Charting and Disrupting the Gene-Regulatory Function of CDK6

The proposal Charting and Disrupting the Gene-Regulatory Function of CDK6 outlines a multi- disciplinary approach to elucidate the role of the cyclin dependent kinase 6 (CDK6) in sustaining leukemic gene-regulatory circuits. Initially, CDK6 and the closely related CDK4 have mostly been implicated in controlling how and when cells divide. Deregulation of this regulatory checkpoint has often been associated with aberrant cell cycle progression and cellular proliferation found in cancer. Small molecule inhibitors that block isolated functions of CDK4 and CDK6 have thus been innovated, clinically approved and are successfully used in the treatment of certain cancer types. Recently, CDK6 has also been shown to directly control gene regulation and gene activity. Interestingly, this role is specific for CDK6 over CDK4. Among others, it has been implicated in sustaining transcriptional networks in different blood cancers as well as blood cancer stem cells. Intriguingly, pharmacologic interference with the gene-regulatory role of CDK6 would thus represent a novel therapeutic approach in treating aberrant transcriptional programs driving leukemia pathogenesis. However, in contrast to the role of CDK6 in promoting cell cycle progression, the gene-regulatory function of CDK6 cannot be blocked with currently available small molecule drugs. This is due to the fact that CDK6 does not influence gene regulation via its enzymatic function but rather by acting as a recruitment factor for downstream transcriptional effectors. This lack of chemical tractability not only stalls translational development but also severely limits our understanding of the mechanistic basis of gene-regulation executed by CDK6. Thus, we hypothesize that novel inhibitors that would be capable of blocking all associated functions of CDK6 would not only have potentially far reaching therapeutic implications but would also significantly improve our understanding of the gene-regulatory circuits controlled by CDK6. To that goal, we propose to create small molecules that can hijack effectors of the cellular protein clearance system to induce the immediate and specific degradation of CDK6. Based on the generalizable approach of phthalimide conjugation, we thus propose to create bifunctional small molecules that will modulate the activity of the E3 ligase receptor cereblon in a targeted manner in order to induce the proteasomal degradation of CDK6. We propose to deploy these pharmacologic degraders to probe the gene-regulatory function of CDK6 in leukemia cell lines and primary patient samples by measuring implications of acute CDK6 loss on global chromatin assembly, accessibility and gene activity. Moreover, we will assay the therapeutic potential of targeted CDK6 degradation in vivo in primary AML derived xenografts.

In the proposal Charting and Disrupting the Gene-Regulatory Function of CDK6, we developed a multi-disciplinary approach to understand the role of CDK6 in aggressive blood cancers. Initially, CDK6 and the closely related CDK4 have mostly been implicated in controlling the cell cycle. Deregulation of this regulatory checkpoint has often been associated with cancer. Small molecules that block the activity of CDK4/6 are successfully used in the treatment of certain cancers. Recently, CDK6 has also been shown to directly control gene regulation and gene activity. Interestingly, this role is specific for CDK6. Unfortunately, currently available pharmacologic principles do not allow to develop selective inhibitors of CDK6 that would not simultaneously also inhibit CDK4. In addition, genetic evidence points to a kinase-independent function of CDK6 in gene control, which is again not amenable to current inhibitor-centric paradigms. To solve these issues, the goal of this proposal was to develop small molecules that can lead to the targeted degradation of CDK6, while sparing CDK4. This was achieved by systematically developing heterobifunctional degraders (so-called "PROTACs") that were building off known CDK4/6 inhibitors. We identified a compound that induces a very profound destabilization of CDK6 by recruiting it to an E3 ubiquitin ligase called CRBN. Interestingly, this compound did not induce the degradation of the closely related CDK4, even though it binds CDK4 and CDK6 with comparable affinities. To understand this selectivity, we developed a novel life-cell proximity assay that reports on the drug-induced dimerization between CDK4/6 and CRBN. This assay revealed that our compound only induced a productive dimerization between CDK6 and CRBN, while it failed to dimerize CDK4 and CRBN. We hypothesized that this has to do with steric differences between CDK4 and CDK6 that are at the binding interface to CRBN. Interestingly, this prototypic CDK6 degrader allowed us to very selectively eliminate cancer cells that are known to depend on CDK6 activity, while sparing cells that depend on the activity of CDK4. This is of particular relevance as selective drugs tend to increase a potential therapeutic index. To further study the selectivity of our compound beyond the CDK4/6 comparison, we coupled cellular treatment with global expression proteomics, which is an experiment that allows us to monitor expression levels of thousands of proteins. Encouragingly, CDK6 was-on a proteome-wide scale-the only destabilized protein, confirming an unmatched selectivity profile. Having this highly selective tool, we further studied the role of CDK6 in acute myeloid leukemia cells by coupling acute degradation to phosphoproteomics and RNA-sequencing. Interestingly, our study uncovered several previously unknown downstream targets that will be subject of future research. Future research will also be required to unambiguously assay the therapeutic potential of pharmacologic CDK6 degradation for the treatment of blood cancers and beyond.