Faculty Bibliography

Mechanisms of translational regulation are poorly understood in hematopoietic stem cells (HSCs) and committed progenitors (MP). In order to investigate the impact of translational regulation on early mouse hematopoietic development, we characterized the translatome using a combination of polysome profiling, RNA-sequencing (RNA-seq) of polysome-associated and total cellular mRNA, and whole proteome evaluation. Comparison of RNA-seq data from HSC-enriched LSK (Lin Sca-1 c-Kit) and MP (Lin Sca 1-c-Kit) cells showed more differentially expressed mRNAs in polysomal RNA than total RNA (412 vs 280 mRNAs, respectively) among ~15,000 mRNAs analyzed. In addition, polysomal mRNAs were enriched for a unique set of functional pathways (e.g. inflammatory response, apoptosis and p53) compared to total RNA in both LSK and MP cells. Interestingly, although LSK cells showed ~20% lower global translation than MPs as demonstrated by polysome profiling (Figure 1A), they exhibited significantly higher translational efficiency (TE = polysome RNA abundance/total RNA abundance) for mRNAs that support HSC maintenance (e.g. glycolysis, fatty acid metabolism, oxidative phosphorylation, mTOR signalling). Integration of proteomic and RNA-seq data demonstrated that 605 out of 784 (77.2%) differentially expressed genes (DEGs) between LSK and MP cells identified based on total RNA-seq data (Groups I & III; Fig 1B) also showed a corresponding change in protein expression, while remaining 179 DEGs (22.8%; Groups II & IV; Fig. 1B) showed an anti-correlation. Remarkably, in the latter group, expression of 129 proteins (72.1% of all differentially expressed proteins in LSKs vs MPs) correlated with their TEs (Figure 1C). While gene set enrichment analysis of published HSC regulators showed an enrichment in LSKs in total RNA-seq data, such an enrichment was not observed when evaluating mRNAs with differential TEs. However, mRNAs with high TE confirmed a surprising enrichment in mTOR-responsive genes independent of their total RNA expression in LSKs, but not in MP cells (Figure 1D). To investigate the biochemical basis of this observation, we performed western blot analysis of LSK and MP cells and observed decreased mTOR protein expression and signaling in purified MP cells, despite their higher global levels of translation. In addition, despite abundant expression of mTOR protein in LSK cells, 4E-BP1, a known target of mTOR, was only phosphorylated at the priming residues Thr-37/46 but not at the downstream Ser-65, a residue that initiates cap-dependent translation. In contrast, MP cells phosphorylated Ser-65, consistent with its increased translation despite the absence of mTOR signalling. mTOR inhibition with Torin-1 did not alter 4E-BP Ser-65 phosphorylation or translation in MPs ex vivo. The presence of mTOR-independent translation in MPs was corroborated by in vivo rapamycin treatment studies, which induced increased colony formation by LSKs, but not MPs. Decreased mTOR activity in MPs was due to degradation of mTOR protein mediated by the proteasome since mTOR protein expression was restored following treatment with the proteasome inhibitors bortezomib and MG132, as well as deletion of the E3 ubiquitin ligase, c-Cbl. Indeed, LSKs and MPs exhibit differential dependencies on mTOR signaling for translation, as mTOR protein is post-translationally downregulated in MPs by a previously undescribed mechanism for mTOR proteosomal degradation mediated by c-Cbl. These findings establish the presence of developmental stage-specific mechanisms of translational regulation in early hematopoiesis. Figure legend. (A) Representative polysome profiles from LSK (Lin Sca-1 c-Kit) and MP (Lin Sca-1 c-Kit) cells. Polysome/subpolysome ratios (poly/subpoly) were calculated by dividing total RNA abundance from polysomes (fractions 5-10) by subpolysomes (fractions 1-4) (n=3, p < 0.05). (B) Comparison of total RNA versus protein expression in LSK versus MP cells. Four groups of mRNAs were identified based on comparisons of their total mRNA and protein expression. Percentage of total analyzed mRNAs is indicated in each quadrant. (C) Comparison of TEs versus protein expression in LSK/MP cells (D) Enrichment plots comparing total RNA and TE in LSK and MP cells, using previously validated mTOR gene sets. (Figure presented)

INTRODUCTION/BACKGROUND:Acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS) are clonal hematopoietic neoplasms that arise from leukemia stem cells (LSCs) and hematopoietic stem cells (HSCs), respectively. Standard chemotherapy can efficiently eliminate the bulk of neoplastic cells, however, LSCs and MDS HSCs are relatively resistant to these therapies and can reinitiate and maintain disease. CD99 is a 32-kDa transmembrane polypeptide that is highly expressed on disease stem cells in the vast majority of AML and MDS. Areas covered: In this editorial, we focus on the current literature surrounding the identification of CD99 as a marker of MDS and AML stem cells and preclinical studies revealing the therapeutic efficacy of targeting CD99 in these diseases. Expert opinion/commentary: Cytotoxic CD99 monoclonal antibodies represent promising stem cell-directed therapies that have the potential to markedly improve clinical outcomes for these difficult-to-treat hematologic malignancies.

Three recent studies independently identified the m6A RNA modifying enzymes METTL3 and METTL14 as critical regulators of differentiation in both normal hematopoiesis and AML pathogenesis. These studies expand the described roles of the epitranscriptome in maintaining the undifferentiated state in somatic stem cells and human cancer.

