Dados do Resumo

Título

Telomerase inhibition mediates cell death in ALL in vitro model and antagonizes cytotoxicity induced by PARP inhibition

Introdução

Adult patients afflicted with Acute Lymphoblastic Leukemia (ALL) still represent a subset of poor prognosis, with a 5-year overall survival of around 46%. We have previously demonstrated the effectiveness of targeted poly-ADP-ribose polymerase (PARP) inhibition in the treatment of Philadelphia positive (Ph+) ALL cell models and we believe that further exploring genomic instability pathways has translational potential in this leukemic subset.

Objetivo

In this study, we aim to investigate the cytotoxic potential of a telomerase inhibitor in a Ph+ ALL cell model with high hTERT expression and correlate this activity with the use of PARP inhibitors, exploring synthetic lethality through induced genomic instability.

Métodos

4 cell lines were screened for their PARP1 and hTERT mRNA expression through qPCR. Inhibition of their metabolic activity was screened through Alamar Blue assay (Invitrogen™) after treatment with either a Telomerase inhibitor, MST-312, 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. For synergism, drugs were screened in pairs, treating cells in a treatment matrix, with constant dose of drug A and a variable dose of drug B. Matrices were uploaded to the web-based software of SynergyFinder version 3.0 (https://synergyfinder.fimm.fi), and analyzed through zero interaction potency (ZIP) reference model for evaluation of interaction level. 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.

Resultados

Of the initially screened cell lines, only SUP-B15 and Raji significantly overexpressed PARP1 and hTERT when compared to the control non-neoplastic cell line, MRC5. While all models responded to Doxorubicin treatment, only SUP-B15 had significant metabolic inhibition when treated with PARPi and was thus prioritized in the investigation of telomerase inhibition. A 72-hour cytotoxicity for MST-312 was also found in SUP-B15, with IC50 of 3.279 µM. Cell death measured by DAPI confirmed the cytotoxicity of the proposed treatments, beyond only metabolic inhibition. In synergy analysis, however, targeting PARP and Telomerase demonstrated significant antagonism, with synergy scores of -10 at sub-inhibitory doses of either treatment. As such, cell death markers for both treatments were investigated through flow cytometry and statistically different caspase-3/7 activation profiles were seen, suggesting different pathways for induced cell death.

Conclusões

While targeting either PARP or Telomerase may be a feasible option for treating Ph+ ALL cell models, combination of both treatments seems to reduce cytotoxic potential and compromise overall efficacy. The explanation for the observed antagonism might lie in understanding the different modes of cell death induced by the investigated treatments. Further analyses are being conducted in order to better characterize treatment-induced phenotypes and their pharmacological interactions.

Financiador do resumo

This study is supported by Brazilian funding agencies: Coordination for the Improvement of Higher Education Personnel (CAPES; to C.B.M., M.B., M.S.M.A, F.M.C.P.P. and B.M.D.N.), National Council of Technological and Scientific Development (CNPq; grant 404213/2021-9 to C.A.M-N and PQ Scholarships to M.E.A.d.M. and C.A.M.-N) and São Paulo Research Foundation (FAPESP; to K.S.E. (grant 2023/15546-0), S.C.G.L. (grants 2020/02043-2, 2021/09900-0), I.P.F. (grant 22/09926-2) and L.E.B.S. (grant 2013/08135-2))

Palavras Chave

Targeted Molecular Therapy; Telomerase Reverse Transcriptase; PARP Inhibitors