Our recent studies of amplification in rhabdomyosarcoma (RMS) focused on the 12q13-q14 amplicon, which occurs preferentially in PAX3-FOXO1-positive RMS. We previously localized the minimal amplified 12q13-q14 region to a 0.5 Mb segment containing 20 genes, including the CDK4 proto-oncogene. We focused our subsequent analysis on CDK4 and previously utilized inducible shRNA constructs to show that CDK4 knockdown abrogates proliferation and transformation of both amplified and non-amplified fusion-positive RMS cells via a G1-phase cell cycle arrest. To extend these findings, we measured susceptibility of RMS cells to pharmacologic CDK4/6 inhibition (LEE011 and PD0332991). Cell culture studies in five fusion-positive RMS lines showed variable sensitivity to CDK4/6 inhibitors; in particular, these findings suggested an inverse correlation of antiproliferative effects with CDK4 protein expression in the RMS lines. Similar to our knockdown studies, there was a G1-phase cell cycle arrest associated with reduced RB phosphorylation and reduced E2F target expression in RMS cells treated with inhibitor. To understand why CDK4-amplified/overexpressing cells were relatively insensitive to pharmacologic CDK4/6 inhibition, we investigated whether CDK4 or CDK6 overexpression attenuates sensitivity to CDK4/6 inhibitors in fusion-positive RMS. In particular, we transduced non-amplified RMS cells with doxycycline-inducible CDK4 or CDK6 constructs and treated these cells with inhibitor and/or doxycycline. Whereas the CDK4/6 inhibitor decreased viability of control cells and CDK4- or CDK6-transduced cells without doxycycline treatment, we found that CDK4 or CDK6 induction rescued the viability of inhibitor-treated RMS cells. These data provide evidence that CDK4 or CDK6 overexpression renders cells less sensitive to pharmacologic CDK4/6 inhibition. Finally we examined the activity of this inhibitor on fusion-positive RMS cells in vivo. In mice with xenografts derived from two RMS cell lines, CDK4/6 inhibition reduced RB phosphorylation and delayed tumor progression similarly to our findings in cell culture. These studies therefore show that fusion-positive RMS cells exhibit an overall sensitivity to pharmacologic CDK4/6 inhibition with the important caveat that there is an inverse relationship between CDK4 expression and inhibitor sensitivity.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011378-05
Application #
9153882
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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