Gabriela Wright

A Novel Element and a TEF-2-like Element Activate the Major Histocompatibility Complex Class II Transactivator in B-lymphocytes

Major histocompatibility complex (MHC) class II molecules play a central role in immune responses, and transcription of this family of genes requires the MHC class II transactivator (CIITA). CIITA has four promoters, which are transcribed in a tissue-specific manner. CIITA promoter III is constitutively active in mature B-lymphocytes. This report now describes the minimal 319-base pair promoter region necessary for maximal transcriptional activity in B-lymphocytes. Ultraviolet light and dimethylsulfate in vivo genomic footprinting analyses reveal five occupied DNA sequence elements present in intact B-lymphocytes. Functional analysis of these elements using promoter deletions and site-specific mutations demonstrates that at least two of the sites occupied in vivo are critical for transcriptional activity. In vitro protein/DNA analysis suggests that one of the sites is a TEF-2-like element and the other is occupied by a novel transcription activator. In addition, nuclear factor-1 associates with the promoter both in vivo and in vitro. In myeloma cell lines, loss of CIITA transcription correlates with a completely unoccupied CIITA promoter III. These findings suggest that CIITA transcription in B-lymphocytes is activated through at least two strong promoter elements, while loss of expression in myeloma cells is mediated through changes in promoter assembly.

Critical Role of the Tumor Suppressor Tuberous Sclerosis Complex 1 in Dendritic Cell Activation of CD4 T Cells by Promoting MHC Class II Expression via IRF4 and CIITA

Dendritic cell (DC) maturation is characterized by upregulation of cell-surface MHC class II (MHC-II) and costimulatory molecules, and production of a variety of cytokines that can shape both innate and adaptive immunity. Paradoxically, transcription of the MHC-II genes, as well as its activator, CIITA, is rapidly silenced during DC maturation. The mechanisms that control CIITA/MHC-II expression and silencing have not been fully understood. We report in this article that the tumor suppressor tuberous sclerosis complex 1 (TSC1) is a critical regulator of DC function for both innate and adaptive immunity. Its deficiency in DCs results in increased mammalian target of rapamycin (mTOR) complex 1 but decreased mTORC2 signaling, altered cytokine production, impaired CIITA/MHC-II expression, and defective Ag presentation to CD4 T cells after TLR4 stimulation. We demonstrate further that IFN regulatory factor 4 can directly bind to CIITA promoters, and decreased IFN regulatory factor 4 expression is partially responsible for decreased CIITA/MHC-II expression in TSC1-deficient DCs. Moreover, we identify that CIITA/MHC-II silencing during DC maturation requires mTOR complex 1 activity. Together, our data reveal unexpected roles of TSC1/mTOR that control multifaceted functions of DCs.

CDKN1C Negatively Regulates RNA Polymerase II C-terminal Domain Phosphorylation in an E2F1-dependent Manner

CDKN1C is a cyclin-dependent kinase inhibitor and is a candidate tumor suppressor gene. We previously found that the CDKN1C protein represses E2F1-driven transcription in an apparent negative feedback loop. Herein, we explore the mechanism by which CDKN1C represses transcription. We find that adenoviral-mediated overexpression of CDKN1C leads to a dramatic reduction in phosphorylation of the RNA polymerase II (pol II) C-terminal domain (CTD). RNA interference studies demonstrate that this activity is not an artifact of CDKN1C overexpression, because endogenous CDKN1C mediates an inhibition of RNA pol II CTD phosphorylation in HeLa cells upon treatment with dexamethasone. Surprisingly, we find that CDKN1C-mediated repression of RNA pol II phosphorylation is E2F1-dependent, suggesting that E2F1 may direct CDKN1C to chromatin. Chromatin immunoprecipitation assays demonstrate that CDKN1C is associated with E2F1-regulated promoters in vivo and that this association can dramatically reduce the level of RNA pol II CTD phosphorylation at both Ser-2 and Ser-5 of the C-terminal domain repeat. In addition, we show that CDKN1C interacts with both CDK7 and CDK9 (putative RNA pol II CTD kinases) and that CDKN1C blocks their ability to phosphorylate a glutathione S-transferase-CTD fusion protein in vitro. E2F1 and CDKN1C are found to form stable complexes both in vivo and in vitro. Molecular studies demonstrate that the E2F1-CDKN1C interaction is mediated by two E2F domains. A central E2F1 domain interacts directly with CDKN1C, whereas a C-terminal E2F1 domain interacts with CDKN1C via interaction with Rb. The results presented in this report highlight a novel mechanism of tumor suppression by CDKN1C.

