RUNX1 C-terminal mutations impair blood cell differentiation by perturbing specific enhancer-promoter networks.

ABSTRACT: The transcription factor RUNX1 is a master regulator of hematopoiesis and is frequently mutated in myeloid malignancies. Mutations in its runt homology domain (RHD) frequently disrupt DNA binding and result in loss of RUNX1 function. However, it is not clearly understood how other RUNX1 mutations contribute to disease development. Here, we characterized RUNX1 mutations outside of the RHD. Our analysis of the patient data sets revealed that mutations within the C-terminus frequently occur in hematopoietic disorders. Remarkably, most of these mutations were nonsense or frameshift mutations and were predicted to be exempt from nonsense-mediated messenger RNA decay. Therefore, this class of mutation is projected to produce DNA-binding proteins that contribute to the pathogenesis in a distinct manner. To model this, we introduced the RUNX1R320∗ mutation into the endogenous gene locus and demonstrated the production of RUNX1R320∗ protein. Expression of RUNX1R320∗ resulted in the disruption of RUNX1 regulated processes such as megakaryocytic differentiation, through a transcriptional signature different from RUNX1 depletion. To understand the underlying mechanisms, we used Global RNA Interactions with DNA by deep sequencing (GRID-seq) to examine enhancer-promoter connections. We identified widespread alterations in the enhancer-promoter networks within RUNX1 mutant cells. Additionally, we uncovered enrichment of RUNX1R320∗ and FOXK2 binding at the MYC super enhancer locus, significantly upregulating MYC transcription and signaling pathways. Together, our study demonstrated that most RUNX1 mutations outside the DNA-binding domain are not subject to nonsense-mediated decay, producing protein products that act in concert with additional cofactors to dysregulate hematopoiesis through mechanisms distinct from those induced by RUNX1 depletion.

Reprogramming of tumor-associated macrophages via NEDD4-mediated CSF1R degradation by targeting USP18.

Tumor-associated myeloid cells modulate the tumor microenvironment and affect tumor progression. Type I interferon (IFN-I) has multiple effects on tumors and immune response, and ubiquitin-specific peptidase 18 (USP18) functions as a negative regulator of IFN-I signal transduction. This study aims to examine the function of IFN-I in myeloid cells during tumor progression. Here, we show that deletion of USP18 in myeloid cells suppresses tumor progression. Enhanced IFN-I signaling and blocked USP18 expression prompt downregulation of colony stimulating factor 1 receptor (CSF1R) and polarization of tumor-associated macrophages toward pro-inflammatory phenotypes. Further in vitro experiments reveal that downregulation of CSF1R is mediated by ubiquitin-proteasome degradation via E3 ligase neural precursor cell-expressed, developmentaly downregulated 4 (NEDD4) and the IFN-induced increase in ubiquitin E2 ubiquitin-conjugating enzyme H5. USP18 impairs ubiquitination and subsequent degradation of CSF1R by interrupting NEDD4 binding to CSF1R. These results reveal a previously unappreciated role of IFN-I in macrophage polarization by regulating CSF1R via USP18 and suggest targeting USP18 in myeloid-lineage cells as an effective strategy for IFN-based therapies.

Expansion of interferon inducible gene pool via USP18 inhibition promotes cancer cell pyroptosis.

While immunotherapy has emerged as a breakthrough cancer therapy, it is only effective in some patients, indicating the need of alternative therapeutic strategies. Induction of cancer immunogenic cell death (ICD) is one promising way to elicit potent adaptive immune responses against tumor-associated antigens. Type I interferon (IFN) is well known to play important roles in different aspects of immune responses, including modulating ICD in anti-tumor action. However, how to expand IFN effect in promoting ICD responses has not been addressed. Here we show that depletion of ubiquitin specific protease 18 (USP18), a negative regulator of IFN signaling, selectively induces cancer cell ICD. Lower USP18 expression correlates with better survival across human selected cancer types and delays cancer progression in mouse models. Mechanistically, nuclear USP18 controls the enhancer landscape of cancer cells and diminishes STAT2-mediated transcription complex binding to IFN-responsive elements. Consequently, USP18 suppression not only enhances expression of canonical IFN-stimulated genes (ISGs), but also activates the expression of a set of atypical ISGs and NF-κB target genes, including genes such as Polo like kinase 2 (PLK2), that induce cancer pyroptosis. These findings may support the use of targeting USP18 as a potential cancer immunotherapy.

