A cyclin-D1 interaction with BAX underlies its oncogenic role and potential as a therapeutic target in mantle cell lymphoma

Abstract

The chromosomal translocation t(11;14)(q13;q32) leading to cyclin-D1 overexpression plays an essential role in the development of mantle cell lymphoma (MCL), an aggressive tumor that remains incurable with current treatment strategies. Cyclin-D1 has been postulated as an effective therapeutic target, but the evaluation of this target has been hampered by our incomplete understanding of its oncogenic functions and by the lack of valid MCL murine models. To address these issues, we generated a cyclin-D1-driven mouse model in which cyclin-D1 expression can be regulated externally. These mice developed cyclin-D1-expressing lymphomas capable of recapitulating features of human MCL. We found that cyclin-D1 inactivation was not sufficient to induce lymphoma regression in vivo; however, using a combination of in vitro and in vivo assays, we identified a novel prosurvival cyclin-D1 function in MCL cells. Specifically, we found that cyclin-D1, besides increasing cell proliferation through deregulation of the cell cycle at the G 1 -S transition, sequestrates the proapoptotic protein BAX in the cytoplasm, thereby favoring BCL2's antiapoptotic function. Accordingly, cyclin-D1 inhibition sensitized the lymphoma cells to apoptosis through BAX release. Thus, genetic or pharmacologic targeting of cyclin-D1 combined with a proapoptotic BH3 mimetic synergistically killed the cyclin-D1-expressing murine lymphomas, human MCL cell lines, and primary lymphoma cells. Our study identifies a role of cyclin-D1 in deregulating apoptosis in MCL cells, and highlights the potential benefit of simultaneously targeting cyclin-D1 and survival pathways in patients with MCL. This effective combination therapy also might be exploited in other cyclin-D1-expressing tumors.antle cell lymphoma (MCL) is a distinct lymphoma entity that accounts for ∼6-8% of all cases of lymphoma (1, 2). MCL is thought to be derived from naïve pregerminal center B lymphocytes localized in primary follicles or in the mantle region of secondary follicles, and thus most tumors do not show somatic hypermutation of the Ig heavy-chain coding (IGH) genes. Cytologically, two major MCL subsets can be distinguished, the classical and blastoid/pleomorphic variants, which share a characteristic CD19 + CD5 + CD23 − immunophenotype (2). Almost all MCL cases show the chromosomal translocation t(11;14)(q13;q32), which juxtaposes the CCND1 gene with IGH gene enhancers and causes overexpression of the cyclin-D1 protein. The best-known function ascribed to cyclin-D1 is in positive regulation of cell cycling. In MCL cells, constitutive cyclin-D1 activation maintains retinoblastoma (RB) protein in a phosphorylated state and promotes cell proliferation, thus likely initiating tumorigenesis (3). However, whether cyclin-D1 has additional oncogenic functions in the lymphoma cells has not been well addressed. In B lymphocytes, cyclin-D1 deregulation seems insufficient to induce neoplastic transformation, given that other genetic changes are required for the development of malignancy (3, 4). Numero...

Acquired mutations in BCL2 family proteins conferring resistance to the BH3 mimetic ABT-199 in lymphoma

“…Establishment and characterization of mouse lymphoma models of resistance to To ascertain whether ABT-199 might induce mutations in target proteins and thus confer therapeutic resistance, we chose MCL as a model disease, because this aggressive cyclin-D1-expressing B-cell lymphoma frequently shows constitutive expression of BCL2 and responds to 27 We used human-like MCL cell lines developed by stably coexpressing cyclin-D1 and Bcl2 in mouse B lymphocytes (supplemental Figure 1). 27 Two mouse cell lines, LyBcl2-6 and LyBcl2-9, were cultured with increasing concentrations of ABT-737, followed by sustained exposure to ABT-199 for 5 months ( Figure 1A).…”

“…The fragment coding amino acids 1 to 204 of mBcl2 (lacking transmembrane domain) was obtained by standard PCR using the pBABE-puro-mBCL2 plasmid as template 27 and then was subcloned into the expression vector pETM-14 using the GIBSON Assembly Cloning Kit (New England BioLabs) according to the manufacturer's instruction. F101L and F101C mutants were generated using site-directed mutagenesis as describe above.…”

“…27 Briefly, CyD1-4 cells were established by cloning the human CCND1 gene into the Combit-TA vector, which was stably transfected into mouse interleukin (IL)-3-dependent BaF3 pro-B lymphocytes. Then, the murine Bcl2 complementary DNA (cDNA) cloned in the pBABE-puro vector (Addgene) was transduced by retroviral infection into CyD1-4 cells, which were also transduced with the SFG-nesTGL expression vector containing a fusion protein with GFP, luciferase, and HSV1-tk.…”

Emerging role of SOX11 in mantle cell lymphoma

“…28,29 In MCL, it has been assumed that Cyclin D1 primarily acts through its role in the cell cycle, controlling the transition of G 1 to S phase by binding and activating cyclin-dependent kinase (CDK) 4 and CDK6 followed by phosphorylation of the retinoblastoma protein and release of the transcription factor E2F. 30 However, in other malignancies several CDK-independent functions of Cyclin D1 have been identified, including interaction with the pro-apoptotic protein BAX 21 and RAD51, 31 the latter impacting DNA repair.…”

“…20 However, although Cyclin D1 is overexpressed in MCL, it has been shown that Cyclin D1 does not induce MCL development in vivo. 21 Furthermore, the lack of prognostic information related to Cyclin D1 22 and the existence of Cyclin D1 negative MCL (7%-15%) challenges the requisite of this protein for tumorigenesis. 18,23 Knockdown of Cyclin D1 has also been reported to only have a limited effect on cell proliferation and apoptosis.…”