Double-hit Lymphoma Cell Research Articles

Imatinib mesylate reduces c-MYC expression in double-hit lymphoma cells by suppressing inducible cytidine deaminase

BackgroundDouble-hit lymphoma (DHL) with c-MYC gene translocation is highly aggressive and has a poor prognosis. In DHL cells, activation-induced cytidine deaminase (AID) promotes antibody class switch recombination (CSR), ultimately leading to c-MYC gene translocation caused by Myc/IgH DNA double-strand breaks. However, currently there is still no method to suppress the expression of AID.MethodsIn this study, we compared the clinical significance of AID expression in DHL, Additionally, two human double-hit lymphoma cell lines were used to analyze the effect of imatinib mesylate on c-MYC in vitro, and the therapeutic effect was also evaluated in xenograft mouse models.ResultsImatinib mesylate downregulated the AID and c-MYC proteins in patients with chronic myelogenous leukemia associated with DHL. In addition, imatinib mesylate reduced AID and c-MYC expression in SU-DHL-4 and OCI-Ly18DHL cells. Imatinib mesylate exerted significant inhibitory effects on the proliferation and metastasis of SU-DHL-4 and OCI-Ly18 cells. Finally, imatinib mesylate reduced not only tumor burden in DHL mouse models, but also AID and c-MYC expression in vivo.ConclusionThese findings reveal that imatinib mesylate effectively reduces the carcinogenic function of c-MYC in DHL, providing novel strategies for developing therapies targeting c-MYC-driven DHL.Graphical abstract

Double-hit primary central nervous system lymphoma with histogenetically proven bone marrow infiltration: a case report and a review of the literature.

Double-hit lymphoma (DHL) formerly referred to high-grade B-cell lymphoma with concurrent MYC and BCL2 or BCL6 rearrangements, however, the updated 2022 World Health Organization Classification (5th edition online) excludes those with MYC and BCL 6 rearrangements from the high-grade category. DHL confined to the central nervous system (CNS), known as double-hit primary CNS lymphoma (DH-PCNSL), is rare with poorly understood clinical features. Here, we report a case of a 64-year-old man with multiple brain tumors diagnosed with DH-PCNSL who showed bone marrow (BM) infiltration early in the clinical course. The histological diagnosis was high-grade B-cell lymphoma with MYC and BCL6 rearrangements. Fluorodeoxyglucose positron emission tomography (FDG-PET) revealed no abnormal accumulation except in the CNS. The patient received whole-brain radiotherapy following the failure of high-dose methotrexate. After completion of radiotherapy, the patient developed thrombocytopenia, and BM biopsy showed infiltration of DHL cells, which were not detected by repeated FDG-PET. This is the first report of DH-PCNSL where identical gene rearrangements were confirmed in both the resected CNS tumor and BM tissue. Patients with DH-PCNSL require careful follow-up because they may be at a potential risk of BM infiltration, which may be undetectable by FDG-PET, particularly early in the disease course.

Single-cell RNA sequencing in double-hit lymphoma: IMPDH2 induces the progression of lymphoma by activating the PI3K/AKT/mTOR signaling pathway

BackgroundIMPDH2 is the rate-limiting enzyme of the de novo GTP synthesis pathway and has a key role in tumors; however, the specific mechanism underlying IMPDH2 activity in diffuse large B cell lymphoma (DLBCL) is still undetermined. This study aims to explore the potential mechanism of IMPDH2 in DLBCL, and its possible involvement in double-hit lymphoma (DHL), i.e., cases with translocations involving MYC and BCL2 and/or BCL6. MethodsUsing single-cell sequencing and bioinformatics analysis to screen for IMPDH2. Exploring the differential expression of IMPDH2 and its correlation with prognosis through multiplexed immunofluorescence analysis. Using CCK8, EdU, clone formation assay, and animal model to analyze biological behavior changes after inhibiting IMPDH2. Explaining the potential mechanism of IMPDH2 in DLBCL by Western blot and multiplexed immunofluorescence. ResultsPrognostic risk model was constructed by single-cell sequencing, which identified IMPDH2 as a DHL-related gene. IMPDH2 was highly expressed in cell lines and tissues, associated with poor patient prognosis and an independent prognostic factor. In vitro and in vivo experiments showed that IMPDH2 inhibition significantly inhibited DHL cell proliferation. Flow cytometry showed apoptosis and cycle arrest. Western blot results suggested that c-Myc regulated the activation of PI3K/AKT/mTOR signaling pathway by IMPDH2 to promote tumor development in DHL. Moreover, multiplex immunofluorescence revealed decreased T-cell infiltration within the tumor microenvironment exhibiting concurrent high expression of IMPDH2 and PD-L1. ConclusionsOur results suggest that IMPDH2 functions as a tumor-promoting factor in DHL. This finding is expected to generate novel insights into the pathogenesis of these patients, thereby identifying potential therapeutic targets.

