Exploring pharmacokinetics of talazoparib in ABCB1/ABCG2-deficient mice using a novel UHPLC-MS/MS method.

A rapid, sensitive, and simple UHPLC-MS/MS method for the determination of the PARP inhibitor talazoparib in mouse plasma was developed and validated using [13C,2H4]-talazoparib as an internal standard (IS). The assay procedure involved extraction of talazoparib and the IS from plasma using a single-step deproteination and separation of the analytes was achieved on an ACQUITY UPLC RP18 HSS T3 column with a mobile phase gradient at a flow rate of 0.4 mL/min in a run time of 5 min. The calibration curve was linear (r2 > 0.99) over the concentration range of 0.5-100 ng/mL, and 10-fold dilution of samples could be accurately quantitated. The matrix effect and mean extraction recovery for talazoparib were between 93.7-109% and 87.7-105%, respectively. Precision and percent bias of quality control samples were always less than ±15%, indicating reproducibility and accuracy of the method. Talazoparib demonstrated bench-top stability at room temperature for 6 h, auto-sampler and reinjection stability at 4 °C for at least 24 h, and no significant degradation was observed after three freeze-thaw cycles. The developed method was successfully applied to pharmacokinetic studies involving serial blood sampling after oral administration of talazoparib to wild-type mice and animals with a genetic deficiency of the efflux transporters ABCB1 (P-gp) and ABCG2 (BCRP). Together, our results demonstrate the successful development of a suitable analytical method for talazoparib in mouse plasma and suggest that mice are a useful model to evaluate transporter-mediated drug-drug interactions involving therapy with talazoparib.

A Phase I Expansion Cohort Study Evaluating the Safety and Efficacy of the CHK1 Inhibitor LY2880070 with Low-dose Gemcitabine in Patients with Metastatic Pancreatic Adenocarcinoma.

PURPOSE: Combining gemcitabine with CHK1 inhibition has shown promise in preclinical models of pancreatic ductal adenocarcinoma (PDAC). Here, we report the findings from a phase I expansion cohort study (NCT02632448) investigating low-dose gemcitabine combined with the CHK1 inhibitor LY2880070 in patients with previously treated advanced PDAC. PATIENTS AND METHODS: Patients with metastatic PDAC were treated with gemcitabine intravenously at 100 mg/m2 on days 1, 8, and 15, and LY2880070 50 mg orally twice daily on days 2-6, 9-13, and 16-20 of each 21-day cycle. Pretreatment tumor biopsies were obtained from each patient for correlative studies and generation of organoid cultures for drug sensitivity testing and biomarker analyses. RESULTS: Eleven patients with PDAC were enrolled in the expansion cohort between August 27, 2020 and July 30, 2021. Four patients (36%) experienced drug-related grade 3 adverse events. No objective radiologic responses were observed, and all patients discontinued the trial by 3.2 months. In contrast to the lack of efficacy observed in patients, organoid cultures derived from biopsies procured from two patients demonstrated strong sensitivity to the gemcitabine/LY2880070 combination and showed treatment-induced upregulation of replication stress and DNA damage biomarkers, including pKAP1, pRPA32, and γH2AX, as well as induction of replication fork instability. CONCLUSIONS: No evidence of clinical activity was observed for combined low-dose gemcitabine and LY2880070 in this treatment-refractory PDAC cohort. However, the gemcitabine/LY2880070 combination showed in vitro efficacy, suggesting that drug sensitivity for this combination in organoid cultures may not predict clinical benefit in patients.

TGFß pathway is required for viable gestation of Fanconi anemia embryos.

