Poster Presentation Advances in Neuroblastoma Research Congress 2016

Targeting cell cycle and transcriptional CDKs using Roniciclib leads to significant high cell death in MYCN/MYC-activated neuroblastoma cells (#291)

Sina Gogolin 1 , Denise Brünig 1 , Jochen Kreth 1 , Volker Ehemann 2 , Xiaoqi Jiang 3 , Gerhard Siemeister 4 , Frank Westermann 1
  1. Neuroblastoma Genomics, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
  2. Division of Cytometry Pathology, University Heidelberg, 69120 Heidelberg, Germany
  3. Division of Biostatistics, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
  4. Global Drug Discovery,TRG Oncology / Gyn. Therapy, Bayer HealthCare (BHC), Berlin, Germany

Background

Poor overall survival of high-risk neuroblastoma patients requires novel molecularly targeted therapies. Since MYCN itself is still undruggable, we identified a group of MYCN direct target genes being strongly activated by MYCN in high-risk neuroblastoma tumors and inhibited them using the small molecule pan-CDK inhibitor, Roniciclib.

Methods

A gene expression-based classifier of 702 neuroblastoma patients revealed high expression of cell cycle genes, especially the group of cyclin-dependent kinases (CDKs) and cyclins, in MYCN-amplified high-risk neuroblastoma tumors. The small molecule compound, Roniciclib, was used to simultaneously inhibit the following CDKs and cyclins: CDK1/CycB, CDK2/CycE, CDK3/CycE, CDK4/CycD, CDK5/p35, CDK7/CycH and CDK9/CycT. About 30 neuroblastoma cell lines established from advanced stage disease harboring different characteristic genetic aberrations affecting MYCN/MYC, ALK and/or members of the p53/pRB pathway (e.g. TP53, MDM2, CDK4, CCND1 and CDKN2A) were treated with different concentrations of Roniciclib and tested for viability reduction, IC50/EC50 values, cell cycle arrest (flow cytometry), cell death induction (nicoletti and BrdU staining, caspase assay), anchorage independent growth inhibition (soft agar assay) and MYCN expression.

Results

Morphologically, Roniciclib treatment induces early cell detachment (~24h) being associated with rapid and strong viability reduction at significant lower nanomolar concentrations in MYCN/MYC-activated neuroblastoma cell lines compared to cells with normal MYCN/MYC expression (IC50/EC50 16.6/16.1 nM MYCN/MYC activated vs 36.1/31.0 nM MYCN/MYC normal). Viability reduction is caused by a prominent G2/M arrest followed by caspase 3/7 activation resulting in cell death induction from all cell cycle phases. Anchorage independent growth was completely inhibited at 10 nM concentration of Roniciclib in 11/24 cell lines and in 23/24 cell lines at 25 nM. Reduction of MYCN/MYC protein expression was observed in 22/24 cell lines 72h after treatment with Roniciclib.

Conclusion

Simultaneous inhibition of cell cycle and transcriptional CDKs using the small molecule compound, Roniciclib, significantly induces high levels of cell death in MYCN/MYC activated neuroblastoma cell cultures opening a novel therapeutic approach for high-risk neuroblastomas.