Hematopoietic stem cell function and survival depend on c-Myc and N-Myc activity

Abstract

Myc activity is emerging as a key element in acquisition and maintenance of stem cell properties. We have previously shown that c-Myc deficiency results in accumulation of defective hematopoietic stem cells (HSCs) due to niche-dependent differentiation defects. Here we report that immature HSCs coexpress c-myc and N-myc mRNA at similar levels. Although conditional deletion of N-myc in the bone marrow does not affect hematopoiesis, combined deficiency of c-Myc and N-Myc (dKO) results in pancytopenia and rapid lethality. Interestingly, proliferation of HSCs depends on both myc genes during homeostasis, but is c-Myc/N-Myc independent during bone marrow repair after injury. Strikingly, while most dKO hematopoietic cells undergo apoptosis, only self-renewing HSCs accumulate the cytotoxic molecule Granzyme B, normally employed by the innate immune system, thereby revealing an unexpected mechanism of stem cell apoptosis. Collectively, Myc activity (c-Myc and N-Myc) controls crucial aspects of HSC function including proliferation, differentiation, and survival.

Figure 1. N-myc Is Highly Expressed in HSCs, but Its Conditional Deletion Does Not Impair Steady-State Hematopoiesis

(A and B) qRT-PCR assessment of the expression levels of c-myc, N-myc, and L-myc in selected bone marrow populations. LT-HSCs (LSK CD150⁺CD48⁻CD34⁻ and LSK CD150⁺CD48⁻CD34⁺). Cell surface marker definition of the other populations is indicated in Table S1. (A) Relative expression of each gene after TBP normalization. (B) Percent contribution of c-myc, N-myc, and L-myc to the total Myc pool in each population. (C) Conditional deletion of c-myc and N-myc by pIpC treatment of MxCre;c-myc^flox/flox;N-myc^flox/flox mice. pIpC triggers IFNα production that, in turn, leads to activation of the Mx promoter, which drives the Cre recombinase. (D–F) Analysis of N-myc-deficient hematopoietic cells at homeostasis 5 weeks after pIpC treatment. (D) Total numbers of BM, thymus, or spleen cells in N-Myc-deficient animals (Nmyc^fl/flCre⁺) compared to normal littermates (Nmyc ^fl/flCre⁻). (E) Total numbers of BM stem and progenitor cell populations Lin⁻Sca-1⁺c-kit⁺ (LSK), LSK-SP, and LSK-CD150⁺CD48⁻ (LSK-SLAM) and (F) of major BM differentiated cell types B cells (CD19⁺B220⁺), Myeloid cells (GR1⁺ and/or F480⁺), Erythroid (Ter119⁺), and Megakaryocytes (CD41⁺GR1⁻). Results are mean ± SD.

Figure 2. Simultaneous Deletion of c-myc and N-myc Results in Rapid BM Failure and Depletion of the HSC Pool

(A) Kaplan-Meyer survival curve of MxCre;c-myc^flox/flox;N-myc^flox/flox (dKO) mice after pIpC-induced deletion. (B) Kinetic analysis of dKO BM cells relative to control mice from 4 to 16 days after the first pIpC injection. Days 4 and 6, n > 8; and days 14 and 16, n = 3. Data are from at least two separate experiments per time point. Data represent mean ± SD. (C) Hematoxylin-eosin staining of transversal sections of the femur trabecular zone 14 days after the first pIpC injection. 10× magnification. Control on the left; dKO on the right. (D) Representative FACS profiles of early hematopoietic stem/progenitor cells isolated from control and dKO mice 6 days after pIpC treatment (bottom). BM cells were stained with Lineage, cKit (CD117), Sca1, CD150, and CD48 antibodies and gated as indicated. cKit⁺ Sca1⁺ labeling alone would erroneously predict a complete loss of stem cells and early precursors (left panel). The use of the SLAM markers CD150 and CD48 shows that the dKO Lin⁻Sca1⁺ compartment downregulates cKit cell-surface expression (middle panel) but still contains a subset of HSCs (Lin⁻Sca1⁺CD150⁺CD48⁻, right panel). (E) Quantification of the number of SLAM-HSCs post pIpC induction. Data are from at least two experiments per time point, 2 < n < 4 per genotype and time point.