Overexpression of the TGFß pathway impairs the proliferation of the hematopoietic stem and progenitor cells (HSPCs) pool in Fanconi anemia (FA). TGFß promotes the expression of NHEJ genes, known to function in a low-fidelity DNA repair pathway, and pharmacological inhibition of TGFß signaling rescues FA HSPCs. Here, we demonstrate that genetic disruption of Smad3, a transducer of the canonical TGFß pathway, modifies the phenotype of FA mouse models deficient for Fancd2. We observed that the TGFß and NHEJ pathway genes are overexpressed during the embryogenesis of Fancd2-/- mice and that the Fancd2-/-Smad3-/- double knockout (DKO) mice undergo high levels of embryonic lethality due to loss of the TGFß-NHEJ axis. Fancd2-deficient embryos acquire extensive genomic instability during gestation which is not reversed by Smad3 inactivation. Strikingly, the few DKO survivors have activated the non-canonical TGFß-ERK pathway, ensuring expression of NHEJ genes during embryogenesis and improved survival. Activation of the TGFß-NHEJ axis was critical for the survival of the few Fancd2-/-Smad3-/- DKO newborn mice but had detrimental consequences for these surviving mice, such as enhanced genomic instability and ineffective hematopoiesis.

Targeting DNA Repair with Combined Inhibition of NHEJ and MMEJ Induces Synthetic Lethality in TP53-Mutant Cancers.

DNA repair pathway inhibitors are a new class of anticancer drugs that are advancing in clinical trials. Peposertib is an inhibitor of DNA-dependent protein kinase (DNA-PK), which is a key driver of nonhomologous end-joining (NHEJ). To identify regulators of response to peposertib, we performed a genome-wide CRISPR knockout screen and found that loss of POLQ (polymerase theta, POLθ) and other genes in the microhomology-mediated end-joining (MMEJ) pathway are key predictors of sensitivity to DNA-PK inhibition. Simultaneous disruption of two DNA repair pathways via combined treatment with peposertib plus a POLθ inhibitor novobiocin exhibited synergistic synthetic lethality resulting from accumulation of toxic levels of DNA double-strand break end resection. TP53-mutant tumor cells were resistant to peposertib but maintained elevated expression of POLQ and increased sensitivity to novobiocin. Consequently, the combination of peposertib plus novobiocin resulted in synthetic lethality in TP53-deficient tumor cell lines, organoid cultures, and patient-derived xenograft models. Thus, the combination of a targeted DNA-PK/NHEJ inhibitor with a targeted POLθ/MMEJ inhibitor may provide a rational treatment strategy for TP53-mutant solid tumors. SIGNIFICANCE: Combined inhibition of NHEJ and MMEJ using two nontoxic, targeted DNA repair inhibitors can effectively induce toxic DNA damage to treat TP53-deficient cancers.

Isolation of human and murine hematopoietic stem cells for DNA damage and DNA repair assays.

Hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow and supply blood cells. Efficient methods for isolation of HSPCs are required. Here, we present protocols for the isolation of human and murine HSPCs using manual and FACS-assisted techniques. Isolated HSPCs can be used for downstream applications, including colony forming unit assays and DNA damage and repair assays. For complete details on the use and execution of this protocol, please refer to Rodríguez et al. (2021a) and (2021b).

Phase 1 Combination Study of the CHK1 Inhibitor Prexasertib and the PARP Inhibitor Olaparib in High-grade Serous Ovarian Cancer and Other Solid Tumors.

