Project 2: To determine the consequences of activating Rb function in cancer cells

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PROJECT SUMMARY In human cancers, the gene coding for the Rb tumor suppressor is frequently silenced, deleted, mutated, or functionally inactivated by phosphorylation by over-expressed Cyclin D-Cdk4/6 kinase complexes. Due to its importance in cancer, the Cyclin D-Cdk4/6-Rb axis is the target of FDA-approved Cdk4/6 inhibitors. The underlying assumption of this effort is that Cdk4/6 inhibition leads to Rb re-activation and tumor suppression in cancer cells where the gene coding for Rb has not been disabled. While a large number of studies have investigated the consequences of Rb loss of function, few studies have investigated the consequences of Rb re- activation in cells, even though this re-activation is highly relevant in the clinic in patients treated with Cdk4/6 inhibitors. Here, we propose to tackle the central question of the mechanisms by which Rb activity can suppress cancer progression using a combination of genetic, cell biological, biochemical, and structural approaches. First, we will determine the effects of Rb re-activation in Rb-inactive cancer cells in vivo using a novel inducible mouse allele in which Rb function can be turned on and off. Using this allele, we will re-activate Rb in tumor cells to determine the molecular consequences of Rb re-introduction as a way to better understand how Rb wild-type cancer cells respond to Cdk4/6 inhibitors. Second, we will investigate the possible tumor suppressor role of the Rb family members p107 and p130 in response to Cdk4/6 inhibitors in cancer cells that are mutant for Rb. The goal is to identify ways to enhance cell cycle inhibition by p107 and p130. Third, we will investigate post-translational modifications that control Rb interactions with E2F and its tumor suppressive effects in cells, including acetylation events that functionally interact with phosphorylation of Rb. The identification of such key residues that are modified in Rb may open new avenues of research to prevent its functional inactivation or enhance its tumor suppressive activity. Taken together, these experiments will provide an in-depth analysis of the functional activation of Rb and its family members in contexts relevant to cancer and may point to new effector mechanisms downstream of and parallel to Rb. Ultimately, these experiments will help identify new ways to enhance the tumor suppressive effects of FDA-approved Cdk4/6 inhibitors such as palbociclib, ribociclib, or abemaciclib.