Antitumor Activity In Lymphoma Research Articles

Artesunate synergistically promotes sorafenib‑induced apoptosis and ferroptosis in non‑Hodgkin lymphoma cells through inhibition of the STAT3 pathway.

Despite the development of advanced therapies, the prognosis of non‑Hodgkin lymphoma (NHL) remains unsatisfactory due to refractory and relapsed cases. Artesunate (ART) and sorafenib (SOR) both exert potential antitumor activity in lymphoma. The present study aimed to investigate whether ART and SOR produce synergistic anti‑lymphoma effects, and to determine the potential underlying mechanisms. Cell viability assay, flow cytometry, malondialdehyde assay, GSH assay and western blotting were performed to evaluate cell viability, and changes in apoptosis, autophagic vacuoles, reactive oxygen species, mitochondrial membrane potential, lipid peroxidation and protein expression. The results demonstrated that ART and SOR synergistically inhibited the viability of NHL cells. ART and SOR also synergistically induced apoptosis, and markedly increased the expression levels of cleaved caspase‑3 and poly (ADP‑ribose) polymerase. Mechanistically, ART and SOR synergistically induced autophagy, and rapamycin enhanced the ART‑ or SOR‑induced inhibition of cell viability. Furthermore, it was demonstrated that ferroptosis promoted ART‑ and SOR‑induced cell death through increasing lipid peroxides. Erastin enhanced the inhibitory effects of ART and SOR on cell viability, whereas ferrostatin‑1 reduced the ART‑ and SOR‑induced apoptosis of SU‑DHL4 cells. Further studies revealed that signal transducer and activator of transcription3 (STAT3) contributed to ferroptosis induced by ART and SOR in NHL cells, and genetic inhibition of STAT3 promoted ART/SOR‑induced ferroptosis and apoptosis, concomitantly reducing the expression levels of glutathione peroxidase4 and myeloid cell leukemia‑1. Moreover, the combined treatment of ART and SOR exerted inhibitory effects on tumor growth, as well as antiangiogenic activity, resulting in the inhibition of CD31 expression in a xenograft model. Collectively, these findings indicated that ART acted synergistically with SOR to inhibit cell viability, and to induce apoptosis and ferroptosis through regulating the STAT3 pathway in NHL. Notably, ART and SOR may act as potential therapeutic agents for the treatment of lymphoma.

Abstract 1817: EG-011 is a first-in-class Wiskott-Aldrich syndrome protein (WASp) activator with anti-tumor activity

Abstract Background. Novel therapeutic targets are needed to improve the outcome of individuals with cancer. EG-011 is a small molecule with in vitro and in vivo anti-tumor activity in lymphoma and acute leukemias, but, at least so far, no activity in solid tumor models (Gaudio et al AACR 2019). Since no information was available on its target, we have now performed experiments showing that EG-011 targets WASp. Methods. Target identification: kinase screens with DiscoverX KINOMEscan, ProQinase Wildtype-Profiler; thermal proteomic profiling. Target validation: pyrene actin polymerization assay. In vitro drug treatment of cell lines followed for changes in actin filaments (F-actin) by confocal imaging with Alexa Fluor 488 Phalloidin. Results. Extensive kinome screens excluded kinases as targets of EG-011. We then applied thermal proteomic profiling to identify new protein targets interacting with EG-011. Over 3,300 proteins from the soluble proteome from the EG-011 sensitive mantle cell lymphoma cell line REC1 were analyzed and 48 possible protein targets were initially identified. Among the proteins undergoing a thermal shift, WASp was among the most highly destabilized by EG-011. Due to the pattern of expression of WASp, compatible with the anti-tumor activity observed only in hematological cancers (Gaudio et al AACR 2019), we performed further experiments to confirm the possibility that EG-011 targets WASp. One of the main functions of WASp is the regulation of actin filaments formation. Pyrene actin polymerization assays demonstrated that EG-011 activated the auto-inhibited form of WASP with strong actin polymerization. Further confirmation was obtained using confocal imaging of cell lines exposed to DMSO or EG-011 (500 nM, 5 μM) and stained for F-actin. An increase in actine polymerization was seen in EG-011 sensitive (VL51) and not in resistant (Z138) cell lines at 4, 8 and 24h with both concentrations. Conclusions. These data demonstrate that EG-011 is the “first-in-class” activator of the auto-inhibited form of WASp with selective anti-tumor activity in lymphomas. Citation Format: Filippo Spriano, Laura Barnabei, Ana Maria Carrasco Del Amor, Meagan R. Tomasso, Chiara Tarantelli, Eugenia Riveiro, Natalina Pazzi, Shae B. Padrick, Susana Cristobal, Franco Cavalli, Eugenio Gaudio, Francesco Bertoni. EG-011 is a first-in-class Wiskott-Aldrich syndrome protein (WASp) activator with anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1817.

