Abstract P109: Targeting BRD4 in T cells with self-delivering RNAi PH-894 for immunotherapy
Authors: Maxwell Melissa , Ujiie Daisuke , Cuiffo Benjamin , Rivest Brianna , Yan Dingxue , Cardia James , Melief Jeroen , Kiessling Rolf , Fricker Simon P. 
Published: December 2021

Abstract
BRD4 is a member of the family of BET proteins that act as epigenetic readers and regulators of gene transcription. BRD4 is a known driver of tumor growth via enhancement of transcription of oncogenes such as Myc. More recent evidence also suggests a role of BRD4 in T cells. T cell differentiation and T cell exhaustion is epigenetically regulated and BRD4 has been associated with T cell differentiation. Available small molecule inhibitors of BET proteins do not target BRD4 specifically, but also inhibit other members of the BET protein family. We therefore investigated the effects of BRD4 silencing on T cells with the INTASYL™ compound PH-894, a self-delivering RNAi specifically targeting BRD4. INTASYL self-delivering RNAi molecules consist of an asymmetric duplex structure, a small duplex region (≤ 15 base pairs), a single-stranded phosphorothioate tail, and chemical modifications to confer stability, hydrophobicity, and efficient cellular uptake. Primary human T cells from healthy donors, either unstimulated or activated by OKT3 or anti-CD3/CD28 beads, were treated with PH-894. Additional studies were performed with CD8+ T cells magnetically sorted from primary human T cells. Silencing of BRD4 mRNA was assessed by RT-qPCR, and protein by flow cytometry. Phenotypic markers of T cell activation, differentiation and function were assessed by flow cytometry and cytokine secretion by cytometric bead array. Expanded and PH-894-treated CD8+ T cells were studied for IFNγ production upon coculture with allogeneic melanoma cell lines. Silencing of BRD4 mRNA and protein by PH-894 was concentration dependent and durable for up to 10 days in activated and naïve T cells. Silencing was specific for BRD4 with no effect on BRD2 or BRD3 expression. In activated T cells there was a suppression of IL-10 secretion and no effect on inflammatory cytokine levels. Markers of T cell activation were elevated on naïve PH-894-treated T cells with a shift to an increased CD8+/CD4+ ratio. In addition, BRD4 silencing in activated CD8+ T cells coincided with a decreased expression of transcription factors typically induced immediately upon TCR activation and are known drivers of T cell differentiation towards an effector phenotype. Moreover, CD8+ T cells expanded in the presence of PH-894 produced increased levels of IFNγ when cocultured with two allogeneic melanoma cell lines. These data translated in vivo with PH-894 treatment eliciting a dose associated inhibition of tumor growth in a CT26 murine colon carcinoma model. Ex vivo analysis of tumors found PH-894 efficacy was associated with decreased expression of BRD4 in tumor infiltrating CD3 T cells, and a reduction of Foxp3hi CD25hi CD4+ CD3+ Tregs, compared with control tumors. Specific silencing of BRD4 with PH-894 resulted in phenotypic changes on T cells associated with T cell activation and reduced immunosuppression. These data suggest that BRD4 not only plays a role on tumor cells but can also regulate T cell function and that PH-894 can reprogram T cells to provide enhanced immunotherapeutic activity.
Citation Format: Melissa Maxwell, Daisuke Ujiie, Benjamin Cuiffo, Brianna Rivest, Dingxue Yan, James Cardia, Jeroen Melief, Rolf Kiessling, Simon P. Fricker. Targeting BRD4 in T cells with self-delivering RNAi PH-894 for immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P109.