Abstract 1824

Leukaemic stem and progenitor cells (LSPC) are relatively resistant to current chemotherapy and considered to contribute to disease progression and relapse in Acute Myeloid Leukemia (AML) patients following initial treatment. We have developed a model to study the in vitro targeting of AML LSPC by novel agents which have the potential to target LSPC whilst in a resistant niche microenvironment. Using this model, we showed that under ‘niche-like’ conditions CD34+CD38-CD123+ LSPC are less sensitive to cytarabine and to the Flt3 inhibitor AG1296. In contrast, LSPC when grown under niche support are extremely sensitive to Mylotarg (Gemtuzumab Ozogamicin), one of a few successful chemotherapeutic agents that is currently used in the clinic. We are now proposing that the combination of Mylotarg (10ng/ml) and the farnesyltransferase inhibitor, Tipifarnib/Zarnestra (5microM) is successful at further targeting the LSPC subset resulting in 70% LSPC kill (n=29 AML primary cells) after 48hr in vitro treatment compared to 31% and 12% LSPC kill in Mylotarg and Tipifarnib treated cells, respectively (p<0.0001). Moreover, stromal MS5 co-culture experiments (used to depict drug-induced cell survival in a niche-like setting) with the Tipifarnib/Mylotarg combination showed enhanced cytotoxicity even in such a resistant setting. Because activation of alternative signaling pathways maybe one mechanism that cancer cells use to evade drug-induced cell kill, we used a protein profiler assay to depict the expression of 46 known human phosphokinase proteins when treated with single and drug combination. Using the CD34+CD38- TF1A cell line, 34/46 (74%) of phosphokinase proteins that are essential for functional survival pathways (e.g. p38MAPK, ERK, JNK, AKT, STAT1/2/4/5, GSK3beta, beta-catenin, p53 etc.) were maximally reduced by the drug combination compared to housekeeping phosphoproteins, 12/46 (26%) proteins were unaffected by the drug combination and only 1/46 (the cell cycle checkpoint regulator and putative tumor suppressor Chk2) increased maximally with the drug combination. We next show that the drug combination's success is indeed characterized by the activation of DNA damage response (increase in H2AX foci by immunoflourescence), an induction of phosphoATM (by flow cytometry) leading to the activation of Chk2 and G2/M cell cycle arrest (using BrdU assay). Finally, we demonstrate that Tipifarnib may be acting as a potent P-glycoprotein inhibitor by allowing the retention of Mylotarg in cells that would otherwise efflux the drug out of the cell. In conclusion, the in vitro data presented here clearly shows why we think the Tipifarnib/Mylotarg combination can be successful in the treatment of AML and warrants further investigation to confirm its efficacy in in vivo models.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.