Decitabine Priming Enhances The Antileukemic Effects Of The Selective Inhibitor Of Nuclear Export (SINE) CRM1/XPO1 Antagonist (Selinexor) In Acute Myeloid Leukemia (AML)

AML is a clonal hematopoietic disorder characterized by genetic and epigenetic alterations. The prognosis of AML is poor highlighting the urgent need for novel therapeutic approaches. Targeting aberrant DNA hypermethylation by using hypomethylating drugs such as decitabine has been recently investigated in AML. Our group reported a relatively effective (47% complete response) single agent decitabine treatment schedule for older AML patients. These results suggested the opportunity to capitalize on this relatively effective and non-toxic treatment by incorporating this compound into novel molecularly targeted approaches. Recent data indicate that targeting nuclear exporter proteins is a novel therapeutic strategy to overcome cancer. In particular, CRM1/XPO1 is the only nuclear exporter involved in the active transport of the majority of tumor suppressor proteins (TSP) [e.g. p53 and FOX03A] out of the nucleus resulting in their inactivation. We recently reported the anti-leukemic activity of oral SINE CRM1/XPO1 Antagonists in AML. SINEs displayed potent anti-proliferative properties, induced apoptosis, cell-cycle arrest and myeloid differentiation in AML cell lines and patient blasts. In addition, treatment of leukemic mice with oral SINE significantly prolongs their survival. By crossing the lists of genes known to be regulated by DNA methylation in AML with the ones whose nuclear transport is XPO1 dependent, we identified important TSPs such as FOXO3A and p21. Here we hypothesize that treatment of AML cells with decitabine will increase the transcription and expression of a subset of TSPs (including FOX03A and p21) whose nuclear anti-leukemic effects could be enhanced by blocking their XPO1 mediated nuclear export using the clinical stage oral SINE (Selinexor). Thus, we expect that the sequential treatment of decitabine followed by Selinexor will be more effective than each drug alone. To confirm this hypothesis first we treated the AML line OCI-AML3 cells with decitabine (500nM) overnight (ovn) followed by Selinexor (200nM, 2 fold lower than IC50) for an additional 24 hours (hrs) and measured cell proliferation using WST-1 assay. Controls include: 1) DMSO ovn +DMSO 24 hours, 2) Selinexor ovn +selinexor 24 hours (200 nM) and 3) decitabine ovn + decitabine 24 hrs (500nM). AML cells that were treated first with decitabine followed by Selinexor exhibited a higher cytotoxicity (absorbance (Abs) 0.7) than cells treated with either decitabine (Abs 1.36), Selinexor (Abs 1, p=0.006) or DMSO (Abs 1.7). Similar results were observed with the MV4-11 cell line. Next we measured the candidate TSPs (FOX03A and p21) mRNA and protein expression in OCI-AML-3 and MV4-11 cell lines after 24 hrs of decitabine treatment. We found a significant up-regulation of p21 in decitabine versus DMSO treated cells (MV4-11, Fold change (FC) 4.67±1.4; OCI-AML3, FC 3.98±1.19, p<0.05). We also detected a modest up-regulation of FOXO3A in both cell lines treated with decitabine when compared to the DMSO controls (MV4-11, FC 2.56±0.74 and OCI-AML3, FC 1.5±0.23, p<0.05). These results were confirmed also at the protein level by using western blot. Next, we asked whether ectopic up-regulation of p21 (mimicking decitabine effects) in OCI-AML3 cells could re-capitulate the decitabine enhancing antileukemic effects of Selinexor. Overexpression of p21 followed by Selinexor (200nM) for 24 hrs resulted in a larger decrease of cell proliferation (Abs 0.5) with respect to controls (Abs 0.7, p<0.05) using the WST assay. Similar results were observed for the MV4-11 cell line. Finally, we tested the efficacy of the sequential decitabine–Selinexor in vivo using the MV4-11 xenograft model. Treatment began one week after leukemic cell inoculation in 4 different cohorts; 1) Vehicle, 2) decitabine i.p. twice weekly (BIW) (0.4mg/kg); 3) Selinexor BIW (20mg/kg by oral gavage) and 4) decitabine BIW i.p (0.4 mg/kg) followed by Selinexor (10 mg/kg BIW). We found no difference in median survival time (MST) between vehicle and decitabine only treated mice. As expected, Selinexor alone treated mice have significantly improved MST at 36.5 days vs. 28.5 days, vehicle, p=<0.01). Most importantly, the sequential treatment of decitabine followed by Selinexor significantly improved MST compared to Selinexor alone 47 vs 36.5, p=0.008). These pre-clinical results hold great promise for the use of this combination in human clinical trials in AML.