DNA Repair Programs Research Articles

DNA damage-induced proteasome phosphorylation controls substrate recognition and facilitates DNA repair

Upon DNA damage, numerous proteins are targeted for ubiquitin-dependent proteasomal degradation, which is an integral part of the DNA repair program. Although details of the ubiquitination processes have been intensively studied, little is known about whether and how the 26S proteasome is regulated in the DNA damage response (DDR). Here, we show that human Rpn10/PSMD4, one of the three ubiquitin receptors of the 26S proteasome, is rapidly phosphorylated in response to different types of DNA damage. The phosphorylation occurs at Rpn10-Ser266 within a conserved SQ motif recognized by ATM/ATR/DNA-PK. Blockade of S266 phosphorylation attenuates homologous recombination-mediated DNA repair and sensitizes cells to genotoxic insults. In vitro and in cellulo experiments indicate that phosphorylation of S266, located in the flexible linker between the two ubiquitin-interacting motifs (UIMs) of Rpn10, alters the configuration of UIMs, and actually reduces ubiquitin chain (substrate) binding. As a result, essential DDR proteins such as BRCA1 are spared from premature degradation and allowed sufficient time to engage in DNA repair, a scenario supported by proximity labeling and quantitative proteomic studies. These findings reveal an inherent self-limiting mechanism of the proteasome that, by controlling substrate recognition through Rpn10 phosphorylation, fine-tunes protein degradation for optimal responses under stress.

Single-Cell RNA-Seq Analysis Reveals Distinct Tumor and Immunosuppressive T Cell Phenotypes in CLL Patients Treated with Ibrutinib

Introduction: The development of Bruton tyrosine kinase inhibitors (BTKIs) and their introduction into clinical practice represents a major advance in the treatment of chronic lymphocytic leukemia (CLL). Monotherapy with ibrutinib or other BTKIs generally do not induce complete remissions or undetectable minimal residual disease (uMRD) even with extended therapy. The reason why some patients relapse, and some have minimal residual disease is unknown. Therefore, there is a need to understand the differences between ibrutinib sensitive and resistant CLL cells along with the immune microenvironment to identify novel therapeutic targets. Methods: Here, we investigate the cellular heterogeneity of peripheral blood mononuclear cells from patients with CLL treated with ibrutinib using single-cell RNA sequencing. To understand ibrutinib resistance mechanisms, samples were collected from 8 patients at baseline (before treatment) (1), where BTK C481S is present with low variant allele frequency (VAF) (2) and at time of relapse (3). Data from these were compared to ibrutinib sensitive patient samples at baseline (1) (before treatment), 3yr (2) and 5yr (3) on treatment timepoints. Single cells were isolated using the 10X Genomics 5' immune profiling kit. Data were processed with CellRanger v3.1.0. Poor quality cells were filtered out based on a high proportion of mitochondrial reads and low overall feature count. Sample variation was reduced using Batchelor to aid clustering. Clustering was performed using the partitioning and Leiden methods. Cluster top markers were calculated using functions from monocle3. Identity mapping was performed using Seurat and a published PBMC reference dataset. Results: Single-cell RNA sequencing revealed transcriptional heterogeneity within the B cell cluster (Fig 1A). Three subpopulations of B cells were defined based on differential representation in the ibrutinib-sensitive and resistant patients. Ibrutinib sensitive cells showed enrichment of B cell populations with upregulation of MHC I molecules and TNF family members. We also identified that inflammatory response and metabolic-related pathways were decreased, whereas cellular response to stress and DNA repair programs were increased in the ibrutinib resistance samples. Gene module analysis showed that ibrutinib sensitive samples had an increase in T cell activation, RNA splicing, cell adhesion and migration programs during therapy. We hypothesized that the emergence of BTK C481S mutation in ibrutinib-resistant cells would lead to a distinct transcriptional profile. Long-read sequencing was performed using Oxford Nanopore technology. BTK-mutant cells were identified based on the presence of two or more unique RNA molecules detected with the C481S mutation and these were predominantly identified in the low-VAF and relapse samples. However, there was no significant difference between BTK-mutant cells and the remaining B cells. To see if T cell phenotypes reflected the differences observed in B cell transcriptional state, we characterized and quantified T cell populations according to ibrutinib response and investigated the changes in the T cell population. Ibrutinib markedly increased CD4+ and CD8+ T cell numbers in both resistant and sensitive CLL patients. However, there was a significant (p<0.05) enrichment of Tregs in the ibrutinib-resistant samples. We found a connection between the immunosuppressive phenotype in the microenvironment demonstrating tumor's intrinsic immunological reactive program with exhausted T cells. Also, we found significant difference in the expression of checkpoint molecules and suppressive genes in T cells (Fig 1B) and NK cells. Conclusion: Our findings demonstrate that the B cell population in ibrutinib-resistant and ibrutinib sensitive CLL patients exhibit distinct transcriptional states. At the single-cell level, our findings demonstrate a systematic picture of diverse malignancy and microenvironmental changes in CLL. Overall, these findings provide an insight into the complex molecular and cellular interactions of CLL that may have a crucial role in BTKI resistance.