Cell-cycle-gated feedback control mediates desensitization to interferon stimulation.

Cells use molecular circuits to interpret and respond to extracellular cues, such as hormones and cytokines, which are often released in a temporally varying fashion. In this study, we combine microfluidics, time-lapse microscopy, and computational modeling to investigate how the type I interferon (IFN)-responsive regulatory network operates in single human cells to process repetitive IFN stimulation. We found that IFN-α pretreatments lead to opposite effects, priming versus desensitization, depending on input durations. These effects are governed by a regulatory network composed of a fast-acting positive feedback loop and a delayed negative feedback loop, mediated by upregulation of ubiquitin-specific peptidase 18 (USP18). We further revealed that USP18 upregulation can only be initiated at the G1/early S phases of cell cycle upon the treatment onset, resulting in heterogeneous and delayed induction kinetics in single cells. This cell cycle gating provides a temporal compartmentalization of feedback loops, enabling duration-dependent desensitization to repetitive stimulations.

The RUNX1-ETO target gene RASSF2 suppresses t(8;21) AML development and regulates Rac GTPase signaling.

Large-scale chromosomal translocations are frequent oncogenic drivers in acute myeloid leukemia (AML). These translocations often occur in critical transcriptional/epigenetic regulators and contribute to malignant cell growth through alteration of normal gene expression. Despite this knowledge, the specific gene expression alterations that contribute to the development of leukemia remain incompletely understood. Here, through characterization of transcriptional regulation by the RUNX1-ETO fusion protein, we have identified Ras-association domain family member 2 (RASSF2) as a critical gene that is aberrantly transcriptionally repressed in t(8;21)-associated AML. Re-expression of RASSF2 specifically inhibits t(8;21) AML development in multiple models. Through biochemical and functional studies, we demonstrate RASSF2-mediated functions to be dependent on interaction with Hippo kinases, MST1 and MST2, but independent of canonical Hippo pathway signaling. Using proximity-based biotin labeling we define the RASSF2-proximal proteome in leukemia cells and reveal association with Rac GTPase-related proteins, including an interaction with the guanine nucleotide exchange factor, DOCK2. Importantly, RASSF2 knockdown impairs Rac GTPase activation, and RASSF2 expression is broadly correlated with Rac-mediated signal transduction in AML patients. Together, these data reveal a previously unappreciated mechanistic link between RASSF2, Hippo kinases, and Rac activity with potentially broad functional consequences in leukemia.

Type I Interferon Regulates a Coordinated Gene Network to Enhance Cytotoxic T Cell-Mediated Tumor Killing.

Type I interferons (IFN), which activate many IFN-stimulated genes (ISG), are known to regulate tumorigenesis. However, little is known regarding how various ISGs coordinate with one another in developing antitumor effects. Here, we report that the ISG UBA7 is a tumor suppressor in breast cancer. UBA7 encodes an enzyme that catalyzes the covalent conjugation of the ubiquitin-like protein product of another ISG (ISG15) to cellular proteins in a process known as "ISGylation." ISGylation of other ISGs, including STAT1 and STAT2, synergistically facilitates production of chemokine-receptor ligands to attract cytotoxic T cells. These gene-activation events are further linked to clustering and nuclear relocalization of STAT1/2 within IFN-induced promyelocytic leukemia (PML) bodies. Importantly, this coordinated ISG-ISGylation network plays a central role in suppressing murine breast cancer growth and metastasis, which parallels improved survival in patients with breast cancer. These findings reveal a cooperative IFN-inducible gene network in orchestrating a tumor-suppressive microenvironment. SIGNIFICANCE: We report a highly cooperative ISG network, in which UBA7-mediated ISGylation facilitates clustering of transcription factors and activates an antitumor gene-expression program. These findings provide mechanistic insights into immune evasion in breast cancer associated with UBA7 loss, emphasizing the importance of a functional ISG-ISGylation network in tumor suppression.This article is highlighted in the In This Issue feature, p. 327.

