Metformin Affects the Proliferation Cell Nuclear Antigen (PCNA) and p21 Protein Interaction Resulting in Direct Anti-Tumor Activity and Enhances the Cytotoxic/Biological Effects of Chemotherapy Agents or Rituximab in Lymphoma in Vitro and in Vivo Pre-Clinical Models

Abstract Most cancer cells predominantly produce adenosine triphosphate (ATP) by a high rate of glycolysis followed by lactic acid fermentation in the cytosol, rather than by a comparatively low rate of glycolysis followed by oxidation of pyruvate in mitochondria. This shift in cellular metabolism is know as the Warburg effect and is primarily observed in rapidly growing tumors including aggressive B-cell lymphoma and is thought to be a consequence of the progression to cancer rather than the cause of it. Altering the glucose metabolism in cancer cells appears to be an attractive strategy in cancer medicine. Previously, we demonstrated that the acquirement of rituximab resistance was associated with an increase in the Warburg effect leading to concomitant chemotherapy resistance in lymphoma pre-clinical models. The use of metformin, an oral biguanide widely used to treat insulin resistance conditions, during chemotherapy has been associated with improved clinical outcomes in solid tumor patients receiving chemotherapy. In a retrospective analysis, we demonstrated that the use of metformin during front-line chemo-immunotherapy (i.e. R+CHOP) improved the clinical outcome of diffuse large B-cell lymphoma (DLBCL). In an attempt to characterize the mechanism by which metformin affects the biology of DLBCL, we studied the metabolic and signaling changes in rituximab-sensitive (RSCL) and rituximab-resistant cell lines (RRCL) exposed to metformin. A panel of RSCL and RRCL were exposed to metformin. Changes in Ki67, proliferation cell nuclear antigen (PCNA), and its regulator (p21) were determined by flow cytometry or Western blotting respectively. Ki67/PCNA/p21 changes were correlated to cell cycle distribution, cell viability and chemotherapy sensitivity. For in vivo studies, SCID mice were inoculated via tail vein injection (iv) with Raji cells (day 0) and assigned to observation, rituximab (at 10mg/kg/dose on Day 3,7,10,14), metformin (at 2ug/ml in drinking water from day 3 till the end of experiment) or metformin with rituximab. Differences in survival (measured as the time for limb paralysis development) were evaluated by log-rank test between treatment arms. Metformin in combination with rituximab (mean survival not reached at 69+/- 5.3 days) lead to an improved survival than rituximab (mean survival 57.1+/-4.2 days) (P=0.05). In vitro exposure of RSCL/RRCL to metformin inhibited cell proliferation (measure by, MTT assay, alamar blue reduction, cell titer glow assay, and ki67 staining) in a dose-dependent manner and enhanced the anti-tumor activity of chemotherapy drugs. Perhaps related to this effect, metformin exposure induced G1 phase cell cycle arrest in both RSCL and RRCL. Using the flow cytometry technology (FITC-labeled anti-KI67 antibody/PI-DNA staining), we detected a decrease in Ki67 of RSCL/RRCL distributed in the G1 and S phase following metformin exposure. In vitro exposure of RSCL and RRCL to metformin increased p21 and reduced PCNA levels. Immuno-precipitation of p21 following metformin drug exposure increased in the interaction between p21 and PCNA. Our data suggests that metformin inhibits the proliferation of RSCL or RRCL and enhances the anti-tumor activity of chemotherapy agents or rituximab in lymphoma pre-clinical models (in vitro and in vivo). Perhaps related to the biological effects observed, p21 and PCNA, play a pivotal role on the anti-tumor activity of metformin in lymphoma pre-clinical models. Our finding highlights a potential role for metformin in the treatment of B-cell malignancies. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures No relevant conflicts of interest to declare.

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Abstract 4182: The two novel BTK-inhibitors M2951 and M7583 show in vivo anti-tumor activity in pre-clinical models of B cell lymphoma

10.1158/1538-7445.am2017-4182 ◽

2017 ◽

Cited By ~ 1

Author(s):

Eugenio Gaudio ◽

Chiara Tarantelli ◽

Emanuele Zucca ◽

Davide Rossi ◽

Anastasios Stathis ◽

...