PURPOSE: Checkpoint kinase 1 (CHK1) plays a central role in the response to replication stress through modulation of cell-cycle checkpoints and homologous recombination (HR) repair. In BRCA-deficient cancers with de novo or acquired PARP inhibitor resistance, the addition of the CHK1 inhibitor prexasertib to the PARP inhibitor olaparib compromises replication fork stability, as well as HR proficiency, allowing for sensitization to PARP inhibition. PATIENTS AND METHODS: This study followed a 3+3 design with a 7-day lead-in of olaparib alone, followed by 28-day cycles with prexasertib administered on days 1 and 15 in combination with an attenuated dose of olaparib on days 1-5 and 15-19. Pharmacokinetic blood samples were collected after olaparib alone and following combination therapy. Patients enrolled to the expansion phase of the study underwent paired tumor biopsies for pharmacodynamic (PD) assessments. RESULTS: Twenty-nine patients were treated. DLTs included grade 3 neutropenia and grade 3 febrile neutropenia. The MTD/recommended phase 2 dose (RP2D) was prexasertib at 70 mg/m2 i.v. with olaparib at 100 mg by mouth twice daily. Most common treatment-related adverse events included leukopenia (83%), neutropenia (86%), thrombocytopenia (66%), and anemia (72%). Four of 18 patients with BRCA1-mutant, PARP inhibitor-resistant, high-grade serous ovarian cancer (HGSOC) achieved partial responses. Paired tumor biopsies demonstrated reduction in RAD51 foci and increased expression of γ-H2AX, pKAP1, and pRPA after combination exposure. CONCLUSIONS: Prexasertib combined with olaparib has preliminary clinical activity in BRCA-mutant patients with HGSOC who have previously progressed on a PARP inhibitor. PD analyses show that prexasertib compromises HR with evidence of induction of DNA damage and replication stress.

MYC Promotes Bone Marrow Stem Cell Dysfunction in Fanconi Anemia.

Bone marrow failure (BMF) in Fanconi anemia (FA) patients results from dysfunctional hematopoietic stem and progenitor cells (HSPCs). To identify determinants of BMF, we performed single-cell transcriptome profiling of primary HSPCs from FA patients. In addition to overexpression of p53 and TGF-ß pathway genes, we identified high levels of MYC expression. We correspondingly observed coexistence of distinct HSPC subpopulations expressing high levels of TP53 or MYC in FA bone marrow (BM). Inhibiting MYC expression with the BET bromodomain inhibitor (+)-JQ1 reduced the clonogenic potential of FA patient HSPCs but rescued physiological and genotoxic stress in HSPCs from FA mice, showing that MYC promotes proliferation while increasing DNA damage. MYC-high HSPCs showed significant downregulation of cell adhesion genes, consistent with enhanced egress of FA HSPCs from bone marrow to peripheral blood. We speculate that MYC overexpression impairs HSPC function in FA patients and contributes to exhaustion in FA bone marrow.

Inhibition of TGFß1 and TGFß3 promotes hematopoiesis in Fanconi anemia.

Fanconi anemia (FA) is a chromosome instability syndrome with congenital abnormalities, cancer predisposition and bone marrow failure (BMF). Although hematopoietic stem and progenitor cell (HSPC) transplantation is the recommended therapy, new therapies are needed for FA patients without suitable donors. BMF in FA is caused, at least in part, by a hyperactive growth-suppressive transforming growth factor ß (TGFß) pathway, regulated by the TGFß1, TGFß2, and TGFß3 ligands. Accordingly, the TGFß pathway is an attractive therapeutic target for FA. While inhibition of TGFß1 and TGFß3 promotes blood cell expansion, inhibition of TGFß2 is known to suppress hematopoiesis. Here, we report the effects of AVID200, a potent TGFß1- and TGFß3-specific inhibitor, on FA hematopoiesis. AVID200 promoted the survival of murine FA HSPCs in vitro. AVID200 also promoted in vitro the survival of human HSPCs from patients with FA, with the strongest effect in patients progressing to severe aplastic anemia or myelodysplastic syndrome (MDS). Previous studies have indicated that the toxic upregulation of the nonhomologous end-joining (NHEJ) pathway accounts, at least in part, for the poor growth of FA HSPCs. AVID200 downregulated the expression of NHEJ-related genes and reduced DNA damage in primary FA HSPC in vitro and in in vivo models. Collectively, AVID200 exhibits activity in FA mouse and human preclinical models. AVID200 may therefore provide a therapeutic approach to improving BMF in FA.

CHK1 Inhibitor Blocks Phosphorylation of FAM122A and Promotes Replication Stress.

