PARP inhibitor olaparib induces DNA damage and acts as a drug sensitizer in an in vitro model of canine hematopoietic cancer

PARP inhibitor olaparib induces DNA damage and acts as a drug sensitizer in an in vitro model of canine hematopoietic cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article PARP inhibitor olaparib induces DNA damage and acts as a drug sensitizer in an in vitro model of canine hematopoietic cancer Jayson Cagadas Pasaol, Ewa Dejnaka, Greta Mucignat, Joanna Bajzert, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6148132/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Jul, 2025 Read the published version in BMC Veterinary Research → Version 1 posted 10 You are reading this latest preprint version Abstract Background The introduction of genetic tests based on next-generation sequencing techniques into veterinary cancer diagnostics provides information on molecularly targeted therapies useful for dogs. However, there is still a lack of in vitro studies describing the effect and mechanism of action of such anticancer drugs in companion animals. Our study aimed to demonstrate in vitro activity of a commonly used PARP inhibitor, olaparib, in canine lymphoma and leukemia cells as well as to indicate its potential uses in anti-cancer therapy based on the mutational status of DNA damage related genes. Canine lymphoma and leukemia cell lines were incubated with olaparib alone and in combination with doxorubicin, and the impact of a single drug and combinations on cell viability, proliferation, induction of apoptosis, and DNA damage were assessed. Results The study showed that olaparib acts as a single agent and also as a chemosensitizer, due to its ability to potentiate cytotoxic effects of doxorubicin. Conclusions Olaparib may be an interesting oral therapy alternative to classic chemotherapy in dogs with known DNA repair disorders and may also be effective in adjuvant therapy due to its synergistic effect with doxorubicin. canine lymphoma targeted therapy DNA repair doxorubicin synergy mutational analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background The search for innovative targeted anti-cancer treatments is critical for modern oncology. As access to genetic tests for dogs becomes widespread, physicians will gather more data providing indications for the use of novel medications targeting specific molecules. Interestingly, common availability of modern molecular methods for cancer diagnosis in animals has significantly outpaced the rate of research on the use of molecularly targeted drugs, thus creating a gap between information about dedicated treatment and the actual possibility of using specific substances in animals. An example may be the use of poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis), drugs with already proven effectiveness in humans, for treatment of patients with DNA damage repair-deficient cancers, mainly with Breast Cancer Associated 1 and 2 (BRCA1 and BRCA2) mutations ( 1 ). PARP1 plays a crucial role in repairing DNA damage. Following DNA insult, PARP1 is rapidly recruited to single-strand breaks (SSBs) and double-strand breaks (DSBs) in DNA. Subsequently, BRCA1 and BRCA2 are recruited as they regulate the major pathway for DSB repair, homologous recombination (HR)( 1 ). Pharmacological inhibition of PARP1 leads to DNA damage and, in the absence of functional BRCA1 and BRCA2 genes, to critical levels of genomic instability provoking cell death ( 2 ). Cell death observed after PARPi treatment of HR-deficient cells is based on synthetic lethality. This occurs when mutation-induced dysfunctions in two genes do not affect cell viability separately, but simultaneous disorders in both genes lead to cell death( 3 ). Thanks to this effect, PARPis quickly became a novel class of anti-cancer drugs, first demonstrating efficiency in treating HR-deficient tumors with a BRCA1 and BRCA2 mutation( 4 ). Interestingly, patients with genetic HR dysfunction are not the only candidates for PARPi treatment. Some sporadic cancers are also characterized by HR deficiency but without BRCA1-2 mutations. The broad range of DNA substrates and various processes targeted by PARPi imply that PARPis may also reduce survival of other DNA repair-deficient cells ( 5 ), a phenomenon referred to as BRCAness ( 6 ).This observation supports current clinical trials of PARPis in patients with other HR-deficient, BRCA-dependent cancers ( 7 ). Despite the success of PARPis in human oncology, these drugs are not extensively researched in veterinary medicine ( 8 , 9 ). Considering the current wide indications for PARPis and increasing data on HR disorders in various dog cancers, we decided to investigate the activity of the first and best-studied PARPi, olaparib, in canine hematopoietic cancers. We assessed the antiproliferative and proapoptotic effect of olaparib, and its ability to induce DNA damage. Additionally, we investigated the synergistic effect of olaparib and doxorubicin to determine their potential utility in chemotherapy for hematological malignancies. Finally, we analyzed the mutational profile of key genes involved in the DNA damage response to assess their impact on olaparib sensitivity in vitro . Results Olaparib shows activity as a single agent in canine lymphoma/leukemia cells Inhibition of cell metabolic activity To check if PARPi may be a potential therapeutic option for dogs with lymphoma or leukemia, we first investigated the effect of olaparib on the metabolic activity of established canine cancer cell lines using the MTT assay. Data obtained indicated that olaparib alone inhibits the cell metabolic activity of all lymphoma/leukemia cell lines used in our study in both concentration- and time-dependent manners. Concentration-dependent curves presenting the effects of olaparib on the metabolic activity of the tested cell lines are shown in Fig. 1 . We identified some differences in the sensitivity of individual cell lines to olaparib, with the GL-1 cell line being less susceptible compared to the others. As expected, the cytotoxic effect became more evident with prolonged incubation, reaching the concentration that inhibited cell metabolic activity by 50% (IC50 value) below 3 µM after 72 hours for all the sensitive cell lines. This concentration is much lower than the maximum one achievable in human patients with solid tumors treated with 200–600 mg olaparib twice daily 19,20. A comparison of IC50 values for each cell line is presented in Table 1 . Table 1 The cytotoxic effects of olaparib after 48, 72, and 96 hours of incubation on the canine lymphoma/leukemia cell lines, expressed as IC 50 values. The results are presented as mean ± standard deviation (SD) of 3 independent experiments, each performed in triplicates. Statistical differences were analyzed using a one-way ANOVA followed by the Tukey’s multiple comparison test. Values without common letters (a, b, c) in the superscript differ statistically (P 33.3 b 72h 2.68 a ± .81 3.03 a ± 1.37 2.03 b ± .28 6.83 c ± 1.57 96h 1.2 a ± .17 2.09 a ± 1.44 1.50 a ± .63 5.57 b ± 1.34 Antiproliferative effect To investigate the antiproliferative effect of olaparib on canine lymphoma/leukemia cells, the expression of Ki-67 (a common proliferation marker) was determined in cells treated with olaparib for 48 hours. The percentage of Ki-67 positive cells (as compared to the untreated control) decreased significantly (P < 0.05) after incubation with 25 and 50 µM of olaparib in all tested cell lines (Fig. 2 ). The strongest effect was observed for the CLBL-1 cell line, where Ki-67 expression dropped below 20% after 48 hours of incubation with 50 µM of olaparib, indicating a decrease in the cell proliferation potential. DNA damage induction Having confirmed that olaparib alone exerts cytotoxic and antiproliferative effects on canine lymphoma/leukemia cells, we decided to investigate if olaparib causes DNA damage in sensitive cells. We sought to determine whether and after what incubation time of the cells with olaparib, DNA damage accumulates in the cell and can be detected as phosphorylation of histone H2A.X on serine 139, (γH2A.X), a verified and common marker of DNA damage. The results of the histone H2A.X phosphorylation analysis using the Western blot technique are shown in Fig. 3 . Olaparib indeed induces the formation of DNA damage, even after 24 hours of incubation, also in the cells with limited sensitivity to olaparib (see MTT results). This result clearly shows that the accumulation of DNA damage in the cells disturbs cell proliferation. Olaparib acts as a chemosensitizer due to its ability to potentiate the cytotoxic effects of doxorubicin – a commonly used cytostatic drug for lymphoma/leukemia therapy Olaparib – doxorubicin combinatory effects To evaluate the potential benefit of adding olaparib to a conventional anti-cancer treatment in dogs, the effects of a combined exposure of lymphoma/leukemia cells to olaparib and doxorubicin were analyzed. The resulting CI values (Fig. 4 B) indicated that doxorubicin and olaparib showed a moderate synergy in most combinations in the CLBL-1 cell line, and slight to moderate synergy in the GL-1 cell line, except for the combination of the lowest concentrations, where the effect was rather additive. It is worth mentioning, that although the synergy was more evident in the CLBL-1 cell line, especially at the lowest concentrations of both drugs, it increased in the GL-1 cell line with increasing drug concentrations, which was also associated with higher fraction affected (Fa) values. Most importantly, as shown in Fig. 4 A, combinations of both agents at concentrations affecting at least 50% of the cells in monotherapy decreased cell metabolic activity by more than 70% at the highest olaparib concentration. This effect was again more pronounced in the CLBL-1 cells (Fig. 4 B). The study indicated that olaparib can also be used as a drug that sensitizes cells to the action of doxorubicin. Genes involved in DNA repair are mutated in the CLBL-1 and GL-1 cell lines Identification of exonic variants from RNA-seq data The complete list of detected SNVs and INDELs is reported in Supplementary Table 1. In particular, INDELs and SNVs were found on 3'/5' UTRs together with 11 and 14 synonymous SNVs in CLBL-1 and GL-1 cells, respectively. The Transeq tool was used to examine each INDEL, and premature stop codons were never identified. A focus on missense SNVs was also provided in Table 2 , which reports for each SNV the CDS position, the exon, the frequency of the alternate allele, and the VEP/Fido-SNP effect prediction. Table 2 Missense SNVs identified in DNA-damage response genes of CLBL1 and GL1 cell lines. Cell line CDS position Exon Frequency of the alternate allele VEP prediction Fido-SNP prediction BRCA1 GL-1 c.604A > G 9 HE Tolerated Benign c.715G > A 10 HE Tolerated Benign c.1329A > C 10 HE Deleterious Pathogenic c.3619A > G 10 HE Tolerated Benign c.5186G > A 13 HE Tolerated Benign BRCA2 CLBL-1 c.428A > G a 5 HE Tolerated Pathogenic c.1158T > G a 10 HO Tolerated Benign c.2401A > C 11 HE Deleterious Benign c.4304A > G a 11 HE Tolerated Benign GL-1 c.428A > G a 5 HE Tolerated Pathogenic c.1158T > G a 10 HE Tolerated Benign c.4304A > G a 11 HE Tolerated Benign c.6686G > T 11 HE Tolerated Benign TP53 CLBL-1 c.374C > T 5 HE Deleterious Pathogenic c.764G > A 8 HE Tolerated* Pathogenic GL-1 c.709C > T 7 HO Deleterious Benign ATM CLBL-1 c.3431A > T a 25 HE Tolerated Benign c.5267T > C a 36 HO Tolerated Benign GL-1 c.3431A > T a 25 HO Tolerated Benign c.5267T > C a 36 HO Tolerated Benign ATR CLBL-1 c.539G > A 4 HO Tolerated** Pathogenic a SNVs shared by CLBL-1 and GL-1 cell lines; *SIFT = 0.09; **SIFT = 0.05; HE: heterozygous; HO: homozygous. It is worth noting that some SNVs shared by GL-1 and CLBL-1 cells had a different rate of expression, suggesting a differential impact on the two cell lines; as an example, c.9995_9996insAAA and c.1158T > G in BRCA2 were homozygous in CLBL-1 and heterozygous in GL-1 cells; conversely, c.3431A > T in ATM was homozygous in GL-1 and heterozygous in CLBL-1 cells. More specifically, TP53 seems the most biologically affected target gene; it showed two heterozygous adverse SNVs (c.374C > T; c.764G > A) in CLBL-1 cells and a homozygous one (c.709C > T) in GL-1 cells. BRCA1 was mutated in the GL-1 cell line only; among the 5 missense SNVs, only c.1329A > C was predicted as deleterious/pathogenic by VEP and Fido-SNP tools. Both cell lines carried mutations on the BRCA2 transcript, but the CLBL-1 cell line seemed to be the most affected one; three SNVs were in common, while a deleterious SNV (c.2401A > C) and a deletion (c.6916_6918del) were specifically identified in CLBL-1 cells. ATR carried a common deletion (c.1550_1561del) in both cell lines. Moreover, in the CLBL-1 cell line, a homozygous missense SNV (c.539G > A) was predicted pathogenic by Fido-SNP and tolerated at the limit of tolerance (SIFT = 0.05) by VEP. Interestingly, for RAD51, a common silent SNV predicted pathogenic by Fido-SNP was noted. Likewise, c.1050G > A (for GL-1 cells) and c.1359G > A (for CLBL-1 cells) in the ATM gene were predicted pathogenic even if synonymous. In the STK11 coding sequence, only one common synonymous SNV was observed, showing a different frequency of alternate alleles between the two cell lines; conversely, no variants were found in CHEK1 and CHEK2 transcripts. Discussion Over the last few decades, biotechnological breakthroughs have led to the identification of complex and unique biologic features associated with carcinogenesis and thus cancer