Clinical Characteristics of Patients with TP53 Multihit MDS and AML. a Single-Center Experience

Introduction TP53 is a tumor suppressor protein encoded by the TP53 gene that plays a pivotal role in maintaining genomic stability in response to DNA damage, activates DNA repair programs and triggers cell-cycle arrest. Mutations in TP53 (TP53-mut) are present in approximately 5-10% of de novo myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), and confer an extremely poor prognosis, irrespectively of the treatment administered. The frequency of TP53 abnormalities increases up to 25-40% in therapy-related MDS and AML, up to 70%-80% in patients with complex karyotype and in patients with loss of chromosome 17/17p, 5/5q, or 7/7q. “Multihit” TP53-mut present with the loss of both wildtype TP53-alleles, either through point mutations or larger aberrations to chromosome 17, in complex monosomal karyotypes. Recent data strongly support the concept that TP53mut, particularly multi-hit TP53, results in similarly poor clinical outcomes, regardless of its classification as MDS or AML, arguing for a revised TP53 mutant myeloid entity that includes both MDS and AML. The aim of our analysis is to assess the impact of the multihit TP53-mut on clinical outcomes according to the type of treatment received. We will particularly focus on the presence of mono or biallelic mutation status of TP53 mutation. Methods We retrospectively evaluated 42 patients with TP53 mutated MDS or AML diagnosed from October 2016 to June 2023 in our department at the Catalan Institute of Oncology-L'Hospitalet in Spain. We analyze the prognosis of these two diseases, focusing on single-hit or multi-hit status of TP53 mutation and treatments received. Results Median age at diagnosis was 71 years (range, 44-87 years) with a male predominance (67%). Thirty-five (84%) patients were diagnosed of AML and 7 (16%) of MDS. Among AML patients, 17% had a prior diagnosis of dysplasia, 78% were diagnosed of AML with dysplasia-related changes, 13% with therapy-related neoplasms, and 9% were AML with maturation and/or AML with mutated NPM1. Regarding MDS patients, 57% had an excess of blast-2 (EB2) MDS, 43% MDS multilineage dysplasia (MLD) and MDS with excess of blast-1(EB1). Complex karyotype was present in 79% of the patients. The median variant allele frequency (VAF) of TP53mut was 36%. Multi-hit TP53 status was observed in 24 patients (57%). In this group of multi-hit TP53mut median age was 72 years (range,44-81 years) with a male predominance (67%), the median VAF of TP53mut was 40%. Twenty (83%) patients had MDS diagnosis and 4 (17%) had AML diagnosis; all of them presented complex karyotype. In the whole group the most concurrent mutations associated with TP53 were TET2 in 17%, ASXL1 in 14% and DNMT3A, NRAS y CREBBP in 12% of patients. Intensive chemotherapy (IC) was administrated as first-line treatment in 17% of patients, 45% received hypomethylating agents (HMA) monotherapy, 24% venetoclax-based combination with HMA, and 14% exclusively supportive treatment. 16 patients (38%) achieved a complete response (CR), 44%, 31% and 25% with HMA+venetoclax, IC and with HMA monotherapy, respectively. Eight patients (19%) achieved a partial response (PR), 88% with HMA and 12% with IC, 7 patients (17%) stable disease, 86% with HMA monotherapy and 14% with HMA+venetoclax and 2 patients (5%) were refractory to IC. In 2 patients (5%), disease was not evaluable. The treatment related mortality was 4.2%. Median overall survival (OS) was 12.5 months [IC 95 % (0.0, 23.54)] and median progression free survival (PFS) was 9, 2 months [IC 95% (3.2, 32.6)]. Median OS of patients with multihit TP53 was 9.8 months, vs 25.7 months in patients with single-hit TP53-mut (P=0.026) (Figure1). Median PFS of the patients with multi-hit and single-hit TP53 was 9.1 and 11.6 months (P=0.07), respectively. The impact of multi-hit TP53 on OS and PFS according to the treatment received (HMA monotherapy vs HMA + venetoclax vs IC) was not statistically significant, just like in the single-hit TP53 (Figure1). Conclusions The presence of multihit TP53 -mut is a prognostic variable for survival in our group of patients analyzed with TP53-Mut. Survival is poor, around one year, in this group of patients independently on the treatment strategy received; this fact would call into question the benefit of IC with the toxicity it entails, in this group of patients. Further development of new effective therapies for multihit TP53-mut AML and MDS is needed.

DDDR-16. GBP3-STING INTERACTION IN GLIOBLASTOMA COORDINATES AUTOPHAGY, ANTI-OXIDATIVE, AND DNA REPAIR PROGRAMS IN RESPONSE TO TEMOZOLOMIDE