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Clinical Models ◽

Anti Tumor Activity ◽

Btk Inhibitors

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The Synergistic Anti-Tumor Activity of EZH2 Inhibitor SHR2554 and HDAC Inhibitor Chidamide through ORC1 Reduction of DNA Replication Process in Diffuse Large B Cell Lymphoma

Cancers ◽

10.3390/cancers13174249 ◽

2021 ◽

Vol 13 (17) ◽

pp. 4249

Author(s):

Xing Wang ◽

Dedao Wang ◽

Ning Ding ◽

Lan Mi ◽

Hui Yu ◽

...

Keyword(s):

B Cell ◽

Hdac Inhibitor ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Replication Process ◽

Large B Cell Lymphoma ◽

Anti Tumor Activity ◽

Large B Cell

Background: Upregulation of H3K27me3 induced by EZH2 overexpression or somatic heterozygous mutations were implicated in lymphomagenesis. It has been demonstrated that several EZH2-target agents have notable therapeutic effects in EZH2-mutant B-cell lymphoma patients. Here we present a novel highly selective EZH2 inhibitor SHR2554 and possible combination strategy in diffuse large B-cell lymphoma (DLBCL). Methods: Cell proliferation, cell cycle and apoptosis were analyzed by CellTiter-Glo Luminescent Cell Viability Assay and flow cytometry. Western Blot was used to detect the expression of related proteins. The gene expression profiling post combination treatment was analyzed by RNA-Seq. Finally, CDX and PDX models were used to evaluate the synergistic anti-tumor effects of the combination treatment in vivo. Results: The novel EZH2 inhibitor SHR2554 inhibited proliferation and induced G1 phase arrest in EZH2-mutant DLBCL cell lines. The combination of EZH2 inhibitor SHR2554 with histone deacetylase (HDAC) inhibitor chidamide (hereafter referred to as HBI8000) exerted synergistic anti-proliferative activity in vitro and in vivo. Gene expression profile analysis revealed dramatic inhibition of the DNA replication process in combined treatment. Conclusions: SHR2554, a potent, highly selective small molecule inhibitor of EZH2, inhibited EZH2-mutant DLBCL more significantly in vitro and in vivo. The combination of HDAC inhibitor HBI8000 with EZH2 inhibitor SHR2554 exhibited dramatic anti-tumor activity in both mutant and wild-type DLBCL, which may become a potential therapeutic modality for the treatment of DLBCL patients.

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Blockade of the CD47/SIRPα Checkpoint Potentiates the Anti-Tumor Efficacy of Tafasitamab

Blood ◽

10.1182/blood-2020-139582 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 11-12

Author(s):

Doris Mangelberger ◽

Christian Augsberger ◽

Karin Landgraf ◽

Christina Heitmüller ◽

Stefan Steidl

Keyword(s):

