The non-homologous end-joining activity is required for Fanconi anemia fetal HSC maintenance

Background Recent studies have shown that deficiency in the Fanconi anemia (FA) DNA repair pathway enhances the error-prone non-homologous end-joining (NHEJ) repair, leading to increased genomic instability, and that genetic or pharmacological inhibition of the NHEJ pathway could rescue the FA phenotype. Methods First, we exposed LSK cells from WT and Fanca−/− mice to DNA-PKcs inhibitor NU7026 or Ku70 knockdown to examine whether inhibition of NHEJ sensitizes Fanca−/− HSPCs to PARP inhibitor (PARPi)- or interstrand crosslinking (ICL)-induced cell death and genomic instability. We then generated DNA-PKcs3A/3AFanca−/− mice to investigate the effect of specific inactivation of NHEJ on fetal HSCs. Lastly, we used two p53 mutant models to test whether specific inactivation of the p53 function in apoptosis is sufficient to rescue embryonic lethality and fetal HSC depletion in Fanca−/− DNA-PKcs3A/3A mice. Results Inhibition of NHEJ sensitizes HSPCs from Fanca−/− mice to PARP inhibition- and ICL-induced cell death and genomic instability and further decreases Fanca−/− HSPC proliferation and hematopoietic repopulation in irradiated transplant recipients. Specific inactivation of NHEJ activity by the knockin DNA-PKcs3A/3A mutation in two FA mouse models, Fanca−/− and Fancc−/−, leads to embryonic lethality. DNA-PKcs3A/3A causes fetal HSC depletion in developing Fanca−/− embryos due to increased HSC apoptosis and cycling. Both p53−/− and a knockin p53515C mutation, which selectively impairs the p53 function in apoptosis, can rescue embryonic lethality and fetal HSC depletion in Fanca−/− DNA-PKcs3A/3A mice. Conclusion These results demonstrate that the NHEJ pathway functions to maintain Fanconi anemia fetal HSCs.


Background
Fanconi anemia (FA) is a genetic disorder associated with bone marrow (BM) failure and malignancies including leukemia and solid cancers [1][2][3][4]. Mutations in any of the 22 FA genes (FANCA-W) lead to clinical manifestations characterized by developmental abnormalities, progressive bone marrow failure (BMF), and a high risk of developing cancer including leukemia [5][6][7][8]. At the cellular level, FA is characterized by chromosomal instability and DNA cross-linker sensitivity, which serves as a clinical diagnostic hallmark of FA [1][2][3][4]. At the molecular level, eight FA proteins (FANCA, -B, -C, -E, -F, -G, -L, and -M), along with other associated factors, form the FA core complex in response to DNA damage or replicative stress, which acts in part as an ubiquitin ligase. This FA core complex monoubiquitinates two downstream FA proteins, FANCD2 and FANCI, which then recruit other downstream FA proteins including several key proteins involved in homologous recombination (HR) repair, and possibly other DNA repair factors, to nuclear loci containing damaged DNA and consequently influence important cellular processes such as DNA replication, cell-cycle control, and DNA damage response and repair [9][10][11].
Recent studies suggested that the FA pathway promotes the error-free HR repair pathway while suppressing the error-prone non-homologous end-joining (NHEJ) pathway [12][13][14][15]. Using FA-deficient Caenorhabditis elegans, chicken and human cells, two studies demonstrated that FA deficiency enhanced the error-prone NHEJ repair, leading to increased genomic instability [12,15]. These studies also showed that genetic or pharmacological inhibition of the NHEJ pathway could rescue the FA phenotype. Another similar study showed that inhibition of the NHEJ ligase, LIG4, ameliorated the FA phenotype, but had no effect on BRCA1 deficiency [16]. It appears the FA pathway may act to prevent inappropriate recruitment of NHEJ factors to sites of DNA damage. However, the exact mechanism by which the FA pathway counteracts the NHEJ pathway is largely unknown.
A clinical application of HR-NHEJ interaction is synthetic lethality induced by poly (ADP-ribose) polymerase (PARP) inhibition in BRCA1/2-mutated cancer [17,18]. Since PARP functions as a critical sensor of single-strand breaks (SSBs) in base-excision repair, as a mediator for restarting stalled replication forks of HR-mediated doublestrand break (DSB) repair, and as a means of preventing the binding of Ku proteins to DNA ends in NHEJ pathway [19][20][21][22], therefore, blocking the ADP-ribosylation activity with small molecules can achieve synthetic lethality with DNA-damaging agents in the treatment of certain cancers [23][24][25][26][27][28][29]. It has been shown that PARP inhibitors could selectively target cancer cells with a defective HR repair of DSB [25]. For example, BRCA1-, BRCA2-, and ATM-deficient cells show hypersensitivity to PARP inhibitors, leading to genomic instability and eventual cell death due to the development of non-viable genetic errors generated by the error-prone NHEJ repair [26][27][28].
In the current study, we show that inhibition of NHEJ sensitizes Fanca −/− HSPCs from mice to PARP inhibitioninduced cell death and genomic instability and leads to a further decrease in the proliferation and hematopoietic repopulation of the Fanca −/− HSPCs. We also show that simultaneous inactivation of DNA-PKcs and Fanca or Fancc causes embryonic lethality in mice, which can be rescued by the apoptosis-defective p53 mutation. Furthermore, using the knockin DNA-PKcs 3A/3A model, which specifically inactivates the NHEJ activity of DNA-PKcs, we demonstrate that the NHEJ activity of DAN-PKcs is required for FA fetal HSC maintenance.