Hippo kinase loss contributes to del(20q) hematologic malignancies through chronic innate immune activation.

Heterozygous deletions within chromosome 20q, or del(20q), are frequent cytogenetic abnormalities detected in hematologic malignancies. To date, identification of genes in the del(20q) common deleted region that contribute to disease development have remained elusive. Through assessment of patient gene expression, we have identified STK4 (encoding Hippo kinase MST1) as a 20q gene that is downregulated below haploinsufficient amounts in myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN). Hematopoietic-specific gene inactivation in mice revealed Hippo kinase loss to induce splenomegaly, thrombocytopenia, megakaryocytic dysplasia, and a propensity for chronic granulocytosis; phenotypes that closely resemble those observed in patients harboring del(20q). In a JAK2-V617F model, heterozygous Hippo kinase inactivation led to accelerated development of lethal myelofibrosis, recapitulating adverse MPN disease progression and revealing a novel genetic interaction between these 2 molecular events. Quantitative serum protein profiling showed that myelofibrotic transformation in mice was associated with cooperative effects of JAK2-V617F and Hippo kinase inactivation on innate immune-associated proinflammatory cytokine production, including IL-1ß and IL-6. Mechanistically, MST1 interacted with IRAK1, and shRNA-mediated knockdown was sufficient to increase IRAK1-dependent innate immune activation of NF-κB in human myeloid cells. Consistent with this, treatment with a small molecule IRAK1/4 inhibitor rescued the aberrantly elevated IL-1ß production in the JAK2-V617F MPN model. This study identified Hippo kinase MST1 (STK4) as having a central role in the biology of del(20q)-associated hematologic malignancies and revealed a novel molecular basis of adverse MPN progression that may be therapeutically exploitable via IRAK1 inhibition.

The probacterial effect of type I interferon signaling requires its own negative regulator USP18.

Type I interferon (IFN-I) signaling paradoxically impairs host immune responses during many primary and secondary bacterial infections. Lack of IFN-I receptor reduces bacterial replication and/or bacterial persistence during infection with several bacteria. However, the mechanisms that mediate the adverse IFN-I effect are incompletely understood. Here, we show that Usp18, an interferon-stimulated gene that negatively regulates IFN-I signaling, is primarily responsible for the deleterious effect of IFN-I signaling during infection of mice with Listeria monocytogenes or Staphylococcus aureus Mechanistically, USP18 promoted bacterial replication by inhibiting antibacterial tumor necrosis factor-α (TNF-α) signaling. Deleting IFNAR1 or USP18 in CD11c-Cre+ cells similarly reduced bacterial titers in multiple organs and enhanced survival. Our results demonstrate that inhibiting USP18 function can promote control of primary and secondary bacterial infection by enhancing the antibacterial effect of TNF-α, which correlates with induction of reactive oxygen species (ROS). These findings suggest that USP18 could be targeted therapeutically in patients to ameliorate disease caused by serious bacterial infections.

Identification of Compounds That Prolong Type I Interferon Signaling as Potential Vaccine Adjuvants.

Vaccines are reliant on adjuvants to enhance the immune stimulus, and type I interferons (IFNs) have been shown to be beneficial in augmenting this response. We were interested in identifying compounds that would sustain activation of an endogenous type I IFN response as a co-adjuvant. We began with generation of a human monocytic THP-1 cell line with an IFN-stimulated response element (ISRE)-ß-lactamase reporter construct for high-throughput screening. Pilot studies were performed to optimize the parameters and conditions for this cell-based Förster resonance energy transfer (FRET) reporter assay for sustaining an IFN-α-induced ISRE activation signal. These conditions were confirmed in an initial pilot screen, followed by the main screen for evaluating prolongation of an IFN-α-induced ISRE activation signal at 16 h. Hit compounds were identified using a structure enrichment strategy based on chemoinformatic clustering and a naïve "Top X" approach. A select list of confirmed hits was then evaluated for toxicity and the ability to sustain IFN activity by gene and protein expression. Finally, for proof of concept, a panel of compounds was used to immunize mice as co-adjuvant with a model antigen and an IFN-inducing Toll-like receptor 4 agonist, lipopolysaccharide, as an adjuvant. Selected compounds significantly augmented antigen-specific immunoglobulin responses.