Synergistic disruption of BTK and BCL-2 causes apoptosis while inducing ferroptosis in double-hit lymphoma

Double-hit lymphoma (DHL) is an aggressive subset of Diffuse Large B-cell Lymphoma (DLBCL) with poor outcomes and without satisfying treatment options. BTK inhibitor monotherapy is ineffective to suppress aggressive lymphoma. Hence, combination with other potential agents is warranted. Here, we demonstrated the second generation of BTK inhibitor, zanubrutinib, and a BCL-2 inhibitor, navitoclax, worked in synergistic manner to suppress DHL. Comprehensive in silico approach by interrogating single-cell to bulk-level profiling was employed along with in vitro and in vivo validation in DHL cell lines. Ablation of BTK enhanced sensitivity to navitoclax and suppressed proliferation of DHL cells. Combination of second generation of BTK inhibitor with navitoclax synergistically suppressed DLBCL cells with higher synergy score in DHL subset. The drug combination triggered apoptosis and ferroptosis, with the latter being characterized by reactive oxygen species (ROS) accumulation, extensive lipid peroxidation, and depletion of reduced glutathione. Moreover, ablation of BTK sensitized DHL cells to ferroptosis. Mechanistically, disruption of BTK and BCL-2 triggered ferroptosis by downregulating NRF2 and HMOX1, while deactivating GPX4. Combination of zanubrutinib and navitoclax effectively suppressed tumor growth in vivo. Our data suggest that zanubrutinib and navitoclax synergistically suppressed DHL by inducing apoptosis and ferroptosis.

Orelabrutinib and venetoclax synergistically induce cell death in double-hit lymphoma by interfering with the crosstalk between the PI3K/AKT and p38/MAPK signaling.

Double-hit lymphoma (DHL) is a rare and aggressive mature B-cell malignancy with concurrentMYCandBCL2rearrangements. When DHL becomes relapsed or refractory, it becomes resistant to the majority of therapeutic approaches and has subpar clinical results. Therefore, innovative therapeutics for this particular patient population are urgently needed. Orelabrutinib, a new oral BTK inhibitor, combined with the Bcl-2 inhibitor venetoclax, was used to confirm the antitumor effect of DHL. Cell counting kit-8 and Annexin V-FITC/PI assays were used to examine the interaction of this combined regimen on DHL cell lines and primary lymphoma cells. RNA sequencing, EdU incorporation assay, mitochondrial membrane potential assay, and western blotting were employed to explore the molecule mechanism for the cytotoxicity of orelabrutinib with or without venetoclax against DHL cell lines. In this study, orelabrutinib combined with venetoclax synergistically induced DHL cell death, as evidenced by the inhibition of cell proliferation, the induct of cell cycle arrest, and the promotion of cell apoptosis via the mitochondrial pathway. Orelabrutinib treatment alters genome-wide gene expression in DHL cells. The combined regimen decreases the expression of BTK and Mcl-1, potentially interfering with the activity and crosstalk of PI3K/AKT signaling and p38/MAPK signaling. In addition, the combination of orelabrutinib and venetoclax shows cytotoxic activity in primary B-lymphoma cells. In summary, these findings reveal a novel therapy targeting BCR signaling and the Bcl-2 family for DHL patients with a poor prognosis.

Venetoclax enhances the efficacy of therapeutic antibodies in B-cell malignancies by augmenting tumor cell phagocytosis

AbstractImmunotherapy has evolved as a powerful tool for the treatment of B-cell malignancies, and patient outcomes have improved by combining therapeutic antibodies with conventional chemotherapy. Overexpression of antiapoptotic B-cell lymphoma 2 (Bcl-2) is associated with a poor prognosis, and increased levels have been described in patients with “double-hit” diffuse large B-cell lymphoma, a subgroup of Burkitt's lymphoma, and patients with pediatric acute lymphoblastic leukemia harboring a t(17;19) translocation. Here, we show that the addition of venetoclax (VEN), a specific Bcl-2 inhibitor, potently enhanced the efficacy of the therapeutic anti-CD20 antibody rituximab, anti-CD38 daratumumab, and anti-CD19-DE, a proprietary version of tafasitamab. This was because of an increase in antibody-dependent cellular phagocytosis by macrophages as shown in vitro and in vivo in cell lines and patient-derived xenograft models. Mechanistically, double-hit lymphoma cells subjected to VEN triggered phagocytosis in an apoptosis-independent manner. Our study identifies the combination of VEN and therapeutic antibodies as a promising novel strategy for the treatment of B-cell malignancies.

