Identification of Novel Combination Therapies Active in BCL2 Inhibitor-Resistant Patient-Derived AML Models

Acute myeloid leukemia (AML) is an aggressive, heterogeneous malignancy. AML patients whose disease relapses on chemotherapy or are unfit for aggressive induction regimens have limited therapeutic options. Many patients benefit from the combination of venetoclax (BCL2i) and a hypomethylating agent (HMA) but this regimen is rarely curative. The addition of novel agents could provide improved benefit for relapsed/refractory patients. To identify such regimens, we screened a panel of 10 AML cell lines with combinations of venetoclax and novel targeted agents. The agents used spanned multiple mechanisms of action (e.g. DNA damage response, kinase signaling, pro-apoptotic agents) and are all in early clinical development. Cells were treated for 72hrs and viability was assessed by CellTiter-Glo. In several of the cell lines that were insensitive or partially sensitive to venetoclax (OCI-AML3, KG1a, MonoMac6, THP1), combinations with inhibitors of MCL1 (AZD5991), AURKB (AZD2811), and BRD4 (AZD5153) showed synergistic activity (Loewe synergy score >5, growth inhibition > 180%) (Table 1). We next asked if these combinations were active in patient-derived xenograft (PDX) models of AML. We established an ex vivo co-culture assay using the HS-5 bone marrow stromal cell line. AML PDX cells were isolated from mouse spleens and plated in 96-well format in direct co-culture with HS-5 cells or in HS-5-derived conditioned media. Cells were treated with three doses of each monotherapy and three doses of fixed ratio combination. Replicate screens using cells from individual mice on different days confirmed data were reproducible (r2=0.687) across animals engrafted with the same PDX. Drug response was similar between conditioned media and direct co-culture assays (r2=0.81). Venetoclax sensitivity varied across PDX models ex vivo. Notably, 2/5 PDX models screened (DFAM-68555 and DFAM-10360) were insensitive to both venetoclax and the combination of venetoclax + 5-azacytidine (HMA) ex vivo. Both models were established from untreated/1L patients and harbor TP53 mutations. Combination treatments did not add additional benefit over venetoclax monotherapy in the DFAM-10360 model. However, in DFAM-68555, AZD5153, AZD5991, and AZD2811 showed improved activity over venetoclax alone (67%, 54%, and 67% vs. 26% decrease in viability for venetoclax alone, respectively). Since combination strategies will likely be most impactful in patients refractory to or relapsed after venetoclax, we chose this venetoclax insensitive model to prioritize in vivo. To confirm the translatability of these findings, we designed a pilot in vivo study using DFAM-68555. Mice were randomized to receive vehicle, venetoclax + HMA, or venetoclax + AZD5153 when peripheral blood disease reached ~5% (hCD45+hCD33+ cells by flow cytometry). After two weeks of dosing, animals were sacrificed to evaluate disease burden in bone marrow (sternum), spleen, and peripheral blood. The model remained insensitive to venetoclax + HMA in vivo. The combination of AZD5153 with venetoclax decreased disease burden in blood and spleen compared to vehicle (30% and 42% hCD45+CD33+ cells by flow cytometry vs 70% and 95%, respectively) with similar efficacy seen by immunohistochemistry in the bone. Finally, we screened these venetoclax combinations in additional aggressive AML PDX models which were resistant or only partially responsive to venetoclax in vivo. Addition of AZD2811NP and AZD5991 to venetoclax was more effective than venetoclax alone and venetoclax + HMA in the bone marrow. The most active combination varied from model to model. Efficacy screening in additional models is ongoing to further build ex vivo to in vivo translation and prioritize development of specific combinations. Also ongoing is genomic and transcriptomic profiling of these PDXs to identify potential predictive biomarkers of combination activity. In summary, we developed an ex vivo screening platform to test clinically actionable combinations for activity in clinically relevant models. Using this platform and subsequent in vivo efficacy, we identified venetoclax combinations across multiple mechanisms (pro-apoptotic, cell cycle regulation, transcriptional regulation, DNA damage response) with activity in venetoclax-insensitive models. These results suggest potential therapeutic options to explore clinically for AML patients. Disclosures Andersen: AstraZeneca: Employment. Christie:AstraZeneca: Employment. Rosen:Astrazeneca: Employment. Maratea:AstraZeneca: Employment. Hattersley:AstraZeneca: Employment. Travers:AstraZeneca: Employment. Cidado:AstraZeneca: Employment. Pulukuri:AstraZeneca: Employment. Saeh:AstraZeneca: Employment. Clark:AstraZeneca: Employment, Equity Ownership. Reimer:AstraZeneca: Employment. Mettetal:AstraZeneca: Employment.