treatment. Genomic analysis, especially NGS has revealed a complex truth about cancer, necessitating a transition in therapy from tumor type-centered to gene-directed personalized treatment based on biomarkers( 10 ). Nowadays, veterinary oncology is transitioning to a genomics-based strategy for informing treatment of the patient. Individual mutations in cancer-bearing dogs are better understood, which encourages the use of genomics for diagnosis, prognosis, and even treatment of canine patients. Such progress in research is also crucial for the translational importance of veterinary oncology. Due to numerous similarities between canine and human cancers, genetic data from canine tumors can be exploited to find therapeutic response signals to inform and guide human drug development ( 11 ). On the other hand, an improved understanding of cancer’s molecular foundation has profoundly transformed the therapy landscape for veterinary oncology patients. For example, single gene-based assays have been available for years for diagnosing KIT mutations in mast cell tumors (MCTs), while assays for BRAF mutations in urothelial carcinomas have just been identified ( 12 , 13 ). An increasing number of companies and tools enabling genomic diagnostic testing are providing a powerful data source for biomarker discovery and cancer profiling for dogs. The most popular of them are SearchLight DNA cancer genomic diagnostic assay ( 14 ) and The FidoCure® Precision Medicine Platform ( 15 ). The SearchLight DNA assay is a cancer NGS gene panel for dogs that uses hybrid capture-based enrichment of 120 canine cancer-related genes to detect single nucleotide variants (SNVs), small insertions or deletions (INDELs), copy number variants (CNVs), and internal tandem duplications (ITDs) using tumor-only sequencing ( 14 ). The panel of 120 genes also includes those for which PARPis are molecularly targeted drugs if a mutation is detected. These genes are primarily those involved in the DNA damage repair process, mainly related to the HR repair pathway: ATM, CHEK2, BRCA1, BRCA2, FANCL, PALB2 , and CDK12 ( 16 ). In their paper from 2023, the author used clinical and genomic data from 134 cancer-bearing dogs profiled by the SearchLight DNA assay to identify mutation-level predictive relationships and evaluate the influence of therapy decisions based on patient-specific mutations ( 17 ). The indication for olaparib treatment was found in dogs with hepatic sarcoma, melanoma, osteosarcoma, renal carcinoma and sarcoma, soft tissue sarcoma, squamous cell carcinoma, and thymic carcinoma. The study showed improved survival in dogs receiving genomically informed olaparib treatment. Despite a lack of research on the mechanism of action, cytotoxicity, and effectiveness of olaparib in the abovementioned types of cancer in dogs, this drug was used and proved to be effective. It was administered at a dose ranging from 1.4 to 3 mg/kg per day ( 14 ). In another study, using the FidoCure® Precision Medicine Platform, the authors analyzed the prognostic effects of treatments based on 5 tumor genomic alterations in the TP53, PIK3CA, NRAS, ATM , and KIT genes. Based on the NGS results, olaparib was recommended for 59 patients with ATM mutations. In our study, we used RNA-seq data to identify various mutational burden differences in genes involved in the DNA damage response in CLBL-1 and GL-1 cell lines that may explain the differential in vitro sensitivity to olaparib. Focus was placed on variants that could alter the amino acid sequence (missense SNVs and INDELs) and in target genes showing the most significant results. However, it should be noted that even synonymous SNVs, albeit not fully taken into consideration, might also sometimes impact protein activity, as demonstrated by Kimchi-Sarfaty and colleagues in the case of mdr1 ( 18 ). In the presented study, we identified missense SNVs in selected DNA damage response genes in both tested canine lymphoma/leukemia cell lines. We started our analysis with the two most important genes associated with sensitivity to PARPis: BRCA1 and BRCA2 . Both genes' germline mutations have been linked to an increased risk of several human malignancies, including breast and ovarian cancers. The repair of DNA DSBs by homologous recombination depends on BRCA1/2; specifically, BRCA1 is fundamental for the recruitment of BRCA2 to the site of DSBs, and in turn, BRCA2 is essential for the recruitment of RAD51 ( 19 ). All BRCA1 missense and synonymous SNVs we observed in the GL-1 cells were also found in canine normal and tumor mammary samples ( 20 ). Additionally, the same authors confirmed all the BRCA2 variants found in our study, except for c.6686G > T (p.Cys2229Phe). In the case of BRCA2 , most of the work was focused on variants located in exon 11, a region that is largely conserved across different species. In fact, BRC repeats that are involved in RAD51 binding are present in this exon and variations that impact RAD51 binding are crucial and may hinder HR-mediated DNA repair ( 21 ). Interestingly, both GL-1 and CLBL-1 cell lines disclosed specific SNVs in this region. The c.4304A > G (p.Lys1435Arg) was already reported in canine mammary tumors and it is precisely located in BRC3 ( 22 ). The same SNV was previously reported together with c.2401A > C (p.Lys801Gln), being the most frequent SNV registered in both canine mammary tumors and normal mammary tissue ( 23 ) ( 24 ). Moreover, both were predicted as deleterious ( 25 ). Another finding concerning BRCA2 is the insertion c.9995_9996insAAA in exon 27, which is heterozygous in GL-1 cells and homozygous in CLBL-1 cells. Exon 27 encodes for the nuclear localization signals, one of RAD51 binding sites, and a cyclin-dependent kinase (CDK) phosphorylation site ( 26 , 27 ). This insertion was previously detected in dogs, but its pathogenicity is still controversial ( 22 , 24 , 28 ). However, it is known that it enhances the nuclear localization of the BRCA2 protein, possibly boosting the DNA damage repair machinery( 29 ). Considering that both cell lines carry deleterious mutations on BRCA1 and/or BRCA2 genes, it’s quite challenging to affirm that this could be the main or unique cause of a differential response to olaparib in vitro . According to the authors, differences in the TP53 and ATR genes, and not BRCA1/2 , may be the primary source of the observed differential sensitivity. TP53 is a tumor suppressor gene, also known as the “guardian of the genome”. It is sensitized by DNA damage and leads to cell cycle arrest in the G1 phase. ATR responds to a wide range of genotoxic stimuli and is mainly activated by DNA single-strand breaks. Upon activation, ATR phosphorylates multiple downstream proteins, especially serine/threonine protein kinase CHK1 (CHK1; coded by CHEK1 ), having this pathway a crucial role in cell-cycle arrest ( 30 – 32 ). In the present study, we found in TP53 CDS two heterozygous deleterious SNVs and a homozygous one in CLBL-1 and GL-1 cells, respectively. All of which were located in the DNA binding domain of P53 ( 33 ). TP53 mutations are common in cancerous cells and could impair the G1 checkpoint, avoiding cell cycle exit in case of DNA damage( 34 ). Therefore, BRCA1/2 mutations often coexist with TP53 ones ( 35 – 37 ) and mutations on TP53 could induce resistance to PARPi treatment. In this respect, in human metastatic castration-resistant prostate cancer it has been described that the differential response to PARPi between BRCA1 and BRCA2 mutated cancers was due to the concomitant presence of TP53 mutation as well as the allelic representation ( 37 , 38 ). Nevertheless, this assumption cannot be applied to all cases, as each mutation and tumor could have specific mechanisms behind a therapeutic response. In our case, both cell lines have different defects in the TP53 gene. It’s reasonable to think that a cell with an impaired ATM/CHK2/P53 axis must rely on other mechanisms to handle replicative stress, such as the previously described ATR/CHK1/WEE1 axis ( 34 ). Compared to GL-1 cells, CLBL-1 cells are more likely to rely on the ATR axis according to previous studies ( 39 ), but interestingly ATR also showed deleterious mutations in this cell line. Overall, since the mutational status of these two cell lines is lacking, except for the study of Das and colleagues (2019) on CLBL-1 and other cancer cell lines, the present investigation partially fills the knowledge regarding these two cell models. Nevertheless, a limitation of the present study is that the detection of mutations was made exclusively on coding and UTR sequences of DNA damage response genes, thus intronic variants remain unknown. Moreover, it has been already described that the impact of a single variant might be compensated by other mechanisms. Thus, more in-depth studies are needed to better define the potential causative association between mutations on DNA damage response genes and PARPi susceptibility. The availability of genetic tests for dogs, as well as frequent indications for olaparib highlight the need to determine the effectiveness of PARP inhibition in canine cancer cells. In the present study, we showed that olaparib, even as a single agent, can negatively affect vital functions of canine lymphoma and leukemia cells. The cytotoxic effect of olaparib was particularly visible after at least 72 hours of incubation, with the IC 50 value of around 3 µM for sensitive lymphoma/leukemia cells. According to published data, the drug has similar potency against numerous human cancer lines, such as Ewing's sarcoma, medulloblastoma, neuroblastoma, rhabdomyosarcoma, colon carcinoma, or osteosarcoma showing various potential applications ( 40 , 41 ). In canine lymphoma/leukemia cell lines, the antiproliferative effect of olaparib in vitro correlated with the expression of Ki-67, an important marker of cell proliferation. The effect was particularly visible for the CLBL-1 cell line, where Ki-67 levels dropped to 13% after 48 hours of incubation with 50 µM olaparib. This effect was also observed in various cancer types with defects in their DNA repair pathways, for example in cancers with BRCA2 mutations ( 42 ). Sensitivity to PARP inhibition in canine leukemia/lymphoma cells is also evidenced by the presence of DNA damage. Our study found that olaparib causes DNA damage as soon as 24 hours after incubation, even in the cells with low sensitivity to olaparib in the MTT test. This result indicates that DNA damage is the reason for the toxic effect of olaparib and suggests disruptions in the DNA repair pathway. Multiple studies show that olaparib as a single drug causes DNA damage in various cancer cell types, such as osteosarcoma ( 43 ), pancreatic cancer ( 44 ), prostatic cancer ( 45 ), lymphocytic and myeloid leukemia ( 46 , 47 ) and lymphoma ( 48 ). This validates the use of olaparib, even as a monotherapy, in cancers with suspected DNA repair dysfunction. At the same time, ATM mutations and ℽH2A.X expression profiles can serve as biomarkers of the response to PARPis, helping clinicians choose the optimal therapy for their animal patients ( 47 ). Offering dog owners affordable oral therapy for their pets may be an alternative when they choose not to pursue intravenous treatment with classic therapeutic protocols. Even greater expectations in the context of improving therapeutic effects are associated with the simultaneous use of PARPis and other drugs. This approach allows for sensitizing cancer cells to the action of a selected drug, to which the cells are resistant in monotherapy. It is also a way to overcome resistance to PARPi, which may appear during treatment. Our study showed that adding olaparib to doxorubicin monotherapy brings better therapeutic results due to the synergistic effect of these two compounds (Fig. 4 ). The synergistic effect of doxorubicin and olaparib shall allow for the development of a less toxic therapy with increased anti-cancer effectiveness. Such a synergistic effect of the mentioned combination has already been demonstrated for other types of cancers such as ovarian cancer, breast cancer, and sarcomas ( 49 , 50 ). In osteosarcoma, the synergistic effect of the combined use of these two drugs was associated with increased apoptosis of cells as indicated by flow cytometry analysis and western blotting, which showed increased expression of cleaved PARP1, cleaved caspase 3, and BAX, and decreased expression of BCL2 ( 43 ). In breast cancer it has been shown that one of the mechanisms through which PARP inhibition can chemosensitize cancer cells in vivo , is targeting Snail expression thus promoting apoptosis ( 51 ). Many studies are also focused on examining the effects of combined use of olaparib with liposomal doxorubicin ( 52 ) , ( 53 ), also in the context of limited cardiotoxic effect of doxorubicin with preserved antitumor activity ( 54 ) , ( 55 ). Combinations with doxorubicin do not exhaust all therapeutic possibilities, because various genotoxic drugs can be combined with PARPis. For example, combinations with platinum derivatives, alkylating compounds, or antimetabolites (all targeting DNA) are successfully used in humans ( 56 ). All these demonstrate the therapeutic potential of employing PARPis and their combinations with synergistic compounds to generate new, molecularly focused therapeutics for malignancies where DNA damage/repair systems play a critical role (Fig. 5 ). Conclusion Understanding DNA damage disorders in canines will contribute to the development of a reliable method for identifying suitable candidates for PARPi therapy. In the