Targeting MYC and BCL2 by a natural compound for "double-hit" lymphoma.

Concurrent translocations of MYC and BCL2 lead to abnormal expression of both oncoproteins, which contribute to the aggressive clinical characteristics of double-hit lymphoma (DHL). An effective therapy for DHL remains an unmet clinical need. In this study, we showed that both Ca2+/calmodulin-dependent protein kinase II δ (CAMKIIδ) and γ (CAMKIIγ) were highly expressed in DHL. Both isoforms of CAMKII stabilize c-Myc protein by phosphorylating it at Ser62, increase BCL2 expression, and promote DHL tumor growth. Inhibition of CAMKIIδ and CAMKIIγ by either berbamine (BBM) or one of its derivatives (PA4) led to the down regulation of c-Myc and BCL2 proteins. BBM/PA4 also exhibited anti-tumor efficacy in DHL cell lines and NSG xenograft models. Altogether, CAMKIIδ and CAMKIIγ appear to be critical for DHL tumor development and are promising therapeutic targets for DHL.

Pharmacological targeting PIKfyve and tubulin as an effective treatment strategy for double-hit lymphoma

Double-hit lymphoma is one of the most aggressive and refractory lymphoma subtypes with recurrent genetic abnormalities of MYC and BCL-2 or BCL6 rearrangement, leading to a poor prognosis in the present clinical practice. Therefore, new therapeutic strategies for eliminating double-hit lymphomas are urgently needed. Here, we reported that HZX-02-059, a novel PIKfyve and tubulin dual-target inhibitor, showed a highly cytotoxic activity against double-hit lymphoma cell lines in vitro and in vivo through a noncanonical caspase-independent cell death, methuosis. Mechanistically, the cytotoxicity triggered by HZX-02-059 was contributed to the PIKfyve/TFEB axis-induced cell death of methuosis, as well as the inhibition of tubulin and mTOR/Myc axis-induced cell cycle arrest. In summary, the present findings suggest that HZX-02-059 represents a good starting point for developing targeted therapeutics against double-hit lymphomas.

ZCL-082, a boron-containing compound, induces apoptosis of non-Hodgkin's lymphoma via targeting p90 ribosomal S6 kinase 1/NF-κB signaling pathway

IntroductionDespite the rapid progress in the diagnosis and treatment, the prognosis of some types of non-Hodgkin's lymphoma (NHL), especially those with double-hit or double-expressor genotypes, remains poor. Novel targets and compounds are needed to improve the prognosis of NHL. MethodsWe investigated the effect of ZCL-082, a novel boron-containing compound with anti-proliferating activity against ovarian cancer cells, on NHL cells and human peripheral blood mononuclear cells by CCK-8 assay, Annexin V/PI double staining assay, RH123/PI double staining, Western blot, and immunohistochemistry. NF-κB pathway activity was analyzed using luciferase reporter gene assay and RT-PCR. The location of p65 was detected by immunofluorescence and nuclear/cytoplasmic fractionation assay. Immunoprecipitation and chromatin immunoprecipitation assays were used to detect the binding between p65 and p300. CETSA and molecular docking assay were carried out to test the interaction between ZCL-082 and p90 ribosomal S6 kinase 1 (RSK1). Kinase reaction was conducted to examine the inhibition of RSK1 kinase activity by ZCL-082. ResultsWe found that ZCL-082 can induce the apoptosis of various NHL cell lines in vitro and in vivo. ZCL-082 significantly inhibits TNFα- or LPS-induced NF-κB activation without disturbing TNFα-induced IκBα degradation or the nuclear translocation and DNA-binding ability of p65. However, ZCL-082 markedly suppresses the phosphorylation of p65 on Ser536 and the interaction between p65 and p300. The overexpression of the phosphomimetic mutant of p65 at Ser536 partially abrogates ZCL-082-induced cell death. We further found that ZCL-082 directly binds to and inhibits the activity of RSK1. RSK1 can phosphorylate RelA/p65 on Ser536 and its overexpression is associated with the poor prognosis of lymphoma. The overexpression of RSK1 partially rescues ZCL-082-induced cell death. Molecular docking studies show that ZCL-082 fits well with the N-terminal kinase domain of RSK1. Furthermore, the combination of ZCL-082 and BCL-2 inhibitor ABT-199 has a synergistic apoptosis-inducing effect against double-hit lymphoma cell line OCI-Ly10. DiscussionWe found that ZCL-082 is a highly promising anti-lymphoma compound that targets RSK1 and interferes with the RSK1/NF-κB signaling pathway. The combination of ZCL-082 with BCL-2 inhibitor may represent a novel strategy to improve the outcome of double-hit or double-expressor lymphoma.

