# Capacity for Compensatory Cyclin D2 Response Confers Trametinib Resistance in Canine Mucosal Melanoma

## TL;DR

Dogs with mucosal melanoma can develop resistance to trametinib by switching from cyclin D1 to cyclin D2, which helps cancer cells continue growing.

## Contribution

The study reveals that compensatory cyclin D2 expression is a novel mechanism of resistance to MEK inhibitors in mucosal melanoma.

## Key findings

- Trametinib-resistant cells upregulate cyclin D2 to maintain proliferation.
- Inhibiting cyclin D2 or the PI3K/AKT/mTOR pathway restores drug sensitivity.
- Cyclin D2 overexpression in sensitive cells promotes survival under trametinib.

## Abstract

Cancers can acquire resistance to treatments through multiple means, and therapeutic resistance continues to be a serious challenge to successful patient outcomes. Escape from cancer therapy is accompanied by continued tumor growth and possible metastasis in the most aggressive forms. For a better understanding of mucosal melanoma resistance to the MEK inhibitor, trametinib was studied by examining cell cycle-initiating cyclin D expression. Mucosal melanoma cancer cells capable of mediating the compensatory switch from cyclin D1 to cyclin D2, important molecules that cells employ to replicate, exhibited greater resistance to trametinib’s downstream suppressive effects on cyclin D1. Mucosal melanoma cells lacking sufficient cyclin D2 compensation were more trametinib-sensitive. Inhibiting the cyclin D subtype switch by targeting the PI3K/AKT/mTOR pathway in resistant cells helped maintain a better degree of drug sensitivity. Our findings can provide insight for more precise treatment of this drug resistance and cancer treatment crosstalk.

Background/objective: Mucosal melanoma (MM) is a poorly responsive, rare and aggressive subtype with few cases having targetable recurrent driver mutations, although Ras/MAPK and PI3K/AKT/mTOR signaling pathway activations are common. Eventual tumor evasion of targeted therapy continues to limit treatment success. Adequate models are necessary to address therapeutic resistance. The relatively greater incidence of naturally occurring MM in dogs, as well as its comparable clinical and pathological characteristics to human MM, represents an opportunity for study as a human MM patient surrogate. Resistance-promoting crosstalk between Ras/MAPK and PI3K/AKT/mTOR signaling under trametinib inhibition of MEK was studied in canine MM. Emphasis was placed on the suppressive effect of trametinib on cell cycle entry and its potential role in drug resistance. Methods: D-type cyclins were investigated following trametinib treatment of five MM cell lines exhibiting differential drug sensitivities. Signaling pathway activation, proliferation, survival, cell death, and cell cycle were analyzed in the context of D-type cyclin expression. Cyclin D2 expression was manipulated using siRNA knockdown or inducible recombinant overexpression. Results: Trametinib diminished cyclin D1 in all cell lines. While relatively trametinib-resistant MM cells exhibited capacity to upregulate cyclin D2, which promoted proliferation, sensitive MM cells lacked similar cyclin D2 compensation. Inhibition of the compensatory cyclin D2 in resistant cells conferred sensitivity. Induced cyclin D2 overexpression in otherwise trametinib-sensitive MM cells promoted survival. Upregulated PI3K/AKT/mTOR signaling under trametinib treatment was suppressed by mTORC1/2 inhibition, which similarly diminished cyclin D2 response. Conclusions: The compensatory switch from preferential reliance on cyclin D1 to D2 plays a role in MM resistance to MEK inhibition.