Abstract A08: Signaling pathways involvement in acute myeloid leukemia resistance to BRD4 inhibitor
Authors: Marcela Latancia, Lilian Inoue, Jussara Souza, Fernanda Koyama, Paula Asprino, Welbert de Oliveira Pereira, Niels Olsen Saraiva Câmara, Luiz Fernando Lima Reis, Mariane Tami Amano
Journal: Clinical Cancer Research (2018)

Abstract Acute myeloid leukemia (AML) is a very aggressive hematologic neoplasy, whose tumoral cells progress at a rapid pace. AML has myeloid lineage progenitors that present abnormal proliferation and are incapable to maturate, thus generating undifferentiated blasts. Its cause and mechanism are not completely understood and this is a molecular and clinical heterogeneous disease, which difficult diagnosis and the establishment of an efficient treatment. Although it has a low incidence, the mortality ratio is high, therefore, the seek for new therapies is relevant. AML development is associated with mutations in epigenetic regulators, and recently, BRD4 was found a promising target for AML therapy (Zuber et al., 2011). BRD4 is a protein of Bromodomain and Extraterminal domain (BET) family, these bromodomains read post-translational acetylation modifications and BRD4, specifically, facilitates transcriptional factors recruitment and transcription elongation. It was found to be involved in BCL2, CDK6 and proto oncogene MYC expression. JQ1 is a BRD4 inhibitor, which binds competitively to acetyl-lysine recognition motifs, and it has shown anti-leukemic effects in pre-clinical assays. Although the literature has pointed BRD4 inhibition as an interesting approach to treat AML patients, recently, it was observed on a mouse leukemia model resistance to JQ1 treatment. The objective of this study is to verify the role of cell signaling pathways on BRD4 inhibitor resistance. We used HL60, U937, HEL and K562 cell lines. Briefly, our experimental procedures were: cell counting with trypan blue to determine proliferation and viability during 4 days, we performed qPCR with 6h of JQ1 treatment, and the phospho array analysis to determine different cell signaling activation. We used two doses of JQ1, 200nM and 800nM. We observed that HL60 and U937 were sensitive cell lines and HEL and K562 were resistant. We performed qPCR of some genes that are BRD4 target. We observed gene expression of MYC, which was diminished in sensitive cell lines and had no significant alteration in resistant cell lines. CDK6 expression was not significantly altered in any of the cell lines, while BCL2 expression was diminished in sensitive cells, but also in resistant cell line HEL, which suggests that these two latter BRD4 target genes cannot be used as parameter of JQ1 resistance. We then analyzed cell signaling activation on sensitive HL60 cell line, and we observed that after JQ1 treatment, phosphorylation of AMPKa1 and HSP60 were diminished, while CREB, a metabolic signaling pathway component, as well as p38, ERK 1/2 and MSK1/2 were increased. Since CREB is activated by AKT, we focused on metabolic pathways to look for possible targets. We used the mTOR inhibitor rapamycin (RAPA), another AKT target, to evaluate whether JQ1 resistance could be broken in AML cell lines. We observed that combined treatment of JQ1 and RAPA was able to reduce resistant cells proliferation and had a synergistic effect on sensitive cells. Here, we established an AML cell line model of sensitivity and resistance to JQ1. As partial conclusion, we can infer that cell signaling pathways involved in cell metabolism seem to play an important role on JQ1 resistance, and blocking such pathways could be an alternative to overcome this resistance. Citation Format: Marcela Latancia, Lilian Inoue, Jussara Souza, Fernanda Koyama, Paula Asprino, Welbert de Oliveira Pereira, Niels Olsen Saraiva Câmara, Luiz Fernando Lima Reis, Mariane Tami Amano. Signaling pathways involvement in acute myeloid leukemia resistance to BRD4 inhibitor [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A08.