Targeting DNA damage and repair mechanism in FLT3-ITD acute myeloid leukemia : a mechanistic and therapeutic study

Abstract

Internal tandem duplication (ITD) of fms-like tyrosine kinase 3 (FLT3) is one of the most common mutations in acute myeloid leukemia (AML), occurring in nearly 30% of cases. FLT3-ITD involves in-frame duplication of 3-400 base-pairs at the juxta-membrane, resulting in ligand-independent activation of FLT3 signaling. Downstream effectors include activation of STAT5 via SRC kinase, phosphorylation of FOXO3A, down-regulation of the equilibrative nucleoside transporter 1 (ENT1) for cytarabine, and induction of reactive oxygen species (ROS) production. These aberrant signals result in increased DNA damage and defective repair, increased cellular proliferation and resistance to apoptosis.

Induction of ROS and DNA damage in FLT3-ITD AML has led to investigation of their mechanistic link and exploration of potential therapeutic targets. By examining gene expression associated with DNA repair in primary AML samples, BRCA2 expression was shown to be down-regulated in FLT3-ITD AML when compared with AML with wild-type FLT3 as well as normal hematopoietic cells. BRCA2 is an important protein in mediating homologous recombination (HR), providing a possible explanation for defective DNA damage response (DDR) in this AML subtype. A double-stranded break (DSB) DNA repair assay was used to measure the fidelity of DSB repair, either via error-free HR or error-prone non-homologous end joining (NHEJ). The results showed that HR was down-regulated in murine Ba/F3 cells transduced with FLT3-ITD while NHEJ remained active.

DDR pathway as a target for therapeutic intervention in human cancers is exemplified by the use of poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) in BRCA mutant breast and ovarian cancers. In Ba/F3 FLT3-ITD cells and knockin Flt3 ITD/+ Npm1 c/+ mouse leukemic cells, PARPi Olaparib suppressed leukemia growth in vitro. Combination of chemotherapy and Olaparib worked synergistically to eradicate leukemic cells in MOLM-13 murine xenograft model. Biochemically, Olaparib inhibited base excision repair and increased the DSB damage. Olaparib also increased intracellular ROS, resulting in positive feedback that accentuated DNA damage. To identify potential therapeutic targets that may be exploited in combination treatment with Olaparib, a DDR shRNA library screening was performed. Potential candidate genes included those associated with checkpoint factors and DNA replication factors, for instance, Atr kinase and members of the Family B DNA Polymerase.

In summary, FLT3-ITD AML showed defective HR and higher levels of intracellular ROS and DSB, and Olaparib induced genomic instability and apoptosis. Targeting defective DNA repair in FLT3-ITD AML using PARPi might be considered as a novel therapeutic strategy.