Abstract 2222: Engineered variants of Neo-2/15 potently expand CAR-T cells and promote antitumor activity in lymphoma and solid tumor mouse models

Abstract Chimeric antigen receptor (CAR) T cells have achieved remarkable success as therapies for B cell malignancies, but failure might occur due to insufficient CAR-T cell expansion. Furthermore, CAR-T cells have been less effective in treating epithelial cancers, which is believed to be due to poor CAR-T cell accumulation at the tumor site. Interleukin-2 (IL-2) is a potent signaling molecule that induces T cell proliferation and survival. Therefore, it has been suggested that IL-2 co-administration might be effective in promoting CAR-T expansion, persistence, and tumor infiltration. Natural IL-2 acts by binding to the trimeric IL-2 receptor complex consisting of the IL-2Rα, IL-2Rβ and common-γ subunits. Although the IL-2Rα subunit is not directly involved in the signaling cascade, when present, it greatly increases the binding (and signaling) potency of IL-2. An obstacle to applying IL-2 to CAR-T therapy is the expression of IL-2Rα on many target-cells, such as immunosuppressive regulatory T cells (Treg) and endothelial cells. Consequently, high doses of IL-2 cause severe toxicities, including vascular leak syndrome, while low doses are therapeutically suboptimal due to preferential expansion of Treg cells. In addition, as a therapeutic, IL-2 has poor pharmacokinetics, necessitating frequent dosing or continuous infusion. Neoleukin-2/15 (Neo-2/15), a recently reported de novo IL-2 and IL-15 mimetic, effectively agonizes the IL-2 receptor IL-2Rβγ and has no dependency (or interaction) with IL-2Rα. Therefore, Neo-2/15 can activate T cells with high potency regardless of IL-2Rα expression. Here, we studied the combination of CAR-T cells with two reengineered versions of Neo-2/15. In the first strategy, NL-201, a half-life-extended version of Neo-2/15, was dosed weekly (300μg/kg) in a B cell lymphoma xenograft model. NL-201 induced potent CD19 CAR-T cell expansion and prolonged mice survival. NL-201 also induced potent ROR1 CAR-T cell expansion in a solid tumor syngeneic model of mice engrafted with the mutant K-ras/p53-/- tumor line overexpressing hROR1. NL-201 significantly increased the frequency of CAR-T cells at the tumor site compared to IL-2 and also dramatically increased the CD8:Treg ratio in the tumor. In the second approach, Neo-2/15 was targeted to engineered CAR-T cells by fusing the molecule to a DARPin domain specific for a transduction marker co-expressed with the CAR. In a B cell tumor xenograft model, targeted Neo-2/15 effectively increased CAR-T cell expansion and prolonged survival compared to treatment with CAR-T cells and non-targeted Neo-2/15. Together, these results show that engineered versions of Neo-2/15 induce robust expansion of CAR-T cells and can enhance their antitumor activity. Citation Format: Isabel Leung, Megan Templeton, Alfredo Rubio, Carl Walkey, Daniel-Adriano Silva, Stanley Riddell. Engineered variants of Neo-2/15 potently expand CAR-T cells and promote antitumor activity in lymphoma and solid tumor mouse models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2222.

Targeting CD205 with the antibody drug conjugate MEN1309/OBT076 is an active new therapeutic strategy in lymphoma models.

