Leukemia is a group of hematological malignancies, presenting a high clonal expansion of malignant cells, originating from myeloid and lymphoid lineages (1). Despite the higher frequency of the t(9;22)(q34;q11) chromosomal translocation in patients with chronic myeloid leukemia (CML) (2), the worst prognosis is observed in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), when they harbor the Philadelphia chromosome (Ph+), being classified as high risk, which tends to worsen with age (3,4).

This cytogenetic alteration leads to creation of the chimeric gene BCR activator of RhoGEF and GTPase::ABL proto-oncogene 1 (BCR::ABL1). The gene has three primary isoforms, namely p190^BCR::ABL1, p210^BCR::ABL1, and p230^BCR::ABL1, depending on the different gene breakpoints (5,6). The p210^BCR::ABL1 is the main isoform responsible for pathogenesis in CML patients (7). Although p190 is detected in only 1-2% of CML patients (8,9), the p190 transcript confers a higher risk of treatment failure in chronic-phase patients, as well as being associated with distinct clinical characteristics (9). Among the acute leukemias, ALL exhibits the highest frequency of p190^BCR::ABL1 (10). Although treatment with tyrosine kinase inhibitors (TKIs) has shown a significant benefit for Ph+ patients, adult patients still suffer from low response of the p190 isoform to TKIs (10-12).

Regardless of leukemia type, the isoform p190^BCR::ABL1 produces a protein with 190 kDa, the lowest molecular weight compared to the others. This is associated with a more aggressive leukemia phenotype, due to shorter transcripts causing the loss of important BCR regulatory sequences (13,14).

The presence of BCR::ABL1 induces genomic instability in neoplastic clones through deregulation of mitochondrial membrane potential by the accumulation of reactive oxygen species. In addition, BCR::ABL1 induces greater dependence on non-homologous repair pathways, contributing to increased genomic instability and decreasing the ability of these neoplastic clones to reliably repair damage to the DNA molecule (15-17).

The enzyme poly-ADP-ribose polymerase-1 (PARP1) has emerged as a potential therapeutic target in certain types of solid tumors (18–21) with excessive dependence on non-conservative repair pathways, with the aim of exploiting a distinctive feature of cancer proposed by Hanahan, genomic instability (22). Among a family of 17 enzymes, PARP1 is the main enzyme responsible for molecular signaling for DNA repair by adding ADP-ribose chains in a process known as PARylation (23).

PARP inhibitors (PARPi) act by the principle of synthetic lethality, increasing damage to the genetic material through the accumulation of DNA damage and genomic instability. In addition to the cytotoxicity exerted by direct inhibition of the PARylation process, PARPi acts on the PARP trapping process, forming insoluble complexes between DNA molecules and PARP. This trapping prevents DNA repair and induces greater genetic damage to neoplastic clones (23,24).

PARP1 expression may play an important role in the pathogenesis of adult patients with BCR::ABL1 p190+, as the Ph+ chromosome confers a profile of greater genetic instability in tumor cells by inducing DNA damage (15,16). In this study, we measured PARP1 expression levels in patients and correlated its expression with BCR::ABL1 p190+, to assess its influence on the clinical outcome of adult patients and propose PARP1 as a potential molecular biomarker for future therapeutic applications.

Patients and ethical issues. The study group consisted of a total of 59 adult patients diagnosed with CML, AML and ALL who had the BCR::ABL1 p190+ from the Ophir Loyola Hospital, Para, Brazil and Fortaleza General Hospital, Ceara, Brazil. Peripheral blood and bone marrow samples from the patients were used. The analysis also included a control group with 10 peripheral blood samples from healthy individuals, for statistical comparison of gene expression. The patients were brought into the study only after understanding and accepting the terms and informed written consent was obtained from each of them. All methods were carried out in accordance with the Helsinki guidelines and regulations. The research was submitted to the Brazil Platform and was approved by the Research Ethics Committee under registration number 5.207.521.

Expression analysis. Total RNA was extracted from the leucocyte layer using TRIzol Reagent^® (Invitrogen™, Carlsbad, CA, USA). Extracted RNA was quantified utilizing the equipment NanoDrop (Thermo Scientific, Carlsbad, CA, USA) and 20 ng were used for cDNA confection using the High-Capacity cDNA Reverse Transcriptase kit (Life Technologies, Carlsbad, CA, USA). The samples were quantified by quantitative real-time PCR (qPCR) using TaqMan^® Gene expression assays (Applied Biosystems^®, Foster City, CA, USA) for the PARP1 (Hs00242302_m1), BCR::ABL1 p190+ (e1a2) (Hs03024844_ft) and ABL (Hs_01104728_m1) and ACTB (Hs01060665_g1) genes, which were used as endogenous controls.

