ALBERT

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: Myelodysplastic syndromes (MDS) and leukemias require the acquisition of multiple mutations during disease development resulting in clonal diversity and different responses. Splicing factors, transcription factors, epigenetic regulators, and cell signaling proteins are the common molecular events mutated during disease evolution and those events rarely occur alone. However, it remains unclear how the combinations of mutations in different categories may have cooperative effects in gene regulation and disease etiology. Mutations in the splicing factor SRSF2 and the transcription factor RUNX1 are closely associated in MDS patients, and their co-existence is linked to poor prognosis. To understand the functional contribution of the coexistence in vivo, we utilized Mx1-Cre based conditional knock-in Srsf2-P95H mutation (P95H/+) mice, and Mx1-Cre based Runx1 conditional knockout mice (Runx1 f/f). We crossed these two strains to establish a new mouse model with inducible double mutations (Srsf2 P95H/+ Runx1Δ/Δ). Double mutant mice showed pancytopenia with MDS features including severe leukopenia in multiple lineages, macrocytic anemia, thrombocytopenia, and dysplastic morphology in peripheral blood. Double mutant mice also displayed more dramatic skewing toward the myeloid lineage at the expense of the B cell lineage when compared to single mutant mice. In competitive bone marrow transplantation assays, SRSF2 P95H cooperated with RUNX1 deficiency to confer a competitive disadvantage in vivo. To investigate the mechanistic basis of this cooperation, differential splicing and gene expression were assessed by RNA sequencing of Lineage- c-kit+ cells isolated from WT, SRSF2 P95H, RUNX1 KO, and Double mutant bone marrow cells. Interestingly, deletion of the Runx1 gene alone resulted in significant changes to RNA splicing in 1120 genes, while the SRSF2 P95H mutation itself induced splicing changes in 935 genes. Furthermore, 2468 splice junctions in 1677 genes showed splicing changes in double mutant samples compared to wildtype controls. Among these altered splicing events, intriguingly, exon skipping was the major alteration in single and double mutants. Furthermore, the double mutant demonstrated increased aberrant splicing events when compared to the single mutants alone. We performed pathway analysis using the differentially spliced genes identified in double mutant cells. Pathways in cancer, DNA replication/repair, cell death and survival, hematological disease and inflammatory response were enriched. Splicing changes were detected in genes recurrently mutated in blood malignancies, including Fanca, Fance, Fancl, Ezh2, Atm, Gnas, Braf, Bcor, Fyn, and Wsb1 as well as in genes critical for splicing regulation, such as Srsf6, Fus, Hnrnpa2b1, and Srrm2. Gene expression analysis revealed 869 significantly differentially expressed genes in double mutant cells. Within the events in the double mutant population, 60% of the differentially expressed genes were also observed in RUNX1 single mutant cells, while only 2% of the differentially expressed genes were observed in SRSF2 single mutant cells, and 38% of the differentially expressed genes were uniquely presented in the double mutant cells. These results suggest that the gene expression program is heavily affected by loss of RUNX1 and the coexistence of an SRSF2 mutation contributes to certain synergistic effects in transcriptional regulation. Furthermore, we identified 101 genes that showed both differential splicing and expression, including Jak3, Jag2, Csf3r, Fcer1g, CD244 which are important in hematologic disorders. Together, these results suggest that the deficiency of compound RUNX1 and SRSF2 P95H mutations impairs multi-lineage hematopoiesis and exacerbates the disease phenotypes caused by single mutations alone. At the genome-wide level, loss of the transcription factor RUNX1 itself dysregulates splicing outcomes and cooperates with the splicing factor SRSF2 P95H mutation to further perturb the expression and splicing of key regulators involved in hematopoietic stem/progenitor cell development, inflammatory responses, DNA damage, and RNA splicing. 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.