Abstract IA31: Modeling and targeting double-hit lymphoma

Abstract High-grade B-cell lymphoma with concurrent activation of MYC and BCL2, also known as double-hit lymphoma (DHL), shows dismal prognosis with current front-line therapies (e.g., R-CHOP), calling for the development of new therapeutic regimens. The synergy between MYC and BCL2 in lymphomagenesis is explained by the ability of BCL2 to block the proapoptotic activity of MYC, while leaving intact its proliferative potential; on this basis, we reasoned that compounds that exacerbate MYC-induced apoptosis might cooperate with BCL2 inhibitors in killing DHL cells. We previously observed that genes encoding components of the mitochondrial ribosome were coordinately activated in MYC-driven lymphoma (1) and were critical for tumor maintenance in transgenic mice (2). In line with the genetic data, inhibition of mitochondrial translation with the antibiotic tigecycline was synthetic-lethal with MYC activation and extended lifespan in lymphoma-bearing mice (2). Combining tigecycline with the BCL2 inhibitor venetoclax revealed synergy in the induction of apoptosis in human DHL cell lines, as well as marked antitumoral activity in xenografted mice (3). These preclinical data warrant the repurposing of venetoclax and tigecycline for the treatment of refractory and/or relapsed DHL. By blocking the synthesis of mitochondrially encoded proteins, tigecycline impairs the assembly of the electron-transport chain (ETC) complexes and oxidative phosphorylation. We thus hypothesized that direct ETC inhibitors might also preferentially kill MYC-overexpressing cells and synergize with venetoclax against DHL. We will present data that verify these predictions for IACS-010759, a small-molecule inhibitor of mitochondrial complex I. A current limitation for further preclinical development and mechanistic analysis lies in the lack of a bona fide mouse model of DHL. While the cooperation between MYC and BCL2 in lymphomagenesis was amply documented over almost three decades, the models described so far led to tumor onset at early stages of B-cell ontogeny; as a consequence, no model is available that reliably reproduces the development of DHL from germinal center (GC) B-cells, as observed in the clinic. In order to overcome this limitation, we combined CRE-activated alleles of MYC and BCL2 with Cgamma1-CRE, a CRE-expressing transgene that is specifically expressed in GC B cells. Concerted activation of both oncogenes led to increased expansion of GC B cells upon immunization, followed within a few months by the onset of aggressive, invasive B-cell lymphomas. Our progress in characterizing this disease model will be presented at the meeting.

Abstract 3702: Establishment and characterization of a novel MYC/BCL2 double-hit high-grade B-cell lymphoma cell line HLA-3

Abstract The majority of MYC/BCL2 double hit lymphoma (DHL) morphologically resembles diffuse large B-cell lymphoma (DLBCL) or unclassified B-cell lymphoma, with intermediate features of DLBCL and Burkitt’s lymphoma. DHL also typically results in shorter survival time than other lymphomas. To further understand DHL’s biology and identify potential effective agents against DHL, we established a unique MYC/BCL2 high-grade B-cell lymphoma cell line with morphological DLBCL features. The HLA-3 DHL cell line was derived from a MYC/BCL2 high-grade B-cell lymphoma patient-derived xenograft (PDX) model. We performed flow cytometry immunophenotypic analysis and established the HLA-3 cell line after 7 months of continuous culture. We utilized molecular and cellular techniques, including flow cytometry and polymerase chain reaction (PCR) analysis, to characterize the HLA-3 cell line. We performed reverse phase protein array (RPPA) analysis to determine its protein signaling profile. To identify agents with anti-DHL activity, we screened the effects of a panel of agents alone and in combination with ibrutinib on the cell viability of this newly established cell line. The HLA-3 cell line was optimally maintained at a density between 1 and 2 × 106 cells/mL and successfully proliferated in a single-cell suspension without cellular clump formation. The HLA-3 cells were EBV-negative and possessed a germinal center B cell immunophenotype. Moreover, the immunophenotypes of the primary patient tumor cells and HLA-3 cells were virtually identical. The HLA-3 cells were positive for CD19, CD45, CD35, CD10 and HLA-ABC, negative for CD3, CD5, CD40, CD34 and TdT, and were dim for CD22, CD56, CD8, CD4 and CD23. Representative flow cytometry histograms showed 99% of the HLA-3 cells were positive for CD19 and CD45, with no detectable surface kappa and lambda light chains. The HLA-3 cells expressed a high level of CD38, an indicator of MYC rearrangement. Similar to the original lymphoma cells, the HLA-3 cells showed dim/low CD20 expression. Based on RPPA data, we constructed a diagram showing activation of the ATM/Chk2 DNA repair and the IGF1R/PI3K/mTOR signaling pathways in the HLA-3 cells. In support of this upregulated activity, we found that 5/7 tested commercial PI3K/mTOR inhibitors significantly reduced the viability of the HLA-3 cells in a dose-dependent manner, with AZD8055 the most sensitive of the tested inhibitors against the cell line. Further, DNA repair pathway inhibitors BMN673 and RG7112 were effective against the HLA-3 cell line. The newly characterized high-grade B-cell lymphoma cell line will provide useful in vitro and in vivo models for biological studies related to human DHL, which is refractory to current therapies and urgently needs novel therapeutic approaches. This work suggests the PI3K/mTOR and DNA repair pathways may be therapeutic targets for MYC/BCL2 double-hit high-grade B-cell lymphoma. Citation Format: Hui Guo, Hui Zhang, Krystle Nomie, Liang Zhang, Kelley P. Murfin, Michael Wang. Establishment and characterization of a novel MYC/BCL2 double-hit high-grade B-cell lymphoma cell line HLA-3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3702.