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HSC Exit From Dormancy Provokes De Novo DNA Damage, Leading To Bone Marrow Failure If Unresolved By The Fanconi Anemia Pathway

Blood ◽

10.1182/blood.v122.21.799.799 ◽

2013 ◽

Vol 122 (21) ◽

pp. 799-799

Author(s):

Dagmar Walter ◽

Amelie Lier ◽

Anja Geiselhart ◽

Sina Huntscha ◽

David Brocks ◽

...

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Aplastic Anemia ◽

Dna Damage Response ◽

Fanconi Anemia ◽

De Novo ◽

Bone Marrow Failure ◽

Damage Response ◽

Fold Induction

Abstract Long-term quiescence has been proposed to preserve the genomic stability of hematopoietic stem cells (HSCs) during aging. The current models of HSC aging are limited in their ability to observe both DNA damage in vivo and the consequences of this damage upon hematopoiesis. Fanconi Anemia (FA) is a hereditary multisystem disorder, characterized by defective DNA damage response and progressive bone marrow failure in most patients. However, the existing genetic models of FA do not develop aplastic anemia, suggesting that cell-extrinsic factors may play a causal role. We sought to identify whether physiologic mediators of HSC activation could be used as agonists to provoke DNA damage and HSC attrition in vivo. Mice were treated with a range of agonists that promote the in vivo exit of HSC from a dormant state into active cycling (polyI:polyC; Interferon-α; G-CSF; TPO; and serial bleeding). Highly purified HSC demonstrated a rapid 3-5-fold induction of DNA damage after treatment with all agonists (p<0.01), as assessed by both enumerating γ-H2AX foci and by alkaline comet assay. Mechanistically, stress-induced exit from quiescence correlated with increased mitochondrial metabolism in HSC, as evaluated by elevated mitochondrial membrane potential (2-fold increased, p<0.01) and superoxide levels (1.5-fold increased, p<0.05). Critically, we could directly implicate these reactive oxygen species in DNA damage as we observed a 1.4-fold increase in 8-Oxo-dG lesions in HSC that had been activated into cycle in vivo(p<0.05). At 48 h post-treatment, γ-H2AX levels began to decrease and this repair was concomitant with an induction of the FA signaling pathway in HSC, as demonstrated by both increased levels of FA gene expression and elevated FANCD2 foci (4-fold induction, p<0.01). Treatment of Fanca-/- mice with polyI:polyC led to a HSC proliferative response comparable to wild type (WT) mice but resulted in a 2-fold higher level of activation-induced DNA damage (p<0.05), demonstrating that this repair pathway is involved in resolving activation-induced DNA damage. Four rounds of serial in vivo activation led to a permanent depletion of the most primitive label-retaining Fanca-/- HSC and this correlated with a 4-fold depletion of functional HSC (p<0.01) as defined by competitive repopulation assays. Subsequent rounds of HSC activation with polyI:polyC resulted in the onset of a severe aplastic anemia (SAA) in 33% of treated Fanca-/- mice but not in any of the WT controls. SSA was characterized by a dramatic reduction in bone marrow (BM) cellularity, profound thrombocytopenia (21-246x106 platelets/ml), leukocytopenia (0.4-0.5x106 WBC/ml), neutropenia (0.03-0.1x106/ml) and anemia (1.5-2.3 g/dL Hb). Examination of BM HSC/progenitors demonstrated nearly complete loss of HSC, MPP, CMP and CLP (depletion of ≥33x, 8x, 4x and 12x respectively compared to PBS-treated Fanca-/-controls). Taken together, these data demonstrates that enforced exit from dormancy in vivo leads to de novo DNA damage in HSC, which is repaired by activation of a FA-dependent DNA damage response. Furthermore, the highly penetrant bone marrow failure observed in Fanconi anemia patients can be recapitulated by the serial application of a physiologic HSC activating signal to Fanca-/- mice. This suggests that the BM failure in FA may be caused by an aberrant response to HSC activation, most likely during exposure to infection or other physiologic stressors. These data provides a novel link between pro-inflammatory cytokines, DNA damage and HSC dysfunction and may have important clinical implications relevant to both prevention of BM failure in FA and in the study of age-related hematopoietic defects in non-FA patients. Moreover, these data provide the first evidence that FA knockout mouse models accurately recapitulate and provide novel insights into the etiology of BM failure in patients with FA. Disclosures: No relevant conflicts of interest to declare.