Negative regulation of type I IFN signaling.

Type I IFNs (α, ß, and others) are a family of cytokines that are produced in physiological conditions as well as in response to the activation of pattern recognition receptors. They are critically important in controlling the host innate and adaptive immune response to viral and some bacterial infections, cancer, and other inflammatory stimuli. However, dysregulation of type I IFN production or response can contribute to immune pathologies termed "interferonopathies", pointing to the importance of balanced activating signals with tightly regulated mechanisms of tuning this signaling. Here, we summarize the recent advances of how type I IFN production and response are controlled at multiple levels of the type I IFN signaling cascade.

STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling.

Type I interferons (IFNs) are multifunctional cytokines that regulate immune responses and cellular functions but also can have detrimental effects on human health. A tight regulatory network therefore controls IFN signaling, which in turn may interfere with medical interventions. The JAK-STAT signaling pathway transmits the IFN extracellular signal to the nucleus, thus resulting in alterations in gene expression. STAT2 is a well-known essential and specific positive effector of type I IFN signaling. Here, we report that STAT2 is also a previously unrecognized, crucial component of the USP18-mediated negative-feedback control in both human and mouse cells. We found that STAT2 recruits USP18 to the type I IFN receptor subunit IFNAR2 via its constitutive membrane-distal STAT2-binding site. This mechanistic coupling of effector and negative-feedback functions of STAT2 may provide novel strategies for treatment of IFN-signaling-related human diseases.

Elevated Response to Type I IFN Enhances RANKL-Mediated Osteoclastogenesis in Usp18-Knockout Mice.

A balance between bone formation and bone resorption is critical for the maintenance of bone mass. In many pathological conditions, including chronic inflammation, uncontrolled activation of osteoclast differentiation often causes excessive bone resorption that results in osteoporosis. In this study, we identified the osteopenia phenotype of mice lacking Usp18 (also called Ubp43), which is a deISGylating enzyme and is known as a negative regulator of type I IFN signaling. The expression of Usp18 was induced in preosteoclasts upon receptor activator of NF-κB ligand (RANKL) treatment. In an in vitro osteoclast-differentiation assay, bone marrow macrophages from Usp18-deficient mice exhibited an enhanced differentiation to multinucleated cells, elevated activation of NFATc1, and an increased expression of osteoclast marker genes upon RANKL treatment. Furthermore, in vitro quantification of bone resorption revealed a great increase in osteoclastic activities in Usp18-deficient cells. Interestingly, proinflammatory cytokine genes, such as IP-10 (CXCL10), were highly expressed in Usp18-deficient bone marrow macrophages upon RANKL treatment compared with wild-type cells. In addition, serum cytokine levels, especially IP-10, were significantly high in Usp18-knockout mice. In sum, we suggest that, although type I IFN is known to restrict osteoclast differentiation, the exaggerated activation of the type I IFN response in Usp18-knockout mice causes an osteopenia phenotype in mice.

Type I IFN induces protein ISGylation to enhance cytokine expression and augments colonic inflammation.

Type I IFNs have broad activity in tissue inflammation and malignant progression that depends on the expression of IFN-stimulated genes (ISGs). ISG15, one such ISG, can form covalent conjugates to many cellular proteins, a process termed "protein ISGylation." Although type I IFNs are involved in multiple inflammatory disorders, the role of protein ISGylation during inflammation has not been evaluated. Here we report that protein ISGylation exacerbates intestinal inflammation and colitis-associated colon cancer in mice. Mechanistically, we demonstrate that protein ISGylation negatively regulates the ubiquitin-proteasome system, leading to increased production of IFN-induced reactive oxygen species (ROS). The increased cellular ROS then enhances LPS-induced activation of p38 MAP kinase and the expression of inflammation-related cytokines in macrophages. Thus our studies reveal a regulatory role for protein ISGylation in colonic inflammation and its related malignant progression, indicating that targeting ubiquitin-activating enzyme E1 homolog has therapeutic potential in treating inflammatory diseases.

Identification and characterization of a novel ISG15-ubiquitin mixed chain and its role in regulating protein homeostasis.

