Abstract 1742: Ibrutinib plus proteasome or MALT1 inhibitors overcome resistance to BCR antagonists in CARD11 mutant-expressing B-lymphoma cells
Authors: Xue Ling , Apatira Mutiah , Sirisawad Mint , Chang Betty 
Published: August 2015

Abstract
Background: Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with poor prognosis. Ibrutinib, a covalent first in-human Bruton's tyrosine kinase inhibitor, approved for second line treatment for MCL patients, has an overall response rate of nearly 70% in patients with relapsed and refractory MCL (Wang, 2013). However, combination strategies with novel therapies remain an important option for the 30% of non-responding or progressing patients. Caspase recruitment domain-containing protein (CARD)11 is a critical component of NF-κB signaling in B and T cells (Stepensky, 2013). Mutations in the coiled-coil domain of CARD11 can result in constitutive NF-κB activation and resistance to B-cell receptor (BCR) antagonists in ABC-DLBCL (Lenz, 2008) and MCL cell lines (Rahal, 2014). This study investigated the effect of CARD11 mutations on MCL tumor growth, and evaluated combination strategies to overcome resistance to BCR antagonists.
Methods: Jeko cells (an ibrutinib-sensitive MCL cell line [IC50 = 0.5 μM]) were co-infected with lentiviral vector pCDH-EF1 expressing either wildtype (WT) or CARD11 mutants (mut2 [L244P] or mut10 [L225LI], Lenz, 2008), generated by site-directed mutagenesis, and with lentiviral vector pGreenPuro expressing an endogenous knockdown small hairpin (sh)RNA targeting the 3′-untranslated region of CARD11. Stable transfectants were compared for their sensitivity to ibrutinib. Pharmacological inhibitors of NF-κB, PI3K/SYK, proteasomes, or apoptosis pathways were also evaluated in combination with ibrutinib.
Results: The mutant stable cell lines have similar levels of CARD11 expression, which are comparable to levels of endogenous CARD11. CARD11 shRNA inhibited 70% of endogenous CARD11 expression resulting in cell death. Jeko cells expressing WT or mutant CARD11 were protected from shRNA toxicity, and cells expressing mut10 CARD11 proliferated 40% faster than WT-expressing cells. Mutant cells were less sensitive to ibrutinib (IC50 = 0.8 μM [WT]; IC50 = 1.9 [mut2] or 2.6 μM [mut10]). In mutant CARD11-expressing Jeko cells, ibrutinib inhibited the activity of downstream BCR signaling molecules ERK and AKT, but not NF-κB. Addition of the proteasome inhibitors (carfilzomib or bortezomib) or the MALT1 inhibitor MI2 sensitized CARD11 mutant cells to ibrutinib treatment (IC50 = 2.6 μM [ibrutinib]; IC50 = 0.2 μM [ibrutinib+bortezomib]; IC50 = 0.1 μM [ibrutinib+MI2]). MI2 also facilitated the degradation of CARD11 protein, and synergized with ibrutinib for complete inhibition of the NF-κB pathway. Analysis of the ibrutinib and MI2 combination in murine models is ongoing.
Conclusion: This study showed that the combination of ibrutinib with proteasome or MALT1 inhibitors can sensitize CARD11 mutant cells to treatment and may provide a strategy to overcoming resistance to BCR-antagonists in-vivo.
Note: This abstract was not presented at the meeting.
Citation Format: Ling Xue, Mutiah Apatira, Mint Sirisawad, Betty Chang. Ibrutinib plus proteasome or MALT1 inhibitors overcome resistance to BCR antagonists in CARD11 mutant-expressing B-lymphoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1742. doi:10.1158/1538-7445.AM2015-1742