Activity of ABT-199 and Acquired Resistance in Follicular Lymphoma Cells

ABT-199, a platelet-sparing pro-apoptotic BH3 mimetic shows promise in the treatment of B-cell malignancy such as chronic lymphocytic leukemia (CLL). Given the central role of BCL2 expression in follicular lymphoma (FL), we studied the effects of ABT-199 on proliferation, induction of apoptosis, and signaling pathways in the t(14;18)+ FL cell lines WSU-FSCCL and FC-TxFL2 and in primary FL cells. We also developed an ABT-199-resistant FC-TxFL2 cell line by continuous treatment with ABT-199.

The growth of tested cell lines and primary FL cells was significantly inhibited with nanomolar concentrations of ABT-199. FC-TxFL2 cells (IC50 = 7 nM) were more sensitive to ABT-199 treatment than WSU-FSCCL cells (IC50 = 110 nM). The increased sensitivity was associated with higher levels of BIM pro-apoptotic protein. ABT-199 induced rapid phosphorylation of JNK kinase and substantial decrease of mitochondrial potential in FC-TxFL2 cells. Inhibition of JNK activation further augmented the apoptosis induced with ABT-199. All other tested anti-apoptotic (such as BCL-2, BCL-xL, MCL-1) and pro-apoptotic proteins (such as Bax, Puma, Bak, Bad, Noxa, Bid, Bok) were similarly expressed in both cell lines and did not change upon ABT-199 treatment. Surprisingly, cleavage of Bid was observed, which points also to activation of extrinsic apoptotic pathway. In cells with acquired resistance after continuous ABT-199 exposure, we identified increased levels of MCL-1, and elevated phosphorylation of BCL-2 on T56 and of AKT on S473, but also evidence for increased autophagy, as assessed by LC3BI/II expression, compared to parental cells. These cells remained resistant several weeks after omitting ABT-199 from culture medium.

In a FL murine xenograft model, ABT-199 showed anti-tumor activity. Treatment significantly decreased tumors volumes compared to untreated mice. Subsequently isolated xenograft FC-TxFL2 cells previously exposed to ABT-199 showed increased resistance to ABT-199. However, unlike in vitro exposed cells, the resistance quickly diminished after cultivation without ABT-199. Next, the ability to overcome ABT-199 resistance with epigenetic modifiers such as decitabine and vorinostat was tested. A strong synergistic effect in potentiation of apoptosis with these therapeutics was observed.

Despite the theoretic rationale for BH3 mimetic activity in FL, activity in patients with FL has not been as dramatic as has been observed for CLL. Relatively easily acquired resistance observed in our model may explain this failure. Therefore, combinations that can prevent resistance acquisition should be intensively studied and moved quickly to clinical trials.