Monoclonal Antibody ◽

Tumor Growth ◽

B Cell ◽

Tumor Model ◽

B Cell Lymphoma ◽

Checkpoint Blockade ◽

Subcutaneous Tumor ◽

Anti Tumor Activity

Introduction Tafasitamab (MOR208) is an Fc-enhanced, humanized, monoclonal antibody that targets CD19 and has shown promising clinical activity in patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL). CD19 is homogeneously expressed among different B-cell malignancies, and the binding of tafasitamab to CD19 directly mediates cell death, induces antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. Aiming to potentiate the tafasitamab-mediated "eat me" signal, we tested a combination with a CD47-directed monoclonal antibody (mAb) to inhibit the CD47/SIRPα "don't eat me" signal and further enhance macrophage-mediated phagocytosis. Preclinical studies demonstrated that blocking the CD47/SIRPα checkpoint in combination with antibodies, such as rituximab, increased phagocytosis by macrophages, resulting in effective anti-tumor effects in non-Hodgkin lymphoma (NHL) (Chao, et al. 2010). Additionally, the combination of the anti-CD47, magrolimab, and the anti-CD20, rituximab, demonstrated beneficial outcomes for patients with refractory NHL (Advani, et al. 2019). Here, we present in vitro and in vivo data on the combinatory effect of tafasitamab and an anti-CD47 mAb in preclinical models of Burkitt's lymphoma (BL). Methods During in vitro studies, CD14+ monocytes were isolated from the whole blood of healthy volunteers and differentiated with 50 ng/mL M-CSF for 6 days. ADCP was analyzed by flow cytometry in co-culture experiments with Ramos cells (BL) after 3 hours of treatment with tafasitamab and anti-CD47 mAb (clone B6H12). In vivo, the combination of tafasitamab with an anti-CD47 mAb was tested in a Ramos disseminated survival and subcutaneous tumor model in SCID and NOD-SCID mice, respectively. In both models, tafasitamab was administered therapeutically twice a week either at 3 mg/kg (disseminated) or 10 mg/kg (subcutaneous) for max. 4 weeks. The anti-CD47 mAb was administered at 4 mg/kg three times per week. Main study readouts were to assess animal survival and any delays in tumor growth. Results The combination of tafasitamab + CD47/SIRPα checkpoint blockade enhanced ADCP activity of primary M2 macrophages on BL-derived Ramos cells, in comparison with the anti-CD47 mAb or tafasitamab monotherapies (Figure 1A). In vivo, a significant increase in anti-tumor activity was observed with the combination of tafasitamab + anti-CD47 mAb. In the Ramos disseminated survival model, the combination showed an increased life span (ILS) of >182% compared with tafasitamab monotherapy control, with an overall survival of all animals treated with the combination (15/15) until the end of the study (Day 99 post-cell injection). Additionally, pronounced anti-tumor efficacies were detected in the Ramos subcutaneous tumor model. Here, the combination resulted in a significant delay in tumor growth compared with the tafasitamab or anti-CD47 mAb monotherapies (ILS >175% tafasitamab and ILS >72% anti-CD47 mAb vs tafasitamab + B6H12) (Figure 1B). Conclusions The ADCP activity of primary macrophages was increased by combining tafasitamab with an anti-CD47 mAb in vitro, resulting in enhanced anti-tumor activity compared with tafasitamab or anti-CD47 mAb monotherapies in vivo. Overall, results indicate the combination of tafasitamab with a CD47/SIRPα checkpoint blockade may be a promising novel combination approach for lymphoma therapy. Disclosures Mangelberger: MorphoSys AG: Current Employment. Augsberger:MorphoSys AG: Current Employment. Landgraf:MorphoSys AG: Current Employment. Heitmüller:MorphoSys AG: Current Employment. Steidl:MorphoSys AG: Current Employment.

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The SMAC Mimetic Lcl-161 Augments the in Vitro and in Vivo anti-Tumor Activity of Rituximab and Chemotherapy in Rituximab Relapse/Refractory Lymphoma Models

Blood ◽

10.1182/blood.v124.21.3110.3110 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3110-3110 ◽

Cited By ~ 2

Author(s):

