J. Jessica Yu

BCL6 enables Ph + acute lymphoblastic leukaemia cells to survive BCR–ABL1 kinase inhibition

Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR–ABL1 (ref. 1) and other oncogenic tyrosine kinases. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs), which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones.

BCL6 is critical for the development of a diverse primary B cell repertoire

BCL6 protects germinal center (GC) B cells against DNA damage–induced apoptosis during somatic hypermutation and class-switch recombination. Although expression of BCL6 was not found in early IL-7–dependent B cell precursors, we report that IL-7Rα–Stat5 signaling negatively regulates BCL6. Upon productive VH-DJH gene rearrangement and expression of a μ heavy chain, however, activation of pre–B cell receptor signaling strongly induces BCL6 expression, whereas IL-7Rα–Stat5 signaling is attenuated. At the transition from IL-7–dependent to –independent stages of B cell development, BCL6 is activated, reaches expression levels resembling those in GC B cells, and protects pre–B cells from DNA damage–induced apoptosis during immunoglobulin (Ig) light chain gene recombination. In the absence of BCL6, DNA breaks during Ig light chain gene rearrangement lead to excessive up-regulation of Arf and p53. As a consequence, the pool of new bone marrow immature B cells is markedly reduced in size and clonal diversity. We conclude that negative regulation of Arf by BCL6 is required for pre–B cell self-renewal and the formation of a diverse polyclonal B cell repertoire.

IL-21 and CD40L Synergistically Promote Plasma Cell Differentiation through Upregulation of Blimp-1 in Human B Cells

After undergoing Ig somatic hypermutation and Ag selection, germinal center (GC) B cells terminally differentiate into either memory or plasma cells (PCs). It is known that the CD40L and IL-21/STAT3 signaling pathways play critical roles in this process, yet it is unclear how the B cell transcription program interprets and integrates these two types of T cell–derived signals. In this study, we characterized the role of STAT3 in the GC-associated PC differentiation using purified human tonsillar GC B cells and a GC B cell-like cell line. When primary GC B cells were cultured under PC differentiation condition, STAT3 inhibition by AG490 prevented the transition from GC centrocytes to preplasmablast, suggesting that STAT3 is required for the initiation of PC development. In a GC B cell-like human B cell line, although IL-21 alone can induce low-level Blimp-1 expression, maximum Blimp-1 upregulation and optimal PC differentiation required both IL-21 and CD40L. CD40L, although having no effect on Blimp-1 as a single agent, greatly augmented the amplitude and duration of IL-21–triggered Jak-STAT3 signaling. In the human PRDM1 locus, CD40L treatment enhanced the ability of STAT3 to upregulate Blimp-1 by removing BCL6, a potent inhibitor of Blimp-1 expression, from a shared BCL6/STAT3 site in intron 3. Thus, IL-21 and CD40L collaborate through at least two distinct mechanisms to synergistically promote Blimp-1 activation and PC differentiation.

BCL-6 Negatively Regulates Expression of the NF-κB1 p105/p50 Subunit

BCL-6 is a transcription repressor frequently deregulated in non-Hodgkin’s B cell lymphomas. Its activity is also critical to germinal center development and balanced Th1/Th2 differentiation. Previous studies have suggested that NF-κB activity is suppressed in germinal center and lymphoma B cells that express high levels of BCL-6, and yet the reason for this is unknown. We report in this study that BCL-6 can bind to three sequence motifs in the 5′ regulatory region of NF-κB1 in vitro and in vivo, and repress NF-κB1 transcription both in reporter assays and in lymphoma B cell lines. BCL-6−/− mice further confirm the biological relevance of BCL-6-dependent regulation of NF-κB1 because BCL-6 inactivation caused notable increase in p105/p50 proteins in several cell types. Among these, BCL-6−/− macrophage cell lines displayed a hyperproliferation phenotype that can be reversed by NF-κB inhibitors, e.g., N-tosyl-l-phenylalanine chloromethyl ketone and SN50, a result that is consistent with increased nuclear κB-binding activity of p50 homodimer and p50/p65 heterodimer. Our results demonstrate that BCL-6 can negatively regulate NF-κB1 expression, thereby inhibiting NF-κB-mediated cellular functions.

CtBP Is an Essential Corepressor for BCL6 Autoregulation

ABSTRACT The transcription repressor BCL6 plays an essential role in the formation and function of germinal centers (GCs). While normal B cells promptly shut off BCL6 when they exit the GC, many GC-derived B-cell lymphomas sustain BCL6 expression through chromosomal translocations and activating mutations. We have previously shown that a common effect of lymphoma-associated BCL6 gene alterations is to bypass a negative autoregulatory loop that controls its transcription. In this study, we report that BCL6 autoregulation is independent of several known corepressor complexes including silencing mediator for retinoid and thyroid hormone receptors, nuclear receptor coreceptor, BCL6 corepressor, and MTA3/NuRD. Furthermore, we show that BCL6 can interact with the CtBP (C-terminal binding protein) corepressor both in vitro and in vivo and that CtBP is recruited by BCL6 to its 5′ regulatory region. In lymphoma cell lines carrying BCL6 translocations, small interfering RNA-mediated CtBP knock-down selectively relieved the previously silenced wild-type BCL6 allele but not the translocated alleles, which are driven by heterologous promoters. These results demonstrate that CtBP is a novel BCL6 corepressor and suggest that a unique corepressor requirement for BCL6 autoregulation may allow GC B cells to differentially control the expression of BCL6 and other BCL6 target genes in response to environmental stimuli during the GC stage of B cell development.

