Extracellular vesicles impose quiescence on residual hematopoietic stem cells in the leukemic niche
2019; Springer Nature; Volume: 20; Issue: 7 Linguagem: Inglês
10.15252/embr.201847546
ISSN1469-3178
AutoresSherif Abdelhamed, John T. Butler, Ben Doron, Amber Halse, Eneida R. Nemecek, Phillip A. Wilmarth, Daniel L. Marks, Bill H. Chang, Terzah M. Horton, Peter Kurre,
Tópico(s)Acute Myeloid Leukemia Research
ResumoArticle17 June 2019free access Source Data Extracellular vesicles impose quiescence on residual hematopoietic stem cells in the leukemic niche Sherif Abdelhamed Department of Pediatrics, Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR, USA Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author John T Butler Department of Pediatrics, Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR, USA Department of Biomedical Engineering, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Ben Doron Department of Pediatrics, Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Amber Halse Department of Pediatrics, Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Eneida Nemecek Department of Pediatrics, Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR, USA Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Phillip A Wilmarth Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR, USA Proteomics Shared Resources, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Daniel L Marks Department of Pediatrics, Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR, USA Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA Brenden-Colson Center for Pancreatic Care, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Bill H Chang Department of Pediatrics, Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR, USA Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Terzah Horton Texas Children's Cancer and Hematology Centers, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Peter Kurre Corresponding Author [email protected] orcid.org/0000-0003-2747-0099 Children's Hospital of Philadelphia, Comprehensive Bone Marrow Failure Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Sherif Abdelhamed Department of Pediatrics, Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR, USA Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author John T Butler Department of Pediatrics, Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR, USA Department of Biomedical Engineering, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Ben Doron Department of Pediatrics, Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Amber Halse Department of Pediatrics, Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Eneida Nemecek Department of Pediatrics, Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR, USA Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Phillip A Wilmarth Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR, USA Proteomics Shared Resources, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Daniel L Marks Department of Pediatrics, Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR, USA Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA Brenden-Colson Center for Pancreatic Care, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Bill H Chang Department of Pediatrics, Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR, USA Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Terzah Horton Texas Children's Cancer and Hematology Centers, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Peter Kurre Corresponding Author [email protected] orcid.org/0000-0003-2747-0099 Children's Hospital of Philadelphia, Comprehensive Bone Marrow Failure Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Author Information Sherif Abdelhamed1,2, John T Butler1,3, Ben Doron1, Amber Halse1, Eneida Nemecek1,2, Phillip A Wilmarth4,5, Daniel L Marks1,2,6, Bill H Chang1,2, Terzah Horton7 and Peter Kurre *,8 1Department of Pediatrics, Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR, USA 2Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA 3Department of Biomedical Engineering, Oregon Health & Science University, Portland, OR, USA 4Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR, USA 5Proteomics Shared Resources, Oregon Health & Science University, Portland, OR, USA 6Brenden-Colson Center for Pancreatic Care, Oregon Health & Science University, Portland, OR, USA 7Texas Children's Cancer and Hematology Centers, Baylor College of Medicine, Houston, TX, USA 8Children's Hospital of Philadelphia, Comprehensive Bone Marrow Failure Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA *Corresponding author. Tel: +1 215 590 0205; E-mail: [email protected] EMBO Rep (2019)20:e47546https://doi.org/10.15252/embr.201847546 