Distinct signaling events promote resistance to mitoxantrone and etoposide in pediatric AML: a Children's Oncology Group report.

Xin Long,Michele S Redell,Todd A Alonzo,Robert B Gerbing

doi:10.18632/oncotarget.21363

Abstract

Despite aggressive chemotherapy including mitoxantrone and etoposide, relapse occurs for almost half of children with acute myeloid leukemia (AML). Since both drugs inhibit topoisomerase II and cause DNA double strand breaks, resistance could be achieved by enhanced DNA damage repair (DDR), via homologous recombination (HR) and/or non-homologous end joining (NHEJ). An important source of extrinsic chemoresistance is the bone marrow stroma. We aimed to reveal intrinsic and stroma-induced signaling pathways that contribute to chemoresistance. Sixty diagnostic pediatric AML samples were cultured on or off stromal cells, with or without chemotherapy. We measured apoptosis, DNA damage signaling, and NHEJ/HR pathway activity by FACS analysis of intracellular cleaved PARP, γH2AX, pDNA-PKcs and pATM, respectively. Mitoxantrone strongly increased γH2AX and pDNA-PKcs. Neither chemotherapy drug induced pATM. DNA-PK inhibition alleviated mitoxantrone resistance for AML cells on and off stromal cells. Regarding stroma-induced signaling pathways, ERK1/2 was most consistently activated in primary AML cells by stromal cells. ERK1/2 inactivation partially restored chemosensitivity to AML cells on stromal cells. Additionally, low stroma-induced STAT3 activity and strong stroma-induced mitoxantrone resistance were associated with inferior clinical outcome. Taken together, the NHEJ DDR and ERK1/2 pathways are potential targets for reducing intrinsic and extrinsic chemotherapy resistance in pediatric AML.

Highlights

Despite aggressive treatments, recurrence still occurs for almost half of children with acute myeloid leukemia (AML) due to resistance to chemotherapy
Since DNA damage repair may contribute to chemotherapy resistance, we compared the DNA damage signaling marker, histone H2AX phosphorylated at Ser 139 (γH2AX), and the apoptosis marker, cleaved PARP (cPARP), in primary AML cells on or off stroma, treated with mitoxantrone for either 4 or 24 hours
For mitoxantrone-treated primary AML cells on stroma, the upregulation of DNA damage signaling was associated with protection from apoptosis (Supplementary Figure 1B, 1D), supporting the hypothesis that DNA damage repair contributes to mitoxantrone resistance

Summary

Introduction

Recurrence still occurs for almost half of children with AML due to resistance to chemotherapy. Interactions between AML blasts and the bone marrow stromal microenvironment are known to protect blasts from chemotherapy, but the mechanisms remain largely unknown [1, 2]. Mitoxantrone and etoposide are standard chemotherapy agents used in upfront treatment regimens for pediatric AML. Both are topoisomerase II inhibitors and cause double strand breaks (DSBs) [3], stroma-mediated resistance occurs through different mechanisms. We found that stromal CYR61, a small protein in the extracellular matrix that binds integrins, promotes resistance to mitoxantrone, but not etoposide, in pediatric AML [4]

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