Multifactorial mechanisms of resistance to the aminopeptidase inhibitor prodrug CHR 2863 provoke marked sensitization to the mTOR inhibitor rapamycin

Submitted 8 Multifactorial mechanisms of resistance to the aminopeptidase inhibitor prodrug CHR2863 provoke marked sensitization to the mTOR inhibitor rapamycin 176 Abstract Carboxylesterases (CES) play a pivotal role in intracellular metabolic processes of drug detoxification or activation as well as lipid/cholesterol homeosta-sis. Here we report the molecular basis of acquired resistance to CHR2863, an orally available hydrophobic aminopeptidase inhibitor (AP) prodrug with an esterase-sensitive motif. CHR2863 can enter target cells by diffusion and is retained therein upon CES-mediated conversion to its active hydrophilic metabo-lite drug CHR6768 to exert amino acid depletion. From CHR2863-sensitive human U937 myeloid leukemia cells, one subline U937/CHR2863(200) with a low level (14-fold) and another, U937/CHR2863(5uM), with a high level (270-fold) of CHR2863 resistance were selected for further characterization. The main features of CHR2863-resistant cells included: (1) a marked down-regulation of CES1 mRNA (3-5 fold) and CES1 protein, mirrored by a 4-7 fold increase in CES2 mRNA, a modest increase in CES2 protein, along with a gain of sensitivity to the CES2-activated prodrug CPT11/irinotecan; (2) loss of CES1 immunoreactivity and colocalization with lipid droplets as observed in parental U937/WT cells by 3D digital imaging fluorescence microscopy; (3) a 40-50% increase in lipid droplets count and intracellular sequestration of CHR2863, associated with a loss of conversion to its active metabolite, and; (4) a progressive activation of pro-survival Akt/mTOR pathway, coinciding with a dramatic gain of sensitivity to the mTOR inhibitor rapamycin in U937/CHR2863(200) cells (54-fold) and U937/CHR2863(5uM) cells (>1,000-fold). In conclusion, impaired CES1-mediated drug activation, sequestration in lipid droplets and Akt/mTOR activation, contribute to a multifactorial mechanism of resistance to CHR2863, which can be overcome by rapamycin or CES2-activated prodrugs.