Shengjian Huang

B-Cell Lymphoma Patient-Derived Xenograft Models Enable Drug Discovery and Are a Platform for Personalized Therapy

Purpose: Patients with B-cell lymphomas often relapse after frontline therapy, and novel therapies are urgently needed to provide long-term remission. We established B-cell lymphoma patient-derived xenograft (PDX) models to assess their ability to mimic tumor biology and to identify B-cell lymphoma patient treatment options. Experimental Design: We established the PDX models from 16 patients with diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, or Burkitt lymphoma by inoculating the patient tumor cells into a human bone chip implanted into mice. We subjected the PDX models to histopathologic and phenotypical examination, sequencing, and drug efficacy analysis. Primary and acquired resistance to ibrutinib, an oral covalent inhibitor of Bruton tyrosine kinase, were investigated to elucidate the mechanisms underlying ibrutinib resistance and to identify drug treatments to overcome resistance. Results: The PDXs maintained the same biological, histopathologic, and immunophenotypical features, retained similar genetic mutations, and produced comparable drug responses with the original patient tumors. In the acquired ibrutinib-resistant PDXs, PLC-γ2, p65, and Src were downregulated; however, a PI3K signaling pathway member was upregulated. Inactivation of the PI3K pathway with the inhibitor idelalisib in combination with ibrutinib significantly inhibited the growth of the ibrutinib-resistant tumors. Furthermore, we used a PDX model derived from a clinically ibrutinib-relapsed patient to evaluate various therapeutic choices, ultimately eliminating the tumor cells in the patients peripheral blood. Conclusions: Our results demonstrate that the B-cell lymphoma PDX model is an effective system to predict and personalize therapies and address therapeutic resistance in B-cell lymphoma patients. Clin Cancer Res; 23(15); 4212–23. ©2017 AACR.

Dual inhibition of PI3K signaling and histone deacetylation halts proliferation and induces lethality in mantle cell lymphoma

The dysregulation of PI3K signaling has been implicated as an underlying mechanism associated with resistance to Bruton’s tyrosine kinase inhibition by ibrutinib in both chronic lymphocytic leukemia and mantle cell lymphoma (MCL). Ibrutinib resistance has become a major unmet clinical need, and the development of therapeutics to overcome ibrutinib resistance will greatly improve the poor outcomes of ibrutinib-exposed MCL patients. CUDC-907 inhibits both PI3K and HDAC functionality to exert synergistic or additive effects. Therefore, the activity of CUDC-907 was examined in MCL cell lines and patient primary cells, including ibrutinib-resistant MCL cells. The efficacy of CUDC-907 was further examined in an ibrutinib-resistant MCL patient-derived xenograft (PDX) mouse model. The molecular mechanisms by which CUDC-907 dually inhibits PI3K and histone deacetylation were assessed using reverse protein array, immunoblotting, and chromatin immunoprecipitation (ChIP) coupled with sequencing. We showed evidence that CUDC-907 treatment increased histone acetylation in MCL cells. We found that CUDC-907 caused decreased proliferation and increased apoptosis in MCL in vitro and in vivo MCL models. In addition, CUDC-907 was effective in inducing lethality in ibrutinib-resistant MCL cells. Lastly, CUDC-907 treatment increased histone acetylation in MCL cells. Overall, these studies suggest that CUDC-907 may be a promising therapeutic option for relapsed or resistant MCL.

Strategic therapeutic targeting to overcome venetoclax resistance in aggressive B-cell lymphomas