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Progressive changes in chromatin structure and DNA damage response signals in bone marrow and peripheral blood during myelomagenesis

Leukemia ◽

10.1038/leu.2013.284 ◽

2013 ◽

Vol 28 (5) ◽

pp. 1113-1121 ◽

Cited By ~ 8

Author(s):

M Gkotzamanidou ◽

E Terpos ◽

C Bamia ◽

S A Kyrtopoulos ◽

P P Sfikakis ◽

...

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Dna Damage Response ◽

Chromatin Structure ◽

Peripheral Blood ◽

Damage Response

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A MODEL to STUDY the Function of NPM-MLF1 IN MYELODYSPLASIA.

Blood ◽

10.1182/blood.v116.21.1571.1571 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1571-1571

Author(s):

Wen-hsin Lee

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Dna Damage Response ◽

Conflicts Of Interest ◽

Mouse Bone Marrow ◽

Self Renewal ◽

Damage Response ◽

Elevated Expression

Abstract Abstract 1571 Myelodysplastic syndromes (MDS) are bone marrow disorders characterized by ineffective haematopoiesis and peripheral cytopenia(s) with frequent evolution to acute myeloid leukemia (AML). Apoptosis is significantly deregulated in early MDS whereas advanced MDS is characterized by deregulation of DNA damage response. As MDS proceeds to AML, the ratio of apoptosis to proliferation decreases, resulting in clonal outgrowth of abnormal cells. The t(3;5)(q25;q34) translocation, creating the NPM-MLF1 fusion, has been found as a sole cytogenetic abnormality in MDS. It is recurrent, with poor prognosis but the precise mechanism through which NPM-MLF1 induces malignant transformation remains unknown. We aimed to model this disease in vitro and in vivo by expressing NPM-MFL1 in mouse bone marrow hematopoietic progenitor cells (HPCs) and analyzing any changes in HPC self-renewal and response to DNA damage. NPM-MLF1 did not impair haematopoiesis in vitro and in vivo. FLT3/ITD was frequently associated with NPM mutant in AML patients; however, NPM-MLF1 did not collaborate with FLT3/ITD in our system. To recapitulate NPM hemizygosity in t(3;5)-MDS patients, we have expressed NPM-MLF1 in HPCs derived from Npm+/− mice. A transient increase in the self-renewal of the NPM-MLF1-expressing Npm+/− HPCs was seen. These cells did not exhibit enhanced proliferation as confirmed by growth curve and analysis of DNA synthesis. Interestingly, unlike control cells, NPM-MLF1-expressing Npm+/− HPCs showed prolonged self-renewal ability in vitro, and an elevated expression of c-Myc, Hoxa9, Hoxa10 and Meis1 genes. In addition to altering HPC self-renewal, NPM-MLF1 was also found to modulate their DNA damage response. This study suggests that the ability of NPM-MLF1 to maintain HPC self-renewal and impaired DNA damage responses may favour the accumulation and outgrowth of the aberrant HPCs, contributing to the abnormal haematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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mTOR Regulates DNA Damage Response of Hematopoietic Stem and Progenitor Cells Through Modulation of Fanconi Anemia Core Complex

Blood ◽

10.1182/blood.v118.21.864.864 ◽

2011 ◽

Vol 118 (21) ◽

pp. 864-864 ◽

Cited By ~ 1

Author(s):