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Progressive remodeling of the bone marrow microenvironment is recognized as an integral aspect of leukemogenesis. Expanding acute myeloid leukemia (AML) clones not only alter stroma composition, but also actively constrain hematopoiesis, representing a significant source of patient morbidity and mortality. Recent studies revealed the surprising resistance of long-term hematopoietic stem cells (LT-HSC) to elimination from the leukemic niche. Here, we examine the fate and function of residual LT-HSC in the BM of murine xenografts with emphasis on the role of AML-derived extracellular vesicles (EV). AML-EV rapidly enter HSC, and their trafficking elicits protein synthesis suppression and LT-HSC quiescence. Mechanistically, AML-EV transfer a panel of miRNA, including miR-1246, that target the mTOR subunit Raptor, causing ribosomal protein S6 hypo-phosphorylation, which in turn impairs protein synthesis in LT-HSC. While HSC functionally recover from quiescence upon transplantation to an AML-naive environment, they maintain relative gains in repopulation capacity. These phenotypic changes are accompanied by DNA double-strand breaks and evidence of a sustained DNA-damage response. In sum, AML-EV contribute to niche-dependent, reversible quiescence and elicit persisting DNA damage in LT-HSC. Synopsis Acute myeloid leukemia (AML) functionally remodels the bone marrow niche. AML cells constitutively release extracellular vesicles (EVs) enriched in miR-1246, that traffic to long-term hematopoietic stem cells (LT-HSC) and induce quiescence and DNA damage. Following EV uptake into LT-HSC, miR-1246 downregulates the Regulatory-associated protein of mTOR (Raptor) leading to the hypo-phosphorylation and deregulation of S6 ribosomal protein (S6RP). Deregulation of the S6RP mediates protein synthesis suppression associated with P53-dependent quiescence. While quiescence and protein synthesis suppression are reversible upon transfer to naïve recipients, cells accrue double-strand DNA-breaks that persist, and generate a long-lasting DNA-damage response (DDR) while conferring gains in proliferative capacity. Introduction Acute myeloid leukemia (AML) is a genetically heterogeneous disease that arises from mutations in hematopoietic stem and progenitor cells (HSPC) 1. The characteristic, and often disproportionate, suppression of native hematopoiesis that develops in the bone marrow (BM) during disease progression and post-treatment relapse accounts for significant morbidity and mortality 2-6. Substantial experimental evidence supports the malignant transition of BM function during AML invasion, and its role in disrupting hematopoiesis and sustaining AML 7-12. Several prior studies of BM niche-conversion emphasize leukemia-induced alterations in stromal and vascular function 8, 13-15. Cell–cell interactions also target the hematopoietic components in the BM and contribute to the functional suppression and displacement of the hematopoietic progenitors predominantly responsible for steady-state hematopoiesis 15-20. The fate of residual hematopoietic stem cells (HSC) under leukemic stress, however, has been more elusive. Unlike the depletion of highly susceptible HSPC, HSC have proved to be more resilient during leukemic invasion, and multiple groups reported the relative accumulation of primitive hematopoietic cells in both murine models and xenograft studies 8, 15, 16, 21-23. Intriguingly, HSC in the leukemic niche enter quiescence through an unidentified process, yet appear to retain their repopulation capacity upon subsequent re-transplantation 22. Extracellular vesicles (EV) comprise multiple populations of nano-sized vesicles, which carry protein and nucleic acids, participate in the regulation of BM function 24-26. We recently showed that AML-EV, including exosomes, are highly abundant in microRNA (miR)-150 and miR-155, which both target the transcription factor c-Myb to suppress HSPC clonogenicity 17, 27-29. Here, we test the hypothesis that EV impact the fate of residual HSC in the AML niche via a distinct mechanism, since HSC function does not rely on c-Myb expression at high levels 30. Our studies in immunodeficient mice confirm the relative accumulation and quiescence of residual HSC previously observed 16, 18, 22, 23, and reveal that AML-EV suppress protein synthesis in LT-HSC. Mechanistically, AML-EV transfer miR-1246 to LT-HSC to cause the translational suppression of the mTOR subunit Raptor, which in turn facilitates the hypo-phosphorylation of S6RP with ensuing deficits in protein synthesis. Intriguingly, while these changes are resolved upon transfer to a naïve BM niche, we show that AML-EV