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

BMC Veterinary Research volume 21, Article number: 439 (2025) Cite this article

Abstract

Background

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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 anti-cancer 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, inhibiting the metabolic activity of canine lymphoma (CLBL-1, CNK-89) and leukemia (CLB70, GL-1) cells, affecting cell proliferation rates and causing DNA damage. In the tested cells, olaparib also worked as a chemosensitizer, due to its ability to potentiate cytotoxic effects of doxorubicin. Finally, RNA-seq data 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 observed in vitro sensitivity differences to olaparib.

Conclusions

Olaparib may be an interesting oral therapy alternative to classic chemotherapy or adjuvant option in dogs with hematopoietic cancer with known DNA repair disorders.

Peer Review reports

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 PARPis 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 [10], 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 PARPis 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 (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. Obtained data 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.

Fig. 1
figure 1

Concentration-dependent curves displaying the effects of olaparib. Metabolic activity (measured by MTT assay) of CLBL-1, CLB70, CNK-89, and GL-1 cell lines after 24, 48, 72, and 96 h of incubation

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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 h for all the sensitive cell lines. This concentration is much lower than the maximum one achievable in human patients with solid tumors treated with 10–400 mg olaparib twice daily [11]. 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 h of incubation on the canine lymphoma/leukemia cell lines, expressed as IC50 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 <.05)
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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 h. The percentage of Ki-67 positive cells (as compared to the untreated control) decreased significantly (P <.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 h of incubation with 50 µM of olaparib, indicating a decrease in the cell proliferation potential.

Fig. 2
figure 2

Histograms presenting percentages of proliferating (Ki-67 positive cells). 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

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DNA damage induction

Having confirmed that olaparib, as a single agent, exerts cytotoxic and antiproliferative effects on canine lymphoma/leukemia cells, we decided to investigate if it also causes DNA damage in sensitive cells. We sought to determine if and after what incubation time olaparib causes DNA damage to accumulate 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 h of incubation, also in the cells with limited sensitivity to olaparib (see MTT results, Fig. 1). This result clearly shows that the accumulation of DNA damage in the cells disturbs cell proliferation.

Fig. 3
figure 3

Western blot analysis for phosphorylated histone H2A.X. The CLBL-1, CLB70, CNK-89, and GL-1 (A-D) cell lines after 24 h 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 <.05; **P <.01; ***P <.001; ****P <.0001; ns– not significant (P >.05)

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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 (Combination Index) values (Fig. 4B) 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. 4A, 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. 4B). The study indicated that olaparib can also be used as a drug that sensitizes cells to the action of doxorubicin.

Fig. 4
figure 4

Analysis of synergistic effect of olaparib with doxorubicin. Cell 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 the 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 <.05; **P <.01; ****P <.0001; ns– not significant (P >.05)

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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 (single-nucleotide variants) and INDELs (small insertions or deletions) 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 coding sequence (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 CLBL-1 and GL-1 cell lines
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Among the genes considered for the present analysis, TP53 appeared to be the most biologically affected; it showed two heterozygous adverse SNVs (c.374 C > T; c.764G > A) in CLBL-1 cells and a homozygous one (c.709 C > T) in GL-1 cells.

BRCA1 was mutated in the GL-1 cell line only; among the 5 missense SNVs, only c.1329 A > 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.2401 A > C) and a deletion (c.6916_6918del) were specifically identified in CLBL-1 cells. 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 (Supplementary Table 1); conversely, c.3431 A > T in ATM was homozygous in GL-1 and heterozygous 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. All these data are presented in Supplementary Table 1.

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 [12]. 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 [13]. 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 [14, 15]. 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. Examples of such tools are: are SearchLight DNA cancer genomic diagnostic assay [16] and The FidoCure® Precision Medicine Platform [17].

