ß-cell deletion of the PKm1 and PKm2 isoforms of pyruvate kinase in mice reveals their essential role as nutrient sensors for the KATP channel.

Pyruvate kinase (PK) and the phosphoenolpyruvate (PEP) cycle play key roles in nutrient-stimulated KATP channel closure and insulin secretion. To identify the PK isoforms involved, we generated mice lacking ß-cell PKm1, PKm2, and mitochondrial PEP carboxykinase (PCK2) that generates mitochondrial PEP. Glucose metabolism was found to generate both glycolytic and mitochondrially derived PEP, which triggers KATP closure through local PKm1 and PKm2 signaling at the plasma membrane. Amino acids, which generate mitochondrial PEP without producing glycolytic fructose 1,6-bisphosphate to allosterically activate PKm2, signal through PKm1 to raise ATP/ADP, close KATP channels, and stimulate insulin secretion. Raising cytosolic ATP/ADP with amino acids is insufficient to close KATP channels in the absence of PK activity or PCK2, indicating that KATP channels are primarily regulated by PEP that provides ATP via plasma membrane-associated PK, rather than mitochondrially derived ATP. Following membrane depolarization, the PEP cycle is involved in an 'off-switch' that facilitates KATP channel reopening and Ca2+ extrusion, as shown by PK activation experiments and ß-cell PCK2 deletion, which prolongs Ca2+ oscillations and increases insulin secretion. In conclusion, the differential response of PKm1 and PKm2 to the glycolytic and mitochondrial sources of PEP influences the ß-cell nutrient response, and controls the oscillatory cycle regulating insulin secretion.

Targeting RET Kinase in Neuroendocrine Prostate Cancer.

The increased treatment of metastatic castration-resistant prostate cancer (mCRPC) with second-generation antiandrogen therapies (ADT) has coincided with a greater incidence of lethal, aggressive variant prostate cancer (AVPC) tumors that have lost dependence on androgen receptor (AR) signaling. These AR-independent tumors may also transdifferentiate to express neuroendocrine lineage markers and are termed neuroendocrine prostate cancer (NEPC). Recent evidence suggests kinase signaling may be an important driver of NEPC. To identify targetable kinases in NEPC, we performed global phosphoproteomics comparing several AR-independent to AR-dependent prostate cancer cell lines and identified multiple altered signaling pathways, including enrichment of RET kinase activity in the AR-independent cell lines. Clinical NEPC patient samples and NEPC patient-derived xenografts displayed upregulated RET transcript and RET pathway activity. Genetic knockdown or pharmacologic inhibition of RET kinase in multiple mouse and human models of NEPC dramatically reduced tumor growth and decreased cell viability. Our results suggest that targeting RET in NEPC tumors with high RET expression could be an effective treatment option. Currently, there are limited treatment options for patients with aggressive neuroendocrine prostate cancer and none are curative. IMPLICATIONS: Identification of aberrantly expressed RET kinase as a driver of tumor growth in multiple models of NEPC provides a significant rationale for testing the clinical application of RET inhibitors in patients with AVPC.

Human cytomegalovirus-encoded viral cyclin-dependent kinase (v-CDK) UL97 phosphorylates and inactivates the retinoblastoma protein-related p107 and p130 proteins.

The human cytomegalovirus (HCMV)-encoded viral cyclin-dependent kinase (v-CDK) UL97 phosphorylates the retinoblastoma (Rb) tumor suppressor. Here, we identify the other Rb family members p107 and p130 as novel targets of UL97. UL97 phosphorylates p107 and p130 thereby inhibiting their ability to repress the E2F-responsive E2F1 promoter. As with Rb, this phosphorylation, and the rescue of E2F-responsive transcription, is dependent on the L1 LXCXE motif in UL97 and its interacting clefts on p107 and p130. Interestingly, UL97 does not induce the disruption of all p107-E2F or p130-E2F complexes, as it does to Rb-E2F complexes. UL97 strongly interacts with p107 but not Rb or p130. Thus the inhibitory mechanisms of UL97 for Rb family protein-mediated repression of E2F-responsive transcription appear to differ for each of the Rb family proteins. The immediate early 1 (IE1) protein of HCMV also rescues p107- and p130-mediated repression of E2F-responsive gene expression, but it does not induce their phosphorylation and does not disrupt p107-E2F or p130-E2F complexes. The unique regulation of Rb family proteins by HCMV UL97 and IE1 attests to the importance of modulating Rb family protein function in HCMV-infected cells.