Abstract INTRODUCTION Guanylate-binding proteins (GBPs) are a group of dynamin-related large (~65 kDa) GTPases expressed in response to interferon and mediate intracellular immunity. In contrast to the Ras- or Rho family of small GTPases (~20 kDa) or the trimeric GTPases, little is known about the function of GBPs beyond their role in innate cellular immunity. METHODS Informatic analysis of clinically annotated glioblastoma datasets, laboratory studies of protein-protein interaction, functional characterization after depletion or exogenous expression RESULTS Of the seven known members of human GBPs, only GBP3 showed significant up-regulation in response to temozolomide, the standard-of-care DNA-alkylating chemotherapy for glioblastoma. Importantly, high levels of glioblastoma GBP3 expression was associated with worsened survival after temozolomide treatment. Consistent with this observation, exogenous expression of GBP3 induced temozolomide resistance in independent patient-derived glioblastoma neurosphere lines, while GBP3 silencing conferred temozolomide sensitivity, both in vitro and in vivo. This sensitivity was associated with the accumulation of cytoplasmic DNA fragments, suggesting the involvement of STING. Supporting this hypothesis, the N-terminal of GBP3 physically interacted with STING to prevent proteasome-mediated degradation of STING. Since cytoplasmic DNA bound STING activates key transcriptional programs, we profiled transcription programs requiring both GBP3 and STING expression and identified an autophagy program mediated by p62 (autophagy), a defense program against oxidative insults mediated by nuclear factor erythroid 2 like 2 (NFE2L2, NRF2), and a DNA repair program mediated by O6-methylguanine-DNA-methyltransferase (MGMT). These programs were previously shown to play central roles in cellular resistance to DNA alkylating agents. CONCLUSION In addition to its central role in innate immunity, GBP3 mediates cellular response to DNA damage through coordination of autophagy, anti-oxidative, and DNA repair programs.

Aspirin-Mediated Reset of Preeclamptic Placental Stem Cell Transcriptome - Implication for Stabilized Placental Function.

Preeclampsia (PE) is a pregnancy-specific disease, occurring in ~ 2-10% of all pregnancies. PE is associated with increased maternal and perinatal morbidity and mortality, hypertension, proteinuria, disrupted artery remodeling, placental ischemia and reperfusion, and inflammation. The mechanism of PE pathogenesis remains unresolved explaining limited treatment. Aspirin is used to reduce the risk of developing PE. This study investigated aspirin's effect on PE-derived placenta mesenchymal stem cells (P-MSCs). P-MSCs from chorionic membrane (CM), chorionic villi, membranes from the maternal and amniotic regions, and umbilical cord were similar in morphology, phenotype and multipotency. Since CM-derived P-MSCs could undergo long-term passages, the experimental studies were conducted with this source of P-MSCs. Aspirin (1mM) induced significant functional and transcriptomic changes in PE-derived P-MSCs, similar to healthy P-MSCs. These include cell cycle quiescence, improved angiogenic pathways, and immune suppressor potential. The latter indicated that aspirin could induce an indirect program to mitigate PE-associated inflammation. As a mediator of activating the DNA repair program, aspirin increased p53, and upregulated genes within the basic excision repair pathway. The robust ability for P-MSCs to maintain its function with high dose aspirin contrasted bone marrow (M) MSCs, which differentiated with eventual senescence/aging with 100 fold less aspirin. This difference cautions how data from other MSC sources are extrapolated to evaluate PE pathogenesis. Dysfunction among P-MSCs in PE involves a network of multiple pathways that can be restored to an almost healthy functional P-MSC. The findings could lead to targeted treatment for PE.

Determinants of Homologous Recombination Deficiency in Pancreatic Cancer.

Simple SummaryA subset of patients with pancreatic cancer demonstrate heightened response rates and prolonged survival to platinum chemotherapy and PARP inhibitors. Deficient homologous recombination (HR), a critical DNA repair program, is a major driver of this susceptibility. Furthermore, the clinical impact of mutations in distinct HR genes is variable and is modified by diverse tumor intrinsic and extrinsic factors. In this review, we discuss the determinants of homologous recombination deficiency (HRD) in pancreatic cancer. We also highlight emerging methods for identifying and inducing HRD in cancer.Pancreatic cancer is a treatment-resistant malignancy associated with high mortality. However, defective homologous recombination (HR), a DNA repair mechanism required for high-fidelity repair of double-strand DNA breaks, is a therapeutic vulnerability. Consistent with this, a subset of patients with pancreatic cancer show unique tumor responsiveness to HR-dependent DNA damage triggered by certain treatments (platinum chemotherapy and PARP inhibitors). While pathogenic mutations in HR genes are a major driver of this sensitivity, another layer of diverse tumor intrinsic and extrinsic factors regulate the HR deficiency (HRD) phenotype. Defining the mechanisms that drive HRD may guide the development of novel strategies and therapeutics to induce treatment sensitivity in non-HRD tumors. Here, we discuss the complexity underlying HRD in pancreatic cancer and highlight implications for identifying and treating this distinct subset of patients.