Figure 3. Simultaneous Deletion of c-myc and N-myc Affects Proliferation and Survival of Hematopoietic Cells in a Context-Dependent Manner

(A and B) Cell-cycle status (DNA content; Hoechst 33342 staining) of control and dKO cells 6 days after pIpC treatment. Representative examples of granulocytic (Gr1⁺CD11b⁺) cells (A) and of SLAM-HSCs (Lin⁻Sca1⁺CD150⁺CD48⁻) (B) are shown in the left panels. Mean percent of cells in S/G₂/M phase (±SD) is shown in the corresponding right panel. n = 3. (C and D) c-Myc/N-Myc expression levels were determined by immunofluorescence on cytospins from FACS sorted BrdU⁺Lin⁻Sca1⁺CD150⁺ cells. BrdU was administered to the mice via the drinking water 15 hr prior to analysis. Representative pictures are shown in (C), with BrdU in green and c-Myc/N-Myc in red. Scale bar, 20 μm. (D) c-Myc/N-Myc expression levels were blindly scored on two control and three dKO mice. A minimum of 25 cells per genotype was analyzed, and each cell was assigned an intensity of Myc expression as exemplified in (C). Genetic controls for the antibodies used are presented in Figure S6. (E–G) Quantitative analysis (FACS) of apoptosis (TUNEL) in the granulocyte (E), erythroblast (F), and SLAM-HSC (G) populations. Data represent mean ± SD, n > 3 per time point.

Figure 4. Bone Marrow Failure in dKO Mice Is Hematopoietic Autonomous, and dKO HSCs Show Decreased Proliferation and Survival

(A) Competitive bone marrow chimeras. Mice were reconstituted with equal amounts of c-myc^flox/flox;N-myc^flox/flox cells and undeleted MxCre;c-myc^flox/flox; N-myc^flox/flox BM cells. After stable engraftment (8 weeks), pIpC was administered to the mice to induce deletion. The chimeras were analyzed 14 or 21 days after the first pIpC injection. (B) Kinetics of loss of donor dKO cells in mixed chimeras. Results are mean ± SD. D14, n = 8; D21, n = 3. (C) dKO HSCs present in mixed chimeras do not downregulate cKit (CD117) cell-surface expression. BM from competitive chimeras 14 days after pIpC treatment was analyzed and gated on Lin⁻Sca1⁺CD150⁺CD48⁻. The expression levels of cKit in the CD45.2⁺ (WT) rescue and in the CD45.1⁺/2⁺ (dKO) population were compared. Representative FACS plots from a total of six mice are shown. (D) Degree of relative chimerism on progenitor and stem cell populations. CLPs (Lin⁻cKit^intSca1^int CD127⁺), CMPs (Lin⁻cKit⁺Sca1⁻); n > 3. (E) Degree of relative chimerism of SLAM-HSCs. Triangles, individual mice; horizontal bar, the mean value for each genotype. D14, n = 8; D21, n = 3. (F) Chimeras 14 days after the first pIpC treatment: BM was stained with stem cell markers, and apoptosis was assessed by TUNEL using FACS. Mean ± SD is shown; n = 3. (G) Chimeras 14 days after the first pIpC treatment were administered BrdU for 15 hr before percent BrdU incorporation was assessed. Mean ± SD is shown; n = 3.

Figure 5. Proliferating HSCs, but Not Quiescent HSCs, Are Preferentially Lost in dKO Mice

(A) Secondary transplantation. BM from primary transplanted mice was harvested 14 days after deletion (see Figure 4A) and transferred into lethally irradiated CD45.2⁺ hosts. Percent CD45.1⁺/2⁺ myeloid cells (B) or SLAM-HSCs (C) in BM of secondary transplanted recipients 84 days after transplantation. (D) Label-Retaining Cell (LRC) assay. Undeleted mixed chimeric mice were administered BrdU in their drinking water for 10 days, followed by 70 days of chase (no BrdU). During the chase period, rapidly dividing cells dilute out the BrdU label, while long-lived quiescent cells (among which are dormant HSCs) retain this marker. Deletion was induced so that the day of analysis corresponded to both 70 days of chase and 14 days after the first pIpC injection. (E) BM from LRC mice was isolated and stained for stem cell markers. Relative chimerism (mean ± SD) of BrdU⁻ or BrdU⁺ SLAM-HSCs is shown. n = 3.

