NF Kappa B as a Therapeutic Target in AML.

Abstract

NF Kappa B is an inducible eukaryotic transcription factor, which is highly conserved throughout nature, and regulates a large number of genes which are essential for immune and inflammatory responses. Inappropriate expression has been implicated in a number of inflammatory and malignant conditions. We have investigated the expression of NF Kappa B in normal mobilised peripheral blood cells, AML cell lines (HL60, NB4, K562, U937) and 27 primary AML cells, with a view with it being a target for NF Kappa B inhibition. The cells were also treated with a range of doses of LC1 (Leuchemix Inc) which includes NF-KB inhibition in its activity profile. The expression of NF Kappa B was determined by Affymetrix Gene Chip Array (22,283 genes) and confirmed by RT-PCR. Overall the leukaemic cells had a greater than a two-fold expression compared with normal cells. Expression was significantly greater in the FAB M2 and M4 subgroups and tended to increase with the degree of morphological differentiation, and with poor risk cytogenetics. There was no relationship between expression age, gender or FLT3 mutation status. Since increased NF Kappa B expression confers resistance to apoptosis, the level of spontaneous apoptosis of cells after 24 hours in vitro was assessed by annexin V expression. Apoptosis was inversely correlated with expression.LC1 (Leuchemix Inc) is a novel agent with NF Kappa B inhibition among its properties. Cells were treated with LC1 in a dose range of 0.002 to 20μmol, and the LC50 determined in an MTS assay. For the cell lines HL60, NB4 and U937 the average LC50 was 6.1μmol. K562 cells have a lower expression of NF Kappa B (40%) compared with the other cell lines and this was reflected in a lower in vitro sensitivity (LC50 > 100μmol). The LC50s for the 27 primary AML stem cell samples ranged from 0.61μmol to > 100μmol with a mode of 7.1μmol. The majority of the cases (24/27) were between 0.61 and 13.2μmol with a mean of 5.9μmol while the 3 outliers have an LC50 which was 4–10 fold higher. In order to assess whether LC1 enhance the cytotoxicity of Ara-C co-incubation experiments were undertaken using the MTS assay. Significant synergy was observed in 8 or 27 cases, and antagonism in 12 of 27 cases.These data suggest that NF Kappa B inhibition with LC1 merits further evaluation as targeted treatment alone, or in combination, in AML.