Sorting protein VPS33B regulates exosomal autocrine signaling to mediate hematopoiesis and leukemogenesis

Abstract

Certain secretory proteins are known to be critical for maintaining the stemness of stem cells through autocrine signaling. However, the processes underlying the biogenesis, maturation, and secretion of these proteins remain largely unknown. Here we demonstrate that many secretory proteins produced by hematopoietic stem cells (HSCs) undergo exosomal maturation and release that is controlled by vacuolar protein sorting protein 33b (VPS33B). Deletion of VPS33B in either mouse or human HSCs resulted in impaired exosome maturation and secretion as well as loss of stemness. Additionally, VPS33B deficiency led to a dramatic delay in leukemogenesis. Exosomes purified from either conditioned medium or human plasma could partially rescue the defects of HSCs and leukemia-initiating cells (LICs). VPS33B co-existed in exosomes with GDI2, VPS16B, FLOT1, and other known exosome markers. Mechanistically, VPS33B interacted with the GDI2/RAB11A/RAB27A pathway to regulate the trafficking of secretory proteins as exosomes. These findings reveal an essential role for VPS33B in exosome pathways in HSCs and LICs. Moreover, they shed light on the understanding of vesicle trafficking in other stem cells and on the development of improved strategies for cancer treatment.

Figure 1. Stemness-related secretory proteins exist in exosomes.

(A and B) 293T cells transfected with TPO, ANGPTL2, and ANGPTL3 were costained with either α-tubulin (A) or CD63 (B) using immunofluorescence staining to evaluate the colocalization, as shown in the merged images of each row. Nuclei were counterstained with DAPI. Scale bars: 10 μm. (C) Exosomes were purified from TPO-, ANGPTL2- and ANGPTL3-conditioned medium (CM) of 293T cells and immunoblotted with antibodies against V5 (for TPO), Flag (for ANGPTL2 and ANGPTL3), and TSG101 and FLOT1 (exosome markers). Ctrl, empty vector; BE, before extraction; AE, after extraction. (D) Representative transmission electron micrographs of exosomes purified from ANGPTL2-conditioned medium of 293T cells (scale bars: 100 nm). (E) Quantitative data for the levels of TPO-, ANGPTL2-, and ANGPTL3-containing exosomes in supernatant after differential centrifugation (AE/BE, n = 3). (F) Exosomes were purified from human plasma and assayed to detect TPO, ANGPTL2, ANGPTL3, TSG101, FLOT1, VPS16B, and VPS33B. (G) Quantitative data for the levels of TPO-, ANGPTL2-, and ANGPTL3-containing exosomes in human plasma after differential centrifugation (AE/BE, n = 3). Experiments were conducted 3 to 5 times for validation.

Figure 2. VPS33B regulates the maturation and secretion of exosomes.

(A) 293T cells transfected with TPO, ANGPTL2, and ANGPTL3 were costained with VPS33B to evaluate colocalization as shown in the merged images of each row. Nuclei were counterstained with DAPI. Scale bars: 10 μm. (B) VPS33B was knocked down by a validated shRNA (shVPS33B) or scramble control (SCR) in 293T cells stably expressing TPO, ANGPTL2, and ANGPTL3. TPO (V5-tagged), ANGPTL2, and ANGPTL3 (Flag-tagged) levels in the conditioned medium (CM) and cells were measured by immunoblot analysis. The VPS33B levels upon knockdown were also confirmed. (C) Quantification of immunoblot image intensities for TPO, ANGPTL2, and ANGPTL3 levels in B. (D) Exosomes were purified from TPO-, ANGPTL2-, and ANGPTL3-conditioned medium upon knockdown of VPS33B in 293T cells and immunoblotted with antibodies against V5, Flag, TSG101, FLOT1, VPS16B, and VPS33B. (E) Quantitative data for the levels of TPO-, ANGPTL2-, and ANGPTL3-containing exosomes in D (shVPS33B/SCR) (n = 3). (F) Number of exosomes purified from TPO-, ANGPTL2-, and ANGPTL3-conditioned medium upon knockdown of VPS33B in 293T cells, as determined by NanoSight (n = 3; *P < 0.05, ***P < 0.001 using Student’s t test). (G) Representative immunoelectron microscopy images of MVBs from ANGPTL2-Flag–expressing 293T cells upon knockdown with scrambled or VPS33B-targeted shRNAs. Immunostaining was performed using an anti-Flag antibody (10 nm gold particles in MVB and cytoplasm indicated by blue and green arrows, respectively). Experiments were conducted 3 to 5 times for validation.

Figure 3. VPS33B maintains HSC functions.