While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging. Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A/PABIR1 confers cellular resistance to CHK1 inhibitors (CHK1is) and cross-resistance to ATR inhibitors. Knockout of FAM122A results in activation of PP2A-B55α, a phosphatase that dephosphorylates the WEE1 protein and rescues WEE1 from ubiquitin-mediated degradation. The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1is. Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55α phosphatase and increased WEE1 expression. A combination of a CHK1i plus a WEE1 inhibitor can overcome CHK1i resistance of these tumor cells, thereby enhancing anti-cancer activity. The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1i sensitivity or resistance.

Cooperation of the ATM and Fanconi Anemia/BRCA Pathways in Double-Strand Break End Resection.

Cells deficient in ataxia telangiectasia mutated (ATM) are hypersensitive to ionizing radiation and other anti-cancer agents that induce double-strand DNA breaks. ATM inhibitors may therefore sensitize cancer cells to these agents. Some cancers may also have underlying genetic defects predisposing them to an ATM inhibitor monotherapy response. We have conducted a genome-wide CRISPR screen to identify genetic vulnerabilities that sensitize lung cancer cells to ATM inhibitors. Knockout of genes in the Fanconi anemia (FA)/BRCA pathway results in hypersensitivity to the ATM inhibitor M3541. Knockdown of either an FA gene or of ATM results in reduced double-strand break end resection, enhanced non-homologous end joining (NHEJ) repair, and decreased homologous recombination repair. Knockout of both the FA/BRCA pathway and ATM strongly inhibits end resection and generates toxic levels of NHEJ, thereby elucidating a mechanism of cellular death by synthetic lethality. ATM inhibitors may therefore be useful for the treatment of tumors with a defective FA/BRCA pathway.

The CHK1 Inhibitor Prexasertib Exhibits Monotherapy Activity in High-Grade Serous Ovarian Cancer Models and Sensitizes to PARP Inhibition.

PURPOSE: PARP inhibitors are approved for the treatment of high-grade serous ovarian cancers (HGSOC). Therapeutic resistance, resulting from restoration of homologous recombination (HR) repair or replication fork stabilization, is a pressing clinical problem. We assessed the activity of prexasertib, a checkpoint kinase 1 (CHK1) inhibitor known to cause replication catastrophe, as monotherapy and in combination with the PARP inhibitor olaparib in preclinical models of HGSOC, including those with acquired PARP inhibitor resistance. EXPERIMENTAL DESIGN: Prexasertib was tested as a single agent or in combination with olaparib in 14 clinically annotated and molecularly characterized luciferized HGSOC patient-derived xenograft (PDX) models and in a panel of ovarian cancer cell lines. The ability of prexasertib to impair HR repair and replication fork stability was also assessed. RESULTS: Prexasertib monotherapy demonstrated antitumor activity across the 14 PDX models. Thirteen models were resistant to olaparib monotherapy, including 4 carrying BRCA1 mutation. The combination of olaparib with prexasertib was synergistic and produced significant tumor growth inhibition in an olaparib-resistant model and further augmented the degree and durability of response in the olaparib-sensitive model. HGSOC cell lines, including those with acquired PARP inhibitor resistance, were also sensitive to prexasertib, associated with induction of DNA damage and replication stress. Prexasertib also sensitized these cell lines to PARP inhibition and compromised both HR repair and replication fork stability. CONCLUSIONS: Prexasertib exhibits monotherapy activity in PARP inhibitor-resistant HGSOC PDX and cell line models, reverses restored HR and replication fork stability, and synergizes with PARP inhibition.

A Fanci knockout mouse model reveals common and distinct functions for FANCI and FANCD2.