Kyle L. Runckel ◽

Joseph Skitzki ◽

Francisco Hernandez ◽

Myron S. Czuczman

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

Chemotherapy Resistance ◽

B Cell Lymphoma ◽

Over Expression ◽

Tail Vein Injection ◽

Refractory Lymphoma ◽

Anti Tumor Activity

Abstract The addition of rituximab to front-line therapy regimens in diffuse large B-cell lymphoma (DLBCL) has greatly improved clinical outcomes, but is also associated with a disease that is more resistant to salvage chemotherapy in the second-line setting, reinforcing the need for new therapies targeted at the overlapping resistance pathways between rituximab and chemotherapy. To better understand the mechanisms responsible for rituximab/chemotherapy cross-resistance we developed several rituximab resistance cell lines which exhibited significant concurrent chemotherapy resistance. These multi-therapy resistant cell lines (TRCL) exhibit decreased expression of the pro-apoptotic Bcl-2 family proteins Bak and Bak, along with over-expression of several anti-apoptotic proteins, including the inhibitor of apoptosis proteins (IAP) survivin and livin (determined by Western blot). High IAP expression has been associated with inferior clinical outcomes in a range of hematological malignancies, and solid tumors. To determine the impact of IAP over-expression on TRCL rituximab/chemotherapy resistance we utilized a transient siRNA knockdown of both survivn and livin. TRCLs with livin knockdown had a statistically significant improvement in response to several chemotherapy agents including doxorubicin, vincristine, and the proteasome inhibitor carfilzomib (measured at 48 hours with the Cell Titer-Glo viability assay). These results support livin over-expression as a key lymphoma therapy resistance mechanism, and establish IAPs as potential therapeutic targets. Small molecule IAP inhibitors, like LCL-161 (obtained from Novartis), are chemical mimetics of the endogenous IAP antagonist termed the second mitochondrial inhibitor of caspases (SMAC). Western blot analysis indicated that TRCLs treated in vitro with LCL-161 exhibited a dose dependent decrease in the expression of several IAPs, including livin. In addition, LCL-161 increased rates of TRCL apoptosis, and produced synergistic anti-tumor activity when combined with cytarabine, gemcitabine, and carfilzomib in vitro. LCL-161 also enhanced the ex vivo anti-tumor activity of carfilzomib against primary tumor cells isolated from lymphoma patients with both de novo, and relapse/refractory disease. Cell viability and apoptosis induction were determined at 48 hours with CellTiter-Glo viability assays and flow cytometry respectively. To evaluate the anti-tumor effect of LCL-161 in vivo severe combined immunodeficiency (SCID) mice were inoculated with the TRCL Raji-4RH via tail vein injection (iv), and assigned to observation or treatment arms 7 days after inoculation. Treatments were LCL-161 alone (60mg/kg), the combination of rituximab: 10mg/kg, gemcitabine: 120mg/kg, and vinorelbine: 8mg/kg (RGV), or LCL-161 and RGV together. LCL-161 was administered on day 7 as one dose given p.o. by gavage; RGV was also administered on day 7 as a single i.v. dose given by tail vein injection. Differences in survival (measured as the time to the development of limb paralysis) were evaluated with the Log-rank, Breslow, and Tarone-Ware tests across treatment arms. As a single agent LCL-161 was ineffective in controlling Raji-4RH tumor growth in vivo. However, the combination of LCL-161 with RGV (median survival 133 days) resulted in a statistically significant (P=0.002 with each test) improvement in overall survival when compared to RGV alone (median survival 53 days). In summary, IAPs, especially livin, contribute to rituximab/chemotherapy resistance in relapse/refractory B-cell lymphoma models. However, the IAP inhibitor LCL-161 can disrupt this resistance and augment the effect of chemotherapy in resistant lymphoma cell line models, as well as relapse/refractory lymphoma patient samples. In addition, LCL-161 can improve the anti-tumor activity of the RGV chemotherapy regimen, and increase overall survival in a mouse in vivo model of human rituximab/chemotherapy resistant lymphoma. Our data supports the continued investigation of LCL-161 as a novel and effective targeted agent for the treatment of aggressive rituximab relapse/refractory B-cell lymphomas. (Supported by a NHI SPORE Lymphoma grant: 5 P50 CA130805-04, a NIH grant R01 CA136907-01A1 and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures No relevant conflicts of interest to declare.

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Development of CD19 CAR Engineered Human Placental CD34 +-Derived Natural Killer Cells (CAR19-CYNK) As an Allogeneic Cancer Immunotherapy

Blood ◽

10.1182/blood-2021-150593 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2779-2779

Author(s):

Marina Gergues ◽

Irene Raitman ◽

Joseph Gleason ◽

Valentina Rousseva ◽

Shuyang He ◽

...