(A) WT and VPS33B-null BM CD45.2 cells along with CD45.1 competitor cells were injected into lethally irradiated CD45.1 recipient mice (n = 5; **P < 0.01, ***P < 0.001 using Student’s t test). Repopulation was analyzed at 3, 8, and 16 weeks after transplantation. (B) Multilineage contribution of donor cells in the primary recipients at 16 weeks post-transplantation (n = 5; *P < 0.05 using Student’s t test). (C) Secondary transplantation was performed with FACS-purified donor CD45.2 BM cells from WT and VPS33B-null primary recipients. Repopulation was analyzed at 4, 8, and 16 weeks after transplantation (n = 5; **P < 0.01, ***P < 0.001 using Student’s t test). (D) Multilineage contribution of donor cells in secondary recipients 16 weeks after transplantation (n = 5; *P < 0.05 using Student’s t test). (E) Vps33b^wt/wt Scl-Cre-ER (VPS33B^+/+) and Vps33b^fl/fl Scl-Cre-ER (VPS33B^–/–) mice were intraperitoneally administered 150 mg/kg 5-FU weekly for 3 times (arrows), and the survival rates were analyzed (n = 7; *P < 0.05 using log-rank test). (F) Vps33b^fl/fl Scl-Cre-ER⁺ BM cells or Vps33b^wt/wt Scl-Cre-ER⁺ BM cells (3 × 10⁵ cells) along with competitor cells were transplanted into lethally irradiated CD45.1 recipient mice, followed by the treatment with tamoxifen 8 weeks after transplantation and analysis for repopulation from 2 to 24 weeks after treatment. Vps33b deletion (arrow) was shown after 2-week treatment (n = 5; **P < 0.01, ***P < 0.001 using Student’s t test). (G) Multilineage contribution of donor cells 24 weeks after transplantation (n = 5; *P < 0.05 using Student’s t test). (H and I) WT and VPS33B-null LT-HSCs were analyzed for cell cycle stage by staining with Hoechst33342/Pyronin Y (H), and the frequencies of the G0, G1, and S-G2-M fractions were quantified (n = 3; *P < 0.05, ***P < 0.001 using Student’s t test) (I). (J) Apoptosis was measured in LT-HSCs from VPS33B^+/+ and VPS33B^–/– mice by using annexin V/7-AAD staining (n = 5; ***P < 0.001 using Student’s t test). (K) Representative images of LT-HSCs from VPS33B^+/+ and VPS33B^–/– mice 10 days after culturing in basic medium (SCF+TPO) (n = 6). (L–N) Cell numbers (L), percentages of LSK cells (M), and percentages of apoptotic cells (N) were evaluated in cultured WT and VPS33B-null LT-HSCs (n = 6; *P < 0.05, **P < 0.01, ***P < 0.001 using Student’s t test). Experiments were conducted 3 to 5 times for validation.

Figure 4. VPS33B collaborates with GDI2 to regulate exosome maturation and secretion.

(A) Representative transmission electron micrographs of LT-HSCs from VPS33B^+/+ and VPS33B^–/– mice showing morphologies of EVs, MVB I (blue arrows) and MVB II (green arrows). Scale bars: 2 μm; 500 nm (insets). (B) Quantification of organelles (EVs, MVB I, and MVB II) present in WT and VPS33B-null LT-HSCs (n = 20; *P < 0.05, ***P < 0.001 using Student’s t test). (C) Flag-tagged VPS33B was transfected into 293T cells and immunoprecipitated. Western blotting was performed with antibodies against GDI2, FLOT1, VPS16B, RAB11A, and RAB27A. The backbone empty vector (Ctrl) was used as the control. (D) Converse co-immunoprecipitation experiments were performed in 293T cells via pulldown of StrepII-tagged GDI2, Fc-tagged RAB11A, and Fc-tagged RAB27A, followed by Western blotting to detect VPS33B levels. (E) VPS33B was either overexpressed (OE) or knocked down (shVPS33B) in 293T cells, followed by exosome extraction and immunoblotting with GDI2, TSG101 and FLOT1. (F) GDI2 levels were measured in human plasma-derived exosomes by western blotting. TSG1 and FLOT1 served as exosome markers (n = 3). (G) Protein levels and colocalization of VPS33B and GDI2 were evaluated between WT and VPS33B-null LT-HSCs using immunostaining (n = 5). Scale bars: 5 μm. Experiments were conducted 3 to 5 times for validation.

Figure 5. Supplement of exogenous ANGPTL2 and ANGPTL3 proteins or overexpression of GDI2 restores the impaired activities of VPS33B-null HSCs.

(A) Representative images of LT-HSCs from VPS33B^+/+ and VPS33B^–/– mice cultured in basic medium (SCF+TPO), ANGPTL2-, and ANGPT3-conditioned medium (referred as Ctrl, A2, and A3, respectively) for 8 days (n = 5). (B and C) Cell numbers and apoptosis were measured in cultured WT and VPS33B-null LT-HSCs in A (n = 5; **P < 0.01, ***P < 0.001 using Student’s t test). (D) ANGPTL2 and ANGPTL3 were overexpressed in VPS33B-null HSCs, which were subjected to competitive reconstitution analysis. Engraftment was analyzed at 4, 8, and 16 weeks after transplantation (n = 5; ***P < 0.001 using Student’s t test). (E) GDI2 was overexpressed in VPS33B-null HSCs, which were subjected to competitive reconstitution analysis. Engraftment was analyzed at 3, 8, and 16 weeks after transplantation (n = 5; ***P < 0.001 using Student’s t test). (F) GDI2 was knocked down in WT HSCs, which were subjected to competitive reconstitution analysis. Engraftment was analyzed at 3, 8, and 16 weeks after transplantation (n = 5; ***P < 0.001 using Student’s t test). Experiments were conducted 3 times for validation.

