Xiaohong Zhao

Coordinated Silencing of MYC-Mediated miR-29 by HDAC3 and EZH2 as a Therapeutic Target of Histone Modification in Aggressive B-Cell Lymphomas

We investigated the transcriptional and epigenetic repression of miR-29 by MYC, HDAC3, and EZH2 in mantle cell lymphoma and other MYC-associated lymphomas. We demonstrate that miR-29 is repressed by MYC through a corepressor complex with HDAC3 and EZH2. MYC contributes to EZH2 upregulation via repression of the EZH2 targeting miR-26a, and EZH2 induces MYC via inhibition of the MYC targeting miR-494 to create positive feedback. Combined inhibition of HDAC3 and EZH2 cooperatively disrupted the MYC-EZH2-miR-29 axis, resulting in restoration of miR-29 expression, downregulation of miR-29-targeted genes, and lymphoma growth suppression in vitro and in vivo. These findings define a MYC-mediated miRNA repression mechanism, shed light on MYC lymphomagenesis mechanisms, and reveal promising therapeutic targets for aggressive B-cell malignancies.

Disruption of the MYC-miRNA-EZH2 loop to suppress aggressive B-cell lymphoma survival and clonogenicity.

c-MYC (hereafter MYC) overexpression has been recognized in aggressive B-cell lymphomas and linked to adverse prognosis. MYC activation results in widespread repression of micro-RNA (miRNA) expression and associated with lymphoma aggressive progression. Our recent study identified a MYC-miRNA-EZH2 feed-forward loop linking overexpression of MYC, EZH2 and miRNA repression. Here, using a novel small-molecule BET bromodomain inhibitor, JQ1, and the EZH2 inhibitor, DZNep, we demonstrated that combined treatment of JQ1 and DZNep cooperatively disrupted MYC activation, resulting in a greater restoration of miR-26a expression and synergistically suppressed lymphoma growth and clonogenicity in aggressive lymphoma cells. Furthermore, CHIP assay demonstrated that MYC recruited EZH2 to miR-26a promoter and cooperatively repressed miR-26a expression in aggressive lymphoma cell lines, as well as primary lymphoma cells. Loss- or gain-of-function approaches revealed that miR-26a functioned as a tumor suppressor miRNA and mediated the combinatorial effects of JQ1 and DZNep. These findings represent a novel promising approach for silencing MYC-miRNA-EZH2 amplification loop for combinatorial therapy of aggressive B-cell lymphomas.

A microenvironment-mediated c-Myc/miR-548m/HDAC6 amplification loop in non-Hodgkin B cell lymphomas

A dynamic interaction occurs between the lymphoma cell and its microenvironment, with each profoundly influencing the behavior of the other. Here, using a clonogenic coculture growth system and a xenograft mouse model, we demonstrated that adhesion of mantle cell lymphoma (MCL) and other non-Hodgkin lymphoma cells to lymphoma stromal cells confers drug resistance, clonogenicity, and induction of histone deacetylase 6 (HDAC6). Furthermore, stroma triggered a c-Myc/miR-548m feed-forward loop, linking sustained c-Myc activation, miR-548m downregulation, and subsequent HDAC6 upregulation and stroma-mediated cell survival and lymphoma progression in lymphoma cell lines, primary MCL and other B cell lymphoma cell lines. Treatment with an HDAC6-selective inhibitor alone or in synergy with a c-Myc inhibitor enhanced cell death, abolished cell adhesion–mediated drug resistance, and suppressed clonogenicity and lymphoma growth ex vivo and in vivo. Together, these data suggest that the lymphoma-stroma interaction in the lymphoma microenvironment directly impacts the biology of lymphoma through genetic and epigenetic regulation, with HDAC6 and c-Myc as potential therapeutic targets.

Unification of de novo and acquired ibrutinib resistance in mantle cell lymphoma

The novel Brutons tyrosine kinase inhibitor ibrutinib has demonstrated high response rates in B-cell lymphomas; however, a growing number of ibrutinib-treated patients relapse with resistance and fulminant progression. Using chemical proteomics and an organotypic cell-based drug screening assay, we determine the functional role of the tumour microenvironment (TME) in ibrutinib activity and acquired ibrutinib resistance. We demonstrate that MCL cells develop ibrutinib resistance through evolutionary processes driven by dynamic feedback between MCL cells and TME, leading to kinome adaptive reprogramming, bypassing the effect of ibrutinib and reciprocal activation of PI3K-AKT-mTOR and integrin-β1 signalling. Combinatorial disruption of B-cell receptor signalling and PI3K-AKT-mTOR axis leads to release of MCL cells from TME, reversal of drug resistance and enhanced anti-MCL activity in MCL patient samples and patient-derived xenograft models. This study unifies TME-mediated de novo and acquired drug resistance mechanisms and provides a novel combination therapeutic strategy against MCL and other B-cell malignancies.