Fanconi Anemia (FA) clinical phenotypes are heterogenous and rely on a mutation in one of the 22 FANC genes (FANCA-W) involved in a common interstrand DNA crosslink-repair pathway. A critical step in the activation of FA pathway is the monoubiquitination of FANCD2 and its binding partner FANCI. To better address the clinical phenotype associated with FANCI and the epistatic relationship with FANCD2, we created the first conditional inactivation model for FANCI in mouse. Fanci -/- mice displayed typical FA features such as delayed development in utero, microphtalmia, cellular sensitivity to mitomycin C, occasional limb abnormalities and hematological deficiencies. Interestingly, the deletion of Fanci leads to a strong meiotic phenotype and severe hypogonadism. FANCI was localized in spermatocytes and spermatids and in the nucleus of oocytes. Both FANCI and FANCD2 proteins co-localized with RPA along meiotic chromosomes, albeit at different levels. Consistent with a role in meiotic recombination, FANCI interacted with RAD51 and stimulated D-loop formation, unlike FANCD2. The double knockout Fanci-/- Fancd2-/- also showed epistatic relationship for hematological defects while being not epistatic with respect to generating viable mice in crosses of double heterozygotes. Collectively, this study highlights common and distinct functions of FANCI and FANCD2 during mouse development, meiotic recombination and hematopoiesis.

USP1 Is Required for Replication Fork Protection in BRCA1-Deficient Tumors.

BRCA1-deficient tumor cells have defects in homologous-recombination repair and replication fork stability, resulting in PARP inhibitor sensitivity. Here, we demonstrate that a deubiquitinase, USP1, is upregulated in tumors with mutations in BRCA1. Knockdown or inhibition of USP1 resulted in replication fork destabilization and decreased viability of BRCA1-deficient cells, revealing a synthetic lethal relationship. USP1 binds to and is stimulated by fork DNA. A truncated form of USP1, lacking its DNA-binding region, was not stimulated by DNA and failed to localize and protect replication forks. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of cell death in the absence of USP1. Taken together, USP1 exhibits DNA-mediated activation at the replication fork, protects the fork, and promotes survival in BRCA1-deficient cells. Inhibition of USP1 may be a useful treatment for a subset of PARP-inhibitor-resistant BRCA1-deficient tumors with acquired replication fork stabilization.

Amelioration of Head and Neck Radiation-Induced Mucositis and Distant Marrow Suppression in Fanca-/- and Fancg-/- Mice by Intraoral Administration of GS-Nitroxide (JP4-039).

Squamous cell carcinomas of the head and neck are appearing with increased frequency in both marrow transplanted and non-transplanted Fanconi anemia (FA) patients. FA patients commonly display radiosensitivity of epithelial tissues, complicating effective radiotherapy. Fancd2-/- mice (C57BL/6J and 129/Sv background) demonstrate epithelial tissue sensitivity to single-fraction or fractionated irradiation to the head and neck and distant marrow suppression (abscopal effect), both ameliorated by intraoral administration of the mitochondrial-targeted antioxidant, GS-nitroxide, JP4-039. We now report that mice of two other FA genotypes, Fancg-/- (B6) and the most prevalent human genotype Fanca-/- (129/Sv), also demonstrate: 1. reduced longevity of hematopoiesis in long-term bone marrow cultures; 2. radiosensitivity of bone marrow stromal cell lines; and 3. head and neck radiation-induced severe mucositis and abscopal suppression of distant marrow hematopoiesis. Intraoral administration of JP4-039/F15, but not non-mitochondrial-targeted 4-amino-Tempo/F15 or F15 alone, prior to each radiation treatment ameliorated both local and abscopal radiation effects. Head and neck irradiated TGF-ß-resistant SMAD3-/- (129/Sv) mice and double-knockout SMAD3-/- Fancd2-/- (129/Sv) mice treated daily with TGF-ß receptor antagonist, LY364947, still displayed abscopal bone marrow suppression, implicating a non-TGF-ß mechanism. Thus, amelioration of both local normal tissue radiosensitivity and distant marrow suppression by intraoral administration of JP4-039 in Fancg-/- and Fanca-/- mice supports a clinical trial of this locally administered normal tissue radioprotector and mitigator during head and neck irradiation in FA patients.

Aldehydes Pose a Threat to BRCA2 Mutation Carriers.