The commercial kit TaqMan^® Expression Master Mix (Applied Biosystems^®, Foster City, CA, USA) was used to detect expression analysis by qPCR begin performed using QuantStudio 5 Real-Time PCR system (Applied Biosystems^®). The experiments were performed in triplicate and the standard requirements for performing the technique were followed (25). For calculating relative expression levels of PARP1 the 2^–∆∆CQ (delta delta cycle quantification) method was used (26), using the health donor samples as the calibrator/control, considering that the amplification of the target gene and the amplification efficiency of the internal control should be approximately equal. A standard curve was applied to determine the p190/ng copy number values.

Statistical analysis. The Chi-square test was conducted to assess the correlation between clinical variables. The Shapiro–Wilk test was used to evaluate the sample’s distribution. Comparison tests were performed by Analysis of Variance (ANOVA) Kruskal–Wallis test and Dunn’s Multiple Comparison post-test were compared in non-parametric tests. The data is based on the mean±standard deviation of the mean of the experimental groups in biological triplicates. The Spearman rank test was applied for correlation between PARP expression and p190/ng copy number values. All statistics were performed using GraphPad Prism (version 8.0.1) and significant differences were considered with an interval of confidence of 95% (p<0.05).

A total of 59 samples from adults diagnosed with BCR::ABL1 p190+ leukemia were used, distributed as ALL (n=28), AML (n=7) and CML (n=24). Among the patients with ALL, 21 were males (75.0%) and 7 females (25.0%), with an average age of 46 years. The average leukometry in peripheral blood was 75.36 (10⁹/l) with an average of 50.1% blasts in the bone marrow.

The patients with AML were 4 males (57.14%) and 3 females (42.85%), with a mean age of 47.5 years. The average leukometry in peripheral blood was 10.59 (10⁹/l) with an average of 72% blasts in the bone marrow. Of the patients with CML, 12 were males (50.0%) and 12 females (50.0%), with a mean age of 56 years. The average leukometry in peripheral blood was 7.15 (10⁹/l) with an average of 3.6% blasts in the bone marrow.

Using the Chi-Square test, we evaluated the influence of clinical variables on the type of leukemia. A significant correlation was identified between age (p=0.0111) on the types of ALL, AML, and CML. However, no significant correlations were found within the other comparisons (Table I).

Due to the high prevalence of the p190 transcript in the ALL population (10), we decided to evaluate the clinical variables of this isolated group. We stratified these patients into two groups according to PARP1 expression (Table II), to assess the influence of the patient clinical status on PARP1 expression. Similarly, no significant correlations were found between the variables analyzed.

The differential expression of PARP1 (F [2,56] = 5,949; p < 0.005; ŋp², = 0,175 "large effect") concerning the types of leukemia was analyzed and statistically significant values were found between the expression in the group with ALL and CML (Δ: 19,29 [37,91; 18,63] p=0.0002) and between AML and CML (Δ: 18,73 [37,36; 18,63] p=0.0334) (Figure 1).

Subsequently, to analyze the correlation between PARP1 and p190 transcript, we stratified the patients who had a PARP1 expression higher than 1.5-Fold Change. All these patients were diagnosed with ALL and AML, representing 22.03% of the total patients used in the study. We found that in this group with a Fold-Change greater than 1.5, the number of copies of p190/ng of RNA transcripts did not show direct proportionality with PARP1 expression.

In CML, all the patients had a Fold Change of less than 1.5, showing reduced PARP1 expression. In addition, most of these patients had a low concentration of p190 copies/ng RNA.

Using the Spearman’s rank test, we compared all the PARP1, p190/ng copies, age and white blood cell (WBC) variables with each other. No statistically significant correlations were found between PARP1 relative expression and the number of p190/ng copies (r=–0.093), age (r=–0.099) and WBC (r=0.247) (Table III).

BCR::ABL1 p190+ leukemia continues to be a complex disease with the worst prognosis in ALL, AML and CML, posing a greater risk and aggressiveness to patients due to p190 (3,4,9). ALL patients exhibit the highest frequency of p190 (10). Due to various advances in the diagnosis and treatment of pediatric ALL, mortality rates have fallen (27,28). However, adult patients continue to suffer from a poor prognosis that tends to worsen with age and the lack of targeted therapies (29).