meantime, characterization of the molecular effects of PARPi combinations with other drugs in different cancer types will be the first step to develop new, molecular-based targeted therapeutic strategies in veterinary oncology. The proposed research outputs might also be translated to human medicine. Here, we showed that olaparib may be an effective therapeutic option for canine lymphomas and leukemias. In vitro experiments showed that olaparib inhibits the proliferation of cancerous dog lymphocytes by causing cell DNA damage. The anticancer effect of olaparib was visible when the drug was used alone, which indicates DNA repair disorders, but it was particularly pronounced in combination with doxorubicin. Thus, olaparib may be an option for simple oral therapy in canine lymphomas and leukemias and may also be a valuable addition to standard therapeutic protocols. Materials and methods Cell lines and cell culture The study involved a panel of 4 different canine lymphoma and leukemia cell lines: CLBL-1 (B-cell lymphoma), GL-1 (B-cell leukemia), CLB70 (B-cell chronic lymphocytic leukemia), and CNK-89 (natural killer-cell lymphoma). The CLBL-1 cell line was obtained from Barbara C. Ruetgen (Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria) ( 57 ); the GL-1 cells were obtained from Yasuhito Fujino and Hajime Tsujimoto (University of Tokyo, Department of Veterinary Internal Medicine) ( 58 ), while the CLB70 ( 59 ) and CNK-89 ( 60 ) cell lines were established in our laboratory. The CLBL-1 and GL-1 cell lines were maintained in the RPMI (Roswell Park Memorial Institute) 1640 medium (Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland), and the CLB70 and CNK-89 cells were cultured in Gibco™ Advanced RPMI 1640 medium (Gibco, Grand Island, New York, USA). All media were supplemented with 2 mM L-glutamine (Sigma Aldrich, Steinheim, Germany), 100 U/mL penicillin, 100 µg/mL streptomycin (Sigma Aldrich), and 10–20% heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, New York, USA). Chemicals and Reagents Olaparib (AZD2281) was obtained from Selleckchem (Cologne, Germany) and dissolved in dimethyl sulfoxide (DMSO) (Sigma Aldrich, Steinheim, Germany) to final concentration of 50 mM immediately prior to the experiments. Doxorubicin, propidium iodide (PI), 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT), RIPA buffer, and SigmaFAST Protease Inhibitor Cocktail were purchased from Sigma-Aldrich (Steinheim, Germany). Annexin V-FITC was purchased from Immunostep (Salamanca, Spain). Cell metabolic activity assay The metabolic activity of canine cells treated with olaparib was determined using the MTT test. In brief, 1 × 10 5 cells per well were seeded in a 96-well plate (Thermo Fisher Scientific, Roskilde, Denmark), and olaparib was added at increasing concentrations (0.1, 0.4, 1.2, 3.7, 11.1, and 33.3 µM). The range of concentrations tested was selected based on literature data on the sensitivity of various cell lines to the cytotoxic effects of olaparib and corresponds to the concentrations achievable in vivo ( 61 , 62 ). Cells only incubated with DMSO were used as controls. After incubation for 24, 48, 72, and 96 hours, 10 µL of MTT solution (5 mg/mL) was added to each well for 4 hours. After dissolving the content, the optical density of the wells was measured with a spectrophotometric microplate reader (Spark, Tecan, Singapore) at a reference wavelength of 570 nm. The results were then expressed as the mean of 3 independent experiments (different plates, different days) each one performed in triplicate. The MTT assay was also used to assess the synergistic effect of olaparib and doxorubicin by comparing cell metabolic activity after treatment with both compounds alone or in combination. To this end, cells seeded at a density of 1.5 x 10 5 /mL (GL-1 cell line) and 3 x 10 5 /mL (CLBL-1) were treated with either olaparib (2.5, 5, and 10 µM for GL-1 and 1.25, 2.5, and 5 µM for CLBL-1 cells) for 96 hours or with doxorubicin (25, 50, and 100 nM) for 48 hours as single drugs and, for combinatorial treatment, cells were pre-incubated with olaparib for 48 hours followed by the addition of doxorubicin for the next 48 hours. Olaparib and doxorubicin concentrations were selected based on MTT assay results for single compounds. The data obtained from 3 independent experiments were used to calculate Combination Index (CI) values according to the method established by Chou and Talalay ( 63 ) using CompuSyn Software (Informer Technologies, Inc). The Chou-Talalay method is based on a median-effect equation, derived from the mass-action law principle, which allows for determining synergy according to the CI theorem, where CI = 1 means addition, CI 1 indicates antagonism( 63 , 64 ). Ki-67 flow cytometry proliferation assay For this cell proliferation assay, cells were plated at a density of 1 × 105/mL in 96-well plates (TPP, Trasadingen, Switzerland), and incubated for 48 hours with two concentrations of olaparib (25 and 50 µM) selected according to MTT test results. After that time, the cells were collected, washed with PBS, labeled using the Ki-67 Proliferation Kit BD Pharmingen (BD Biosciences) according to the manufacturer’s instructions, and then analyzed in a flow cytometer (Cytoflex, Beckman Coulter). Percentages of proliferating (Ki-67 positive) cells were used to calculate the means ± SD for each cell line. The presented results were obtained from 3 independent experiments. Western blotting A total of 5 × 106 cells was rinsed with cold PBS and lysed with RIPA buffer (50m MTris-HCl pH 7.5, 100 mM NaCl, 1% NP-40, protease inhibitors set) and incubated for 20 min on ice. Then, after centrifuging at 10,000 rpm at 4 ℃ for 12 min SDS sample buffer was added to clear the supernatants and the samples were boiled at 95 ℃ for 5 minutes and subjected to SDS-polyacrylamide gel electrophoresis in a 12% gel (BioRad Mini-PROTEAN Tetra Vertical Electrophoresis Cell system, Hercules, USA). After the electrophoresis, the samples were transferred to a nitrocellulose membrane using a BioRad Mini Trans-Blot® Cell for wet transfer and Western Blot enhancer treatment was performed according to manufacturer’s protocol with Pierce™ Western Blot Signal Enhancer (Thermo Scientific). Then, the membranes were blocked with 3% BSA in TBST at room temperature for 1 hour. After blocking, the membranes were incubated overnight at 4 ℃ with the following murine monoclonal primary antibodies: anti-γH2A.X clone 9F3 (ab26350; Abcam, Cambridge, United Kingdom), dilution 1:1000 and anti-β-actin clone C4 (sc47 778; Santa Cruz, California, USA), dilution 1:2000. Goat AntiMouse Immunoglobulins/HRP (#P0447 at 1:20000) was used as secondary antibody. The membranes were incubated with the secondary antibody for 90 minutes at room temperature. The reaction was developed using Blotting substrate - Pierce™ ECL Western Blotting Substrate (Thermo Scientific) as a substrate. Membrane visualization was performed using ChemiDoc Touch Instruments (exposure: first image, 5 second; last image, 120 seconds; images, 5; BioRad). For protein expression quantification, Western blot normalization with a single housekeeping protein (β-actin) was performed using Image LabTM software (version 6.1.0; BioRad). Next-generation RNA sequencing (NGS) RNA extractions First, 1 mL of TRIzol Reagent was used to lyse and homogenize the samples and then the material was mixed with 200 µL of chloroform. Samples were centrifuged at 4 ℃ for 20 minutes at 13,000 rpm following a 2-minute incubation period. After that, the top aqueous phase was gathered, mixed with 70% ice cold ethanol, and moved into a RNeasy spin column. The next steps of the protocol for the RNeasy mini Kit (Qiagen, Hilden, Germany) were carried out according to the manufacturer's instructions. The Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) were used to measure total RNA. The RNA Integrity Number (RIN) value for each sample was greater than 7. RNA-Seq Library Preparation and Sequencing Library preparation and sequencing were performed by Novogene Biotechnology (Cambridge, UK). A total of 8 tagged RNA-seq libraries were prepared and sequenced using a 150 bp strand-specific paired-end strategy on an Illumina Novaseq 6000. Identification of exonic variants from RNA-seq data RNA-seq data were used to evaluate the exonic mutational profile of CLBL-1 and GL-1 cell lines, the most susceptible and resistant to olaparib, respectively. Nine target genes implicated in DNA damage response (Table 3 ) were selected. For every cell line, four biological replicates were considered. Raw reads underwent a preliminary quality control with FastQC software (v.0.11.9) before proceeding with the other analyses. Reads were aligned to the reference cDNA sequences retrieved from Ensembl (release 112; GCA_000002285.4; canFam6) (Table 2 ). Bowtie2 (v.2.5.4) was used to build the indexes and align the reads. Table 3 Target genes selected for the exonic mutational profiling of GL-1 and CLBL-1 cell lines: gene symbol, gene description and Ensembl transcript ID. Gene symbol Gene description Transcript ID BRCA1 BRCA1 DNA Repair Associated ENSCAFT00000065506.2 BRCA2 BRCA2 DNA Repair Associated ENSCAFT00000010309.5 TP53 Tumor Protein P53 ENSCAFT00000026465.5 RAD51 RAD51 Recombinase ENSCAFT00000073671.2 ATM ATM Serine/Threonine kinase ENSCAFT00000048574.3 ATR ATR Serine/Threonine Kinase ENSCAFT00000012571.5 CHEK1 Checkpoint Kinase 1 ENSCAFT00000016788.5 CHEK2 Checkpoint Kinase 2 ENSCAFT00000018932.5 STK11 Serine/Threonine Kinase 11 ENSCAFT00000031055.5 Following alignment, samtools (v.1.19) were used to sort the SAM outputs and convert them to BAM files. An index was also prepared with samtools to visualize them on Integrative Genomic Viewer (v.2.18.2). Thanks to IGV visualization, possible single nucleotide variants (SNVs) and insertions-deletions mutations (INDELs) were recognized, and each variant was checked for possible strand biases. The corresponding coding sequence (CDS), and the encoded protein position were retrieved from the Ensembl Genome Browser. To check for the possible biological consequences of SNVs, the Ensembl Variant Effect Predictor (VEP) and Fido-SNP platforms were used. For INDELs that couldn’t be evaluated with the previous softwares, the nucleotide sequence was converted into the corresponding amino acid sequence checking the possible effect of mutations on the reading frame using EMBOSS Transeq. Statistical analysis All data are shown as means with SD. Statistical differences were analyzed using one-way analysis of variance (ANOVA) followed by the Dunnett's multiple comparison test (Ki-67 expression marker analysis) and unpaired T test (DNA damage induction assessment). Statistical analysis was performed using either STATISTICA software version 13.3 (TIBCO Software Inc., Palo Alto, CA, USA) or GraphPad Prism v.9 (GraphPad Software, San Diego, CA, USA). The results were considered significant at P < .05. Abbreviations ANOVA, Analysis of Variance ATM, Ataxia Telangiectasia Mutated BRCA1, Breast Cancer Associated 1 BRCA2, Breast Cancer Associated 2 CDK12, Cyclin-Dependent Kinase 12 CI, Combination Index CLB70, Canine B-cell Chronic Lymphocytic Leukemia Cell Line CLBL-1, Canine B-cell Lymphoma Cell Line CNK-89, Canine Natural Killer-cell Lymphoma Cell Line DMSO, Dimethyl Sulfoxide DSBs, Double-Strand Breaks FBS, Fetal Bovine Serum FITC, Fluorescein Isothiocyanate γH2AX, Phosphorylated Histone H2AX GL-1, B-cell Leukemia Cell Line HR, Homologous Recombination IC50, Half Maximal Inhibitory Concentration INDELs, Insertions or Deletions ITDs, Internal Tandem Duplications MAPK12, Mitogen-Activated Protein Kinase 12 MCTs, Mast Cell Tumors MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide NGS, Next-Generation Sequencing PARP, Poly(ADP-ribose) Polymerase PARPi, PARP Inhibitors PI, Propidium Iodide PI3K, Phosphoinositide 3-Kinases PLK3, Polo-Like Kinase 3 PNKP, Polynucleotide Kinase-Phosphatase SSBs, Single-Strand Breaks STK22C, Serine/Threonine-Protein Kinase 22C STK36, Serine/Threonine Kinase 36 USP1, Ubiquitin Specific Peptidase 1 USP7, Ubiquitin Specific Peptidase 7 USP11, Ubiquitin Specific Peptidase 11 Declarations Supplementary materials Supplementary Table 1. Complete list of variants identified in DNA-damage response genes of CLBL-1 and GL-1 cell lines. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials Raw Illumina sequencing data have been deposited in GenBank (SRA) under the BioProject accession PRJNA1242827. Additional supporting information can be found online in the Supporting Information section at the end of this article (Supplementary Table 1 with complete list of variants identified in DNA-damage response genes of CLBL-1 and GL-1 cell lines). Competing interests The authors declare no competing interests. Funding Funding provided by the: Polish National Agency for Academic Exchange, Grant/Award Number: PPI/APM/2019/1/00044/U/00001 and by the Wrocław University of Environmental and Life Sciences (Poland) as part of research project no N070/0012/23. The article is part of a PhD dissertation titled “A Novel Therapeutic Approach Using PolyA DP Ribose Polymerase (PARP) Inhibitors in Canine Cancer Cells”, prepared during Doctoral School at the Wrocław University of Environmental and Life Sciences. The APC is financed by Wrocław University of Environmental and Life Sciences. Authors' contributions AP and MD designed the study and contributed to the implementation of the research; JCP, ED, GM, JB, MH and AP organized and performed the experiments; JCP, ED, GM, MG, MP, MD and AP analysed the data. JP, ED, GM, BOM, MG, MP, CZ, MD and AP wrote and revised the manuscript. All authors read and approved the final manuscript. Acknowledgements We are grateful to B.C. Ruetgen (Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine, Vienna) for providing the CLBL-1 and Y. Fujino and H. Tsujimoto (Department of Veterinary Internal Medicine, University of Tokyo) for providing the GL-1 cell line. 