We have designed ROS-activated cytotoxic agents (RACs) which are active against Acute Myeloid Leukemia (AML) cells harboring common oncogenes. In this study the mechanism and synergistic effects of treated AML cells was investigated. We used dual oncogene expression of MLL-AF9 and FLT3-ITD, which is characteristic of poor prognosis AML. Treatment of these AML cells led to an IC50 value of 1.8±0.3 micromolar with a selectivity of 9-fold compared to untransformed cord blood cells. Furthermore, transformation led to a statistically significant 32% reduction in the key anti-oxidant catalase and a 71% increase in the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine. Treatment also leads to DNA strand breaks and apoptosis. To gain an unbiased view of the mechanism of action we examined treatment in these AML cells by quantitative proteomics on isolated nuclei and RNA-seq. RNA-seq showed expression of more than 1300 mRNAs and alignment by ToppGene showed common signatures of damage while proteomics revealed key features including VCP an early stage protein in double strand break repair when in the nucleus and TOLDO1 part of the pentose phosphate pathway that supplies ribose-phosphate for DNA synthesis. Western blotting further confirmed DNA double strand break repair by homologous recombination since gamma-H2AX, phopho-ATM, and phopho-BRCA1 showed enhanced concentration in the nucleus. Further supporting this, an ATM inhibitor (Ku-55933), an ATR inhibitor (VE-821), a DNA double strand damaging agent (doxorubicin), a DNA anti-metabolite (AraC) all showed synergistic effects in all cases at 0.5 dose-effects. Taken together, these results show that this RAC can selectively target poor prognosis AML and do so by creating double strand DNA breaks that require homologous recombination.<br/>

ROS-activatable agent elicits homologous recombination DNA repair and synergizes with pathway compounds

251st National Meeting (2016)

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