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Vol. 46. Núm. S4.
HEMO 2024
Páginas S352-S353 (outubro 2024)
Vol. 46. Núm. S4.
HEMO 2024
Páginas S352-S353 (outubro 2024)
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PARP AND PARG INHIBITION SELECTIVELY IMPAIR METABOLISM OF B-ALL CELL LINES AND INDUCE CYTOTOXICITY THROUGH DISTINCT CELL-DEATH PATHWAYS
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CB Machadoa,b, KS Estevesb, SCG Limab, IP Furtadob, M Brandãob, FMCP Pessoaa, BMD Nogueiraa, MEA Moraesa, LEB Souzab, CFA Moreira-Nunesa
a Department of Medicine, Núcleo de Pesquisa e Desenvolvimento de Medicamentos (NPDM), Universidade Federal do Ceará (UFC), Fortaleza, Brazil
b Center for Cell-based Therapy, Fundação Hemocentro de Ribeirão Preto (FUNDHERP), Faculdade de Medicina de Ribeirão Preto (FMRP), Universidade de São Paulo (USP), Ribeirão Preto, Brazil
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Vol. 46. Núm S4

HEMO 2024

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Background

Adult patients afflicted with Acute Lymphoblastic Leukemia (ALL) still represent a subset of poor prognosis, with a 5-year overall survival of around 46%. The development of new therapeutic approaches is of utmost importance to the oncologic routine and the search for targeted therapies is ever growing in the translational field.

Objective

In this study, we aim to investigate an agonistic effect of poly-ADP-ribose polymerase 1 (PARP1) in inducing parthanatos, a PARP1-dependent and programed mode of cell death, in leukemic cell lines overexpressing PARP1, through inhibition of its antagonist, poly-ADP-ribose glicohydrolase (PARG).

Methods and materials

We screened 6 cell lines for their PARP1 mRNA expression through qPCR. Inhibition of their metabolic activity was screened through Alamar Blue assay (Invitrogen™) after treatment with either a PARG inhibitor, JA2131, a PARP inhibitor, AZD2461, or a cytotoxic control in Doxorubicin. An IC50 that considered only cell death was assayed through treatment in serial dilutions followed by DAPI solution staining and analysis in flow cytometry. The profile of early death was determined by co-staining of treated cells with CellEvent™Caspase-3/7 Green Reagent (Invitrogen™), APC Annexin V (BD Pharmingen™) and DAPI Solution.

Results and discussion

Of the initially screened cell lines, only B-ALL models significantly overexpresed PARP1 when compared to PBMC of healthy donors. Also, while all models responded to Doxorubicin treatment, only B-ALL models had measurable metabolic inhibition when incubated with PARGi and PARPi treatment. Cell death measured by DAPI has only been conducted in one cell line as of this moment, SUP-B15, a B-ALL model. While PARPi and Doxorubicin had IC50 values similar to those observed in metabolic inhibition by Alamar Blue, PARGi only induced cell death in treatment values approximately 3-fold higher than the necessary for metabolic inhibition. Nevertheless, cell death induced by the treatments demonstrate different early death profiles, with PARGi treatment not inducing any effector caspase activity previously to cell death, a hallmark of parthanatos, while PARPi and Doxorubicin had clear increase in caspase activity and annexin-V staining in early apoptotic cells.

Conclusion

Targeted therapeutics with PARG or PARP inhibitors may be a feasible option for B-ALL treatment, with different modes of cell death induction. However, further analysis needs to be conducted in order to better characterize treatment-induced phenotypes and their cytotoxicity over non-neoplastic cells.

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Idiomas
Hematology, Transfusion and Cell Therapy
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