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: 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: The t(8;21) chromosomal translocation is among the most frequent recurring cytogenetic abnormalities associated with acute myeloid leukemia (AML), found in 8-12% of de novo AML patients. The t(8;21) results in the stable fusion of the RUNX1 and RUNX1T1 genes, and formation of the oncofusion protein RUNX1-ETO (AML1-ETO). RUNX1-ETO is composed of the N-terminal DNA-binding domain of RUNX1 and nearly the entire ETO protein. RUNX1-ETO promotes leukemia development via the recruitment of transcription factor/transcriptional repression complexes (including NCOR, HDACs, p300, etc.) to regulatory regions of RUNX1 target genes known to be critical for myeloid differentiation and function, such as CEBPA, SPI1 (PU.1), NFE2, and CSF1R. Despite this knowledge, additional RUNX1-ETO target genes remain poorly characterized, and the complete molecular mechanism through which RUNX1-ETO leads to leukemic transformation remains to be elucidated. We propose that a better understanding of additional RUNX1-ETO target genes will lead to the potential for development of novel therapeutics to treat these patients. One such gene that we initially identified as markedly downregulated in RUNX1-ETO leukemia cells using a mouse model of t(8;21) AML is RASSF2 (Lo et al, Blood, 2012). Assessment of publicly available gene expression data revealed that RASSF2 is specifically downregulated in the bone marrow of t(8;21) AML patients compared to patients of different cytogenetic subtypes or to non-t(8;21) FAB subtype M2 AML patients. Additionally, RT-qPCR analysis confirmed that RASSF2 transcript is downregulated 10-100-fold in the t(8;21) AML cell lines, Kasumi-1 and SKNO-1, compared to non-t(8;21) AML cell lines and normal CD34+ hematopoietic cells. Expression of RUNX1-ETO in a non-t(8;21) AML cell line led to a reduction in RASSF2 mRNA expression, while knockdown of RUNX1-ETO in Kasumi-1 cells resulted in a ~5-fold increase in RASSF2 expression. Assessment of published ChIP-seq data showed that RUNX1-ETO directly binds at two regulatory regions within the RASSF2 genomic locus in t(8;21) AML cell lines and patient samples. Re-expression of RASSF2 at physiological levels in t(8;21) AML cell lines resulted in a modest negative growth phenotype, and greatly sensitized these cells to apoptosis following stimulation with various pro-apoptotic agents. Re-expression of RASSF2 in RUNX1-ETO-transduced primary mouse bone marrow caused these cells to lose their long-term self-renewal ability after 3 weeks in a serial replating/colony formation assay. This loss of self-renewal ability in co-transduced cells was accompanied by a marked increase in apoptosis during each of the first three weeks of replating. Mechanistically, re-expression of full-length RASSF2, but not of a deletion mutant lacking the SARAH heterodimerization domain (RASSF2ΔSARAH), in t(8;21) AML cell lines resulted in increased protein amount of the pro-apoptotic kinase, MST1. This suggests that RASSF2 may be a critical regulator of MST1 protein stability in AML cells. Importantly, modest (2-3-fold) overexpression of MST1 in t(8;21) AML cell lines resulted in a significant increase in apoptosis and caused growth arrest. The effects of RASSF2 or MST1 expression in non-t(8;21) AML cell lines were greatly reduced, suggesting that the cellular context of RUNX-ETO-driven leukemias makes them highly susceptible to MST1-dependent apoptosis. Overall, we have identified the importance of a MST1-driven pro-apoptotic signaling axis in t(8;21) leukemia. RUNX1-ETO-dependent transcriptional repression of RASSF2 may be essential for evasion of this apoptosis signaling during leukemic transformation via reduction of MST1 protein stability. MST1, perhaps better known as the mammalian orthologue of the drosophila Hippo kinase, is a critical tumor suppressor in many solid tumor types; and we believe our studies warrant the continued investigation of this pathway in hematological malignancy. Disclosures No relevant conflicts of interest to declare.