Fukun Guo ◽

Jie Li ◽

Wei Du ◽

Shuangmin Zhang ◽

Wei Liu ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Dna Damage ◽

Dna Damage Response ◽

Fanconi Anemia ◽

Hematopoietic Stem ◽

Damage Response ◽

Stem And Progenitor Cells

Abstract Abstract 864 The mammalian target of rapamycin (mTOR) integrates signals from nutrients, growth factors, and cellular energy status to control protein synthesis, cell growth, proliferation, survival and metabolism in various cancer cells, but its physiological function in the hematopoiesis process and signaling role in hematopoietic stem cell (HSC) regulation remain unknown. By using the inhibitor rapamycin, mTOR has previously been suggested to regulate megakaryocyte and dendritic cell proliferation and differentiation. Hyperactivation of mTOR by deletion of the negative regulators of mTOR, TSC1/TSC2 or PTEN, causes a loss of quiescence and long-term exhaustion of HSCs. Since conventional gene targeting of mTOR leads to early embryonic lethality, a conditional mTOR knockout mouse model has recently been generated. We have produced mTORflox/flox; Mx-Cre compound mice that allow interferon-induced mTOR deletion in bone marrow (BM) following a transplantation and polyI:C induction protocol. We found that depletion of mTOR drastically affected hematopoiesis: the mTORflox/flox;Mx-Cre BM recipient mice showed a marked reduction in total BM cellularity and in the numbers and frequency of myeloid and lymphoid cells, erythrocytes, and platelets in peripheral blood, bone marrow, and thymus, after induced mTOR deletion, resulting in bone marrow failure and lethality. Interestingly, the numbers of hematopoietic stem and progenitor cells (HSPCs; Lin−Sca-1+c-Kit+) and HSCs (CD150+ CD41−CD48− Lin−Sca-1+c-Kit+) in bone marrow increased transiently by approximately 5- and 8-fold, respectively, while the numbers of early progenitors (CMP, GMP, MEP, CLP) were mildly affected in the mutant mice 7–14 days after polyI:C treatment. While the more mature lineage committed mTOR−/− blood cells showed a cell cycle blockage and significantly increased apoptosis, mTOR−/− HSPCs and HSCs displayed a loss of quiescence and increased proliferation, as assessed by 5-bromodeoxyuridine incorporation assays, and a normal survival index. Transplantation of mTOR−/− BM cells into immunodeficient or syngeneic mice demonstrated that the mTOR−/− HSPCs failed to engraft and repopulate in the recipients. At the molecular level, mRNA microarray, quantitative real-time PCR and immunoblotting analyses of mTOR−/− HSPCs or Lin− cells revealed that the cell cycle inhibitor Rb was downregulated while the positive regulator of cell cycle E2F5 and pro-survival regulators MCL1 and BCL-xL were upregulated. mTOR deficiency abolished the activation of translational regulators S6K and 4E-BP but led to an increased activation of Akt. In addition, mTOR deficiency sensitized Lin− cells to DNA damage induced in vitro or in vivo by melphalan or mitomycin C (MMC), evidenced by a marked increase in γH2AX foci as well as DNA double-strand breaks (comet-tailed value of 30.2 ± 7.6 in mTOR−/− cells treated in vitro with melphalan and 37.6 ± 3.4 in mTOR−/− cells treated in vivo with MMC compared to 7.6 ± 2.1 in melphalan-treated WT cells and 17.3 ± 6.7 in MMC-treated WT cells, respectively). The increased DNA damage response can be attributed to an ∼300-fold reduction of the expression of FANCD2, a key component of the Fanconi DNA damage repair complex. Significantly, the effect of mTOR deficiency on Fanconi gene expression was specific to FANCD2, because the expression of other Fanconi proteins such as FANCA and FANCC was not affected in mTOR−/− Lin− cells. Intriguingly, the mTOR−/− Lin− cells phenocopied the DNA damage response of FANCD2−/− Lin− cells in vitro and in vivo. Similar effects of reduced FANCD2 expression and dampened DNA damage response were observed in human lymphoblasts treated with pp242, a mTOR kinase inhibitor. FANCD2-deficient human Fanconi anemia patient cells recapitulated the pp242-induced DNA damage phenotypes that could be rescued by FANCD2 reconstitution. Taken together, these results demonstrate that mTOR is a critical regulator of HSC quiescence and engraftment through the regulation of cell cycle machinery and is essential in multiple stages of hematopoiesis. Moreover, mTOR is required for maintaining genomic stability of HSPCs through modulation of the Fanconi anemia DNA damage response pathway. Disclosures: No relevant conflicts of interest to declare.

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The DNA damage inducible lncRNA SCAT7 regulates genomic integrity and topoisomerase 1 turnover in lung adenocarcinoma

NAR Cancer ◽

10.1093/narcan/zcab002 ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Luisa Statello ◽

Mohamad M Ali ◽

Silke Reischl ◽

Sagar Mahale ◽

Subazini Thankaswamy Kosalai ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Cancer Biology ◽