Mice and treatment
Fanca −/− and Fancc −/− mice [30,31] were generated by interbreeding the heterozygous Fanca +/− (Dr. Madeleine Carreau at Laval University) or Fancc +/− mice (Dr. Manuel Buchwald, University of Toronto), respectively. p53 515C/515C mice (provided by Dr. Guillermina Lozano at University of Texas M.D. Anderson Cancer Center) [32] or DNA-PKcs 3A/3A mice (provided by Dr. Benjamin P. C. Chen at University of Texas Southwestern Medical Center) [33] were generated by interbreeding heterozygous p53 +/515C or DNA-PKcs +/3A mice, respectively. All the animals including BoyJ mice were maintained in the animal barrier facility at Cincinnati Children's Hospital Medical Center. All animal experiments were performed in accordance with the institutional guidelines and approved by the Institutional Animal Care and Use Committee of Cincinnati Children's Hospital Medical Center (IACUC2018-0006).

Isolation of bone marrow cells and flow cytometry analysis
The femora and tibiae were harvested from the mice immediately after their sacrifice with CO 2 . Bone marrow (BM) cells were flushed from bones into Iscove's modified Dulbecco's medium (IMDM; Invitrogen) containing 10% FCS, using a 21-gauge needle and syringe. Low-density BM mononuclear cells (LDBMMNCs) were separated by Ficoll Hypaque density gradient (Sigma-Aldrich, St. Louis, MO) and washed with IMDM medium.

Chromosomal breakage analysis
Chromosome breakage analysis was performed on LSK cells as previously described [34]. Briefly, cells were treated with 0.05 mg/ml colcermid (Gibco, Grand Island, NY, USA) for 90 min, followed by 0.4% KCl hypotonic solution at 37°for 20 min, fixed with methanol and acetic acid at 4°for 15 min, and dropped onto microscope slides. The cells were then rinsed with isoton, stained with Giemsa for 5 min, and rinsed with Gurr Buffer (CTL Scientific, Deer Park, NY, USA) and Milli-Q-filtered deionized water. A total of 50 cells from each sample were scored for chromosome aberrations.

Bone marrow transplantation (BMT)
One thousand to 2000 LSK cells (CD45.2 + ), along with 200,000 c-Kit-depleted protector cells, were transplanted into lethally irradiated BoyJ (CD45.1 + ) mice. The recipients were subjected to flow cytometric analysis for donor-derived LSK cells 16 weeks after BMT. In other experiments, 2000 GFP-sorted scramble shRNA or Ku70 shRNA lentiviral vector-transduced LSK cells, along with 200,000 c-Kit-depleted protector cells, were transplanted into lethally irradiated BoyJ mice. The recipients were subjected to flow cytometric analysis for donor-derived LSK cells 16 weeks after BMT.

Cell-cycle and apoptosis analysis
To analyze the cell-cycle status of the HSC subsets, bone marrow cells were initially stained with antibodies against Lin + cells, C-KIT, SCA-1, CD150, and CD48 as described above. After incubation with these cell surface antibodies, the cells underwent fixation and permeabilization with transcription factor buffer set (BD Biosciences, #562725) according to the manufacturer's instruction. After fixation, cells were incubated with APC-anti-Ki67 (BD Biosciences, #558615), washed and stained with PI. Cells were analyzed by flow cytometry. For the apoptosis detection, bone marrow cells were stained with the antibodies for the HSC surface markers and then stained with APC-Annexin V (BD Biosciences, #550474) and 7 AAD. Annexin V-positive populations were determined as apoptotic cells using the FACS LSR II (BD Biosciences).

Colony-forming unit assay
For the in vitro colony-forming unit (CFU) assay, 1000 sorted LSK cells were seeded in MethoCult GF M3434 (STEMCELL Technologies) according to the manufacturer's recommendations. Colonies were visualized and counted at day 7. The experiment was performed in triplicate for each sample.

