Therapeutic targeting of mutant p53 in pediatric acute lymphoblastic leukemia.

Alterations of the tumor suppressor gene TP53 are found in different cancers, in particular in carcinomas of adults. In pediatric acute lymphoblastic leukemia (ALL), TP53 mutations are infrequent but enriched at relapse. As in most cancers, mainly DNA-binding domain missense mutations are found, resulting in accumulation of mutant p53, poor therapy response, and inferior outcome. Different strategies to target mutant p53 have been developed including reactivation of p53's wildtype function by the small molecule APR-246. We investigated TP53 mutations in cell lines and 62 B-cell precursor ALL samples and evaluated the activity of APR-246 in TP53-mutated or wildtype ALL. We identified cases with TP53 missense mutations, high (mutant) p53 expression and insensitivity to the DNA-damaging agent doxorubicin. In TP53-mutated ALL, APR-246 induced apoptosis showing strong anti-leukemia activity. APR-246 restored mutant p53 to its wildtype conformation, leading to pathway activation with induction of transcriptional targets and re-sensitization to genotoxic therapy in vitro and in vivo In addition, induction of oxidative stress contributed to APR-246-mediated cell death. In a preclinical model of patient-derived TP53-mutant ALL, APR-246 reduced leukemia burden and synergized strongly with the genotoxic agent doxorubicin, leading to superior leukemia-free survival in vivo Thus, targeting mutant p53 by APR-246, restoring its tumor suppressive function, seems to be an effective therapeutic strategy for this high-risk group of TP53-mutant ALL.

Biallelic germline BRCA1 mutations in a patient with early onset breast cancer, mild Fanconi anemia-like phenotype, and no chromosome fragility.

BACKGROUND: Biallelic BRCA1 mutations are regarded either embryonically lethal or to cause Fanconi anemia (FA), a genomic instability syndrome characterized by bone marrow failure, developmental abnormalities, and cancer predisposition. We report biallelic BRCA1 mutations c.181T > G (p.Cys61Gly) and c.5096G > A (p.Arg1699Gln) in a woman with breast cancer diagnosed at the age of 30 years. The common European founder mutation p.Cys61Gly confers high cancer risk, whereas the deleterious p.Arg1699Gln is hypomorphic and was suggested to confer intermediate cancer risk. METHODS AND RESULTS: Aside from significant toxicity from chemotherapy, the patient showed mild FA-like features (e.g., short stature, microcephaly, skin hyperpigmentation). Chromosome fragility, a hallmark of FA patient cells, was not present in patient-derived peripheral blood lymphocytes. We demonstrated that the p.Arg1699Gln mutation impairs DNA double-strand break repair, elevates RAD51 foci levels at baseline, and compromises BRCA1 protein function in protecting from replication stress. Although the p.Arg1699Gln mutation compromises BRCA1 function, the residual activity of the p.Arg1699Gln allele likely prevents from chromosome fragility and a more severe FA phenotype. CONCLUSION: Our data expand the clinical spectrum associated with biallelic BRCA1 mutations, ranging from embryonic lethality to a mild FA-like phenotype and no chromosome fragility.

The C-terminal domain of p53 orchestrates the interplay between non-covalent and covalent poly(ADP-ribosyl)ation of p53 by PARP1.

The post-translational modification poly(ADP-ribosyl)ation (PARylation) plays key roles in genome maintenance and transcription. Both non-covalent poly(ADP-ribose) binding and covalent PARylation control protein functions, however, it is unknown how the two modes of modification crosstalk mechanistically. Employing the tumor suppressor p53 as a model substrate, this study provides detailed insights into the interplay between non-covalent and covalent PARylation and unravels its functional significance in the regulation of p53. We reveal that the multifunctional C-terminal domain (CTD) of p53 acts as the central hub in the PARylation-dependent regulation of p53. Specifically, p53 bound to auto-PARylated PARP1 via highly specific non-covalent PAR-CTD interaction, which conveyed target specificity for its covalent PARylation by PARP1. Strikingly, fusing the p53-CTD to a protein that is normally not PARylated, renders this a target for covalent PARylation as well. Functional studies revealed that the p53-PAR interaction had substantial implications on molecular and cellular levels. Thus, PAR significantly influenced the complex p53-DNA binding properties and controlled p53 functions, with major implications on the p53-dependent interactome, transcription, and replication-associated recombination. Remarkably, this mechanism potentially also applies to other PARylation targets, since a bioinformatics analysis revealed that CTD-like regions are highly enriched in the PARylated proteome.

ATM Deficiency Generating Genomic Instability Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Therapy-Induced DNA Damage.

Pancreatic ductal adenocarcinomas (PDAC) harbor recurrent functional mutations of the master DNA damage response kinase ATM, which has been shown to accelerate tumorigenesis and epithelial-mesenchymal transition. To study how ATM deficiency affects genome integrity in this setting, we evaluated the molecular and functional effects of conditional Atm deletion in a mouse model of PDAC. ATM deficiency was associated with increased mitotic defects, recurrent genomic rearrangements, and deregulated DNA integrity checkpoints, reminiscent of human PDAC. We hypothesized that altered genome integrity might allow synthetic lethality-based options for targeted therapeutic intervention. Supporting this possibility, we found that the PARP inhibitor olaparib or ATR inhibitors reduced the viability of PDAC cells in vitro and in vivo associated with a genotype-selective increase in apoptosis. Overall, our results offered a preclinical mechanistic rationale for the use of PARP and ATR inhibitors to improve treatment of ATM-mutant PDAC. Cancer Res; 77(20); 5576-90. ©2017 AACR.

