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Objective To investigate the role of PF-06650833, a highly potent and selective small-molecule inhibitor of interleukin-1-associated kinase 4 (IRAK4), in autoimmune pathophysiology in vitro, in vivo, and in the clinical setting. Methods Rheumatoid arthritis (RA) inflammatory pathophysiology was modeled in vitro through 1) stimulation of primary human macrophages with anti-citrullinated protein antibody immune complexes (ICs), 2) RA fibroblast-like synoviocyte (FLS) cultures stimulated with Toll-like receptor (TLR) ligands, as well as 3) additional human primary cell cocultures exposed to inflammatory stimuli. Systemic lupus erythematosus (SLE) pathophysiology was simulated in human neutrophils, dendritic cells, B cells, and peripheral blood mononuclear cells stimulated with TLR ligands and SLE patient ICs. PF-06650833 was evaluated in vivo in the rat collagen-induced arthritis (CIA) model and the mouse pristane-induced and MRL/lpr models of lupus. Finally, RNA sequencing data generated with whole blood samples from a phase I multiple-ascending-dose clinical trial of PF-06650833 were used to test in vivo human pharmacology. Results In vitro, PF-06650833 inhibited human primary cell inflammatory responses to physiologically relevant stimuli generated with RA and SLE patient plasma. In vivo, PF-06650833 reduced circulating autoantibody levels in the pristane-induced and MRL/lpr murine models of lupus and protected against CIA in rats. In a phase I clinical trial (NCT02485769), PF-06650833 demonstrated in vivo pharmacologic action pertinent to SLE by reducing whole blood interferon gene signature expression in healthy volunteers. Conclusion These data demonstrate that inhibition of IRAK4 kinase activity can reduce levels of inflammation markers in humans and provide confidence in the rationale for clinical development of IRAK4 inhibitors for rheumatologic indications.

The transcription factor IFN regulatory factor 5 (IRF5) is a central mediator of innate and adaptive immunity. Genetic variations within IRF5 are associated with a risk of systemic lupus erythematosus (SLE), and mice lacking Irf5 are protected from lupus onset and severity, but how IRF5 functions in the context of SLE disease progression remains unclear. Using the NZB/W F1 model of murine lupus, we show that murine IRF5 becomes hyperactivated before clinical onset. In patients with SLE, IRF5 hyperactivation correlated with dsDNA titers. To test whether IRF5 hyperactivation is a targetable function, we developed inhibitors that are cell permeable, nontoxic, and selectively bind to the inactive IRF5 monomer. Preclinical treatment of NZB/W F1 mice with an inhibitor attenuated lupus pathology by reducing serum antinuclear autoantibodies, dsDNA titers, and the number of circulating plasma cells, which alleviated kidney pathology and improved survival. Clinical treatment of MRL/lpr and pristane-induced lupus mice with an inhibitor led to significant reductions in dsDNA levels and improved survival. In ex vivo human studies, the inhibitor blocked SLE serum-induced IRF5 activation and reversed basal IRF5 hyperactivation in SLE immune cells. We believe this study provides the first in vivo clinical support for treating patients with SLE with an IRF5 inhibitor.

Genetic variations within interferon regulatory factor 5 (IRF5) associate with risk of systemic lupus erythematosus (SLE) and mice lacking Irf5 are protected from lupus onset and severity. Exactly how IRF5 functions in the context of SLE disease progression remains unclear. We sought to determine how IRF5 genetic risk contributes to SLE disease onset and severity, and whether targeting IRF5 with select inhibitors would alleviate disease severity and mortality. Studies were performed in blood from genotyped healthy donors, SLE patients and mouse models of murine lupus. In NZB/W F1 lupus mice, we found that Irf5 is already hyper-activated before clinical onset in a cell type-specific manner. In healthy donors carrying IRF5 genetic risk, we detected IRF5 hyper-activation in the myeloid compartment that drives a pre-symptomatic SLE immune-phenotype. In SLE patients, IRF5 hyper-activation correlates with SLEDAI and dsDNA titers. To test whether IRF5 hyper-activation is a targetable function, we developed novel inhibitors that are cell permeable, non-toxic and selectively bind to the inactive IRF5 monomer. Treatment of NZB/W F1 and MRL/lpr mice with inhibitor attenuated lupus pathology by reducing serum ANA and dsDNA titers. In NZB/W F1 mice, we detected significant reductions in the number of circulating plasma cells and age- or autoimmune-associated B cells (ABCs), which alleviated kidney pathology and improved overall survival. In ex vivo human studies, the inhibitor blocked SLE serum-induced IRF5 activation in healthy immune cells and reversed basal IRF5 hyper-activation in SLE immune cells. This study provides the first in vivo pre-clinical support for treating SLE patients with an IRF5 inhibitor.

