB-286638 (0–100nM) or 1.5ug/ml puromycin for 48h, and viability was assayed using MTT. Data represent means (+/−SD) of triplicate cultures. P53 and p-RNAPII S2 were examined by western blotting of whole cell lysates of parental MM.1S cells and MM.1S cells transduced with p53 shRNA or with empty vector.(B) RGB-286638 reduced transcription in mutant-p53 MM cell lines. U266, OPM1, and RPMI cells were treated for 2 and 24 h with RGB-286638 (0–100nM); [5′-3H]uridine was then added, and RNA synthesis measured. Data represent means (+/−SD) of triplicate cultures.(D) RGB-286638 triggered apoptosis in MM cell lines expressing mutant-p53. U266, OPM1, and RPMI cells incubated with or without 50nM RGB-286638 for 1, 4 and 8 h were examined by western blotting with anti-Mcl-1, -PARP, and - GAPDH antibodies.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3928098&req=5

Figure 5: RGB-286638 Mediated p53-Independent Apoptosis in MM Cells(A) RGB-286638 induced cytotoxicity in p53 knockdown MM.1S. P53 shRNA- or empty vector-transduced MM.1S were cultured with RGB-286638 (0–100nM) or 1.5ug/ml puromycin for 48h, and viability was assayed using MTT. Data represent means (+/−SD) of triplicate cultures. P53 and p-RNAPII S2 were examined by western blotting of whole cell lysates of parental MM.1S cells and MM.1S cells transduced with p53 shRNA or with empty vector.(B) RGB-286638 reduced transcription in mutant-p53 MM cell lines. U266, OPM1, and RPMI cells were treated for 2 and 24 h with RGB-286638 (0–100nM); [5′-3H]uridine was then added, and RNA synthesis measured. Data represent means (+/−SD) of triplicate cultures.(D) RGB-286638 triggered apoptosis in MM cell lines expressing mutant-p53. U266, OPM1, and RPMI cells incubated with or without 50nM RGB-286638 for 1, 4 and 8 h were examined by western blotting with anti-Mcl-1, -PARP, and - GAPDH antibodies.

Mentions: We next tested whether RGB-286638-induced cell death was independent of p53 pathway activation. Knockdown of p53 by p53-targeting shRNA lentiviral constructs rescued 14% cells from RGB-286638-induced cell death (Fig. 5A). No significant effect on p-RNAPII was observed, suggesting that RNAPII activity was independent of p53 status. P53-independent RGB-286638 activity was further validated by its cytotoxic effects in p53-mutated U266, OPM1, and RPMI MM cell lines. As measured by [5′-3H]uridine incorporation, 2h exposure to increasing doses of RGB-286638 reduced RNA synthesis in p53-mutated MM cell lines, with significant inhibition at 24h (Fig. 5B). As expected, western blot studies of p53-mutated U266, OPM1 and RPMI cells showed that RGB-286638 treatment consistently reduced Mcl-1 levels, which preceded PARP cleavage (Fig. 5C).


Small-molecule multi-targeted kinase inhibitor RGB-286638 triggers P53-dependent and -independent anti-multiple myeloma activity through inhibition of transcriptional CDKs.

Cirstea D, Hideshima T, Santo L, Eda H, Mishima Y, Nemani N, Hu Y, Mimura N, Cottini F, Gorgun G, Ohguchi H, Suzuki R, Loferer H, Munshi NC, Anderson KC, Raje N - Leukemia (2013)

RGB-286638 Mediated p53-Independent Apoptosis in MM Cells(A) RGB-286638 induced cytotoxicity in p53 knockdown MM.1S. P53 shRNA- or empty vector-transduced MM.1S were cultured with RGB-286638 (0–100nM) or 1.5ug/ml puromycin for 48h, and viability was assayed using MTT. Data represent means (+/−SD) of triplicate cultures. P53 and p-RNAPII S2 were examined by western blotting of whole cell lysates of parental MM.1S cells and MM.1S cells transduced with p53 shRNA or with empty vector.(B) RGB-286638 reduced transcription in mutant-p53 MM cell lines. U266, OPM1, and RPMI cells were treated for 2 and 24 h with RGB-286638 (0–100nM); [5′-3H]uridine was then added, and RNA synthesis measured. Data represent means (+/−SD) of triplicate cultures.(D) RGB-286638 triggered apoptosis in MM cell lines expressing mutant-p53. U266, OPM1, and RPMI cells incubated with or without 50nM RGB-286638 for 1, 4 and 8 h were examined by western blotting with anti-Mcl-1, -PARP, and - GAPDH antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3928098&req=5

