NF Kappa B as a Therapeutic Target in AML.

NF Kappa B is an inducible transcription factor that regulates the expression of a large number of genes essential for the normal immune and inflammatory responses. However, inappropriate NF Kappa B expression has been implicated in a number of malignancies. In this study, a two fold increase in NF Kappa B1 expression in AML samples, as compared to normal cell populations was shown by Affymetrix gene array. In addition, constitutive activation of the p65 protein component of NF Kappa B was demonstrated by ELISA in 70% of nuclear extracts derived from primary AML samples. In comparison there was no constitutive activation in the normal marrow samples tested. Elevated levels of AML nuclear p65 were inverse correlated with the proportion of cells undergoing spontaneous apoptosis, indicating that increased basal activation of NF Kappa B confers a cytoprotective effect on primary AML blasts. This data strongly suggest that NF Kappa B is a potential therapeutic target in AML. Consequently, the effects of the novel NF Kappa B inhibitor, LC1, were investigated in AML cells in-vitro. Leukemic cells were treated with LC1, at a range of 0.002 – 20mM, and the LC₅₀ values for each sample were determined by MTS assay. The AML cell lines NB4, HL60 and U937 were also evaluated in the same way and demonstrated LC₅₀ values of 2.98, 7.5, and 5.9 μM respectively. K562 cells were remarkably resistant to LC1, and were also shown to have the lowest NF Kappa B expression by Affymetrix gene array. Primary diagnostic AML (n=48) samples were analysed and an LC₅₀ range of between 0.61μM and >100μM was detected with a median of 6.9μM. Annexin V and cell cycle analysis confirmed increased apoptosis as the mode of cell death. A time- and dose-dependent reduction in nuclear p65 NF Kappa B protein levels by LC1 was proven by ELISA. This preceded the induction of apoptosis, demonstrating that the inhibition of nuclear p65 NF Kappa B was not simply a result of cell death. The levels of NF Kappa B expression were correlated with the LC1 in-vitro LC₅₀ response, demonstrating a significant inverse relationship. In addition, the downstream consequences of LC1-induced NF Kappa B inhibition were shown with a decrease in cytokine expression as demonstrated by cytokine array. The potential for synergy between LC1 and the conventional chemotherapeutic agent Ara-C and the proteasome inhibitor Velcade were also measured. Synergy was demonstrated in 60% of cell samples with Ara-C, and 80% with Velcade. This study suggests that LC1 is a potentially useful novel agent for the molecular therapy of acute myeloid leukemia.