Interleukin-1 receptor (IL1R)-associated kinase 4 (IRAK4) is a central regulator of innate immune signaling, controlling IL1R and Toll-like receptor (TLR)-mediated responses and containing both scaffolding and kinase activities. Humans deficient in IRAK4 activity have autosomal recessive primary immune deficiency (PID). Here, we characterized the molecular mechanism of dysfunction of two IRAK4 PID variants, G298D and the compound variant R12C (R12C/R391H/T458I). Using these variants and the kinase-inactive D329A variant to delineate the contributions of IRAK4's scaffolding and kinase activities to IL1R signaling, we found that the G298D variant is kinase-inactive and expressed at extremely low levels, acting functionally as a null mutation. The R12C compound variant possessed WT kinase activity, but could not interact with myeloid differentiation primary response 88 (MyD88) and IRAK1, causing impairment of IL-1–induced signaling and cytokine production. Quantitation of IL-1 signaling in IRAK4-deficient cells complemented with either WT or the R12C or D329A variant indicated that the loss of MyD88 interaction had a greater impact on IL-1–induced signaling and cytokine expression than the loss of IRAK4 kinase activity. Importantly, kinase-inactive IRAK4 exhibited a greater association with MyD88 and a weaker association with IRAK1 in IRAK4-deficient cells expressing kinase-inactive IRAK4 and in primary cells treated with a selective IRAK4 inhibitor. Loss of IRAK4 kinase activity only partially inhibited IL-1–induced cytokine and NF-κB signaling. Therefore, the IRAK4–MyD88 scaffolding function is essential for IL-1 signaling, but IRAK4 kinase activity can control IL-1 signal strength by modulating the association of IRAK4, MyD88, and IRAK1.

Clinical studies using prognostic and predictive signatures have shown that an immune signal emanating from whole tumors reflects the level of immune cell infiltration--a high immune signal linked to improved outcome. Factors regulating immune cell trafficking to the tumor, however, are not known. Previous work has shown that expression of interferon regulatory factor 5 (IRF5), a critical immune regulator, is lost in ~80% of invasive ductal carcinomas examined. We postulated that IRF5-positive and -negative breast tumors would differentially regulate immune cell trafficking to the tumor. Using a focused tumor inflammatory array, differences in cytokine and chemokine expression were examined between IRF5-positive and -negative MDA-MB-231 cells grown in three-dimensional culture. A number of cytokines/chemokines were found to be dysregulated between cultures. CXCL13 was identified as a direct target of IRF5 resulting in the enhanced recruitment of B and T cells to IRF5-positive tumor-conditioned media. The ability of IRF5 to regulate mediators of cell migration was confirmed by enzyme-linked immunosorbent assay, chromatin immunoprecipitation assay, small interfering RNA knockdown and immunofluorescence staining of human breast tumor tissues. Analysis of primary immune cell subsets revealed that IRF5 specifically recruits CXCR5(+) B and T cells to the tumor; CXCR5 is the receptor for CXCL13. Analysis of primary breast tumor tissues revealed a significant correlation between IRF5 and CXCL13 expression providing clinical relevance to the study. Together, these data support that IRF5 directly regulates a network of genes that shapes a tumor immune response and may, in combination with CXCL13, serve as a novel prognostic marker for antitumor immunity.

In the present work, we find a set of reliability functionals to fix up an allocation strategy among K(≥2) treatments when the response distributions, conditionally dependent on some continuous prognostic variable, are exponential with unknown linear regression functions as the means of the respective conditional distributions. Targeting such reliability functionals, we propose a covariate-adjusted response-adaptive randomization procedure for the multi-treatment single-period clinical trial under the Koziol–Green model for informative censoring. We compare the proposed procedure with its competitive covariate-eliminated procedure.