Venetoclax as a single agent and in combination with PI3K‐MTOR1/2 kinase inhibitors against ibrutinib sensitive and resistant mantle cell lymphoma

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Resistance to Ibrutinib in B Cell Malignancies: One Size Does Not Fit All

Ibrutinib resistance, as a result of coordinated rewiring of signaling networks and enforced tumor microenvironment (TME)-lymphoma interactions, drives unrestrained proliferation and disease progression. To combat resistance mechanisms, we must identify the compensatory resistance pathways and the central modulators of reprogramming events. Targeting the transcriptome and kinome reprogramming of lymphoma cells represents a rational approach to mitigate ibrutinib resistance in B cell malignancies. However, with the apparent heterogeneity and plasticity of tumors shown in therapy response, a one size fits all approach may be unattainable. To this end, a reliable and real-time drug screening platform to tailor effective individualized therapies in patients with B cell malignancies is warranted. Here, we describe the complexity of ibrutinib resistance in B cell lymphomas and the current approaches, including a drug screening assay, which has the potential to further explore the mechanisms of ibrutinib resistance and to design effective individualized combination therapies to overcome resistance and disable aggressive lymphomas (see Outstanding Questions).

PLK1 stabilizes a MYC-dependent kinase network in aggressive B cell lymphomas

Concordant activation of MYC and BCL-2 oncoproteins in double-hit lymphoma (DHL) results in aggressive disease that is refractory to treatment. By integrating activity-based proteomic profiling and drug screens, polo-like kinase-1 (PLK1) was identified as an essential regulator of the MYC-dependent kinome in DHL. Notably, PLK1 was expressed at high levels in DHL, correlated with MYC expression, and connoted poor outcome. Further, PLK1 signaling augmented MYC protein stability, and in turn, MYC directly induced PLK1 transcription, establishing a feed-forward MYC-PLK1 circuit in DHL. Finally, inhibition of PLK1 triggered degradation of MYC and of the antiapoptotic protein MCL-1, and PLK1 inhibitors showed synergy with BCL-2 antagonists in blocking DHL cell growth, survival, and tumorigenicity, supporting clinical targeting of PLK1 in DHL.

Abstract 4944: Combined treatment of BTK and PI3K inhibitors synergistically disrupts BCR-signaling, overcomes microenviroment-mediated survival and drug resistance in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is recognized as an aggressive lymphoma characterized by overexpression of cyclin D1 as a result of t (11:14) translocation. It carries poor prognosis due to the emergence of drug resistance and currently no effective cure is available for this disease. Dynamic interactions between the lymphoma cell and its microenvironment play a critical role in lymphoma development and response to therapy. Recently, inhibitors of BCR signaling have become an area of substantial clinical interest. B-cell receptor (BCR) provides essential growth and survival signals to lymphomas. We therefore studied the role of BCR signal pathways in stroma mediated survival and drug resistance of mental cell lymphoma. We demonstrate that all mantle cell lymphoma cell lines and primary MCL cells express key BCR downstream signaling molecules, BTK (Bruton9s tyrosine kinase), AKT and ERK, in activated (phosphorylated) form. Phosphorylation statutes of pBTK, pAKT and pERK are further up regulated upon adhesion to lymph node stroma cells. Cell adhesion between lymphoma cells and tumor stroma cells is essential for tumor cell survival and drug resistance. Inhibition of BTK by PCI 32765 or PI3Kδ by CAL101 abolishes intrinsic as well as stroma induced activation of BTK, AKT, ERK. It also significantly disrupts tumor - stromal cell adhesion and induces cell apoptosis. Moreover, combined treatment of PCI 32765 and CAL101 synergetically inhibit BCR signaling and induce cell apoptosis in absence and presence of HK stroma cells. CXCR4 has been reported to play a critical role in lymphoma cell homing, migration, and adhesion. We evaluated CXCR4 expression upon MCL adhesion to stroma cells. We demonstrate that the CXCR4 level was significantly increased in Jeko-1 and HBL2 cell lines and primary MCL cells when co-cultured with HK stroma cells. The stroma cells-induced CXCR4 increase was significantly abolished by BCR signaling inhibitors, PCI 32765 and Cal101. We further explored the effect of those inhibitors on cyclin D1. We show that cyclin D1 is constitutively expressed in MCL cell lines and primary MCL cells and that its expression was further increased when co-cultured with HK stoma cells. Both PCI 32765 and Cal101 decreased intrinsic cyclin D1 expression in the absence and presence of HK cells. Furthermore, combined treatment of PCI 32765 and Cal101 more strikingly attenuated cyclin D1 expression than either agent alone. Collectively, Our study demonstrates (1) the constitutive expression and biological function of BCR signaling in MCL, (2) the essential role of BCR signaling in microenvironment (stroma cell) mediated MCL survival, adhesion and drug resistance, and (3) the therapeutic potential of using novel BCR inhibitors, PCI 32765 and Cal101, in single or combine for MCL therapy. Citation Format: Jiangchuan Tao, Tint Lwin, Xiaohong Zhao, Bijal Shah, Ling Zhang, Lynn Moscinski, William Dalton, Eduardo M. Sotomayor, Jianguo Tao. Combined treatment of BTK and PI3K inhibitors synergistically disrupts BCR-signaling, overcomes microenviroment-mediated survival and drug 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 4944. doi:10.1158/1538-7445.AM2013-4944