Abstract SP125: Rescue DNA repair pathway and tumor immunity

Abstract Defects in DNA damage response (DDR) is a major factor that predispose normal cells to acquire oncogenic mutations. However, after a tumor develops, cancer cells manage their survival by repairing DNA damage resulting from unchecked DNA replication. Cancer cells can use the alternative or backup (rescue) DNA repair programs to overcome their DDR defects and cope with genotoxic lesions. For example, breast cancer cells with deficiency in homologous recombination (HR) can repair their DNA by either relying on other highly expressed HR-related proteins (such as RAD51 or PARP1), or by using other DNA repair mechanisms such as break-induced replication, alternative nonhomologous end joining (ALT-NHEJ) and basal excision repair (BER). Since DNA repair pathways operate in related networks in cancers in general and breast cancers in particular, it is likely that factor/s that play important roles in multiple DNA repair pathways may serve as a critical component of backup DNA repair pathways that breast cancer cells get addicted to survive and to become therapy resistant. One way these rescue DNA repair pathway factor/s may promote breast cancer progression is by shaping tumor immune microenvironment. Consistent with that, DNA damage and DNA repair pathways in general have been shown to regulate multiple arms of the immune response including antigenicity or tumor mutational burden (TMB) and regulation of immune checkpoint protein levels. Our studies suggest that rescue DNA repair pathways may support tumor progression and therapy response by promoting tumor cell intrinsic and extrinsic mechanisms of immunosuppression. Our results reveal that factors associated with backup DNA repair pathwaysmay promote immunosuppression by altering cGAS-STING and Type I Interferon (IFN) signaling pathways. Furthermore, using a specific inhibitor that targets rescue DNA repair pathway signaling, we show altered levels of several proteins involved in antigen processing and presentation, including proteasome/Immunoproteasome subunits and MHC I protein subunits, which are essential for the detection of cancer cells by cytotoxic T cells. Importantly, our pre-clinical and clinical data suggest that approaches aimed at successfully targeting rescue DNA repair factor(s) may promote anti-tumor immunity and will have promising clinical outcomes for treating breast cancer patients. Citation Format: M Rao. Rescue DNA repair pathway and tumor immunity [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP125.

Durable Response of Enzalutamide-resistant Prostate Cancer to Supraphysiological Testosterone Is Associated with a Multifaceted Growth Suppression and Impaired DNA Damage Response Transcriptomic Program in Patient-derived Xenografts

BackgroundAndrogen deprivation therapy improves the survival of castration-resistant prostate cancer (CRPC) patients, yet ultimately fails with debilitating side effects. Supraphysiological testosterone (SPT)-based therapy produces clinical responses with improved quality of life in a subset of patients. Currently, no information defines a durable response to SPT. ObjectiveTo identify key molecular phenotypes underlying SPT response to improve patient selection and guide combination treatment to achieve a durable response. Design, setting, and participantsA patient-derived xenograft (PDX) preclinical trial was performed with 13 CRPC PDXs to identify molecular features associated with SPT response. Comprehensive intratumoral androgen, tumor growth, and integrated transcriptomic and protein analyses were performed in three PDXs resistant to the newer androgen receptor (AR) pathway inhibitor enzalutamide (ENZ) to define SPT response and resistance. InterventionTestosterone cypionate. Outcome measurements and statistical analysisSPT efficacy was evaluated by PDX growth, prostate-specific antigen (PSA) change, and survival. Intratumoral androgens were analyzed using mass spectrometry. Global transcriptome analysis was performed using RNA sequencing, and confirmed by quantitative real-time polymerase chain reaction and immunohistochemistry. Log-rank and Mann-Whitney tests were used for survival and molecular analyses, respectively. Results and limitationsA durable SPT responder was identified, presenting robust repressions of ARv7 and E2F transcriptional outputs, and a DNA damage response (DDR) transcriptomic program that were altogether restored upon SPT resistance in the transient responder. ENZ rechallenge of SPT-relapsed PDXs resulted in PSA decreases but tumor progression. ConclusionsSPT produces a durable response in AR-pathway inhibitor ENZ CRPC that is associated with sustained suppression of ARv7 and E2F transcriptional outputs, and the DDR transcriptome, highlighting the potential of combination treatments that maintain suppression of these programs to drive a durable response to SPT. Patient summaryPatients with ENZ-resistant prostate cancer have very limited treatment options. Supraphysiological testosterone presents a prominent option for improved quality of life and a potential durable response in patients with sustained suppression on ARv7/E2F transcriptional outputs and DNA repair program.

Prolonged Idasanutlin (RG7388) Treatment Leads to the Generation of p53-Mutated Cells.

The protein p53 protects the organism against carcinogenic events by the induction of cell cycle arrest and DNA repair program upon DNA damage. Virtually all cancers inactivate p53 either by mutations/deletions of the TP53 gene or by boosting negative regulation of p53 activity. The overexpression of MDM2 protein is one of the most common mechanisms utilized by p53wt cancers to keep p53 inactive. Inhibition of MDM2 action by its antagonists has proved its anticancer potential in vitro and is now tested in clinical trials. However, the prolonged treatment of p53wt cells with MDM2 antagonists leads to the development of secondary resistance, as shown first for Nutlin-3a, and later for three other small molecules. In the present study, we show that secondary resistance occurs also after treatment of p53wt cells with idasanutlin (RG7388, RO5503781), which is the only MDM2 antagonist that has passed phase II and entered phase III clinical trials, so far. Idasanutlin strongly activates p53, as evidenced by the induction of p21 expression and potent cell cycle arrest in all the three cell lines tested, i.e., MCF-7, U-2 OS, and SJSA-1. Notably, apoptosis was induced only in SJSA-1 cells, while MCF-7 and U-2 OS cells were able to restore the proliferation upon the removal of idasanutlin. Moreover, idasanutlin-treated U-2 OS cells could be cultured for long time periods in the presence of the drug. This prolonged treatment led to the generation of p53-mutated resistant cell populations. This resistance was generated de novo, as evidenced by the utilization of monoclonal U-2 OS subpopulations. Thus, although idasanutlin presents much improved activities compared to its precursor, it displays the similar weaknesses, which are limited elimination of cancer cells and the generation of p53-mutated drug-resistant subpopulations.