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 [16]. This panel identifies mutated genes that inform sensitivity to PARPis. 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 [10]. Chon et al. 2023 showed 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 [18]. 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 [16]. 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 [19]. 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 [20]. All BRCA1 missense and synonymous SNVs we observed in the GL-1 cells were also found in canine normal and tumor mammary samples [21]. 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 [22]. Interestingly, both GL-1 and CLBL-1 cell lines disclosed specific SNVs in this region. The c.4304 A > G (p.Lys1435Arg) was already reported in canine mammary tumors and it is precisely located in BRC3 [23]. The same SNV was previously reported together with c.2401 A > C (p.Lys801Gln), being the most frequent SNV registered in both canine mammary tumors and normal mammary tissue [24, 25]. Moreover, both were predicted as deleterious [26]. 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 [27, 28]. This insertion was previously detected in dogs, but its pathogenicity is still controversial [23, 25, 29]. However, it is known that it enhances the nuclear localization of the BRCA2 protein, possibly boosting the DNA damage repair machinery [30]. 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 [31]. 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), which is a pathway that plays a crucial role in cell-cycle arrest [32,33,34]. In GL-1 we found one homozygous and in CLBL-1 we found two heterozygous deleterious SNVs in the TP53 CDS. All of which were located in the DNA binding domain of P53 [35]. TP53 mutations are common in cancerous cells and could impair the G1 checkpoint, avoiding cell cycle exit in case of DNA damage [36]. Therefore, BRCA1/2 mutations often coexist with TP53 ones [37,38,39] 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 a TP53 mutation as well as the allelic representation [39, 40]. 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 [36]. Compared to GL-1 cells, CLBL-1 cells are more likely to rely on the ATR axis according to previous studies [41], 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) [42] 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 already been described that the impact of a single variant might be compensated by other mechanisms [43,44,45]. 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 the metabolic activity and proliferation of canine lymphoma and leukemia cells. The cytotoxic effect of olaparib was particularly visible after at least 72 h of incubation, with the IC50 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 [46, 47]. 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 h 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 [48].

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 h after incubation, even in the cells with low sensitivity to olaparib in the MTT test. This result indicates that DNA damage may be one of the reasons 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 [49], pancreatic cancer [50], prostatic cancer [51], lymphocytic and myeloid leukemia [52, 53] and lymphoma [54]. 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 [53]. 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. Our study showed that adding olaparib to doxorubicin monotherapy brings better therapeutic results due to the synergistic effect of these two compounds (Fig. 4), giving an opportunity for the development of a less toxic therapy with increased anti-cancer effectiveness. The synergism demonstrated by these two substances used simultaneously will allow for the reduction of the therapeutic dose of doxorubicin compared to the dose used in monotherapy [55]. This is of particular importance because doxorubicin has a cardiotoxic effect, which limits the dose that can be used in the treatment of dogs (it is not recommended to exceed 240 mg/m2 cumulative dose) [56]. The possibility of using a lower dose of doxorubicin with preserved anti-cancer activity may be an opportunity for safe use of the drug also in dogs predisposed to the development of dilated cardiomyopathy (DCM), which show a five-fold higher prevalence of anthracycline-induced circulatory failure than other dogs [57]. Breeds predisposed to the development of DCM, and thus anthracycline-induced cardiomyopathy (AICM), include Dobermans, Great Danes, Rottweilers, and Boxers [57].

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 [49]. 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 [58]. Many studies are also focused on examining the effects of combined use of olaparib with liposomal doxorubicin [59, 60], also in the context of limited cardiotoxic effect of doxorubicin with preserved antitumor activity [61, 62]. 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 [62]. 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).