Description: Introduction The t(8;21) chromosomal translocation is one of the most common chromosomal translocations associated with acute myeloid leukemia (AML), present in greater than 10% of de novo AML cases. Most of these t(8;21) AML cases are classified as FAB subtype M2. This translocation results in the formation of a stable fusion protein made up of portions of the RUNX1 (aka AML1) and ETO (aka MTG8 and RUNX1T1) proteins. RUNX1 is a transcription factor that is essential for regulating the differentiation of hematopoietic cells, and the fusion protein retains its DNA-binding domain. Additionally, ETO contains four Nervy homology (NH) domains which facilitate a number of protein-protein interactions, notably with the NCOR2/SMRT co-repressor complex. The identification of individual genes or biological pathways which are specifically disrupted in the presence of RUNX1-ETO will provide further molecular insight into the pathogenesis of t(8;21)+ AML and lead to the possibility for improved treatment for these patients. Methods/Results We analyzed publicly available gene expression microarray datasets (Oncomine, TCGA) to search for genes whose expression was significantly altered in the blood of t(8;21)+ AML patients as compared to non-t(8;21) FAB subtype M2 AML and to CD34+ cells in healthy controls. One such gene that was consistently significantly downregulated in t(8;21)+ patients was Ras-association domain family member 2 (RASSF2). RASSF2 is a putative tumor suppressor that is capable of mediating apoptosis (in a Ras dependent manner) through its interactions with the MST1/2 kinases and the cancer-specific apoptotic protein Par-4. RASSF2 has previously been shown to be frequently downregulated via hypermethylation in a wide variety of solid tumors, however little is known about its function in leukemia. Here we demonstrate that RASSF2 is a potentially interesting target for downregulation by the RUNX1-ETO fusion protein. Gene expression analysis by RT-qPCR in leukemia cell lines confirmed that RASSF2 is significantly downregulated in both Kasumi-1 and SKNO t(8;21)+ cell lines as compared to a similar non-t(8;21) HL-60 line. We found that exogenous expression of AML1-ETO in HL-60 leukemia cells induces a rapid downregulation of RASSF2, further supporting that it is a target of this leukemogenic fusion protein. Over-expression of RASSF2 in leukemia cells significantly inhibits their proliferative capability, indicating an important biological effect of RASSF2 in blood cells. Finally, over-expression of RASSF2 significantly inhibits the long-term self-renewal capability of RUNX1-ETO expressing hematopoietic cells as measured by their serial replating ability in a colony formation assay. Discussion Based on the analysis of patient data and our own experiments it appears that RASSF2 is a direct target for downregulation by the AML1-ETO fusion protein. Due to its potential involvement as a mediator of apoptosis in important oncogenic signaling pathways RASSF2 is a strong candidate for further investigation in the context of t(8;21)+ AML pathogenesis. In particular, it will be interesting to continue to investigate the relationship between RASSF2 and apoptotic protein Par-4, as several lines of evidence suggest Par-4 to be therapeutically relevant due to its ability to selectively induce apoptosis in cancer cells. Disclosures: No relevant conflicts of interest to declare.

