Metabolic Adaptations to MEK and CDK4/6 Cotargeting in Uveal Melanoma

Abstract

Frequent GNAQ and GNA11 mutations in uveal melanoma hyperactivate the MEK-ERK signaling pathway, leading to aberrant regulation of cyclin-dependent kinases (CDK) and cell-cycle progression. MEK inhibitors (MEKi) alone show poor efficacy in uveal melanoma, raising the question of whether downstream targets can be vertically inhibited to provide long-term benefit. CDK4/6 selective inhibitors are FDA-approved in patients with estrogen receptor (ER)-positive breast cancer in combination with ER antagonists/aromatase inhibitors. We determined the effects of MEKi plus CDK4/6 inhibitors (CDK4/6i) in uveal melanoma. In vitro, palbociclib, a CDK4/6i, enhanced the effects of MEKi via downregulation of cell-cycle proteins. In contrast, in vivo CDK4/6 inhibition alone led to cytostasis and was as effective as MEKi plus CDK4/6i treatment at delaying tumor growth. RNA sequencing revealed upregulation of the oxidative phosphorylation (OxPhos) pathway in both MEKi-resistant tumors and CDK4/6i-tolerant tumors. Furthermore, oxygen consumption rate was increased following MEKi + CDK4/6i treatment. IACS-010759, an OxPhos inhibitor, decreased uveal melanoma cell survival in combination with MEKi + CDK4/6i. These data highlight adaptive upregulation of OxPhos in response to MEKi + CDK4/6i treatment in uveal melanoma and suggest that suppression of this metabolic state may improve the efficacy of MEKi plus CDK4/6i combinations.

Figure 1.. Enhanced effects of combined MEK and CDK4/6 inhibition in UM cell lines.

A MTT cell viability assays of UM cell lines treated with MEKi (trametinib, 5 nM) alone, CDK4/6i (palbociclib, 0.5 μM) alone, or the combination (trametinib + palbociclib) for 7 days (*p<0.05, **p<0.005) (n=3). B Downregulation of cell cycle proteins following MEKi + CDK4/6i treatment. UM cell lines treated with single agent or combination of MEKi (PD0325901, 5nM) and CDK4/6i (palbociclib, 0.5 μM) for 48 hours. Heatmap of median centered log2-transformed average group expression RPPA data for antibodies both differentially expressed (Storey q-value < 0.05, ratio >50%) and up or downregulated in at least two cell lines between combination or DMSO treated samples. C Western blot analysis of UM cell lines treated with single agent or combination of MEKi (PD0325901, 5 nM) and CDK4/6i (palbociclib, 0.5 μM) for 48 hours. Representative blots from triplicate experiments are shown.

Figure 2.. CDK4/6 inhibition in the metastatic UM line, UM001, leads to cytostatic tumors.

A UM001 xenografts were treated with control, MEKi (PD0325901), CDK4/6i (palbociclib), or the combination (combo) (*p=0.02) (n=3). Palbociclib was dosed intermittently, 3 weeks on, 1 week off. B Quantification of Ki67 positive tumor cells by IHC staining (*p<0.05).

Figure 3.. Enrichment of OxPhos signature in CDK4/6i-tolerant (CDK4/6i-T) and MEKi-resistant (MEKi-R) tumors.

A Heatmap of unsupervised hierarchical clustering of samples based on genes differentially expressed between CDK4/6i-T and control (CTL1, CTL2) (green) or MEKi-R and control (CTL1, CTL2) (purple) (BHFDR < 0.05 and absolute log2(FC) > 1). B Radar plot showing normalized enrichment score (NES) values for CDK4/6i-T or MEKi-R vs control samples for the most commonly enriched gene sets as well as most variable between CDK4/6i-T and MEKi-R. C Gene set enrichment plots of the top-ranked Hallmark OxPhos gene set for comparison of CDK4/6i-T (top) or MEKi-R (bottom) to control samples. D Top ranked genes within the OxPhos pathway in both CDK4/6i-T and MEKi-R samples.

Figure 4.. Induction of metabolic activity by CDK4/6 and MEK inhibition is functional and can be targeted with an OxPhos inhibitor.

A OCR of UM001 treated with control (DMSO), MEKi (PD0325901, 5 nM), CDK4/6i (palbociclib, 0.5 μM), or the combination (PD0325901 + palbociclib) (n=3). B as A, but WM3618F cells were utilized. C Incucyte analysis of UM001 and WM3618F cell lines treated with control (DMSO), MEKi (PD0325901, 5 nM), CDK4/6i (palbociclib, 0.5 μM), OxPhosi (IACS-010759, 50 nM), MEKi + CDK4/6i (combo), or MEKi + CDK4/6i + OxPhosi (combo + OxPhosi) (*p=0.05, **p<0.005) (n=3). D Percentage annexin-V positive cells following treatment of UM001 and WM3618F with control (DMSO), MEKi (PD0325901, 5 nM), CDK4/6i (palbociclib, 0.5 μM), OxPhosi (IACS-010759, 50 nM), MEKi + CDK4/6i (combo), or MEKi + CDK4/6i + OxPhosi (combo + OxPhosi) for 48 hours (*p=0.05) (n=3).