B-cell non-Hodgkin Lymphoma Cell Research Articles

Combination of CDK4/6 Inhibitor Palbociclib and PI3K Inhibitor Idelalisib Synergistically Induces an Anti-Tumor Effect in B-Cell Lymphoma and Overcomes Ibrutinib Resistance

Introduction: Aberrant activity of cyclin-dependent kinases 4/6 (CDK4/6) is frequently detected in B-cell non-Hodgkin's lymphoma (B-NHL), indicating the therapeutic implications of CDK4/6 inhibitors for B-cell malignancies. What's more, the introduction of agents targeting B cell receptor signaling, especially phosphatidylinositol 3-kinase (PI3K) isoform-specific inhibitors, is rapidly changing how B cell malignancies are treated. In this study, we aim to investigate whether the combination of CDK4/6 inhibitor palbociclib (PAL) and PI3Kδ inhibitor idelalisib (IDE) has synergistic anti-tumor effects in B cell lymphoma. Besides, we also evaluated the effect of co-administration PAL and IDE in Bruton's tyrosine kinase inhibitor (BTKi) resistant model. Methods: In vitro, cell viability was analyzed using Cell Titer-Glo ®️ Luminescent Cell Viability Assay. Flow cytometry was used to detect apoptosis and cell cycle arrest induction. Western Blotting analysis was used to detect the changes in essential proteins in related signaling pathways. We also conducted RNA-seq to evaluate the whole transcriptome changes brought by co-treatment with PAL and IDE. The synergistic anti-tumor effects of PAL and IDE were also evaluated in vivo. BTKi secondary resistant cell lines were generated by long-time exposure to ibrutinib and BTK C481S mutation cell line was conducted by lentivirus infection and puromycin selection. Results: Firstly, we analyzed diffuse large cell lymphoma (DLBCL) gene expression profile results from GEO database, and there were more than 50% patients carried cell cycle-related genes alterations, especially for CDKN2A-CDK4/6/CCND1-Rb machinery. Then, it was noteworthy that the high level of CDK4/6 mRNA and protein expression correlated with worse survival of DLBCL patients. Next, we illustrated that pharmacological inhibition of CDK4/6 kinase activity induced inhibition of cell proliferation, tumor cell apoptosis, and cell senescence. Meanwhile, PAL treatment upregulated the expression of PI3Kδ, p-AKT (S473), and p-mTOR (S2448), suggesting compensatory activation of the PI3K-AKT-mTOR signaling pathway. Thus, combination treatment of PAL and PI3K inhibitor IDE was assessed, and this combination synergistically induced a more potent anti-proliferative effect in several B-NHL cell lines, including mantle cell lymphoma cell lines (Z-138 and Jeko-1), DLBCL cell lines (U2932 and OCI-Ly8) and Burkitt lymphoma cell line (Raji). Meanwhile, co-administration treatment could also trigger synergistic anti-tumor activity in BTK inhibitor ibrutinib-resistant HBL-1 BTK C481S and HBL-1 IR cells (long time exposure to ibrutinib). Next, the flow cytometry results indicated that the B-NHL cells became apoptotic after the PAL and IDE treatment. The activation of apoptosis-related proteins (cleaved-caspase3 and cleaved-PARP) and decrease of anti-apoptosis proteins expression (BCL-2, BCL-xL, XIAP, and MCL-1) were induced by combination of CDK4/6 and PI3K inhibitor treatment. The combination of PAL and IDE also substantially increased the cell population of G0/G1 phase and elevated expression of CDK4, CDK6, and CyclinD1. Furthermore, the gene expression profile analysis demonstrated that the mRNA expression level of PLK1 was significantly decreased by co-administration treatment, rather than in PAL or IDE treatment alone. Finally, the combination treatment also showed synergistic anti-tumor activity in multiple tumor-bearing mice models. Conclusion: The combination of CDK4/6 inhibitor palbociclib and PI3K inhibitor idelalisib synergistically induced anti-tumor activity in B-cell lymphoma through downregulation of PLK1 expression, suggested a new combination direction for the treatment of B-NHL and even BTK inhibitor-resistant patients.

The Potent and Selective Antitumor Activity of CO-1 and Fully Humanized CO-1bi in Treatment of B Cell Non-Hodgkin's Lymphoma

Recent advancements have identified CD47 blockade as a promising approach for the treatment of B cell Non-Hodgkin's Lymphoma (B-NHL). CD47 blockade inhibits the CD47-SIRPα axis, which inhibits a “don't eat me” signal that facilitates the phagocytosis of tumor cells by macrophages [1,2]. While the effect of anti-CD47 monoclonal antibodies (mAbs) as monotherapy has been limited in B-NHL, combining CD47 mAbs with conventional treatment such as rituximab has demonstrated more encouraging outcomes. This combinatory approach may be attributed to the need for additional cytotoxic mechanisms to achieve clinically meaningful responses with CD47-mediated therapy in B-NHL [3]. Additionally, CD47 blockage has raised concerns about off-target effects and red blood cell depletion [4]. To address these challenges and improve treatment possibilities we developed a fully humanized CO-1bi based on the chimeric CO-1. The bifunctional CO-1 is an agonistic IgG4 anti-CD47 mAb designed to induce direct and rapid programmed cancer cell death (PCCD) and stimulate phagocytosis through blockage of CD47-SIRPα binding. CO-1bi is a humanized bivalent scFv-Fc fusion of CO-1 with superior safety (no red blood cell hemagglutination) while maintaining the functionality of CO-1. In this study, we evaluated the antitumor activity of CO-1 and CO-1bi on different B-NHL cell lines in vitro as well as in xenografts of B-NHL established in NOD- scid IL2Rγ null (NSG) mice. Both constructs demonstrated potent and fast cell death induction in lymphoma cell lines while sparing normal B cells. The robust antitumor activity demonstrated by CO-1 and CO-1bi as single agents in xenograft models of Burkitt's lymphoma underscores the significance of their additional agonistic PCCD capabilities compared to other anti-CD47 agents. The emergence of CO-1bi offers a unique strategy with dual functionality for the treatment of B-NHL and other malignancies without red blood cell depletion characterized by current anti-CD47 mAb in clinical trials. This innovative approach provides an improved and promising avenue for cancer treatment.

