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Breast cancer is the second leading cause of death for women in the United States. However, only 10% of breast cancers have been linked to inherited genomic mutations. Thus, identifying biomarkers to predict cancer progression and metastasis remains a clear need in the research and medical world. Here, we report a novel role for Interferon Regulatory Factor 5 (IRF5) in mammary gland development, tumorigenesis, and metastasis. Historically, IRF5 has been studied as a transcription factor in the context of genetic risk for autoimmune diseases. However, mining of The Cancer Genome Atlas revealed that loss of IRF5 expression in human breast cancer is significantly linked with progression to high grade carcinoma, increased metastasis, and decreased overall and recurrence-free survival. Supporting these analyses, we demonstrated that female Irf5-/- BALB/c mice have higher incidence of spontaneous atypical ductal hyperplasia (ADH), increased progression to DCIS, and ultimately, increased incidence of IDC. Using qPCR, FISH and IHC, we confirmed that IRF5 is expressed in both luminal and basal myoepithelial cells, but expression is higher in basal cells. Histologic analysis of whole mount preparations of Irf5-/- mammary glands revealed aberrant ductal morphogenesis, characterized by expansion of luminal and basal myoepithelial cells with a loss of organized glandular structure. RNAseq of primary mammary epithelial cells from wild type and Irf5-/- littermate mice showed Irf5-/- mammary epithelial cells to be enriched in ribosome biogenesis pathways, the physiologic consequences of which were demonstrated through increased rates of protein synthesis. Transferring our studies in vivo, we demonstrated that loss of tumor IRF5 expression resulted in decreased tumor-infiltrating lymphocytes and increased pulmonary metastasis in the murine orthotopic 4T1 implantation model. Mechanistically, we found that—as in our studies utilizing primary mammary epithelial cells—IRF5 expression in tumors regulated protein translation and ribosome biogenesis. In light of these findings, we propose IRF5 as a novel prognostic biomarker, loss of which alters mammary gland development, drives tumor initiation and metastasis, and dysregulates mammary epithelial cell protein synthesis. Citation Format: Betsy Barnes, Zarina Brune, Matthew Rice, Carter Somerville. Rediscovering IRF5: A prognostic indicator with novel roles in mammary gland development, ribosome biogenesis, tumor initiation, and metastasis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-15-01.

SummaryDespite the progress made in identifying cellular factors and mechanisms that predict progression and metastasis, breast cancer remains the second leading cause of death for women in the US. Using The Cancer Genome Atlas and mouse models of spontaneous and invasive mammary tumorigenesis, we identified that loss of function of interferon regulatory factor 5 (IRF5) is a predictor of metastasis and survival. Histologic analysis ofIrf5-/-mammary glands revealed expansion of luminal and myoepithelial cells, loss of organized glandular structure, and altered terminal end budding and migration. RNA-seq and ChIP-seq analyses of primary mammary epithelial cells fromIrf5+/+andIrf5-/-littermate mice revealed IRF5-mediated transcriptional regulation of proteins involved in ribosomal biogenesis. Using an invasive model of breast cancer lackingIrf5, we demonstrate that IRF5 re-expression inhibits tumor growth and metastasis via increased trafficking of tumor infiltrating lymphocytes and altered tumor cell protein synthesis. These findings uncover a new function for IRF5 in the regulation of mammary tumorigenesis and metastasis.HighlightsLoss of IRF5 is a predictor of metastasis and survival in breast cancer.IRF5 contributes to the regulation of ribosome biogenesis in mammary epithelial cells.Loss of IRF5 function in mammary epithelial cells leads to increased protein translation.

The mechanisms by which cells adapt to proteotoxic stress are largely unknown, but are key to understanding how tumor cells, particularly in vivo, are largely resistant to proteasome inhibitors. Analysis of cancer cell lines, mouse xenografts and patient-derived tumor samples all showed an association between mitochondrial metabolism and proteasome inhibitor sensitivity. When cells were forced to use oxidative phosphorylation rather than glycolysis, they became proteasome-inhibitor resistant. This mitochondrial state, however, creates a unique vulnerability: sensitivity to the small molecule compound elesclomol. Genome-wide CRISPR-Cas9 screening showed that a single gene, encoding the mitochondrial reductase FDX1, could rescue elesclomol-induced cell death. Enzymatic function and nuclear-magnetic-resonance-based analyses further showed that FDX1 is the direct target of elesclomol, which promotes a unique form of copper-dependent cell death. These studies explain a fundamental mechanism by which cells adapt to proteotoxic stress and suggest strategies to mitigate proteasome inhibitor resistance.

