Abstract Introduction: Mantle cell lymphoma (MCL) is not curable and novel therapeutic agents and their rational combinations are needed. The ubiquitin-proteasome pathway is an attractive target for antitumor therapy, as its persistent activity is associated with tumor formation, growth, metastasis, and drug resistance in many cancer types, including B-cell non-Hodgkin lymphoma. 20S proteasome activity is regulated by the catalytic subunits β1, β2, and β5 or in the case of the immunoproteasome (i20S) by the inducible catalytic subunits LMP2 (β1i), MECL-1(β2i), and LMP7 (β5i). Carfilzomib (CFZ), a proteasome inhibitor, inhibits the proteasome by binding specifically and irreversibly to the catalytic subunit β5/β5i. Materials and Methods: CFZ was supplied by Onyx Pharmaceuticals (South San Francisco, CA). The Btk inhibitor ibrutinib (PCI-32765) was provided by Pharmacyclics (Sunnyvale, CA). Human MCL cell lines (Mino, Jeko-1, Z138, and Rec-1) were either created by our laboratory or obtained from American Type Culture Collection. Primary cells were obtained from newly diagnosed MCL patients with informed consent. Cell viability was assessed by an MTT assay, apoptosis by Annexin V binding assays, and proteasome expression by Western blotting. Results: We found that MCL cells had different responses to CFZ. For the CFZ-sensitive MCL cell lines Mino and Z138 and freshly isolated MCL cells from 6 patients, CFZ significantly induced the growth inhibition and apoptosis at a low dose (IC50 2-6 nM). In contrast, for the CFZ-resistant MCL cell line (Rec-1) and fresh primary MCL cells from patients with clinical resistance, CFZ did not induce cell death (IC50 not reached at 100 nM). Next, Western blot analysis showed that the CFZ-resistant cells lacked expression of the i20S subunits LMP2, LMP7, and MECL-1. By contrast, the CFZ-sensitive cells showed high levels of LMP2, LMP7, and MECL-1. These results suggest that an intact immunoproteasome is necessary for CFZ-induced anti-MCL activity. In an attempt to overcome the CFZ resistance, we studied the effect of ibrutinib in CFZ-resistant MCL cells. Ibrutinib alone was toxic to both CFZ-sensitive and CFZ-resistant MCL cells with IC50 values ranging from 3 to 12.5 μM. Furthermore, we found that ibrutinib synergized with CFZ in CFZ-sensitive cell lines, and primary MCL cells. The Chou-Talay combination index was < 1, indicating synergism between the two drugs. Most importantly, the CFZ-resistant MCL cells Rec-1 were sensitive to ibrutinib, with an IC50 of 6 uM. Conclusion: Our data suggest that LMP2, LMP7, and MECL-1 may serve as potential biomarkers for patients who are likely to be sensitive to CFZ. Our data also suggest that ibrutinib may be useful in patients with CFZ-resistant MCL. Our preclinical data provide a strong rationale for testing the ibrutinib-CFZ combination clinically. Citation Format: Zhishuo Ou, Liang Zhang, Kate Newberry, Luhong Sun, Kirk J. Christopher, Alex Rollo, Archito Tamayo, John Lee, Richard J. Ford, Michael Wang, Lan V. Pham. The bruton's tyrosine kinase inhibitor ibrutinib synergized with the proteasome inhibitor carfilzomib and overcame immunoproteasome-mediated carfilzomib resistance in mantle cell lymphoma . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2432. doi:10.1158/1538-7445.AM2013-2432
Abstract We used paired samples to address whether long-term storage of cells in TRIzol has a significant effect on microarray-based gene expression profiling (GEP). The TRIzol reagent is widely used for the preservation and isolation of RNA, but there is suspicion that prolonged storage of tissues prior to RNA isolation, even at −80 C., can cause chemical modification (depurination) of RNA. This could result in early termination during reverse transcription (RT) of mRNA molecules, potentially affecting GEP more strongly for transcripts with probes located farther from the 3’ end. The Myeloma and Leukemia Tissue Banks at M. D. Anderson Cancer Center routinely collect and store primary tumor samples for research under informed consent. From these banks we selected primary tumor samples of multiple myeloma (MM; CD138+) and acute myeloid leukemia (AML; Ficoll-purified, CD3- and CD19-depleted), for which paired aliquots had been stored frozen in TRIzol (“Tri”) or cryopreservation medium (RPMI + 20% FCS + 10% DMSO, “Cryo”) since the time of initial isolation (range, 2-9 years). Cryo aliquots were quickly thawed, washed, and placed in TRIzol, then total RNA was isolated from all aliquots as per the TRIzol manufacturer's instructions. RNA quality was assessed with a Bioanalyzer 2100 (Agilent); the RNA integrity number (RIN) was > 9 for 12/12 AML Tri, 6/12 AML Cryo, 6/6 MM Tri, and 3/6 MM Cryo samples. RNA from 12 sample pairs (6 AML, 6 MM, all with RIN > 7) was further purified with Qiagen RNAeasy columns, and the Illumina® TotalPrep RNA Amplification Kit (Ambion) was used to generate amplified, biotinylated cRNA after RT by the Eberwine procedure. Bioanalyzer-generated histograms of cRNA were similar for all sample aliquots, suggesting no early termination of RT due to TRIzol. GEP was performed with cRNA on Illumina HT-12 BeadArrays. Analysis of “probe-level” data with GenomeStudio software (Illumina), after median normalization, showed excellent concordance between sample Tri/Cryo pairs for all but probes with low intensities (<50). For the 48795 probes on the arrays, no more than 320 gave DiffScores < −13 or > 13 for any sample pair, and most pairs had <50 probes that differed. After exclusion of low-intensity values, hierarchical clustering associated all Tri aliquots with their corresponding Cryo aliquot. We conclude that long-term TRIzol storage has little effect on microarray-based GEP, and preserves RNA quality better than does cryopreservation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2177.
Abstract Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma (NHL) with poor prognoses; novel agents are needed for its therapy. Bruton's tyrosine kinase (Btk) has been identified as an essential kinase for B-cell survival and it is activated through the B-cell receptor (BCR) pathway. Btk has recently emerged as a promising target in MCL, as demonstrated by recent clinical trials on the Btk inhibitor PCI-32765 (Pharmacyclics, Sunnyvale, CA), suggesting that elucidating critical signaling pathways emanating through Btk will hold an important key to deciphering the pathogenesis of MCL that can lead to the development of more effective targeted therapies. In this study, we showed that Btk is constitutively phosphorylated in most MCL cell lines except Rec1 and DB SP53 and is variable among primary cells from patients. We demonstrated that knockdown of Btk by siRNA diminished Btk expression, reduced constitutive NF-kB activation by luciferase assays, leading to the cell growth inhibition and induction of apoptosis in MCL cell lines. MCL cells treated with the Btk inhibitor PCI-32765 effectively inhibits Btk activity, leading to reduced MCL cell growth with IC50 values range from 2-6 uM in Mino, Jeko1, Z138 and JMP1. Interestingly, the IC50 value in DB SP53, which lacks both phosphorylated Btk and membrane immunoglobulins, is 35 uM, suggesting that BCR signaling is not active in these cell lines. PCI-32765 also induced caspase-dependent apoptosis in a dose- and time-dependent manner in representative MCL cell lines and in patient primary cells. We further demonstrated that PCI-32765 down-regulates NF-kB activity through both, the canonical and alternative NF-kB pathways. Since previous studies have indicated synergy between proteasome inhibitors and tyrosine kinase inhibitors, we evaluated whether there is synergism between PCI-32765 and the next generation proteasome inhibitor Carfilzomib (Onyx Pharmaceuticals, South San Francisco, CA). Our data suggest potential synergy between Carfilzomib and PCI-32765 in represented MCL cell lines in terms of inhibiting cell growth and induction of apoptosis. Both compounds also synergize to inhibit NF-kB activation in MCL cells. In summary, our data suggest that Btk is a key survival kinase in MCL and strategic targeting of growth/survival Btk-mediated NF-kB pathways with novel therapeutic agents such as PCI-32765 should provide a novel therapy regimen for MCL patients. Combining PCI-32765 with the proteasome inhibitor Carfilzomib can synergize its effect in MCL and may be a useful therapeutic strategy, particularly for patients with relapsed/refractory MCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2854. doi:1538-7445.AM2012-2854
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