Diversity of intratumoral regulatory T cells in B-cell non-Hodgkin lymphoma

AbstractTumor-infiltrating regulatory T cells (Tregs) contribute to an immunosuppressive tumor microenvironment. Despite extensive studies, the prognostic impact of tumor-infiltrating Tregs in B-cell non-Hodgkin lymphomas (B-NHLs) remains unclear. Emerging studies suggest substantial heterogeneity in the phenotypes and suppressive capacities of Tregs, emphasizing the importance of understanding Treg diversity and the need for additional markers to identify highly suppressive Tregs. Here, we applied single-cell RNA sequencing and T-cell receptor sequencing combined with high-dimensional cytometry to decipher the heterogeneity of intratumoral Tregs in diffuse large B-cell lymphoma and follicular lymphoma (FL), compared with that in nonmalignant tonsillar tissue. We identified 3 distinct transcriptional states of Tregs: resting, activated, and unconventional LAG3+FOXP3− Tregs. Activated Tregs were enriched in B-NHL tumors, coexpressed several checkpoint receptors, and had stronger immunosuppressive activity compared with resting Tregs. In FL, activated Tregs were found in closer proximity to CD4+ and CD8+ T cells than other cell types. Furthermore, we used a computational approach to develop unique gene signature matrices, which were used to enumerate each Treg subset in cohorts with bulk gene expression data. In 2 independent FL cohorts, activated Tregs was the major subset, and high abundance was associated with adverse outcome. This study demonstrates that Tregs infiltrating B-NHL tumors are transcriptionally and functionally diverse. Highly immunosuppressive activated Tregs were enriched in tumor tissue but absent in the peripheral blood. Our data suggest that a deeper understanding of Treg heterogeneity in B-NHL could open new paths for rational drug design, facilitating selective targeting to improve antitumor immunity.

Targeted chitosan nanobubbles as a strategy to down-regulate microRNA-17 into B-cell lymphomamodels.

MicroRNAs represent interesting targets for new therapies because their altered expression influences tumor development and progression. miR-17 is a prototype of onco-miRNA, known to be overexpressed in B-cell non-Hodgkin lymphoma (B-NHL) with peculiar clinic-biological features. AntagomiR molecules have been largely studied to repress the regulatory functions of up-regulated onco-miRNAs, but their clinical use is mainly limited by their rapid degradation, kidney elimination and poor cellular uptake when injected as naked oligonucleotides. To overcome these problems, we exploited CD20 targeted chitosan nanobubbles (NBs) for a preferential and safe delivery of antagomiR17 to B-NHL cells. Positively charged 400 nm-sized nanobubbles (NBs) represent a stable and effective nanoplatform for antagomiR encapsulation and specific release into B-NHL cells. NBs rapidly accumulated in tumor microenvironment, but only those conjugated with a targeting system (antiCD20 antibodies) were internalized into B-NHL cells, releasing antagomiR17 in the cytoplasm, both in vitro and in vivo. The result is the down-regulation of miR-17 level and the reduction in tumor burden in a human-mouse B-NHL model, without any documented side effects. Anti-CD20 targeted NBs investigated in this study showed physico-chemical and stability properties suitable for antagomiR17 delivery in vivo and represent a useful nanoplatform to address B-cell malignancies or other cancers through the modification of their surface with specific targeting antibodies.

TRIAL IN PROGRESS: ODRONEXTAMAB, A CD20×CD3 BISPECIFIC ANTIBODY, FOR THE TREATMENT OF RELAPSED/REFRACTORY MARGINAL ZONE LYMPHOMA (R/R MZL)—A COHORT FROM THE ELM‐2 STUDY

Introduction: MZL is a heterogeneous disease comprising 3 subtypes, extra-nodal MZL of mucosa-associated lymphoid tissue, nodal MZL, and splenic MZL. Treatment of R/R MZL is similar to other indolent B-cell non-Hodgkin lymphoma (B-NHL) subtypes (e.g., follicular lymphoma [FL]), comprising rituximab-based immunochemotherapy regimens that achieve an objective response rate (ORR) of ∼45%–80%. Options after progression on rituximab-based regimens include the BTK inhibitor ibrutinib, which demonstrated an ORR of 46%, but a compete response (CR) rate of only 3%, in its pivotal trial in R/R MZL. Overall, limited options are available after progression on current therapies, and a need remains for more effective treatments for R/R MZL. Odronextamab is a bispecific antibody that binds CD20-expressing B-NHL cells and CD3 on T cells. In the Phase 1 ELM-1 study, odronextamab monotherapy showed antitumor activity and a manageable safety profile in a range of R/R B-NHL subtypes, including MZL (n = 6; ORR 67%) (Bannerji et al. Lancet Haematol, 2022). In the Phase 2 ELM-2 study, odronextamab elicited an ORR of 82% and CR rate of 75% in patients with heavily pretreated R/R FL (Kim, et al. ASH, 2022). These positive data in an indolent B-NHL subtype support evaluation of odronextamab in R/R MZL. Methods: ELM-2 (NCT03888105) is an open-label study of odronextamab in patients with R/R B-NHL comprising 5 disease-specific cohorts: FL, diffuse large B-cell lymphoma, mantle cell lymphoma, MZL, and other B-NHLs. Each cohort will be evaluated separately, and patients recruited from sites across North America, Europe, and Asia-Pacific regions. The MZL cohort is planned to include 78 patients, who will receive IV odronextamab monotherapy until disease progression or other protocol-defined reason for treatment discontinuation. Odronextamab is administered according to a step-up regimen during the first 21-day cycle (C), consisting of 0.7 mg split over C1 Day (D) 1 (0.2 mg) and C1D2 (0.5 mg), 4 mg split over C1D8 and C1D9, and 20 mg split over C1D15 and C1D16. The full 80 mg dose is given QW during C2 to C4, then 160 mg Q2W from C5 onwards. If a patient achieves a CR and has a durable response for ≥9 months after initial determination of the CR, then dosing interval will be decreased from Q2W to Q4W. Patients eligible for the MZL cohort will be ≥18 years of age; relapsed/refractory to ≥2 prior lines of systemic therapy; ECOG performance status ≤1; and have adequate bone marrow and hepatic functions. Patients with prior allogeneic stem cell transplant or CAR T treatment will be excluded. Primary endpoint is ORR (Lugano classification; assessed by independent central review). Key secondary endpoints include ORR (investigator evaluation); CR rate; progression-free survival; overall survival; duration of response; safety; pharmacokinetics; and patient-reported quality of life outcomes. As of January 2023, 19 patients with MZL had enrolled. Encore Abstract—previously submitted to ASCO 2023 and EHA 2023 The research was funded by: Regeneron Pharmaceuticals, Inc. Keywords: indolent non-Hodgkin lymphoma, immunotherapy, ongoing trials Conflicts of interests pertinent to the abstract. T. M. Kim Consultant or advisory role Boryung, AstraZeneca/MedImmune, Takeda, Novartis, Janssen, Hanmi, and Regeneron Pharmaceuticals, Inc Honoraria: Takeda, Janssen, Regeneron Pharmaceuticals, Inc, Samsung Bioepis, and AstraZeneca Research funding: Celgene, Boryung, Boehringer-Ingelheim, BMS, Bayer, AstraZeneca/MedImmune, Takeda, Roche/Genentech, Novartis, Janssen, Hanmi, Genmab, Merck Serono, Merck Sharp & Dohme, Regeneron Pharmaceuticals, Inc, and Sanofi M. Taszner Consultant or advisory role Roche and Takeda Honoraria: Roche, Takeda, Novartis, and Servier Educational grants: Roche A. Chaudhry Employment or leadership position: Regeneron Pharmaceuticals, Inc Stock ownership: Regeneron Pharmaceuticals, Inc M. Ufkin Employment or leadership position: Regeneron Pharmaceuticals, Inc Stock ownership: Regeneron Pharmaceuticals, Inc A. Uppala Employment or leadership position: Regeneron Pharmaceuticals, Inc Stock ownership: Regeneron Pharmaceuticals, Inc A. Sabir Employment or leadership position: Regeneron Pharmaceuticals, Inc Stock ownership: Regeneron Pharmaceuticals, Inc H. Mohamed Employment or leadership position: Regeneron Pharmaceuticals, Inc Stock ownership: Regeneron Pharmaceuticals, Inc S. Ambati Employment or leadership position: Regeneron Pharmaceuticals, Inc Stock ownership: Regeneron Pharmaceuticals, Inc E. Bachy Consultant or advisory role Roche, Gilead, ADC Therapeutics, Takeda, Novartis, and Incyte Honoraria: Kite, Gilead, Novartis, Roche, Incyte, Miltenyi Biotech, Takeda, and Sanofi Research funding: Amgen and BMS