Antibody drug conjugates represent an important class of anti-cancer drugs in both solid tumors and hematologic cancers. Here, we report preclinical data on the anti-tumor activity of the first-in-class antibody drug conjugate MEN1309/OBT076 targeting CD205. The study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination, and validation experiments on in vivomodels. CD205 was first shown frequently expressed in lymphomas, leukemias and multiple myeloma by immunohistochemistry on tissue microarrays. Anti-tumor activity of MEN1309/OBT076 as single agent was then shown across 42 B-cell lymphoma cell lines with a median IC50 of 200 pM and induction of apoptosis in 25 of 42 (59.5%) of the cases. The activity appeared highly correlated with its target expression. After in vivo validation as the single agent, the antibody drug conjugate synergized with the BCL2 inhibitor venetoclax and the anti-CD20 monoclonal antibody rituximab. The first-in-class antibody drug targeting CD205, MEN1309/OBT076, demonstrated strong pre-clinical anti-tumor activity in lymphoma, warranting further investigations as a single agent and in combination.

Targeting glycogen synthase kinase 3 for therapeutic benefit in lymphoma

AbstractTargeting the B-cell receptor and phosphatidylinositol 3-kinase/mTOR signaling pathways has shown meaningful, but incomplete, antitumor activity in lymphoma. Glycogen synthase kinase 3 (GSK3) α and β are 2 homologous and functionally overlapping serine/threonine kinases that phosphorylate multiple protein substrates in several key signaling pathways. To date, no agent targeting GSK3 has been approved for lymphoma therapy. We show that lymphoma cells abundantly express GSK3α and GSK3β compared with normal B and T lymphocytes at the messenger RNA and protein levels. Utilizing a new GSK3 inhibitor 9-ING-41 and by genetic deletion of GSK3α and GSK3β genes using CRISPR/CAS9 knockout, GSK3 was demonstrated to be functionally important to lymphoma cell growth and proliferation. GSK3β binds to centrosomes and microtubules, and lymphoma cells treated with 9-ING-41 become arrested in mitotic prophase, supporting the notion that GSK3β is necessary for the progression of mitosis. By analyzing recently published RNA sequencing data on 234 diffuse large B-cell lymphoma patients, we found that higher expression of GSK3α or GSK3β correlates well with shorter overall survival. These data provide rationale for testing GSK3 inhibitors in lymphoma patient trials.

Abstract 5179: The first in class FLI1 inhibitor TK-216 presents both in vitro and in vivo anti-tumor activity in lymphoma

Abstract Background. ETS transcription factors, such as FLI1 and SPIB, are recurrently deregulated in human lymphomas (Bonetti et al, Blood 2013; Lenz et al, PNAS 2008). The small molecule YK-4-279 inhibits binding of EWS1-FLI1 fusion protein to RHA resulting in growth arrest and apoptosis in Ewing sarcoma cells (Erkizan et al, Nat Med 2009) and we previously showed that YK-4-279 has in vitro anti-lymphoma activity (Chung et al, AACR 2015). TK-216 is a YK-4-279 clinical derivative that is in phase 1 for patients with relapsed or refractory Ewing sarcoma (NCT02657005). Here, we present extensive preclinical results obtained with TK-216 in lymphoma models. Methods. 56 cell lines [27 diffuse large B cell lymphoma (DLBCL); 10 mantle cell lymphoma; 6 marginal zone lymphoma; 5 anaplastic large T-cell lymphoma; 8 others] were exposed to TK-216 increasing doses for 72h using a Tecan D300e Digital Dispenser and 384well plates; cell proliferation was measured with MTT. In vivo studies were performed in NOD-SCID mice and treatments started with approximately sc 60mm3 tumor volumes. Results. TK-216 displayed high activity: median IC50 was 449 nM (95%CI: 367-506). Sensitivity was not affected by the lymphoma cell of origin [B vs T; activated B cell type (ABC) vs germinal center type DLBCL] or MYC and TP53 status. There was a non-statistically significant trend for lower sensitivity in cell lines bearing BCL2 chromosomal translocation (P=0.07, DLBCL only; P=0.06, all cell lines). Anti-tumor activity was mainly cytotoxic as confirmed by performing cell cycle analysis and Annexin V staining in 6 DLBCL cell lines (TMD8, U2932, HBL1, OCI-LY-18, WSU-DLCL2, DOHH2 for 24, 48, 72h), in which a time-dependent apoptosis was preceded by G2/M arrest. Antitumor activity was confirmed in DLBCL TMD8 xenografts. Compared with control group (n=10), mice treated with TK-216 (100 mg/Kg, BID; n=9) clearly presented a reduction in tumor growth, already evident at day 3 and becoming much stronger with time (D3, D5, D8, D11: P<0.01; D13, P not available since control group had to be stopped due to tumor volume) and a 4 times reduction in tumor volume at D11 (P<0.01). TK-216 was tested in combination with other targeted agents in DLBCL cell lines. A benefit was observed with the combination of TK-216 with the immunomodulator lenalidomide (synergism in 2/2 ABC DLBCL), with the BET inhibitor OTX015 (MK-8628) (synergism in 2/4 cells and additive effect in 1/4), the anti-CD20 monoclonal antibody rituximab (synergism in 2/3 cells) and the BCL2 inhibitor venetoclax (synergism in 3/4 cells). The latter synergism could be linked to the previously mentioned negative trend between TK-216 IC50 values and the presence of BCL2 translocation. Conclusions. The novel small molecule TK-216 presented strong preclinical anti-lymphoma activity, which provides evidence for further preclinical and clinical development as single agent and in combination. Citation Format: Filippo Spriano, Chiara Tarantelli, Eugenio Gaudio, Elaine YL Chung, Alberto J. Arribas, Luciano Cascione, Sara Napoli, Ivo Kwee, Andrea Rinaldi, Davide Rossi, Emanuele Zucca, Anastasios Stathis, Katti Jessen, Brian Lannutti, Jeffrey Toretsky, Francesco Bertoni. The first in class FLI1 inhibitor TK-216 presents both in vitro and in vivo anti-tumor activity in 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 5179. doi:10.1158/1538-7445.AM2017-5179