Background: Health care systems in the United States are continuously expanding and contracting spaces to treat patients with coronavirus disease 2019 (COVID-19) in intensive care units (ICUs). As a result, hospitals must effectively decontaminate and contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in constructed and deconstructed ICUs that care for patients with COVID-19. We assessed decontamination of a COVID-19 ICU and examined the containment efficacy of combined contact and droplet precautions in creating and maintaining a SARS-CoV-2–negative ICU “antechamber”. Methods: To examine the efficacy of chemical decontamination, we used high-density, semi-quantitative environmental sampling to detect SARS-CoV-2 on surfaces in a COVID-19 ICU and COVID-19 ICU antechamber. Quantitative real-time polymerase chain reaction was used to measure viral RNA on surfaces. Viral location mapping revealed the distribution of viral RNA in the COVID-19 ICU and COVID-19 ICU antechamber. Results were further assessed using loop-mediated isothermal amplification. Results: We collected 224 surface samples pre-decontamination and 193 samples post-decontamination from a COVID-19 ICU and adjoining COVID-19 ICU antechamber. We found that 46% of antechamber objects were positive for SARS-CoV-2 pre-decontamination despite the construction of a swinging door barrier system, implementation of contact precautions, and installation of high-efficiency particulate air filters. The object positivity rate reduced to 32.1% and viral particle rate reduced by 95.4% following decontamination. Matched items had an average of 432.2 ± 2729 viral copies/cm2 pre-decontamination and 19.2 ± 118 viral copies/cm2 post-decontamination, demonstrating significantly reduced viral surface distribution (p &lt, 0.0001). Conclusions: Environmental sampling is an effective method for evaluating decontamination protocols and validating measures used to contain SARS-CoV-2 viral particles. While chemical decontamination effectively removes detectable viral RNA from surfaces, our approach to droplet/contact containment with an antechamber was not highly effective. These data suggest that hospitals should plan for the potential of aerosolized virions when creating strategies to contain SARS-CoV-2.

Interferon Regulatory Factor 5 (IRF5) is one of nine members of the IRF family of transcription factors. Although initially discovered as a key regulator of the type I interferon and pro-inflammatory cytokine arm of the innate immune response, IRF5 has now been found to also mediate pathways involved in cell growth and differentiation, apoptosis, metabolic homeostasis and tumor suppression. Hyperactivation of IRF5 has been implicated in numerous autoimmune diseases, chief among them systemic lupus erythematosus (SLE). SLE is a heterogeneous autoimmune disease in which patients often share similar characteristics in terms of autoantibody production and strong genetic risk factors, yet also possess unique disease signatures. IRF5 pathogenic alleles contribute one of the strongest risk factors for SLE disease development. Multiple models of murine lupus have shown that loss of Irf5 is protective against disease development. In an attempt to elucidate the regulatory role(s) of IRF5 in driving SLE pathogenesis, labs have begun to examine the function of IRF5 in several immune cell types, including B cells, macrophages, and dendritic cells. A somewhat untouched area of research on IRF5 is in T cells, even though Irf5 knockout mice were reported to have skewing of T cell subsets from T helper 1 (Th1) and T helper 17 (Th17) toward T helper 2 (Th2), indicating a potential role for IRF5 in T cell regulation. However, most studies attributed this T cell phenotype in Irf5 knockout mice to dysregulation of antigen presenting cell function rather than an intrinsic role for IRF5 in T cells. In this review, we offer a different interpretation of the literature. The role of IRF5 in T cells, specifically its control of T cell effector polarization and the resultant T cell-mediated cytokine production, has yet to be elucidated. A strong understanding of the regulatory role(s) of this key transcription factor in T cells is necessary for us to grasp the full picture of the complex pathogenesis of autoimmune diseases like SLE.