PO-490 Treatment of P53 wild-type cells with P53-inducer idasanutlin® (RG7388) generates stable drug-resistant clones

IntroductionThe protein p53 is activated in response to DNA damage. The active protein induces cell cycle arrest and DNA repair program and thus protects the organism against carcinogenic events. Inactivation of p53 gives the cancer cell a profit of genetic instability, resulting in enhanced cancer cell microevolution. Apart from gaining direct mutations in p53-coding gene, overexpression of Mdm2 protein, which is a natural negative regulator of p53, is the most common method utilised by cancer to keep p53 inactive. Mdm2 binds to p53, blocks its transactivation domain and directs p53 to degradation. The re-activation of p53 protein by forced dissociation of Mdm2-p53 complexes using small molecule Mdm2 antagonists is currently believed to be a promising therapeutic strategy of the treatment of cancers expressing wild-type p53. Several representatives of this class of molecules, such as Idasanutlin (RG7388), are currently evaluated in clinical trials. However no data concerning the generation of secondary resistance is available for those modern, optimised compounds.Material and methodsHuman osteosarcoma U-2 OS cells were treated with Idasanutlin, followed by the analysis of viability (MTT assay), p53 activation (western blotting), the induction of apoptosis (annexin V staining) and cell cycle distribution (double staining with BrdU/FITC-anti-BrdU and propidium iodide). Homogenous populations of U-2 OS cells were established by expanding the monoclonal populations from single U-2 OS cells. For the generation of drug-resistant subpopulations the cells were treated with 5 µM Idasanutlin for 12–20 days. The resistant populations were visualised by in situ staining of the growing cells with BrdU/FITC-anti-BrdU and Hoechst 33 342.Results and discussionsThe treatment of U-2 OS cells with Idasanutlin leads to a strong induction of p53 expression level and the expression of p53-target gene encoding p21 protein. As a result Idasanutlin induces tremendous cell cycle arrest. However no apoptosis is induced and Idasanutlin -treated U-2 OS cells can be cultured for long time periods in the presence of the drug. This prolonged treatment of U-2 OS cells leads to de novo generation of cell populations that escape from the cytostatic effect evoked by Idasanutlin.ConclusionAlthough modern Mdm2 antagonists have much improved activities compared to their precursors (i.e. nutlin-3), they suffer from similar weaknesses, which are limited elimination of cancer cells and the generation of p53-mutated drug resistant subpopulations.

PhytoNet: comparative co-expression network analyses across phytoplankton and land plants

Phytoplankton consists of autotrophic, photosynthesizing microorganisms that are a crucial component of freshwater and ocean ecosystems. However, despite being the major primary producers of organic compounds, accounting for half of the photosynthetic activity worldwide and serving as the entry point to the food chain, functions of most of the genes of the model phytoplankton organisms remain unknown. To remedy this, we have gathered publicly available expression data for one chlorophyte, one rhodophyte, one haptophyte, two heterokonts and four cyanobacteria and integrated it into our PlaNet (Plant Networks) database, which now allows mining gene expression profiles and identification of co-expressed genes of 19 species. We exemplify how the co-expressed gene networks can be used to reveal functionally related genes and how the comparative features of PhytoNet allow detection of conserved transcriptional programs between cyanobacteria, green algae, and land plants. Additionally, we illustrate how the database allows detection of duplicated transcriptional programs within an organism, as exemplified by two putative DNA repair programs within Chlamydomonas reinhardtii. PhytoNet is available from www.gene2function.de.

A threshold of endogenous stress is required to engage cellular response to protect against mutagenesis.

Endogenous stress represents a major source of genome instability, but is in essence difficult to apprehend. Incorporation of labeled radionuclides into DNA constitutes a tractable model to analyze cellular responses to endogenous attacks. Here we show that incorporation of [3H]thymidine into CHO cells generates oxidative-induced mutagenesis, but, with a peak at low doses. Proteomic analysis showed that the cellular response differs between low and high levels of endogenous stress. In particular, these results confirmed the involvement of proteins implicated in redox homeostasis and DNA damage signaling pathways. Induced-mutagenesis was abolished by the anti-oxidant N-acetyl cysteine and plateaued, at high doses, upon exposure to L-buthionine sulfoximine, which represses cellular detoxification. The [3H]thymidine-induced mutation spectrum revealed mostly base substitutions, exhibiting a signature specific for low doses (GC > CG and AT > CG). Consistently, the enzymatic activity of the base excision repair protein APE-1 is induced at only medium or high doses. Collectively, the data reveal that a threshold of endogenous stress must be reached to trigger cellular detoxification and DNA repair programs; below this threshold, the consequences of endogenous stress escape cellular surveillance, leading to high levels of mutagenesis. Therefore, low doses of endogenous local stress can jeopardize genome integrity more efficiently than higher doses.

