ALBERT

Description: N 6-methyladenosine (m6A) modification is the most abundant internal RNA modification in eukaryotes. Recent studies have shown that the dynamic and reversible regulation of m6A modifications in mRNAs or non-coding RNAs plays critical roles in tissue development, stem cell self-renewal and differentiation, control of heat shock response, and circadian clock controlling, as well as in RNA metabolism and processing. However, little is known about the functions of m6A and m6A regulators in malignant hematopoiesis. METTL14 is a major m6A writer which together with METTL3 forms the core of the methyltransferase complex that catalyzes the conversion of adenosine (A) to m6A. Through qPCR assays, we found that METTL14 was aberrantly up-regulated in mononuclear cells (MNC) from acute myeloid leukemia (AML) patients with t(11q23), t(15;17), or t(8;21) relative to those from healthy donors. To investigate the pathological role of METTL14 in AML, we transduced lineage negative (Lin-) bone marrow (BM) progenitor cells from Mettl14fl/flCreERT mice with MLL-AF9, AML1-ETO9a, or PML-RARa fusion genes and performed colony-forming/replating assays with or without addition of 4-hydroxytamoxifen (4-OHT). Induction of genetic knockout of Mettl14 by 4-OHT treatment remarkably impaired the colony-forming ability of all these AML-related fusion genes after replating. After the first round of plating, we harvested MLL-AF9-transduced cells that were not treated with 4-OHT and transplanted them into lethally irradiated recipient mice. As expected, tamoxifen (TAM) treatment of transplanted mice significantly delayed leukemogenesis compared to mice treated with vehicle (MLL-AF9+TAM, with median survival of 91 days; MLL-AF9+vehicle, with median survival of 71 days; P=0.0012) (Fig.1A). In addition, specific knockdown of Mettl14 with shRNAs showed similar patterns to Mettl14 knockout. Thus our data demonstrate that Mettl14 is crucial for cell transformation and leukemogenesis. Further, to determine the role of Mettl14 in the maintenance of leukemia, we transduced leukemic BM cells from primary MLL-AF9 leukemic mice with shRNAs against Mettl14 or scramble shRNA and transplanted these cells into lethally irradiated recipient mice. Again, a significantly prolonged survival was observed in Mettl14 knockdown groups compared to that in the control group (MLL-AF9+shRNA1, with median survival of 32 days; MLL-AF9+shRNA2, with median survival of 32 days; MLL-AF9+shScramble, with median survival of 23.5 days; P〈 0.001 for both knockdown groups) (Fig.1B). Noticeable, mice in Mettl14 knockdown groups showed less c-kit+ cells in BM than mice in the control group (Fig.1C). In addition to the mouse model, we used human leukemia cell lines to investigate the function of METTL14 in human AML cells. Silencing of METTL14 with shRNAs significantly inhibited cell viability, induced apoptosis as well as terminal differentiation of MONOMAC6 and NB4 cell lines (Fig.1D, E, F). Moreover, xenograft model showed that repression of METTL14 significantly inhibited the engraftment of MONOMAC6 cells and thus delayed the onset of leukemia in NSG-SGM3 (NSGS) immunodeficient mice (Fig.1G). Furthermore, knockdown of METTL14 sensitized MONOMAC cells to ATRA or PMA-induced differentiation. Taken together, our results support the oncogenic role of METTL14 in AML and highlight METTL14 as a novel therapeutic target in AML. Figure 1 Oncogenic roles of METTL14 in AML. Figure 1. Oncogenic roles of METTL14 in AML. Disclosures No relevant conflicts of interest to declare.

