Targeted Repression of TP53 Promotes Erythropoiesis in Del(5q) MDS and Overcomes Clinical Resistance to Lenalidomide

Stabilization of p53 as a result of ribosomal stress is a key effector of the hypoplastic anemia in del(5q)MDS. We recently reported that lenalidomide (LEN) stabilizes MDM2 to promote p53 degradation, fostering cell cycle transition and arrest of del(5q) progenitors in G2/M (Wei S, et. al. Oncogene 2012). P53 expression in erythroid precursors (EP) decreased in responders, but was up-regulated upon emergence of resistance to LEN (Wei S, et. al. PNAS 2010). We hypothesized that targeted suppression of p53 may be an effective strategy to restore erythropoiesis in del(5q)MDS EP after LEN-treatment failure.

To test this hypothesis, we first investigated the in vitro effects of cenersen (Aezea®, Eleos Inc. Plymouth, MN), a 20mer phosphorothioate antisense oligonucleotide complementary to the exon 10 coding sequence of the TP53 gene transcript, on cellular p53 expression and colony forming capacity (CFC) in EP. Cenersen, through RNase H-mediated site specific mRNA cleavage, down regulates wild type & mutant TP53 mRNA and protein expression. Using a 2-stage EP differentiation assay, CD34+ cells from MDS patient specimens (del5q, n=11; non-del5q, n=4; normal donor, n=5) were cultured in StemSpan^® SFEM expansion medium (StemCell Technologies, Vancouver, Canada) supplemented with SCF, EPO, and dexamethasone for 3–10 days, then transfected with 40nM cenersen or scrambled oligodeoxynucleotides using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA). Cells were harvested after 3 days for analysis of p53 expression by fluorescence microscopy, FISH and methylcellulose cultures. CFC was assessed after 7–14 days incubation.

Del(5q) EP displayed greater nuclear p53 fluorescence intensity (FI) than non-del(5q)MDS or controls [del(5q) mean =82.12±11.4, non-del(5q)=41.55±13.05, controls=63.58±10.14, P=0.07; del(5q) vs. controls, P=0.33]. Treatment with cenersen significantly reduced nuclear (> 50%) and cytoplasmic p53 expression (> 59%) in EP in 9 of 11 del(5q) specimens, (mean nuclear FI, cenersen=41.75±9.02 vs. non-sense=86.84±13.12, P=0.01; mean cytoplasmic FI, cenersen= 23.38±3.14 vs. non-sense= 41.34±6.21, P=0.02). Similar but non-significant trends in nuclear p53 were observed in control (P=0.19) and non-del5qMDS (P=0.29) EP, whereas cytoplasmic p53 was significantly reduced only in controls (>52%, P=0.03; non-del5q, P=0.35). Cenersen treatment significantly increased recovery of BFU-E and CFU-E (n=15, P=0.01; n=13,P=0.0001, respectively) in MDS specimens, whereas there were no consistent changes in granulocyte-macrophage or mixed lineage colony yields. Similar but less robust improvements in CFC were observed in control samples with cenersen treatment (CFU-E, n=4, P=1.0; BFU-E, n=5, P=0.43). The magnitude of improvement in BFU-E in MDS specimens highly correlated with the extent of p53 suppression (nuclear Spearman r= -0.64, P=0.009; cytoplasmic r= -0.65, P=0.008). Changes in CFU-E showed no significant correlation with changes in nuclear (P=0.65) or cytoplasmic (P= 0.89) p53 expression. FISH analysis of cytospin preparations following oligonucleotide treatment of del(5q) EP using the EGR1 5q31/5p15.31 deletion probe (Cytocell, Cambridge UK) showed no significant differences between cenersen and non-sense treated cells (P=0.55, n=10). To determine if in vivo p53 suppression can restore transfusion independence (TI) after secondary LEN treatment failure, we treated 8 LEN-resistant, transfusion-dependent del(5q) MDS patients with dexamethasone (Dex) 20 mg weekly and continued LEN 5–10 mg/day. The glucocorticoid receptor acts as a ligand-dependent transcription factor and mutual antagonist of p53. All patients had <5% myeloblasts and Int-1 IPSS, whereas 3 patients had del(5q)+1 additional chromosome abnormality. Five of 8 patients achieved TI with LEN-Dex treatment. There was no significant reduction in the proportion of del(5q) metaphases after achievement of TI in responders. Median duration of LEN-Dex treatment was 4 months, and 9 months in responders (n=5). None of the patients experienced AML progression.

Targeted suppression of p53 restores effective erythropoiesis in del(5q) MDS in the absence of clonal suppression. A clinical trial testing treatment with cenersen +Dex in LEN-resistant patients is currently in development.

Smith:Smith Holdings LLC: Equity Ownership, Smith Holdings owns rights to the drug involved in this study. Patents & Royalties; Eleos, Inc: Eleos has licensed rights to the drug from Smith Holdings. Patents & Royalties. Daugherty:Eleos, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Wells:Alexion: Honoraria; Janssen: Honoraria; Celgene : Honoraria; Novartis: Honoraria, Research Funding. List:Celgene: Consultancy. Off Label Use: lenalidomide combined with Dexamethasone in lenalidomide refractory patients .