Retraction: In Vitro and In Vivo Interactions between the HDAC6 Inhibitor Ricolinostat (ACY1215) and the Irreversible Proteasome Inhibitor Carfilzomib in Non-Hodgkin Lymphoma Cells

Interactions between the HDAC6 inhibitor ricolinostat (ACY1215) and the irreversible proteasome inhibitor carfilzomib were examined in non-Hodgkin lymphoma (NHL) models, including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and double-hit lymphoma cells. Marked in vitro synergism was observed in multiple cell types associated with activation of cellular stress pathways (e.g., JNK1/2, ERK1/2, and p38) accompanied by increases in DNA damage (γH2A.X), G2–M arrest, and the pronounced induction of mitochondrial injury and apoptosis. Combination treatment with carfilzomib and ricolinostat increased reactive oxygen species (ROS), whereas the antioxidant TBAP attenuated DNA damage, JNK activation, and cell death. Similar interactions occurred in bortezomib-resistant and double-hit DLBCL, MCL, and primary DLBCL cells, but not in normal CD34+ cells. However, ricolinostat did not potentiate inhibition of chymotryptic activity by carfilzomib. shRNA knockdown of JNK1 (but not MEK1/2), or pharmacologic inhibition of p38, significantly reduced carfilzomib–ricolinostat lethality, indicating a functional contribution of these stress pathways to apoptosis. Combined exposure to carfilzomib and ricolinostat also markedly downregulated the cargo-loading protein HR23B. Moreover, HR23B knockdown significantly increased carfilzomib- and ricolinostat-mediated lethality, suggesting a role for this event in cell death. Finally, combined in vivo treatment with carfilzomib and ricolinostat was well tolerated and significantly suppressed tumor growth and increased survival in an MCL xenograft model. Collectively, these findings indicate that carfilzomib and ricolinostat interact synergistically in NHL cells through multiple stress-related mechanisms, and suggest that this strategy warrants further consideration in NHL. Mol Cancer Ther; 13(12); 2886–97. ©2014 AACR .

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.

Generation and characteristics of a novel “double-hit” high grade B-cell lymphoma cell line DH-My6 with MYC/IGH and BCL6/IGH gene arrangements and potential molecular targeted therapies

“Double-hit” lymphoma (DHL) is a high-grade B-cell lymphoma that harbors concurrent MYC and BCL2 or BCL6 rearrangements. Because cases of MYC/BCL6 DHL are uncommon, most reported conclusions have been based on cases of MYC/BCL2 DHL. Lack of experimental MYC/BCL6 DHL models continues to hinder the pathophysiologic and therapeutic investigations of this disorder. We herein describe a novel MYC/BCL6 DHL cell line, designated DH-My6, carrying both the MYC–IGH and BCL6–IGH fusion genes. Interruptions of MYC and BCL6 expressions using short interfering RNAs and chemical inhibitors led to significant attenuation of DH-My6 cell growth. Greater antitumor effects were found when the cells were treated with a combination of MYC and BCL6 inhibitors. Moreover, the PLK1 inhibitor volasertib and the HDAC inhibitor vorinostat synergized strongly when combined with the bromodomain inhibitor JQ1. DH-My6 is a new well-validated MYC/BCL6 DHL cell line that will provide a useful model for studies of the pathogenesis and therapeutics for the less common DHL tumor type. The rationale for approaches targeting both MYC and BCL6, and in combination with PLK1 or HDAC inhibitors for superior suppression of the aggressive MYC/BCL6 DHL warrants further in vivo testing in a preclinical model.

