High-Risk AML, Including Flt3ITD+, Exhibits Resistance To Conventional Cytarabine Induction Associated With Diminished ENT1 and p16INK Expression: Evidence For Epigenetic Repression and HDAC Inhibitor Modulated De-Repression

Resistance to conventional induction chemotherapy involving continuous infusion cytarabine, 100mg/m2 X 7 days, has been observed in several poor-risk AML phenotypes including normal karyotype with Flt3ITD or DNMT3A mutations (JP Patel, NEJM; 2012). However, escalated anthracycline dosing along with conventional cytarabine was determined to confer superior survival among certain subgroups, including mDNMT3A, in the randomized E1900 trial. We asked whether optimal therapy among other AML molecular subgroups, where the former combination may be inferior, might preferentially require intermediate/high-dose cytarabine within induction regimens containing conventional anthracycline. This hypothesis was generated in recognition of diminished cytosine analog (cytarabine, azacytidine) transport by the rate-limiting equilibrative nucleoside transporter (ENT1) in poor-risk groups (J Hummel-Eisenbeiss, Mol Pharm, 2013) and observation of disparate cytarabine LC50’s among known AML subgroups: normal karyotype (NK), =/> 10 micromolar; CBF+ve, 1-4 micromolar. In an attempt to identify an impact of diminished ENT1 expression on successful remission induction at day+14 of conventional “7+3” therapy and/or associated severe limitation of event-free survival, an unselected group of 20 AML patients’ blasts were comparatively studied for ENT1 by immunoblot and/or quantitative RT-PCR, and as well were subjected to parallel analysis of p16INK4a transcripts, whose diminution, accompanying an epigenetic mechanism, is also implicated in poor treatment outcomes (HJ DeJonge, Blood; 2009). Median follow-up of the 20 patients exceeded 12 months. Among patients with high ENT1 expression by immunoblot (50kd species) were included CBF+ve and NK AMLs, of whom 7/8 pts. overall (2 CBF+ve, 1 NK-mNPM1, 1 NK-absent Flt3ITD, 2 NK Flt3ITD+, 1 complex/trisomy 13) had no evidence of leukemia on a day +14 marrow, and the event-free survival exceeded the 12 month mark. By contrast, among patients with 2-5-fold lower blast cell ENT1 expression were included a higher fraction of poor-risk (complex) karyotype or normal karyotype with Flt3ITD mutation (62%). These patients either had persistent disease at day +14, or had less than 3-month event-free survival. There was correspondence for differing ENT1 protein expressions with transcript quantity, and coupling between low/nondetection or high/detection of both ENT1/p16INK4, respectively, predicted divergent outcome of therapy from usual prognostic groups. Because the mechanism of repressed ENT1 transcriptional expression resulting from transduction of Flt3ITD gene in model AML systems is linked to transfactor c-jun interaction on the ENT1/SLC29A1 promoter, we tested the activity of tyrosine kinase/FLT3 inhibitor or HDAC inhibitor (SAHA), or their combination, on upmodulation of ENT1 in cultured primary blasts. In patient blasts in which phospho-c-jun expression was associated with diminished ENT1 expression, application of inhibitor(s) yielded downregulation of p-jun and upregulation of ENT1. This result supports an initiative to introduce both epigenetic agent(s) along with intermediate-dose cytarabine into induction therapy of certain poor-risk AML subgroups, including Flt3ITD+.