Purpose: B-cell lymphoma-2 (BCL-2), an antiapoptotic protein often dysregulated in B-cell lymphomas, promotes cell survival and provides protection from stress. A recent phase I first-in-human study of the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma showed an overall response rate of 44%. These promising clinical results prompted our examination of the biological effects and mechanism of action underlying venetoclax activity in aggressive B-cell lymphoma, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Experimental Design: MCL and DLBCL cell lines, primary patient samples, and in vivo patient-derived xenograft (PDX) models were utilized to examine venetoclax efficacy. Furthermore, the mechanisms underlying venetoclax response and the development of venetoclax resistance were evaluated using proteomics analysis and Western blotting. Results: Potential biomarkers linked to venetoclax activity and targeted combination therapies that can augment venetoclax response were identified. We demonstrate that DLBCL and MCL cell lines, primary patient samples, and PDX mouse models expressing high BCL-2 levels are extremely sensitive to venetoclax treatment. Proteomics studies showed that venetoclax substantially alters the expression levels and phosphorylation status of key proteins involved in cellular processes, including the DNA damage response, cell metabolism, cell growth/survival, and apoptosis. Short- and long-term exposure to venetoclax inhibited PTEN expression, leading to enhanced AKT pathway activation and concomitant susceptibility to PI3K/AKT inhibition. Intrinsic venetoclax-resistant cells possess high AKT activation and are highly sensitive to PI3K/AKT inhibition. Conclusions: These findings demonstrate the on-target effect of venetoclax and offer potential mechanisms to overcome acquired and intrinsic venetoclax resistance through PI3K/AKT inhibition. Clin Cancer Res; 24(16); 3967–80. ©2018 AACR.

The CD20-specific engineered toxin antibody MT-3724 exhibits lethal effects against mantle cell lymphoma

Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma (NHL) subtype with aggressive clinical demonstration characterized by the expression of neoplastic Bcell markers such as CD5, CD19, CD20, CD79, and BSAP/ PAX5. Notably, CD20 is strongly expressed by neoplastic B cells, enabling this cell surface protein to be exploited as a therapeutic target. Targeting CD20 with anti-CD20 monoclonal antibodies such as rituximab has shown clinically meaningful outcomes. To enhance antibody-mediated therapy, immunotoxins are utilized as an innovative cancer therapy tool that links an antibody or antibody fragment with a toxin, selectively localizing the toxin to the target cells to induce lethality. MT-3724, an engineered toxin body (ETB) comprised of a modified cytotoxic Shiga-like toxin 1A (3F7) and a CD20-specific single-chain variable fragment (scFv), recognizes CD20-expressing cells and triggers protein synthesis inhibition and apoptosis. Although targeted therapy such as Bruton’s tyrosine kinase (BTK) inhibition by ibrutinib has achieved high response rates (68%) in relapsed/refractory MCL, therapeutic resistance has emerged as a barrier to improved patient outcomes and survival. MT-3724 has the potential to bypass possible resistance mechanisms mediated via acquired BTK mutations or the activation of alternative survival signaling pathways by inhibiting tumor growth and survival through toxin-mediated activity. To assess the antiMCL effects of MT-3724, we tested its in vitro and in vivo efficacy in MCL cell lines and patient-derived xenograft (PDX) mouse models. To correlate MT-3724 cytotoxicity with CD20 expression, CD20 surface expression was examined across 8 MCL cell lines (Supplementary Fig. S1A), and the CD20 MFI varied among different cell lines (Supplementary Fig. S1B and Supplementary Table S1). Four cell lines were treated with two MT-3724 doses for 24 h, resulting in undetectable CD20 expression, suggesting complete occupation of CD20 with MT-3724 (Supplementary Fig. S1C). We next verified whether MT-3724 induces cytotoxic activity against MCL. Indeed, MT-3724 inhibited the growth of MCL cell lines dose dependently (Fig. 1a), with the MT-3724 IC50 value ranging from 78 to 1383 ng/mL (Supplementary Table S1). No negative correlation between the IC50 and CD20 MFI was observed among the MCL cell lines (Supplementary Fig. S1D). However, no significant difference in the MT-3724 IC50 values was observed among the ibrutinib-sensitive and ibrutinibresistant cell lines (Fig. 1b). Furthermore, 300 ng/mL MT3724 was sufficient to reduce cell growth over time (Fig. 1c). Shiga toxin triggers mitochondrial stress via various cellular mechanisms such as decreasing anti-apoptotic protein levels, including MCL-1 and BCL-2. To investigate whether MT-3724 induces apoptosis or cell cycle arrest in MCL, one pair of cell lines (Jeko-1 and Jeko-R) was treated with different MT-3724 doses for 24 h. As previously reported, Jeko-R is an acquired ibrutinibresistant MCL cell line generated through chronic exposure to low ibrutinib concentrations. MT-3724 induced apoptosis, and the percentage of apoptotic cells (Fig. 1d–e) and caspase 3/7 expression (Supplementary Fig. S2A-B) correlated with dosage in both cell lines. MT-