PROTEOMIC PROFILING IDENTIFIES GRANZYME B INHIBITOR SERPIN B9 AS MEDIATOR OF RESISTANCE TO CAR T‐CELL AND BISPECIFIC ANTIBODY TREATMENT IN NODAL B‐CELL LYMPHOMA

Introduction: Treatment options for relapsed or refractory (r/r) B-cell non-Hodgkin lymphomas (B-NHL) have broadened towards T-cell engaging therapies, including CD19-targeting chimeric antigen receptor T-cells (CD19-CAR) and bispecific antibodies (CD19-BsAb). Although CD19-CAR and CD19-BsAb induce durable responses in some r/r B-NHL patients, response remains heterogenous and a significant proportion of patients experience insufficient responses or relapse. A thorough understanding of tumor-intrinsic mechanisms of resistance is crucial for the clinical improvement of T-cell immunotherapy. Methods: Aiming to identify lymphoma cell-inherent mechanisms that impair response to T-cell engaging therapy, we quantified the in-vitro response of 46 B-NHL cell lines to 3rd generation CD19-CAR and CD19-BsAb. Response was measured using a high-throughput flow cytometry-based assay. In parallel, cell lines were subjected to proteomic profiling and protein abundance was regressed on in-vitro response in order to identify proteomic determinants of response. We further combined CD19-CAR treatment with clinically relevant drugs and assessed how killing of lymphoma cells and expansion of T-cells was altered. Results: Response to CD19-CAR and CD19-BsAb was highly heterogeneous across cell lines and B-NHL entities, but not significantly linked to their proliferation rate or CD19 expression. Integration of the proteomic profiles revealed that abundance of Serpin B9, endogenous inhibitor of T-cell effector molecule granzyme B, was associated with poor response to CD19-CAR and CD19-BsAb. This finding was validated in overexpression and knock-out models. Overexpression of SERPINB9 indeed attenuated response to CD19-CAR and CD19-BsAb, while its knock-out rendered cell lines more sensitive. The observed changes in susceptibility to T-cell-mediated cytotoxicity upon modulation of SERPINB9 expression were independent of T-cell donor and expression of T-cell activation and proliferation markers. We sought to improve T-cell-mediated cytotoxicity specifically in samples with high SERPINB9 expression and found that immune checkpoint inhibitors and lenalidomide enhanced expansion of CAR T-cells and increased killing of lymphoma cells. In addition, vorinostat and antibody-drug-conjugate polatuzumab vedotin specifically killed lymphoma cells without affecting T-cell expansion. However, the observed improvement in T-cell-mediated cytotoxicity was independent of SERPINB9 expression. Keywords: aggressive B-cell non-Hodgkin lymphoma, immunotherapy, indolent non-Hodgkin lymphoma No conflicts of interests pertinent to the abstract.

G protein-coupled receptor 183 mediates the sensitization of Burkitt lymphoma tumors to CD47 immune checkpoint blockade by anti-CD20/PI3Kδi dual therapy.

Immunotherapy-based regimens have considerably improved the survival rate of B-cell non-Hodgkin lymphoma (B-NHL) patients in the last decades; however, most disease subtypes remain almost incurable. TG-1801, a bispecific antibody that targets CD47 selectively on CD19+ B-cells, is under clinical evaluation in relapsed/refractory (R/R) B-NHL patients either as a single-agent or in combination with ublituximab, a new generation CD20 antibody. A set of eight B-NHL cell lines and primary samples were cultured in vitro in the presence of bone marrow-derived stromal cells, M2-polarized primary macrophages, and primary circulating PBMCs as a source of effector cells. Cell response to TG-1801 alone or combined with the U2 regimen associating ublituximab to the PI3Kδ inhibitor umbralisib, was analyzed by proliferation assay, western blot, transcriptomic analysis (qPCR array and RNA sequencing followed by gene set enrichment analysis) and/or quantification of antibody-dependent cell death (ADCC) and antibody-dependent cell phagocytosis (ADCP). CRISPR-Cas9 gene edition was used to selectively abrogate GPR183 gene expression in B-NHL cells. In vivo, drug efficacy was determined in immunodeficient (NSG mice) or immune-competent (chicken embryo chorioallantoic membrane (CAM)) B-NHL xenograft models. Using a panel of B-NHL co-cultures, we show that TG-1801, by disrupting the CD47-SIRPα axis, potentiates anti-CD20-mediated ADCC and ADCP. This led to a remarkable and durable antitumor effect of the triplet therapy composed by TG-1801 and U2 regimen, in vitro, as well as in mice and CAM xenograft models of B-NHL. Transcriptomic analysis also uncovered the upregulation of the G protein-coupled and inflammatory receptor, GPR183, as a crucial event associated with the efficacy of the triplet combination. Genetic depletion and pharmacological inhibition of GPR183 impaired ADCP initiation, cytoskeleton remodeling and cell migration in 2D and 3D spheroid B-NHL co-cultures, and disrupted macrophage-mediated control of tumor growth in B-NHL CAM xenografts. Altogether, our results support a crucial role for GPR183 in the recognition and elimination of malignant B cells upon concomitant targeting of CD20, CD47 and PI3Kδ, and warrant further clinical evaluation of this triplet regimen in B-NHL.

