Faculty Bibliography

The myelodysplastic syndromes (MDS) represent a group of clonal disorders characterized by ineffective hematopoiesis, resulting in peripheral cytopenias and frequent transformation to acute myeloid leukemia (AML). We and others have demonstrated that MDS arises in, and is propagated by malignant stem cells (MDS-SCs), that arise due to the sequential acquisition of genetic and epigenetic alterations in normal hematopoietic stem cells (HSCs). This review focuses on recent advancements in the cellular and molecular characterization of MDS-SCs, as well as their role in mediating MDS clinical outcomes. In addition to discussing the cell surface proteins aberrantly upregulated on MDS-SCs that have allowed the identification and prospective isolation of MDS-SCs, we will discuss the recurrent cytogenetic abnormalities and genetic mutations present in MDS-SCs and their roles in initiating disease, including recent studies demonstrating patterns of clonal evolution and disease progression from pre-malignant HSCs to MDS-SCs. We also will discuss the pathways that have been described as drivers or promoters of disease, including hyperactivated innate immune signaling, and how the identification of these alterations in MDS-SC have led to investigations of novel therapeutic strategies to treat MDS. It is important to note that despite our increasing understanding of the pathogenesis of MDS, the molecular mechanisms that drive responses to therapy remain poorly understood, especially the mechanisms that underlie and distinguish hematologic improvement from reductions in blast burden. Ultimately, such distinctions will be required in order to determine the shared and/or unique molecular mechanisms that drive ineffective hematopoiesis, MDS-SC maintenance, and leukemic transformation.

Hematopoietic stem cells (HSCs) require highly regulated rates of protein synthesis, but it is unclear if they or lineage-committed progenitors preferentially recruit transcripts to translating ribosomes. We utilized polysome profiling, RNA sequencing, and whole-proteomic approaches to examine the translatome in LSK (Lin^-Sca-1⁺c-Kit⁺) and myeloid progenitor (MP; Lin^-Sca-1^-c-Kit⁺) cells. Our studies show that LSKs exhibit low global translation but high translational efficiencies (TEs) of mRNAs required for HSC maintenance. In contrast, MPs activate translation in an mTOR-independent manner due, at least in part, to proteasomal degradation of mTOR by the E3 ubiquitin ligase c-Cbl. In the near absence of mTOR, CDK1 activates eIF4E-dependent translation in MPs through phosphorylation of 4E-BP1. Aberrant activation of mTOR expression and signaling in c-Cbl-deficient MPs results in increased mature myeloid lineage output. Overall, our data demonstrate that hematopoietic stem and progenitor cells (HSPCs) undergo translational reprogramming mediated by previously uncharacterized mechanisms of translational regulation.

Inflammatory signals arising from the microenvironment have emerged as critical regulators of hematopoietic stem cell (HSC) function during diverse processes including embryonic development, infectious diseases, and myelosuppressive injuries caused by irradiation and chemotherapy. However, the contributions of cellular subsets within the microenvironment that elicit niche-driven inflammation remain poorly understood. Here, we identify endothelial cells as a crucial component in driving bone marrow (BM) inflammation and HSC dysfunction observed following myelosuppression. We demonstrate that sustained activation of endothelial MAPK causes NF-κB-dependent inflammatory stress response within the BM, leading to significant HSC dysfunction including loss of engraftment ability and a myeloid-biased output. These phenotypes are resolved upon inhibition of endothelial NF-κB signaling. We identify SCGF as a niche-derived factor that suppresses BM inflammation and enhances hematopoietic recovery following myelosuppression. Our findings demonstrate that chronic endothelial inflammation adversely impacts niche activity and HSC function which is reversible upon suppression of inflammation.

Background/UNASSIGNED:Glioma is a family of primary brain malignancies with limited treatment options and in need of novel therapies. We previously demonstrated that the adhesion G protein-coupled receptor GPR133 (ADGRD1) is necessary for tumor growth in adult glioblastoma, the most advanced malignancy within the glioma family. However, the expression pattern of GPR133 in other types of adult glioma is unknown. Methods/UNASSIGNED:We used immunohistochemistry in tumor specimens and non-neoplastic cadaveric brain tissue to profile GPR133 expression in adult gliomas. Results/UNASSIGNED:We show that GPR133 expression increases as a function of WHO grade and peaks in glioblastoma, where all tumors ubiquitously express it. Importantly, GPR133 is expressed within the tumor bulk, as well as in the brain-infiltrating tumor margin. Furthermore, GPR133 is expressed in both isocitrate dehydrogenase (IDH) wild-type and mutant gliomas, albeit at higher levels in IDH wild-type tumors. Conclusion/UNASSIGNED:The fact that GPR133 is absent from non-neoplastic brain tissue but de novo expressed in glioma suggests that it may be exploited therapeutically.