Bromodomain inhibitor OTX015 (MK-8628) combined with targeted agents shows strong in vivo antitumor activity in lymphoma.

The bromodomain inhibitor OTX015 (MK-8628) has shown anti-lymphoma activity as a single agent in both the preclinical and clinical settings, as well as in vitro synergism with several anticancer agents. Here, we report in vivo data for OTX015 in combination with the histone deacetylase inhibitor vorinostat, the Bruton's tyrosine kinase inhibitor ibrutinib, the anti-CD20 monoclonal antibody rituximab, and the mTOR inhibitor everolimus in a diffuse large B cell lymphoma model. The antitumor effect of OTX015-containing combinations in SU-DHL-2 xenografts in mice was much stronger than the activity of the corresponding single agents with almost complete tumor eradication for all four combinations. Pharmacokinetic analyses showed similar OTX015 levels in plasma and tumor samples of approximately 1.5 μM, which is equivalent to the concentration showing strong in vitro activity. For all four combinations, mean terminal levels of the bromodomain inhibitor differed from those in mice exposed to single agent OTX015, indicating a need for thorough pharmacokinetic investigations in phase I combination studies. In conclusion, our results provide a strong rationale to explore OTX015-containing combinations in the clinical lymphoma setting.

Abstract 4700: A novel selective EZH2 inhibitor exhibits anti-tumor activity in lymphoma with EZH2 activating mutations

Abstract EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) responsible for methylating histone H3 on lysine 27 (H3K27) and repressing transcription of target genes. Dysregulation of H3K27 methylation is implicated in tumorigenesis in both solid and hematopoietic tumors and occurs through multiple mechanisms including elevation of EZH2 due to loss of regulating miRNAs; inactivating mutations in UTX, an H3K27 demethylase which acts in opposition to EZH2; and somatic mutations in EZH2. Point mutations identified in EZH2 in both follicular lymphoma (FL) and GCB diffuse large B cell lymphoma (DLBCL) exhibit increased H3K27 tri-methylation due to an altered histone substrate preference. This suggests that inhibitors of EZH2 activity may be effective in treating lymphomas carrying activating mutations in EZH2. We have identified first-in-class inhibitors of EZH2 which are highly-potent, reversible, SAM competitive, selective and equipotent against both WT and mutant EZH2. Cellular mechanistic studies demonstrate that inhibition of EZH2 decreases global H3K27me3 in both WT and EZH2 mutant cell lines; however, the cell proliferation response is quite different. Overall, DLBCL cell lines harboring activating mutations in EZH2 are highly sensitive to EZH2 inhibition, while DLBCL cell lines with WT EZH2 are moderately sensitive or resistant. Transcriptional and ChIP-seq studies reveal differences in the transcriptional response of EZH2 target genes and methylation patterns among cell lines providing insight into the mechanism of sensitivity. In a mouse model bearing EZH2 mutant DLBCL tumor xenografts, inhibition of EZH2 methyltransferase activity reduces global H3K27me3, increases expression of EZH2 target genes and inhibits the growth of tumors in a dose dependent manner. Together, these data demonstrate for the first time that direct inhibition of EZH2 methyltransferase activity is effective at inhibiting tumor growth and suggests a promising path forward in the clinic for the treatment of EZH2 mutant lymphoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4700. doi:1538-7445.AM2012-4700

Abstract A157: Targeting MYC dependence in cancer by inhibiting BET bromodomains.