Description: The transforming growth factor beta (TGF-β) signaling pathway controls hematopoietic stem cell (HSC) behavior in the marrow niche; however, TGF-β signaling becomes chronic in early-stage myelodysplastic syndrome (MDS). Although TGF-β signaling normally induces negative feedback, in early-stage MDS, high levels of microRNA-21 (miR-21) contribute to chronic TGF-β signaling. We found that a TGF-β signal–correlated gene signature is sufficient to identify an MDS patient population with abnormal RNA splicing (eg, CSF3R) independent of splicing factor mutations and coincident with low HNRNPK activity. Levels of SKI messenger RNA (mRNA) encoding a TGF-β antagonist are sufficient to identify these patients. However, MDS patients with high SKI mRNA and chronic TGF-β signaling lack SKI protein because of miR-21 activity. To determine the impact of SKI loss, we examined murine Ski−/− HSC function. First, competitive HSC transplants revealed a profound defect in stem cell fitness (competitive disadvantage) but not specification, homing, or multilineage production. Aged recipients of Ski−/− HSCs exhibited mild phenotypes similar to phenotypes in those with macrocytic anemia. Second, blastocyst complementation revealed a dramatic block in Ski−/− hematopoiesis in the absence of transplantation. Similar to SKI-high MDS patient samples, Ski−/− HSCs strikingly upregulated TGF-β signaling and deregulated expression of spliceosome genes (including Hnrnpk). Moreover, novel single-cell splicing analyses demonstrated that Ski−/− HSCs and high levels of SKI expression in MDS patient samples share abnormal alternative splicing of common genes (including those that encode splicing factors). We conclude that miR-21–mediated loss of SKI activates TGF-β signaling and alternative splicing to impair the competitive advantage of normal HSCs (fitness), which could contribute to selection of early-stage MDS-genic clones.

Description: Efforts to understand the genomic impact of human-disease-relevant genetic lesions and how they disrupt the normal sequence of cell-state transitions is hampered by a lack of defined hierarchical cellular states and corresponding networks of regulatory genes (transcription factors). Severe congenital neutropenia (SCN) patients display inherited and de novo mutations in Growth factor independent-1 (GFI1), which encodes a zinc-finger transcription factor. We identified known (e.g. N382S in zinc finger 5) and novel GFI1 sequence changes in SCN patients, then used lentiviral mediated expression to functionally evaluate them. GFI1-N382S, GFI1-K403R and GFI1-R412X mutations (in zinc finger 6) significantly elevated the expression of the Gfi1 target gene, Irf8. We generated mice with these patient-derived SCN-associated mutations in the murine Gfi1 locus. Neonatal and adult Gfi1N382S/- and Gfi1R412X/-mice are neutropenic, but Gfi1K403R/- mice have normal steady-state neutrophil levels. The resulting steady-state dysgranulopoiesis in adult mice was further pronounced in neonates. We noted that Gfi1R412X/-mice accumulate less Gfi1 protein than Gfi1+/+, while Gfi1R412X/R412Xhomozygous alleles genetically rescued both the hypomorphic protein defect and substantially restored neutrophil numbers (though not to normal). In contrast, functional challenge with neutrophil-dependent pathogens in vivo revealed a broad susceptibility for all Gfi1-mutant mice. To determine the underlying mechanism of neutropenia and immune defects, we first used novel flow cytometry analyses and Fluidigm C1 single cell RNA-Seq to establish the successive genomic states encompassing normal granulocyte specification and commitment. Independent CITE-Seq/10x sequencing analysis provided direct correlation between flow cytometry populations and genomic information, while also establishing the trajectory through genomic states traversed during terminal granulopoiesis. Next, using a novel bioinformatics algorithm (cellHarmony) we assigned Gfi1-mutant cells to their respective wild-type cell states and then determined differential gene expression. We find few genes deregulated across granulopoiesis, and that the bulk of transcriptional impact on Gfi1 target genes is specific to successive granulopoietic cell states. These insights facilitated Gfi1R412X/- Irf8+/-genetic rescue of granulocytic specification, but not post-commitment defects. We noted that a portion of Gfi1R412X/-gene deregulation unrepaired by genetic rescue was enriched for chromosome organization, proteolysis, and innate immune effectors. Electron microscopy revealed uncondensed chromatin in mature Gfi1R412X/-neutrophils while SWATH proteomics identified a loss of neutrophil granule proteins and members of the NADPH oxidase complex (potentially linking SCN with chronic granulomatous disease genes). To this end, we functionally validated impaired NADPH oxidase complex function in neutrophils from Gfi1-mutant mice. We noted Gfi1 mutant mice have consistently elevated levels of granulocyte colony stimulating factor (but not other cytokines), and so we extended our analysis of oxidative burst to GCSF-rescued human SCN patients to find profound defects; underscoring the inability of genetic or cytokine rescued specification to resolve post-commitment defects. We illustrate a work flow that can be broadly applied to molecularly dissect translationally relevant mouse models of disease, and underscores the necessity of evaluating mutations within the context of relevant cell states. Disclosures Dwivedi: Abbvie: Employment. Myers:Bellicum Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Nazor:BioLegend: Employment.