Clinical and Molecular Characterization of p53-Mutated Acute Myeloid Leukemia

P53 is a multifunctional tumor suppressor protein encoded by the TP53 gene, located on the short arm of chromosome 17. Among its functions, it plays a role in maintaining genomic stability by reacting to DNA damage, activating DNA repair programs, and triggering cell cycle arrest. Over half of human malignancies contain mutations or deletions in TP53 that compromise its function. Mutations in TP53 have been described in 8-10% of cases of acute myeloid leukemia (AML) and are associated with an adverse prognosis.We examined a cohort of 293 patients (pts) with newly diagnosed, non-CBF, non-APL AML treated at our institution in whom mutation screening was performed. Mutation testing was performed using a validated 28-gene, whole-exome sequencing panel. Our aim was to characterize the clinico-pathologic characteristics of AML pts with TP53 mutations (mut-P53), their response to different therapies, and their survival compared to those with wild-type TP53 (wt-P53). Pts were treated with several different therapies that could be classified has high-dose cytarabine-based (HDAC), hypomethylating agent (HYPO), or low-intensity therapy other than single-agent HYPO (LOW-INT).Of the 293 pts studied, 97 (33%) and 196 (67%) were < 60 years and ≥ 60 years, respectively. Overall, 53 (18%) had TP53 mutations, 17 (18%) pts < 60 years and 36 (18%) pts ≥ 60 years. 95% of the mutations were found in exons 5-8 of TP53, the DNA-binding domain. Patient characteristics by age and TP53 mutation status are summarized in Table 1. In pts < 60 years, those with mut-P53 had significantly lower peripheral blasts, bone marrow (BM) blasts, and fibrinogen compared to those with wt-P53. For pts ≥ 60 years, those with mut-P53 were older, had lower WBC counts, lower platelet counts, and significantly lower BM blast counts than those with wt-P53. Significantly more pts with mut-P53 had complex karyotype (p<0.001), abnormalities of chromosomes 5 and/or 7 (p<0.001), abnormalities of chromosome 17 (p<0.001), and a diagnosis of therapy-related AML (p=0.004) compared to pts with wt-P53. 81% of pts with mut-P53 had a complex karyotype and 55% of pts with a complex karyotype had mut-P53. A 28-gene mutation screen that was performed detected concomitant mutations and delineated the molecular landscape of AML pts with mut-P53 (Figure 1A). Mutations in FLT3 (p=0.023), RAS (p=0.037), and NPM1 (p=0.029) were significantly underrepresented in pts with mut-P53 compared to wt-P53. Rates of CR were lower in pts with mut-P53 compared to those with wt-P53 in the younger and older cohorts (Table 1). The OS of pts younger and older than 60 years by p53 status is shown in Figure 1B. Among pts with mut-P53, the CR rates for HDAC, HYPO, and LOW-INT were 60%, 29%, and 50%, respectively compared with 72%, 35%, and 63% in wt-P53 pts, respectively. When divided into cohorts by age < and ≥ 60yrs, there was no difference in OS for patients with mutated TP53 treated with HDAC, HYPO, or LOW-INT.TP53 mutations in AML are associated with low blast counts, low WBC counts, complex karyotype, therapy-related disease, and inferior outcomes compared to those with wt-P53. In our cohort there was no difference in outcome among different intensity therapies. Newer therapies are needed to improve outcome in this difficult cohort of pts.Table 1Age <60 (N=97)Characteristicmut-p53wt-p53p-valueMedian Age49 (20-59)51 (22-59)0.8Median WBC2.7 (0.9 - 30)4.9 (0.5 - 103)0.46Median Platelet32 (4 - 153)39 (1 - 708)0.35Median periph blasts6 (0 - 63)27 (0 - 97)0.03Median LDH937 (392 - 10298)717 (231 - 11952)0.25Median Fibrinogen330 (43 - 558)413 (67 - 1117)0.06Median BM Blasts27 (12 - 91)54 (1 - 96)0.008CR Rate (%)53700.255Age >60 (N=196)Characteristicmut-p53wt-p53p-valueMedian Age74 (62-90)71 (60-92)0.02Median WBC2.3 (0.7-17.5)2.9 (0.2-164.5)0.09Median Platelet34 (8-321)45 (1-1069)0.07Median periph blasts10 (0-86)8 (0-96)0.8Median LDH535 (286-3616)616 (284-17486)0.4Median Fibrinogen407 (213-753)388 (102-923)0.5Median BM Blasts32 (3-97)47 (4-93)0.009CR Rate (%)36500.196 [Display omitted] DisclosuresCortes:Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding; BerGenBio AS: Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. DiNardo:Novartis: Research Funding.

Cellular stress responses in cancer and cancer therapy.

Cellular stress responses in cancer and cancer therapy.