Abstract The MYC transcription factor is a master regulator of diverse cellular functions and has long been considered a compelling therapeutic target because of its role in a wide range of human malignancies. However, pharmacologic inhibition of MYC function has proven challenging due to both the diverse mechanisms driving its aberrant expression and the challenge of disrupting protein-DNA interactions. Here we demonstrate the rapid and potent abrogation of MYC gene transcription by representative small molecule inhibitors of the BET family of chromatin adaptors. MYC transcriptional suppression was observed in a variety of genetic contexts. Inhibition of BET bromodomain-promoter interactions and subsequent reduction of MYC transcript and protein levels resulted in G1 arrest and extensive apoptosis in a variety of cell lines. Genetic reconstitution experiments indicate that MYC predominantly mediates the phenotypes observed with BET inhibition. MYC suppression was also accompanied by deregulation of the MYC transcriptome. Treatment with a BET inhibitor resulted in significant anti-tumor activity in lymphoma and leukemia xenograft models. These findings demonstrate that pharmacologic inhibition of MYC is achievable through targeting BET bromodomains. Such inhibitors may have clinical utility given the widespread pathogenetic role of MYC in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A157.

Therapeutic Potential of Anti-CD137 Antibody in Lymphoma.

Abstract 722Immunomodulating monoclonal antibodies (mAb) directed against immune cell targets can be used to enhance antitumor immune responses. CD137 (4-1BB) is a costimulatory molecule expressed on a variety of activated immune cells, including T and NK cells. Anti-CD137 agonistic mAb has demonstrated antitumor activity in various tumor models and has now entered clinical trials for the treatment of solid tumors (BMS-663513). Here, we investigate the therapeutic potential of anti-CD137 mAb in lymphoma.Using microarray gene expression data, we compared CD137 mRNA expression across 17 different types of cancer. We found that lymphoma tumor specimens significantly over-expressed CD137 mRNA compared to other tumors (p<0.0001). Single cell analysis of primary lymphoma samples of various histologies (follicular lymphoma [n=29], mantle cell lymphoma [n=14], and diffuse large B cell lymphoma [n=11]) revealed that CD137 was expressed by a significant proportion of tumor-infiltrating T cells, particularly in the CD8 subset (mean[range]=6.1%[2.5-11.5], 15.4%[2.7-40.9] and 18.7%[6.7-51.7], respectively). In contrast, CD137 was not expressed by the tumor cells. These results suggest that the target of anti-CD137 mAb is present and selectively expressed on cells with potential antitumor activity in patients with lymphoma.Using a murine model, we then investigated the anti-lymphoma activity of anti-CD137 agonistic mAb in vivo. BALB/c mice, bearing large and established A20 lymphoma tumors, were treated with anti-CD137 mAb or other immunomodulating mAbs (anti-OX40, anti-CTLA4, anti-GITR) for comparison. The mAbs were given i.p. on days 5 and 10 post tumor inoculation. Anti-CD137 mAb demonstrated potent anti-lymphoma activity in vivo, and appeared to be significantly superior to the other immunomodulating mAbs tested (Figure 1). The antitumor effect of anti-CD137 mAb was not due to direct targeting of the malignant cells as A20 tumor cells do not express CD137. [Display omitted] Depletion experiments revealed that anti-CD137 therapy required both NK and CD8 T cells. Analysis of tumor-bearing mice showed that anti-CD137 therapy increased the number of CD8 T cells and reduced the number of Tregs at the tumor site. Anti-CD137 therapy also induced long-lasting antitumor immunity as cured mice harbored antitumor IFN-g producing memory CD8 T cells and were protected from tumor re-challenge more than 100 days after initial therapy.In conclusion, this study demonstrates for the first time that anti-CD137 agonistic mAb has potent antitumor activity in lymphoma. These results support the evaluation of anti-CD137 mAb in clinical trials for patients with lymphoma. Disclosures:No relevant conflicts of interest to declare.