Description: DNA cytosine methylation is one of the best-characterized epigenetic modifications that play important roles in diverse cellular and pathological processes. The mechanism underlying the dynamic regulation of the level and distribution of 5-methylcytosine (5mC) as well as the biological consequence of DNA methylation deregulation have been interesting research topics in recent years. TET1, first identified as a fusion partner of the histone H3 Lys4 (H3K4) methyltransferase MLL (mixed-lineage leukemia) in acute myeloid leukemia (AML), is the founding member of the Ten-Eleven-Translocation (TET) family of DNA hydroxylases which are capable of converting 5mC to 5hmC (5-hydroxymethylcytosine) and lead to gene activation. Our group has previously demonstrated that TET1 plays an oncogenic role in MLL-rearranged leukemia (Huang H, et al. PNAS 2013; 110(29):11994-9). The expression of the TET1 protein and the global level of its enzymatic product, 5hmC, are significantly up-regulated in MLL-rearranged leukemia, whereas the opposite has been reported in other cancers where TET1 functions as a tumor suppressor. Therefore, a global understanding of the targets of TET1 in MLL-rearranged leukemia would greatly help to understand the role of TET1 in this specific type of AML. To this end, we performed proteomics study in parallel with RNA-seq to systematically explore the functional targets of TET1 in a genome-wide and unbiased way. Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomic profiling showed that when Tet1 was knocked down in MLL-ENL-estrogen receptor inducible (ERtm) mouse myeloid leukemia cells, a total of 123 proteins were down-regulated whereas 191 were up-regulated with a fold-change cutoff of 1.2 (Fig. 1A and B), representing positively and negatively regulated targets of TET1, respectively. Most of the proteins with altered expression upon Tet1 knock-down showed a corresponding change at the mRNA level as reflected by the RNA-seq data. Interestingly, gene ontology (GO) analysis indicated enrichment on genes associated with DNA replication and cell cycle progression. Among these genes, the minichromosome maintenance complex genes, including MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7, showed significant downregulation when Tet1 expression was depleted. We further conducted chromatin immunoprecipitation (ChIP) assays and demonstrated that TET1 binds directly to the CpG islands in the promoters of these MCM genes, suggesting that the regulation of the MCM genes by TET1 may occur at the transcriptional level. The six main minichromosome maintenance proteins (MCM2-7) are recruited to DNA replication origins in early G1 phase of the cell cycle and constitute the core of the replicative DNA helicase. We showed that not only the total levels of the MCM2-7 proteins, but also their binding to chromatin (Fig. 1C), were decreased by shRNAs against TET1 in human leukemia cell lines. Examination on cell cycle distribution revealed a significant decrease in the S phase population upon TET1 knockdown (Fig. 1D), which could be phenocopied by silencing of individual MCM genes. Consistently, incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into newly synthesized DNA in the S phase can be inhibited by TET1 shRNAs (Fig. 1E), indicating the inhibition on DNA replication by TET1 silencing. Furthermore, DNA combing assays suggest that TET1 knockdown inhibits new origin firing (Fig. 1F) but does not influence replication fork speed. Collectively, our findings reveal a novel role of TET1 on regulating DNA replication in MLL-rearranged leukemia through targeting of MCM genes and highlight the therapeutic implication of targeting the TET1/MCM signaling. Figure 1 Role of TET1 in regulate DNA replication by controlling expression of MCM genes Figure 1. Role of TET1 in regulate DNA replication by controlling expression of MCM genes Disclosures No relevant conflicts of interest to declare.

