In Fanconi anemia, impaired accumulation of bone marrow neutrophils during emergency granulopoiesis induces hematopoietic stem cell stress

Abstract

Fanconi anemia (FA) is an inherited disorder of DNA repair due to mutation in one of 20+ interrelated genes that repair intrastrand DNA crosslinks and rescue collapsed or stalled replication forks. The most common hematologic abnormality in FA is anemia, but progression to bone marrow failure (BMF), clonal hematopoiesis, or acute myeloid leukemia may also occur. In prior studies, we found that Fanconi DNA repair is required for successful emergency granulopoiesis; the process for rapid neutrophil production during the innate immune response. Specifically, Fancc^-/- mice did not develop neutrophilia in response to emergency granulopoiesis stimuli, but instead exhibited apoptosis of bone marrow hematopoietic stem cells and differentiating neutrophils. Repeated emergency granulopoiesis challenges induced BMF in most Fancc^-/- mice, with acute myeloid leukemia in survivors. In contrast, we found equivalent neutrophilia during emergency granulopoiesis in Fancc^-/-Tp53^+/- mice and WT mice, without BMF. Since termination of emergency granulopoiesis is triggered by accumulation of bone marrow neutrophils, we hypothesize neutrophilia protects Fancc^-/-Tp53^+/- bone marrow from the stress of a sustained inflammation that is experienced by Fancc^-/- mice. In the current work, we found that blocking neutrophil accumulation during emergency granulopoiesis led to BMF in Fancc^-/-Tp53^+/- mice, consistent with this hypothesis. Blocking neutrophilia during emergency granulopoiesis in Fancc^-/-Tp53^+/- mice (but not WT) impaired cell cycle checkpoint activity, also found in Fancc^-/- mice. Mechanisms for loss of cell cycle checkpoints during infectious disease challenges may define molecular markers of FA progression, or suggest therapeutic targets for bone marrow protection in this disorder.

Keywords: DNA damage response; DNA repair; cell cycle; hematopoiesis; inflammasome; innate immunity; neutrophil.

Figure 1

Blocking bone marrow neutrophil accumulation during emergency granulopoiesis induces cytopenias in Fancc^−/−Tp53^+/− mice. Fancc^−/−, Fancc^−/−Tp53^+/− or WT mice were injected IP with Alum to induce emergency granulopoiesis. Some cohorts were injected with Ly6G-antibody to prevent bone marrow neutrophil accumulation (versus isotype control antibody). Red numbers indicate weeks with injection of Alum or saline (as a steady state control). A, Ly6G-antibody treatment induced progressive decrease in peripheral blood neutrophils in Fancc^−/−Tp53^+/− mice during multiple episodes of emergency granulopoiesis. Statistically significant differences in circulating neutrophils indicated by ∗p = 0.0005, ∗∗p = 0.002 or ∗∗∗p = 0.0004. Error bars represent standard deviation and n = 6 for all cohorts. B, Ly6G-antibody treatment prevented bone marrow neutrophil accumulation during emergency granulopoiesis, but did not cause absolute neutropenia. Statistically significant differences in bone marrow neutrophils indicated by ∗p = 0.0002, ∗∗p = 0.014, ∗∗∗p = 0.005, #p = 0.003, ##p = 0.00008, ###p = 0.004 or &p = 0.0002. Error bars represent standard deviation and n = 6 for all cohorts. Photomicrograph of sternal bone marrow at 40X magnification. C, Hgb concentration progressively decreased in Fancc^−/−Tp53^+/− mice treated with Ly6G-antibody during emergency granulopoiesis. Statistically significant differences in Hgb concentration by ^#p = 0.009, n = 6. Error bars represent standard deviation and n = 6 for all cohorts. IP, intraperitoneal.