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Pawlak","email":"data:image/png;base64,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","orcid":"","institution":"Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Norwida 31, 50-375 Wroclaw","correspondingAuthor":true,"prefix":"","firstName":"Aleksandra","middleName":"","lastName":"Pawlak","suffix":""}],"badges":[],"createdAt":"2025-03-03 16:53:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6148132/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6148132/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12917-025-04880-z","type":"published","date":"2025-07-05T15:56:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80856663,"identity":"97f37420-7ccb-4271-a9f4-53144b0a76b8","added_by":"auto","created_at":"2025-04-17 21:39:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":391574,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConcentration-dependent curves displaying the effects of olaparib.\u003c/strong\u003e Metabolic activity (measured by MTT assay) of CLBL-1, CLB70, CNK-89, and GL-1 cell lines after 24, 48, 72, and 96 hours of incubation.\u003c/p\u003e","description":"","filename":"Figure1color170mm.png","url":"https://assets-eu.researchsquare.com/files/rs-6148132/v1/09962f581e6ba8d75eda0350.png"},{"id":80856900,"identity":"3e975172-211b-4c37-995a-d0b7a076d2b8","added_by":"auto","created_at":"2025-04-17 21:47:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":247805,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistograms presenting percentages of proliferating (Ki-67 positive cells).\u003c/strong\u003e Cells after 48-hour treatment with 25 and 50 µM of olaparib with reference to the control (A). Graphs displaying the statistical analysis of the results (B). Values are presented as means ± standard deviation (SD) of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure2color170mm.png","url":"https://assets-eu.researchsquare.com/files/rs-6148132/v1/2532b5daa18cdcf528f3e312.png"},{"id":80856320,"identity":"467bfedd-5140-4971-af13-2d59e7e4cc9c","added_by":"auto","created_at":"2025-04-17 21:31:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":264815,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWestern blot analysis for phosphorylated histone H2A.X.\u003c/strong\u003e The CLBL-1, CLB70, CNK-89, and GL-1 (A-D) cell lines after 24 hours of incubation with different concentrations of olaparib (25 and 50 µM). Quantification was performed by normalizing the expression level of the protein of interest to the expression level of the loading control, β-actin. Mean and standard deviations were calculated based on three independent experiments. The asterisks (*) are used to represent comparative statistically significant results. *P \u0026lt; .05; **P \u0026lt; .01; ***P \u0026lt; .001; ****P \u0026lt; .0001. The statistical analysis was done with the unpaired T test using GraphPad Prism.\u003c/p\u003e","description":"","filename":"Figure3BMC170mm.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6148132/v1/0677a01ebc308c4d3f2ac011.jpg"},{"id":80856325,"identity":"0ffc4b75-3dc1-4149-95f5-778da2838e74","added_by":"auto","created_at":"2025-04-17 21:31:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":304762,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of synergistic effect of olaparib with doxorubicin. \u003c/strong\u003eCell metabolic activity after incubation with olaparib (96 h), doxorubicin (48 h), or preincubation with olaparib for 48 h and following exposure to the combination of doxorubicin and olaparib for next 48 h (MTT assay) (A). Combination indexes (CI) of olaparib and doxorubicin in GL-1 and CLBL-1 cells (B). ± indicates nearly additive, + slight synergism, ++ moderate synergism, +++ synergism. The asterisks (*) are used to represent comparative statistically significant results. *P \u0026lt; .05; **P \u0026lt; .01; ****P \u0026lt; .0001; ns – not significant (P \u0026gt; .05).\u003c/p\u003e","description":"","filename":"Figure4color170mm.png","url":"https://assets-eu.researchsquare.com/files/rs-6148132/v1/94524528daf0005f64e22925.png"},{"id":80856667,"identity":"48765a20-a2ad-4085-bcc0-40584903ddec","added_by":"auto","created_at":"2025-04-17 21:39:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":686511,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representations of the potential of PARPi in cancer therapy. \u003c/strong\u003eCharacterization of the molecular effects of PARPi combinations with additional drugs in various cancer types as a first step to develop new, molecular-based targeted therapeutic strategies in veterinary oncology.\u003c/p\u003e","description":"","filename":"Figure5color170mm.png","url":"https://assets-eu.researchsquare.com/files/rs-6148132/v1/00d0568d55f26163e4b419ef.png"},{"id":86178875,"identity":"76c14896-0488-494f-8769-dd49f0ff4371","added_by":"auto","created_at":"2025-07-07 16:05:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3311229,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6148132/v1/f08cc76b-6a1c-40bb-a04c-b01f16b0c555.pdf"},{"id":80856319,"identity":"5c23de82-6605-4ea1-9636-9fc82f874589","added_by":"auto","created_at":"2025-04-17 21:31:51","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16770,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6148132/v1/dc864360080746c93a0d1f6e.xlsx"},{"id":80856666,"identity":"efecbfb6-0215-44ae-9381-62a87e31d249","added_by":"auto","created_at":"2025-04-17 21:39:51","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1406688,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformationWesternblotsv2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6148132/v1/5112e9566bad7d24508f0ab3.docx"},{"id":80856327,"identity":"c0d6ce7a-b951-42e2-a109-63cd22a1bbcf","added_by":"auto","created_at":"2025-04-17 21:31:51","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1663755,"visible":true,"origin":"","legend":"","description":"","filename":"Olafarmakologia2024112914h14m52s.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6148132/v1/2d621653f1fdd980009654b6.jpg"},{"id":80856329,"identity":"288e655b-9161-4ade-8a36-1ce67384daf7","added_by":"auto","created_at":"2025-04-17 21:31:51","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1697159,"visible":true,"origin":"","legend":"","description":"","filename":"Olafarmakologia2024112815h06m04s.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6148132/v1/835dda2fc27d6381b60252ae.jpg"},{"id":80856331,"identity":"006713ad-bec2-4c88-925e-9179903147e2","added_by":"auto","created_at":"2025-04-17 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21:39:51","extension":"scn","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":3033116,"visible":true,"origin":"","legend":"","description":"","filename":"Olafarmakologia2024112815h06m04s.scn","url":"https://assets-eu.researchsquare.com/files/rs-6148132/v1/2246e737db4ca967a0419172.scn"},{"id":80856671,"identity":"88a4fa05-3500-4334-87a4-dbbed3c7ce6b","added_by":"auto","created_at":"2025-04-17 21:39:51","extension":"scn","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":3033114,"visible":true,"origin":"","legend":"","description":"","filename":"Olafarmakologia2024120914h20m14s.scn","url":"https://assets-eu.researchsquare.com/files/rs-6148132/v1/0c8901360da26d7a696b31ab.scn"},{"id":80856341,"identity":"5e996d3c-2d57-455d-93c5-fe39413a4225","added_by":"auto","created_at":"2025-04-17 21:31:51","extension":"scn","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":3033116,"visible":true,"origin":"","legend":"","description":"","filename":"Olafarmakologia2024112914h14m52s.scn","url":"https://assets-eu.researchsquare.com/files/rs-6148132/v1/3580d4be79dc8c190fd70475.scn"},{"id":80856672,"identity":"c16249fe-cc18-49c7-9cd6-0653608ce82e","added_by":"auto","created_at":"2025-04-17 21:39:51","extension":"scn","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":3033114,"visible":true,"origin":"","legend":"","description":"","filename":"Olafarmakologia2024120514h34m47s.scn","url":"https://assets-eu.researchsquare.com/files/rs-6148132/v1/5b83994d9c61ee754b17e93b.scn"}],"financialInterests":"No competing interests reported.","formattedTitle":"PARP inhibitor olaparib induces DNA damage and acts as a drug sensitizer in an in vitro model of canine hematopoietic cancer","fulltext":[{"header":"Background","content":"\u003cp\u003eThe search for innovative targeted anti-cancer treatments is critical for modern oncology. As access to genetic tests for dogs becomes widespread, physicians will gather more data providing indications for the use of novel medications targeting specific molecules. Interestingly, common availability of modern molecular methods for cancer diagnosis in animals has significantly outpaced the rate of research on the use of molecularly targeted drugs, thus creating a gap between information about dedicated treatment and the actual possibility of using specific substances in animals. An example may be the use of poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis), drugs with already proven effectiveness in humans, for treatment of patients with DNA damage repair-deficient cancers, mainly with Breast Cancer Associated 1 and 2 (BRCA1 and BRCA2) mutations (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePARP1 plays a crucial role in repairing DNA damage. Following DNA insult, PARP1 is rapidly recruited to single-strand breaks (SSBs) and double-strand breaks (DSBs) in DNA. Subsequently, BRCA1 and BRCA2 are recruited as they regulate the major pathway for DSB repair, homologous recombination (HR)(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Pharmacological inhibition of PARP1 leads to DNA damage and, in the absence of functional \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e genes, to critical levels of genomic instability provoking cell death (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Cell death observed after PARPi treatment of HR-deficient cells is based on synthetic lethality. This occurs when mutation-induced dysfunctions in two genes do not affect cell viability separately, but simultaneous disorders in both genes lead to cell death(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Thanks to this effect, PARPis quickly became a novel class of anti-cancer drugs, first demonstrating efficiency in treating HR-deficient tumors with a BRCA1 and BRCA2 mutation(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Interestingly, patients with genetic HR dysfunction are not the only candidates for PARPi treatment. Some sporadic cancers are also characterized by HR deficiency but without \u003cem\u003eBRCA1-2\u003c/em\u003e mutations. The broad range of DNA substrates and various processes targeted by PARPi imply that PARPis may also reduce survival of other DNA repair-deficient cells (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), a phenomenon referred to as BRCAness (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).This observation supports current clinical trials of PARPis in patients with other HR-deficient, BRCA-dependent cancers (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite the success of PARPis in human oncology, these drugs are not extensively researched in veterinary medicine (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Considering the current wide indications for PARPis and increasing data on HR disorders in various dog cancers, we decided to investigate the activity of the first and best-studied PARPi, olaparib, in canine hematopoietic cancers. We assessed the antiproliferative and proapoptotic effect of olaparib, and its ability to induce DNA damage. Additionally, we investigated the synergistic effect of olaparib and doxorubicin to determine their potential utility in chemotherapy for hematological malignancies. Finally, we analyzed the mutational profile of key genes involved in the DNA damage response to assess their impact on olaparib sensitivity \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOlaparib shows activity as a single agent in canine lymphoma/leukemia cells\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eInhibition of cell metabolic activity\u003c/h2\u003e \u003cp\u003eTo check if PARPi may be a potential therapeutic option for dogs with lymphoma or leukemia, we first investigated the effect of olaparib on the metabolic activity of established canine cancer cell lines using the MTT assay. Data obtained indicated that olaparib alone inhibits the cell metabolic activity of all lymphoma/leukemia cell lines used in our study in both concentration- and time-dependent manners. Concentration-dependent curves presenting the effects of olaparib on the metabolic activity of the tested cell lines are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe identified some differences in the sensitivity of individual cell lines to olaparib, with the GL-1 cell line being less susceptible compared to the others. As expected, the cytotoxic effect became more evident with prolonged incubation, reaching the concentration that inhibited cell metabolic activity by 50% (IC50 value) below 3 \u0026micro;M after 72 hours for all the sensitive cell lines. This concentration is much lower than the maximum one achievable in human patients with solid tumors treated with 200\u0026ndash;600 mg olaparib twice daily 19,20. A comparison of IC50 values for each cell line is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe cytotoxic effects of olaparib after 48, 72, and 96 hours of incubation on the canine lymphoma/leukemia cell lines, expressed as IC\u003csub\u003e50\u003c/sub\u003e values. The results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of 3 independent experiments, each performed in triplicates. Statistical differences were analyzed using a one-way ANOVA followed by the Tukey\u0026rsquo;s multiple comparison test. Values without common letters (a, b, c) in the superscript differ statistically (P\u0026thinsp;\u0026lt;\u0026thinsp;.05).