In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis.

Dysfunction of homologous recombination is a common denominator of changes associated with breast cancer-predisposing mutations. In our previous work, we identified a functional signature in peripheral blood lymphocytes from women who were predisposed that indicated a shift from homologous recombination to alternative, error-prone DNA double-strand break (DSB) repair pathways. To capture both hereditary and nonhereditary factors, we newly established a protocol for isolation and ex vivo analysis of epithelial cells, epithelial-mesenchymal transition cells (EMTs), and fibroblasts from breast cancer specimens (147 patients). By applying a fluorescence-based test system, we analyzed the error-prone DSB repair pathway microhomology-mediated end joining in these tumor-derived cell types and peripheral blood lymphocytes. In parallel, we investigated DNA lesion processing by quantitative immunofluorescence microscopy of histone H2AX phosphorylated on Ser139 focus after radiomimetic treatment. Our study reveals elevated histone H2AX phosphorylated on Ser139 damage removal in epithelial cells, not EMTs, and poly(ADP-ribose)polymerase inhibitor sensitivities, which suggested a DSB repair pathway shift with increasing patient age. Of interest, we found elevated microhomology-mediated end joining in EMTs, not epithelial cells, from patients who received a treatment recommendation of adjuvant chemotherapy, that is, those with high-risk tumors. Our discoveries of altered DSB repair activities in cells may serve as a method to further classify breast cancer to predict responsiveness to adjuvant chemotherapy and/or therapeutics that target DSB repair-dysfunctional tumors.-Deniz, M., Kaufmann, J., Stahl, A., Gundelach, T., Janni, W., Hoffmann, I., Keimling, M., Hampp, S., Ihle, M., Wiesmüller, L. In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis.

DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression.

DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.

Modulation of the poly (ADP-ribose) polymerase inhibitor response and DNA recombination in breast cancer cells by drugs affecting endogenous wild-type p53.

Synthetic lethal interactions between poly (ADP-ribose) polymerase (PARP) and homologous recombination (HR) repair pathways have been exploited for the development of novel mono- and combination cancer therapies. The tumor suppressor p53 was demonstrated to exhibit indirect and direct regulatory activities in DNA repair, particularly in DNA double-strand break (DSB)-induced and replication-associated HR. In this study, we tested a potential influence of the p53 status on the response to PARP inhibition, which is known to cause replication stress. Silencing endogenous or inducibly expressing p53 we found a protective effect of p53 on PARP inhibitor (PARPi)-mediated cytotoxicities. This effect was specific for wild-type versus mutant p53 and observed in cancer but not in non-transformed cell lines. Enhanced cytotoxicities after treatment with the p53-inhibitory drug Pifithrinα further supported p53-mediated resistance to PARP inhibition. Surprisingly, we equally observed increased PARPi sensitivity in the presence of the p53-activating compound Nutlin-3. As a common denominator, both drug responses correlated with decreased HR activities: Pifithrinα downregulated spontaneous HR resulting in damage accumulation. Nutlin-3 induced a decrease of DSB-induced HR, which was accompanied by a severe drop in RAD51 protein levels. Thus, we revealed a novel link between PARPi responsiveness and p53-controlled HR activities. These data expand the concept of cell and stress type-dependent healer and killer functions of wild-type p53 in response to cancer therapeutic treatment. Our findings have implications for the individualized design of cancer therapies using PARPi and the potentially combined use of p53-modulatory drugs.

Characterization of the nonallelic homologous recombination hotspot PRS3 associated with type-3 NF1 deletions.

Nonallelic homologous recombination (NAHR) is the major mechanism underlying recurrent genomic rearrangements, including the large deletions at 17q11.2 that cause neurofibromatosis type 1 (NF1). Here, we identify a novel NAHR hotspot, responsible for type-3 NF1 deletions that span 1.0 Mb. Breakpoint clustering within this 1-kb hotspot, termed PRS3, was noted in 10 of 11 known type-3 NF1 deletions. PRS3 is located within the LRRC37B pseudogene of the NF1-REPb and NF1-REPc low-copy repeats. In contrast to other previously characterized NAHR hotspots, PRS3 has not developed on a preexisting allelic homologous recombination hotspot. Furthermore, the variation pattern of PRS3 and its flanking regions is unusual since only NF1-REPc (and not NF1-REPb) is characterized by a high single nucleotide polymorphism (SNP) frequency, suggestive of unidirectional sequence transfer via nonallelic homologous gene conversion (NAHGC). By contrast, the previously described intense NAHR hotspots within the CMT1A-REPs, and the PRS1 and PRS2 hotspots underlying type-1 NF1 deletions, experience frequent bidirectional sequence transfer. PRS3 within NF1-REPc was also found to be involved in NAHGC with the LRRC37B gene, the progenitor locus of the LRRC37B-P duplicons, as indicated by the presence of shared SNPs between these loci. PRS3 therefore represents a weak (and probably evolutionarily rather young) NAHR hotspot with unique properties.