Inhibition of NHEJ sensitizes Fanca −/− HSPCs to PARPiinduced cell death and genomic instability
To understand the mechanism by which the FA pathway counteracts NHEJ in genomic maintenance in HSPCs, we exposed BM LSK (Lin − Sca1 + c-kit + ; Fig. 1a) cells from WT and Fanca −/− mice to DNA-PKcs inhibitor NU7026 or Ku70 knockdown in the presence of PARP inhibitor KU58948. The reason for PARP inhibition was that we and others have shown that PARP inhibition could greatly boost NHEJ activity in HR-deficient cells including FA HSPCs [28,29,35]. Both WT and Fanca −/− LSK cells were not sensitive to the PARP inhibitor (Fig. 1b). However, treatment with the DNA-PKcs inhibitor NU7026 sensitized the Fanca −/− LSK cells to PARPi-induced cell death at low doses (0.1-1 μM), which had no effect on WT cells (Fig. 1b). Furthermore, inhibition of DNA-PKcs exacerbated genomic instability (chromosome and chromatid breaks, and radial chromosomes) in Fanca −/− LSK cells (Fig. 1c). We also genetically inhibited NHEJ by knocking down Ku70 expression using lentiviral shRNAs (Fig. 1d). We found that knockdown of Ku70 caused much higher levels of cell death (Fig. 1d) and chromosome aberrations (Fig. 1e) in Fanca −/− LSK cells than in WT cells. Furthermore, we treated BM LSK cells from WT and Fanca −/− mice with DNA cross-linker mitomycin C (MMC), which induces interstrand crosslinking (ICL), and found that knockdown of Ku70 caused much higher levels of cell death (Fig. 1d) and chromosome aberrations (Fig. 1e) in Fanca −/− LSK cells compared to Fanca −/− mock control cells. Together, these results suggest that the NHEJ pathway actually contributes to cell survival and genomic maintenance in Fanca −/− HSPCs.

Inhibition of NHEJ further decreases Fanca −/− HSPC renewal and repopulation
We next determined the effect of NHEJ inhibition on the proliferation of Fanca −/− HSPCs using the in vitro colony-forming unit (CFU) assay and the in vivo hematopoietic repopulation assay. Inhibition of NHEJ by the DNA-PKcs inhibitor NU7026 further reduced the capacity of Fanca −/− LSK cells to produced colony formation units when plated in methylcellulose supplemented with hematopoietic cytokines (Fig. 2a) and decreased the potential of these cells to proliferate in irradiated transplant recipients (Fig. 2b). Similar results were obtained with the Fanca −/− LSK cells that had been subjected to knockdown of Ku70 (Fig. 2c, d). Specifically, knocking down Ku70 further compromised the ability of Fanca −/− LSK cells to form colony in the absence of stromal support (Fig. 2c) and to repopulate the transplant recipient mice (Fig. 2d). Taken together, these results indicate a crucial role of NHEJ in maintaining Fanca −/− HSPC proliferation.
Inactivation of the NHEJ activity of DNA-PKcs in Fanca −/− or Fancc −/− mice leads to embryonic lethality The observation that inhibition of NHEJ exacerbated genomic instability in Fanca −/− HSPCs appears to be conflict with previous reports that inhibition of the key NHEJ factors such as Ku, Lig4, or DNA-PKcs could ameliorate the sensitivity of FA cells to interstrand crosslinking agents [12,15]. This prompted us to determine the in vivo effect of NHEJ inhibition in Fanca −/− mice. We crossed the Fanca −/− mice with a strain carrying the knockin DNA-PKcs 3A/3A mutation, which selectively inactivates the NHEJ activity but does not affect the kinase activity of DNA-PKcs [33]. To exclude the probability that the identified phenotypes might be due to a specific effect of a particular FA complementation group, we also employed an additional FA (Fancc −/− ) mouse model. Screening more than 160 E10.5 embryos and 270 pups showed that while we were able to obtain DNA-PKcs +/3A Fanca −/− and DNA-PKcs +/3A Fancc −/− pups, we found that  (Tables 1  and 2). Thus, these results indicate that simultaneous inactivation of DNA-PKcs and Fanca or Fancc causes embryonic lethality in mice.
DNA-PKcs 3A/3A causes fetal HSC depletion in Fanca −/− embryos due to increased HSC apoptosis and cycling We next investigated the effect of DNA-PKcs-Fanca deficiencies on fetal hematopoiesis by examining the frequency of fetal HSCs (CD150 + CD48 − Lin − Mac-1 + Sca-1 + ) in the E14.5 fetal liver of the mice, which has been shown to include all fetal liver HSC activity and are highly enriched for HSCs [36]. As shown in Fig. 3a, the frequency of fetal HSCs was more than four-to fivefold lower in DNA-PKcs 3A/3A Fanca −/− fetal livers compared to control samples from WT or single-deficient (Fanca −/− or DNA-PKcs 3A/3A ) mice (Fig. 3a), indicating a phenotype of fetal HSC depletion.
Because we observed exacerbated cell death in Fanca −/− LSK cells upon NHEJ inhibition (Fig. 1b, d), we wondered if increased apoptosis played a causal role in the depletion of fetal HSCs in DNA-PKcs 3A/3A Fanca −/− mice. To examine this possibility, we measured the apoptosis of fetal liver cells in WT, Fanca −/− , DNA-PKcs 3A/3A , and DNA-PKcs 3A/3A Fanca −/− embryos at E14.5 by Annexin V staining. Low levels (approximately 5%) of apoptotic cells were observed in the livers of both WT and Fanca −/− embryos (Fig. 3b). Whereas there was a significant increase in apoptotic fetal HSCs in DNA-PKcs 3A/3A embryos compared to WT and Fanca −/− embryos, this increase was greatly exacerbated in DNA-PKcs 3A/3A Fanca −/− fetal livers (Fig. 3b). These results suggest that fetal HSC depletion observed in DNA-PKcs 3A/3A Fanca −/− mice may be caused by increased apoptosis. We also performed cell-cycle analysis to evaluate the effect of DNA-PKcs 3A/3A on quiescence of Fanca −/− fetal HSCs. We observed a statistically significant reduction of quiescent fetal HSCs in DNA-PKcs 3A/3A and Fanca −/− embryos compared with WT embryos (Fig. 3c). Interestingly, a more dramatic decrease in quiescent fetal HSCs was detected in DNA-PKcs 3A/3A Fanca −/− embryos compared with the other three groups (Fig. 3c). These results suggest that the NHEJ activity of DNA-PKcs and Fanca may play a quantitative or collaborative functional role in the cell cycle of fetal HSCs.

