USP7 Cooperates with NOTCH1 to Drive the Oncogenic Transcriptional Program in T-Cell Leukemia.

PURPOSE: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes. EXPERIMENTAL DESIGN: To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models. RESULTS: We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth in vitro and in vivo. CONCLUSIONS: These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.

Loss of Mll3 Catalytic Function Promotes Aberrant Myelopoiesis.

Two of the most common myeloid malignancies, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), are associated with exceedingly low survival rates despite recent therapeutic advances. While their etiology is not completely understood, evidence suggests that certain chromosomal abnormalities contribute to MDS and AML progression. Among the most frequent chromosomal abnormalities in these disorders are alterations of chromosome 7: either complete loss of one copy of chromosome 7 (-7) or partial deletion of 7q (del(7q)), both of which increase the risk of progression from MDS to AML and are associated with chemoresistance. Notably, 7q36.1, a critical minimally deleted region in 7q, includes the gene encoding the histone methyltransferase mixed-lineage leukemia 3 (MLL3), which is also mutated in a small percentage of AML patients. However, the mechanisms by which MLL3 loss contributes to malignancy are unknown. Using an engineered mouse model expressing a catalytically inactive form of Mll3, we found a significant shift in hematopoiesis toward the granulocyte/macrophage lineage, correlating with myeloid infiltration and enlargement of secondary lymphoid organs. Therefore, we propose that MLL3 loss in patients may contribute to the progression of MDS and AML by promoting myelopoiesis.

TRAF6 is a critical regulator of LMP1 functions in vivo.

EBV-encoded latent membrane protein 1 (LMP1) is critical for EBV-driven B-cell transformation and most EBV-associated malignancies and is also implicated in exacerbation of autoimmunity. LMP1 functionally mimics the TNFR superfamily member CD40, but LMP1-induced signals and downstream B-cell functions are amplified and sustained compared with those mediated by CD40. CD40 and LMP1 both depend upon TNFR-associated factor (TRAF) adaptor molecules to mediate signaling but use them differently. LMP1 is dependent upon TRAFs 3 and 5 to deliver B-cell activation signals, while CD40 predominantly uses TRAFs 2 and 6 for this purpose. Both LMP1 and CD40 functions in B cells require TRAF6, which physically associates with both receptors but via different binding sites. In B-cell CD40 signaling, TRAF6 is required for a particular subset of CD40-dependent immune functions in vivo. Inasmuch as CD40 and LMP1 use other TRAFs differentially, we predicted that TRAF6 is critical for a specific subset of LMP1 functions in vivo and that this subset will be overlapping but distinct from the TRAF6-requiring functions of CD40. This study tests this prediction using a B-cell-specific TRAF6-deficient mouse model. We found that B-cell TRAF6 is important for LMP1-mediated antibody and autoantibody production in mice, as well as germinal center formation, but not the secondary lymphoid organ enlargement that results from LMP1 transgenic expression. Results highlight differential TRAF6 requirements for specific B-cell functions by LMP1 versus CD40. These differences may make important contributions to the contrasts between normally regulated CD40 versus pathogenic LMP1-mediated signals.

TRAF binding is required for a distinct subset of in vivo B cell functions of the oncoprotein LMP1.

EBV-encoded latent membrane protein 1 (LMP1) is important for EBV contributions to B cell transformation and many EBV-associated malignancies, as well as EBV-mediated exacerbation of autoimmunity. LMP1 functionally mimics TNF receptor (TNFR) superfamily member CD40, but LMP1 signals and downstream effects are amplified and sustained compared with CD40. CD40 and LMP1 both use TNFR-associated factor (TRAF) adaptor proteins, but in distinct ways. LMP1 functions require TRAFs 3, 5, and 6, which interact with LMP1. However, TRAFs can also contribute to signaling in the absence of direct interactions with cell surface receptors, so we investigated whether their roles in LMP1 in vivo functions require direct association. We show in this study that the LMP1 TRAF binding site was required for LMP1-mediated autoantibody production, the germinal center response to immunization, and optimal production of several isotypes of Ig, but not LMP1-dependent enlargement of secondary lymphoid organs in transgenic mice. Thus, LMP1 in vivo effects can be mediated via both TRAF binding-dependent and -independent pathways. Together with our previous findings, these results indicate that TRAF-dependent receptor functions may not always require TRAF-receptor binding. These data suggest that TRAF-mediated signaling pathways, such as those of LMP1, may be more diverse than previously appreciated. This finding has significant implications for receptor and TRAF-targeted therapies.