Figure 2

Blocking bone marrow neutrophil accumulation during emergency granulopoiesis in Fancc^−/−Tp53^+/−mice shortens survival, but delays leukemogenesis. Fancc^−/−, Fancc^−/−Tp53^+/− or WT mice were injected with Alum to induce emergency granulopoiesis, and some cohorts were also injected with Ly6G antibody to prevent neutrophil accumulation during this process. Mice were injected every 4 weeks with Alum injection indicated in red. A, Ly6G-antibody treatment during emergency granulopoiesis shortened survival of Fancc^−/−Tp53^+/− mice. Statistically significant difference between survival in Fancc^−/−Tp53^+/− mice with Alum versus Alum plus Ly6G treatment indicated by ∗p = 0.025 with n = 6 for all cohorts. B, Ly6G-antibody treatment during emergency granulopoiesis delayed AML in Fancc^−/−Tp53^+/− mice. AML was defined as >10% circulating myeloid blasts. Statistically significant differences indicated by ∗p = 0.00006, ∗∗p = 0.000032, ∗∗∗p = 0.001, ^#p = 0.000026, or ^##p = 0.003. Error bars represent standard deviation and n = 6 for all cohorts. AML, acute myeloid leukemia.

Figure 3

Episodes of neutropenia at steady state did not induce progressive cytopenias in Fancc^−/−or Fancc^−/−Tp53^+/−mice.Fancc^−/−, Fancc^−/−Tp53^+/− or WT mice were injected with Ly6G antibody every 4 weeks to induce absolute neutropenia at steady state. Red numbers indicate weeks with initiation of Ly6G-injection. Statistically significant differences in (A) circulating neutrophils or (B). Hgb concentration are indicated by ∗p = 0.00005, ∗∗p = 0.004 or ∗∗∗p = 0.0003. Error bars represent standard deviation and n = 6 for all cohorts.

Figure 4

Blocking bone marrow neutrophil accumulation during emergency granulopoiesis increases bone marrow apoptosis and DNA damage in Fancc^−/− or Fancc^−/−Tp53^+/− mice. Fancc^−/−, Fancc^−/−Tp53^+/− or WT mice were injected with Alum to induce emergency granulopoiesis. Cohorts were injected with Ly6G antibody to prevent neutrophil accumulation (or isotype control antibody). Mice were injected every 4 weeks and bone marrow was obtained for analysis two or four weeks after the first injection. A, Ly6G-antibody treatment during emergency granulopoiesis enhances apoptosis of LSK cells in Fancc^−/−Tp53^+/− mice and Lin-ckit+ cells in Fancc^−/− mice. Statistically significant differences indicated by ∗p = 0.018, ∗∗p = 0.037, ∗∗∗p = 0.002, ^#p = 0.0022, ^##p = 0.024, ^###p = 0.008, ^&p = 0.008, ^&&p = 0.033, ^&&&p = 0.007, ^@p = 0.003, ^@@p = 0.005, ^@@@p = 0.008. Error bars represent standard deviation and n = 3 independent experiments for all cohorts. B, Ly6G-antibody treatment during emergency granulopoiesis enhances DNA damage in proliferating Fancc^−/−Tp53^+/− or Fancc^−/− LSKs. Statistically significant differences indicated by ∗p = 0.005, ∗∗p = 0.006, ∗∗∗p = 0.0002, ^#p = 0.023. Error bars represent standard deviation and n = 3 independent experiments for all cohorts. For both panels, representative histograms are shown with the percent increase with versus without Ly6G-antibody indicated. LSK, Lin^-Sca1⁺ckit⁺.

Figure 5

Transcriptome differences identify common pathway alterations associated with impaired neutrophil accumulation during emergency granulopoiesis. Fancc^−/−, Fancc^−/−Tp53^+/− and WT mice were injected with Alum to induce emergency granulopoiesis. Some cohorts were also treated with Ly6G-antibody to block neutrophil accumulation (or isotype antibody control). Mice were sacrificed and bone marrow LSKs isolated two or four weeks after Alum-injection for RNA-sequencing. Transcriptomes were compared and pathway activity suggested by gene ontology. Common pathway differences in various cohort comparisons are indicated by different X colors. A, during emergency granulopoiesis, Fancc^−/− LSK transcriptomes had decreased activity of pathways involved in phagocyte differentiation and function, the innate immune response, cellular response to stress, oxidative phosphorylation, protein glycosylation and cell cycle regulation in comparison to Fancc^−/−Tp53^+/− LSK transcriptomes, but increased activity of pathways involved in apoptosis, downregulation of cytokine production and growth factor signaling. B, during emergency granulopoiesis, Ly6G-antibody treatment of Fancc^−/−Tp53^+/− mice induced LSK transcriptomes with decreased activity of pathways involved in the innate immune response, cellular response to stress, DNA repair and cell cycle regulation, but increased activity of pathways involved in apoptosis versus cohorts treated with isotype control antibody. C, during emergency granulopoiesis, Ly6G-antibody treatment of WT mice induced LSK transcriptomes with decreased activity of pathways involved in phagocyte differentiation and function, the innate immune response, and cellular response to stress, but increased regulation of cytokine response pathways versus cohorts treated with isotype control antibody. D, during emergency granulopoiesis, Fancc^−/− LSK transcriptomes had increased activity of pathways involved in metabolism of RNA and proteins and Tgfβ signaling, but decreased activity of cell cycle regulatory pathways in comparison to LSK transcriptomes from Ly6G-treated WT mice. LSK, Lin^-Sca1⁺ckit⁺.