elicit DNA damage that persists in vitro and in vivo through serial progenitor replating and transplantation, respectively. Results AML-EV are taken up by hematopoietic cells, including LT-HSC We previously showed 17, 28, 29, 31 and herein confirmed that AML cells (Molm-14 and U-937) predominantly release nano-sized, lipid bilayer vesicles with a diameter of 50–130 nm, as demonstrated by Cryo-TEM imaging (Fig 1A). To investigate the quantitative uptake of AML-EV in HSC, we relied on a set of AML cell lines (Molm-14, U-937, and HL-60) that were transduced with a lentiviral vector to constitutively express green fluorescence protein with a myristoyl group (mGFP) (Fig 1B). The resulting GFP-tag was incorporated into the lipid bilayer of both the cell and the released EV, allowing measurement of uptake in vivo and in vitro, as previously reported 17. As modeled in Fig 1C, we then injected these engineered AML cells into NSG mice for 3–6 weeks to allow the AML cells to reach to 20–40% of the BM. We targeted low levels of chimerism to minimize cell–cell contact driving the AML-HSC crosstalk. GFP+ EV purified from the peripheral blood plasma of Molm-14 and the U-937 xenografts were visualized by fluorescence microscopy (Fig 1D). Live-cell imaging of xenograft-derived KSL and LT-HSC demonstrated the uptake of mGFP+ EV into the intracellular space (Fig 1E). Next, we measured the kinetics of EV uptake by exposing KSL and LT-HSC to EV harvested from Molm-14-mGFP or U-937-mGFP cells in vitro. By capturing live-cell 3D z-stacks at 0, 30, and 150 min, we found that both KSL and LT-HSC bind and internalize numerous mGFP+ EV within 30 min of exposure with continued accumulation at 150 min (Fig EV1A). We also analyzed KSL from wild-type Molm-14 xenografts to rule out confounding autofluorescence. HSPC harvested from non-transduced Molm-14 xenografts contained no mGFP+ foci and exhibited relative background fluorescence similar to non-xenografted controls (Fig EV1B), confirming that mGFP+ foci were membrane-derived vesicles originating from Molm-14-mGFP and U-937-mGFP cells. Figure 1. In vivo uptake of AML-EV in hematopoietic stem cells Cryo-TEM images demonstrate the lipid bilayer EV purified from Molm-14 and U-937 cells. Scale bars are 100 nm. A schematic diagram of the myristoylated GFP (mGFP)-expressing lentiviral construct and its incorporation into the cell membrane and EV. Long terminal repeat (LTR), poly-adenylate (pA), cytomegalovirus (CMV). Schematic diagram of the workflow. Cells were injected via tail-vein injection into NSG mice. After 21 days, bone marrow (BM) cells were flushed to sort GFP+ cells by flow cytometry and perform imaging of sorted HSC. Peripheral blood (PB) plasma of control animals contains no mGFP+ foci (top); however, Molm-14-mGFP (middle) and U-937-mGFP (bottom) xenografts contain numerous mGFP-labeled EV (green) detectible without vesicle concentration. Scale bars are 5 μm. Live-cell microscopy of KSL cells (left panel) LT-HSC cells (right panel). Cells were sorted from control mice (top), Molm-14-mGFP (middle), and U-937-mGFP (bottom), stained with Cell Mask (red) and nuclear stain Hoechst (blue), imaged using the GE/API Deltavision (DV) widefield microscope (60× objective) to show the uptake of the GFP+ EV (green). Scale bars are 5 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. The kinetics of AML-EV interaction with BM hematopoietic cells Microscopic images using the GE/API Deltavision widefield microscope (60× objective) of live LT-HSC (top rows), or KSL (bottom rows), in vitro exposed to EV from Molm-14-mGFP and U-937-mGFP cells for 0, 30, and 150 min. Green: mGFP+ EV, red: plasma membrane surface. Scale bars are 5 μm. Quantification of mGFP+ EV foci in KSL FACS purified from AML xenografts: wild-type Molm-14 (n = 40), Molm-14-mGFP (n = 247), and U-937-mGFP (n = 107). The background autofluorescence was measured in non-engrafted controls, and this threshold value was then subtracted from xenografted mice. Individual mGFP+ foci were counted in individual KSL from xenografted animals using Imaris software. Microscopic quantification of EV from hCD34 (3 × 107 cells) or Molm-14 (3 × 107 cells). The purified EV were stained with the lipid dye, FM1-43, imaged using the Yokogawa CSU-W1 spinning disk microscope (100× objective) and quantified using Imaris software. Separate measurements for hCD34, 11; Molm14 EV, 8. Download figure Download PowerPoint AML-EV increase the relative frequency of LT-HSC, upregulate P53, and confer quiescence To study the effect of AML-EV on residual HSC fate, we relied on the Molm-14-xenograft model complemented with intrafemoral (IF) injection of EV from Molm-14, HL-60, and U-937 