ALBERT

Description: 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.

Description: 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.

Description: Stable and permanent hematopoiesis is established from the most primitive long-term self-renewing hematopoietic stem cells (LT-HSC), which can give rise to more differentiated short-term (ST-HSC) and multi-potent progenitors (MPP). Progenitors further differentiate into more committed cells that can generate the mature lymphoid and myeloid lineages. In order to maintain a normal hematopoietic system, HSCs must be properly regulated. We previously cloned Ubiquitin Specific Protease 18 (USP18/UBP43) during analysis of hematopoietic cells of t(8;21) AML fusion protein AML1-ETO knock-in mice (Liu et al, 1999 Mol Cell Biol 19:3029-3038; Schwer et al, 2000 Genomics 65, 44-52). However, its function in hematopoiesis, especially in hematopoietic stem cells, has not been carefully examined. We show here that depletion of Usp18 in C57/BL6 mice leads to death at embryonic days 13.5-14.5 with less fetal liver cellularity. To examine the precise role of Usp18 in vivo, we generated Usp18 conditional knockout mice (Usp18f/f). Survival analyses of Usp18f/- crossed with Usp18f/+Vav-iCre revealed that the embryonic lethality of Usp18 -deficient mice is due to defects in hematopoiesis. To examine the hematopoietic potential of fetal liver cells of Usp18-deficient mice, we conducted a colony forming assay using the E12.5 fetal livers. All types of colonies as well as the number of total cells from colonies were substantially reduced in Usp18-/- fetal liver compared to control, indicating that the blood progenitor cells of Usp18-/- fetal liver are not fully functional. To assess whether Usp18 is required for fetal liver HSC maintenance, we determined the frequency of HSCs in the fetal liver of Usp18+/+, Usp18+/-, and Usp18-/-. We detected the Lin- Sca-1+ c-Kit+ (LSK) cell population, which is HSC-enriched population in fetal livers, in mice of all three genotypes. Recent studies indicate that the most primitive LT-HSC population in fetal livers includes ESAM positive (LSK CD48- CD150+ ESAM+) stem cells (Ooi et al, 2009 Stem Cells 27:653-661; Pietras et al, 2014 JEM 211:245-262). Both the frequency and absolute numbers of the LT-HSC population in Usp18 -/- fetal livers were appreciably reduced compared to wild-type. Taken together, we conclude that Usp18 is indispensable for fetal liver HSC maintenance. We then addressed whether Usp18 is required for the HSC maintenance in adult mice by analyzing the frequency of HSCs in UBCER-Cre negative or positive Usp18 f/- bone marrow cells. After tamoxifen injections, we observed a significant reduction in the frequency of the LT-HSC population in Usp18f/-UBCER-Cre positive bone marrow cells compared to Usp18 f/-UBCER-Cre negative ones. Consistent with these results, Usp18 f/-UBCER-Cre positive bone marrow cells were much less competitive than Cre negative cells by competitive bone marrow transplantation assay. Finally, to examine whether the suppression of Usp18 in the leukemic cells provides a survival benefit, we used secondary-transplanted mice receiving Usp18f/fUBCER-Cre positive AML1-ETO9a leukemia cells (5 × 10 5 EGFP+ cells) isolated from primary transplanted mice. The tamoxifen treatment was initiated 3 weeks after transplantation. All the mice in the vehicle injected group (n = 7) succumbed to leukemia within a week after treatment started. However, mice treated with tamoxifen (n = 7) showed a longer survival time. Five of seven mice are still alive after 5 weeks of bone marrow transplantation, demonstrating the critical role of USP18 in maintenance of leukemia stem cells. Collectively, we conclude that Usp18 is essential for hematopoietic stem cell maintenance, and specific modulating activity of USP18 in leukemic cells may be considered as an effective therapeutic approach. Disclosures No relevant conflicts of interest to declare.