Activation of MYC, a bona fide client of HSP90, contributes to intrinsic ibrutinib resistance in mantle cell lymphoma

The BTK inhibitor ibrutinib has demonstrated a remarkable therapeutic effect in mantle cell lymphoma (MCL). However, approximately one-third of patients do not respond to the drug initially. To identify the mechanisms underlying primary ibrutinib resistance in MCL, we analyzed the transcriptome changes in ibrutinib-sensitive and ibrutinib-resistant cell lines on ibrutinib treatment. We found that MYC gene signature was suppressed by ibrutinib in sensitive but not resistant cell lines. We demonstrated that MYC gene was structurally abnormal and MYC protein was overexpressed in MCL cells. Further, MYC knockdown with RNA interference inhibited cell growth in ibrutinib-sensitive as well as ibrutinib-resistant cells. We explored the possibility of inhibiting MYC through HSP90 inhibition. The chaperon protein is overexpressed in both cell lines and primary MCL cells from the patients. We demonstrated that MYC is a bona fide client of HSP90 in the context of MCL by both immunoprecipitation and chemical precipitation. Furthermore, inhibition of HSP90 using PU-H71 induced apoptosis and caused cell cycle arrest. PU-H71 also demonstrates strong and relatively specific inhibition of the MYC transcriptional program compared with other oncogenic pathways. In a MCL patient-derived xenograft model, the HSP90 inhibitor retards tumor growth and prolongs survival. Last, we showed that PU-H71 induced apoptosis and downregulated MYC protein in MCL cells derived from patients who were clinically resistant to ibrutinib. In conclusion, MYC activity underlies intrinsic resistance to ibrutinib in MCL. As a client protein of HSP90, MYC can be inhibited via PU-H71 to overcome primary ibrutinib resistance.

Abstract 4099: Targeting the paracaspase MALT1: A potential therapy to overcome ibrutinib resistance in relapsed/refractory MCL patients

Mantle cell lymphoma (MCL) is an aggressive B cell malignancy that is not yet curable. Ibrutinib was FDA-approved in 2013 to treat relapsed/refractory MCL; however, ibrutinib resistance inevitably develops. Once patients relapse after ibrutinib treatment, the 1-year survival rate is only 22%; therefore, there is an urgent unmet need to overcome ibrutinib resistance and to study alternative treatment options. Constitutive NF-κB activation is the hallmark of MCL. Indeed, next generation sequencing analysis of 110 MCL patient samples revealed that genes in the NF-κB signaling pathway had the highest mutation rates (28.8%), indicating the significant contribution of NF-κB signaling to MCL malignancy. Mucosa-associated lymphoid tissue transformation protein (MALT1) plays a crucial role in NF-κB signaling. MALT1 is a unique paracaspase within the human genome, and the proteolytic activity of MALT1 has been found to be constitutively active in many MCL samples, suggesting MALT1 may be a potential therapeutic target without significant off-target side effects. MI-2 is a specific inhibitor of MALT1 and its efficacy and safety are currently being evaluated in a clinical trial with ABC-type diffuse large B cell lymphoma patients. Mice treated with MI-2 did not have detectable physiological, histological or biochemical signs of toxicity. However, whether MALT1 activity contributes to ibrutinib resistance and whether targeting MALT1 can overcome ibrutinib resistance in relapsed/refractory MCL patients remain unclear. In this study, we found that both canonical and non-canonical NF-κB signaling is activated in ibrutinib-resistant MCL cells, which correlates with constitutive MALT1 activity. Interestingly, we found that MALT1 is highly mutated in four clusters, including the death domain, TRAF6-binding site, Caspase-like domain, and IKKγ-binding site in MCL samples. Occurrence of L79P, K80R, E319D, L445P and N446S was also highly correlated with ibrutinib resistance and disease progression, which requires more detailed investigation. Treatment with MI-2 significantly reduced cell viability in several MCL cell lines with nanomolar activity. MI-2 treatment inhibited NF-κB activation, IL-6 production and its downstream STAT3 activation. Combining MI-2 with ibrutinib resulted in synergistic growth inhibition in both ibrutinib-resistant MCL cell lines and primary MCL cells. These findings suggest that targeting MALT1 catalytic activity in MCL is a promising therapy to overcome ibrutinib resistance in relapsed/refractory MCL patients. The effect of MI-2 in in vivo PDX models is currently under investigation. This work and follow-up in vitro and in vivo studies will provide strong evidence that targeting MALT1 with MI-2 may be an effective novel therapeutic approach to overcome ibrutinib resistance. Citation Format: Changying Jiang, Shengjian Huang, Xin Lin, Michael Wang, Liang Zhang. Targeting the paracaspase MALT1: A potential therapy to overcome ibrutinib resistance in relapsed/refractory MCL patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4099. doi:10.1158/1538-7445.AM2017-4099