Aging hematopoiesis is characterized by increased numbers of immunophenotypic HSCs that exhibit impaired self-renewal and long-term reconstitution potential, both in competitive and noncompetitive settings. We previously demonstrated that normal young mouse HSCs (CD34-CD150+LSK) can be fractionated into subsets based on expression of c-Kit surface expression, with c-KitXX HSCs exhibiting reduced self-renewal and megakaryocytic biased differentiation (Shin et al., 2014). We therefore hypothesized that the expansion of c-KitXX HSCs in old mice could potentially explain the age-related decline in immunophenotypically defined old HSC function. Evaluation of the bone marrow of 24-month-old C57Bl/6 mice revealed that the frequency of c-KitXXHSCs (out of total HSCs) is 1.5-fold higher in old mice than in 3-month old mice (P=0.04), while the frequency of c-KitXX HSCs was 1.5-fold lower in old mice (P=0.007; Fig 1A). This finding is consistent with our previous observation of a megakaryocytic-bias in c-KitXXHSCs, since peripheral blood analysis of old mice revealed a 2.1-fold increase in platelets compared to young mice (p<0.01) (Fig 1B). To test the long-term reconstitution potential of aging HSCs, we competitively transplanted 400 c-KitXX HSCs from 24-month old mice, along with 300,000 competitor bone marrow cells, into lethally irradiated young recipients. Sixteen weeks post-transplantation, mice receiving old c-KitXX HSCs exhibited significantly lower donor peripheral blood chimerism levels compared to old c-KitXX HSC recipients (9.4% vs 57.1%, P=0.02) (Fig 1C). Both old c-KitXXand old c-KitXX HSCs exhibited similar myeloid-reconstituting potential (Fig 1D). Furthermore, mice transplanted with old c-KitXX HSCs exhibited 78% donor HSC chimerism, achieving 6.4-fold higher chimerism levels than mice transplanted with old c-KitXX HSCs, this was comparable to the differences observed with young c-KitXX transplanted HSCs (Fig 1E). To quantify the self-renewal capacity of old HSCs, we calculated the "self-renewal quotient" (Challen et al., 2010). This analysis showed that the self-renewal potential in old c-KitXX HSCs were 0.8 and 7.8 respectively, indicating higher self-renewal potential in c-KitXX HSCs (Fig 1F). Collectively, these data suggest that myeloid-biased differentiation is an age-associated change in hematopoiesis that may not be associated with decreased self-renewal in all HSCs. To gain mechanistic insights underlying these qualitative differences, we interrogated transcriptional profiles of microarray data from c-KitXX HSCs, to identify potential pathways critical for HSC maintenance. Gene Ontology and pathway analyses showed several differentially expressed pathways between c-KitXXHSCs, of which genes related to protein translation and mitochondrial activity was significantly enriched in c-KitXX HSCs (Fig 1G). Given the underrepresentation of translation-related genes in c-KitXX HSCs, we tested whether they exhibit reduced global translation using OP-Puro incorporation assays. These studies confirmed that old c-KitXX HSCs show lower global translation levels than c-KitXXHSCs (Fig 1H). Overall, our studies demonstrate functional heterogeneity among old HSCs and identify a novel strategy to identify old HSCs with preserved self-renewal and long-term reconstitution capacity. The ability to identify and prospectively fractionate old HSCs offers a novel approach to investigate the molecular mechanisms underlying HSC aging. Figure legend. (A) Frequency of c-KitXXor c-KitXX HSCs was assessed by flow cytometry. (B) Circulating platelet numbers were assessed using a Hemavet counter. Competitive transplants of old c-KitXX and c-KitXX HSCs into lethally irradiated recipients (C-F). Donor chimerism (C) and lineage potential (D) was evaluated in the peripheral blood of primary recipients. Bone marrow was analyzed at 16 weeks, for donor-derived HSC chimerism (E) and self-renewal quotient (F). (G) Enrichment plots comparing microarray data generated from c-KitXX HSCs, using pathways translation-related gene sets. (H) OP-Puro incorporation assays in 24-month old mice. Results are representative of three independent experiments, and shown as mean +/- SEM. n = 4-5 mice. *, P < 0.05; **, P < 0.01. [Formula presented] Disclosures: No relevant conflicts of interest to declare.XXCopyright

