Cyclin-dependent kinase 4 and 6 (CDK4/6) are critical components of the cell cycle and have been implicated in carcinogenesis and, more recently, immunosurveillance. This effect may be mediated by upregulation of PD-L1 expression as well as cell death via reactive oxygen species (ROS). Indeed, CDK4/6 inhibition alters mitochondrial function to release ROS. Merkel cell carcinoma (MCC) is an aggressive form of skin cancer that can be controlled with PD-1-based immune therapy in about half of cases. Treating MCC cells with a CDK4/6 inhibitor, palbociclib, increased transcription from the PD-L1 gene and elevated PD-L1 cell surface expression. The increase in PD-L1 gene expression was due to induction of HIF2a, a transcriptional activator of PD-L1. A HIF2a-specific inhibitor (TC-S7009), was used to test the importance of blocking the activity of HIF2a, which prevented palbociclib-induced PD-L1 transcription and, interestingly, potently enhanced MCC cell death. We further studied the mechanism of cell death and found that targeting both CDK4/6 and HIF2a caused suppression of a cysteine uptake regulatory protein, SLC7A11, critical for cellular redox balance. This was mediated by upregulation of an antisense long noncoding RNA known to block expression of the SLC7A11 redox regulatory protein. Reduction in SLC7A11 level has been associated with ferroptosis, an immunogenic, iron-dependent cell death. Indeed, when co-treatment was tested in several MCC cell lines, the level of SLC7A11 was dramatically reduced, and cell death that was immunogenic in nature was markedly augmented. Thus, our studies indicate that targeting CDK4/6 plus HIF2a can promote immunogenic cell death in MCC cells. Taken together, these findings suggest that hypoxia induced by cell cycle inhibition could be exploited to augmented effective cancer immunotherapy.

Background Merkel cell carcinoma (MCC) is an aggressive, high-grade, cutaneous neuroendocrine tumour (NET). Agents blocking programmed death 1/programmed death ligand 1 have efficacy in metastaticMCC(mMCC), but half of patients do not derive durable benefit. Somatostatin analogues (SSAs) are commonly used to treat low- and moderate-gradeNETs that express somatostatin receptors (SSTRs). Objectives To assessSSTRexpression and the efficacy ofSSAs inmMCC, a high-gradeNET. MethodsIn this retrospective study of 40 patients withmMCC,SSTRexpression was assessed radiologically by somatostatin receptor scintigraphy (SRS;n= 39) and/or immunohistochemically when feasible (n= 9). Nineteen patients (18 hadSRSuptake inMCCtumours) were treated withSSA. Disease control was defined as progression-free survival (PFS) of >= 120 days after initiation ofSSA. Results Thirty-three of 39 patients (85%) had some degree (low 52%, moderate 23%, high 10%) ofSRSuptake. Of 19 patients treated withSSA, seven had a response-evaluable target lesion; three of these seven patients (43%) experienced disease control, with a medianPFSof 237 days (range 152-358). Twelve of 19 patients did not have a response-evaluable lesion due to antecedent radiation; five of these 12 (42%) experienced disease control (medianPFSof 429 days, range 143-1757). The degree ofSSTRexpression (determined bySRSand/or immunohistochemistry) did not correlate significantly with the efficacy endpoints. Conclusions In contrast to other high-gradeNETs,mMCCtumours appear frequently to expressSSTRs.SSAs can lead to clinically meaningful disease control with minimal side-effects. Targeting ofSSTRs usingSSAor other novel approaches should be explored further formMCC.

TopBP1 plays important roles in chromosome replication, DNA damage response, and other cellular regulatory functions in vertebrates. Although the roles of TopBP1 have been studied mostly in cancer cell lines, its physiological function remains unclear in mice and untransformed cells. We generated conditional knock-out mice in which exons 5 and 6 of the TopBP1 gene are flanked by loxP sequences. Although TopBP1-deficient embryos developed to the blastocyst stage, no homozygous mutant embryos were recovered at E8.5 or beyond, and completely resorbed embryos were frequent at E7.5, indicating that mutant embryos tend to die at the peri-implantation stage. This finding indicated that TopBP1 is essential for cell proliferation during early embryogenesis. Ablation of TopBP1 in TopBP1(flox/flox) mouse embryonic fibroblasts and 3T3 cells using Cre recombinase-expressing retrovirus arrests cell cycle progression at the G(1), S, and G(2)/M phases. The TopBP1-ablated mouse cells exhibit phosphorylation of H2AX and Chk2, indicating that the cells contain DNA breaks. The TopBP1-ablated mouse cells enter cellular senescence. Although RNA interference-mediated knockdown of TopBP1 induced cellular senescence in human primary cells, it induced apoptosis in cancer cells. Therefore, TopBP1 deficiency in untransformed mouse and human primary cells induces cellular senescence rather than apoptosis. These results indicate that TopBP1 is essential for cell proliferation and maintenance of chromosomal integrity.

Transglutaminase 2 knockout (TGase2(-/-)) mice show significantly reduced inflammation with decreased myofibroblasts in a unilateral ureteral obstruction (UUO) model, but the mechanism remains to be clarified. Nuclear factor-kappa B (NF-kappa B) activation plays a major role in the progression of inflammation in an obstructive nephropathy model. However, the key factors extending the duration of NF-kappa B activation in UUO are not known. In several inflammatory diseases, we and others recently found that TGase 2 plays a key role in extending NF-kappa B activation, which contributes to the pathogenesis of disease. In the current study, we found that NF-kappa B activity in mouse embryogenic fibroblasts (MEFs) from TGase2(-/-) mice remained at the control level while the NF-kappa B activity of wild-type (WT) MEFs was highly increased under hypoxic stress. Using the obstructive nephropathy model, we found that NF-kappa B activity remained at the control level in TGase2(-/-) mouse kidney tissues, as measured by COX-2 expression, but was highly increased in WT tissues. We conclude that TGase 2 gene ablation reduces the duration of NF-kappa B activation in ischemic injury. (C) 2010 Elsevier Inc. All rights reserved.

We investigated the cellular localization of ectopically-expressed CIS, SOCS1, SOCS2 and SOCS3 proteins. We found that SOCS proteins localize to the nucleus where they reduce Stat3 proteins and that the presence of proteasome inhibitors increased SOCS nuclear localization. Our results indicate that increased nuclear localization resulted from increased levels of SOCS proteins in the cytoplasm. Finally, we demonstrate that the same effect occurs with endogenously-expressed SOCS proteins. These observations suggest that increased cytoplasmic levels of proteins in the SOCS family are regulated through nuclear translocation.