Association between CHFR and PARP-1, and Their Roles in Regulation of Proliferation and Apoptosis of B Cell Lymphoma

Background. Aberrant methylation of checkpoint with forkhead and ring finger domains (CHFR) was found in B-cell non-Hodgkin lymphoma (NHL), whereas its role in carcinogenesis is not clear. CHFR can control poly (ADP-ribose) polymerase levels by causing its degradation. The study was aimed to explore the roles and mechanisms of CHFR in the pathogenesis of B-cell NHL. Methods. Short hairpin ribonucleic acid (ShRNAs) targeting CHFR and poly (ADP-ribose) polymerase 1 (PARP-1) were transduced into Raji cells, and real-time polymerase chain reaction (PCR) and western blotting were carried out to determine their expression. Afterwards, the CCK-8 assay and flow cytometry were used to evaluate the cell growth and apoptosis. Tumor size and weight were determined using a xenograft model, and decitabine (5-Aza-dC) was used to further determine the methylation status of CHFR through a methylation specificity-PCR assay. Results. 5-Aza-dC-treatment promoted the expression of CHFR and decreased the expression of PARP-1 at both messenger ribonucleic acid (mRNA) and protein levels. 5-Aza-dC also accelerated Raji-cell apoptosis and restrained its growth in vitro and in vivo ( P < 0.05 ). These results were contrary to those observed in the shRNA-CHFR group but consistent with those observed in the shRNA-PARP-1 group. The expression profiles of CHFR and PARP-1 in the xenograft model were consistent with those in the cellular model. Treatment with 5-Aza-dC led to demethylation of CHFR in nude mice. Besides, there may be a negative correlation between CHFR and PARP-1 in B-cell NHL cells. Conclusion. Our findings indicated that 5-Aza-dC could lead to the demethylation of the CHFR promoter and suppress Raji cell growth.

Galunisertib synergistically potentiates the doxorubicin-mediated antitumor effect and kickstarts the immune system against aggressive lymphoma.

In clinical practice, major efforts are underway to identify appropriate drug combinations to boost anticancer activity while suppressing unwanted adverse effects. In this regard, we evaluated the efficacy of combination treatment with the widely used chemotherapeutic drug doxorubicin along with the TGFβRI inhibitor galunisertib (LY2157299) in aggressive B-cell non-Hodgkin lymphoma (B-NHL). The antiproliferative effects of these drugs as single agents or in combination against several B-NHL cell lines and the synergism of the drug combination were evaluated by calculating the combination index. To understand the putative molecular mechanism of drug synergism, the TGF-β and stress signaling pathways were analyzed after combination treatment. An aggressive lymphoma model was used to evaluate the anticancer activity and post-therapeutic immune response of the drug combination in vivo. Galunisertib sensitized various B-NHL cells to doxorubicin and in combination synergistically increased apoptosis. The antitumor activity of the drug combinations involved upregulation of p-P38 MAPK and inhibition of the TGF-β/Smad2/3 and PI3K/AKT signaling pathways. Combined drug treatment significantly reduced tumor growth and enhanced survival, indicating that the synergism between galunisertib and Dox observed in vitro was most likely retained in vivo. Based on the tumor-draining lymph node analysis, combination therapy results in better prognosis, including disappearance of disease-exacerbating regulatory T cells and prevention of CD8+ T-cell exhaustion by downregulating MDSCs. Galunisertib synergistically potentiates the doxorubicin-mediated antitumor effect without aggravating the toxic effects and the ability to kickstart the immune system, supporting the clinical relevance of targeting TGF-βRI in combination with doxorubicin against lymphoma.

Characterization of anti-CD79b/CD3 bispecific antibody, a potential therapy for B cell malignancies.

High rates of relapse and poor prognosis confer an urgent need for novel therapeutic agents for B cell non-Hodgkin lymphomas (B-NHLs). Herein, we describe a human IgG-like anti-CD79b/CD3 bispecific antibody (IBI38D9-L) that selectively depletes antigen-positive malignant B cells as an alternative treatment option for relapsed or refractory NHL patients. The antitumor activity and mechanism of action of IBI38D9-L were investigated in vitro using B-NHL cell lines and human primary effector cells and in vivo using xenograft models reconstituted with human PBMCs (peripheral blood mononuclear cells). Pharmacokinetic (PK) properties and preclinical toxicology were evaluated in cynomolgus monkeys and HSC-NPG mice. IBI38D9-L exerted potent B cell killing as well as T cell activation and proliferation in a tumor cell-dependent manner in vitro and was active against B-NHL cell lines with various CD79b expression levels. Subcutaneous xenograft tumors in NOG mice engrafted with human PBMCs were eradicated by IBI38D9-L treatment. Moreover, IBI38D9-L-treated mice showed a strong infiltration of activated T cells. In HSC-NPG mice, IBI38D9-L resulted in potent B cell depletion in peripheral blood and induced only slight body weight loss and cytokine release syndrome without significant toxicological findings. In cynomolgus monkeys, IBI38D9-L was well tolerated with good pharmacokinetic profiles. Collectively, these preclinical efficacy and safety data provide strong scientific rationales for using anti-CD79b/CD3 bispecific antibody as a promising therapeutic agent for B cell malignancies.