cell lines versus EV from expanded healthy donor BM CD34+ cell (hCD34) followed by flow cytometric analysis (Fig 2A). To ensure a valid comparison of the injected EV, we normalized the numbers of the EV-producing cells (EV from 3 × 107 cells/femur) and confirmed their EV concentration by microscopic quantification using a lipid dye, FM1-43, finding no significant differences (Fig EV1C). Figure 2. AML-EV increase the relative frequency of LT-HSC, upregulate P53 expression, and confer quiescence A. Schematic diagram of methods: the in vivo AML xenografts (tail-vein injection of 105 Molm-14 cells or vehicle per mouse) and the intrafemoral (IF) injection of AML-EV into one femur with a contralateral control vehicle-injected femur of the same mouse. EV were isolated by serial high-speed centrifugation (at 2×, 10×, 100 × 103 g) from AML cell-line culture media or AML patient plasma. Bone marrow was flushed from long bones at the indicated time points, and immunophenotypic analysis was performed by flow cytometry. B, C. Flow cytometric analysis showing the frequency of LK and KSL cells in lineage-negative cells (left panel) and LT-HSC in KSL (right panel) in: (B) Molm-14 xenograft (red, n = 10) versus control (black, n = 10). Data were obtained from at least two independent experiments. (C) IF injection of EV from Molm-14 cells (red, n = 8), AML plasma EV (orange, n = 6), and human CD34 EV (blue, n = 4) relative to the vehicle-injected contralateral femurs. Data were obtained from at least two independent experiments. Statistics: Student's t-test (*P < 0.05, ***P < 0.001). D. Cell-cycle histograms of KSL (upper panel) and LT-HSC (lower panel) from Molm-14 xenografts or control using Hoechst-33342 staining. E. Flow cytometric analysis of the Ki67-ve percentage of LT-HSC representing the G0 phase of cell cycle in: (left panel) Molm-14 xenografts (red, n = 7) versus control (black, n = 9); (right panel) IF injection of Molm-14-EV, U-937 EV, HL-60 EV (red, n = 5,4,4) versus human CD34 EV (blue, n = 3) versus controls (black). Data were obtained from at least two independent experiments Statistics: Student's t-test (*P < 0.05, **P < 0.01). F, G. Flow cytometric analysis of intracellular P53 levels shown in histograms (F) and MFI (G) of LT-HSC in: (left panel) Molm-14 xenograft, n = 6 red versus non-engrafted control, n = 4 black, or (right panel) IF injection of EV from Molm-14, U-937, HL-60 (red, n = 5,5,3) or human CD34 cells (blue, n = 6) normalized to vehicle-injected contralateral femurs. Data were obtained from at least two independent experiments. Statistics: Student's t-test (*P < 0.05, **P < 0.01). Download figure Download PowerPoint We first assessed the frequencies of myeloid progenitor LK (lineage−/cKit+); the early progenitor/stem pool KSL (Lin−/cKit+/Sca-1+); and LT-HSC (KSL/CD48−/CD150+) (Fig 2A). While there was no change in KSL frequency or absolute cell number, AML-EV caused a relative reduction in myeloid progenitor LK cells (Figs 2B and C, and EV2A). More importantly, we observed a significant increase in LT-HSC frequency and absolute cell number in Molm-14 xenografts and after IF injection of Molm-14-EV, but not after control CD34+ EV (Figs 2B and C, and EV2A). In addition to cell-line-derived EV, we also tested EV from the plasma of six AML patients (Appendix Table S2). IF injection of patient plasma EV confirmed the observed reduction in LK and a concomitant increase in LT-HSC (Fig 2C). Together, the data suggest suppressed progenitor differentiation with proportional accumulation in LT-HSC after exposure to AML-EV. Click here to expand this figure. Figure EV2. AML-EV induce a p53-dependent quiescence in hematopoietic cells with no evidence of apoptosis Flow cytometric analysis showing the absolute cell number of LK and KSL cells in lineage-negative cells (left panel) and LT-HSC (right panel) in Molm-14 xenograft (red, n = 10) versus control (black, n = 10). Statistics: Student's t-test (*P < 0.05). Flow cytometric analysis of KSL percentage in G0 phase from Molm-14 xenografts (red, n = 7) versus control (black, n = 9). Statistics: Student's t-test (*P < 0.05). Flow cytometric analysis of P53 MFI in KSL in: (left panel) Molm-14 xenograft (red, n = 6) versus control (black, n = 4), or (right panel) IF injection of EV from Molm-14, U-937, HL-60 (red, n = 5,5,3), or CD34 cells (blue, n = 6) normalized to vehicle-injected femurs. Statistics: Student's t-test (*P < 0.05, **P < 0.01). Flow cytometric analysis of pP53ser15 MFI of LT-HSC in: (upper panel) Molm-14 xenografts, n = 9 red versus control, n = 7 black, or (lower panel) LT-HSC and KSL after IF injection of Molm-14 EV (red, n = 4) normalized to vehicle-injected