Fig. 5
figure 5

Schematic representations of the potential of PARPi in cancer therapy. Characterization 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

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Conclusion

Understanding DNA damage disorders in canines will contribute to the development of a reliable method for identifying suitable candidates for PARPis therapy. In the meantime, characterization of the molecular effects of PARPis 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 anti-cancer 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) [63]; the GL-1 cells were obtained from Yasuhito Fujino and Hajime Tsujimoto (University of Tokyo, Department of Veterinary Internal Medicine) [64], while the CLB70 [65] and CNK-89 [66] 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 a 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 × 105 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 [11, 67]. Cells only incubated with DMSO were used as controls. After incubation for 24, 48, 72, and 96 h, 10 µL of MTT solution (5 mg/mL) was added to each well for 4 h. 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 × 105/mL (GL-1 cell line) and 3 × 105/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 h or with doxorubicin (25, 50, and 100 nM) for 48 h as single drugs and, for combinatorial treatment, cells were pre-incubated with olaparib for 48 h followed by the addition of doxorubicin for the next 48 h. 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 [68] 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 means synergism, and CI > 1 indicates antagonism [68, 69].

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 h 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 were rinsed with cold PBS and lysed with RIPA buffer (50 mM Tris-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 min 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 h. 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; Dako, Agilent; Santa Clara, USA, at 1:20000) was used as secondary antibody. The membranes were incubated with the secondary antibody for 90 min 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 s; last image, 120 s; 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 min 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. This evaluation was performed on control, untreated CLBL-1 and GL-1 cells. 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
Full size table

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 (IGV)(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 CDS, and the encoded protein position were retrieved from the Ensembl Genome Browser.

To check for the possible biological consequences of SNVs, VEP and Fido-SNP platforms were used. The prediction of the biological effect of VEP is based on SIFT score [70], while Fido-SNP is a binary classifier based on the Gradient Boosting algorithm [71]. Both tools allow for the prediction of the pathogenicity of SNVs in both coding and non-coding regions of the dog genome. SIFT-VEP scores only missense SNVs, while Fido-SNP scores both synonymous and non-synonymous SNVs. For INDELs that couldn’t be evaluated with the previous software, 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, 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.

Data availability

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).

Abbreviations

AICM:

Anthracycline-induced Cardiomyopathy

ANOVA:

Analysis of Variance

ATM:

Ataxia Telangiectasia Mutated

ATR:

Ataxia Telangiectasia and Rad3-related

BRCA1:

Breast Cancer Associated 1

BRCA2:

Breast Cancer Associated 2

AKD:

Cyclin-dependent Kinase

CDK12:

Cyclin-dependent Kinase 12

CDS:

Coding Sequence

CI:

Combination Index

CHK1:

Checkpoint kinase 1

CHK2:

Checkpoint kinase 2

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

DCM:

dilated cardiomyopathy

DMSO:

Dimethyl Sulfoxide

DSBs:

Double-Strand Breaks

FANCL:

Fanconi Anemia Complementation Group L

FBS:

Fetal Bovine Serum

FITC:

Fluorescein Isothiocyanate

γH2AX:

Phosphorylated Histone H2AX

GL-1:

B-cell Leukemia Cell Line

HE:

Heterozygous

HO:

Homozygous

HR:

Homologous Recombination

IC50 :

Half Maximal Inhibitory Concentration

IGV:

Integrative Genomic Viewer

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

RAD51:

RAD51 Recombinase

SIFT:

Sorting Intolerant From Tolerant

SSBs:

Single-Strand Breaks

SNVs:

Single-nucleotide Variants

STK22C:

Serine/Threonine-Protein Kinase 22 C

STK36:

Serine/Threonine Kinase 36

TP53:

Tumor Protein P53

References

  1. Dias MP, Moser SC, Ganesan S, Jonkers J. Understanding and overcoming resistance to PARP inhibitors in cancer therapy. Nat Reviews Clin Oncol. 2021;18(12):773–91.

    Article  Google Scholar 

  2. Ashworth A, Lord CJ. Synthetic lethal therapies for cancer: what’s next after PARP inhibitors? Nat Reviews Clin Oncol. 2018;15(9):564–76.