Interferon regulatory factor 5 (IRF5) is a member of the IRF family of transcription factors. IRF5 was first identified and characterized as a transcriptional regulator of type I interferon expression after virus infection. In addition to its critical role(s) in the regulation and development of host immunity, subsequent studies revealed important roles for IRF5 in autoimmunity, cancer, obesity, pain, cardiovascular disease and metabolism. Based on these important disease-related findings, a large number of commercial antibodies have become available to study the expression and function of IRF5. Here we validate a number of these antibodies for the detection of IRF5 by immunoblot, flow cytometry and immunofluorescence or immunohistochemistry using well-established positive and negative controls. Somewhat surprising, the majority of commercial antibodies tested were unable to specifically recognize human or mouse IRF5. We present data on antibodies that do specifically recognize human or mouse IRF5 in a particular application. These findings reiterate the importance of proper controls and molecular weight standards for the analysis of protein expression. Given that dysregulated IRF5 expression has been implicated in the pathogenesis of numerous diseases, including autoimmune and cancer, results indicate that caution should be used in the evaluation and interpretation of IRF5 expression analysis.

The use of ridit, as a probability score, is a very common practice to compare discrete random variables in discrete data analysis. In the present work we formulate ridit reliability functionals for some comparison of K independent binary random variables. We use such functionals to provide a generalized response-adaptive design (GRAD) on K(≥ +2) treatment-arms for dichotomous response variables. We exhibit some properties of the proposed design and compare it with some of the existing competitors by computing its various performance measures. We also provide a discussion towards a possible modification of the GRAD in the presence of covariates.

Upon recognition of antigen, B cells undergo rapid proliferation followed by differentiation to specialized antibody secreting cells (ASCs). Increased levels of ASCs are seen in autoimmune diseases, such as systemic lupus erythematosus (SLE), and some B cell-associated malignancies. Studies suggest that altered transcription factor (TF) expression and/or activation play a role in the imbalance of B cell subsets in these diseases. Interferon regulatory factor 5 (IRF5) is one such TF, as polymorphisms in IRF5 associate with risk of numerous autoimmune diseases and correlate with elevated IRF5 expression in SLE patients. Recent findings from IRF5 knockdown (KD) in purified naïve B cells identified IRF5 as an early intrinsic regulator of B cell activation, proliferation and plasma cell differentiation in response to TLR9 activation. In this study, IRF4 was identified as a new IRF5 target gene by ChIP-Seq and RNA-Seq analysis. IRF4 expression is high in ASCs and murine studies show that it plays an essential, cell-intrinsic role in the generation of germinal center B cells. To date, little is known of IRF4 function in human ASC differentiation or whether these two factors (IRF4 and 5) cooperate. Activation of TLR9 and B cell receptor (BCR) revealed distinct kinetic changes in IRF4 and IRF5 during the early transition of naïve B cells to ASCs. Moreover, IRF4 KD of human primary naïve B cells resulted in significant IgD retention and reduced plasmablast (PB) differentiation without loss of early B cell activation (CD86) or key regulatory factors of class switch recombination in response to TLR9/BCR stimulation. Together, these results show that IRF4 and IRF5 are kinetically distinct TFs that have non-redundant roles in human ASC differentiation.

The transcription factor interferon regulatory factor 5 (IRF5) is a central mediator of innate and adaptive immunity. Genetic variations within IRF5 associate with risk for systemic lupus erythematosus (SLE) and mice lacking Irf5 are protected from lupus onset and severity. Here we show that IRF5 genetic variants confer risk by increasing basal IRF5 activation in plasmacytoid dendritic cells resulting in elevated IFNα production. IRF5 hyperactivation in SLE immune cells correlates with SLEDAI and dsDNA titers. Upon characterization of immune cells from NZB/W F1 lupus-prone mice, we found that Irf5 was hyperactivated at 19 weeks-old, which precedes clinical onset. To test whether IRF5 hyperactivation is a driver of SLE, we developed novel therapeutics targeting IRF5 activation. Inhibitors are cell permeable, non-toxic and selectively bind to full-length inactive IRF5 monomer. In vivo analysis revealed functional mimicry with Irf5 knockout mice and treatment of NZB/W F1 mice for a two-week period prior to clinical onset gave significantly reduced dsDNA titers, anti-nuclear antibody (ANA) staining, and improved survival. The IRF5 inhibitor also blocked ex vivo SLE serum-induced IRF5 activation and reversed IRF5 hyperactivation detected in SLE immune cells. Results implicate IRF5 hyperactivation as a risk factor that drives SLE and provide the first pre-clinical support for treating SLE patients or those at risk of SLE with IRF5 inhibitors.

