Abstract
Mantle cell lymphoma (MCL), which overexpresses cyclin-D1 through an alteration in the t(11;14)(q13;q32) chromosomal region, is associated with brief disease-free and overall survival durations characteristic of aggressive B-cell lymphomas. Bruton tyrosine kinase (BTK) has been identified as a key component of the B-cell antigen receptor (BCR) signaling pathway and is implicated in the pathogenesis of certain B-cell malignancies. Phase III clinical trials of BKT inhibitor ibrutinib in MCL patients have demonstrated clinical responses characterized by mobilization of tissue-resident MCL cells into the peripheral blood. However, since the time to maximum response with ibrutinib is relatively long and patients may become resistant to BTK inhibition, combination regimens that accelerate time to remission and increase depth of remission are of considerable interest.
We hypothesized that combinations of ibrutinib with proapoptotic drugs that function independently of BCR signaling could yield synergistic anti-lymphoma interactions. Thus we investigated the antitumor effects and molecular mechanisms of simultaneous treatment with ibrutinib and selexinor, an oral selective inhibitor of nuclear export (SINE)(KPT-330, Karyopharm), or ABT-199, a selective Bcl-2 inhibitor. SINE agents exhibit antiproliferative and proapoptotic activities against MCL cells via inhibition of nuclear export of tumor suppresor proteins, transcription factors and oncogenic mRNAs and repression of ribosomal biogenesis (Tabe et al. ASH 2013). Selinexor showed promising anti tumor activity in agrresive lymphoma as part of ongoing Phase 1 study (ASCO 2014). ABT-199 has promising proapoptotic activity in relapsed/refractory CLL and NHL without inducing thrombocytopenia.
In this study, we utilized four MCL cell lines: MINO, Z138, Jeko-1, and JVM2. Inhibition of BTK activity by ibrutinib resulted in reduction of cell proliferation in a dose-dependent manner with G0/G1 cell cycle arrest but no apoptosis induction (IC50 at 48 hrs by MTT: 5.4 mM for MINO, 3.5 mM for Z138, 0.5 mM for Jeko-1, 3.1 mM for JVM2). Western blot analysis demonstrated ibrutinib-induced downregulation of phospho-(p-)BTK, p-Akt, mTORC1 substrates p-S6K and p-4EBP1, and cyclin D1 expression. Single-agent selinexor induced cell growth inhibition with G0/G1 cell cycle arrest in a dose-dependent manner (IC50 ranging from 10 nM to 130 nM). The ibrutinib/selinexor combination resulted in further decrease of p-4EBP1 and cyclin D1 expression and downregulation of p-Rb, c-Myc, and Mcl-1, which translated into synergistic reduction of cell proliferation in three of the four tested cell lines (combination index [CI]: 0.4 for MINO, 0.2 for Jeko-1, 0.2 for JVM2). ABT-199 inhibited cell proliferation with apoptosis induction in MINO and Z138 cells (IC50: 1.5 nM for MINO, 17.5 nM for Z138), which synergistically enhanced the antiproliferative effects of ibrutinib (CI: 0.6 for MINO, 0.8 for Z138) with striking reductions of p-4EBP1, cyclin D1, p-Rb, and c-Myc expression along with induction of Bax and cleaved caspase-3.
To investigate the molecular modifications of the cellular pathway network in response to BTK blockade by ibrutinib alone or in combination with selinexor or ABT-199, we employed the proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ). In MINO cells, iTRAQ identified 1,401 unique proteins. Ibrutinib induced downregulation of isoform BTK (p=0.02) and the cell cycle initiation of mitosis pathway (p=0.003) with decreases of ribosomal proteins and elongation factors. Combination with selinexor upregulated the apoptosis and oxidative stress–associated pathways with increases of cytochrome c, voltage-dependent anion channels, HSP10, and histone H1, all of which function as dynamic initiators of mitochondria-mediated apoptosis (p < 0.05). ABT-199 by itself induced upregulation of the apoptosis and oxidative stress–associated pathways (p < 0.001), and these effects were significantly enhanced by combination with ibrutinib.
Taken together, our findings suggest that treatment with combinations of ibrutinib and selinexor or ABT-199 exerts synergistic antiproliferative effects through inhibition of mTOR signaling, downregulation of ribosomal biosynthesis, and induction of mitochondria-mediated apoptosis. These combinations warrant further evaluation in clinical trials in MCL patients.
Disclosures
Andreeff: Karyopharm: Research Funding.
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