Figure 6. VPS33B regulates the release of growth factor–containing exosomes to maintain mouse and human HSC functions.

(A) Representative flow cytometric analyses for the binding activity of ANGPTL2-containing exosomes (ANGPTL2-Exo) and control exosomes (Ctrl-Exo) to LILRB2-transfected 293T cells. ANGPTL2-conditioned medium (ANGPTL2-CM) and control conditioned medium (Ctrl-CM) served as the positive and negative controls, respectively (n = 3). (B) Representative images of LT-HSCs from VPS33B^+/+ and VPS33B^–/– mice cultured in basic medium (SCF+TPO; Ctrl), ANGPTL2-conditioned medium (A2), and ANGPTL3-conditioned medium (A3); in medium containing purified control exosomes (CExo), ANGPTL2-containing exosomes (A2Exo), and ANGPTL3-containing exosomes (A3Exo) from conditioned medium; and in human plasma–derived exosomes (hExo) for 6 days (n = 4). (C) Multilineage contribution of CD34⁺ cells from human umbilical cord blood upon VPS33B knockdown. Representative flow cytometric profiles from 1 transplanted NOD-SCID mouse of each group. The IgG isotype served as a negative control. The middle set of profiles shows results for myeloid cells, and the right set of profiles shows results for lymphoid (CD19⁺/CD20⁺) and HSCs/progenitor cells (CD19^–/CD20^–CD34⁺). (D) Repopulation of human CD45⁺/CD71⁺ in the BM of NOD-SCID mice as shown in C at 2 months after transplantation (n = 7; ***P < 0.001 using Student’s t test). (E) Summary of the multilineage contributions as described in C (n = 7; **P < 0.01, ***P < 0.001 using Student’s t test). Experiments were conducted 3 times for validation.

Figure 7. VPS33B supports mouse AML development.

(A) Percentages of YFP⁺ leukemia cells in the peripheral blood of the recipients 4 weeks after primary transplantation (n = 5; **P < 0.01 using Student’s t test). (B) The representative flow cytometric analysis of Mac-1⁺c-Kit⁺ LICs in the BM of primary recipient mice is shown. (C) Quantification of the percentages of Mac-1⁺c-Kit⁺ cells in WT and VPS33B-null recipient mice (n = 5; ***P < 0.001 using Student’s t test). (D) Mice transplanted with MLL-AF9–infected VPS33B-null HSCs/progenitors had significantly extended survival upon primary transplantation compared with the survival of mice transplanted with control cells (n = 5; **P < 0.01 using log-rank test). (E) Secondary transplantation of 1 × 10⁴ leukemia cells displayed a significantly delayed onset of leukemogenesis by VPS33B-null cells compared with control cells (n = 6; ***P < 0.001 using log-rank test). (F) Representative transmission electron micrographs of Mac-1⁺c-Kit⁺ WT and VPS33B-null LICs showing representative images of EVs, MVB I (blue arrows), and MVB II (green arrows). Scale bars: 2 μm; 500 nm (insets). (G) Quantification of organelles (EVs, MVB I, and MVB II) present in WT and VPS33B-null Mac-1⁺c-Kit⁺ LICs (n = 20; ***P < 0.001 using Student’s t test). (H) Representative images of Mac-1⁺c-Kit⁺ WT and VPS33B-null LICs cultured in basic medium (SCF+IL-3+IL-6) with or without ANGPTL2- and ANGPTL3-conditioned medium (Ctrl, A2, and A3) or their purified exosomes (CExo, A2Exo, and A3Exo), as well as human plasma–derived exosomes (hExo) for 6 days (n = 5). Experiments were conducted 3 times for validation.

Figure 8. VPS33B supports human AML development.

(A–C) The number of human CD34⁺ LICs from 3 different patients was counted on the indicated days after VPS33B knockdown (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 using Student’s t test). (D) Apoptosis of AML samples in A–C were evaluated by the staining with annexin V/7-AAD (n = 3; ***P < 0.001 using Student’s t test). (E and F) Representative images and colony numbers were measured in human CD34⁺ LICs after VPS33B knockdown (n = 3; ***P < 0.001 using Student’s t test). (G) Representative images of human CD34⁺ LICs cultured in basic medium (SCF+IL-3+IL-6+FLT3 ligand) with or without ANGPTL2- and ANGPTL3-conditioned medium (Ctrl, A2, and A3) or their purified exosomes (CExo, A2Exo, and A3Exo) as well as human plasma–derived exosomes (hExo) for 6 days (n = 3). (H) Working model for the roles of VPS33B in the regulation of exosome maturation, secretion, and stemness maintenance in both HSCs and LICs. Experiments were conducted 3 times for validation.