Description: Recurring chromosome abnormalities are frequent events in cancer and are especially prevalent in hematologic neoplasms. Somatic heterozygous deletions on chromosome 20q are detected in a variety of hematopoietic malignancies including myelodysplastic syndrome (MDS), classical myeloproliferative neoplasm (MPN), MDS/MPN overlap disorders such as chronic myelomonocytic leukemia (CMML), and acute leukemias. Del(20q) is especially prevalent in MPN patients (~10-15%), where it is the most commonly detected cytogenetic abnormality associated with primary myelofibrosis (PMF) and post-polycythemia vera myelofibrosis (MF). This suggests that heterozygous loss of genes in the del(20q) common deleted region (CDR) may contribute to adverse MPN progression. Despite these observations, relatively few genes located within the CDR have been unambiguously implicated, highlighting a significant need for further investigation. To identify genes that may play an important role in the biology of del(20q)-associated malignancies we utilized a published gene expression dataset of bone-marrow derived CD34+ cells from MDS patients and healthy controls (Gerstung et al, 2015). Comparison of the patients harboring del(20q) to healthy controls revealed STK4 (encoding Hippo kinase MST1) to be the most significantly downregulated gene (mean: 3.5-fold) among those located within the chromosome 20q CDR. We therefore set out to assess the role of Hippo kinase inactivation in hematologic malignancy using conditional gene inactivation in mice. We found that complete inactivation of both Hippo kinases (Stk4 and Stk3) within the hematopoietic system using Vav1-Cre (Stk4-/-Stk3-/-) resulted in a lethal bone marrow failure (median survival: 7 weeks) associated with myelodysplastic features and frequent extramedullary hematopoiesis in the spleen. A single copy of Stk4 rescued the lethality due to bone marrow failure, however sub-haploinsufficient mice displayed thrombocytopenia with a trend towards mild anemia; phenotypes that closely resemble those observed in MDS patients with isolated del(20q). Both a reduced number of mature megakaryocytes and the presence of dysplastic megakaryocytes were apparent in bone marrow sections. Inducible Hippo kinase inactivation in adult mice using the Mx1-Cre system similarly recapitulated several phenotypic features of both MDS and MPN. In competitive bone marrow transplant assays we found that Stk4-/-Stk3-/- hematopoietic stem cells (HSC) completely lacked engraftment potential and failed to reconstitute normal hematopoiesis, revealing a potential role for Hippo kinase function in HSC homing and retention in the bone marrow. Heterozygous HSCs maintained relatively normal steady-state hematopoiesis in peripheral blood and bone marrow for up to 48 weeks in primary and secondary transplantations, although upon aging these mice were prone to development of thrombocytopenia with increased mean platelet volume. Given the high frequency of del(20q) in MPN, especially PMF, we asked whether heterozygous Hippo kinase inactivation may cooperate with the common driver mutation JAK2-V617F to accelerate disease progression. Using an HSC-enriched retroviral transduction/transplantation model in C57BL/6 recipient mice, we monitored MPN progression for 36 weeks in heterozygous Stk4+/-Stk3+/-, or control (Vav1-Cre-), cells with or without expression of JAK2-V617F. While both JAK2-V617F groups initially displayed a similar degree of polycythemia relative to controls, we found heterozygous Hippo kinase inactivation to promote accelerated disease progression towards lethal bone marrow fibrosis during the course of observation. Recipients in this group showed significantly reduced overall survival, which was associated with higher grade fibrosis in bone marrow, elevated peripheral granulocyte counts, enhanced splenomegaly, and increased frequencies of hematopoietic stem and progenitor populations in the spleen. Together, these findings implicate aberrant Hippo kinase loss-of-function in the pathogenesis of del(20q)-associated hematologic malignancies, and shed new light on the molecular events that contribute to adverse MPN progression. Disclosures Bejar: Genoptix: Consultancy; Modus Outcomes: Consultancy; Celgene: Consultancy, Honoraria; Takeda: Research Funding; Astex/Otsuka: Consultancy, Honoraria; AbbVie/Genentech: Consultancy, Honoraria; Foundation Medicine: Consultancy. Guan:Vivace: Equity Ownership.

Description: Deletions of chromosome 20q are frequent abnormalities in myelodysplastic syndrome (MDS) and myeloproliferative neoplasms. Stoner et al identify STK4, which encodes Hippo kinase MST1, as the candidate gene from the deleted region that, when deleted, promotes features of MDS and induces myelofibrosis in the presence of JAK2V617F through modulation of inflammatory pathways.