DNA Repair Meets the RNA World

The ability to repair damaged DNA and to maintain genome stability is the utmost importance for the survival of any species. Hence, it is not surprising to find that DNA repair mechanisms are evolutionarily conserved and are expected to evolve to maintain the existence of species. In the last few years, there has been an exponential increase in the evidence linking RNA processing with DNA repair programs. For instance, the well-studied DNA base excision repair (BER) enzyme apurinic/apyrimidinic endonuclease 1 can cleave RNA molecules, regulate mRNA levels, and associate physically with proteins involved in RNA processing. It is now clear that not only the expression of noncoding RNAs are changed upon DNA damage, they can modulate the expression of genes involved in the genome stability programs. The five reviews in this Forum provide the up-to-date knowledge on DNA repair, with a focus on BER, and a perspective on how the two ancient biochemical pathways are linked. The contributions demonstrate the complexity of such interactions, but also pointed out the opportunities for new therapeutic interventions. Future in vivo studies on the link between DNA repair processes and RNA metabolism should contribute to our basic understanding of physiology, disease, and treatment strategies.

Loss Of p53 Rescues The Defective Function Of Foxo3-/- Hematopoietic Stem Cells But Enhances Their Predisposition To Malignancy

Leukemic stem cells (LSCs) share many of the same properties of normal hematopoietic stem cells (HSCs) including their highly quiescent state, capacity to self-renew, low levels of reactive oxygen species (ROS) and enhanced DNA repair program. These properties make the efficient and specific eradication of these cells challenging. Foxo3 and p53 are two transcription factors essential for the modulation of HSC quiescence and self-renewal. While Foxo3 is inhibited by signaling from several oncoproteins but crucial for the maintenance of the LSCs in both chronic and acute myeloid leukemia (CML and AML respectively), mutations of p53 although rare, are associated with poor prognosis in advanced stages of these diseases. In vivo ROS-mediated activation of p53 is known to lead to loss of quiescence, alterations of cell cycle and exhaustion of the Foxo3-/- HSC pool. Seeking to understand the contribution of p53 to Foxo3-/- HSC cycling defects, we crossed p53+/- and Foxo3+/- mice. To our surprise we found the bone marrow (BM) frequency of both p53+/-Foxo3-/- and p53-/-Foxo3-/- LSK (Lin-Sca1+cKit+) and long-term-HSC (LT-HSC, LSK Flk2-CD34-) populations greatly increased as compared to their Foxo3-/- counterparts (n=5 mice per genotype; p<0.05). Using Ki67 and DAPI staining we found that loss of one or both alleles of p53 gradually rescued the cell cycle defect of Foxo3-/- HSC and increased the frequency of LSK cells in Go by 2-fold. Loss of p53 also rescued the impaired capacity of Foxo3-/- LSK cells to competitively repopulate multilineage blood over 16 weeks, as shown by the higher frequency of p53+/-Foxo3-/- and p53-/-Foxo3-/- donor-derived cells in the peripheral blood of recipient animals (∼47% recipients of double-mutant cells versus 20% in Foxo3-/- recipients, n=5 per group). Furthermore, loss of p53 significantly improved the compromised self-renewal of Foxo3 mutant HSC in serial BM transplantations. In our quest to identify mechanisms whereby p53 depletion improves Foxo3-/- HSC function, we noticed that the DNA damage accumulated in Foxo3-/- HSC at the steady-state was remarkably ameliorated by removal of one or both alleles of p53 from Foxo3-/- HSCs, as measured by flow cytometry levels of phospho-H2AX (gamma-H2AX) and DNA breaks by comet assay (n=3, p<0.05). Unexpectedly, ROS levels were also significantly reduced by 30% in p53+/-Foxo3-/- in comparison to Foxo3-/- LSK cells, while ROS levels in p53+/- LSK cells were similar to that in WT cells. Consistent with these results, the expression of several anti-oxidant enzymes including Sod1, Sod2, Catalase, Gpx1, Sesn1 and Sesn2 (n≥2), was highly upregulated while a number of genes implicated in mitochondrial generation of ROS were significantly deregulated as a result of loss of one or both alleles of p53. These combined findings suggest that a switch from anti-oxidant to pro-oxidant activity of p53 contributes to Foxo3-/- HSC defects. Despite their apparent normal stem cell function, p53+/-Foxo3-/- HSC were highly altered in their gene expression profile. Interestingly, Gene Set Enrichment Analysis (GSEA) of the microarray analysis (Illumina bead chip mouse-Ref8) of WT, p53+/-, Foxo3-/-, and p53+/-Foxo3-/- LSK cells showed that a cluster of genes associated with fatty acid metabolism was highly enriched in p53+/-Foxo3-/- HSCs (ES=0.746; p<0.01). In addition, from 3976 genes exclusively deregulated in p53+/-Foxo3-/- LSK cells, 201 (out of 1051) overlapped with genes downregulated, while 9 (out of 14) overlapped with genes exclusively upregulated in a LSC-gene signature. To evaluate whether this pre-leukemic profile was associated with increased susceptibility to malignancy, we compared the potential and timeline of BCR-ABL-transformed p53+/-Foxo3-/- HSC as compared to controls in establishing CML in mice. We found a shorter time to the onset of the disease and decreased survival of the recipients of p53+/-Foxo3-/- transformed HSCs (n=4 per group, p<0.05) as compared to WT and Foxo3-/- controls. We propose that the p53+/-Foxo3-/- double-mutant HSCs are enriched for preleukemic stem cells based on their quiescence and self-renewal capacity, low ROS, robust DNA repair, susceptibility to transformation and aberrant gene expression profile. These findings raise the possibility that the coordinated Foxo3 and p53 regulation of ROS wires together the stem cell program. Disclosures:No relevant conflicts of interest to declare.