Abstract 5442: Dose-dependent toxicity of FTY720, a sphingosine-1-phosphate receptor agonist, on double hit lymphoma cells via PP2A-mediated dephosphorylation of c-myc and bcl-2

Abstract High grade B cell lymphoma with translocations involving MYC and rearrangements involving BCL2 and/or BCL6 define an aggressive subtype of diffuse large B cell lymphoma (DLBCL) with poor prognosis and high rates of relapse. The standard treatment for DLBCL is not considered effective and targeted molecular therapy has yet to be identified for this disease. The mechanisms that make this type of lymphoma particularly aggressive and refractory to treatment include cell growth signals from the c-myc transcriptional regulator working together with the anti-apoptotic activity of the bcl-2 protein. Protein Phosphatase 2A (PP2A) is a ubiquitous cellular enzyme that acts as a tumor suppressor through dephosphorylation of specific cellular phosphoproteins. In fact, PP2A has been shown to dephosphorylate bcl-2 at ser70 leading to inhibition of its function. Further, PP2A-mediated dephosphorylation of c-myc at ser62 promotes its degradation. In many cancers, PP2A activity is inhibited due to overexpression of binding proteins such as SET and CIP2A. Small molecules can act as PP2A activators by releasing PP2A from these inhibitory binding proteins. In the present study we investigated the efficacy of FTY720, a high-affinity agonist of the sphingosine 1-phosphate receptor-1 and PP2A activator, on the double hit lymphoma derived cell line, WSU DLCL2. WSU DLCL2 cells were treated with various concentrations of FTY720, a derivative of myriocin, a fungal metabolite of the Chinese herb Iscaria sinclarii known to promote PP2A activation. Cell viability and PP2A activity were measured 24 hours after FTY720 treatment. In addition, cell lysates were assayed by Western blot using antibodies to detect PP2A subunits, as well as total and phosphorylated c-myc and bcl-2 proteins. A dose-dependent decrease in cell viability was observed over the range of FTY720 concentrations from 1 to 10 micromolar with concomitant increase in in vitro PP2A activity. Western blots showed decreases in c-myc protein levels with FTY720 treatment as well as decreases in ser70 phosphorylation levels of bcl-2 and ser62 phosphorylation levels of c-myc. The results indicate that PP2A activation via FTY720, a small molecule activator of PP2A, leads to a dose-dependent induction of cell death in double hit lymphoma. The data are consistent with an underlying cell death mechanism that involves the targeted dephosphorylation of c-myc and bcl2. Taken together, these data suggest that FTY720 may act as a targeted treatment with therapeutic potential as a single agent or as a combination with other therapies in the treatment of this subtype of lymphoma. There is also the potential of a therapeutic role in other lymphoma subtypes with poor prognosis including transformed follicular lymphoma and relapsed burkitt's lymphoma that rely individually on bcl-2 and c-myc, respectively, for their growth. Citation Format: Geoffrey P. Shouse, Rosalia de Necochea-Campion, Saied Mirshahidi, Chien-Shing Chen, Kimberly J. Payne. Dose-dependent toxicity of FTY720, a sphingosine-1-phosphate receptor agonist, on double hit lymphoma cells via PP2A-mediated dephosphorylation of c-myc and bcl-2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5442.

The BET bromodomain inhibitor CPI203 overcomes resistance to ABT-199 (venetoclax) by downregulation of BFL-1/A1 in in vitro and in vivo models of MYC+/BCL2+ double hit lymphoma.

High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, mostly known as double-hit lymphoma (DHL), is a rare entity characterized by morphologic and molecular features between Burkitt lymphoma and the clinically manageable diffuse large B-cell lymphoma (DLBCL). DHL patients usually undergo a rapidly progressing clinical course associated with resistance to standard chemo-immunotherapy. As a consequence, the prognosis of this entity is particularly poor with a median overall survival inferior to 1 year. ABT-199 (venetoclax) is a potent and selective small-molecule antagonist of BCL-2 recently approved for the treatment of a specific subtype of lymphoid neoplasm. In this study, we demonstrate that single-agent ABT-199 efficiently displaces BAX from BCL-2 complexes but fails to maintain a significant antitumor activity over time in most MYC+/BCL2+DHL cell lines and primary cultures, as well as in a xenograft mouse model of the disease. We further identify the accumulation of the BCL2-like protein BFL-1 to be a major mechanism involved in acquired resistance to ABT-199. Noteworthy, this phenomenon can be counteracted by the BET bromodomain inhibitor CPI203, since gene expression profiling identifies BCL2A1, the BFL-1 coding gene, as one of the top apoptosis-related gene modulated by this compound. Upon CPI203 treatment, simultaneous downregulation of MYC and BFL-1 further overcomes resistance to ABT-199 both in vitro and in vivo, engaging synergistic caspase-mediated apoptosis in DHL cultures and tumor xenografts. Together, these findings highlight the relevance of BFL-1 in DH lymphoma-associated drug resistance and support the combined use of a BCL-2 antagonist and a BET inhibitor as a promising therapeutic strategy for patients with aggressive DHL.

