Dissecting the Role of Individual Bcl-2 Members in Response and Resistance to Ibrutinib or Venetoclax in CLL

Introduction. BCL-2 family members are crucial determinants for survival of normal and malignant B cells. Venetoclax, the BCL-2 targeting drug, is now successfully applied in Chronic Lymphocytic Leukemia (CLL). CLL cells cycle between the lymph node (LN) and peripheral blood (PB), and in those compartments display major changes in expression of BCL-XL, MCL-1 and BFL-1, which are reportedly not targeted by Venetoclax. Indeed, prolonged in vitro CD40 engagement also induces expression of these BCL-2 family members and renders CLL cells resistant to Venetoclax (Thijssen et al Haematologica 2015). The BTK inhibitor Ibrutinib drives CLL cells out of the LN and deprives them of survival and proliferative signals. After prolonged therapy, Ibrutinib resistance develops in certain patients, who then show fast disease progression. It is currently unknown how LN expulsion or development of resistance affects expression of BCL-2 members.

In order to design effective, long-term therapies including rational combinations, it is crucial to understand the contribution of distinct BCL-2 family members to resistance against these single drugs. Recent LN emigrants (CD5hi/CXCR4lo) can be distinguished from 'old' returning cells (CD5lo/CXCR4hi; Calissano et al Mol med 2011), which allows for differential analysis of the two compartments using only PB samples. In addition, new BH3 mimetics targeting BCL-XL and MCL-1, and cysteine-reactive stapled BH3 peptides specific for BFL-1 (Huhn et al Cell Chem Biol 2016), constitute new tools for functional profiling of individual pro-survival BCL-2 members.

Aims . We measured changes in prosurvival BCL-2 members in LN emigrants versus returning cells in PB samples of responding and relapsed patients on Ibrutinib treatment, and during Venetoclax ramp-up treatment. We also determined the contribution of individual BCL-2 members in determining CD40-induced resistance to Venetoclax by BH3 mimetic profiling (Peperzak, et al. CDD 2017), and by stapled peptides targeting BFL-1.

Experimental procedures. Surface CD19/CD5/CXCR4 staining was combined with intracellular FACS staining for BCL-2, BCL-XL and MCL-1. Combinations of BH3 mimetics (ABT-199, WEHI-539, A1331852, A1210477, S63845), and a stapled BIM BH3 helix bearing an acrylamide warhead allowing selective covalent engagement of BFL-1, were applied in vitro to dissect Venetoclax resistance. Furthermore, RNA and siRNA nucleofection for individual BCL-2 members into CLL cells was applied.

Results. In pretreatment blood samples, recent LN emigrants have higher BCL-XL and MCL-1 expression than returning cells. This clear distinction collapses in response to Ibrutinib (currently N=15), indicating that although peripheral blood cell counts rise, blocking access to the LN effectively lowers expression of protective BCLXL and MCL-1 in vivo . However, in patients that develop Ibrutinib resistance (N=9), the MCL-1 or BCL-XL pre-treatment profile reappears. For such patients the next therapy might be Venetoclax, and we are investigating retrospectively to what extent Ibrutinib resistance and the rebound in BCL-XL or MCL-1 levels affects sensitivity for Venetoclax in vitro .

Venetoclax treatment associates with rapid reduction of CLL cells from PB and LN (N=8), but in contrast to Ibrutinib no changes in BCL-2 members in LN emigrants versus immigrants were observed. In fact, their levels can even increase in patients that show residual cells at T = 1-1.5 months (N=2).

To dissect the role of BCL-XL, MCL-1 and BFL-1 in CD40-induced maximal resistance to Venetoclax, we combined BH3 mimetics with a stapled peptide targeting BFL-1. We found that Venetoclax resistance in CD40-treated CLL cells can be reverted to almost pre-stimulation susceptibility by combined antagonism of BCL-XL and BFL-1. This pharmacologic approach was complemented with siRNA suppression or RNA overexpression for individual BCL-2 members. Combined, our data indicate that BCL-XL and also BFL-1 contribute strongly to Venetoclax resistance, while MCL-1 unexpectedly seems less dominant.

Conclusions . 1) BCL-2 members can be used not only as drug targets but also as markers for response and relapse. 2) Strategies that combine Venetoclax with Ibrutinib to prevent access to the protective LN environment, or tailored combinations with other BH3 mimetics, hold promise for successful long-term treatment of CLL.