Maintenance and pharmacologic targeting of ROR1 protein levels via UHRF1 in t(1;19) pre-B-ALL.

Expression of the transmembrane pseudokinase ROR1 is required for survival of t(1;19)-pre-B-cell acute lymphoblastic leukemia (t(1;19) pre-B-ALL), chronic lymphocytic leukemia, and many solid tumors. However, targeting ROR1 with small-molecules has been challenging due to the absence of ROR1 kinase activity. To identify genes that regulate ROR1 expression and may, therefore, serve as surrogate drug targets, we employed an siRNA screening approach and determined that the epigenetic regulator and E3 ubiquitin ligase, UHRF1, is required for t(1;19) pre-B-ALL cell viability in a ROR1-dependent manner. Upon UHRF1 silencing, ROR1 protein is reduced without altering ROR1 mRNA, and ectopically expressed UHRF1 is sufficient to increase ROR1 levels. Additionally, proteasome inhibition rescues loss of ROR1 protein after UHRF1 silencing, suggesting a role for the proteasome in the UHRF1-ROR1 axis. Finally, we show that ROR1-positive cells are twice as sensitive to the UHRF1-targeting drug, naphthazarin, and undergo increased apoptosis compared to ROR1-negative cells. Naphthazarin elicits reduced expression of UHRF1 and ROR1, and combination of naphthazarin with inhibitors of pre-B cell receptor signaling results in further reduction of cell survival compared with either inhibitor alone. Therefore, our work reveals a mechanism by which UHRF1 stabilizes ROR1, suggesting a potential targeting strategy to inhibit ROR1 in t(1;19) pre-B-ALL and other malignancies.

Super-Enhancers Promote Transcriptional Dysregulation in Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is an invasive cancer with particularly high incidence in Southeast Asia and Southern China. The pathogenic mechanisms of NPC, particularly those involving epigenetic dysregulation, remain largely elusive, hampering clinical management of this malignancy. To identify novel druggable targets, we carried out an unbiased high-throughput chemical screening and observed that NPC cells were highly sensitive to inhibitors of cyclin-dependent kinases (CDK), especially THZ1, a covalent inhibitor of CDK7. THZ1 demonstrated pronounced antineoplastic activities both in vitro and in vivo An integrative analysis using both whole-transcriptome sequencing and chromatin immunoprecipitation sequencing pinpointed oncogenic transcriptional amplification mediated by super-enhancers (SE) as a key mechanism underlying the vulnerability of NPC cells to THZ1 treatment. Further characterization of SE-mediated networks identified many novel SE-associated oncogenic transcripts, such as BCAR1, F3, LDLR, TBC1D2, and the long noncoding RNA TP53TG1. These transcripts were highly and specifically expressed in NPC and functionally promoted NPC malignant phenotypes. Moreover, DNA-binding motif analysis within the SE segments suggest that several transcription factors (including ETS2, MAFK, and TEAD1) may help establish and maintain SE activity across the genome. Taken together, our data establish the landscape of SE-associated oncogenic transcriptional network in NPC, which can be exploited for the development of more effective therapeutic regimens for this disease. Cancer Res; 77(23); 6614-26. ©2017 AACR.

Identification of a Novel SYK/c-MYC/MALAT1 Signaling Pathway and Its Potential Therapeutic Value in Ewing Sarcoma.

Purpose: Ewing sarcoma (EWS) is a devastating soft tissue sarcoma affecting predominantly young individuals. Tyrosine kinases (TK) and associated pathways are continuously activated in many malignancies, including EWS; these enzymes provide candidate therapeutic targets.Experimental Design: Two high-throughput screens (a siRNA library and a small-molecule inhibitor library) were performed in EWS cells to establish candidate targets. Spleen tyrosine kinase (SYK) phosphorylation was assessed in EWS patients and cell lines. SYK was inhibited by a variety of genetic and pharmacological approaches, and SYK-regulated pathways were investigated by cDNA microarrays. The transcriptional regulation of MALAT1 was examined by ChIP-qPCR, luciferase reporter, and qRT-PCR assays.Results: SYK was identified as a candidate actionable target through both high-throughput screens. SYK was highly phosphorylated in the majority of EWS cells, and SYK inhibition by a variety of genetic and pharmacologic approaches markedly inhibited EWS cells both in vitro and in vivo Ectopic expression of SYK rescued the cytotoxicity triggered by SYK-depletion associated with the reactivation of both AKT and c-MYC. A long noncoding RNA, MALAT1, was identified to be dependent on SYK-mediated signaling. Moreover, c-MYC, a SYK-promoted gene, bound to the promoter of MALAT1 and transcriptionally activated MALAT1, which further promoted the proliferation of EWS cells.Conclusions: This study identifies a novel signaling involving SYK/c-MYC/MALAT1 as a promising therapeutic target for the treatment of EWS. Clin Cancer Res; 23(15); 4376-87. ©2017 AACR.

Benzo[a]pyrene increases the Nrf2 content by downregulating the Keap1 message.

We employed the suppressive subtractive hybridization to identify 41 up- and downregulated transcripts in Jurkat cells after benzo[a]pyrene (BaP) treatment. Among the 21 downregulated transcripts, we found that BaP suppresses the Keap1 transcript by 7.5-fold. Subsequent analyses revealed that BaP significantly suppresses the Keap1 message and protein levels to about 40 and 60%, respectively, of the vehicle controls in Jurkat cells without reactive oxygen species involvement. In addition, the nuclear Nrf2 (nuclear factor erythroid 2-related factor) protein content is significantly increased by 2.6-fold. The same BaP treatment to Hepa1c1c7 cells also downregulates the Keap1 message and protein levels to a similar extent. When we treated Jurkat cells with 3-(4-morpholinyl)propyl isothiocyanate, which is known to increase the amount of the Nrf2 content, we found that there is no change in the Keap1 message, but the amount of the Keap1 (kelch-like ECH-associated protein 1) protein is reduced to 75% of the vehicle controls. Although both Nrf2 target messages nqo1 and gstp1 are upregulated by BaP in Jurkat cells, only GSTP1 is upregulated at the protein level. Unlike Hepa1c1c7 cells, Jurkat cells have no detectable aryl hydrocarbon receptor and BaP metabolites, minimal CYP1A1 activity, and no quinone oxidoreductase 1 (NQO1) activity. We concluded that BaP, but not its metabolites, increases the amount of the nuclear Nrf2 protein by downregulating the Keap1 message in Jurkat cells.