Description: To understand the molecular pathogenesis of human disease, precision analyses to define alterations within and between disease-associated cell populations are desperately needed. Single-cell genomics represents an ideal platform to enable the identification and comparison of normal and diseased transcriptional cell populations. We created cellHarmony, an integrated solution for the unsupervised analysis, classification, and comparison of cell types from diverse single-cell RNA-Seq datasets. cellHarmony efficiently and accurately matches single-cell transcriptomes using a community-clustering and alignment strategy to compute differences in cell-type specific gene expression over potentially dozens of cell populations. Such transcriptional differences are used to automatically identify distinct and shared gene programs among cell-types and identify impacted pathways and transcriptional regulatory networks to understand the impact of perturbations at a systems level. cellHarmony is implemented as a python package and as an integrated workflow within the software AltAnalyze. We demonstrate that cellHarmony has improved or equivalent performance to alternative label projection methods, is able to identify the likely cellular origins of malignant states, stratify patients into clinical disease subtypes from identified gene programs, resolve discrete disease networks impacting specific cell-types, and illuminate therapeutic mechanisms. Thus, this approach holds tremendous promise in revealing the molecular and cellular origins of complex disease.

Description: The transforming growth factor-beta (TGFβ) signaling pathway controls hematopoietic stem cell (HSC) behavior in the marrow niche; however, TGFb signaling becomes chronic in early-stage myelodysplasia (MDS) where it may select MDS-genic HSC clones. In MDS, the level of TGFb signaling may have prognostic value while TGFβ-receptor inhibitors improve hematopoiesis in MDS samples. Thus, to better understand MDS pathobiology, it is vital to understand the mechanisms underlying chronic TGFb signaling. Normally, TGFβ signaling is tightly controlled by antagonists (e.g. SMAD7, SKI) which block promiscuous activity. Upon ligand-receptor engagement, these antagonists are transiently eliminated to amplify the signal; however, they are re-induced by TGFβ signaling and subsequently terminate the signal (a negative feedback loop). Early-stage MDS marrow cells have significantly diminished expression of SMAD7 and elevated levels of microRNA-21 (miR-21), which targets SMAD7. Thus, miR-21 is one factor that interferes with the TGFβ negative feedback loop to generate a chronic TGFβ signal in MDS. We bioinformatically reanalyzed a recently published RNA-Seq dataset of MDS patient samples and find that a TGFb-signal-correlated gene signature is sufficient to identify a population of MDS patients with abnormal RNA splicing (e.g. CSF3R) independent of splicing factor mutations, and coincident with low HNRNPK activity. Elevated levels of SKI mRNA, encoding a transcriptional corepressor and TGFb-antagonist, are sufficient to identify these patients. We questioned why elevated SKI mRNA (encoding a TGFβ-antagonist) would be associated with chronic activation of TGFβ. We show that miR-21 targets SKI to block translation, and that event is associated with slight elevation in SKI mRNA abundance. Moreover, SKI protein is reduced in primary bone marrow samples from early-stage MDS patients with elevated miR-21 and chronic TGFβ signaling. To determine the impact of SKI loss, we examined murine Ski-/- HSC. Ski-/- HSC are initially specified and rescue transplant recipients in the absence of competitors, where they participate in multilineage hematopoiesis. However, when challenged with wild-type competitors, Ski-/- HSC display a profound defect in HSC fitness that can be rescued by increasing the number of Ski-/- HSC transplanted. Ski wild type and null embryonic stem cell - blastocyst complementation assays confirm an intrinsic Ski-/- HSC defect in the absence of transplantation. Using single-cell RNA-Seq, Ski-/- HSC exhibited striking upregulation of TGFb signaling, including Tgfb1 itself. Novel bioinformatics single-cell-splicing analyses revealed splicing alterations in Ski-/- HSC concordant with low HNRNPK activity. Strikingly, a large fraction of the differentially expressed genes and alternative splicing events in Ski-/- HSC are found in SKI-high MDS patients. We conclude that miR-21-mediated loss of SKI contributes to early stage MDS pathogenesis by activating TGFβ signaling and alternative splicing while hindering HSC fitness. Disclosures No relevant conflicts of interest to declare.