Epigenetic regulators in normal and malignant hematopoiesis have been shown to be important in normal and malignant stem cell self-renewal function and myeloid leukemogenesis. While epigenetic dysregulation can occur through activating and/or loss-of-function mutations, these regulators can also be modulated by other regulators, such as microRNAs. Specifically, miR-29 has been previously identified as an "epi-mir" for contributing to epigenetic regulation by altering expression of DNMT3a and TET. We and others have previously shown that in the hematopoietic system, miR-29 is a positive regulator of hematopoietic stem cell (HSC) self-renewal, is upregulated in AML blasts, and when over-expressed in transplanted HSCs and immature progenitors, leads to a myeloproliferative-like disorder that progresses to acute myeloid leukemia (AML). To investigate how miR-29 shapes the leukemic stem cell (LSC) epigenome, we transduced the MLL-AF9 fusion oncogene into WT and mir29a/b1 null Lin-ckit+sca1+ (LSK) cells and transplanted them into recipient mice. Transplantation of MLL-AF9+ miR-29 null cells into lethally irradiated recipients resulted in an increased disease latency compared to recipients of WT MLL-AF9+ cells (median: 56 vs 151 days, p<0.001). Characterization of miR-29 null blasts revealed increased expression of myeloid differentiation markers (CD11b, CD14), increased apoptosis, and reduced CFU capacity, consistent with decreased LSC self-renewal. To gain molecular insights into this phenotype, we interrogated RNA-seq data generated from WT and miR-29 null blasts. Compared to WT blasts, miR-29 null blasts showed loss of LSC gene signatures and enrichment for myeloid lineage genes, consistent with increased differentiation (Figure A). In addition, GSEA showed enrichment of H3K27me3, H3K4me2, and gene-specific polycomb group (PcG) associated pathways. On further validation, ChIP-Seq showed overall genome-wide reduction of DNA modification markers such as H3K27me3, H3K9me3, H3K36me3, H3K79me2, and H3K4me3 in miR-29 null blasts compared to WT. Hence, we hypothesized that miR-29 likely targets epigenetic regulators critical for LSC maintenance, and that loss of miR-29 rewires the LSC epigenetic landscape to induce differentiation and abrogate LSC self-renewal. In order to identify downstream mediators of the miR-29 null blast epigenetic phenotype, we used an shRNA library against 500 known epigenetic regulators. The top enriched genes in miR-29 null blasts were members of the PcG family, and as predicted, mRNAs encoding these proteins were upregulated in miR-29 null blasts, including CBX2. These findings were further corroborated by comparing the transcriptomes of AML patient samples to normal hematopoietic stem/progenitor cells (https://gexc.riken.jp

