PARP1 IS DIFFERENTIALLY EXPRESSED IN BCR-ABL P190+ ALL PATIENT SAMPLES AND TARGETING PARP INHIBITION INDUCES CELL DEATH COMPARABLE TO THAT OF TYROSINE-KINASE GOLDEN STANDARD IN PRE-CLINICAL MODELS

Abstract

Objective: BCR-ABL1 p190 translocation represents a worse prognosis for acute lymphoblastic leukemia (ALL) patients, and development of new therapeutic approaches is of utmost importance to the oncologic routine. This study aims to evaluate the cytotoxic effect of inhibiting poly-ADP-ribose (PARP) in an ALL Philadelphia positive (Ph+) cell model and validate biomarker differential expression in patient samples. Methodology: Cell lines SUP-B15, Raji and Namalwa were screened for PARP1 expression through qPCR. Non-neoplastic cell line MCR5 was used as a control and ACTB was chosen as the housekeeping gene. Data was compared through one-way ANOVA followed by Bonferroni's post-test. Cells were then treated with either AZD2461, Imatinib or Doxorubicin through serial dilutions and, after 72 hours of treatment, cells were incubated with Alamar Blue and read for fluorescence. Viability results were analyzed through non-linear regression. Finally, samples from 31 p190+ ALL patients were quantified for PARP1 expression and compared to 10 healthy controls. The study was approved by the Ethics Committee of the Federal University of Ceará (5.207.521) and of the Ophir Loyola Hospital (9611320.0.0000.5550), informed written consent was obtained from the patient, being brought into the study only after accepting the terms, and all methods were carried out in accordance with Helsinki guidelines and regulations. Statistical comparison went as previously described for qPCR analysis. All statistical analysis were made using GraphPad Prism (v. 5.01). Results: SUP-B15 considerably overexpresses PARP1 in comparison to both Raji and Namalwa, as well as to the control MRC5 (p < 0.0001), and treatment with AZD2461 showed cytotoxic activity comparable to that of Imatinib in SUP-B15, represented by an IC50 at 72 hours of treatment of 344.3 nM and 329.2 nM, respectively, while showing little to no efficacy when treating either of the remaining cell lines. Validation of biomarker in patient samples reveals an approximate 2-fold increase in PARP1 levels compared to healthy controls (p < 0.01). Discussion: The presence of BCR-ABL1 translocation is highly associated with increased chromosomal instability through intracellular accumulation of reactive oxygen species and promotion of non-conservative repair pathways. The major cytotoxic effect of AZD2461 over SUP-B15, in levels comparable to those of Imatinib, shows that ALL p190+ models seem to be over reliant on pathways that regulate genomic damage and minimize the instability brought by BCR-ABL1, specifically PARP. The overexpression of PARP1 in our experimental model reinforces its translational credibility due to the differential expression of PARP1 detected in p190+ ALL patient samples and sets PARP1 as a potential new target for therapeutic of BCR-ABL1 positive ALL. Conclusion: The use of PARPis shows great effect over the viability of ALL Ph+ models and PARP1 represents a potential new target for investigation in BCR-ABL1 positive tumors. However, studies with larger cohorts of patient samples, as well as in vivo investigations, are still necessary to further elaborate on this hypothesis.