Abstract CT127: Trial in progress: A phase II, open-label, multicenter study of capivasertib, a potent, oral pan-AKT inhibitor in patients with relapsed or refractory B-cell non-hodgkin lymphoma (CAPITAL)

Abstract Background: The PI3K/AKT/mTOR pathway is an established therapeutic target in indolent B-cell non-Hodgkin lymphoma (B-NHL). Several PI3K inhibitors are approved to treat relapsed/refractory (R/R) follicular lymphoma (FL) and, more recently, marginal zone lymphoma (MZL). Nonetheless, potential class-specific and PI3K-isoform-related toxicities may limit their clinical utility. Capivasertib is an oral, potent, selective inhibitor of the 3 AKT isoforms that demonstrated a manageable safety profile (n>1500) and survival benefit in combination with other agents in patients (pts) with solid tumors. Unlike PI3K inhibitors, colitis or pneumonitis have not been a concern with monotherapy. Preclinically, capivasertib was active both in vitro in a large panel of B-NHL cell lines and in vivo in PTEN-null GCB-DLBCL PDX models, where it downregulated the oncogenic factor MYC. In the JVM2 mantle cell lymphoma (MCL) cell line, capivasertib showed superior activity vs PI3Ka/d (AZD8835) and PI3Kb/d (AZD8186) inhibitors. Of note, AKT activation following dysregulation of PTEN has been frequently described in FL (where >85% of tFL or FL/DLBCL exhibited a GCB phenotype). Therefore, capivasertib may represent an effective treatment option for pts with R/R B-NHL. Trial Design: This modular phase II study will enroll pts in 3 cohorts according to B-NHL type. Core inclusion criteria include: age ≥18 years and ECOG PS ≤2. Module-specific criteria include: cohort 1A: R/R FL after ≥2 prior systemic lines (max 5), including an anti-CD20 monoclonal antibody (mAb) and an alkylating agent; cohort 1B: R/R MZL of splenic, nodal, and extranodal subtypes after ≥2 prior systemic lines (max 5) including ≥1 anti-CD20 mAb; cohort 1C: R/R MCL after ≥2 prior systemic lines (max 4), previously exposed to anti-CD20 mAb and a BTK inhibitor. Pts with FL grade 3B, transformed disease, blastoid or TP53 mutation MCL, active CNS disease, diabetes requiring insulin, or being <3 months post-CD19-directed CAR-T therapy will be excluded.Capivasertib will be administered orally at 480 mg BID with an intermittent schedule (4 days on/3 days off; 28-day cycles) until disease progression or unacceptable toxicity. The primary efficacy endpoint in AACR 2022_Capivasertib TiP Abstracteach cohort is objective response rate based on 2014 Lugano criteria per blinded independent central review. Secondary endpoints include duration of response, progression-free survival, overall survival, patient-reported outcomes, safety, and pharmacokinetics. An interim analysis of safety will occur when approximately 30 pts across all cohorts have been treated for 8 weeks. An interim analysis of efficacy will occur after 28 pts (cohorts 1A+1B) or 24 pts (cohort 1C) have been treated for 4 months. A total of 272 pts are planned across 28 sites in 7 countries globally (US, Europe, and Asia). Recruitment began in October 2021. Funding: AstraZeneca Clinical Trial Registration: NCT050080552 Citation Format: Michael Wang, Ho-Jin Shin, Alex F. Herrera, Moshe Levy, Wong Seog Kim, Tae Min Kim, Jin Seok Kim, Dok Hyun Yoon, Vincent Ribrag, Miguel Canales, John Radford, Dima El-Sharkawi, Brandy Hemmer, Richa Manwani, Veerendra Munugalavadla, Robert Chen, Andrew Mortlock, Alessandra Forcina, Franck Morschhauser. Trial in progress: A phase II, open-label, multicenter study of capivasertib, a potent, oral pan-AKT inhibitor in patients with relapsed or refractory B-cell non-hodgkin lymphoma (CAPITAL) [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 CT127.

Abstract 6303: Activity and tolerability of combination of venetoclax with the Aurora kinase B Inhibitor AZD2811 in preclinical diffuse large B-cell lymphoma models

Abstract Diffuse large B-cell lymphoma (DLBCL) is an aggressive type of non-Hodgkin lymphoma (NHL) and the most common form of adult lymphoma. Despite efforts to develop new therapies, the standard of care for DLBCL remains chemotherapy based. The current standard of care is comprised of a regimen of 4 drugs (cyclophosphamide, doxorubicin, vincristine, and prednisone) combined with immunotherapy using a chimeric mAb against the protein CD20 (rituximab), R-CHOP. Unfortunately only 50 to 60% of patients with DLBCL achieve and maintain complete remission after first-line therapy and 40 to 50 % relapse or have refractory disease. In addition, the average age at the time of diagnosis for DLBCL is 64 which makes intensive chemotherapy challenging. Therefore, the development of new treatment options is critical. We explored preclinical activity of AZD2811 nanoparticle, a potent and selective inhibitor of Aurora B kinase combined with venetoclax, an inhibitor of BCL2 currently used as standard of care in treatment of Haematological cancers, in B-Cell non-Hodgkin lymphoma models. Twenty five B-cell NHL cell lines including 11 Diffuse Large B-cell Lymphoma (DLBCL) cell lines were exposed to a 7 x 7 matrix of AZD2811 and venetoclax for 72 hrs. Cell viability was assessed using Celltiter-glo. Strong combination activity (HSA > 0.1 and combination Emax > 0.5) was observed in 6 cell lines including 2 DLBCL cell lines (WSU-DLCL2 and KARPAS_422). Moreover, pre-treatment with pan caspase inhibitor Q-VD-OPH, increased cell viability in cells treated with the combination. Next we explored the significant gene expression and mutation biomarkers (effect size > 1, p < 0.05) for this combination associated with Bliss and combination Emax in B-cell NHL cell lines. We found downregulation of 4 genes (FLT4, TIE1, PRKN) and mutations in 2 genes (EP300 and PTEN) to be associated with Bliss score and upregulation of one gene (FGR) associated with combination Emax. In vivo, antitumor activity of AZD2811 combined with venetoclax was assessed in mice bearing WSU-DLCL2luc xenografts. Once weekly intravenous administration of 25 mg/kg AZD2811 resulted in a statistically significant tumor growth inhibition (TGI) of 74%, while daily oral gavage of 100 mg/kg venetoclax resulted in 49% TGI but failed to reach statistical significance. While both monotherapies were unable to prevent progressive tumor growth, a combination dosing regimen of AZD2811 and venetoclax drove progressive tumor regression resulting in statistically significant complete regression (98% regression) by the third week of dosing. These results suggest combining AZD2811 with venetoclax has potential to be active in DLBCL patients. Citation Format: Azadeh Cheraghchi-Bashi, Brandon Willis, Kevin. Mongeon, Jacob Gordon, Krishna Bulusu, Elizabeth Coker, Patricia Jaaks, Mathew Garnett, Jerome Mettetal. Activity and tolerability of combination of venetoclax with the Aurora kinase B Inhibitor AZD2811 in preclinical diffuse large B-cell lymphoma models [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 6303.