Alkylating Agent-Induced ER Stress Overcomes Microenvironmental Resistance to Lymphoma Therapy

Compartment-specific resistance to cancer therapy remains poorly understood. We utilized orthotopic xenografts of human double-hit lymphoma (DHL) to interrogate responses across involved sites. We identified resistance to multiple chemotherapies and the anti-CD52 antibody Alemtuzumab within the bone marrow (BM) that depended on extensive lymphoma involvement and impaired antibody-dependent cellular phagocytosis. This resistance was overcome by high doses of alkylating agents, including cyclophosphamide (CTX), which exhibited >80-fold in vivo synergy with Alemtuzumab. CTX induced ER stress in BM DHL cells leading to ATF4- mediated paracrine secretion of VEGF-A and massive macrophage infiltration. Macrophages from DHL-engrafted, CTX-treated mice had increased phagocytic capacity for lymphoma cells that was reversed by VEGF-A blockade and required SYK phosphorylation. A subset of these macrophages, defined by surface CD36/FcgRIV and a distinct transcriptional state, were “superphagocytic.” Together, our findings define a unique mechanism through which high-dose alkylating agents can overcome therapy-resistant niches by ER stress-induced activation of phagocytosis.

GSK-3β inhibitor, 9-ING-41, reduces cell viability and halts proliferation of B-cell lymphoma cell lines as a single agent and in combination with novel agents.

The complexities of GSK-3β function and interactions with PI3K/AKT/mTOR signaling, cell cycling, and apoptotic pathways are poorly understood in the context of lymphomagenesis and cancer therapeutics. In this study, we explored the anti-tumor effects of the GSK-3β inhibitor, 9-ING-41, in lymphoma cell lines as a single agent and in combination with novel agents comprising BCL-2 inhibitor (Venetoclax), CDK-9 inhibitor (BAY-1143572) and p110δ-PI3K inhibitor (Idelalisib). Treatment of Daudi, SUDHL-4, Karpas 422, KPUM-UH1, and TMD8 lymphoma cell lines with 1 μM 9-ING-41 reduced cell viability by 40-70% (p<0.05) and halted proliferation. Luminex analysis of apoptotic pathways revealed a significant increase in active caspase 3 in all lymphoma cell lines (p<0.001) except TMD8 cells. Co-treating SUDHL-4 and KPUM-UH1 lymphoma cells with 0.5 μM 9-ING-41 showed 8-and 2-fold reduction in IC50 values of Venetoclax, respectively. No significant benefit for this combination was seen in other lymphoma cells tested. The combination of BAY-1143572 with 0.5 μM 9-ING-41 showed an 8-fold reduction in the IC50 value of the former in SUDHL-4 lymphoma cells alone. No significant changes in IC50 values of Idelalisib were measured across all cell lines for the combination of 9-ING-41 and Idelalisib. Further, signaling analysis via Western blot in the double-hit lymphoma cell line, KPUM-UH1, suggests that phospho-c-MYC is modified with 9-ING-41 treatment. Altogether, our data show that 9-ING-41 results in increased apoptosis and decreased proliferation in aggressive B-cell lymphoma cells and enhances the antitumor effects of BCL-2 and CDK-9 antagonists.

Co-targeting Aurora kinase A and Bcl-2 synergistically inhibits the viability in double-hit lymphoma cells