Humans with inherited heterozygous BRCA2 mutations have an increased risk of developing cancer; however, what triggers carcinogenesis in these individuals is unclear. Tan et al. find that environmental and metabolic aldehydes pose a threat to these individuals by promoting degradation of wild-type BRCA2 protein, thereby predisposing them to genomic instability and perhaps to cancer.

Small Molecule Inhibition of the Ubiquitin-specific Protease USP2 Accelerates cyclin D1 Degradation and Leads to Cell Cycle Arrest in Colorectal Cancer and Mantle Cell Lymphoma Models.

Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC50 of 1.1 µm in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines. ML364, and not the inactive analog 2, was antiproliferative in cancer cell lines. Consistent with the role of cyclin D1 in DNA damage response, ML364 also caused a decrease in homologous recombination-mediated DNA repair. These effects by a small molecule inhibitor support a key role for USP2 as a regulator of cell cycle, DNA repair, and tumor cell growth.

TGF-ß Inhibition Rescues Hematopoietic Stem Cell Defects and Bone Marrow Failure in Fanconi Anemia.

Fanconi anemia (FA) is an inherited DNA repair disorder characterized by progressive bone marrow failure (BMF) from hematopoietic stem and progenitor cell (HSPC) attrition. A greater understanding of the pathogenesis of BMF could improve the therapeutic options for FA patients. Using a genome-wide shRNA screen in human FA fibroblasts, we identify transforming growth factor-ß (TGF-ß) pathway-mediated growth suppression as a cause of BMF in FA. Blocking the TGF-ß pathway improves the survival of FA cells and rescues the proliferative and functional defects of HSPCs derived from FA mice and FA patients. Inhibition of TGF-ß signaling in FA HSPCs results in elevated homologous recombination (HR) repair with a concomitant decrease in non-homologous end-joining (NHEJ), accounting for the improvement in cellular growth. Together, our results suggest that elevated TGF-ß signaling contributes to BMF in FA by impairing HSPC function and may be a potential therapeutic target for the treatment of FA.

Intraoral Mitochondrial-Targeted GS-Nitroxide, JP4-039, Radioprotects Normal Tissue in Tumor-Bearing Radiosensitive Fancd2(-/-) (C57BL/6) Mice.

We evaluated normal tissue specific radioprotection of the oral cavity in radiosensitive Fanconi Anemia (FA) Fancd2(-/-) mice with orally established tumors using mitochondrial-targeted GS-nitroxide (JP4-039). Adult (10-12 weeks old) Fancd2(+/+), Fancd2(+/-) and Fancd2(-/-) mice (C57BL/6 background) and subgroups with orally established TC-1 epithelial cell tumors received a single fraction of 28 Gy or four daily fractions of 8 Gy to the head and neck. Subgroups received JP4-039 in F15 emulsion (F15/JP4-039; 0.4 mg/mouse), 4-amino-Tempo in F15 emulsion (F15/4-amino-Tempo; 0.2 mg/mouse) or F15 emulsion alone prior to each irradiation. Oral mucosa of Fancd2(-/-) mice showed baseline elevated RNA transcripts for Sod2, p53, p21 and Rad51 (all P < 0.0012) and suppressed levels of Nfkb and Tgfb, (all P < 0.0020) compared with Fancd2(+/+) mice. The oral mucosa in tumor-bearing mice of all genotypes showed decreased levels of p53 and elevated Tgfb and Gadd45a (P ≤ 0.0001 for all three genotypes). Intraoral F15/JP4-039, but not F15/4-amino-Tempo, modulated radiation-induced normal tissue transcript elevation, ameliorated mucosal ulceration and reduced the depletion of antioxidant stores in oral cavity tissue of all genotypes, but did not radioprotect tumors. Mitochondrial targeting makes F15/JP4-039 an effective normal tissue radioprotector for Fancd2(-/-) mice, as well as wild-type mice.

Serum microRNAs are early indicators of survival after radiation-induced hematopoietic injury.