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;M) values after olaparib treatment of canine lymphoma/leukemia cell lines\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCLBL-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCLB70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCNK-89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGL-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e48h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.60\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.82\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.29\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;33.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e72h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.68\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.03\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.03\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.83\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e96h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.2\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.09\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.50\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.57\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eAntiproliferative effect\u003c/h3\u003e\n\u003cp\u003eTo investigate the antiproliferative effect of olaparib on canine lymphoma/leukemia cells, the expression of Ki-67 (a common proliferation marker) was determined in cells treated with olaparib for 48 hours. The percentage of Ki-67 positive cells (as compared to the untreated control) decreased significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after incubation with 25 and 50 \u0026micro;M of olaparib in all tested cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The strongest effect was observed for the CLBL-1 cell line, where Ki-67 expression dropped below 20% after 48 hours of incubation with 50 \u0026micro;M of olaparib, indicating a decrease in the cell proliferation potential.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eDNA damage induction\u003c/h3\u003e\n\u003cp\u003eHaving confirmed that olaparib alone exerts cytotoxic and antiproliferative effects on canine lymphoma/leukemia cells, we decided to investigate if olaparib causes DNA damage in sensitive cells. We sought to determine whether and after what incubation time of the cells with olaparib, DNA damage accumulates in the cell and can be detected as phosphorylation of histone H2A.X on serine 139, (γH2A.X), a verified and common marker of DNA damage. The results of the histone H2A.X phosphorylation analysis using the Western blot technique are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Olaparib indeed induces the formation of DNA damage, even after 24 hours of incubation, also in the cells with limited sensitivity to olaparib (see MTT results). This result clearly shows that the accumulation of DNA damage in the cells disturbs cell proliferation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOlaparib acts as a chemosensitizer due to its ability to potentiate the cytotoxic effects of doxorubicin \u0026ndash; a commonly used cytostatic drug for lymphoma/leukemia therapy\u003c/b\u003e \u003c/p\u003e\n\u003ch3\u003eOlaparib – doxorubicin combinatory effects\u003c/h3\u003e\n\u003cp\u003eTo evaluate the potential benefit of adding olaparib to a conventional anti-cancer treatment in dogs, the effects of a combined exposure of lymphoma/leukemia cells to olaparib and doxorubicin were analyzed. The resulting CI values (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) indicated that doxorubicin and olaparib showed a moderate synergy in most combinations in the CLBL-1 cell line, and slight to moderate synergy in the GL-1 cell line, except for the combination of the lowest concentrations, where the effect was rather additive. It is worth mentioning, that although the synergy was more evident in the CLBL-1 cell line, especially at the lowest concentrations of both drugs, it increased in the GL-1 cell line with increasing drug concentrations, which was also associated with higher fraction affected (Fa) values. Most importantly, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, combinations of both agents at concentrations affecting at least 50% of the cells in monotherapy decreased cell metabolic activity by more than 70% at the highest olaparib concentration. This effect was again more pronounced in the CLBL-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The study indicated that olaparib can also be used as a drug that sensitizes cells to the action of doxorubicin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGenes involved in DNA repair are mutated in the CLBL-1 and GL-1 cell lines\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eIdentification of exonic variants from RNA-seq data\u003c/h2\u003e \u003cp\u003eThe complete list of detected SNVs and INDELs is reported in Supplementary Table\u0026nbsp;1. In particular, INDELs and SNVs were found on 3'/5' UTRs together with 11 and 14 synonymous SNVs in CLBL-1 and GL-1 cells, respectively. The Transeq tool was used to examine each INDEL, and premature stop codons were never identified. A focus on missense SNVs was also provided in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, which reports for each SNV the CDS position, the exon, the frequency of the alternate allele, and the VEP/Fido-SNP effect prediction.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMissense SNVs identified in DNA-damage response genes of CLBL1 and GL1 cell lines.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell line\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDS position\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExon\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFrequency of the alternate allele\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVEP prediction\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFido-SNP prediction\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eGL-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.604A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTolerated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBenign\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.715G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTolerated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBenign\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.1329A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDeleterious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePathogenic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.3619A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTolerated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBenign\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.5186G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTolerated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBenign\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e \u003cp\u003e\u003cem\u003eBRCA2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eCLBL-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.428A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTolerated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePathogenic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.1158T\u0026thinsp;\u0026gt;\u0026thinsp;G\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTolerated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBenign\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.2401A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDeleterious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBenign\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.4304A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTolerated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBenign\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eGL-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.428A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTolerated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePathogenic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.1158T\u0026thinsp;\u0026gt;\u0026thinsp;G\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTolerated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBenign\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.4304A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTolerated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBenign\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.6686G\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTolerated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBenign\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eTP53\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCLBL-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.374C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDeleterious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePathogenic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.764G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTolerated*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePathogenic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGL-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.709C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDeleterious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBenign\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cem\u003eATM\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCLBL-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.3431A\u0026thinsp;\u0026gt;\u0026thinsp;T\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTolerated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBenign\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.5267T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTolerated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBenign\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGL-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.3431A\u0026thinsp;\u0026gt;\u0026thinsp;T\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTolerated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBenign\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.5267T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTolerated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBenign\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eATR\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCLBL-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.539G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTolerated**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePathogenic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003eSNVs shared by CLBL-1 and GL-1 cell lines; *SIFT\u0026thinsp;=\u0026thinsp;0.09; **SIFT\u0026thinsp;=\u0026thinsp;0.05; HE: heterozygous; HO: homozygous.\u003c/p\u003e \u003cp\u003eIt is worth noting that some SNVs shared by GL-1 and CLBL-1 cells had a different rate of expression, suggesting a differential impact on the two cell lines; as an example, c.9995_9996insAAA and c.1158T\u0026thinsp;\u0026gt;\u0026thinsp;G in BRCA2 were homozygous in CLBL-1 and heterozygous in GL-1 cells; conversely, c.3431A\u0026thinsp;\u0026gt;\u0026thinsp;T in ATM was homozygous in GL-1 and heterozygous in CLBL-1 cells.\u003c/p\u003e \u003cp\u003eMore specifically, TP53 seems the most biologically affected target gene; it showed two heterozygous adverse SNVs (c.374C\u0026thinsp;\u0026gt;\u0026thinsp;T; c.764G\u0026thinsp;\u0026gt;\u0026thinsp;A) in CLBL-1 cells and a homozygous one (c.709C\u0026thinsp;\u0026gt;\u0026thinsp;T) in GL-1 cells.\u003c/p\u003e \u003cp\u003eBRCA1 was mutated in the GL-1 cell line only; among the 5 missense SNVs, only c.1329A\u0026thinsp;\u0026gt;\u0026thinsp;C was predicted as deleterious/pathogenic by VEP and Fido-SNP tools. Both cell lines carried mutations on the BRCA2 transcript, but the CLBL-1 cell line seemed to be the most affected one; three SNVs were in common, while a deleterious SNV (c.2401A\u0026thinsp;\u0026gt;\u0026thinsp;C) and a deletion (c.6916_6918del) were specifically identified in CLBL-1 cells.\u003c/p\u003e \u003cp\u003eATR carried a common deletion (c.1550_1561del) in both cell lines. Moreover, in the CLBL-1 cell line, a homozygous missense SNV (c.539G\u0026thinsp;\u0026gt;\u0026thinsp;A) was predicted pathogenic by Fido-SNP and tolerated at the limit of tolerance (SIFT\u0026thinsp;=\u0026thinsp;0.05) by VEP.\u003c/p\u003e \u003cp\u003eInterestingly, for RAD51, a common silent SNV predicted pathogenic by Fido-SNP was noted. Likewise, c.1050G\u0026thinsp;\u0026gt;\u0026thinsp;A (for GL-1 cells) and c.1359G\u0026thinsp;\u0026gt;\u0026thinsp;A (for CLBL-1 cells) in the ATM gene were predicted pathogenic even if synonymous.