NUT midline carcinoma (NMC) is an aggressive subtype of squamous cell carcinoma that typically harbors BRD4/3-NUT fusion oncoproteins that block differentiation and maintain tumor growth. In 20% of cases NUT is fused to uncharacterized non-BRD gene(s). We established a new patient-derived NMC cell line (1221) and demonstrated that it harbors a novel NSD3-NUT fusion oncogene. We find that NSD3-NUT is both necessary and sufficient for the blockade of differentiation and maintenance of proliferation in NMC cells. NSD3-NUT binds to BRD4, and BRD bromodomain inhibitors induce differentiation and arrest proliferation of 1221 cells. We find further that NSD3 is required for the blockade of differentiation in BRD4-NUT-expressing NMCs. These findings identify NSD3 as a novel critical oncogenic component and potential therapeutic target in NMC.

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Ovarian cancer presents as malignant tumors in the female reproductive system with high mortality. MicroRNAs are involved in the progression of ovarian cancer; however, the regulatory relationship among miRs remains unclear. In our study, we verified that both miR-145 and miR-133b messenger RNA (mRNA) levels in ovarian cancer tissues were lower than in normal ovarian tissues, and their mRNA level in serum of patients with ovarian cancer was reduced. We demonstrated miR-145 targeted c-myc, and c-myc interacted physically with DNMT3A in ovarian cancer cells. We confirmed that c-myc recruited DNMT3A to the miR-133b promoter. miR-133b overexpression also inhibited target gene PKM2 expression along with the Warburg effect. Our results indicate that miR-145 inhibited the Warburg effect through miR-133b/PKM2 pathways, which may improve approaches to ovarian cancer diagnosis and treatment. K E Y W O R D Sc-myc, DNMT3A, miR-133b, miR-145, ovarian cancer, Warburg effect

Naturally occurring regulatory T cells (nTregs; CD4+CD25+Foxp3+) are capable of suppressing the chronic inflammation observed in a variety of different animal models of autoimmune and chronic inflammatory diseases such as inflammatory bowel diseases, diabetes, and arthritis. A major limitation in exploring how and where nTregs exert their suppression in vivo is the relative paucity of these regulatory cells. Although several laboratories have described different methods to expand flow-purified nTregs or convert conventional/naïve T cells (CD4+Foxp3−) to Foxp3-expressing “induced” Tregs (iTregs; CD4+Foxp3+) ex vivo, we have found that many of these approaches are encumbered with their own limitations. Therefore, we sought to develop a relatively simple ex vivo method to generate large numbers of Foxp3-expressing iTregs that can be used to evaluate their trafficking properties, suppressive activity, and therapeutic efficacy in a mouse model of chronic gut inflammation in vivo. We present a detailed protocol demonstrating that polyclonal activation of conventional CD4+ T cells in the presence of IL-2, TGFβ, and all trans retinoic acid induces >90% conversion of these T cells to Foxp3-expressing iTregs as well as promotes a three- to fourfold increase in proliferation following a 4-day incubation period in vitro. This protocol enhances modestly the surface expression of the gut-homing adhesion molecule CCR9 but not α4β7. Furthermore, we provide preliminary data demonstrating that these iTregs are significantly more potent at suppressing T-cell activation in vitro and are equally effective as freshly isolated nTregs at attenuating chronic colitis in vivo. Finally, we report that this protocol has the potential to generate 30–40 million iTregs from one healthy mouse spleen.