Systemic Lupus Erythematosus (SLE) is a devastating autoimmune disease that results from failure of the immune system to distinguish “self” from “non-self”. Studies in our lab and others demonstrated that human SLE CD4+ T cells have elevated levels of IRF5 and increased metabolism, while Irf5 knockout murine CD4+ T cells show diminished utilization of oxidative phosphorylation and glycolysis, respectively. However, how IRF5 contributes to CD4+ T cell support of B cell function and dysfunction has not been fully elucidated. Here, using IRF5 KO C57BL/6J mice, we show that loss of IRF5 in CD4+ T cells directly contributes to defects in plasmablast generation. Examination of the CD40-CD40L interaction shows significant decreases in CD40L expression in Irf5 KO CD4+ T cells. Examination of Cd40lg transcript expression shows no difference in mRNA expression in Irf5 KO murine CD4+ T cells when compared to WT. This finding indicated a novel post-transcriptional regulatory role for IRF5. Examination of the canonical mTOR pathway, a key translational regulator, shows decreased phosphorylation of the S6 ribosomal protein upon loss of Irf5, indicating diminished activity of the mTOR pathway. mTOR inhibition with rapamycin in turn results in decreased CD40L expression, supporting a role for mTOR in CD40L regulation. As mTOR signaling is known to be regulated by cellular metabolism, we examined if loss of IRF5 results in altered T cell metabolism. We found that Irf5 KO T cells have decreased glucose metabolism and increased glutamine metabolism. This data has increased our understanding of how metabolism influences CD4+ T cell function and provided insight into novel post-translational regulatory roles for IRF5 via T cell metabolic regulation.

The transcription factor IRF5 modulates human B cell activation, proliferation, and plasmablast differentiation via an intrinsic mechanism. Targeted knockdown of IRF5 in human primary naïve B cells revealed a significant reduction in CD19+CD86+ B cells, which demarcate early activated B cells, after BCR/TLR9 stimulation, resulting in reduced clonal expansion and plasmablast differentiation. The mechanism(s) by which IRF5 modulates early B cell activation is not known. We previously found that BCR stimulation alone was insufficient to induce IRF5 nuclear translocation (activation), while BCR/TLR9 stimulation induced robust IRF5 activation. Thus, the focus of this study is to determine the mechanism(s) by which IRF5 functions downstream of BCR and dual BCR/TLR9 signaling at the early time points of activation (5 and 15 min). Experiments were performed in human B cells and B cells from wild-type and Irf5−/− mice. Signaling and protein interactions were examined by Western blot, immunoprecipitation, flow cytometry, and biochemical assay. We identified Bruton’s tyrosine kinase (Btk) as a novel interacting partner of IRF5 in both unstimulated and stimulated human and murine B cells. Of note, active phosphorylated Btk was bound to IRF5 only in stimulated cells. Preliminary data also suggest a defect downstream of Btk activation in Irf5−/− B cells. Together, these data indicate a conserved role for IRF5 in the early stages of B cell activation via BCR/TLR9 signaling.

Generation of antibody secreting cells (ASCs) requires multiple B cell activation pathways, including B cell receptor (BCR), T cell receptor (TCR), and Toll-like receptors (TLRs). In human naïve B cells, we reported that knockdown of interferon regulatory factor 5 (IRF5) resulted in IgD retention, reduced proliferation and plasma cell differentiation, and reduced IgG secretion. Defects were due to early impairments in B cell activation and clonal expansion. Conversely, murine Irf5 was reported to regulate antibody production through direct control of class switch recombination (CSR) at the γ2a locus. To further elucidate distinct and overlapping roles between human and murine IRF5 in the regulation of plasma cell differentiation and antibody production, we performed in vivo analysis of B and T cell differentiation and function in wild-type (wt) and Irf5 knockout (ko) littermate mice after immunization. B and T cell subsets were analyzed by multi-color flow cytometry. Naïve B cells were in vitro differentiated to ASCs with CpG-B, anti-IgM, anti-CD40 and IL-21. Kinetics of B cell activation, AID expression and IgG production were determined. BCR and TCR signaling were examined by phospho-flow. A combination of T-dependent/independent and TLR-dependent/independent immunizations were used to study ASC differentiation. RAG−/− mice were used to determine cell type-specific effects. Results indicate distinct roles for human and murine IRF5 at the early stages of BCR signaling and redundant roles at the later stages of CSR, plasma cell differentiation and antibody secretion via regulation of AID.

