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Mediator kinases CDK19 and CDK8, pleiotropic regulators of transcriptional reprogramming, are differentially regulated by androgen signaling, but both kinases are upregulated in castration-resistant prostate cancer (CRPC). Genetic or pharmacological inhibition of CDK8 and CDK19 reverses the castration-resistant phenotype and restores the sensitivity of CRPC xenografts to androgen deprivation in vivo. Prolonged CDK8/19 inhibitor treatment combined with castration not only suppressed the growth of CRPC xenografts but also induced tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplified and modulated the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affected stromal gene expression, indicating that Mediator kinase activity in CRPC molded the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulated the MYC pathway, and Mediator kinase inhibition suppressed a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors showed efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlated with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediated androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.

The outgrowth and stabilization of nascent dendritic spines are crucial processes underlying learning and memory. Most new spines retract shortly after growth; only a small subset is stabilized and integrated into the new circuit connections that support learning. New spine stabilization has been shown to rely upon activity-dependent molecular mechanisms that also contribute to long-term potentiation (LTP) of synaptic strength. Indeed, disruption of the activity-dependent targeting of the kinase CaMKIIα to the GluN2B subunit of the NMDA-type glutamate receptor disrupts both LTP and activity-dependent stabilization of new spines. Yet it is not known which of CaMKIIα's many enzymatic and structural functions are important for new spine stabilization. Here, we used two-photon imaging and photolysis of caged glutamate to monitor the activity-dependent stabilization of new dendritic spines on hippocampal CA1 neurons from mice of both sexes in conditions where CaMKIIα functional and structural interactions were altered. Surprisingly, we found that inhibiting CaMKIIα kinase activity either genetically or pharmacologically did not impair activity-dependent new spine stabilization. In contrast, shRNA knockdown of CaMKIIα abolished activity-dependent new spine stabilization, which was rescued by co-expressing shRNA-resistant full-length CaMKIIα, but not by a truncated monomeric CaMKIIα. Notably, overexpression of phospho-mimetic CaMKIIα-T286D, which exhibits activity-independent targeting to GluN2B, enhanced basal new spine survivorship in the absence of additional glutamatergic stimulation, even when kinase activity was disrupted. Together, our results support a model in which nascent dendritic spine stabilization requires structural and scaffolding interactions mediated by dodecameric CaMKIIα that are independent of its enzymatic activities.

Simple Summary: Patients with treatable cancers all too often return to the clinic with untreatable tumors, requiring highly toxic secondary treatments or palliative care. In this study, we aim to determine whether activation of the Anaphase Promoting Complex (APC) in multiple-drug-resistant (MDR) breast cancer cells will re-sensitize them to chemotherapeutic agents. The APC is required for the targeted degradation of proteins that inhibit passage through mitosis. We found that APC activity is indeed impaired in MDR cells and that chemical activation of the APC increased the sensitivity of these cells to doxorubicin. Mitotic progression was slowed in MDR cells, compared to matched parental drug sensitive cells, with an accumulation of APC substrate proteins. APC activation in nocodazole-arrested cells resulted in increased passage through mitosis with lower APC substrate levels. Mice growing a patient-derived xenografted (PDX) tumor were treated with increasing doses of a chemical APC activator, resulting in a dose-dependent reduction in tumor size. Taken together, our data show that APC activity is reduced in MDR cells, with APC activation resulting in a species- and cancer-type-independent reversal of the MDR phenotype. The development of multiple-drug-resistant (MDR) cancer all too often signals the need for toxic alternative therapy or palliative care. Our recent in vivo and in vitro studies using canine MDR lymphoma cancer cells demonstrate that the Anaphase Promoting Complex (APC) is impaired in MDR cells compared to normal canine control and drug-sensitive cancer cells. Here, we sought to establish whether this phenomena is a generalizable mechanism independent of species, malignancy type, or chemotherapy regime. To test the association of blunted APC activity with MDR cancer behavior, we used matched parental and MDR MCF7 human breast cancer cells, and a patient-derived xenograft (PDX) model of human triple-negative breast cancer. We show that APC activating mechanisms, such as APC subunit 1 (APC1) phosphorylation and CDC27/CDC20 protein associations, are reduced in MCF7 MDR cells when compared to chemo-sensitive matched cell lines. Consistent with impaired APC function in MDR cells, APC substrate proteins failed to be effectively degraded. Similar to our previous observations in canine MDR lymphoma cells, chemical activation of the APC using Mad2 Inhibitor-1 (M2I-1) in MCF7 MDR cells enhanced APC substrate degradation and resensitized MDR cells in vitro to the cytotoxic effects of the alkylating chemotherapeutic agent, doxorubicin (DOX). Using cell cycle arrest/release experiments, we show that mitosis is delayed in MDR cells with elevated substrate levels. When pretreated with M2I-1, MDR cells progress through mitosis at a faster rate that coincides with reduced levels of APC substrates. In our PDX model, mice growing a clinically MDR human triple-negative breast cancer tumor show significantly reduced tumor growth when treated with M2I-1, with evidence of increased DNA damage and apoptosis. Thus, our results strongly support the hypothesis that APC impairment is a driver of aggressive tumor development and that targeting the APC for activation has the potential for meaningful clinical benefits in treating recurrent cases of MDR malignancy. [ABSTRACT FROM AUTHOR]