Potential of NF-κB Inhibition in the Treatment of Chronic Lymphocytic Leukemia

Introduction:

The transcription factor NF-κB plays a major role in various cellular functions such as apoptosis, proliferation, and inflammation and is often deregulated in cancer. It is known to regulate the expression of anti-apoptotic genes (e.g. TRAF1, Mcl-1) and to be constitutively active in CLL. Different mouse models indicate a role for NF-κB in CLL pathogenesisand p65 (RelA), a subunit of NF-κB, appears to have prognostic relevance in CLL. Indeed, high binding activity of p65 to its DNA-binding site is predictive of a short time to first treatment, time to subsequent treatment, and overall survival. These data suggest NF-κB as promising treatment target in CLL. Indeed, several NF-κB inhibitors induced apoptosis of mono-cultured CLL cells in vitro. However, the efficacy of NF-κB inhibition has not been tested on CLL cells in the presence of protective microenvironment or in combination with B-cell receptor (BCR) pathway inhibitors.

Methods:

The specific NF-κB inhibitor Dehydroxymethylepoxyquinomicin (DHMEQ) was used alone (2µg/ml, 5µg/ml) or in combination with the Btk-inhibitor Ibrutinib (10µM), the PI3K-inhibitor Idelalisib (10µM), or the Syk-inhibitor R406 (4µM) on primary CLL cells in mono- or in co-culture with stroma cells (M2-10B4- or HS-5-cell lines, ratio CLL cells : stroma cells = 20:1). Apoptosis measurements were performed using AnnexinV/PI stainings. Protein expression was analyzed by western blot using standard protocols. For measuring the activity of the different NF-κB subunits (p65, p50, c-Rel, p52, RelB) with a DNA-binding ELISA, protein lysates were prepared after 8h of treatment with DHMEQ (5µg/ml).

Results:

DHMEQ significantly induced apoptosis of freshly isolated CLL cells in monoculture compared with untreated cells (19% vs. 70% live cells, 5µg/ml, n=6) but surprisingly this effect was suppressed when co-cultured with HS-5 cells (81% vs. 85%). This observation was confirmed with M2-10B4 cells. DHMEQ treatment of mono-cultured CLL cells for 48h significantly downregulated the protein expression of the anti-apoptotic factors Mcl-1 and TRAF1 by 43% and 68% respectively (n=9) while increasing PARP-cleavage four-fold (n=9). However, in co-culture with HS-5 cells no change in the expression of these proteins was observed. A time course analysis of protein expression showed that TRAF1 down-regulation occured after 2h before the increase in PARP-cleavage after 8h (n=6). Although the activity of all NF-κB subunits was reduced in both mono- and co-culture with M2-10B4 cells upon DHMEQ treatment, this effect was attenuated by stroma cell presence: the latter reduced DNA-binding activity of p50 and p65 by 29% and 60% in mono-cultured and 13% and 35% in M2-10B4 co-cultured CLL cells (c-Rel: 25% vs. 15% ; p52: 34% vs. 17% ; RelB: 32% vs. 19% ; n=3). Finally, the combined use of DHMEQ (5µg/ml) with Ibrutinib (n=10) or R406 (n=12) on CLL cells co-cultured with M2-10B4 cells led to a significantly higher rate of apoptosis than DHMEQ, Ibrutinib, or R406 alone. A similar tendency was observed with Idelalisib but it did not reach statistical significance (n=10).

Conclusions:

In contrast to mono-cultured CLL cells, the specific NF-κB inhibitor DHMEQ did not induce apoptosis in stroma-supported CLL cells, suggesting that apoptosis induction by NF-κB inhibition may be impaired by the microenvironment. As seen in the NF-kB activity assay, this could in part be due to maintained high NF-κB activity in CLL cells co-cultured with stroma cells even in the presence of DHMEQ. However, we could also show that the effect of BCR-pathway inhibitors, which are highly effective in the treatment of CLL, was further increased by the use of DHMEQ. To further evaluate these encouraging results, we are currently investigating the molecular basis of these observations. We are also planning to verify the efficacy of DHMEQ combination treatments in a CLL mouse model.