As a ubiquitin-like modifier, ISG15 is conjugated to many cellular proteins in a process termed protein ISGylation. However, the crosstalk between protein ISGylation and the ubiquitin proteasome system is not fully understood. Here, we report that cellular ubiquitin is a substrate of ISG15 and Lys 29 on ubiquitin is the major ISG15 acceptor site. Using a model substrate, we demonstrate that ISG15 can modify ubiquitin, which is immobilized on its substrate, to form ISG15-ubiquitin mixed chains. Furthermore, our results indicate that ISG15-ubiquitin mixed chains do not serve as degradation signals for a ubiquitin fusion degradation substrate. Accordingly, an ISG15-ubiquitin fusion protein, which mimics an ISG15-ubiquitin mixed chain, negatively regulates cellular turnover of ubiquitylated proteins. In addition, ISG15-ubiquitin mixed chains, which are detectable on endogenously ubiquitylated proteins, dampen cellular turnover of these proteins. Thus, our studies unveil an unanticipated interplay between two protein modification systems and highlight its role in coordinating protein homeostasis.

Murine Herc6 Plays a Critical Role in Protein ISGylation In Vivo and Has an ISGylation-Independent Function in Seminal Vesicles.

ISG15 conjugation (ISGylation) to proteins is a multistep process involving interferon (IFN)-inducible UBE1L (E1), UbcH8 (E2), and ISG15 E3 ligases (E3s). Studies performed over the past several years have shown that ISGylation plays a pivotal role in the host antiviral response against certain viruses. Recent in vitro studies revealed that human Herc5 and mouse Herc6 are major ISG15 E3 ligases, respectively. However, the global function of Herc5/6 proteins in vivo still remains unclear. Here, we report generation and initial characterization of Herc6 knockout mice. Substantial reductions of ISGylation were observed in Herc6-deficient cells after polyinosinic-polycytidylic acid double-stranded RNA injection of mice or IFN treatment of cells. On the other hand, Herc6-deficient cells and wild-type (WT) cells had similar responses to IFN stimulation, Sendai virus (Z strain) infection, and vesicular stomatitis virus infection. These results indicate that Herc6 does not play a critical role in antiviral defense of these viral infections in mice. Interestingly, male Herc6-deficient mice showed seminal vesicle hypertrophy. No such problem was detected in WT and ISG15 activating enzyme Ube1L-deficient mice. These results suggest that in addition to promoting protein ISGylation, Herc6 has a novel and protein ISGylation-independent function in the male reproductive system.

Plakophilin-2 promotes tumor development by enhancing ligand-dependent and -independent epidermal growth factor receptor dimerization and activation.

Epidermal growth factor (EGF) receptor (EGFR) has been implicated in tumor development and invasion. Dimerization and autophosphorylation of EGFR are the critical events for EGFR activation. However, the regulation of EGF-dependent and EGF-independent dimerization and phosphorylation of EGFR has not been fully understood. Here, we report that cytoplasmic protein plakophilin-2 (PKP2) is a novel positive regulator of EGFR signaling. PKP2 specifically interacts with EGFR via its N-terminal head domain. Increased PKP2 expression enhances EGF-dependent and EGF-independent EGFR dimerization and phosphorylation. Moreover, PKP2 knockdown reduces EGFR phosphorylation and attenuates EGFR-mediated signal activation, resulting in a significant decrease in proliferation and migration of cancer cells and tumor development. Our results indicate that PKP2 is a novel activator of the EGFR signaling pathway and a potential new drug target for inhibiting tumor growth.

Interferon-mediated ISG15 conjugation restricts dengue virus 2 replication.

ISGylation, an ubiquitin-like post-translational modification by ISG15, has been reported to participate in the interferon (IFN)-mediated antiviral response. In this study, we analyzed the functional role of ISGylation in dengue virus 2 (DENV-2) replication. Overexpression of ISG15 was found to significantly suppress the amount of extracellular infectious virus released, while intracellular viral RNA was unaffected. This effect was not observed with a conjugation-defective ISG15 mutant. In addition, extracellular virus infectivity was decreased by ISG15 overexpression. To further clarify the role of ISGylation in the anti-DENV-2 response, we depleted endogenous ISG15 by RNA interference and analyzed the virus production in the absence or presence of type-I IFN. Results showed a significant reduction in extracellular DENV-2 RNA levels for cells treated with IFN, and that these DENV-2 RNA levels could be partially restored by the ISG15 knockdown. Among various DENV-2 proteins, NS3 and NS5 were subjected to the ISGylation. These results demonstrate that IFN-inducible ISGylation suppresses DENV-2 particle release, and that ISG15 is one of the mediators of IFN-induced inhibition of DENV-2 replication. ISG15 therefore functions as a host antiviral factor against DENV-2 infection.