Background: Double-hit lymphoma (DHL) is a rare high-grade B-cell lymphoma characterized by MYC and Bcl-2 or Bcl-6 gene translocations. The treatment of DHL remains a substantial clinical challenge due to remarkably undesirable outcomes. Innovative drugs or combination treatment strategies need to be developed to improve the prognosis of DHL patients. The purpose of this study was to investigate the combination treatment of a novel Aurora kinase A (Aurka) inhibitor, named alisertib (MLN8237) and a selective Bcl-2 inhibitor, named ABT-199 in vitro, and to explore the underlying mechanisms in human DHL cell lines. Methods: Cell proliferation was assessed using MTS assay in DHL cells treated with alisertib and ABT-199. Synergistic effects between two drugs were analyzed based on combination index (CI) values. Cell cycle and apoptosis were detected through flow cytometry. The expression of drug-targeted proteins, cell-cycle proteins as well as apoptosis-related proteins were detected by Western blot. Results: Alisertib and ABT-199 alone exhibited a relatively modest cytotoxicity in a concentration- and time-dependent manner, but the combination treatment produced stronger antitumor efficacy and synergistically suppressed the DHL cell growth. Otherwise, the combination treatment between alisertib and ABT-199 arrested the cell cycle in the G2/M phase at respectively lower concentrations (2/5 IC50), while the higher concentrations (3/5 IC50) resulted in a striking increase of cell apoptosis. Further studies demonstrated that both alisertib and ABT-199 downregulated the expression of cyclin-dependent kinase 1 (CDK1) and cyclin B1, but upregulated the p21 and p53 expression. The combination treatment enhanced the expression of cell intrinsic apoptotic proteins, including cleaved Poly ADP-ribose polymerase (PARP) and caspase-3. In addition, the combination treatment almost completely inhibited the expression of drug-targeted proteins, including MYC and Aurka phosphorylation. However, alisertib and ABT-199 did not alter the Bcl-2 expression. Conclusions: Alisertib and ABT-199 had synergistic effects on the inhibition of cell viability, induction of cell G2/M phase arrest and apoptosis in DHL cells. The underlying mechanism of synergism was associated with the p53/p21 and cell intrinsic apoptotic pathways. Our findings suggested that co-targeting Aurka and Bcl-2 might be a potentially therapeutic strategy for DHL lymphoma.

T Cells Bearing Anti-CD19- and/or Anti-CD38-Chimeric Antigen Receptors Effectively Abrogate Primary Double-Hit Lymphoma Cells

Patients with B-cell lymphomas bearing MYC translocation combined with an additional translocation involving other genes, such as BCL2, BCL3, or BCL6, whose category is defined as double-hit lymphoma (DHL), have dismal prognosis, because these cells are refractory to conventional immunochemotherapy. Recent studies have expanded the concept of a such disease entity to include double-expressing lymphomas (DEL) that co-overexpress MYC protein with those proteins, which have also poor prognosis. An adoptive T-cell immunotherapy with a chimeric antigen receptor (CAR) is clinically shown to have a powerful cytotoxicity in refractory neoplasias. Especially, several results showed that T cells transduced with an anti-CD19-CAR have successfully worked well in patients with refractory acute B-cell lymphoblastic leukemia and B-cell lymphoma as well as chronic B-cell lymphocytic leukemia. Thus, CAR T-cell therapy is a clinically promising tool for various refractory hematopoietic disorders. Accordingly, we examined whether anti-CD19- and/or anti-CD38-CAR-T cells, both of which we previously developed, could abrogate DHL cells. Here, we revealed that the remarkable cytotoxicity of anti-CD19- and/or anti-CD38-CAR T cells against DHL cells from patients. Firstly, DHL cell line cells (KPU-H1, a generous gift form Dr. Junya Kuroda, Kyoto Prefectural University of Medicine) were co-cultured with anti-CD19- and/or anti-CD38-CAR T cells at an effector (E) target (T) ratio of 1: 2 for three days. Cells harvested and stained with anti-CD19 and/or anti-CD38 antibody-APC or -PE were subjected to flow cytometry. Flow cytometric analysis showed that anti-CD19- or anti-CD38-CAR T cells almost killed KPU-H1 cells, respectively (specific cytotoxicity was >90%). Intriguingly, T cells expressing anti-CD19-CAR exerted a collaborative cytotoxicity against KPU-H1 cells with anti-CD38-CAR T cells in vitro. CD38-specific T cells were co-cultured with cytogenetic DHL (n=3) or DEL (n=2) cells from five patients carrying a poor prognosis for 3 days. We examined whether T cells retrovirally transduced with anti-CD19- and/or anti-CD38-CAR vector could show cytotoxicity against primary DHL cells obtained from patients. Anti-CD19 and/or ant-CD38-CAR T cells were co-cultured with primary DHL cells at an E: T ratio of 1: 2 for 3 days. Interestingly, anti-CD19 and anti-CD38-CAR T cells completely abolished these DHL cells from patients, respectively. Additionally, anti-CD19- and anti-38-CAR T cells were synergistically effective to eliminate DHL cells. These results showed that DHL cells, which are refractory or resistant to existing chemotherapeutic agents, can be efficiently abrogated by a clinical use of T cells with anti-CD19- and/or anti-CD38-CAR. These results might warrant adoptive immunotherapy with autologous T cells transduced with anti-CD19 and anti-CD38-CAR for patients with refractory DHL. DisclosuresNo relevant conflicts of interest to declare.