Keyword(s):

B Cell ◽

Nk Cells ◽

Cell Lines ◽

B Cell Lymphoma ◽

Publicly Traded ◽

B Cell Malignancies ◽

Cd34 Cells ◽

Anti Tumor Activity

Abstract Background: Natural killer (NK) cells exhibit anti-tumor activity in a non-antigen-specific manner without causing graft-versus-host disease. T cell and cord blood NK cells expressing chimeric antigen receptor (CAR) targeting CD19 have demonstrated remarkable clinical efficacies against B cell lymphomas (Maude et al, N Engl J Med 2018; Neelapu et al, N Engl J Med 2017; Liu et al, N Engl J Med 2020). Celularity has developed a platform for the expansion and differentiation of human placental CD34 + stem cells towards NK cells. The introduction of CD19 CAR enables generation of CAR19-CYNK cells that can be used as an off-the-shelf, cryopreserved, allogeneic cell therapy for CD19 + B cell malignancies. Reported here are the in vitro and in vivo results evaluating anti-tumor activity of CAR19-CYNK against CD19 + B cell malignancies. Methods: CAR19-CYNK cells were generated by retroviral transduction of human placental CD34 + cells with an anti-CD19 CAR (CD19scFv-CD28CD3ζ, Sorrento Therapeutics), followed by culture expansion in the presence of cytokines. CD19 CAR expression and phenotype of CAR19-CYNK cells were characterized by flow cytometry using the following surface markers: CD56, CD3, CD226, CD16, CD11a, CD94, NKG2D, NKp30, NKp44, NKp46. The in vitro anti-tumor activity of CAR19-CYNK against the B cell lymphoma cell lines, Daudi and Nalm-6, was assessed at various effector to target (E:T) ratios using a flow cytometry-based cytotoxicity assay and multiplex Luminex analysis for cytokine profiling. Non-transduced (NT) NK cells were used as control. In vivo efficacy of CAR19-CYNK was assessed using a disseminated B-cell lymphoma xenograft model in B-NDG-hIL15 mice. B-NDG-hIL15 mice lack T, B, and NK cells and are transgenic for human IL-15 to support CAR19-CYNK persistence and maturation. Luciferase expressing Daudi cells (3×10 6) were intravenously (IV) injected on Day 0 three days after the mice were preconditioned with a myeloablative dose of busulfan to allow for better tumor cell engraftment. CAR19-CYNK cells (1x10 7) were IV injected on Day 7. Tumor burden was assessed weekly by bioluminescence imaging (BLI) and the mice were followed for assessment of their survival (n=5 mice per group). Results: Placental CD34 + cells were genetically modified using a retroviral vector and achieved an average of 29.2% ± 12.4% (range 17.5% to 50.1%; n=5 donor lots) CD19 CAR expression on CAR19-CYNK cells at the end of 35-day culture. The average fold expansion of CAR19-CYNK was 6186 ± 2847 with the range of 2692 to 10626 (n=5 donor lots). Post-thaw evaluation of CAR19-CYNK (n=5 donor lots) revealed 93.8 ± 3.9% of CD56 +CD3 - NK cells, and transduction of CD19 CAR on CYNK did not significantly alter NK cell phenotype based on various activation and lineage markers (CD226, CD16, CD11a, CD94, NKG2D, NKp30, NKp44, NKp46). CAR19-CYNK displayed enhanced in vitro cytotoxicity against lymphoma cell lines, Daudi and Nalm-6, compared to that of NT NK cells. At the E:T ratio of 10:1, CAR19-CYNK (n=5 donor lots) elicited significant increased cytotoxicity against Nalm-6 compared to that of NT NK cells, with 75.9 ± 14.8% vs. 0.00 ± 0.00% at 24h (p<0.005). Under the same condition, CAR19-CYNK (n=4 donor lots) showed higher cytotoxicity against Daudi compared to that of NT NK cells with 23.6 ± 18.9% vs. 4.9 ± 4.0%. When cocultured with tumor cell lines, CAR19-CYNK showed increased secretion of the proinflammatory cytokines GM-CSF (p<0.05 for both Nalm-6 and Daudi), IFN-g (p<0.05 for Nalm-6), and TNF-a compared to that of NT NK cells at an E:T ratio of 1:1 for 24h. To evaluate the in vivo efficacy of CAR19-CYNK, a disseminated Daudi xenograft B-NDG-hIL15 model was used. CAR19-CYNK treated mice demonstrated a significant survival benefit with a median survival of 39 days versus a median survival of 28 days for the vehicle treated group (p<0.05). Conclusions: In summary, we have successfully established a process for generating CAR19-CYNK cells from human placental CD34 + cells. CAR19-CYNK demonstrated enhanced in vitro cytotoxicity against CD19 + B cell malignancies and in vivo survival benefit in a disseminated lymphoma xenograft B-NDG-hIL15 model. Further development of CAR19-CYNK for CD19 + B cell malignancies is warranted. Disclosures Gergues: Celularity Inc: Current Employment, Current equity holder in publicly-traded company. Raitman: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Gleason: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Rousseva: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. He: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Van Der Touw: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Ye: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Kang: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang: Sorrento Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Pai: Sorrento Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Guo: Sorrento Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Ji: Sorrento Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Hariri: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang: Celularity Inc.: Current equity holder in publicly-traded company, Ended employment in the past 24 months.

