(P 019) COUMERMYCIN-A1 (CA1) IDENTIFIED AS A FIRST-IN-CLASS CDC45-MCM-GINS HELICASE INHIBITOR OFFERING THERAPEUTIC POTENTIAL FOR OSTEOSARCOMA TREATMENT

Objective: Osteosarcoma (OS) is the most common bone cancer affecting children and young adults with suboptimal outcomes. Hopes for a recurrent, targetable, genomic finding have not materialized. OS is considered too heterogeneous between patients being characterized by vast structural variations. There is a convergence on the cell cycle in OS with common Rb1 loss or gains of oncogenes such as CyclinE or Myc. The Cdc45-MCM-GINS (CMG) replicative helicase is central to the cell cycle, redundantly loaded onto DNA during G phase, and never mutated in cancers since it is essential for tumor growth. The cell cycle convergence point is mismanaged in cancer cells and thus has therapeutic potential. Examples of mismanagement include Cyclin E overexpression causing too few reserve CMG complexes (less redundancy) to be loaded while Myc overexpression activates too many CMG helicases at the same time. We hypothesized that the recently identified CMG inhibitor coumermycin A1 (CA1) would be active in osteosarcoma.

Methods: Two osteosarcoma cells line were utilized (143B and OS252) and treated with CA1 for 18 hours. Cells were collected and processed for total cell and chromatin-bound proteins. Western blots were then performed comparing MCM component expression levels and markers of cellular stress. Cells were also co-treated with CA1 or gemcitabine for 18 hours, allowed to recover for 8 hours, and then treated with the opposite treatment for another 18 hours. Cells were trypsinized and plated in 96-well plates and cell viability was quantified using Cell-Titer Glo 2.0.

Results: CA1 administration inhibits CMG assembly and activity, as seen in decreased Cdc45, Psf2, Psf3, and RPA32 association with chromatin. CA1 also increase cellular stress, indicated by an increase in PARP1. Treatment with gemcitabine followed by CA1 increases treatment efficacy significantly while the reverse appears to offer a protective effect, diminishing the effect of either drug alone. Further combinations and order of addition experiments are being conducted along with matching observations to genomic gains and losses in the individual cell lines

Conclusion: The discovery of these aminocoumarins as first-in-class, effective CMG inhibitor provides an starting point for translating CMG inhibition in combinations in osteosarcoma. Notably, the observed effect is seen across multiple osteosarcoma cell lines. Past work with CA1 provides supportive evidence that inhibitory levels can be administered with acceptable toxicity.