Discussion
In the present study, we used multiple mouse models of closely related DNA damage response (FA, NHEJ, p53) pathways to show that inhibition of NHEJ sensitizes Fanca −/− HSPCs to PARPi-induced cell death and genomic instability. This surprising finding prompted us to propose that inhibition of the NHEJ pathway in FA HSPCs might actually exacerbate their sensitivity to DNA damage, which is the cellular hallmark of FA. In support of this notion, we showed that specific inactivation of the NHEJ activity of DNA-PKcs caused embryonic lethality in mice deficient for two components of the FA core complex Fanca and Fancc. Our results are in strike contrast to the studies reported by Adamo et al. [12] and Pace et al. [15] that hypersensitivity of   [38]. Interestingly, a more recent study shows that deletion of Ku80, another NHEJ factor, also causes embryonic lethality in mice deficient for Fancd2 [39]. The cause of embryonic lethality in DNA-PKcs 3A/ 3A Fanca −/− mice may be due to fetal HSC depletion. In support of this notion, we observed significantly increased HSC apoptosis and cycling in developing embryos of DNA-PKcs 3A/3A Fanca −/− mice compared to those of WT, DNA-PKcs 3A/3A or Fanca −/− mice. It is well known that aberrantly increased cell cycling can lead to the depletion of adult HSCs, which are quiescent under normal conditions [40][41][42]. Our results raise the possibility that abnormally increased cell-cycle progression in fetal HSCs could also lead to their depletion. Interestingly, both p53 null and a knockin p53 515C mutation, which selectively impairs only the p53 function in apoptosis, can rescue embryonic lethality and fetal HSC depletion in Fanca −/− DNA-PKcs 3A/3A mice. This suggests that although DNA-PKcs 3A/3A increases Fanca −/− HSC cycling, the cell-cycle activity of p53 is not the Fetal liver cells from E14.5 embryos with the indicated genotype were subjected to flow cytometric analysis for fetal HSC (CD150 + CD48 − Lin − Mac-1 + Sca-1 + ). ***p < 0.001 vs WT control. c p53 515C rescues embryonic lethality in Fanca −/− DNA-PKcs 3A/3A mice. Graphical representation of expected vs. observed number of pups based on Mendelian inheritance of alleles. d p53 515 rescues fetal HSC depletion in Fanca −/− DNA-PKcs 3A/3A mice. Fetal liver cells from E14.5 embryos with the indicated genotype were subjected to flow cytometric analysis for fetal HSC (CD150 + CD48 − Lin − Mac-1 + Sca-1 + ). ***p < 0.001 vs WT control decisive factor in the regulation of DNA-PKcs 3A/3A HSC maintenance. In this context, our results are consistent with previous studies that show p53-dependent apoptosis in the DNA-PKcs 3A/3A HSCs and FA HSPCs [33,37].

Conclusions
In this study, we employed multiple mouse models of closely related DNA damage response (FA, NHEJ, p53) pathways to demonstrate that the NHEJ pathway is required for cell survival and proliferation of murine FA HSPCs. We further show that the NHEJ pathway functions to maintain Fanconi anemia fetal HSCs.