Organizational commitment to diversity, equity, and inclusion: A strategic path forward.

PURPOSE: To describe an approach to diversity, equity, and inclusion (DEI) strategy development at a school of pharmacy aimed at stakeholder investment and infrastructure that can address systemic challenges in various healthcare settings. SUMMARY: The UNC Eshelman School of Pharmacy utilized an organizational approach focused on infrastructure to produce a diverse and inclusive school community. The Office of Organizational Diversity and Inclusion (ODI) established vision and mission statements to represent the school's commitment and conducted a comprehensive environmental scan to compose a shared vision. Students, faculty, staff, and alumni participated in a series of retreats, symposiums, and focus groups to identify opportunities to cultivate a diverse and inclusive school community. A working group comprised of key leaders in the school developed and launched a 3-year DEI Strategic Plan along with initiatives and metrics for year 1. The plan's 3 priorities were (1) to recruit and retain diverse talent, (2) to prepare culturally intelligent professionals, and (3) to build an inclusive community. The ODI collaborated with internal and external stakeholders, which included students, faculty, staff, postdocs, alumni, and partners from health systems, industry, and other institutions and organizations, to initiate, implement, and monitor progress through an organizational approach to establish accountability and greater commitment. CONCLUSION: An organizational approach to DEI strategy through stakeholder engagement and infrastructure increased commitment and shared ownership among members of the school community. Applications in an organizational approach can be adapted to multiple healthcare settings to contribute to the cultural transformation necessary to develop a diverse and inclusive healthcare workforce.

Coexpression of ABCB1 and ABCG2 in a Cell Line Model Reveals Both Independent and Additive Transporter Function.

Although overexpression of multiple ATP-binding cassette transporters has been reported in clinical samples, few studies have examined how coexpression of multiple transporters affected resistance to chemotherapeutic drugs. We therefore examined how coexpression of ABCB1 (P-glycoprotein) and ABCG2 contributes to drug resistance in a cell line model. HEK293 cells were transfected with vector-encoding full-length ABCB1, ABCG2, or a bicistronic vector containing both genes, each under the control of a separate promoter. Cells transfected with both transporters (B1/G2 cells) demonstrated high levels of both transporters, and uptake of both the ABCB1-specific substrate rhodamine 123 and the ABCG2-specific substrate pheophorbide a was reduced when examined by flow cytometry. B1/G2 cells were also cross-resistant to the ABCB1 substrate doxorubicin, the ABCG2 substrate topotecan, as well as mitoxantrone and the cell cycle checkpoint kinase 1 inhibitor prexasertib, both of which were found to be substrates of both ABCB1 and ABCG2. When B1/G2 cells were incubated with both rhodamine 123 and pheophorbide a, transport of both compounds was observed, suggesting that ABCB1 and ABCG2, when coexpressed, can function independently to transport substrates. ABCB1 and ABCG2 also functioned additively to transport the common fluorescent substrates mitoxantrone and BODIPY-prazosin, as it was necessary to inhibit both transporters to prevent efflux from B1/G2 cells. ABCG2 expression was also found to decrease the efficacy of the ABCB1 inhibitor tariquidar in B1/G2 cells. Thus, ABCB1 and ABCG2 can independently and additively confer resistance to substrates, underscoring the need to inhibit multiple transporters when they are coexpressed.

The Brd4 acetyllysine-binding protein is involved in activation of polyomavirus JC.

Brd4 is an epigenetic reader protein and a member of the BET (bromodomain and extra terminal domain) family of proteins with two bromodomains that recognize acetylated lysine residues. Brd4 specifically binds to acetylated transcription factor NF-κB p65 and coactivates transcription. Polyomavirus JC (JCV) is regulated by a noncoding control region (NCCR) containing promoter/enhancer elements for viral gene expression including a binding site for NF-κB, which responds to proinflammatory cytokines such as TNF-α, the DNA damage response, calcium signaling and acetylation of the NF-κB p65 subunit on lysine residues K218 and K221. Earlier studies indicated that NF-κB is involved in the reactivation of persistent/latent JCV in glial cells to cause progressive multifocal leukoencephalopathy (PML), a severe demyelinating disease of the brain caused by replication of JCV in glial cells. To investigate the mechanism of action of NF-κB acetylation on JCV transcription, we examined Brd4 and found that JCV early transcription was stimulated by Brd4 via the JCV NF-κB site and that p65 K218 and K221 were involved. Treatment with the Brd4 inhibitor JQ1(+) or mutation of either K218 or K221 to glutamine (K218R or K221) inhibited this stimulation and decreased the proportion of p65 in the nucleus. We conclude that Brd4 is involved in the regulation of the activation status of JCV in glial cells.

Epigenetic regulation of polyomavirus JC involves acetylation of specific lysine residues in NF-κB p65.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease caused by neurotropic polyomavirus, JC virus (JCV), a virus that causes lytic infection of CNS glial cells. After primary infection, JCV is controlled by the immune system but virus persists asymptomatically. Rarely, when immune function is impaired, it can reemerge to cause PML. The mechanisms of JCV persistence and reactivation are not well understood but our earlier work implicated epigenetic control by protein acetylation since histone deacetylase inhibitors such as trichostatin A (TSA) strongly stimulate JCV transcription. Since both TNF-α and TSA activate JCV transcription via the same unique NF-κB site in the JCV control region, we investigated a role for acetylation of NF-κB in JCV regulation. A site-directed mutagenesis strategy was employed targeting the known lysine acetylation sites of NF-κB p65: K218, K221, and K310. We individually mutated each lysine to arginine, which cannot be acetylated and retains a positive charge like lysine. K218R and K221R impaired transactivation of JCV early promoter transcription either alone or combined with TSA treatment or coexpression of acetyltransferase transcriptional coactivator p300 but K310R was largely without effect. Mutation of lysine to glutamine gives mutants with a negative charge like acetyllysine. However, K218Q and K221Q showed impaired activity and only K310Q showed enhanced transactivation. NF-κB acetylation can regulate several aspects of the process of activation including complex formation with IκB, translocation to the nucleus, and DNA binding and transcriptional activation. Cell fractionation studies revealed that the mutants had no defect in translocation to the nucleus whereas gel shift studies revealed reduced binding to the JCV NF-κB site. Thus, acetylation regulates NF-κB p65 activity toward JCV at the level of p65 binding to the JCV control region and activation of JCV transcription.