Roles of the kinase TAK1 in TRAF6-dependent signaling by CD40 and its oncogenic viral mimic, LMP1.

The Epstein-Barr virus (EBV)-encoded protein latent membrane protein 1 (LMP1) is essential for EBV-mediated B cell transformation and plays a critical role in the development of post-transplant B cell lymphomas. LMP1 also contributes to the exacerbation of autoimmune diseases such as systemic lupus erythematosus (SLE). LMP1 is a functional mimic of the tumor necrosis factor receptor (TNFR) superfamily member CD40, and relies on TNFR-associated factor (TRAF) adaptor proteins to mediate signaling. However, LMP1 activation signals to the B cell are amplified and sustained compared to CD40 signals. We previously demonstrated that LMP1 and CD40 use TRAF molecules differently. Although associating with CD40 and LMP1 via separate mechanisms, TRAF6 plays a significant role in signal transduction by both. It is unknown whether TRAF6 mediates CD40 versus LMP1 functions via distinct or shared pathways. In this study, we tested the hypothesis that TRAF6 uses the kinase TAK1 to trigger important signaling pathways following both CD40 and LMP1 stimulation. We determined that TAK1 was required for JNK activation and interleukin-6 (IL-6) production mediated by CD40 and LMP1, in both mouse and human B cells. Additionally, TRAF3 negatively regulated TRAF6-dependent, CD40-mediated TAK1 activation by limiting TRAF6 recruitment. This mode of regulation was not observed for LMP1 and may contribute to the dysregulation of LMP1 compared to CD40 signals.

Molecular mechanisms of TNFR-associated factor 6 (TRAF6) utilization by the oncogenic viral mimic of CD40, latent membrane protein 1 (LMP1).

Latent membrane protein 1 (LMP1), encoded by Epstein-Barr virus, is required for EBV-mediated B cell transformation and plays a significant role in the development of posttransplant B cell lymphomas. LMP1 has also been implicated in exacerbation of autoimmune diseases such as systemic lupus erythematosus. LMP1 is a constitutively active functional mimic of the tumor necrosis factor receptor superfamily member CD40, utilizing tumor necrosis factor receptor-associated factor (TRAF) adaptor proteins to induce signaling. However, LMP1-mediated B cell activation is amplified and sustained compared with CD40. We have previously shown that LMP1 and CD40 use TRAFs 1, 2, 3, and 5 differently. TRAF6 is important for CD40 signaling, but the role of TRAF6 in LMP1 signaling in B cells is not clear. Although TRAF6 binds directly to CD40, TRAF6 interaction with LMP1 in B cells has not been characterized. Here we tested the hypothesis that TRAF6 is a critical regulator of LMP1 signaling in B cells, either as part of a receptor-associated complex and/or as a cytoplasmic adaptor protein. Using TRAF6-deficient B cells, we determined that TRAF6 was critical for LMP1-mediated B cell activation. Although CD40-mediated TRAF6-dependent signaling does not require the TRAF6 receptor-binding domain, we found that LMP1 signaling required the presence of this domain. Furthermore, TRAF6 was recruited to the LMP1 signaling complex via the TRAF1/2/3/5 binding site within the cytoplasmic domain of LMP1.

Differential B-lymphocyte regulation by CD40 and its viral mimic, latent membrane protein 1.

CD40 plays a vital role in humoral immunity, via its potent and multifaceted function as an activating receptor of various immune cells, most notably B lymphocytes. The Epstein-Barr virus-encoded transforming protein latent membrane protein 1 (LMP1) serves as a functional mimic of CD40 signals to B cells but lacks key regulatory controls that restrain CD40 signaling. This allows LMP1 to activate B cells in an abnormal manner that can contribute to the pathogenesis of human B-cell lymphoma and autoimmune disease. This review focuses upon a comparative analysis of CD40 versus LMP1 functions and mechanisms of action in B lymphocytes, discussing how this comparison can provide valuable information on both how CD40 signaling is normally regulated and how LMP1 disrupts the normal CD40 pathways, which can provide information of value to therapeutic design.