Taylor Bell

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.

Abstract 4391: Overcoming primary ibrutinib resistance in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a rare and incurable subtype of B-cell lymphoma. In a phase II study of ibrutinib in MCL patients, most of the patients responded and had long durable remissions; however, 22.7% of patients were considered to be non-responsive to ibrutinib, and an additional 22/110 patients displayed initial positive responses to ibrutinib but also experienced disease progression within 12 months of treatment, with both responses classified as primary ibrutinib resistant. Therefore, understanding the mechanisms mediating primary ibrutinib resistance may identify new prognostic and predictive markers and potential therapeutic targets. Bruton9s tyrosine kinase (BTK) plays an important role in B-cell development, activation, and differentiation. Upon B-cell receptor (BCR) activation, BTK is phosphorylated and activated by SYK. Phosphoinositide 3-kinase (PI3K) is recruited to the BCR, and the induction of these signaling activities leads to the downstream activation of multiple effector proteins, including nuclear factor-kB (NF-kB), AKT, RAS, mTOR and mitogen-activated protein kinase (MAPK). BTK and PI3K have been shown to function independently to mediate BCR signaling, suggesting that PI3K signaling activities may underlie ibrutinib resistance independently of BTK. PI3K has also been associated with primary ibrutinib resistance. To further elucidate the mechanisms underlying ibrutinib resistance, we conferred primary ibrutinib resistance using both MCL cell lines and MCL-bearing patient-derived xenograft (PDX) mouse models and used whole exome sequencing (WES) and reverse phase protein analysis (RPPA) to identify any genetic and expression changes associated with primary ibrutinib resistance. WES did not reveal any mutations in BTK or within the proximal BCR pathway, consistent with WES data on primary ibrutinib resistant MCL cases. RPPA analysis showed a significant increase in the PI3K/AKT/mTOR/MCL-1 compensatory pathway component levels in ibrutinib-resistant cell lines when compared with their parental cells as well as ibrutinib-resistant PDXs. To determine whether inhibiting these pathways would overcome primary ibrutinib resistance, we tested various therapeutic combinations targeting these pathways. Ibrutinib plus the PI3K inhibitor idelalisib, the AKT inhibitor ACP-319, the mTOR inhibitors AZD8055 or BEZ235 as well as the proteasome inhibitor carfilzomib inhibited tumor growth in vitro and in vivo in the PDX mouse models, demonstrating that targeting these alternative pathways may overcome ibrutinib resistance. This work strongly suggests that targeting the PI3K/AKT/mTOR/MCL-1 compensatory pathway successfully inhibits the viability of ibrutinib-resistant MCL tumor cells both in vitro and in vivo and identified potential therapies that can be used to treat ibrutinib-resistant patients in clinical practice. Citation Format: Leo Zhang, Lan Pham, Hui Zhang, Jingmeng Xie, Wenjing Tao, Taylor Bell, Zhihong Chen, Krystle Nomie, Bingliang Fang, Michael Wang. Overcoming primary ibrutinib resistance in mantle cell lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4391.

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-

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

Abstract B18: B-cell lymphoma patient-derived xenograft models: The road to personalized therapy

Lymphoma is the most common hematological malignancy, and B-cell lymphoma accounts for 85% of all lymphomas. The current overall cure rate for B-cell lymphoma is estimated at ~30%, even with the development and application of novel therapies, and the majority of patients relapse after treatment due to the development of drug resistance. The evaluation of novel drug targets using established B-cell lymphoma cell lines is limited by the inexact correlation between responsiveness observed in the cell line versus the patient sample. However, patient-derived xenograft (PDX) mouse models have been shown to recapitulate the diversity of growth, metastasis, and histopathology of the original tumor, overcoming the limitations of cell lines. Furthermore, recent studies have indicated that PDXs can recapitulate treatment responses of the parental tumor and can successfully choose a therapeutic target and regimen for the patient. We previously established a SCID-hu in vivo human primary mantle cell lymphoma (MCL) PDX model, the first human primary MCL animal model for biological and therapeutic research, and investigated the disease biology and potential novel human MCL therapies using this model. In this MCL PDX model, the engraftment and growth of patient MCL cells were dependent on the human bone marrow microenvironment supplied by an implanted human fetal bone chip. Our clinical information and reports show that numerous B-cell lymphoma subtypes involve bone marrow; therefore, we expanded our PDX model to include various B-cell lymphomas to study the clonal evolution of tumors and to evaluate novel therapies for the treatments of these diseases. PDX models (n=20) were established for multiple different types of B-cell lymphoma, including MCL (n=12), Burkitt9s lymphoma (n=1), marginal zone lymphoma (n=2), follicular lymphoma (n=2), chronic lymphocytic leukemia (n=1) and diffuse large B-cell lymphoma (n=2). The engraftment rate was high at 95%, the tumor xenografts rapidly grew at 3-4 weeks/generation, and 68% of the tumor xenografts were passaged for multiple generations, even up to 14 generations. Further demonstrating the utility of this model to recapitulate the characteristics of the original patient tumor, the tumor xenograft cells migrated to the lymph nodes, spleen, bone marrow, and gastrointestinal tract of the host mice, mimicking the disease progression observed in humans, and HE Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B18.