    Article  CAS  Google Scholar 

  3. Liu FW, Tewari KS. New targeted agents in gynecologic cancers: synthetic lethality, homologous recombination deficiency, and PARP inhibitors. Curr Treat Options Oncol. 2016;17(3):015–0378.

    Article  Google Scholar 

  4. Rose M, Burgess JT, O’Byrne K, Richard DJ, Bolderson E. PARP inhibitors: clinical relevance, mechanisms of action and tumor resistance. Front Cell Dev Biology. 2020;8.

  5. Hodgson DR, Dougherty BA, Lai Z, Fielding A, Grinsted L, Spencer S, et al. Candidate biomarkers of PARP inhibitor sensitivity in ovarian cancer beyond the BRCA genes. Br J Cancer. 2018;119(11):1401–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Gadducci A, Guerrieri ME. PARP inhibitors alone and in combination with other biological agents in homologous recombination deficient epithelial ovarian cancer: from the basic research to the clinic. Crit Rev Oncol Hematol. 2017;114:153–65.

    Article  PubMed  Google Scholar 

  7. Kim D-S, Camacho CV, Kraus WL. Alternate therapeutic pathways for PARP inhibitors and potential mechanisms of resistance. Exp Mol Med. 2021;53(1):42–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Patatsos K, Shekhar TM, Hawkins CJ. Pre-clinical evaluation of proteasome inhibitors for canine and human osteosarcoma. Vet Comp Oncol. 2018;16(4):544–53.

    Article  CAS  PubMed  Google Scholar 

  9. Saba C, Paoloni M, Mazcko C, Kisseberth W, Burton JH, Smith A et al. A comparative oncology study of Iniparib defines its Pharmacokinetic profile and biological activity in a Naturally-Occurring canine Cancer model. PLoS ONE. 2016;11(2).

  10. Hernández-Suárez B, Gillespie DA, Pawlak A. DNA damage response proteins in canine cancer as potential research targets in comparative oncology. Vet Comp Oncol. 2022;20(2):347–61.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Bundred N, Gardovskis J, Jaskiewicz J, Eglitis J, Paramonov V, McCormack P, et al. Evaluation of the pharmacodynamics and pharmacokinetics of the PARP inhibitor olaparib: a phase I multicentre trial in patients scheduled for elective breast cancer surgery. Investig New Drugs. 2013;31:949–58.

    Article  CAS  Google Scholar 

  12. Tsimberidou AM, Fountzilas E, Nikanjam M, Kurzrock R. Review of precision cancer medicine: evolution of the treatment paradigm. Cancer Treat Rev. 2020;86:102019.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Bhinder B, Gilvary C, Madhukar NS, Elemento O. Artificial intelligence in cancer research and precision medicine. Cancer Discov. 2021;11(4):900–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Thamm DH, Avery AC, Berlato D, Bulman-Fleming J, Clifford CA, Hershey AE, et al. Prognostic and predictive significance of KIT protein expression and c-kit gene mutation in canine cutaneous mast cell tumours: A consensus of the Oncology-Pathology working group. Vet Comp Oncol. 2019;17(4):451–5.

    Article  PubMed  Google Scholar 

  15. Mochizuki H, Shapiro SG, Breen M. Detection of BRAF mutation in urine DNA as a molecular diagnostic for canine urothelial and prostatic carcinoma. PLoS ONE. 2015;10(12):e0144170.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Chon E, Sakthikumar S, Tang M, Hamilton MJ, Vaughan A, Smith A, et al. Novel genomic prognostic biomarkers for dogs with cancer. J Vet Intern Med. 2023;37(6):2410–21.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Wu K, Rodrigues L, Post G, Harvey G, White M, Miller A, et al. Analyses of canine cancer mutations and treatment outcomes using real-world clinico-genomics data of 2119 dogs. Npj Precision Oncol. 2023;7(1):8.

