Combinations of Kinase Inhibitors May Be An Effective Therapeutic Strategy for Acute Myeloid Leukemia (AML) with MLL-AF9 Expression.
Authors: Poiré Xavier , Diaz-Flores Ernesto , Bergerson Rachel Joy , Sher Dorie , Odenike Olatoyosi , Shannon Kevin , Largaespada David , Stock Wendy 
Published: October 2019

Abstract
Abstract 1032
Poster Board I-54
MLL-AF9 translocation is the most common MLL fusion gene found in AML and has been associated with a relatively poor prognosis. The acquisition of MLL-AF9 is a crucial event in leukemogenesis but might require additional genetic events to induce AML. Mutations in cell-signaling molecules including FLT3 and RAS, which confer cell survival and proliferation advantages, occur frequently in MLL leukemias and point to a potential crucial pathogenetic role of the receptor tyrosine kinase (RTK) FLT3 and downstream PI3K/AKT, RAS/MAPK and STAT5 pathways. Targeted inhibition of these pathways can be achieved therapeutically, but efficacy of a single kinase inhibitor (KI) is often limited due to activation of compensatory survival pathways. To explore potential synergy and better efficacy, we have tested a number of KI combinations in MLL-AF9 murine leukemia cells.

METHODS:
AML was induced in MLL-AF9 knock-in mice using the murine leukemia virus MOL4070LTR, and 4 different cell lines have been generated and listed as 467D, 512C, 467B and 417B. The following KIs were tested in various dual combinations: rapamycin, a mTOR inhibitor; PI-103, a PI3K inhibitor; sunitinib, a RTK inhibitor; erlotinib, another RTK inhibitor with off-target effects on STAT5; PD0325901, a MEK inhibitor; and P6, a JAK inhibitor. KIs concentrations for the combination treatments were based on available in vivo pharmacological data. Effects on cell cycle were measured using BrdU incorporation on all 4 cell lines and phospho-protein levels were measured on 512C using flow cytometry 24-hours after drug exposure.

RESULTS:
Each drug alone modestly affected cell proliferation, but some of the combinations exerted marked anti-proliferative and pro-apoptotic effects, suggesting synergy. Interestingly, some heterogeneity in response to the different combinations was seen in the 4 cell lines. Indeed, the most effective drug duets were PI-103 and P6 in 467D, rapamycin and PI-103 in 512C, and sunitinib and PD0325901 in 467B and 417B. The significant inhibitory effect of some of these combinations on cell cycle progression correlated with a decrease of phosphorylated histone 3 and an increase in cleaved caspase. Furthermore, we were able to demonstrate a correlation with intracellular signaling events, including strong inhibition of pS6, pmTOR, p4EBP1, peIF4E, pErk and Bcl-xl. No effect on pSTAT5 was observed. Despite the heterogeneity in response, 3 different drug combinations were identified as being the most effective for all 4 cell lines: rapamycin and PD0325901, rapamycin and sunitinib, and sunitinib and PD0325901 resulted in a mean decrease in S phase of 24.4 ± 6.4% and a mean increase in apoptosis of 7.2 ± 4.9% after 24 hours exposure in all 4 cell lines. Furthermore, the combination of sunitinib with PD0325901 resulted in a prolonged effect on proliferation and apoptosis after 48 and 72-hour exposure, while the other 2 combinations showed only a transient effect.

CONCLUSIONS:
The combination of sunitinib and PD0325901 was active in all 4 cell lines, overcoming the genetic heterogeneity, and will now be tested in vivo in these murine MLL-AF9 leukemias. Our results suggest that combined blockade of RTK and MEK signaling may be an effective and attractive therapeutic strategy for MLL-AF9 AML.

Disclosures:
Sher: invivoscribe: Employment.