The aging hematopoietic system undergoes numerous changes, including reduced production of red blood cells and lymphocytes as well as a relative increase in the production of myeloid cells. Emerging evidence indicates that many of these changes are due to selection pressures from cell-intrinsic and cell-extrinsic factors that result in clonal shifts in the hematopoietic stem cell (HSC) pool, resulting in predominant HSC clones that exhibit the functional characteristics associated with HSC aging. Given the recent descriptions of clonal hematopoiesis in aged populations, the increased risk of developing hematologic malignancies in individuals with clonal hematopoiesis, and the many similarities in hematopoietic aging and acquired bone marrow failure (BMF) syndromes, such as myelodysplastic syndromes (MDS), this raises significant questions regarding the relationship between aging hematopoiesis and MDS, including the factors that regulate HSC aging, whether clonal hematopoiesis is required for the development of MDS, and even whether BMF is an inevitable consequence of aging. In this article, we will review our current understanding of these processes and the potential intersections among them.

The aging hematopoietic system undergoes numerous changes, including reduced production of red blood cells and lymphocytes as well as a relative increase in the production of myeloid cells. Emerging evidence indicates that many of these changes are due to selection pressures from cell-intrinsic and cell-extrinsic factors that result in clonal shifts in the hematopoietic stem cell (HSC) pool, resulting in predominant HSC clones that exhibit the functional characteristics associated with HSC aging. Given the recent descriptions of clonal hematopoiesis in aged populations, the increased risk of developing hematologic malignancies in individuals with clonal hematopoiesis, and the many similarities in hematopoietic aging and acquired bone marrow failure (BMF) syndromes, such as myelodysplastic syndromes (MDS), this raises significant questions regarding the relationship between aging hematopoiesis and MDS, including the factors that regulate HSC aging, whether clonal hematopoiesis is required for the development of MDS, and even whether BMF is an inevitable consequence of aging. In this article, we will review our current understanding of these processes and the potential intersections among them.

Loss-of-function mutations in TET2 occur frequently in patients with clonal hematopoiesis, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and are associated with a DNA hypermethylation phenotype. To determine the role of TET2 deficiency in leukemia stem cell maintenance, we generated a reversible transgenic RNAi mouse to model restoration of endogenous Tet2 expression. Tet2 restoration reverses aberrant hematopoietic stem and progenitor cell (HSPC) self-renewal in vitro and in vivo. Treatment with vitamin C, a co-factor of Fe2+ and alpha-KG-dependent dioxygenases, mimics TET2 restoration by enhancing 5-hydroxymethylcytosine formation in Tet2-deficient mouse HSPCs and suppresses human leukemic colony formation and leukemia progression of primary human leukemia PDXs. Vitamin C also drives DNA hypomethylation and expression of a TET2-dependent gene signature in human leukemia cell lines. Furthermore, TET-mediated DNA oxidation induced by vitamin C treatment in leukemia cells enhances their sensitivity to PARP inhibition and could provide a safe and effective combination strategy to selectively target TET deficiency in cancer.

The microRNA-99 (miR-99) family comprises a group of broadly conserved microRNAs that are highly expressed in hematopoietic stem cells (HSCs) and acute myeloid leukemia stem cells (LSCs) compared with their differentiated progeny. Herein, we show that miR-99 regulates self-renewal in both HSCs and LSCs. miR-99 maintains HSC long-term reconstitution activity by inhibiting differentiation and cell cycle entry. Moreover, miR-99 inhibition induced LSC differentiation and depletion in an MLL-AF9-driven mouse model of AML, leading to reduction in leukemia-initiating activity and improved survival in secondary transplants. Confirming miR-99's role in established AML, miR-99 inhibition induced primary AML patient blasts to undergo differentiation. A forward genetic shRNA library screen revealed Hoxa1 as a critical mediator of miR-99 function in HSC maintenance, and this observation was independently confirmed in both HSCs and LSCs. Together, these studies demonstrate the importance of noncoding RNAs in the regulation of HSC and LSC function and identify miR-99 as a critical regulator of stem cell self-renewal.

Langerhans Cell Histiocytosis (LCH) and the non-LCH neoplasm Erdheim-Chester Disease (ECD) are heterogeneous neoplastic disorders marked by infiltration of pathologic macrophage-, dendritic cell-, or monocyte-derived cells in tissues driven by recurrent mutations activating MAP kinase signaling. Although recent data indicate that at least a proportion of LCH and ECD patients have detectable activating kinase mutations in circulating hematopoietic cells and bone marrow-based hematopoietic progenitors, functional evidence of the cell-of-origin of histiocytosis from actual patient materials has long been elusive. Here we provide evidence for mutations in MAP kinase signaling intermediates in CD34+ cells from patients with ECD and LCH/ECD, including detection of shared origin of LCH and acute myelomonocytic leukemia driven by TET2-mutant CD34+ cell progenitors in one patient. We also demonstrate functional self-renewal capacity for CD34+ cells to drive the development of histiocytosis in xenotransplantation assays in vivo. These data indicate that the cell-of-origin of at least a proportion of patients with systemic histiocytoses resides in hematopoietic progenitor cells prior to committed monocyte/macrophage or dendritic cell differentiation and provide the first example of a patient-derived xenotransplantation model for a human histiocytic neoplasm.

The mechanism of action of oncogenes in acute myeloid leukaemia is poorly understood. A study now shows that the fusion oncoprotein AML1-ETO regulates leukaemogenesis by increasing the expression of small nucleolar RNAs through post-transcriptional mechanisms, resulting in increased ribosomal RNA methylation, protein translation, and promotion of leukaemic-cell self-renewal and growth.