    Article  CAS  Google Scholar 

  18. Chon E, Wang G, Whitley D, Sakthikumar S, Warrier M, Wong S et al. Genomic tumor analysis provides clinical guidance for the management of diagnostically challenging cancers in dogs. J Am Vet Med Assoc. 2023:1–10.

  19. Kimchi-Sarfaty C, Oh JM, Kim I-W, Sauna Z, Calcagno A, Ambudkar S, et al. A silent polymorphism in the MDR1 gene changes substrate specificity. Science. 2007;315:525–8.

    Article  CAS  PubMed  Google Scholar 

  20. Sun Y, McCorvie T, Yates L, Zhang X. Structural basis of homologous recombination. Cell Mol Life Sci. 2019;77:3–18.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Qiu H, Lin D. Roles of DNA mutation in the coding region and DNA methylation in the 5′ flanking region of BRCA1 in canine mammary tumors. J Veterinary Med Sci. 2016;78:943–9.

    Article  CAS  Google Scholar 

  22. Lo T, Pellegrini L, Venkitaraman A, Blundell T. Sequence fingerprints in BRCA2 and RAD51: implications for DNA repair and cancer. DNA Repair. 2003;2 9:1015–28.

    Article  Google Scholar 

  23. Ochiai K, Ishiguro-Oonuma T, Yoshikawa Y, Udagawa C, Kato Y, Watanabe M, et al. Polymorphisms of canine BRCA2 BRC repeats affecting interaction with RAD51. Biomed Res. 2015;36(2):155–8.

    Article  CAS  PubMed  Google Scholar 

  24. Fedets O, Dmytruk K, Adaszek L, Kurlyak I, Dmytruk O, Lisiecka U et al. Polymorphism of glutathione S-transferase P1 of dogs with mammary tumors. Veterinary and comparative oncology. 2023.

  25. Ozmen O, Kul S, Risvanli A, Ozalp G, Sabuncu A, Kul O. Somatic SNPs of the BRCA2 gene at the fragments encoding RAD51 binding sites of canine mammary tumors. Vet Comp Oncol. 2017;15:1479.

    Article  CAS  PubMed  Google Scholar 

  26. Dong C, Wei P, Jian X, Gibbs R, Boerwinkle E, Wang K, et al. Comparison and integration of deleteriousness prediction methods for nonsynonymous SNVs in whole exome sequencing studies. Hum Mol Genet. 2015;24 8:2125–37.

    Article  Google Scholar 

  27. Lim G, Chang Y, Huh W. Phosphoregulation of Rad51/Rad52 by CDK1 functions as a molecular switch for cell cycle–specific activation of homologous recombination. Sci Adv. 2020;6.

  28. Saito K, Kagawa W, Suzuki T, Suzuki H, Yokoyama S, Saitoh H, et al. The putative nuclear localization signal of the human RAD52 protein is a potential sumoylation site. J BioChem. 2010;147 6:833–42.

    Article  Google Scholar 

  29. Szabo C, Wagner L, Francisco L, Roach J, Argonza R, King M, et al. Human, canine and murine BRCA1 genes: sequence comparison among species. Hum Mol Genet. 1996;5 9:1289–98.

    Article  Google Scholar 

  30. Yano KI, Morotomi K, Saito H, Kato M, Matsuo F, Miki Y. Nuclear localization signals of the BRCA2 protein. Biochem Biophys Res Commun. 2000;270(1):171–5.

    Article  CAS  PubMed  Google Scholar 

  31. Lane DP. p53, guardian of the genome. Nature. 1992;358(6381).

  32. Zhao H, Piwnica-Worms H. ATR-Mediated checkpoint pathways regulate phosphorylation and activation of human Chk1. Mol Cell Biol. 2001;21:4129–39.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Walker M, Black E, Oehler V, Gillespie D, Scott M. Chk1 C-terminal regulatory phosphorylation mediates checkpoint activation via derepression of Chk1 catalytic activity. Oncogene. 2009;28:2314–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Leung-Pineda V, Ryan C, Piwnica-Worms H. Phosphorylation of Chk1 by ATR is antagonized by a Chk1-Regulated protein phosphatase 2A circuit. Mol Cell Biol. 2006;26:7529–38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. McKinney K, Prives C. Regulation of p53 DNA binding. 2007:27–51.