contralateral femurs. Data are presented after subtracting the background fluorescence. Statistics: Student's t-test (*P < 0.05). Flow cytometric analysis of the pMDM2 MFI in KSL (upper panel) and LT-HSC (lower panel) in Molm-14 xenografts (red, n = 5) versus control (black, n = 4). Statistics: Student's t-test (*P < 0.05, **P < 0.01). qRT–PCR showing the fold change of Cdkn1a (upper panel) and P16INK4a (lower panel) in KSL from Molm-14 xenografts relative to control mice and normalized to Gapdh endogenous control. Data are mean ± SEM from at least three independent experiments with technical replicates. Statistics: one-way ANOVA with Bonferroni post hoc correction (*P < 0.05). Annexin V+ analysis of KSL (upper panel) and LT-HSC (lower panel), in Molm-14 xenograft (red, n = 4) versus control (black, n = 4). Statistics: Student's t-test (NS = not significant).&!#6; Download figure Download PowerPoint To further test this hypothesis, we investigated the impact of AML-EV on cell-cycle status in the xenograft model and after direct injection of AML-EV. We found that AML-EV consistently induced quiescence in LT-HSC as shown in the histograms after Hoechst staining (Fig 2D), as well as the fraction of cells in G0 cell-cycle phase determined by Ki67 staining (Fig 2E). AML-EV-mediated quiescence was also observed in the bulk KSL population (Fig EV2B) and occurred after IF injection of AML-EV, but not after injection of EV from healthy hCD34 control cultures. Owing to its crucial role in regulating HSC quiescence 32, we evaluated the P53 activation in residual HSC. We found a significant upregulation of P53 in LT-HSC and KSL from the xenografted mice as well as the IF-injected mice with EV from AML cells or patient plasma (Figs 2F–G and EV2C). We also observed hyper-phosphorylation of P53Ser15 in LT-HSC influenced by AML-EV (Fig EV2D). We found a significant hypo-phosphorylation of MDM2ser166, a negative regulator of P53, in both KSL and LT-HSC from xenografted mice and after IF injection of Molm-14-EV (Fig EV2E). In further agreement, we observed transcriptional upregulation of the Cdkn1a (p21), a P53-effector, in KSL from Molm-14 xenografts relative to control (Fig EV2F). We found no evidence of senescence induction via P16INK4a expression in KSL from Molm-14 xenografts (Fig EV2F). Likewise, no evidence of apoptosis was observed in KSL or in LT-HSC from Molm-14 xenografts (Fig EV2G). Together, our data thus far demonstrated a proliferative defect in LT-HSC by AML-EV. AML-EV induces ribosome biogenesis suppression in hematopoietic cells To understand the mechanism by which AML-EV may enforce quiescence, we performed tandem mass tag proteomic profiling of in vitro-cultured cKit+ HSPC (to obtain the minimum required amount of protein lysates) treated with EV from HL-60 or Molm-14 for 48 h versus vehicle-treated controls. We used the differential expression statistical package EdgeR 33 with multiple testing corrections to calculate the false discovery rate (FDR) of differentially expressed proteins between EV- and vehicle-treated controls. Numbers of quantifiable proteins and biological replicates differed between the Molm-14 experiments (4,407 proteins, 2 vehicle replicates, and 4 EV replicates) and the HL-60 experiments (4883 proteins, 4 vehicle replicates, and 4 EV replicates). We chose FDR cutoffs for each experiment (FDR < 10−2 for the Molm-14 experiment and FDR < 10−9 for the HL-60 experiment) to produce lists of differentially abundant candidates that contained ~10% of the quantifiable proteins (Datasets EV1 and EV2). There were 394 differentially regulated proteins in response to the Molm-14-EV (221 upregulated and 173 downregulated), with 491 differentially regulated in response to the HL-60-EV (325 upregulated and 166 downregulated). While there were 111 commonly upregulated proteins, 54 proteins were consistently downregulated in HSPC after treatment with both HL-60-EV and Molm-14-EV relative to vehicle-treated controls (Fig 3A). Functional analysis of these proteins using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) platform identified ribosomal biogenesis as the most highly enriched functionally related gene group among downregulated targets in EV-exposed cells (Fig 3B). This category comprised ribosomal proteins involved in RNA-binding, RNA-splicing, and translation initiation (Fig 3C). Figure 3. AML-EV impair ribosome biogenesis in hematopoietic cells and suppress protein synthesis in LT-HSC A. A Venn diagram showing the most highly up- and downregulated proteins between Molm-14-EV and HL-60 EV-treated cKit+ HSPC determined by isobaric TMT labeling and edgeR analyses to calculate the false discovery rate (FDR) analysis