Topoisomerase I ◽

Cell Cycle Checkpoints ◽

Topoisomerase 1 ◽

Damage Response ◽

Promote Cell Survival

Abstract Despite the rapid improvements in unveiling the importance of lncRNAs in all aspects of cancer biology, there is still a void in mechanistic understanding of their role in the DNA damage response. Here we explored the potential role of the oncogenic lncRNA SCAT7 (ELF3-AS1) in the maintenance of genome integrity. We show that SCAT7 is upregulated in response to DNA-damaging drugs like cisplatin and camptothecin, where SCAT7 expression is required to promote cell survival. SCAT7 silencing leads to decreased proliferation of cisplatin-resistant cells in vitro and in vivo through interfering with cell cycle checkpoints and DNA repair molecular pathways. SCAT7 regulates ATR signaling, promoting homologous recombination. Importantly, SCAT7 also takes part in proteasome-mediated topoisomerase I (TOP1) degradation, and its depletion causes an accumulation of TOP1–cc structures responsible for the high levels of intrinsic DNA damage. Thus, our data demonstrate that SCAT7 is an important constituent of the DNA damage response pathway and serves as a potential therapeutic target for hard-to-treat drug resistant cancers.

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PAICS contributes to gastric carcinogenesis and participates in DNA damage response by interacting with histone deacetylase 1/2

Cell Death and Disease ◽

10.1038/s41419-020-2708-5 ◽

2020 ◽

Vol 11 (7) ◽

Author(s):

Nan Huang ◽

Chang Xu ◽

Liang Deng ◽

Xue Li ◽

Zhixuan Bian ◽

...

Keyword(s):

Dna Damage ◽

Histone Deacetylase ◽

Dna Damage Response ◽

De Novo ◽

Dna Damage Repair ◽

Histone Deacetylase 1 ◽

Damage Repair ◽

Damage Response

AbstractPhosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), an essential enzyme involved in de novo purine biosynthesis, is connected with formation of various tumors. However, the specific biological roles and related mechanisms of PAICS in gastric cancer (GC) remain unclear. In the present study, we identified for the first time that PAICS was significantly upregulated in GC and high expression of PAICS was correlated with poor prognosis of patients with GC. In addition, knockdown of PAICS significantly induced cell apoptosis, and inhibited GC cell growth both in vitro and in vivo. Mechanistic studies first found that PAICS was engaged in DNA damage response, and knockdown of PAICS in GC cell lines induced DNA damage and impaired DNA damage repair efficiency. Further explorations revealed that PAICS interacted with histone deacetylase HDAC1 and HDAC2, and PAICS deficiency decreased the expression of DAD51 and inhibited its recruitment to DNA damage sites by impairing HDAC1/2 deacetylase activity, eventually preventing DNA damage repair. Consistently, PAICS deficiency enhanced the sensitivity of GC cells to DNA damage agent, cisplatin (CDDP), both in vitro and in vivo. Altogether, our findings demonstrate that PAICS plays an oncogenic role in GC, which act as a novel diagnosis and prognostic biomarker for patients with GC.

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Slx4 Regulates DNA Damage Checkpoint-dependent Phosphorylation of the BRCT Domain Protein Rtt107/Esc4

Molecular Biology of the Cell ◽

10.1091/mbc.e05-08-0785 ◽

2006 ◽

Vol 17 (1) ◽

pp. 539-548 ◽

Cited By ~ 59

Author(s):

Tania M. Roberts ◽

Michael S. Kobor ◽

Suzanne A. Bastin-Shanower ◽

Miki Ii ◽

Sonja A. Horte ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Dna Damage Checkpoint ◽

Checkpoint Activation ◽

Alkylation Damage ◽

Binding Partner ◽

Replication Restart ◽

Damage Response ◽

Domain Protein

RTT107 (ESC4, YHR154W) encodes a BRCA1 C-terminal-domain protein that is important for recovery from DNA damage during S phase. Rtt107 is a substrate of the checkpoint protein kinase Mec1, although the mechanism by which Rtt107 is targeted by Mec1 after checkpoint activation is currently unclear. Slx4, a component of the Slx1-Slx4 structure-specific nuclease, formed a complex with Rtt107. Deletion of SLX4 conferred many of the same DNA-repair defects observed in rtt107Δ, including DNA damage sensitivity, prolonged DNA damage checkpoint activation, and increased spontaneous DNA damage. These phenotypes were not shared by the Slx4 binding partner Slx1, suggesting that the functions of the Slx4 and Slx1 proteins in the DNA damage response were not identical. Of particular interest, Slx4, but not Slx1, was required for phosphorylation of Rtt107 by Mec1 in vivo, indicating that Slx4 was a mediator of DNA damage-dependent phosphorylation of the checkpoint effector Rtt107. We propose that Slx4 has roles in the DNA damage response that are distinct from the function of Slx1-Slx4 in maintaining rDNA structure and that Slx4-dependent phosphorylation of Rtt107 by Mec1 is critical for replication restart after alkylation damage.

