Faculty Bibliography

The myelodysplastic syndromes (MDS) represent a group of clonal disorders that result in ineffective hematopoiesis and are associated with an increased risk of transformation into acute leukemia. MDS arises from hematopoietic stem cells (HSCs); therefore, successful elimination of MDS HSCs is an important part of any curative therapy. However, current treatment options, including allogeneic hematopoietic cell transplantation (HCT), often fail to ablate disease-initiating MDS HSCs, and thus have low curative potential and high relapse rates. Here, we demonstrate that human HSCs can be targeted and eliminated by monoclonal antibodies (mAbs) that bind cell surface CD117 (c-Kit). We show that an anti-human CD117 mAb, SR-1, inhibits normal cord blood and bone marrow HSCs in vitro. Further, SR-1 and clinical-grade humanized anti-human CD117 mAb, AMG 191, deplete normal and MDS HSCs in vivo in xenograft mouse models. Anti-CD117 mAbs also facilitate the engraftment of normal donor human HSCs in MDS xenograft mouse models, restoring normal human hematopoiesis and eradicating aggressive pathologic MDS cells. This study is the first to demonstrate that anti-human CD117 mAbs have potential as novel therapeutics to eradicate MDS HSCs and augment the curative effect of allogeneic HCT for this disease. Moreover, we establish the foundation for use of these antibody agents not only in the treatment of MDS but for the multitude of other HSC-driven blood and immune disorders for which transplant can be disease-altering.

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.