to obtain ˜10% of the differentially regulated proteins. We obtained 325 and 221 upregulated proteins in the HL-60 dataset and the Molm-14 dataset, respectively. We also obtained 166 and 173 downregulated protein in the HL-60 dataset and the Molm-14 dataset, respectively. Among them, 54 proteins were commonly downregulated. B. A bar graph showing the functional annotation enrichment analysis by DAVID biostatistical plate form of the 54 commonly downregulated protein identified the indicated pathways; among them, the ribosomal biogenesis pathway showed the highest enrichment. P-values are indicated in white. Modified Fisher's exact test. C. A heatmap showing the highly deregulated ribosomal proteins in AML-EV-exposed cells. D, E. Flow cytometric analysis showing the histograms and MFI of O-propargyl-puromycin (OPP) incorporation in LT-HSC in: (D) Molm-14 xenografts, red n = 6 versus non-engrafted controls, black n = 6, or (E) IF injection of EV from Molm-14, U-937, HL-60 (red, n = 5,4,3), AML patient plasma (orange, n = 6) or human CD34+ cells (blue, n = 3) normalized to vehicle-injected contralateral femurs after subtracting the background fluorescence. Data were obtained from at least two independent experiments. Statistics: Student's t-test (*P < 0.05). Download figure Download PowerPoint AML-EV suppress protein synthesis only in LT-HSC Ribosomal biogenesis is a principal regulatory step for protein homeostasis 34. This prompted us to test the effect of AML-EV on protein synthesis rates among HSPC populations. We used the recently validated OPP Click-iT assay 35 that relies on the incorporation and labeling of a modified puromycin analogue in newly generated proteins, and thus positively correlates gains in fluorescence intensity with protein synthesis. After animal sacrifice, cells were cultured in RPMI with 10% FBS and treated with OPP for 30 min to measure the fluorescently labeled OPP by flow cytometry. Among the different HSPC populations, only the LT-HSC from Molm-14 xenografts showed significant suppression in their protein synthesis, as represented in the histogram and MFI quantification (Figs 3D and EV3A and B). Protein synthesis was similarly suppressed in LT-HSC and KSL cells from IF-injected mice with EV from Molm-14, U-937, and AML patient plasma, but not the hCD34-EV, and more modestly by HL-60-EV (Figs 3E and EV3C). These data suggest that the global EV-mediated ribosome biogenesis impairment in all HSPC most profoundly suppresses protein synthesis in LT-HSC, but not KSL or other progenitors. Together, the data demonstrate protein synthesis suppression and quiescence induction in LT-HSC by AML-EV. Click here to expand this figure. Figure EV3. AML do not suppress protein synthesis in other hematopoietic populations A. Flow cytometric analysis showing MFI of OPP incorporation in MPP3/4, MPP2, and ST-HSC in the Molm-14 xenografts (red, n = 6) versus non-engrafted mice (black, n = 6). Statistics: Student's t-test (not significant "NS"). B, C. OPP flow cytometric analysis of KSL in: (B) Molm-14 xenografts (red, n = 6) versus non-engrafted mice (black, n = 6), or (C) IF injection of EV from Molm-14, U-937, HL-60 (red, n = 5,4,3), AML patient plasma (orange, n = 6), or CD34+ cells (blue, n = 3) normalized to contralateral femurs after subtracting background fluorescence. Download figure Download PowerPoint AML-EV impair protein synthesis in LT-HSC via the mTOR pathway The mTOR pathway is critical for translating extrinsic signals into cell-intrinsic events and, among other functions, governs ribosome biogenesis and protein synthesis 36-38. Because we found a global reduction of ribosome biogenesis in HSPC (Fig 3), we evaluated the EV-mediated regulation of the mTOR pathway using the bulk HSPC, KSL. Our qPCR transcriptional analysis indicated the downregulation of several mTOR-associated targets, including the mTORC1 subunit Raptor, in KSL from Molm-14 xenografts relative to controls (Fig 4A). KSL cells from femurs injected with Molm-14-EV confirmed the suppression of Raptor, and other mTOR-associated genes, relative to cells from vehicle-injected contralateral femurs (Fig 4B). To assess mTOR activity, we tested the effect of AML-EV on S6RP phosphorylation, a downstream mTOR effector 38, and found a consistent reduction of pS6RP in LT-HSC in the xenografts relative to control mice (Fig 4C and D) and after injection of AML-EV but not hCD34-EV (Fig 4C and E). Similarly, AML-EV decreased pS6RP in KSL, suggesting that the AML-EV-mediated mTOR suppression affects the HSPC pool more broadly (Fig EV4A). Consistent with the reported role of Raptor in regulating HSC quies
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