Development and Characterization of 3D Hybrid Spheroids for the Investigation of the Crosstalk Between B-Cell Non-Hodgkin Lymphomas and Mesenchymal Stromal Cells.

PurposeB-cell non-Hodgkin lymphomas (B-NHLs) are the most common lymphoproliferative malignancy. Despite targeted therapies, the bone marrow involvement remains a challenge in treating aggressive B-NHLs, partly due to the protective interactions of lymphoma cells with mesenchymal stromal cells (MSCs). However, data elucidating the relationship between MSCs and B-NHLs are limited and inconclusive due to the lack of reproducible in vitro three-dimensional (3D) models. Here, we developed and described a size-controlled and stable 3D hybrid spheroids of Ri-1 (diffuse large B-cell lymphoma, DLBCL) and RAJI (Burkitt lymphoma, BL) cells with HS-5 fibroblasts to facilitate research on the crosstalk between B-NHL cells and MSCs.Materials and MethodsWe applied the commercially available agarose hydrogel microwells for a fast, low-cost, and reproducible hybrid lymphoma/stromal spheroids formation. Standard histological automated procedures were used for formalin fixation and paraffin embedding (FFPE) of 3D models to produce good quality slides for histopathology and immunohistochemical staining. Next, we tested the effect of the anti-cancer drugs: doxorubicin (DOX) and ibrutinib (IBR) on mono-cultured and co-cultured B-NHLs with the use of alamarBlue and live/dead cell fluorescence based assays to confirm their relevancy for drug testing studies.ResultsWe optimized the conditions for B-NHLs spheroid formation in both: a cell line-specific and application-specific manner. Lymphoma cells aggregate into stable spheroids when co-cultured with stromal cells, of which internal architecture was driven by self-organization. Furthermore, we revealed that co-culturing of lymphoma cells with stromal cells significantly reduced IBR-induced apoptosis compared to the 3D mono-culture.ConclusionThis article provides details for generating 3D B-NHL spheroids for the studies on the lymphoma- stromal cells. This approach makes it suitable to assess in a relevant in vitro model the activity of new therapeutic agents in B-NHLs.

Sting Is Commonly and Differentially Expressed in T- and Nk-Cell but Not B-Cell Non-Hodgkin Lymphomas.

Simple SummaryT/NK-cell non-Hodgkin lymphomas (NHLs) represent approximately 10% of all NHLs and most patients have a poor outcome using current treatment options. Molecules involved in the host response against lymphoma cells are currently being investigated in an effort to develop novel therapeutic strategies combining targeted therapy and immunotherapy. In this study, we show that expression of STING, a key protein in the cGAS–STING immune response pathway, is restricted to lymphomas of T- and NK-cell origin and seems to be down regulated in B-cell NHLs. These results are based on the analysis of 14 lymphoma cell lines of various types at the RNA and protein level and immunohistochemical analysis of a large number of B-cell (n = 265) and T/NK-cell (n = 158) NHLs obtained from previously untreated patients from three institutions. In these patient cohorts, STING is differentially expressed among T/NK-cell NHLs, whereas all B-cell NHLs were negative for STING expression. Thus, STING represents a novel biomarker and therapeutic target in T- and NK-cell lymphomas with direct immunotherapeutic implications, since modulators of cGAS–STING activity are already available for clinical use, and could therefore be used to benefit patients with these difficult-to-treat diseases. The expression patterns of stimulator of interferon genes (STING) were investigated in a cohort of 158 T- and natural killer (NK)-cell and 265 B-cell non-Hodgkin lymphomas (NHLs), as well as in control reactive lymph nodes and tonsils. STING expression was assessed by immunohistochemical methods using diagnostic biopsy specimens obtained prior to treatment. Using an arbitrary 10% cutoff, STING was differentially expressed among T/NK-cell NHLs; positive in 36 out of 38 (95%) cases of ALK+ anaplastic large cell lymphoma (ALCL), 23 out of 37 (62%) ALK-ALCLs, 1 out of 13 (7.7%) angioimmunoblastic T-cell lymphomas, 15 out of 19 (79%) peripheral T-cell lymphomas, not otherwise specified, 20 out of 36 (56%) extranodal NK/T-cell lymphomas of nasal type, 6 out of 7 (86%) T-cell lymphoblastic lymphomas, and 3 out of 4 (75%) mycosis fungoides. STING expression did not correlate with clinicopathological parameters or outcome in these patients with T/NK-cell lymphoma. By contrast, all 265 B-cell NHLs of various types were STING-negative. In addition, STING mRNA levels were very high in 6 out of 7 T-cell NHL cell lines, namely, ALK+ and ALK-ALCL cell lines, and very low or undetectable in 7 B-cell NHL cell lines, suggesting transcriptional downregulation of STING in neoplastic B-cells. At the protein level, using Western blot analysis and immunohistochemistry performed on cell blocks, STING expression was found to be restricted to T-cell NHL cell lines. Taken together, STING expression represents a novel biomarker and therapeutic target in T- and NK-cell lymphomas with direct immunotherapeutic implications since modulators of cGAS–STING activity are already available for clinical use.

Bone Marrow Infiltration Is a Distinctive Risk Factor for Rituximab Infusion-Related Reactions in CD20-Positive B-Cell Non-Hodgkin Lymphoma.