Deficiencies in Cellular Processes Modulated by the Retinoblastoma Protein Do Not Account for Reduced Human Cytomegalovirus Replication in Its Absence.

UNLABELLED: Despite encoding multiple viral proteins that modulate the retinoblastoma (Rb) protein in a manner classically defined as inactivation, human cytomegalovirus (HCMV) requires the presence of the Rb protein to replicate efficiently. In uninfected cells, Rb controls numerous pathways that the virus also commandeers during infection. These include cell cycle progression, senescence, mitochondrial biogenesis, apoptosis, and glutaminolysis. We investigated whether a potential inability of HCMV to regulate these Rb-controlled pathways in the absence of the Rb protein was the reason for reduced viral productive replication in Rb knockdown cells. We found that HCMV was equally able to modulate these pathways in the parental Rb-expressing and Rb-depleted cells. Our results suggest that Rb may be required to enhance a specific viral process during HCMV productive replication. IMPORTANCE: The retinoblastoma (Rb) tumor suppressor is well established as a repressor of E2F-dependent transcription. Rb hyperphosphorylation, degradation, and binding by viral oncoproteins are also codified. Recent reports indicate Rb can be monophosphorylated, repress the transcription of antiviral genes in association with adenovirus E1A, modulate cellular responses to polycomb-mediated epigenetic methylations in human papillomavirus type 16 E7 expressing cells, and increase the efficiency of human cytomegalovirus (HCMV) productive replication. Since Rb function also now extends to regulation of mitochondrial function (apoptosis, metabolism), it is clear that our current understanding of this protein is insufficient to explain its roles in virus-infected cells and tumors. Work here reinforces this concept, showing the known roles of Rb are insufficient to explain its positive impact on HCMV replication. Therefore, HCMV, along with other viral systems, provide valuable tools to probe functions of Rb that might be modulated with therapeutics for cancers with viral or nonviral etiologies.

The retinoblastoma tumor suppressor promotes efficient human cytomegalovirus lytic replication.

UNLABELLED: The retinoblastoma (Rb) tumor suppressor controls cell cycle, DNA damage, apoptotic, and metabolic pathways. DNA tumor virus oncoproteins reduce Rb function by either inducing Rb degradation or physically disrupting complexes between Rb and its myriad binding proteins. Human cytomegalovirus (HCMV), a betaherpesvirus being investigated for potential roles in human cancers, encodes multiple lytic-phase proteins that inactivate Rb in distinct ways, leading to the hypothesis that reduced Rb levels and/or activity would benefit HCMV lytic infection. Paradoxically, we found that Rb knockdown prior to infection, whether transient or constitutive, impaired HCMV lytic infection at multiple stages, notably viral DNA replication, late protein expression, and infectious virion production. The existence of differentially modified forms of Rb, the temporally and functionally distinct means by which HCMV proteins interact with Rb, and the necessity of Rb for efficient HCMV lytic replication combine to highlight the complex relationship between the virus and this critical tumor suppressor. IMPORTANCE: Initial work examining viral protein modulation of cell cycle progression and oncogenic transformation revealed that these proteins inactivated the function of cellular tumor suppressor proteins. However, subsequent work, including experiments described here using human cytomegalovirus, demonstrate a more nuanced interaction that includes the necessity of cellular tumor suppressors for efficient viral replication. Understanding the positive impacts that cellular tumor suppressors have on viral infections may reveal new activities of these well-studied yet incompletely understood proteins. The basis for oncolytic viral therapy is the selective replication of viruses in transformed cells in which tumor suppressor function may be compromised. Understanding how tumor suppressors support viral infections may allow for the generation of modified oncolytic viruses with greater selective tumor cell replication and killing.