In the present work we use ridit, a measure comparing two discrete random variables, to develop a two-treatment response adaptive allocation technique in the presence of a stochastic prognostic factor. Various exact and asymptotic performance measures of the proposed technique are obtained and are compared with that of the existing techniques.

In recent years adaptive designs have been becoming popular in the context of clinical trials. The purpose of the present work is to provide a sequential two-treatment allocation rule for when the response variables are continuous. The rule is ethical as well as sometimes optimal depending upon the nature of the distribution of the study variables. We examine the various properties of the rule.

B cells represent a critical arm of the adaptive immune system, and under normal circumstances, provide long lasting immunity to a wide range of pathogens. To achieve this, B cells differentiate into specialized subpopulations, which perform unique functions in response to antigen. A complex network of transcription factors regulates this transition as well as subsequent effector functions. Dysregulation of these transcription factors can lead to altered B cell biology and potentially autoimmunity. Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by increased autoantibody production and B cell hyper-reactivity. It still remains unclear, however, what factors contribute to disease pathogenesis. Recent work has suggested dysregulation of B cell transcription factors may play a role in disease onset. Given the prominent role B cells play in the pathogenesis of SLE, it is important to have a full understanding of what transcription factors regulate B cell development, and how dysregulation of some of these transcription factors may contribute to the occurrence and/or pathogenesis of SLE. A comprehensive review of where this field currently stands, what new potential targets have recently been identified, and what functional information is still missing will be discussed.

The DNA damage response comprises DNA repair, cell-cycle checkpoint control, and DNA damage-induced apoptosis that collectively promote genomic integrity and suppress tumorigenesis. Previously, we have shown that the Chk2 kinase functions independently of the Mre11 complex (Mre11, Rad50, and Nbs1) and ATM in apoptosis and suppresses tumorigenesis resulting from hypomorphic alleles of Mre11 or Nbs1 . Based on this work, we have proposed that Chk2 limits the oncogenic potential of replication-associated DNA damage. Here we further address the role of Chk2 and damage-induced apoptosis in suppressing the oncogenic potential of chromosome breaks. We show that loss of Chk2 or a mutation in p53 (R172P), which selectively impairs its function in apoptosis, rescued the lethality of mice lacking Lig4, a ligase required for nonhomologous end-joining (NHEJ) repair of DNA double-strand breaks in G0/G1. In contrast to Lig4 −/− p53 −/− mice, Lig4 −/− Chk2 −/− and Lig4 −/− p53 R172P/R172P mice were not prone to organ-specific, rapid tumorigenesis. Although the severe NHEJ deficiency of Lig4 −/− was a less potent initiator of tumorigenesis in the p53 R172P/R172P and Chk2 −/− backgrounds, where p53 cell-cycle functions are largely intact, even mild defects in the intra-S and G2/M checkpoints caused by mutations in Nbs1 are sufficient to influence malignancy in p53 R172P/R172P mice. We conclude that the oncogenic potential of double-strand breaks resulting from NHEJ deficiency is highly restricted by nonapoptotic functions of p53, such as the G1/S checkpoint or senescence, suggesting that the particular facets of the DNA damage response required for tumor suppression are dictated by the proliferative status of the tumor-initiating cell.

