Cross-Talk between Cytokine and NF-ĸB Signaling in the CLL Microenvironment Can Affect Sensitivity for Venetoclax

INTRODUCTION. The Bcl-2 inhibitor Venetoclax provides profound reductions in circulating chronic lymphocytic leukemia (CLL) cells in the majority of patients. The lymph node (LN) responses are less complete, which may be linked with pro-survival signals in the LN. Bcl-XL expression is prominently increased in LN compared to blood. Since Bcl-XL is a prime determinant of resistance to Venetoclax, its regulation is of clinical significance. In the present study, we investigated how candidate LN signals from T helper cytokines IL-21 or IL-4 affect CD40-signalling, specifically in relation to sensitivity/resistance to Venetoclax.

RESULTS In an expression screen of changes in apoptotic regulators following CD40 stimulation, IL-4 and IL-21 had opposing effects specifically on Bcl-XL expression. This was associated with CD40-induced resistance to Venetoclax which was augmented by IL-4 and reversed by IL-21. We investigated the signalling pathway(s) involved and found that upon IL-4 or IL-21 stimulation, STAT3 and -6 were differentially phosphorylated, and pharmacological inhibition of upstream JAK kinases restored Bcl-XL expression. Molecularly, IL-21 or IL-4 signals did not significantly affect (non-)canonical NF-κB activation measured by DNA-binding of p65 or p52. STAT3 and -6 have predicted binding sites near the known p65 and p52 binding sites in the Bcl-XL promoter region. Using reporter assays with Bcl-XL promotor constructs we demonstrate synergy (STAT6) or competition (STAT3) with NF-κB. We next applied novel IKKα and NIK inhibitors to inhibit NF-kB signalling during CD40 stimulation. Especially IKKα inhibition specifically blocked Bcl-XL expression, without affecting overall viability of the CLL cells or normal T cells.Importantly, both types of NF-κB inhibitors significantly reversed CD40-induced resistance for Venetoclax. Averaged LD50 values for Venetoclax increased approximately 2500-fold from 1.8 nM to 4.6 μM after 24 hrs CD40 stimulation, but in the presence of IKKα inhibitor only to 7.4 nM (N=8). This value remains well below the average plasma Venetoclax level in CLL patients undergoing treatment.

CONCLUSIONS. These data show that protective signals from the CLL microenvironment can be tipped towards apoptosis sensitivity by interfering with JAK/STAT and NF-kB signals, providing novel therapeutic clues in case of emerging resistance to targeted drugs such as Venetoclax.