The heat shock protein 90 (Hsp90) chaperone functions as a protein-folding buffer and plays a unique role promoting the evolution of new heritable traits. To investigate the role of Hsp90 in modulating protein synthesis, we screened more than 1200 proteins involved in mRNA regulation for physical interactions with Hsp90 in human cells. Among the top hits was CPEB2, which strongly binds Hsp90 via its prion domain, reminiscent of the prion-like regulation of translation of Aplysia CPEB. In a yeast model of CPEB prion-dependent translation regulation, transient inhibition of Hsp90 amplified CPEB2 prion activity and resulted in persistent translation of the CPEB reporter. Remarkably, inhibition of Hsp90 was sufficient to induce a heritable change in protein translation that persisted for 30 generations, even in the absence of exogenous CPEB. Although we identified a variety of perturbations that enhanced translation of the reporter, only Hsp90 inhibition led to persistent activation. Thus, transient loss of Hsp90 function leads to the non-genetic inheritance of a novel translational state. We propose that, in addition to sculpting the conformational landscape of the proteome, Hsp90 promotes phenotypic variation by modulating protein synthesis.

SUMMARYThe mechanisms used by cancer cells to resist the severe disruption in protein homeostasis caused by proteasome inhibitors remain obscure. Here, we show this resistance correlates with a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS). Employing small molecule screens, we identified a striking overlap between compounds that preferentially impede the growth of proteasome inhibitor-resistant cancer cells and those that block the growth of high OXPHOS cells. Elesclomol potently exhibits both characteristics. Using genome-wide CRISPR/Cas9-based screening, in vitro validation and NMR spectroscopy we identify mitochondrial protein ferredoxin 1 (FDX1), a critical component of mitochondrial iron-sulfur (Fe-S) cluster biosynthesis, as the primary target of elesclomol. In a mouse model of multiple myeloma, inhibition of FDX1 with elesclomol significantly attenuated the emergence of proteasome inhibitor-resistance and markedly prolonged survival. Our work reveals that the mitochondrial Fe-S cluster pathway is a targetable vulnerability in cancers that are resistant to increased proteotoxic burden.

The mechanisms by which cells adapt to proteotoxic stress are largely unknown, but are key to understanding how tumor cells, particularly in vivo, are largely resistant to proteasome inhibitors. Analysis of cancer cell lines, mouse xenografts and patient-derived tumor samples all showed an association between mitochondrial metabolism and proteasome inhibitor sensitivity. When cells were forced to use oxidative phosphorylation rather than glycolysis, they became proteasome-inhibitor resistant. This mitochondrial state, however, creates a unique vulnerability: sensitivity to the small molecule compound elesclomol. Genome-wide CRISPR-Cas9 screening showed that a single gene, encoding the mitochondrial reductase FDX1, could rescue elesclomol-induced cell death. Enzymatic function and nuclear-magnetic-resonance-based analyses further showed that FDX1 is the direct target of elesclomol, which promotes a unique form of copper-dependent cell death. These studies explain a fundamental mechanism by which cells adapt to proteotoxic stress and suggest strategies to mitigate proteasome inhibitor resistance.

The use of proteasome inhibitors to target cancer's dependence on altered protein homeostasis has been greatly limited by intrinsic and acquired resistance. Analyzing data from thousands of cancer lines and tumors, we find that those with suppressed expression of one or more 19S proteasome subunits show intrinsic proteasome inhibitor resistance. Moreover, such proteasome subunit suppression is associated with poor outcome in myeloma patients, where proteasome inhibitors are a mainstay of treatment. Beyond conferring resistance to proteasome inhibitors, proteasome subunit suppression also serves as a sentinel of a more global remodeling of the transcriptome. This remodeling produces a distinct gene signature and new vulnerabilities to the proapoptotic drug, ABT-263. This frequent, naturally arising imbalance in 19S regulatory complex composition is achieved through a variety of mechanisms, including DNA methylation, and marks the emergence of a heritably altered and therapeutically relevant state in diverse cancers.