Accidental radiation exposure is a threat to human health that necessitates effective clinical planning and diagnosis. Minimally invasive biomarkers that can predict long-term radiation injury are urgently needed for optimal management after a radiation accident. We have identified serum microRNA (miRNA) signatures that indicate long-term impact of total body irradiation (TBI) in mice when measured within 24 hours of exposure. Impact of TBI on the hematopoietic system was systematically assessed to determine a correlation of residual hematopoietic stem cells (HSCs) with increasing doses of radiation. Serum miRNA signatures distinguished untreated mice from animals exposed to radiation and correlated with the impact of radiation on HSCs. Mice exposed to sublethal (6.5 Gy) and lethal (8 Gy) doses of radiation were indistinguishable for 3 to 4 weeks after exposure. A serum miRNA signature detectable 24 hours after radiation exposure consistently segregated these two cohorts. Furthermore, using either a radioprotective agent before, or radiation mitigation after, lethal radiation, we determined that the serum miRNA signature correlated with the impact of radiation on animal health rather than the radiation dose. Last, using humanized mice that had been engrafted with human CD34(+) HSCs, we determined that the serum miRNA signature indicated radiation-induced injury to the human bone marrow cells. Our data suggest that serum miRNAs can serve as functional dosimeters of radiation, representing a potential breakthrough in early assessment of radiation-induced hematopoietic damage and timely use of medical countermeasures to mitigate the long-term impact of radiation.

Radiologic differences between bone marrow stromal and hematopoietic progenitor cell lines from Fanconi Anemia (Fancd2(-/-)) mice.

FancD2 plays a central role in the human Fanconi anemia DNA damage response (DDR) pathway. Fancd2(-/-) mice exhibit many features of human Fanconi anemia including cellular DNA repair defects. Whether the DNA repair defect in Fancd2(-/-) mice results in radiologic changes in all cell lineages is unknown. We measured stress of hematopoiesis in long-term marrow cultures and radiosensitivity in clonogenic survival curves, as well as comet tail intensity, total antioxidant stores and radiation-induced gene expression in hematopoietic progenitor compared to bone marrow stromal cell lines. We further evaluated radioprotection by a mitochondrial-targeted antioxidant GS-nitroxide, JP4-039. Hematopoiesis longevity in Fancd2(-/-) mouse long-term marrow cultures was diminished and bone marrow stromal cell lines were radiosensitive compared to Fancd2(+/+) stromal cells (Fancd2(-/-) D0 = 1.4 ± 0.1 Gy, ñ = 5.0 ± 0.6 vs. Fancd2(+/+) D0 = 1.6 ± 0.1 Gy, ñ = 6.7 ± 1.6), P = 0.0124 for D0 and P = 0.0023 for ñ, respectively). In contrast, Fancd2(-/-) IL-3-dependent hematopoietic progenitor cells were radioresistant (D0 = 1.71 ± 0.04 Gy and ñ = 5.07 ± 0.52) compared to Fancd2(+/+) (D0 = 1.39 ± 0.09 Gy and ñ = 2.31 ± 0.85, P = 0.001 for D0). CFU-GM from freshly explanted Fancd2(-/-) marrow was also radioresistant. Consistent with radiosensitivity, irradiated Fancd2(-/-) stromal cells had higher DNA damage by comet tail intensity assay compared to Fancd2(+/+) cells (P < 0.0001), slower DNA damage recovery, lower baseline total antioxidant capacity, enhanced radiation-induced depletion of antioxidants, and increased CDKN1A-p21 gene transcripts and protein. Consistent with radioresistance, Fancd2(-/-) IL-3-dependent hematopoietic cells had higher baseline and post irradiation total antioxidant capacity. While, there was no detectable alteration of radiation-induced cell cycle arrest with Fancd2(-/-) stromal cells, hematopoietic progenitor cells showed reduced G2/M cell cycle arrest. The absence of the mouse Fancd2 gene product confers radiosensitivity to bone marrow stromal but not hematopoietic progenitor cells.