Figure 6. c-Myc and N-Myc Promote Survival of HSCs and Prevent GrB Accumulation

(A) Absolute numbers, relative to control mice, of SLAM-HSCs in dKO and H2K::BCL2 dKO mice, 6 days after pIpC treatment. Mean ± SD is shown; n ≥ 3. (B) SLAM-HSCs from dKO and control animals were stained with DCF-DA to measure the intracellular concentration of ROS. A representative FACS plot is shown; n = 3. (C) Differential expression levels of selected genes as analyzed by microarray analysis 4 days after the first pIpC injection. (D) Intracellular FACS staining to determine expression of Granzyme B (GrB) protein (blue line, control; red line, dKO; tinted, isotype control; n ≥3 for each condition). (E) Quantification of GrB expression at day 4 and day 6. Percent GrB⁺ in gated Lin⁻Sca1⁺CD150⁺CD48⁻ cells. The Lin cocktail includes DX5, NK1.1, CD3, CD4, and CD8 antibodies in order to eliminate any possible contamination from canonical GrB-producing cells, such as CTLs and NK cells. Blue triangles, individual control mice; red circles, dKO mice. Horizontal bars indicate averages of at least 6 mice per genotype. (F) Intracellular GrB staining on indicated cell types 4 days after pIpC treatment. (G) Representative GrB expression profiles gated on LSKCD150⁺CD48⁻ in mixed chimeras (left panel) and in cultured SLAM HSCs 48 hr after IFNα addition. (H) Representative ictures of GrB by immunofluorescence. Lin⁻ cells or activated NK cells (DX5⁺, positive control) were cytospun onto slides and stained for GrB protein and DAPI (blue). Scale bar, 5 μm. (I–K) GrB/TUNEL double staining on gated Lin⁻Sca1⁺CD150⁺CD48⁻ cells at 4 and 6 days after c-myc and N-myc deletion. (I) Representative FACS plots at day 6. (J and K) Quantification of percentage of GrB⁺ cells among TUNEL⁺ SLAM HSCs ([J], mean ± SD) and of TUNEL⁺ cells among GrB⁺ SLAM HSCs (K). Day 4, n = 3; day 6, n = 2 for controls and n = 5 for dKO.

Figure 7. A Model for the Consequ ences of Loss of Myc in the Hematopoietic System

(A) Schematic representation of steady-state hematopoiesis in a normal mouse. The HSC compartment comprises both self-renewing HSCs that contribute to day-to-day hematopoiesis and dormant HSCs that are only activated upon injury. N-myc expression levels contribute significantly to the Myc pool only in the most primitive HSCs. All other hematopoietic cell types are predominantly controlled by the c-Myc homolog. (B) Upon loss of c-myc alone (Wilson et al., 2004), HSCs are unable to differentiate into progenitors. However, the presence of N-Myc allows them to maintain their self-renewal capacity, resulting in HSC accumulation in the bone marrow niche. The majority of early and late progenitors stop proliferating. (C) In the presence of c-Myc, elimination of N-myc does not impact steady-state hematopoiesis. (D and E) Deletion of both c-myc and N-myc reveals that c-Myc and N-Myc are crucial to promote survival throughout the hematopoietic system, as Myc-deficient self-renewing HSCs accumulate GrB and rapidly die. When no wild-type cells are present (“straight” dKO situation) (D), the rapid cell death of most differentiated cell types induces pancytopenia. As a consequence, a positive-feedback loop is generated, leading to activation of dormant HSCs. In the absence of c-Myc and N-Myc, self-renewing HSCs enter apoptosis and are therefore rapidly exhausted. In mixed chimeras (E), pancytopenia does not occur due to the presence of wild-type cells. Thus, no feedback loop is generated and dKO-dormant HSCs are maintained.