Figure 6

Cell cycle checkpoints activation in response to DNA damage is impaired during emergency granulopoiesis in Fancc^−/− mice. Fancc^−/−, Fancc^−/−Tp53^+/− or WT mice were treated with Alum and sacrificed 0, 2, or 4 weeks later for bone marrow analysis. A, gene expression differences identified by RNA-sequencing were confirmed in independent experiments. LSKs from Fancc^−/−, Fancc^−/−Tp53^+/−, or Ly6G-treated Fancc^−/−Tp53^+/− mice were isolated 2 weeks after Alum injection and expression of genes identified by RNA-Seq were quantified by real time PCR. Statistically significant differences are indicated by ∗p = 0.018, ∗∗p = 0.013, ∗∗∗p = 0.004, ^#p = 0.001, ^##p = 0.006, ^###p = 0.002, ^&p = 0.0008, ^&&p = 0.005, ^&&&p = 0.0005, ^∧p = 0.009, ^∧∧p = 0.004, ^∧∧∧p = 0.005, ^>p = 0.002, ^>>p = 0.007, or ^>>>p = 0.0004. Error bars represent standard deviation and n = 3 independent experiments for all cohorts. B, Atr and phospho-Chk1 are decreased in Fancc^−/− bone marrow during emergency granulopoiesis compared to WT. Bone marrow Lin-cells from Fancc^−/− or WT mice were obtained 2 weeks after injection of Alum or saline. Western blots of cell lysates were serially probed with antibodies to Atr, phospho-Chk1, total Chk1, or Tubulin (loading control). Protein expression was quantified by densitometry. C, during emergency granulopoiesis, Fancc^−/− bone marrow LSKs exhibit a relative increase in S phase, but decrease in G0/G1 or G2/M, compared to LSKs from Fancc^−/−Tp53^+/− or WT mice. Cell cycle analysis was performed on LSK cells 0, 2, and 4 weeks post Alum injection and expressed as percent total LSKs. Statistically significant differences indicated by ∗p = 0.0001, ∗∗p = 0.0008, ∗∗∗p = 0.004, ^#p = 0.02, ^##p = 0.002, ^###p = 0.007, ^&p = 0.001, or ^&&p = 0.002. Error bars represent standard deviation and n = 3 independent experiments for all cohorts. D, Fbxw10-knockdown in Fancc^−/− bone marrow increases cells in G1/G0, and decreases those S and G2/M, during emergency granulopoiesis. Fancc^−/− or WT mice were injected with Alum to induce emergency granulopoiesis or saline as a steady state control. Bone marrow harvested 2 weeks later was transduced with vectors to express Fbxw10-specific shRNAs versus scrambled control shRNAs, followed by cell cycle analysis of GFP+LSKs. Statistically significant differences indicated by ∗p = 0.00003, ∗∗p = 0.00001, ∗∗∗p = 0.0001, ^#p = 0.00004, ^##p = 0.00012, ^###p = 0.00018. Error bars represent standard deviation and n = 5 independent experiments for all cohorts. E, transduction of Fancc^−/− bone marrow cells with Fbxw10-specific shRNAs decreases Fbxw10 mRNA during emergency granulopoiesis. GFP+Lin-murine bone marrow cells from the experiment above were analyzed for Fbxw10 mRNA abundance by quantitative real time PCR. Statistically significant differences indicated by ∗p = 0.001 or ∗∗p = 0.0003. Error bars represent standard deviation and n = 3 independent experiments for all cohorts. LSK, Lin^-Sca1⁺ckit⁺.