Requirement for cyclin D3 in germinal center formation and function.

Germinal centers (GC) of secondary lymphoid tissues are critical to mounting a high-affinity humoral immune response. B cells within the GC undergo rapid clonal expansion and selection while diversifying their antibody genes. Although it is generally believed that GC B cells employ a unique proliferative program to accommodate these processes, little is known about how the GC-associated cell cycle is orchestrated. The D-type cyclins constitute an important component of the cell cycle engine that enables the cells to respond to physiological changes. Cell type- and developmental stage-specific roles of D-type cyclins have been described but the cyclin D requirement during GC reaction has not been addressed. In this study, we report that cyclin D3 is largely dispensable for proliferation and Ig class switching of in vitro activated B cells. In contrast, GC development in Ccnd3−/− mice is markedly impaired, as is the T cell-dependent antibody response. Within the GC, although both switched and unswitched B cells are affected by cyclin D3 inactivation, the IgM− pool is more severely reduced. Interestingly, despite a compensatory increase in cyclin D2 expression, a significant number of Ccnd3−/− GC B cells accumulate in quiescent G0 state. Lastly, although cyclin D3 inactivation did not disrupt BCL6 expression in GC B cells, it completely blocked the GC promoting effect of BCL6 overexpression, suggesting that cyclin D3 acts downstream of BCL6 to regulate GC formation. This is the first demonstration that cyclin D3 plays an important and unique role at the GC stage of B cell development.

Abstract 2913: STAT3 inhibition overcomes CHOP-resistance in the activated B-cell subtype of DLBCL by a ROS-dependent mechanism.

Diffuse large B cell lymphoma (DLBCL) is the most common lymphoid malignancy in the adult population and can be subdivided into two main subtypes, e.g. GCB-DLBCL and ABC-DLBCL. Both subtypes are derived from normal germinal center (GC) B cells found in secondary lymphoid organs but differ in their B cell maturation stage, transformation pathway, and clinical behavior. When treated with either the combination chemotherapy CHOP or the immuno-chemotherapy R-CHOP, ABC-DLBCL patients typically have a significantly worse survival outcome compared to those carrying GCB-DLBCLs. The underlying mechanism for this disparity is unclear. We have previously reported that expression of the STAT3 gene is developmentally regulated in GC B cells; in addition, high level and constitutively activated STAT3 is prevalent in ABC-DLBCL but not in GCB-DLBCL. Although STAT3 activation in epithelial cancers is often associated with increased chemo-resistance, it is unclear if and how activated STAT3 contributes to therapy resistance in ABC-DLBCL. Since the principle cytotoxic component of CHOP is Doxorubicin (Dox), often described as a Topo II inhibitor, we began our investigation by examining DNA damage response (DDR) following Dox treatment in GCB-DLBCL and ABC-DLBCL cell lines. When applied at IC50 concentrations, Dox elicited a progressive and robust DDR in GCB-DLBCL cell lines starting from 1 h after treatment. In ABC-DLBCL cells, however, activation of several DDR markers was delayed until 24 h post treatment. Instead, Dox increased the levels of reactive oxygen species (ROS) in ABC-DLBCL cells with the most prominent increase in mitochondria superoxide as measured by MitoSox staining. The importance of ROS in Dox-triggered cytotoxicity in ABC-DLBCL cells is supported by a direct correlation between the dose response to Dox and H2O2 among ABC-DLBCL cell lines and by the ability of N-acetyl cysteine (NAC) to significantly impede Dox-triggered cell death. Interestingly, STAT3 inhibition by either siRNA-mediated gene silencing or the small molecule inhibitor, CPA-7, led to significant increase in MitoSox staining. STAT3 siRNA also enhanced Dox-triggered superoxide production and cell death. These findings implicate a ROS-detoxification role of STAT3 in ABC-DLBCLs which could be explained, at least in part, by its ability to promote the expression of the superoxide dismutase 2 (SOD2) gene. Finally, our results demonstrate that the STAT3 inhibitor, CPA-7, can markedly sensitize ABC-DLBCL cells to killing by Dox both in cell culture and in a xenograft model of ABC-DLBCL. Our study not only uncovers a novel mechanism of action for Dox in ABC-DLBCL, it also implicates STAT3 in cellular redox control and provides the proof-of-concept data for development of STAT3-directed lymphoma therapy for ABC-DLBCLs. Citation Format: J. Jessica Yu, Yun Mai, Enguang Bi, Hongshan Chen, B. Belinda Ding, Bhaskar Das, Samir Parekh, Yiyu Zou, B. Hilda Ye. STAT3 inhibition overcomes CHOP-resistance in the activated B-cell subtype of DLBCL by a ROS-dependent mechanism. [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 2913. doi:10.1158/1538-7445.AM2013-2913