Description: The t(8;21) chromosomal translocation is one of the most common chromosomal translocations associated with acute myeloid leukemia (AML), found in approximately 12% of de novo AML cases. The majority of these cases are classified as FAB-subtype M2 AML. The t(8;21) results in the stable fusion of the AML1 (RUNX1) and ETO (RUNX1T1) genes. The AML1-ETO fusion protein is composed of the N-terminal portion of AML1, which includes the DNA-binding Runt-homology domain, and nearly the full-length ETO protein. The primary accepted mechanism by which AML1-ETO promotes leukemia development is through the aberrant recruitment of transcriptional repression/activation complexes to normal AML1 target genes. Therefore, the identification of individual genes or biological pathways that are specifically disrupted in the presence of AML1-ETO will provide further molecular insight into the pathogenesis of t(8;21) AML and lead to the possibility for improved treatment for these patients. We identified RASSF2 as a gene that is specifically downregulated in (2-4 fold) in total bone marrow of t(8;21) patients compared to non-t(8;21) FAB-subtype M2 AML patients by analyzing publicly available gene expression datasets. Similarly, using a mouse model of t(8;21) AML we found Rassf2 mRNA levels to be nearly 30-fold lower in t(8;21) leukemia cells compared to wild-type Lin-Sca-cKit+ (LK) myeloid progenitors. Gene expression analysis by RT-qPCR in leukemia cell lines confirmed that RASSF2 mRNA levels are significantly downregulated (8-10-fold) in both Kasumi-1 and SKNO-1 t(8;21) cell lines as compared to a similar non-t(8;21) HL-60 cell line and to primary human CD34+ control cells. In addition, expression of AML1-ETO in HL-60 or CD34+ cells results in a decrease in RASSF2 mRNA expression, which further suggests that RASSF2 is a target for regulation by AML1-ETO. Assessment of published ChIP-seq data shows that AML1-ETO binds the RASSF2 gene locus at two distinct regions in both primary t(8;21) AML patient samples and in the Kasumi-1 and SKNO-1 cell lines. These regions are similarly bound by several important hematopoietic transcription factors in primary human CD34+ cells, including AML1, ERG, FLI1, and TCF7L2, implicating these two regions as important for the regulation of RASSF2 expression during blood cell differentiation. Overexpression of RASSF2 in human leukemia cell lines using an MSCV-IRES-GFP (MIG) construct revealed that RASSF2 has a strong negative effect on leukemia cell proliferation and viability. The overall percentage of GFP-positive cells in MIG-RASSF2 transduced cells markedly decreased compared to MIG-control transduced cells over a period of 14 days. This effect was primarily due to significantly increased apoptosis in the RASSF2 expressing cell populations. Similarly, we found that expression of RASSF2 significantly inhibits the long-term self-renewal capability of hematopoietic cells transduced with AML1-ETO in a serial replating/colony formation assay. AML1-ETO transduced hematopoietic cells were normally capable of serial replating for more than 6 weeks. However, AML1-ETO transduced cells co-expressing RASSF2 consistently had reduced colony number and lost their ability to replate after 3-4 weeks. This was due to a dramatically increased rate of apoptosis in RASSF2 expressing cells. RASSF2 is reported to be a tumor suppressor that is frequently downregulated at the transcriptional level by hypermethylation in primary tumor samples, but not healthy controls. Here we have identified RASSF2 as a target for repression, and demonstrated its tumor suppressive function in t(8;21) leukemia cells. Further insights into the molecular mechanisms of RASSF2 function in AML will continue to be explored. Disclosures No relevant conflicts of interest to declare.

Description: Alternative polyadenylation (APA) can alter the three prime untranslated region (3'UTR) length of mRNAs, crucial for regulating mRNA metabolism and gene expression. Despite the prevalence of APA post-transcriptional regulation in cancers, changes in 3'UTR length by APA and its contribution to leukemia development have not been thoroughly studied. In this study, we demonstrated the significance of APA of leukemic fusion genes in acute myelogenous leukemia (AML) development. T(8;21) is the most common chromosomal abnormality in AML and encodes the AML1-ETO (AE) fusion gene. The AE 3'UTR has a full length of 5.2kb that contains 4 canonical polyadenylation sites (PAS), such that APA can result in several mRNA isoforms with varying 3'UTR lengths. However, RNA-seq and absolute quantification qPCR revealed that AE mainly uses 2 PAS: 5.2kb (long 3'UTR) and 3.7kb (short 3'UTR). Moreover, the AE short 3'UTR is the major isoform in both t(8;21) AML primary patients and cell lines. Based on these findings, we next hypothesize that changes in PAS usage and thus AE 3'UTR length, can modulate fusion gene expression. The single cell based dynamic array revealed that AE+ non-leukemic differentiated cells coexisted with AML blasts in the diagnostic bone marrow and these non-leukemic AE+ cells expressed much lower AE compared to AML blasts (monocytes, 0.19-fold and granulocytes, 0.11-fold, p