Background Bone marrow infiltration of lymphoma cells is a candidate risk factor for infusion-related reactions (IRRs) in patients with CD20-positive B-cell non-Hodgkin lymphoma (B-NHL). However, despite with the lack of sufficient data, the effect of bone marrow infiltration of B-NHL cells on the incidence rate of grade 2 or higher IRRs with the administration of rituximab has been retrospectively studied in this paper. Methods Patients with CD20-positive B-NHL who received the rituximab induction therapy for the first time were enrolled in this study. To evaluate the bone marrow infiltration of B-NHL cells, May–Giemsa stain of bone marrow films and flow cytometry examination of bone marrow aspiration samples were performed. IRR grade was determined using the IRR criteria in the Common Terminology Criteria for Adverse Events version 4.0. Results A total of 127 patients were eligible for this study. Grade 2 or higher IRRs were observed in 43 (34%) patients. In univariate analysis, use of glucocorticoid before rituximab infusion was a strong risk-avoiding factor for grade 2 or higher IRRs. Advanced stage of disease (Ann Arbor: stages III and IV) or bone marrow infiltration of B-NHL cells revealed the risk factors, regardless of glucocorticoid premedication. Using multivariate analysis, bone marrow infiltration was found to be an independent risk factor for patients without prior glucocorticoid use. Conclusion Bone marrow infiltration of B-NHL cells is a risk factor for grade 2 or higher IRRs at the first rituximab induction therapy without glucocorticoid premedication.

Antitumor Activity of the Novel BTK Inhibitor TG-1701 Is Associated with Disruption of Ikaros Signaling in Patients with B-cell Non-Hodgkin Lymphoma.

Despite the remarkable activity of BTK inhibitors (BTKi) in relapsed B-cell non-Hodgkin lymphoma (B-NHL), no clinically-relevant biomarker has been associated to these agents so far. The relevance of phosphoproteomic profiling for the early identification of BTKi responders remains underexplored. A set of six clinical samples from an ongoing phase I trial dosing patients with chronic lymphocytic leukemia (CLL) with TG-1701, a novel irreversible and highly specific BTKi, were characterized by phosphoproteomic and RNA sequencing (RNA-seq) analysis. The activity of TG-1701 was evaluated in a panel of 11 B-NHL cell lines and mouse xenografts, including two NF-κB- and BTKC481S-driven BTKi-resistant models. Biomarker validation and signal transduction analysis were conducted through real-time PCR, Western blot analysis, immunostaining, and gene knockout (KO) experiments. A nonsupervised, phosphoproteomic-based clustering did match the early clinical outcomes of patients with CLL and separated a group of "early-responders" from a group of "late-responders." This clustering was based on a selected list of 96 phosphosites with Ikaros-pSer442/445 as a potential biomarker for TG-1701 efficacy. TG-1701 treatment was further shown to blunt Ikaros gene signature, including YES1 and MYC, in early-responder patients as well as in BTKi-sensitive B-NHL cell lines and xenografts. In contrast, Ikaros nuclear activity and signaling remained unaffected by the drug in vitro and in vivo in late-responder patients and in BTKC481S, BTKKO, and noncanonical NF-κB models. These data validate phosphoproteomic as a valuable tool for the early detection of response to BTK inhibition in the clinic, and for the determination of drug mechanism of action.

Abstract 2351: Multiomics integration elucidates onco-metabolic modulators of drug resistance in lymphoma

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma (NHL). B-cell NHLs rely on Bruton's tyrosine kinase (BTK) mediated B-cell receptor signaling for survival and disease progression. However, they are often resistant to BTK inhibitors or soon acquire resistance after drug exposure. Upon reaching drug tolerance, B-cell NHL cells proliferate faster, which suggests increased metabolic activity. In this study, we explored the onco-metabolic regulators of ibrutinib -resistant activated B-cell (ABC) DLBCL using a ‘multi-omics' analysis that integrated metabolomics (using high-resolution mass spectrometry) and transcriptomics (gene expression analysis). We performed unbiased statistical analyses to uncover dysregulated metabolic pathways among the proteomic and transcriptomic datasets. Overlay of the analyses identified two significantly altered metabolic pathways at both the metabolic and transcript level. Gene-metabolite integration revealed interleukin 4 induced 1 (IL4I1) at the crosstalk of two significantly altered metabolic pathways involved in the production of various amino acids. We showed for the first time that a metabolic shift from glycolysis towards oxidative phosphorylation in these lymphomas is activated via the BTK-PI3K-AKT-IL4I1 axis and can be targeted therapeutically. These findings from our unbiased analyses highlight the role of metabolic dysregulation during drug resistance development. Furthermore, our work demonstrates that a multi-omics approach can be a powerful and unbiased strategy to uncover genes and pathways that drive metabolic dysregulation in cancer cells. Citation Format: Satishkumar Singh, Fouad Choueiry, Amber Hart, Anuvrat Sircar, Jiangjiang Zhu, Lalit Sehgal. Multiomics integration elucidates onco-metabolic modulators of drug resistance in lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2351.

High Frequency of CD74 Expression in B-Cell Non-Hodgkin's Lymphoma (NHL) and Targeting with SΤRO-001, a Novel Anti-CD74 Antibody Drug Conjugate (ADC) with Potent I n Vitro Cytotoxicty and In Vivo Anti-Tumor Activity