FOXP3Regulates Sensitivity of Cancer Cells to Irradiation by Transcriptional Repression ofBRCA1

FOXP3 is an X-linked tumor suppressor gene and a master regulator in T regulatory cell function. This gene has been found to be mutated frequently in breast and prostate cancers and to inhibit tumor cell growth, but its functional significance in DNA repair has not been studied. We found that FOXP3 silencing stimulates homologous recombination-mediated DNA repair and also repair of γ-irradiation-induced DNA damage. Expression profiling and chromatin-immunoprecipitation analyses revealed that FOXP3 regulated the BRCA1-mediated DNA repair program. Among 48 FOXP3-regulated DNA repair genes, BRCA1 and 12 others were direct targets of FOXP3 transcriptional control. Site-specific interaction of FOXP3 with the BRCA1 promoter repressed its transcription. Somatic FOXP3 mutants identified in breast cancer samples had reduced BRCA1 repressor activity, whereas FOXP3 silencing and knock-in of a prostate cancer-derived somatic FOXP3 mutant increased the radioresistance of cancer cells. Together our findings provide a missing link between FOXP3 function and DNA repair programs.

Genome-wide profiles of H2AX and γ-H2AX differentiate endogenous and exogenous DNA damage hotspots in human cells

Phosphorylation of the histone variant H2AX forms γ-H2AX that marks DNA double-strand break (DSB). Here, we generated the sequencing-based maps of H2AX and γ-H2AX positioning in resting and proliferating cells before and after ionizing irradiation. Genome-wide locations of possible endogenous and exogenous DSBs were identified based on γ-H2AX distribution in dividing cancer cells without irradiation and that in resting cells upon irradiation, respectively. γ-H2AX-enriched regions of endogenous origin in replicating cells included sub-telomeres and active transcription start sites, apparently reflecting replication- and transcription-mediated stress during rapid cell division. Surprisingly, H2AX itself, prior to phosphorylation, was specifically located at these endogenous hotspots. This phenomenon was only observed in dividing cancer cells but not in resting cells. Endogenous H2AX was concentrated on the transcription start site of actively transcribed genes but was irrelevant to pausing of RNA polymerase II (pol II), which precisely coincided with γ-H2AX of endogenous origin. γ-H2AX enrichment upon irradiation also coincided with actively transcribed regions, but unlike endogenous γ-H2AX, it extended into the gene body and was not specifically concentrated on the pausing site of pol II. Sub-telomeres were less responsive to external DNA damage than to endogenous stress. Our findings provide insight into DNA repair programs of cancer and may have implications for cancer therapy.

BRCA1-associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair.

Following irradiation, numerous DNA-damage-responsive proteins rapidly redistribute into microscopically visible subnuclear aggregates, termed ionising-radiation-induced foci (IRIF). How the enrichment of proteins on damaged chromatin actually relates to DNA repair remains unclear. Here, we use super-resolution microscopy to examine the spatial distribution of BRCA1 and 53BP1 proteins within single IRIF at subdiffraction-limit resolution, yielding an unprecedented increase in detail that was not previously apparent by conventional microscopy. Consistent with a role for 53BP1 in promoting DNA double-strand break repair by non-homologous end joining, 53BP1 enrichment in IRIF is most prominent in the G0/G1 cell cycle phases, where it is enriched in dense globular structures. By contrast, as cells transition through S phase, the recruitment of BRCA1 into the core of IRIF is associated with an exclusion of 53BP1 to the focal periphery, leading to an overall reduction of 53BP1 occupancy at DNA damage sites. Our data suggest that the BRCA1-associated IRIF core corresponds to chromatin regions associated with repair by homologous recombination, and the enrichment of BRCA1 in IRIF represents a temporal switch in the DNA repair program. We propose that BRCA1 antagonises 53BP1-dependent DNA repair in S phase by inhibiting its interaction with chromatin proximal to damage sites. Furthermore, the genomic instability exhibited by BRCA1-deficient cells might result from a failure to efficiently exclude 53BP1 from such regions during S phase.

Human and Mouse Granzyme A Induce a Proinflammatory Cytokine Response

Granzyme A (GzmA) is considered a major proapoptotic protease. We have discovered that GzmA-induced cell death involves rapid membrane damage that depends on the synergy between micromolar concentrations of GzmA and sublytic perforin (PFN). Ironically, GzmA and GzmB, independent of their catalytic activity, both mediated this swift necrosis. Even without PFN, lower concentrations of human GzmA stimulated monocytic cells to secrete proinflammatory cytokines (interleukin-1beta [IL-1beta], TNFalpha, and IL-6) that were blocked by a caspase-1 inhibitor. Moreover, murine GzmA and GzmA(+) cytotoxic T lymphocytes (CTLs) induce IL-1beta from primary mouse macrophages, and GzmA(-/-) mice resist lipopolysaccharide-induced toxicity. Thus, the granule secretory pathway plays an unexpected role in inflammation, with GzmA acting as an endogenous modulator.