The cyclin-dependent kinases (CDKs) that promote cell-cycle progression are targets for negative regulation by signals from damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face of DNA damage implies that there are mechanisms to insulate certain CDKs from checkpoint inhibition. It remains difficult, however, to assign precise functions to specific CDKs in protecting genomic integrity. In mammals, Cdk2 is active throughout S and G2 phases, but Cdk2 protein is dispensable for survival, owing to compensation by other CDKs. That plasticity obscured a requirement for Cdk2 activity in proliferation of human cells, which we uncovered by replacement of wild-type Cdk2 with a mutant version sensitized to inhibition by bulky adenine analogs. Here we show that transient, selective inhibition of analog-sensitive (AS) Cdk2 after exposure to ionizing radiation (IR) enhances cell-killing. In extracts supplemented with an ATP analog used preferentially by AS kinases, Cdk2as phosphorylated the Nijmegen Breakage Syndrome gene product Nbs1—a component of the conserved Mre11-Rad50-Nbs1 complex required for normal DNA damage repair and checkpoint signaling—dependent on a consensus CDK recognition site at Ser432. In vivo, selective inhibition of Cdk2 delayed and diminished Nbs1-Ser432 phosphorylation during S phase, and mutation of Ser432 to Ala or Asp increased IR–sensitivity. Therefore, by chemical genetics, we uncovered both a non-redundant requirement for Cdk2 activity in response to DNA damage and a specific target of Cdk2 within the DNA repair machinery., Author Summary Multiple cyclin-dependent kinases (CDKs) control human cell proliferation, but it remains unclear how functions of different CDKs are coordinated during unperturbed cell division or after dividing cells incur DNA damage. DNA lesions activate checkpoint signaling pathways to inhibit CDK activity, arrest the cell division cycle, and thus prevent loss of genetic information; but an effective response to damage also requires CDK activity to modify components of repair and checkpoint pathways. We took a chemical-genetic approach to ask if a specific CDK, Cdk2, played a specialized, non-redundant role in protecting genomic integrity of human cells. By sensitizing Cdk2 to chemical inhibition, we were able to detect a specific requirement for its catalytic activity in survival of cells after exposure to ionizing radiation (IR). We identified Nbs1, product of the gene mutated in the cancer-predisposing Nijmegen Breakage Syndrome, as a Cdk2 substrate and showed that mutant forms of Nbs1 that cannot be modified by Cdk2 are defective in protecting cells from death due to IR–induced DNA damage. Therefore, our work defines a DNA damage response pathway that depends on catalytic activity of a specific CDK in human cells and suggests a mechanism to promote efficient repair without triggering inappropriate cell division.

The majority of spontaneous chromosome breakage occurs during the process of DNA replication. Homologous recombination is the primary mechanism of repair of such damage, which probably accounts for the fact that it is essential for genome integrity and viability in mammalian cells. The Mre11 complex plays diverse roles in the maintenance of genomic integrity, influencing homologous recombination, checkpoint activation, and telomere maintenance. The complex is essential for cellular viability, but given its myriad influences on genomic integrity, the mechanistic basis for the nonviability of Mre11 complex-deficient cells has not been defined. In this study we generated mice carrying a conditional allele of Rad50 and examined the effects of Rad50 deficiency in proliferative and nonproliferative settings. Depletion of Rad50 in cultured cells caused extensive DNA damage and death within 3 to 5 days of Rad50 deletion. This was not associated with gross telomere dysfunction, suggesting that the telomeric functions of the Mre11 complex are not required for viability. Rad50 was also dispensable for the viability of quiescent liver and postmitotic Purkinje cells of the cerebellum. These findings support the idea that the essential functions of the Mre11 complex are associated with DNA replication and further suggest that homologous recombination is not essential in nondividing cells.

Clinical studies using predictive signatures have shown that a high immune signal from whole tumor expression profiles correlate with improved outcome in some cancers, including breast cancer. Factors regulating immune cell trafficking to the tumor, however, are not known. Utilizing MDA-MB-231 breast cancer cells grown in 3D culture, we examined whether intratumoral expression of interferon regulatory factor 5 (IRF5) regulates the profile of cytokines/chemokines expressed. Previous work has shown that IRF5 expression is lost in ~80% of all invasive ductal carcinomas examined. Using focused arrays, we found a number of cytokines/chemokines that were dysregulated between IRF5-positive and -negative MDA-MB-231 cells. CXCL13 was identified as a direct target of IRF5. Using tumor-conditioned media (TCM) from IRF5-positive and -negative cells, we found a significant increase in B and T cell trafficking to TCM from IRF5-positive cells. Subset analysis revealed that primary CXCR5+ B and T cells showed significantly reduced migration to IRF5-negative TCM or IRF5-positive TCM depleted of CXCL13. CXCR5 is the primary receptor for CXCL13. Analysis of human breast tumor tissues revealed a positive correlation between IRF5 and CXCL13 expression providing clinical relevance to this study. Data support that IRF5 directly regulates a network of genes that shapes a tumor immune response and may serve as a novel prognostic marker for chemotherapy and immunotherapy response.