Abstract 3651: Preclinical examination of the effects of MT-3724, an engineered toxin body targeting CD20, in mantle cell lymphoma

Mantle cell lymphoma (MCL) accounts for 6-8% of all non-Hodgkin lymphoma cases and is a therapeutic challenge. MCL is characterized by the expression of different B-cell markers such as CD-19, CD-20 and BSAP/PAX5, and CD-20 is strongly expressed and can be used as a potential target. MT-3724 was developed by Molecular Templates and is an engineered toxin body (ETB) targeting CD-20. MT-3724 binds CD-20 and forces its own internalization into the target cell where it subsequently self-routes to the cytosol to enzymatically and permanently inhibit protein synthesis via ribosome inactivation. By selectively and specifically targeting CD20-positive cells, MT-3724 may decrease cell proliferation and induce apoptosis in MCL. We tested the effects of MT-3724 by in vitro cell proliferation in 3 ibrutinib-sensitive cell lines and 5 ibrutinib-resistant cell lines (4 primary resistant and 1 acquired resistant). We also measured the levels of apoptotic cells in both ibrutinib-sensitive and -resistant cell lines treated with MT-3724 by Annexin V/ PI staining. Lastly, we conducted an in vivo efficacy assay of MT-3724 in a MCL PDX model resistant to a wide-range of drugs, including ibrutinib. MT-3724 inhibited cell proliferation effectively and efficiently in most ibrutinib-sensitive and ibrutinib-resistant cell lines in a dose-dependent manner. IC50 500 ng/ml was considered resistant to MT-3724. Regarding the ibrutinib-sensitive cell lines, the 3 cell lines (Jeko-1, Mino and Rec-1) were sensitive to MT-3724 with IC50 values of 139.1, 309.3 and 457.7 ng/ml, respectively. Regarding the ibrutinib-resistant cell lines, 4 cell lines (Maver-1, JVM-13, Jeko-R and Granta519) were sensitive to MT-3724 with IC50 values of 124.6, 155.1, 266.2 and 442.4 ng/ml, respectively, and 1 cell line (Z-138) was resistant to MT-3724 (IC50 = 1231 ng/ml). However, no significant differences in IC50 values were found between ibrutinib-sensitive and -resistant cell lines (p = 0.36). In a time-dependent assay, 300 ng/ml MT-3724 also reduced cell proliferation in 2 ibrutinib-sensitive cell lines (Mino and Jeko-1) and 2 ibrutinib-resistant cell lines (Jeko-R and Maver-1) over time. Furthermore, MT-3724 also induced cell apoptosis in both ibrutinib-sensitive (Jeko-1) and -resistant (Jeko-R and Maver-1) cell lines. Lastly, MT-3724 was administered intraperitoneally for three consecutive weeks in a PDX model resistant to a wide-range of targeted agents. Interestingly, MT-3724 dramatically reduced tumor burden and increased survival (median of 27 days) of the PDX mice. MT-3724 is the first toxin engineered body targeting CD-20 used in MCL, which may be a potential therapeutic candidate for MCL, especially for drug-resistant cases. Citation Format: Shengjian Huang, Taylor Bell, Yang Liu, Hui Guo, Carrie Li, Makhdum Ahmed, Laura Lam, Hui Zhang, Zhihong Chen, Michael L. Wang, Leo Zhang, Krystle Nomie. Preclinical examination of the effects of MT-3724, an engineered toxin body targeting CD20, in mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3651. doi:10.1158/1538-7445.AM2017-3651