Introduction: STRO-001 is a novel CD74-targeting ADC comprised of an anti-CD74 human IgG1 antibody (SP7219) genetically incorporating the non-natural amino acid para-azidomethyl-L-phenylalanine (pAMF) with a non-cleavable dibenzocyclooctyne (DBCO)-maytansinoid linker-warhead. Highly efficient and precise site-specific conjugation enabled by cell-free antibody synthesis technology produced a well-defined homogeneous ADC with a drug-antibody ratio (DAR) of 2. Conjugation sites were selected based on highest stability both in vitro and in vivo . The aim of this study was to investigate the expression of CD74 in NHL and explore the therapeutic potential of STRO-001 in B-cell NHL cell lines and xenografts.Methods: The binding affinity of SP7219 to recombinant human CD74 ECD (extracellular domain) and cynomolgus monkey (cyno) CD74 ECD was measured by SPR using a Biacore T200. Biotinylated SP7219 was used for immunohistochemistry (IHC) in NHL tumor specimens. Flow cytometry was used for measuring CD74 expression in NHL cell lines. STRO-001 was used to determine the EC50 and percent span of killing in NHL cell lines. The anti-tumor activity of STRO-001 in NHL xenografts in severe combined immune deficient (SCID) mice was examined.Results: Anti-CD74 antibody SP7219 binds to both human and cyno CD74 ECD with high affinity (KD=0.85nM for human, KD=2.4nM for cyno). Tissue samples from the Department of Pathology at Stanford University were used to evaluate CD74 expression by IHC after appropriate permissions were obtained. Medium to high CD74 expression in >70% of cells was observed in 118/118 (100%) diffuse large B-cell lymphoma (DLBCL), 148/151 (98%) follicular lymphoma and 19/21 (90%) mantle cell lymphoma (MCL) samples. In vitro cytotoxicity assays show potent activity of STRO-001 in a diverse panel of B-cell tumor lines with EC50 values ranging from 0.17-13 nM (Table 1). CD74 cell surface expression is required for STRO-001 cytotoxicity but expression level does not correlate highly with in vitro potency (R2 = 0.4154). STRO-001 exhibits dose-dependent tumor growth inhibition in rituximab-resistant germinal center B-cell-like (GCB)-DLBCL SU-DHL-6 xenografts starting at 2.5 mg/kg weekly x 3 doses. STRO-001 + bendamustine/rituximab (BR) further improves tumor suppression in SU-DHL-6 xenografts compared to vehicle (p = 0.002) or BR alone (p = 0.02). In the activated B-cell (ABC)-DLBCL U2932 model, single doses of STRO-001 produce dose-dependent growth inhibition at 1 and 3 mg/kg and complete tumor regression at 10 mg/kg in 7/7 animals with no tumor re-growth up to > 90 days post-treatment (Figure 1). Studies with an MCL model, Jeko-1, demonstrate robust STRO-001 anti-tumor activity compared to vehicle (p < 0.0001) starting at a single 3 mg/kg dose, with a single 10 mg/kg dose resulting in tumor regression for up to 64 days post treatment (Figure 2). STRO-001 treatment 14 days post tumor inoculation was used to evaluate disease progression in the disseminated Mino MCL model. Vehicle-treated animals developed advanced progressive disease, with median survival of 81 days. In contrast, all Mino xenografts treated with STRO-001 at 3 mg/kg or 10 mg/kg were alive and disease-free at the time of sacrifice 135 days post tumor inoculation (Figure 3).Conclusions: CD74 is expressed with high frequency and intensity in DLBCL, follicular lymphoma and MCL, supporting the investigation of novel biologics targeting this antigen. STRO-001 demonstrates potent in vitro cytotoxicity in multiple NHL cell lines and anti-tumor activity in GCB-DLBCL, ABC-DLBCL and mantle cell lymphoma xenograft models, including prolongation of survival in the disseminated Mino MCL model and prevention of tumor regrowth in the U2932 ABC-DLBCL and Jeko-1 MCL subcutaneous models. Clinical studies of this novel ADC for treatment of B-cell malignancies are under development. [Display omitted] DisclosuresYu:Sutro Biopharma: Employment. Abrahams:Sutro Biopharma: Employment. Embry:Sutro Biopharma: Employment. Li:Sutro Biopharma: Employment. Zhao:Stanford University School of Medicine: Employment. Henningsen:Sutro Biopharma: Employment. DeAlmeida:Sutro Biopharma: Employment. Matheny:Sutro Biopharma: Employment. Kline:Sutro Biopharma: Employment. Yam:Sutro Biopharma: Employment. Stafford:Sutro Biopharma: Employment. Natkunam:Stanford University School of Medicine: Employment. Hallam:Sutro Biopharma: Employment. Lupher:Sutro Biopharma: Employment. Molina:Sutro Biopharma: Employment.

PD-1 blockade enhances cytokine-induced killer cell-mediated cytotoxicity in B-cell non-Hodgkin lymphoma cell lines.

The clinical utility of immune checkpoint inhibitors, such as programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) inhibitors used alone or in combination with other therapies, is currently gaining attention. In this particular scenario, the inclusion of cytokine-induced killer (CIK) cells has proven to be a novel therapeutic approach. CIK cells have shown anticancer activity in various hematopoietic malignancies, but their defined cytotoxicity in B-cell non-Hodgkin lymphoma (B-NHL) remains to be fully elucidated. The present study investigated the role of PD-1/PD-L1 blockades on the cytotoxic efficacy of CIK cells primarily in B-NHL cell lines. The current analysis revealed that CIK cells prompted cytotoxicity against B-NHL cell lines (DAUDI and SU-DHL-4), and a significant increase in PD-L1 expression was observed when CIK cells were co-cultured with B-NHL cells. Additionally, a combination of PD-1 and PD-L1 antibodies with CIK cells significantly decreased cell viability only in DAUDI cells. Furthermore, IFN-γ elevation was observed in both cell lines treated with CIK alone or with PD-1 antibody, but this tendency was not observed for PD-L1. Since PD-1 can suppress immune inactivation, whereas CD40L can promote it, the effects of CD40L blockade were also examined; however, no significant changes in cell viability were observed. Overall, the present in vitro data suggested that CIK cells exerted a cytotoxic function in B-NHL cells, and a combination of PD-1 inhibitors with CIK cells may provide a potential therapeutic option for this type of lymphoma. Nevertheless, in vivo experiments are warranted to undermine the extent to which PD-1 inhibitors may be used to enhance the antitumor activity of CIK cells in B-NHL.

Eupafolin induces autophagy and apoptosis in B-cell non-Hodgkin lymphomas.

Eupafolin, an extract from Artemisia princeps, possesses multiple pharmacological activities. However, the effect of eupafolin on B-cell non-Hodgkin lymphomas is currently unknown. In this study, we report that eupafolin shows anticancer activity against B-cell non-Hodgkin lymphomas cell line, OCI-LY-3. A CCK-8 assay was used to detect the proliferation inhibition of OCI-LY-3 cells treated with additional concentrations of eupafolin. Flow cytometric analysis method of the cell apoptosis was detected after cells stained with Annexin-V-FITC/PI according to the manufacturer's instructions. The proteins in the cell were detected by western blot after treatment with eupafolin. Eupafolin induced apoptosis in this cell line evidenced by the caspases activation, cleavage of PARP and downregulation of Bcl-2 and Bcl-xl. Eupafolin-induced autophagy was verified by accumulation of LC3-II and beclin-1. Eupafolin induced autophagy promoting apoptosis by the treatment of eupafolin combined with autophagy inhibitors 3-methyladenine and bafilomycin A1, respectively. Moreover, we disclose that the expression levels of p-Akt, p-mTOR,p-P70S6K and p-4EBP1 decrease in the Akt/mTOR signalling pathway, and the expression levels of proteins in the NF-ΚB signalling pathway, such as p-p65, p-IκBα, is downregulation. Together, these results provide crucial evidences explaining the antitumour activity of eupafolin in human NHL cell line, OCI-LY-3.