Figure 5: RGB-286638 Mediated p53-Independent Apoptosis in MM Cells(A) RGB-286638 induced cytotoxicity in p53 knockdown MM.1S. P53 shRNA- or empty vector-transduced MM.1S were cultured with RGB-286638 (0–100nM) or 1.5ug/ml puromycin for 48h, and viability was assayed using MTT. Data represent means (+/−SD) of triplicate cultures. P53 and p-RNAPII S2 were examined by western blotting of whole cell lysates of parental MM.1S cells and MM.1S cells transduced with p53 shRNA or with empty vector.(B) RGB-286638 reduced transcription in mutant-p53 MM cell lines. U266, OPM1, and RPMI cells were treated for 2 and 24 h with RGB-286638 (0–100nM); [5′-3H]uridine was then added, and RNA synthesis measured. Data represent means (+/−SD) of triplicate cultures.(D) RGB-286638 triggered apoptosis in MM cell lines expressing mutant-p53. U266, OPM1, and RPMI cells incubated with or without 50nM RGB-286638 for 1, 4 and 8 h were examined by western blotting with anti-Mcl-1, -PARP, and - GAPDH antibodies.
Mentions: We next tested whether RGB-286638-induced cell death was independent of p53 pathway activation. Knockdown of p53 by p53-targeting shRNA lentiviral constructs rescued 14% cells from RGB-286638-induced cell death (Fig. 5A). No significant effect on p-RNAPII was observed, suggesting that RNAPII activity was independent of p53 status. P53-independent RGB-286638 activity was further validated by its cytotoxic effects in p53-mutated U266, OPM1, and RPMI MM cell lines. As measured by [5′-3H]uridine incorporation, 2h exposure to increasing doses of RGB-286638 reduced RNA synthesis in p53-mutated MM cell lines, with significant inhibition at 24h (Fig. 5B). As expected, western blot studies of p53-mutated U266, OPM1 and RPMI cells showed that RGB-286638 treatment consistently reduced Mcl-1 levels, which preceded PARP cleavage (Fig. 5C).

Bottom Line: RGB-286638 displayed caspase-dependent apoptosis in both wt-p53 and mutant-p53 cells that was closely associated with the downregulation of RNA polymerase II phosphorylation and inhibition of transcription.We also demonstrated downregulation of oncogenic miR-19, miR-92a-1 and miR-21.Our data provide the rationale for the development of transcriptional CDKIs as therapeutic agents, which activate p53 in competent cells, while circumventing p53 deficiency through alternative p53-independent cell death mechanisms in p53-mutant/deleted cells.

View Article: PubMed Central - PubMed

Affiliation: 1] MGH Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA [2] Leebow Institute of Myeloma Therapeutics and Jerome Lipper Multiple Myeloma Disease Center, Dana-Farber Cancer Institute, Boston, MA, USA.

ABSTRACT
Small-molecule multi-targeted cyclin-dependent kinase (CDK) inhibitors (CDKIs) are of particular interest due to their potent antitumor activity independent of p53 gene alterations. P53 deletion is associated with a very poor prognosis in multiple myeloma (MM). In this regard, we tested the anti-MM activity of RGB-286638, an indenopyrazole-derived CDKI with Ki-nanomolar activity against transcriptional CDKs. We examined RGB-286638's mode-of-action in MM cell lines with wild-type (wt)-p53 and those expressing mutant p53. RGB-286638 treatment resulted in MM cytotoxicity in vitro associated with inhibition of MM tumor growth and prolonged survival in vivo. RGB-286638 displayed caspase-dependent apoptosis in both wt-p53 and mutant-p53 cells that was closely associated with the downregulation of RNA polymerase II phosphorylation and inhibition of transcription. RGB-286638 triggered p53 accumulation via nucleolar stress and loss of Mdm2, accompanied by induction of p53 DNA-binding activity. In addition, RGB-286638 mediated p53-independent activity, which was confirmed by cytotoxicity in p53-knockdown and p53-mutant cells. We also demonstrated downregulation of oncogenic miR-19, miR-92a-1 and miR-21. Our data provide the rationale for the development of transcriptional CDKIs as therapeutic agents, which activate p53 in competent cells, while circumventing p53 deficiency through alternative p53-independent cell death mechanisms in p53-mutant/deleted cells.

Show MeSH
Related in: MedlinePlus