Plakophilin-2 induced EGFR phosphorylation: a focus on the intracellular activators of EGFR.

The oncogenic role of EGFR in many tumors has attracted a great deal of attention in the recent years and initiated the development of several potent EGFR inhibitors, which are used clinically for cancer treatment. However, the current therapeutic inhibition of EGFR signaling is limited to monoclonal antibodies that bind to the EGFR extracellular domain or tyrosine kinase inhibitors that block EGFR kinase activation directly. Despite the great promise of these inhibitors, a certain percentage of patients develop resistance to these therapies, highlighting the necessity for alternative therapeutic strategies based on our most current knowledge of the mechanisms of EGFR signaling. We recently reported that Plakofilin-2 (PKP2) is a novel ligand-independent cytoplasmic activator of EGFR signaling. Here we focus on recent studies demonstrating important roles of intracellular EGFR activators, and propose targeted disruption of these activators as a novel avenue of therapeutic intervention to inhibit EGFR-mediated cancer development.

Usp18 deficient mammary epithelial cells create an antitumour environment driven by hypersensitivity to IFN-λ and elevated secretion of Cxcl10.

The theory of cancer immunoediting refers to mechanisms by which the immune system can suppress or promote tumour progression. A major challenge for the development of novel cancer immunotherapies is to find ways to exploit the immune system's antitumour activity while concomitantly reducing its protumour activity. Using the PyVmT model of mammary tumourigenesis, we show that lack of the Usp18 gene significantly inhibits tumour growth by creating a tumour-suppressive microenvironment. Generation of this antitumour environment is driven by elevated secretion of the potent T-cell chemoattractant Cxcl10 by Usp18 deficient mammary epithelial cells (MECs), which leads to recruitment of Th1 subtype CD4(+) T cells. Furthermore, we show that Cxcl10 upregulation in MECs is promoted by interferon-λ and that Usp18 is a novel inhibitor of interferon-λ signalling. Knockdown of the interferon-λ specific receptor subunit IL-28R1 in Usp18 deficient MECs dramatically enhances tumour growth. Taken together, our data suggest that targeting Usp18 may be a viable approach to boost antitumour immunity while suppressing the protumour activity of the immune system.

Polyubiquitin conjugation to NEMO by triparite motif protein 23 (TRIM23) is critical in antiviral defense.

The rapid induction of type I IFN is a central event of the innate defense against viral infections and is tightly regulated by a number of cellular molecules. Viral components induce strong type I IFN responses through the activation of toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as an RNA helicase RIG-I and/or MDA5. According to recent studies, the NF-kappaB essential modulator (NEMO, also called IKKgamma) is crucial for this virus-induced antiviral response. However, the precise roles of signal activation by NEMO adaptor have not been elucidated. Here, we show that virus-induced IRF3 and NF-kappaB activation depends on the K(lys)-27-linked polyubiquitination to NEMO by the novel ubiquitin E3 ligase triparite motif protein 23 (TRIM23). Virus-induced IRF3 and NF-kappaB activation, as well as K27-linked NEMO polyubiquitination, were abrogated in TRIM23 knockdown cells, whereas TRIM23 knockdown had no effect on TNFalpha-mediated NF-kappaB activation. Furthermore, in NEMO-deficient mouse embryo fibroblast cells, IFN-stimulated response element-driven reporter activity was restored by ectopic expression of WT NEMO, as expected, but only partial recovery by NEMO K165/309/325/326/344R multipoints mutant on which TRIM23-mediated ubiquitin conjugation was substantially reduced. Thus, we conclude that TRIM23-mediated ubiquitin conjugation to NEMO is essential for TLR3- and RIG-I/MDA5-mediated antiviral innate and inflammatory responses.