NR4A1 inhibition synergizes with ibrutinib in killing mantle cell lymphoma cells

NR4A1 (Nur77, TR3, NGFI-B), a member of the nuclear receptor family, is known as an immediate early or stress response gene. NR4A1 plays a physiological role in development and cellular homeostasis, and is also involved in tumorigenesis. NR4A1 is overexpressed and exhibits oncogenic activity in many solid cancers, whereas it acts as a tumor suppressor in hematologic malignancies. This is likely due to a dual role for NR4A1 in mediating cell proliferation/survival vs apoptosis. As a transcription factor, NR4A1 is primarily localized in the nucleus and regulates gene expression to enhance cell proliferation and survival. When NR4A1 is exported from the nucleus to mitochondria, it binds BCL-2 and subsequently induces cell apoptosis. NR4A1 was first characterized as a tumor suppressor in a report showing the rapid development of acute myeloid leukemia in NR4A1 and NR4A3 double knockout mice but not in single knockout animals. Reduced expression of these two genes is a common feature in human acute myeloid leukemia cells. In diffuse large B-cell lymphoma and high-grade follicular lymphoma, low NR4A1 expression was significantly associated with a non-germinal center B-cell subtype and with poor overall survival. NR4A1 overexpression induced apoptosis of diffuse large B-cell lymphoma cells and inhibited xenografted tumor growth. These findings together with our recent study prompted us to investigate the role of NR4A1 in mantle cell lymphoma (MCL), a currently incurable non-Hodgkin lymphoma. Our RNA sequencing (RNA-seq) analysis in four MCL cell lines identified that NR4A1 is one of six common downregulated genes by Bruton tyrosine kinase (BTK) short hairpin RNA (Fig. 1a). This result was further confirmed by quantitative PCR (Supplementary Fig. 1A). BTK is a key component of the early B-cell antigen receptor (BCR) signaling pathway and its inhibitor ibrutinib has emerged as an effective therapeutic agent for the treatment of MCL. To further verify that NR4A1 is a target gene of BCR/BTK signaling, we stimulated naive B cells from peripheral blood with α-IgM or combined with CD40L. Indeed, both messenger RNA and protein levels of NR4A1 were significantly increased with the BCR stimulation (Supplementary Fig. 1B, C), consistent with a previous study. Next, we determined NR4A1 expression in a tissue microarray of 46 MCL cases by immunohistochemical (IHC) analysis. All these MCL cases bore the typical chromosomal translocation t(11:14) involving cyclin D1 with the morphological characteristics of MCL, as described previously. As shown in Fig. 1b, NR4A1 was mainly localized in the nucleus and the average of nuclear NR4A1 protein expression in MCL cases was significantly higher than that in normal B cells from tonsils or lymph nodes. NR4A1 protein was rarely present in the cytoplasm of both MCL and normal B cells. Also, NR4A1 expression with two main isoforms was detected by immunoblot analysis in all eight MCL cell lines tested, despite at various levels (Fig. 1c). Consistent with the above IHC analysis, immunofluorescence staining in Rec-1 cells displayed nuclear localization of NR4A1 (Fig. 1d). Given that NR4A1 is expressed in MCL, mainly present in the nucleus, and is induced for expression by BCR/BTK signaling, we hypothesized that NR4A1 is not a tumor suppressor but rather a potential oncogene in MCL. To test this hypothesis, we first expressed NR4A1 complementary DNA using an inducible retroviral vector in Mino and Granta-519 cell lines, both of which have relatively low levels of endogenous NR4A1 expression. We confirmed NR4A1 expression by both immunoblot