New Anti-Cancer Drug Compounds to Treat FLT-3 Mutated Leukemia

Around 30 % of the acute myeloid leukemia (AML) patient present a FLT3 mutations, that can be divided in 2 groups: punctual mutations represent 5 % and internal tandem duplicates represent around 25 % (FLT3-ITD). AML with FLT3-ITD mutations lead to significantly poor prognosis often requiring allogenic stem cell transplant. Different drugs are used to treat AML such as midostaurin, azacytidine, and daunorubicin.

In this study, MOLM-13 cells (adult acute myeloid leukemia), expressing the FLT3-ITD mutation, were used to test different drugs to decrease their cell proliferation and increase their apoptosis level, compared to midostaurin (an inhibitor of FLT3), for 24 hours treatment. MOLM-13 cells were cultured in RPMI1640, in presence of 2, 10 and 25 % of fetal bovine serum (FBS). 25 % FBS was tested to be as close as possible with the level of serum present in the blood. Adipose stromal cells (ASC) were used as a reference control, in absence of serum. Compounds A (targeting the FLT3 pathway) was used at various concentrations (0.05 to 100 µM), over 24h of treatment, in combination or not with compound B (targeting the DNA). Compound A has a high affinity for IRAK4, downstream kinase of TLR pathway, and has the potential to block FLT3 pathway. TLR pathway activation in leukemia, through Myd88 and IRAK4, is involved in the pro-inflammation response but it is also inducing cell proliferation and cell survival. Compound B is an inhibitor of the poly (ADP-ribose) polymerase (PARP), which are enzymes that are involved in DNA transcription, cell cycle regulation and DNA repair.

When ASC were treated with the compound A, the caspase 3/7 activity was not different from the control, but the number of cells was lower at 50 and 100 µM, indicating that the compound A was delaying the cell growth. The number of MOLM-13 cells decreased, in a dose response manner with the compound A (1 to 100 µM). The level of caspase 3/7 activity was elevated, in a dose response manner, which is consistent with the decreased number of cells. MOLM-13 cells resisted more to the anti-cancer effect of the compound A at 25 % FBS, indicating that it could work on patients. Midostaurin, from 0.05 to 100 µM, was reducing the number of cells and increasing the caspase 3/7 activity in the cells, in a dose response manner, at 2, 10 and 25 % FBS. However, data suggest that at the lowest doses, midostaurin was more efficient than compound A, but the compound A was more efficient at higher dose than midostaurin. Compound B alone, at 10 µM, was decreasing the number of MOLM-13 cells and increase the apoptotic level for the 2, 10 and 25 % FBS. A synergistic effect was demonstrated with compound B (10 µM) mixed with low concentration of the compound A (from 1 to 50 µM), at 25 % FBS. This latest result suggest that it will be possible to lower the posology of the compound A when it will be given to the patients, in combination with the compound B.

In conclusion, the data showed the compound A and compound B decreased the level of proliferation and increased the level of apoptosis in MOLM-13 cells, cultured with 2 to 25 % of FBS fetal bovine serum, even if MOLM-13 cells were more resistant to the deleterious drugs effect at 25% FBS. Their combination had a synergic effect in increasing the caspase 3/7 activity and decreasing the cell proliferation. Further studies will be necessary to understand more the molecular mechanism affected by the compound A and/or B in the FLT-3 pathway. The compounds A and B demonstrate potential in treatment of AML expressing FLT-ITD.