Given that BCR::ABL1 is an important inducer of genetic instability (15,16), it is hypothesized that patients afflicted with malignant hematological tumors harboring this translocation might benefit from the applicability of PARP as a therapeutic target (30,31). Considering this, we investigated PARP1 expression as a potential biomarker for prognosis in this population.

Although the Chi-square test showed no significant associations between the clinical variables and types of leukemia, the clinical heterogeneity observed suggests that the relationship between these factors may be more complex. The literature describes that patients with p190+ CML have distinct clinical characteristics, increased leukemic progression and, especially high rates of resistance/failure to treatment using first-generation TKIs (32–34).

BCR::ABL1 in AML is still a rare entity (35) and for this reason data on its main clinical characteristics and responses to TKIs are still scarce. Some case reports with the presence of p190+ in adults describe that the presence of mutations and co-expression of other rare transcripts can affect the response to treatment and induce a more aggressive leukemia (36,37).

Epidemiologically corroborating our results in ALL, one study showed a higher frequency of the p190 isoform in adult male patients than in female patients (38). Due to the heterogeneity of clinical characteristics and responses to treatment, we have tried to evaluate alterations in the genome of these patients. The most recurrent alterations are deletions in the IKZF1, PAX5 and CDKN2A/B genes, which are negatively associated with patient outcomes (39,40).

When we evaluated the expression of PARP1 between leukemia types (Figure 1), we found significant differences, showing that the expression in patients with ALL was higher than in patients with AML (p=0.0334) and substantially higher than in patients with CML (p=0.0002). Indeed, the differential expression of PARP1 found a 2-fold increase in ALL compared to healthy patients, and reduced expression in CML (31).

As for AML, studies have shown that high PARP1 expression is associated with complex cytogenetic abnormalities (41). Another study revealed that AML patients with high PARP1 expression had a shorter overall survival and had high levels of blasts, WBC in peripheral blood and were associated with a higher frequency of mutations in FLT3 (42). In addition, a cell culture study showed that PARP2 is also overexpressed in AML cell lines. This suggests that in addition to PARP1, PARP2 may contribute to the pathogenesis of AML (43).

Although PARP1 is overexpressed in CML cell lines (30), our results show that there were no statistically significant correlations between the relative expression of PARP1 and the amount of p190 transcripts in patients with CML, demonstrating that the two events are apparently independent. Although this lack of statistical correlation is evident in this study, the role of PARP1 in BCR::ABL1 leukemia cannot be ruled out, given that cells expressing BCR::ABL1 have homologous recombination (HR) defects and that this expression increases DNA damage and reactive oxygen species (44). In addition, it has been described that this oncogene is capable of inducing leukemia on its own in Ph+ tumors that tend to be extremely dependent on BCR::ABL1 expression (45,46), giving rise to an increased anti-leukemic effect in BCR::ABL1 positive cells in combination with TKI and PARPi therapies (47).

Finally, the characterization of the genetic background of mutations, cytogenetic alterations and DNA repair capacity must be considered so that PARPi therapy can benefit leukemia patients in the future (48).

BCR::ABL1 p190+ leukemia remains a complex disease with a poor prognosis and distinct clinical features. We found that PARP1 is differentially expressed in ALL, AML and CML and that p190 transcripts do not follow a linear pattern in these populations. We also found that PARP1 expression is not correlated with age, white blood cell and the amount of p190 transcripts. Despite the lack of statistical correlation between the variables analyzed, the role of PARP1 in BCR::ABL1 leukemia cannot be ruled out, given the instability profile promoted by this translocation. Finally, further studies involving a larger sample of patients are needed, as well as investigations into other molecular pathways that may impact on the pathogenesis of different BCR::ABL1 leukemic subtypes.

The Authors declare no conflicts of interest regarding this study.

Morais GP, Machado CB and Moreira-Nunes CA, performed the study design; Morais GP, Machado CB and JBSS performed the performed gene expression analysis; Morais GP, Machado CB, FMCPP and Nogueira BMD performed the molecular and statistical analysis; Morais GP, Machado CB, and Nogueira BMD, Moraes-Filho MO, Moraes MEA, and Moreira-Nunes CA wrote the article. All Authors read and approved the final article.

This study was supported by Brazilian funding agencies National Counsel of Technological and Scientific Development (CNPq; to ASK; MEAM, MOMF and CAMN).