\u003c/p\u003e \u003cp\u003eIn the STK11 coding sequence, only one common synonymous SNV was observed, showing a different frequency of alternate alleles between the two cell lines; conversely, no variants were found in CHEK1 and CHEK2 transcripts.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOver the last few decades, biotechnological breakthroughs have led to the identification of complex and unique biologic features associated with carcinogenesis and thus cancer treatment. Genomic analysis, especially NGS has revealed a complex truth about cancer, necessitating a transition in therapy from tumor type-centered to gene-directed personalized treatment based on biomarkers(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Nowadays, veterinary oncology is transitioning to a genomics-based strategy for informing treatment of the patient. Individual mutations in cancer-bearing dogs are better understood, which encourages the use of genomics for diagnosis, prognosis, and even treatment of canine patients. Such progress in research is also crucial for the translational importance of veterinary oncology. Due to numerous similarities between canine and human cancers, genetic data from canine tumors can be exploited to find therapeutic response signals to inform and guide human drug development (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). On the other hand, an improved understanding of cancer\u0026rsquo;s molecular foundation has profoundly transformed the therapy landscape for veterinary oncology patients. For example, single gene-based assays have been available for years for diagnosing \u003cem\u003eKIT\u003c/em\u003e mutations in mast cell tumors (MCTs), while assays for \u003cem\u003eBRAF\u003c/em\u003e mutations in urothelial carcinomas have just been identified (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). An increasing number of companies and tools enabling genomic diagnostic testing are providing a powerful data source for biomarker discovery and cancer profiling for dogs. The most popular of them are SearchLight DNA cancer genomic diagnostic assay (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) and The FidoCure\u0026reg; Precision Medicine Platform (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe SearchLight DNA assay is a cancer NGS gene panel for dogs that uses hybrid capture-based enrichment of 120 canine cancer-related genes to detect single nucleotide variants (SNVs), small insertions or deletions (INDELs), copy number variants (CNVs), and internal tandem duplications (ITDs) using tumor-only sequencing (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The panel of 120 genes also includes those for which PARPis are molecularly targeted drugs if a mutation is detected. These genes are primarily those involved in the DNA damage repair process, mainly related to the HR repair pathway: \u003cem\u003eATM, CHEK2, BRCA1, BRCA2, FANCL, PALB2\u003c/em\u003e, and \u003cem\u003eCDK12\u003c/em\u003e (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). In their paper from 2023, the author used clinical and genomic data from 134 cancer-bearing dogs profiled by the SearchLight DNA assay to identify mutation-level predictive relationships and evaluate the influence of therapy decisions based on patient-specific mutations (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The indication for olaparib treatment was found in dogs with hepatic sarcoma, melanoma, osteosarcoma, renal carcinoma and sarcoma, soft tissue sarcoma, squamous cell carcinoma, and thymic carcinoma. The study showed improved survival in dogs receiving genomically informed olaparib treatment. Despite a lack of research on the mechanism of action, cytotoxicity, and effectiveness of olaparib in the abovementioned types of cancer in dogs, this drug was used and proved to be effective. It was administered at a dose ranging from 1.4 to 3 mg/kg per day (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). In another study, using the FidoCure\u0026reg; Precision Medicine Platform, the authors analyzed the prognostic effects of treatments based on 5 tumor genomic alterations in the \u003cem\u003eTP53, PIK3CA, NRAS, ATM\u003c/em\u003e, and \u003cem\u003eKIT\u003c/em\u003e genes. Based on the NGS results, olaparib was recommended for 59 patients with \u003cem\u003eATM\u003c/em\u003e mutations.\u003c/p\u003e \u003cp\u003eIn our study, we used RNA-seq data to identify various mutational burden differences in genes involved in the DNA damage response in CLBL-1 and GL-1 cell lines that may explain the differential \u003cem\u003ein vitro\u003c/em\u003e sensitivity to olaparib. Focus was placed on variants that could alter the amino acid sequence (missense SNVs and INDELs) and in target genes showing the most significant results. However, it should be noted that even synonymous SNVs, albeit not fully taken into consideration, might also sometimes impact protein activity, as demonstrated by Kimchi-Sarfaty and colleagues in the case of mdr1 (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). In the presented study, we identified missense SNVs in selected DNA damage response genes in both tested canine lymphoma/leukemia cell lines. We started our analysis with the two most important genes associated with sensitivity to PARPis: \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e. Both genes' germline mutations have been linked to an increased risk of several human malignancies, including breast and ovarian cancers. The repair of DNA DSBs by homologous recombination depends on BRCA1/2; specifically, BRCA1 is fundamental for the recruitment of BRCA2 to the site of DSBs, and in turn, BRCA2 is essential for the recruitment of RAD51 (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). All \u003cem\u003eBRCA1\u003c/em\u003e missense and synonymous SNVs we observed in the GL-1 cells were also found in canine normal and tumor mammary samples (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Additionally, the same authors confirmed all the \u003cem\u003eBRCA2\u003c/em\u003e variants found in our study, except for c.6686G\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Cys2229Phe). In the case of \u003cem\u003eBRCA2\u003c/em\u003e, most of the work was focused on variants located in exon 11, a region that is largely conserved across different species. In fact, BRC repeats that are involved in RAD51 binding are present in this exon and variations that impact RAD51 binding are crucial and may hinder HR-mediated DNA repair (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Interestingly, both GL-1 and CLBL-1 cell lines disclosed specific SNVs in this region. The c.4304A\u0026thinsp;\u0026gt;\u0026thinsp;G (p.Lys1435Arg) was already reported in canine mammary tumors and it is precisely located in BRC3 (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The same SNV was previously reported together with c.2401A\u0026thinsp;\u0026gt;\u0026thinsp;C (p.Lys801Gln), being the most frequent SNV registered in both canine mammary tumors and normal mammary tissue (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Moreover, both were predicted as deleterious (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Another finding concerning \u003cem\u003eBRCA2\u003c/em\u003e is the insertion c.9995_9996insAAA in exon 27, which is heterozygous in GL-1 cells and homozygous in CLBL-1 cells. Exon 27 encodes for the nuclear localization signals, one of RAD51 binding sites, and a cyclin-dependent kinase (CDK) phosphorylation site (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). This insertion was previously detected in dogs, but its pathogenicity is still controversial (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). However, it is known that it enhances the nuclear localization of the BRCA2 protein, possibly boosting the DNA damage repair machinery(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Considering that both cell lines carry deleterious mutations on \u003cem\u003eBRCA1\u003c/em\u003e and/or \u003cem\u003eBRCA2\u003c/em\u003e genes, it\u0026rsquo;s quite challenging to affirm that this could be the main or unique cause of a differential response to olaparib \u003cem\u003ein vitro\u003c/em\u003e. According to the authors, differences in the \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003eATR\u003c/em\u003e genes, and not \u003cem\u003eBRCA1/2\u003c/em\u003e, may be the primary source of the observed differential sensitivity.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTP53\u003c/em\u003e is a tumor suppressor gene, also known as the \u0026ldquo;guardian of the genome\u0026rdquo;. It is sensitized by DNA damage and leads to cell cycle arrest in the G1 phase. \u003cem\u003eATR\u003c/em\u003e responds to a wide range of genotoxic stimuli and is mainly activated by DNA single-strand breaks. Upon activation, ATR phosphorylates multiple downstream proteins, especially serine/threonine protein kinase CHK1 (CHK1; coded by \u003cem\u003eCHEK1\u003c/em\u003e), having this pathway a crucial role in cell-cycle arrest (\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In the present study, we found in \u003cem\u003eTP53\u003c/em\u003e CDS two heterozygous deleterious SNVs and a homozygous one in CLBL-1 and GL-1 cells, respectively. All of which were located in the DNA binding domain of P53 (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). \u003cem\u003eTP53\u003c/em\u003e mutations are common in cancerous cells and could impair the G1 checkpoint, avoiding cell cycle exit in case of DNA damage(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Therefore, \u003cem\u003eBRCA1/2\u003c/em\u003e mutations often coexist with \u003cem\u003eTP53\u003c/em\u003e ones (\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) and mutations on \u003cem\u003eTP53\u003c/em\u003e could induce resistance to PARPi treatment. In this respect, in human metastatic castration-resistant prostate cancer it has been described that the differential response to PARPi between \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e mutated cancers was due to the concomitant presence of \u003cem\u003eTP53\u003c/em\u003e mutation as well as the allelic representation (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Nevertheless, this assumption cannot be applied to all cases, as each mutation and tumor could have specific mechanisms behind a therapeutic response. In our case, both cell lines have different defects in the \u003cem\u003eTP53\u003c/em\u003e gene. It\u0026rsquo;s reasonable to think that a cell with an impaired ATM/CHK2/P53 axis must rely on other mechanisms to handle replicative stress, such as the previously described ATR/CHK1/WEE1 axis (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Compared to GL-1 cells, CLBL-1 cells are more likely to rely on the ATR axis according to previous studies (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), but interestingly \u003cem\u003eATR\u003c/em\u003e also showed deleterious mutations in this cell line.\u003c/p\u003e \u003cp\u003eOverall, since the mutational status of these two cell lines is lacking, except for the study of Das and colleagues (2019) on CLBL-1 and other cancer cell lines, the present investigation partially fills the knowledge regarding these two cell models. Nevertheless, a limitation of the present study is that the detection of mutations was made exclusively on coding and \u003cem\u003eUTR\u003c/em\u003e sequences of DNA damage response genes, thus intronic variants remain unknown. Moreover, it has been already described that the impact of a single variant might be compensated by other mechanisms. Thus, more in-depth studies are needed to better define the potential causative association between mutations on DNA damage response genes and PARPi susceptibility.\u003c/p\u003e \u003cp\u003eThe availability of genetic tests for dogs, as well as frequent indications for olaparib highlight the need to determine the effectiveness of PARP inhibition in canine cancer cells. In the present study, we showed that olaparib, even as a single agent, can negatively affect vital functions of canine lymphoma and leukemia cells. The cytotoxic effect of olaparib was particularly visible after at least 72 hours of incubation, with the IC\u003csub\u003e50\u003c/sub\u003e value of around 3 \u0026micro;M for sensitive lymphoma/leukemia cells. According to published data, the drug has similar potency against numerous human cancer lines, such as Ewing's sarcoma, medulloblastoma, neuroblastoma, rhabdomyosarcoma, colon carcinoma, or osteosarcoma showing various potential applications (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). In canine lymphoma/leukemia cell lines, the antiproliferative effect of olaparib \u003cem\u003ein vitro\u003c/em\u003e correlated with the expression of Ki-67, an important marker of cell proliferation. The effect was particularly visible for the CLBL-1 cell line, where Ki-67 levels dropped to 13% after 48 hours of incubation with 50 \u0026micro;M olaparib. This effect was also observed in various cancer types with defects in their DNA repair pathways, for example in cancers with \u003cem\u003eBRCA2\u003c/em\u003e mutations (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSensitivity to PARP inhibition in canine leukemia/lymphoma cells is also evidenced by the presence of DNA damage. Our study found that olaparib causes DNA damage as soon as 24 hours after incubation, even in the cells with low sensitivity to olaparib in the MTT test. This result indicates that DNA damage is the reason for the toxic effect of olaparib and suggests disruptions in the DNA repair pathway. Multiple studies show that olaparib as a single drug causes DNA damage in various cancer cell types, such as osteosarcoma (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e), pancreatic cancer (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e), prostatic cancer (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), lymphocytic and myeloid leukemia (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e) and lymphoma (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). This validates the use of olaparib, even as a monotherapy, in cancers with suspected DNA repair dysfunction. At the same time, \u003cem\u003eATM\u003c/em\u003e mutations and ℽH2A.X expression profiles can serve as biomarkers of the response to PARPis, helping clinicians choose the optimal therapy for their animal patients (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Offering dog owners affordable oral therapy for their pets may be an alternative when they choose not to pursue intravenous treatment with classic therapeutic protocols.