  36. Reinhardt H, Aslanian A, Lees J, Yaffe M. p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell. 2007;11(2):175–89.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Waks A, Cohen O, Kochupurakkal B, Kim D, Dunn C, Buendia B et al. Reversion and non-reversion mechanisms of resistance to PARP inhibitor or platinum chemotherapy in BRCA1/2-mutant metastatic breast cancer. Annals Oncology: Official J Eur Soc Med Oncol. 2020.

  38. Kondrashova O, Nguyen M, Shield-Artin K, Tinker A, Teng N, Harrell M, et al. Secondary somatic mutations restoring RAD51C and RAD51D associated with acquired resistance to the PARP inhibitor Rucaparib in High-Grade ovarian carcinoma. Cancer Discov. 2017;7 9:984–98.

    Article  Google Scholar 

  39. Cruz C, Castroviejo-Bermejo M, Gutiérrez-Enríquez S, Llop-Guevara A, Ibrahim Y, Gris-Oliver A, et al. RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer. Ann Oncol. 2018;29:1203–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Collot T, Niogret J, Carnet M, Chevrier S, Humblin É, Favier L, et al. PARP inhibitor resistance and TP53 mutations in patients treated with Olaparib for BRCA-mutated cancer: four case reports. Mol Med Rep. 2020;23:1.

    Article  Google Scholar 

  41. Hernández-Suárez 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.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Das S, Idate R, Cronise KE, Gustafson DL, Duval DL. Identifying candidate druggable targets in canine cancer cell lines using whole-exome sequencing. Mol Cancer Ther. 2019;18(8):1460–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Al Abo M, Dejsuphong D, Hirota K, Yonetani Y, Yamazoe M, Kurumizaka H, et al. Compensatory functions and interdependency of the DNA-binding domain of BRCA2 with the BRCA1–PALB2–BRCA2 complex. Cancer Res. 2014;74(3):797–807.

    Article  CAS  PubMed  Google Scholar 

  44. Yazinski SA, Comaills V, Buisson R, Genois M-M, Nguyen HD, Ho CK, et al. ATR Inhibition disrupts rewired homologous recombination and fork protection pathways in PARP inhibitor-resistant BRCA-deficient cancer cells. Genes Dev. 2017;31(3):318–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Hu J, Cao J, Topatana W, Juengpanich S, Li S, Zhang B, et al. Targeting mutant p53 for cancer therapy: direct and indirect strategies. J Hematol Oncol. 2021;14:1–19.

    Article  Google Scholar 

  46. Tahara 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;13:1170–80.

    Article  CAS  PubMed  Google Scholar 

  47. Oza A, Cibula D, Benzaquén A, Poole C, Mathijssen R, Sonke G, et al. Olaparib combined with chemotherapy for recurrent platinum-sensitive ovarian cancer: a randomised phase 2 trial. Lancet Oncol. 2015;16 1:87–97.

    Article  Google Scholar 

  48. Kortmann U, McAlpine JN, Xue H, Guan J, Ha G, Tully S, et al. Tumor growth Inhibition by Olaparib in BRCA2 germline-mutated patient-derived ovarian cancer tissue xenografts. Clin Cancer Res. 2011;17(4):783–91.

    Article  CAS  PubMed  Google Scholar 

  49. Park SH, Lee J, Kang MA, Jang KY, Kim JR. Mitoxantrone induces apoptosis in osteosarcoma cells through regulation of the Akt/FOXO3 pathway. Oncol Lett. 2018;15(6):9687–96.

