Faculty Bibliography

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.

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)