\u003c/p\u003e \u003cp\u003eEven greater expectations in the context of improving therapeutic effects are associated with the simultaneous use of PARPis and other drugs. This approach allows for sensitizing cancer cells to the action of a selected drug, to which the cells are resistant in monotherapy. It is also a way to overcome resistance to PARPi, which may appear during treatment. Our study showed that adding olaparib to doxorubicin monotherapy brings better therapeutic results due to the synergistic effect of these two compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The synergistic effect of doxorubicin and olaparib shall allow for the development of a less toxic therapy with increased anti-cancer effectiveness. Such a synergistic effect of the mentioned combination has already been demonstrated for other types of cancers such as ovarian cancer, breast cancer, and sarcomas (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). In osteosarcoma, the synergistic effect of the combined use of these two drugs was associated with increased apoptosis of cells as indicated by flow cytometry analysis and western blotting, which showed increased expression of cleaved PARP1, cleaved caspase 3, and BAX, and decreased expression of BCL2 (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). In breast cancer it has been shown that one of the mechanisms through which PARP inhibition can chemosensitize cancer cells \u003cem\u003ein vivo\u003c/em\u003e, is targeting Snail expression thus promoting apoptosis (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Many studies are also focused on examining the effects of combined use of olaparib with liposomal doxorubicin (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e)\u003csup\u003e,\u003c/sup\u003e(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e), also in the context of limited cardiotoxic effect of doxorubicin with preserved antitumor activity (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e)\u003csup\u003e,\u003c/sup\u003e(\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Combinations with doxorubicin do not exhaust all therapeutic possibilities, because various genotoxic drugs can be combined with PARPis. For example, combinations with platinum derivatives, alkylating compounds, or antimetabolites (all targeting DNA) are successfully used in humans (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). All these demonstrate the therapeutic potential of employing PARPis and their combinations with synergistic compounds to generate new, molecularly focused therapeutics for malignancies where DNA damage/repair systems play a critical role (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eUnderstanding DNA damage disorders in canines will contribute to the development of a reliable method for identifying suitable candidates for PARPi therapy. In the meantime, characterization of the molecular effects of PARPi combinations with other drugs in different cancer types will be the first step to develop new, molecular-based targeted therapeutic strategies in veterinary oncology. The proposed research outputs might also be translated to human medicine. Here, we showed that olaparib may be an effective therapeutic option for canine lymphomas and leukemias. In vitro experiments showed that olaparib inhibits the proliferation of cancerous dog lymphocytes by causing cell DNA damage. The anticancer effect of olaparib was visible when the drug was used alone, which indicates DNA repair disorders, but it was particularly pronounced in combination with doxorubicin. Thus, olaparib may be an option for simple oral therapy in canine lymphomas and leukemias and may also be a valuable addition to standard therapeutic protocols.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and cell culture\u003c/h2\u003e \u003cp\u003eThe study involved a panel of 4 different canine lymphoma and leukemia cell lines: CLBL-1 (B-cell lymphoma), GL-1 (B-cell leukemia), CLB70 (B-cell chronic lymphocytic leukemia), and CNK-89 (natural killer-cell lymphoma). The CLBL-1 cell line was obtained from Barbara C. Ruetgen (Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria) (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e); the GL-1 cells were obtained from Yasuhito Fujino and Hajime Tsujimoto (University of Tokyo, Department of Veterinary Internal Medicine) (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e), while the CLB70 (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e) and CNK-89 (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e) cell lines were established in our laboratory. The CLBL-1 and GL-1 cell lines were maintained in the RPMI (Roswell Park Memorial Institute) 1640 medium (Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland), and the CLB70 and CNK-89 cells were cultured in Gibco\u0026trade; Advanced RPMI 1640 medium (Gibco, Grand Island, New York, USA). All media were supplemented with 2 mM L-glutamine (Sigma Aldrich, Steinheim, Germany), 100 U/mL penicillin, 100 \u0026micro;g/mL streptomycin (Sigma Aldrich), and 10\u0026ndash;20% heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, New York, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and Reagents\u003c/h2\u003e \u003cp\u003eOlaparib (AZD2281) was obtained from Selleckchem (Cologne, Germany) and dissolved in dimethyl sulfoxide (DMSO) (Sigma Aldrich, Steinheim, Germany) to final concentration of 50 mM immediately prior to the experiments. Doxorubicin, propidium iodide (PI), 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT), RIPA buffer, and SigmaFAST Protease Inhibitor Cocktail were purchased from Sigma-Aldrich (Steinheim, Germany). Annexin V-FITC was purchased from Immunostep (Salamanca, Spain).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCell metabolic activity assay\u003c/h2\u003e \u003cp\u003eThe metabolic activity of canine cells treated with olaparib was determined using the MTT test. In brief, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well were seeded in a 96-well plate (Thermo Fisher Scientific, Roskilde, Denmark), and olaparib was added at increasing concentrations (0.1, 0.4, 1.2, 3.7, 11.1, and 33.3 \u0026micro;M). The range of concentrations tested was selected based on literature data on the sensitivity of various cell lines to the cytotoxic effects of olaparib and corresponds to the concentrations achievable \u003cem\u003ein vivo\u003c/em\u003e (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). Cells only incubated with DMSO were used as controls. After incubation for 24, 48, 72, and 96 hours, 10 \u0026micro;L of MTT solution (5 mg/mL) was added to each well for 4 hours. After dissolving the content, the optical density of the wells was measured with a spectrophotometric microplate reader (Spark, Tecan, Singapore) at a reference wavelength of 570 nm. The results were then expressed as the mean of 3 independent experiments (different plates, different days) each one performed in triplicate.\u003c/p\u003e \u003cp\u003eThe MTT assay was also used to assess the synergistic effect of olaparib and doxorubicin by comparing cell metabolic activity after treatment with both compounds alone or in combination. To this end, cells seeded at a density of 1.5 x 10\u003csup\u003e5\u003c/sup\u003e/mL (GL-1 cell line) and 3 x 10\u003csup\u003e5\u003c/sup\u003e/mL (CLBL-1) were treated with either olaparib (2.5, 5, and 10 \u0026micro;M for GL-1 and 1.25, 2.5, and 5 \u0026micro;M for CLBL-1 cells) for 96 hours or with doxorubicin (25, 50, and 100 nM) for 48 hours as single drugs and, for combinatorial treatment, cells were pre-incubated with olaparib for 48 hours followed by the addition of doxorubicin for the next 48 hours. Olaparib and doxorubicin concentrations were selected based on MTT assay results for single compounds. The data obtained from 3 independent experiments were used to calculate Combination Index (CI) values according to the method established by Chou and Talalay (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e) using CompuSyn Software (Informer Technologies, Inc). The Chou-Talalay method is based on a median-effect equation, derived from the mass-action law principle, which allows for determining synergy according to the CI theorem, where CI\u0026thinsp;=\u0026thinsp;1 means addition, CI\u0026thinsp;\u0026lt;\u0026thinsp;1 means synergism, and CI\u0026thinsp;\u0026gt;\u0026thinsp;1 indicates antagonism(\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eKi-67 flow cytometry proliferation assay\u003c/h2\u003e \u003cp\u003eFor this cell proliferation assay, cells were plated at a density of 1 \u0026times; 105/mL in 96-well plates (TPP, Trasadingen, Switzerland), and incubated for 48 hours with two concentrations of olaparib (25 and 50 \u0026micro;M) selected according to MTT test results. After that time, the cells were collected, washed with PBS, labeled using the Ki-67 Proliferation Kit BD Pharmingen (BD Biosciences) according to the manufacturer\u0026rsquo;s instructions, and then analyzed in a flow cytometer (Cytoflex, Beckman Coulter). Percentages of proliferating (Ki-67 positive) cells were used to calculate the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for each cell line. The presented results were obtained from 3 independent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eA total of 5 \u0026times; 106 cells was rinsed with cold PBS and lysed with RIPA buffer (50m MTris-HCl pH 7.5, 100 mM NaCl, 1% NP-40, protease inhibitors set) and incubated for 20 min on ice. Then, after centrifuging at 10,000 rpm at 4 ℃ for 12 min SDS sample buffer was added to clear the supernatants and the samples were boiled at 95 ℃ for 5 minutes and subjected to SDS-polyacrylamide gel electrophoresis in a 12% gel (BioRad Mini-PROTEAN Tetra Vertical Electrophoresis Cell system, Hercules, USA). After the electrophoresis, the samples were transferred to a nitrocellulose membrane using a BioRad Mini Trans-Blot\u0026reg; Cell for wet transfer and Western Blot enhancer treatment was performed according to manufacturer\u0026rsquo;s protocol with Pierce\u0026trade; Western Blot Signal Enhancer (Thermo Scientific). Then, the membranes were blocked with 3% BSA in TBST at room temperature for 1 hour. After blocking, the membranes were incubated overnight at 4 ℃ with the following murine monoclonal primary antibodies: anti-γH2A.X clone 9F3 (ab26350; Abcam, Cambridge, United Kingdom), dilution 1:1000 and anti-β-actin clone C4 (sc47 778; Santa Cruz, California, USA), dilution 1:2000. Goat AntiMouse Immunoglobulins/HRP (#P0447 at 1:20000) was used as secondary antibody. The membranes were incubated with the secondary antibody for 90 minutes at room temperature. The reaction was developed using Blotting substrate - Pierce\u0026trade; ECL Western Blotting Substrate (Thermo Scientific) as a substrate. Membrane visualization was performed using ChemiDoc Touch Instruments (exposure: first image, 5 second; last image, 120 seconds; images, 5; BioRad). For protein expression quantification, Western blot normalization with a single housekeeping protein (β-actin) was performed using Image LabTM software (version 6.1.0; BioRad).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eNext-generation RNA sequencing (NGS)\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003eRNA extractions\u003c/h2\u003e \u003cp\u003eFirst, 1 mL of TRIzol Reagent was used to lyse and homogenize the samples and then the material was mixed with 200 \u0026micro;L of chloroform. Samples were centrifuged at 4 ℃ for 20 minutes at 13,000 rpm following a 2-minute incubation period. After that, the top aqueous phase was gathered, mixed with 70% ice cold ethanol, and moved into a RNeasy spin column. The next steps of the protocol for the RNeasy mini Kit (Qiagen, Hilden, Germany) were carried out according to the manufacturer's instructions. The Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) were used to measure total RNA. The RNA Integrity Number (RIN) value for each sample was greater than 7.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eRNA-Seq Library Preparation and Sequencing\u003c/h2\u003e \u003cp\u003eLibrary preparation and sequencing were performed by Novogene Biotechnology (Cambridge, UK). A total of 8 tagged RNA-seq libraries were prepared and sequenced using a 150 bp strand-specific paired-end strategy on an Illumina Novaseq 6000.