    PubMed  PubMed Central  Google Scholar 

  50. Javle M, Shacham-Shmueli E, Xiao L, Varadhachary G, Halpern N, Fogelman D, et al. Olaparib monotherapy for previously treated pancreatic Cancer with DNA damage repair genetic alterations other than germline BRCA variants: findings from 2 phase 2 nonrandomized clinical trials. JAMA Oncol. 2021;7(5):693–9.

    Article  PubMed  PubMed Central  Google Scholar 

  51. De Felice F, Tombolini V, Marampon F, Musella A, Marchetti C. Defective DNA repair mechanisms in prostate cancer: impact of Olaparib. Drug Des Devel Ther. 2017;11:547–52.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Wu Y, Hu Y, Yu X, Zhang Y, Huang X, Chen S, et al. TAL1 mediates imatinib-induced CML cell apoptosis via the PTEN/PI3K/AKT pathway. Biochem Biophys Res Commun. 2019;519(2):234–9.

    Article  CAS  PubMed  Google Scholar 

  53. Faraoni I, Compagnone M, Lavorgna S, Angelini DF, Cencioni MT, Piras E, et al. BRCA1, PARP1 and γH2AX in acute myeloid leukemia: role as biomarkers of response to the PARP inhibitor Olaparib. Biochim Biophys Acta. 2015;1852(3):462–72.

    Article  CAS  PubMed  Google Scholar 

  54. Weston VJ, Oldreive CE, Skowronska A, Oscier DG, Pratt G, Dyer MJ, et al. The PARP inhibitor Olaparib induces significant killing of ATM-deficient lymphoid tumor cells in vitro and in vivo. Blood. 2010;116(22):4578–87.

    Article  CAS  PubMed  Google Scholar 

  55. Tallarida RJ. Quantitative methods for assessing drug synergism. Genes cancer. 2011;2(11):1003–8.

    Article  PubMed  PubMed Central  Google Scholar 

  56. Banco B, Grieco V, Servida F, Giudice C. Sudden death in a dog after doxorubicin chemotherapy. Vet Pathol. 2011;48(5):1035–7.

    Article  CAS  PubMed  Google Scholar 

  57. Hallman BE, Hauck ML, Williams LE, Hess PR, Suter SE. Incidence and risk factors associated with development of clinical cardiotoxicity in dogs receiving doxorubicin. J Vet Intern Med. 2019;33(2):783–91.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Mariano G, Ricciardi M, Trisciuoglio D, Zampieri M, Ciccarone F, Guastafierro T, et al. PARP inhibitor ABT-888 affects response of MDA-MB-231 cells to doxorubicin treatment, targeting snail expression. Oncotarget. 2015;6:15008–21.

    Article  PubMed  PubMed Central  Google Scholar 

  59. Conte G, Sessa C, Moos R, Viganò L, Digena T, Locatelli A, et al. Phase I study of Olaparib in combination with liposomal doxorubicin in patients with advanced solid tumours. Br J Cancer. 2014;111:651–9.

    Article  PubMed  PubMed Central  Google Scholar 

  60. Perez-Fidalgo JA, Cortés A, Guerra E, García Y, Iglesias M, Bohn Sarmiento U, et al. Olaparib in combination with pegylated liposomal doxorubicin for platinum-resistant ovarian cancer regardless of BRCA status: a GEICO phase II trial (ROLANDO study). ESMO Open. 2021;6(4):100212.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Zhang Q, Yang J-J, Zhang H-S. Carvedilol (CAR) combined with carnosic acid (CAA) attenuates doxorubicin-induced cardiotoxicity by suppressing excessive oxidative stress, inflammation, apoptosis and autophagy. Biomed pharmacotherapy = Biomedecine Pharmacotherapie. 2019;109:71–83.