Dual targeting of WEE1 and PLK1 by AZD1775 elicits single agent cellular anticancer activity

Inhibition of the WEE1 tyrosine kinase enhances anticancer chemotherapy efficacy. Accordingly, the WEE1 inhibitor AZD1775 (previously MK-1775) is currently under evaluation in clinical trials for cancer in combination with chemotherapy. AZD1775 has been reported to display high selectivity and is therefore used in many studies as a probe to interrogate WEE1 biology. However, AZD1775 also exhibits anticancer activity as a single agent although the underlying mechanism is not fully understood. Using a chemical proteomics approach, we here describe a proteome-wide survey of AZD1775 targets in lung cancer cells and identify several previously unknown targets in addition to WEE1. In particular, we observed polo-like kinase 1 (PLK1) as a new target of AZD1775. Importantly, in vitro kinase assays showed PLK1 and WEE1 to be inhibited by AZD1775 with similar potency. Subsequent loss-of-function experiments using RNAi for WEE1 and PLK1 suggested that targeting PLK1 enhances the pro-apoptotic and antiproliferative effects observed with WEE1 knockdown. Combination of RNAi with AZD1775 treatment suggested WEE1 and PLK1 to be the most relevant targets for mediating AZD1775s anticancer effects. Furthermore, disruption of WEE1 by CRISPR-Cas9 sensitized H322 lung cancer cells to AZD1775 to a similar extent as the potent PLK1 inhibitor BI-2536 suggesting a complex crosstalk between PLK1 and WEE1. In summary, we show that AZD1775 is a potent dual WEE1 and PLK1 inhibitor, which limits its use as a specific molecular probe for WEE1. However, PLK1 inhibition makes important contributions to the single agent mechanism of action of AZD1775 and enhances its anticancer effects.

PAXIP1 Potentiates the Combination of WEE1 Inhibitor AZD1775 and Platinum Agents in Lung Cancer

The DNA damage response (DDR) involves a complex network of signaling events mediated by modular protein domains such as the BRCA1 C-terminal (BRCT) domain. Thus, proteins that interact with BRCT domains and are a part of the DDR constitute potential targets for sensitization to DNA-damaging chemotherapy agents. We performed a pharmacologic screen to evaluate 17 kinases, identified in a BRCT-mediated interaction network as targets to enhance platinum-based chemotherapy in lung cancer. Inhibition of mitotic kinase WEE1 was found to have the most effective response in combination with platinum compounds in lung cancer cell lines. In the BRCT-mediated interaction network, WEE1 was found in complex with PAXIP1, a protein containing six BRCT domains involved in transcription and in the cellular response to DNA damage. We show that PAXIP1 BRCT domains regulate WEE1-mediated phosphorylation of CDK1. Furthermore, ectopic expression of PAXIP1 promotes enhanced caspase-3–mediated apoptosis in cells treated with WEE1 inhibitor AZD1775 (formerly, MK-1775) and cisplatin compared with cells treated with AZD1775 alone. Cell lines and patient-derived xenograft models expressing both PAXIP1 and WEE1 exhibited synergistic effects of AZD1775 and cisplatin. In summary, PAXIP1 is involved in sensitizing lung cancer cells to the WEE1 inhibitor AZD1775 in combination with platinum-based treatment. We propose that WEE1 and PAXIP1 levels may be used as mechanism-based biomarkers of response when WEE1 inhibitor AZD1775 is combined with DNA-damaging agents. Mol Cancer Ther; 15(7); 1669–81. ©2016 AACR.