Description: Polyadenylation is a post-transcriptional modification where the 3' end of an mRNA is cleaved and 250-300 adenines are added. It is predicted that 70-75% of human genes have more than one polyadenylation sequence (PAS) and are subject to alternative polyadenylation (APA). APA events affect the coding sequence of a gene when a proximal PAS is located within an intron, constitutive exon, or alternative exon. Gene expression is also affected if there are multiple PAS within the distal 3' untranslated region (UTR); proximal PAS usage shortens the 3'UTR, which can remove cis-regulatory regions such as miRNA and RNA-binding protein (RBP) sites. Furthermore, global changes in APA are linked to cellular state-proximal PAS usage is associated with immature developmental phases, cell proliferation, and cancerous phenotypes. Consequently, APA is a pertinent post-transcriptional modification that regulates gene expression and isoform generation across developmental stages and tissue types. Despite its significance, there are few APA studies in the hematology field, and those that exist have focused on global shifts in PAS usage. In this study, we uniquely focus on the APA mechanism of a single gene, RUNX1, and how this event can alter hematopoietic stem cell (HSC) homeostasis and hematopoiesis. There are three main isoforms of RUNX1 that differ in promoter and/or PAS usage. RUNX1b/c use different promoters, but have identical C-terminal regions. RUNX1a utilizes the same promoter as RUNX1b, but differs from both RUNX1b/c due to usage of a proximal PAS located in alternative exon 7a. RUNX1b/c are robustly expressed in most progenitor populations and differentiated blood cell lineages, whereas RUNX1a is restricted to human CD34+ HSCs. Functionally, RUNX1b/c promote HSC differentiation and lineage commitment, whereas RUNX1a expands HSCs and their engraftment potential, a property with therapeutic advantages but leukemic potential. Due to the difference in expression pattern and distinct functionality of RUNX1a compared to RUNX1b/c, it is relevant to study the APA event that dictates isoform generation. Elucidating this mechanism could provide valuable insight into the transient control of the HSC population for therapeutic benefit and illuminate new leukemogenic pathways. To study RUNX1 APA, we cloned alternative terminal exon 7a (RUNX1a) and constitutive exon 7b (RUNX1b/c) in between the two exons of a split GFP minigene reporter, along with 500 bp of their upstream and downstream flanking introns. We hypothesized that exon 7a would be skipped during processing of the minigene construct because the proximal PAS is rarely used in vivo. Conversely, exon 7b, the penultimate exon in RUNX1b/c, would be spliced in between the GFP exons, disrupting the GFP protein. These constructs were tested in KG-1a and U937 cells. Flow cytometry for GFP fluorescence supported our hypothesis as the exon 7a minigene produced a robust GFP signal and the exon 7b minigene produced no GFP signal. We confirmed that the GFP changes were due to the hypothesized mRNA processing events by performing RT-PCR using primers specific to the two GFP exons. These data show that important cis-regulatory elements that determine RUNX1 APA are located within exon 7a, 7b, and the cloned intronic regions. Next, we altered these minigenes by strategically making chimeric constructs that consist of either exon 7a or 7b with all combinations of upstream/downstream flanking introns. We discovered that replacing the intron upstream of exon 7a confers 2-5 fold greater splicing and polyadenylation of exon 7a, indicative of RUNX1a isoform generation. Therefore, a suppressor cis-element is located in this upstream intronic region. However, placing this intron upstream of exon 7b is not sufficient to reduce its inclusion between the GFP exons. Instead, both the upstream and downstream intronic regions flanking exon 7a are required. This suggests an RNA-looping mechanism that prevents splicing and usage of the exon 7a proximal PAS. Cleavage factor (CFIm) and Polypyrimidine-tract binding protein 1 (PTBP1) are RBPs involved in splicing and polyadenylation that alter mRNA processing by RNA-looping. We aim to narrow down the suppressor region upstream of exon 7a to identify a consensus sequence and the respective RBP that diminishes RUNX1 proximal PAS usage. This knowledge can be leveraged to enhance RUNX1a production and expand HSCs for therapeutic benefit. Disclosures No relevant conflicts of interest to declare.