    Article  CAS  Google Scholar 

  62. Basourakos SP, Li L, Aparicio AM, Corn PG, Kim J, Thompson TC. Combination Platinum-based and DNA damage Response-targeting Cancer therapy: evolution and future directions. Curr Med Chem. 2017;24(15):1586–606.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Rutgen BC, Hammer SE, Gerner W, Christian M, de Arespacochaga AG, Willmann M, et al. Establishment and characterization of a novel canine B-cell line derived from a spontaneously occurring diffuse large cell lymphoma. Leuk Res. 2010;34(7):932–8.

    Article  PubMed  Google Scholar 

  64. Nakaichi M, Taura Y, Kanki M, Mamba K, Momoi Y, Tsujimoto H, et al. Establishment and characterization of a new canine B-cell leukemia cell line. J Vet Med Sci. 1996;58(5):469–71.

    Article  CAS  PubMed  Google Scholar 

  65. Pawlak A, Ziolo E, Kutkowska J, Blazejczyk A, Wietrzyk J, Krupa A, et al. A novel canine B-cell leukaemia cell line. Establishment, characterisation and sensitivity to chemotherapeutics. Vet Comp Oncol. 2017;15(4):1218–31.

    Article  CAS  PubMed  Google Scholar 

  66. Grudzien M, Pawlak A, Kutkowska J, Ziolo E, Wysokińska E, Hildebrand W et al. A newly established canine NK-type cell line and its cytotoxic properties. Vet Comp Oncol. 2021.

  67. Samol J, Ranson M, Scott E, Macpherson E, Carmichael J, Thomas A, et al. Safety and tolerability of the Poly (ADP-ribose) Polymerase (PARP) inhibitor, Olaparib (AZD2281) in combination with Topotecan for the treatment of patients with advanced solid tumors: a phase I study. Investig New Drugs. 2012;30:1493–500.

    Article  CAS  Google Scholar 

  68. Chou T. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 2010;70(2):440–6.

    Article  CAS  PubMed  Google Scholar 

  69. Chou T. Preclinical versus clinical drug combination studies. Leuk Lymphoma. 2008;49:2059–80.

    Article  PubMed  Google Scholar 

  70. McLaren W, Gil L, Hunt SE, Riat HS, Ritchie GR, Thormann A, et al. The ensembl variant effect predictor. Genome Biol. 2016;17:1–14.

    Article  Google Scholar 

  71. Capriotti E, Montanucci L, Profiti G, Rossi I, Giannuzzi D, Aresu L, et al. Fido-SNP: the first webserver for scoring the impact of single nucleotide variants in the dog genome. Nucleic Acids Res. 2019;47(W1):W136–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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.

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.

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Authors and Affiliations

  1. Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Norwida 31, Wroclaw, 50-375, Poland

    Jayson Cagadas Pasaol, Ewa Dejnaka, Marta Henklewska, Bożena Obmińska-Mrukowicz & Aleksandra Pawlak

  2. Department of Comparative Biomedicine and Food Science, Division of Veterinary Pharmacology and Toxicology, University of Padua, Legnaro, 35020, Italy

    Greta Mucignat, Mery Giantin, Marianna Pauletto & Mauro Dacasto

  3. Department of Immunology, Pathophysiology and Veterinary Preventive Medicine, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Norwida 31, Wroclaw, 50-375, Poland

    Joanna Bajzert

  4. Precision One Health Initiative, College of Veterinary Medicine, University of Georgia, 220 Riverbend Road, Athens, GA, 30602, USA

    Christopher Zdyrski & Aleksandra Pawlak

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  1. Jayson Cagadas Pasaol
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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.

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Correspondence to Aleksandra Pawlak.

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Pasaol, J.C., Dejnaka, E., Mucignat, G. et al. PARP inhibitor olaparib induces DNA damage and acts as a drug sensitizer in an in vitro model of canine hematopoietic cancer. BMC Vet Res 21, 439 (2025). https://doi.org/10.1186/s12917-025-04880-z

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BMC Veterinary Research

ISSN: 1746-6148

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