Prior studies in numerous biological systems have shown that alterations in mRNA expression frequently fail to predict changes in protein expression. This may be due to many regulatory mechanisms that occur post-transcriptionally including mRNA recruitment to ribosomes, translational initiation, ribosome processivity, and protein stability, among others. Indeed, several examples of selective translation of mRNAs has been described both in malignant and normal cells. To determine the extent and potential impact of translational reprogramming on early hematopoietic development, we performed an integrated analysis of total RNA, polysome RNA, and whole proteome data generated from HSC-enriched LSK (Lin^-Sca-1⁺c-Kit⁺) and MP (Lin^-Sca^-1-c-Kit⁺) cells from mouse. Our studies revealed that although LSK cells show lower global translation than MPs, they exhibited significantly higher translational efficiency (TE = polysome/total RNA abundance) of mRNAs supporting processes required for HSC maintenance (e.g. glycolysis, fatty acid metabolism, oxidative phosphorylation, mTOR signaling) (Fig 1A). Additionally, integrated analysis of proteomic and RNA expression data showed that, TE changes better predicted protein expression changes for these pathways, than total RNA expression (Fig1B). Biochemical characterization of MP cells revealed markedly decreased mTOR protein expression and signaling in MP cells, especially in GMP and MEP. This is mediated through proteasomal degradation of mTOR protein. An E3 ligase prediction algorithm, identified c-Cbl as a potential candidate, targeting mTOR, which was confirmed by demonstrating the aberrant expression of mTOR in MPs in c-Cbl KO mice. In vitro and in vivo mTOR inhibition studies confirm that the MPN-like phenotype of c-Cbl KO mice, is due to aberrant activation of mTOR signaling in committed myeloid progenitors. Intriguingly, despite decreased expression of mTOR protein in MP cells, 4E-BP1, a known target of mTOR, was still phosphorylated at Ser-65- a critical step for initiating cap-dependent translation. Through a combination of prediction algorithms and candidate gene experimental approaches, we show that the critical phosphorylation event at Ser-65 is mediated by, as immunoprecipitation studies show physical association between CDK1 and 4E-BP, and pharmacological inhibition of CDK1 activity, reduced 4E-BP P-Ser-65 levels. Overall, our data provide the first comprehensive characterization of the translatome in early hematopoiesis and demonstrated that the LSK to MP transition is characterized by significant translational reprogramming. This is, in part, mediated by the activation of a unique, mTOR-independent pathway to activate cap-dependent translation through the concerted action of c-Cbl and CDK1 to induce degradation of mTOR and phosphorylate 4E-BP to activation translation, respectively. Abrogation of the downregulation of mTOR signaling in myeloid progenitors, results in expansions of numerous myeloid lineages including neutrophils, monocytes and platelets (Fig 1C). Thus, our studies demonstrate the importance of proper translational reprogramming in early hematopoiesis. Figure legend. (A) Heatmap showing pathways significantly enriched in LSK and or MP cells based on TE. (B) Comparison of TE to protein expression in LSK cells for genes involved in the indicated biological processes (Blue dots: mRNAs that showed an anticorrelation between total RNA and protein expression; Red dots: mRNAs that showed a positive correlation between total RNA and protein expression). (C) Model for translational reprogramming in early hematopoiesis. Despite lower rates of global translation, LSK cells show preferential translation of mRNAs sensitive to mTOR inhibition and required for HSC maintenance. In contrast, in highly translating MP cells, loss of mTOR expression is mediated by the E3 ubiquitin ligase c-Cbl. When c-Cbl is deleted and mTOR protein is aberrantly expressed, this results in increased mature myeloid output. In the absence of mTOR, eIF4E-cap-dependent translation is maintained through the action of CDK1, which phosphorylates the S65 residue of 4E-BP1 to release eIF4E. [Formula presented] Disclosures: No relevant conflicts of interest to declare.
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Despite significant efforts to improve therapies for acute myeloid leukemia (AML), clinical outcomes remain poor. Understanding the mechanisms that regulate the development and maintenance of leukemic stem cells (LSCs) is important to reveal new therapeutic opportunities. We have identified CD97, a member of the adhesion class of G protein-coupled receptors (GPCRs), as a frequently up-regulated antigen on AML blasts that is a critical regulator of blast function. High levels of CD97 correlate with poor prognosis, and silencing of CD97 reduces disease aggressiveness in vivo. These phenotypes are due to CD97's ability to promote proliferation, survival, and the maintenance of the undifferentiated state in leukemic blasts. Collectively, our data credential CD97 as a promising therapeutic target on LSCs in AML.

The response to systemic infection and injury requires the rapid adaptation of hematopoietic stem cells (HSCs), which proliferate and divert their differentiation toward the myeloid lineage. Significant interest has emerged in understanding the signals that trigger the emergency hematopoietic program. However, the mechanisms that halt this response of HSCs, which is critical to restore homeostasis, remain unknown. Here we reveal that the E3 ubiquitin ligase Speckle-type BTB-POZ protein (SPOP) restrains the inflammatory activation of HSCs. In the absence of Spop, systemic inflammation proceeded in an unresolved manner, and the sustained response in the HSCs resulted in a lethal phenotype reminiscent of hyper-inflammatory syndrome or sepsis. Our proteomic studies decipher that SPOP restricted inflammation by ubiquitinating the innate signal transducer myeloid differentiation primary response protein 88 (MYD88). These findings unearth an HSC-intrinsic post-translational mechanism that is essential for reestablishing homeostasis after emergency hematopoiesis.