The ATM Inhibitor KU60019 Synergizes the Antineoplastic Effect of Romidepsin in Mantle Cell Lymphoma (MCL)

Introduction: Romidepsin (R), an HDAC inhibitor (HDACi) approved for the treatment of relapsed T-cell lymphoma, is thought to induce cell cycle arrest and apoptosis. Central to the block of cell proliferation is the up-regulation of the cdk inhibitor p21^Cip1/Waf1. Interestingly up-regulation of p21^Cip1/Waf1 has also been shown to reduce sensitivity to romidepsin.  HDAC inhibitors as a class appear to activate p21^Cip1/Waf1 expression via ATM.  KU60019, a specific ATM inhibitor, has been shown to decrease the p21^Cip1/Waf1 protein levels in a concentration dependent manner.  We sought to explore the potential synergistic interaction of an ATM inhibitor with R, given the potential complementary effects around p21^{Cip1/Waf1 .}

Methods: For all cytotoxicity assays, luminescent cell viability was performed using CellTiter-Glo^TM . Gene expression analysis was performed via Western blot and semi-quantitative PCR assay. Apoptosis and cell cycle analysis were analyzed via Fluorescence-activated cell sorting (FACS) and Western blot. The efficacy of the compounds as single agent and combination is evaluated using a subcutaneous xenograft MCL mouse model.

Results:  Synergy analyses were performed using Jeko-1, Maver-1 and Z-138 cells. A synergistic cytotoxic effect was observed in all MCL cell lines when the HDACi was combined with KU60019 throughout the range of all tested concentrations. Flow cytometry analysis of all three cell lines treated with single agents and combination indicated a substantial increase in the apoptotic cell fraction when R was combined with KU60019. Furthermore protein expression analysis revealed changes in a host of proteins known to be involved in cell cycle control and apoptosis. Increased activation of the programmed cell death proteins Caspase 8 and Caspase 3 was observed upon combinations of the single agents in all three cell lines, resulting in an increased cleavage of Poly (ADP-ribose) polymerase (PARP-1) and accumulation of the DNA damage marker gammaH2AX. Finally, the abundance of the anti-apoptotic proteins Bcl-XL and BCL-2 showed a significant decrease after treatment with R plus increased concentrations of KU60019 when compared with their abundance in the presence of the single agents.

Romidepsin kill tumor cells by driving premature exit from aberrant mitosis and inducing the rapid onset of apoptosis. The increased p21 protein expression upon addition of R, responsible for cell cycle arrest in the G2/M phase, was not observed when the R treatment was combined with the KU60019 suggesting that the increased apoptosis observed in the combination treatment was due to the ability of the KU60019 to affect the up-regulation of the cdk inhibitor p21^Cip1/Waf1 by the HDACi.  Indeed cell cycle and protein expession analysis of Jeko-1 cells treated with single agents and the combination confirmed that addition of KU60019 results in the inhibition of the p21^Cip1/Waf1 induced G2/M cell cycle arrest. Western blot and transfection analysis of p21 promoter constructs indicated that activation of p21(WAF1/Cip1) transcription by HDACi in Jeko-1 cells occurs through Sp1 sites and KU60019 affected R ability to induce p21 expression at transcriptional level. A decreased in the expression level of SP1 protein upon addition of KU60019 would suggest that KU60019 affect the ability of HDACi to induce p21(WAF1/Cip1) by modulating the expression levels of SP1. A survey of the effect of R in combination with KU60019 in other tumor types derived cell lines (CTCL, DLBCL, TALL and ATLL) also shown an overall synergistic effect similar to the one observed in MCL. The potential therapeutic effect of the HDACi in combination with KU60019 is currently evaluated in a MCL subcutaneous xenograft mouse model using the Z-138 lymphoma derived cell line.

Conclusions: These data support the novel concept that dual targeting of HDAC and ATM inhibition may be a synergistic and effective strategy in MCL.