Abstract B166: Investigating the effect of CDK4/6 and MDM2 inhibition on melanoma
Authors: Slesur Lauren , Vilgelm Anna , Al-Rohil Rami , Richmond Ann 
Published: August 2018

Abstract
Introduction: The purpose was to investigate how inhibition of MDM2 and CDK4/6 would affect growth of melanoma tumors with deleted CDKN2A in vivo. Methods: B16F0 and WM115 cells were cultured with increasing concentrations of MDM2 inhibitors nutlin-3a, RG7112, or RG7388. Viability was monitored by CellTiter-Blue assay. Induction of p21, MDM2, and BAX expression was monitored by qRT-PCR. C57BL/6J mice (n=40) were injected subcutaneously into both flanks with 50,000 B16F0 cells, a CDKN2A-deficient mouse melanoma cell line. Tumors grew for one week, then mice were randomized into four treatment groups: vehicle (100 µL 0.5% methylcellulose) (n=10), CDK4/6 inhibitor palbociclib 100 mg/kg (n=10), RG7388 150 mg/kg (n=10), and combination palbociclib 100 mg/kg + RG7388 150 mg/kg (n=10). Drugs were given daily via oral gavage. Tumor volumes were measured every 2-3 days, and mice with tumors &gt;15 mm in diameter were euthanized. After 12 days of therapy, tumors were excised and weighed. At the time of euthanasia, mice were bled to evaluate AST and ALT levels. Four tumors per group were randomly selected for H&E staining to evaluate necrosis and IHC for CD3 (T cell marker), Ki67 (proliferation marker), and cleaved caspase 3 (apoptosis marker). The percent of necrosis and number of cells that were positive on IHC were evaluated by a pathologist blinded to the groupings. A mixed-effects model was used to compare tumor growth rate and tumor weights. Kruskal-Wallis and post-hoc Wilcoxon were used for other analyses. Results: In vitro experiments showed RG7388 was the most potent inhibitor of B16F0 and WM115 viability and the strongest inducer of p21 and MDM2. In vivo experiments revealed B16F0 tumor growth rate was significantly reduced in mice treated with palbociclib (p&lt;0.001), RG7388 (p=0.0012), and the combination regimen (p&lt;0.001) compared to those that received the vehicle. Comparing tumor weights to the vehicle group, tumors in the RG7388 (p=0.0281) and combination (p=0.0412) groups weighed significantly less. The drugs did not cause toxicity when used alone or combined based on AST and ALT. Tumors in the RG7388 group had fewer Ki67-positive cells than those in the vehicle (p&lt;0.05) and combination (p&lt;0.01) groups at the endpoint. There was no difference in cleaved caspase 3 expression, necrosis, or CD3 cell infiltration between groups at treatment endpoint. Conclusion: Each therapy reduced tumor growth rate compared to the control and reduced the final tumor weight with the exception of palbociclib-alone-treated tumors, which did not weigh less than control tumors after 12 days of treatment. The reduced growth rate was not associated with changes in apoptosis by cleaved caspase 3 staining or necrosis at the endpoint. Only RG7388-treated tumors showed decreased Ki67 staining. It may be that palbociclib and the combination regimen reduced growth by other means or that the contribution of Ki67-positive nontumor cells skewed the results. Palbociclib and the combination therapy group did not show an increase in TIL recruitment based on CD3 IHC analysis at the endpoint. However, it is likely that T-cell recruitment peaks early in the therapy regimen. We conclude that RG7388, and RG7388 combined with palbociclib, reduced B16 melanoma tumor growth in vivo. Future efforts should include earlier time point analyses of multiplex FACS to characterize full immune filtrate in the tumor over time as well as analyses of multiple cell lines.
Citation Format: Lauren Slesur, Anna Vilgelm, Rami Al-Rohil, Ann Richmond. Investigating the effect of CDK4/6 and MDM2 inhibition on melanoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B166.