Description: Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) is a cytokine that regulates various cellular processes including differentiation, proliferation, survival, and leukocyte activation. The receptor for GM-CSF is a dodecamer composed of the CSF2RA and CSF2RB receptor subunits. CSF2RB is also a shared common beta subunit for the IL3 and IL5 receptors, and is the predominant subunit for signaling. CSF2RA is primarily a ligand-binding subunit, with a short 54 amino acid intracellular domain, which provides specificity of signaling. We previously reported that GM-CSF signaling is inhibitory to leukemogenesis in a murine model for t(8;21) acute myeloid leukemia (AML), and aids in promoting myeloid differentiation of leukemic blasts. Interestingly, around 32-59% of t(8;21) AML patients suffer from haploinsufficiency of CSF2RA, a gene located on the pseudoautosomal region (PAR) of the sex chromosomes, due to loss of a sex chromosome (LOS). CSF2RA expression has also been reported to be lower in t(8;21) AML patients compared to non-t(8;21) patients. Although we discovered that GM-CSF signaling is inhibitory to t(8;21) leukemogenesis, we hypothesize that CSF2RA itself may act as a tumor suppressor. Although CSF2RA confers specificity of GM-CSF signaling, very little is known about the role of its expression and signaling in the context of t(8;21) leukemogenesis. To address whether CSF2RA expression has negative effects on the leukemic potential of t(8;21) cells, we restored CSF2RA expression in the t(8;21) leukemia cell lines, Kasumi-1 and SKNO-1. Interestingly, CSF2RA expression specifically inhibited cell proliferation and induced apoptosis in the t(8;21) cell lines, but not in other non-t(8;21) myeloid leukemia cell lines. To further confirm that these effects were specific to the presence of t(8;21), we expressed CSF2RA with RUNX1-ETO, the oncofusion protein generated from t(8;21), in primary murine bone marrow cells. CSF2RA expression in RUNX1-ETO cells had similar effects as was observed in the t(8;21) cell lines. Additionally, this was specific to RUNX1-ETO expression, as control cells did not exhibit these effects. Moreover, we determined that the anti-proliferative and pro-apoptotic effects of CSF2RA expression were ligand-independent, due to the fact that GM-CSF has no cross-species reactivity between humans and mice. CSF2RA expression also reduced the stemness of RUNX1-ETO bone marrow cells and inhibited their colony forming ability. To identify which region of the receptor was mediating these pro-apoptotic effects, we generated truncation mutants and determined that the 25 amino acids at the carboxyl-terminus of the intracellular domain of CSF2RA are required. IL3RA, the gene encoding the IL3 alpha receptor, is also located on the PAR and suffers from haploinsufficiency in the case of LOS. Therefore, IL3RA also has the potential to also serve as a tumor suppressor in t(8;21) leukemogenesis. Additionally, given that IL3RA oligomerizes with CSF2RB for ligand binding and signaling, we investigated whether IL3RA expression could elicit similar effects as with CSF2RA expression. Interestingly, IL3RA expression had no inhibitory effect on t(8;21) cells, indicating that the observed pro-apoptotic and anti-proliferative effects are unique to CSF2RA in t(8;21) cells. Altogether, we have discovered that CSF2RA expression, which would be reduced upon LOS, inhibits the leukemic potential of t(8;21) cells by reducing proliferation and inducing apoptosis in a ligand-independent fashion. These phenotypes were especially surprising given that GM-CSF generally promotes cell proliferation and survival, and indicates that CSF2RA may have a novel role as a tumor suppressor in the pathogenesis of t(8;21) AML. Our findings provide greater insight into how LOS may serve as a critical cooperating event in t(8;21) leukemogenesis. Further mechanistic studies will aid in elucidating which signaling pathways are differentially affected upon CSF2RA expression in t(8;21) cells, and may uncover novel therapeutic targets for treating t(8;21) AML. Disclosures No relevant conflicts of interest to declare.