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Dephosphorylation of the C-terminal Tyrosyl Residue of the DNA Damage-related Histone H2A.X Is Mediated by the Protein Phosphatase Eyes Absent

Journal of Biological Chemistry ◽

10.1074/jbc.c900032200 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16066-16070 ◽

Cited By ~ 88

Author(s):

Navasona Krishnan ◽

Dae Gwin Jeong ◽

Suk-Kyeong Jung ◽

Seong Eon Ryu ◽

Andrew Xiao ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Mammalian Cells ◽

Protein Tyrosine Phosphatases ◽

Histone H2a ◽

Eyes Absent ◽

Damage Repair ◽

Damage Response

In mammalian cells, the DNA damage-related histone H2A variant H2A.X is characterized by a C-terminal tyrosyl residue, Tyr-142, which is phosphorylated by an atypical kinase, WSTF. The phosphorylation status of Tyr-142 in H2A.X has been shown to be an important regulator of the DNA damage response by controlling the formation of γH2A.X foci, which are platforms for recruiting molecules involved in DNA damage repair and signaling. In this work, we present evidence to support the identification of the Eyes Absent (EYA) phosphatases, protein-tyrosine phosphatases of the haloacid dehalogenase superfamily, as being responsible for dephosphorylating the C-terminal tyrosyl residue of histone H2A.X. We demonstrate that EYA2 and EYA3 displayed specificity for Tyr-142 of H2A.X in assays in vitro. Suppression of eya3 by RNA interference resulted in elevated basal phosphorylation and inhibited DNA damage-induced dephosphorylation of Tyr-142 of H2A.X in vivo. This study provides the first indication of a physiological substrate for the EYA phosphatases and suggests a novel role for these enzymes in regulation of the DNA damage response.

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Differential DNA Damage Response of Peripheral Blood Lymphocyte Populations

Frontiers in Immunology ◽

10.3389/fimmu.2021.739675 ◽

2021 ◽

Vol 12 ◽

Author(s):

Kerstin Felgentreff ◽

Catharina Schuetz ◽

Ulrich Baumann ◽

Christian Klemann ◽

Dorothee Viemann ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Ionizing Radiation ◽

Dna Damage Response ◽

Peripheral Blood ◽

Low Dose ◽

Lymphocyte Subsets ◽

Mass Cytometry ◽

Damage Response ◽

Lymphocyte Populations

DNA damage occurs constantly in every cell triggered by endogenous processes of replication and metabolism, and external influences such as ionizing radiation and intercalating chemicals. Large sets of proteins are involved in sensing, stabilizing and repairing this damage including control of cell cycle and proliferation. Some of these factors are phosphorylated upon activation and can be used as biomarkers of DNA damage response (DDR) by flow and mass cytometry. Differential survival rates of lymphocyte subsets in response to DNA damage are well established, characterizing NK cells as most resistant and B cells as most sensitive to DNA damage. We investigated DDR to low dose gamma radiation (2Gy) in peripheral blood lymphocytes of 26 healthy donors and 3 patients with ataxia telangiectasia (AT) using mass cytometry. γH2AX, p-CHK2, p-ATM and p53 were analyzed as specific DDR biomarkers for functional readouts of DNA repair efficiency in combination with cell cycle and T, B and NK cell populations characterized by 20 surface markers. We identified significant differences in DDR among lymphocyte populations in healthy individuals. Whereas CD56+CD16+ NK cells showed a strong γH2AX response to low dose ionizing radiation, a reduced response rate could be observed in CD19+CD20+ B cells that was associated with reduced survival. Interestingly, γH2AX induction level correlated inversely with ATM-dependent p-CHK2 and p53 responses. Differential DDR could be further noticed in naïve compared to memory T and B cell subsets, characterized by reduced γH2AX, but increased p53 induction in naïve T cells. In contrast, DDR was abrogated in all lymphocyte populations of AT patients. Our results demonstrate differential DDR capacities in lymphocyte subsets that depend on maturation and correlate inversely with DNA damage-related survival. Importantly, DDR analysis of peripheral blood cells for diagnostic purposes should be stratified to lymphocyte subsets.

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