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of exonic variants from RNA-seq data\u003c/h2\u003e \u003cp\u003eRNA-seq data were used to evaluate the exonic mutational profile of CLBL-1 and GL-1 cell lines, the most susceptible and resistant to olaparib, respectively. Nine target genes implicated in DNA damage response (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) were selected. For every cell line, four biological replicates were considered. Raw reads underwent a preliminary quality control with FastQC software (v.0.11.9) before proceeding with the other analyses. Reads were aligned to the reference cDNA sequences retrieved from Ensembl (release 112; GCA_000002285.4; canFam6) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Bowtie2 (v.2.5.4) was used to build the indexes and align the reads.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTarget genes selected for the exonic mutational profiling of GL-1 and CLBL-1 cell lines: gene symbol, gene description and Ensembl transcript ID.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene symbol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene description\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTranscript ID\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBRCA1 DNA Repair Associated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eENSCAFT00000065506.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBRCA2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBRCA2 DNA Repair Associated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eENSCAFT00000010309.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTP53\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTumor Protein P53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eENSCAFT00000026465.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRAD51\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRAD51 Recombinase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eENSCAFT00000073671.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eATM\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATM Serine/Threonine kinase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eENSCAFT00000048574.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eATR\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATR Serine/Threonine Kinase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eENSCAFT00000012571.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCHEK1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCheckpoint Kinase 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eENSCAFT00000016788.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCHEK2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCheckpoint Kinase 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eENSCAFT00000018932.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSTK11\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSerine/Threonine Kinase 11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eENSCAFT00000031055.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFollowing alignment, samtools (v.1.19) were used to sort the SAM outputs and convert them to BAM files. An index was also prepared with samtools to visualize them on Integrative Genomic Viewer (v.2.18.2). Thanks to IGV visualization, possible single nucleotide variants (SNVs) and insertions-deletions mutations (INDELs) were recognized, and each variant was checked for possible strand biases. The corresponding coding sequence (CDS), and the encoded protein position were retrieved from the Ensembl Genome Browser.\u003c/p\u003e \u003cp\u003eTo check for the possible biological consequences of SNVs, the Ensembl Variant Effect Predictor (VEP) and Fido-SNP platforms were used. For INDELs that couldn\u0026rsquo;t be evaluated with the previous softwares, the nucleotide sequence was converted into the corresponding amino acid sequence checking the possible effect of mutations on the reading frame using EMBOSS Transeq.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are shown as means with SD. Statistical differences were analyzed using one-way analysis of variance (ANOVA) followed by the Dunnett's multiple comparison test (Ki-67 expression marker analysis) and unpaired T test (DNA damage induction assessment). Statistical analysis was performed using either STATISTICA software version 13.3 (TIBCO Software Inc., Palo Alto, CA, USA) or GraphPad Prism v.9 (GraphPad Software, San Diego, CA, USA). The results were considered significant at P\u0026thinsp;\u0026lt;\u0026thinsp;.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eANOVA, Analysis of Variance\u003c/p\u003e\n\u003cp\u003eATM, Ataxia Telangiectasia Mutated\u003c/p\u003e\n\u003cp\u003eBRCA1, Breast Cancer Associated 1\u003c/p\u003e\n\u003cp\u003eBRCA2, Breast Cancer Associated 2\u003c/p\u003e\n\u003cp\u003eCDK12, Cyclin-Dependent Kinase 12\u003c/p\u003e\n\u003cp\u003eCI, Combination Index\u003c/p\u003e\n\u003cp\u003eCLB70, Canine B-cell Chronic Lymphocytic Leukemia Cell Line\u003c/p\u003e\n\u003cp\u003eCLBL-1, Canine B-cell Lymphoma Cell Line\u003c/p\u003e\n\u003cp\u003eCNK-89, Canine Natural Killer-cell Lymphoma Cell Line\u003c/p\u003e\n\u003cp\u003eDMSO, Dimethyl Sulfoxide\u003c/p\u003e\n\u003cp\u003eDSBs, Double-Strand Breaks\u003c/p\u003e\n\u003cp\u003eFBS, Fetal Bovine Serum\u003c/p\u003e\n\u003cp\u003eFITC, Fluorescein Isothiocyanate\u003c/p\u003e\n\u003cp\u003eγH2AX, Phosphorylated Histone H2AX\u003c/p\u003e\n\u003cp\u003eGL-1, B-cell Leukemia Cell Line\u003c/p\u003e\n\u003cp\u003eHR, Homologous Recombination\u003c/p\u003e\n\u003cp\u003eIC50, Half Maximal Inhibitory Concentration\u003c/p\u003e\n\u003cp\u003eINDELs, Insertions or Deletions\u003c/p\u003e\n\u003cp\u003eITDs, Internal Tandem Duplications\u003c/p\u003e\n\u003cp\u003eMAPK12, Mitogen-Activated Protein Kinase 12\u003c/p\u003e\n\u003cp\u003eMCTs, Mast Cell Tumors\u003c/p\u003e\n\u003cp\u003eMTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide\u003c/p\u003e\n\u003cp\u003eNGS, Next-Generation Sequencing\u003c/p\u003e\n\u003cp\u003ePARP, Poly(ADP-ribose) Polymerase\u003c/p\u003e\n\u003cp\u003ePARPi, PARP Inhibitors\u003c/p\u003e\n\u003cp\u003ePI, Propidium Iodide\u003c/p\u003e\n\u003cp\u003ePI3K, Phosphoinositide 3-Kinases\u003c/p\u003e\n\u003cp\u003ePLK3, Polo-Like Kinase 3\u003c/p\u003e\n\u003cp\u003ePNKP, Polynucleotide Kinase-Phosphatase\u003c/p\u003e\n\u003cp\u003eSSBs, Single-Strand Breaks\u003c/p\u003e\n\u003cp\u003eSTK22C, Serine/Threonine-Protein Kinase 22C\u003c/p\u003e\n\u003cp\u003eSTK36, Serine/Threonine Kinase 36\u003c/p\u003e\n\u003cp\u003eUSP1, Ubiquitin Specific Peptidase 1\u003c/p\u003e\n\u003cp\u003eUSP7, Ubiquitin Specific Peptidase 7\u003c/p\u003e\n\u003cp\u003eUSP11, Ubiquitin Specific Peptidase 11\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary Table 1. Complete list of variants identified in DNA-damage response genes of CLBL-1 and GL-1 cell lines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003eRaw Illumina sequencing data have been deposited in GenBank (SRA) under the BioProject accession\u0026nbsp;PRJNA1242827. Additional supporting information can be found online in the Supporting Information section at the end of this article (Supplementary Table 1 with complete list of variants identified in DNA-damage response genes of CLBL-1 and GL-1 cell lines).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eFunding provided by the: Polish National Agency for Academic Exchange, Grant/Award Number: PPI/APM/2019/1/00044/U/00001 and by the Wrocław University of Environmental and Life Sciences (Poland) as part of research project no N070/0012/23. The article is part of a PhD dissertation titled “A Novel Therapeutic Approach Using PolyA DP Ribose Polymerase (PARP) Inhibitors in Canine Cancer Cells”, prepared during Doctoral School at the Wrocław University of Environmental and Life Sciences. The APC is financed by Wrocław University of Environmental and Life Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAP and MD designed the study and contributed to the implementation of the research; JCP, ED, GM, JB, MH and AP organized and performed the experiments; JCP, ED, GM, MG, MP, MD and AP analysed the data. JP, ED, GM, BOM, MG, MP, CZ, MD and AP wrote and revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to B.C. Ruetgen (Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine, Vienna) for providing the CLBL-1 and Y. Fujino and H. Tsujimoto (Department of Veterinary Internal Medicine, University of Tokyo) for providing the GL-1 cell line.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDias MP, Moser SC, Ganesan S, Jonkers J. Understanding and overcoming resistance to PARP inhibitors in cancer therapy. Nat Rev Clin Oncol. 2021;18(12):773\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAshworth A, Lord CJ. Synthetic lethal therapies for cancer: what\u0026rsquo;s next after PARP inhibitors? Nat Rev Clin Oncol. 2018;15(9):564\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu FW, Tewari KS. New targeted agents in gynecologic cancers: synthetic lethality, homologous recombination deficiency, and PARP inhibitors. Curr Treat Options Oncol. 2016;17(3):15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRose M, Burgess JT, O\u0026rsquo;Byrne K, Richard DJ, Bolderson E. 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Mol Med Rep. 2020;23(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHern\u0026aacute;ndez-Su\u0026aacute;rez B, Gillespie DA, Dejnaka E, Kupczyk P, Obmińska-Mrukowicz B, Pawlak A. Studying the DNA damage response pathway in hematopoietic canine cancer cell lines, a necessary step for finding targets to generate new therapies to treat cancer in dogs. Front Vet Sci. 2023;10:1227683.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTahara M, Inoue T, Sato F, Miyakura Y, Horie H, Yasuda Y, et al. The use of olaparib (AZD2281) potentiates SN-38 cytotoxicity in colon cancer cells by indirect inhibition of Rad51-mediated repair of DNA double-strand breaks. Mol Cancer Ther. 2014;13(5):1170\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOza AM, Cibula D, Benzaqu\u0026eacute;n AO, Poole C, Mathijssen RH, Sonke GS, et al. Olaparib combined with chemotherapy for recurrent platinum-sensitive ovarian cancer: a randomized phase 2 trial. 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Leuk Lymphoma. 2008;49(11):2059\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"canine lymphoma, targeted therapy, DNA repair, doxorubicin synergy, mutational analysis","lastPublishedDoi":"10.21203/rs.3.rs-6148132/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6148132/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e The introduction of genetic tests based on next-generation sequencing techniques into veterinary cancer diagnostics provides information on molecularly targeted therapies useful for dogs. However, there is still a lack of in vitro studies describing the effect and mechanism of action of such anticancer drugs in companion animals. Our study aimed to demonstrate in vitro activity of a commonly used PARP inhibitor, olaparib, in canine lymphoma and leukemia cells as well as to indicate its potential uses in anti-cancer therapy based on the mutational status of DNA damage related genes. Canine lymphoma and leukemia cell lines were incubated with olaparib alone and in combination with doxorubicin, and the impact of a single drug and combinations on cell viability, proliferation, induction of apoptosis, and DNA damage were assessed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e The study showed that olaparib acts as a single agent and also as a chemosensitizer, due to its ability to potentiate cytotoxic effects of doxorubicin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e Olaparib may be an interesting oral therapy alternative to classic chemotherapy in dogs with known DNA repair disorders and may also be effective in adjuvant therapy due to its synergistic effect with doxorubicin.\u003c/p\u003e","manuscriptTitle":"PARP inhibitor olaparib induces DNA damage and acts as a drug sensitizer in an in vitro model of canine hematopoietic cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 21:31:46","doi":"10.21203/rs.3.rs-6148132/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-16T15:02:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-15T08:11:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-13T14:36:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"37176418268806150669599974541636074628","date":"2025-04-19T10:35:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148202098772446842456061131871585138705","date":"2025-04-17T03:57:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-15T21:16:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-15T21:10:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-15T14:04:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-07T17:22:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2025-04-07T17:21:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"09bbff8e-0ffa-41d6-a15f-8b683f073ff7","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T15:59:34+00:00","versionOfRecord":{"articleIdentity":"rs-6148132","link":"https://doi.org/10.1186/s12917-025-04880-z","journal":{"identity":"bmc-veterinary-research","isVorOnly":false,"title":"BMC Veterinary Research"},"publishedOn":"2025-07-05 15:56:59","publishedOnDateReadable":"July 5th, 2025"},"versionCreatedAt":"2025-04-17 21:31:46","video":"","vorDoi":"10.1186/s12917-025-04880-z","vorDoiUrl":"https://doi.org/10.1186/s12917-025-04880-z","workflowStages":[]},"version":"v1","identity":"rs-6148132","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6148132","identity":"rs-6148132","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}