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Epratuzumab (Humanized Anti-CD22 MAb) Conjugated with SN-38, a New Antibody-Drug Conjugate (ADC) for the Treatment of Hematologic Tumors: Preclinical Studies Alone and In Combination with Veltuzumab, a Humanized Anti-CD20 MAb

Blood ◽

10.1182/blood.v116.21.3941.3941 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3941-3941

Author(s):

David M Goldenberg ◽

Serengulam Govindan ◽

Tom M Cardillo ◽

Robert M Sharkey

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

In Vivo Studies ◽

Anti Cd20 ◽

Anti Tumor Activity

Abstract Abstract 3941 Background: Monoclonal antibody (MAb) therapy has had a significant impact on the management of B-cell malignancies, but is most often used in combination with chemotherapy. We developed an ADC that combines SN-38, the active component of irinotecan, a topoisomerase I inhibitor, with the internalizing, humanized, anti-CD22 IgG, epratuzumab, and determined its activity alone and in combination with an anti-CD20 antibody therapy (veltuzumab). Methods: Epratuzumab was conjugated with SN-38 (E-SN-38) at a mole ratio of ∼6:1. The conjugate is designed specifically to be released slowly in the presence of serum (50% released over ∼1.5 days), allowing liberation of the drug when internalized, but also being released locally after being bound to the tumor. In vitro and in vivo studies were performed to assess the activity of the conjugate against several subcutaneously- or intravenously-inoculated B-cell lymphoma cell lines. In vivo studies also examined combination therapy using E-SN-38 and the veltuzumab (V). Results: In vitro studies in 4 B-cell lymphoma cells lines (Daudi, Raji, Ramos, WSU-FSCCL) and 4 acute lymphoblastic lymphoma cell lines (697, REH, MN-60, and RS4;11) expressing varying amounts of CD22 showed an IC50 for E-SN-38 in the nanomolar range, confirming potent activity. Nude mice bearing SC Ramos human lymphoma had significant selective anti-tumor activity compared to a control, non-targeting, IgG-SN-38 conjugate, at a dosing regimen of 75 to 250 μg of the conjugates given twice-weekly for 4 weeks. Significant anti-tumor activity was also found in several other cell lines. When combined with veltuzumab, significant improvement in therapeutic activity was observed. For example, median survival in a WSU-FSCCL human follicular B-cell lymphoma IV model with treatment initiated 5 days after implantation was 42 d (0/10 surviving at 160 d) and 91 d (2/10 surviving) for untreated and veltuzumab-treated animals, respectively; 63d (0/10 surviving after 160 d) and >160 d (with 6/10 surviving) for E-SN-38 and E-SN-38 + V, respectively; and 63 d (0/10) and 91 d (2/10) for non-targeting IgG-SN-38 conjugate alone and combined with V). The E-SN-38 conjugate combined with V was significantly better than all treatment or control groups (P ≤ 0.05). Conclusion: E-SN-38 ADC is a potent therapeutic, even at non-toxic dose levels, and shows significantly enhanced efficacy when combined with anti-CD20 immunotherapy, representing an important new ADC treatment regimen. Disclosures: Goldenberg: Immunomedics, Inc.: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Govindan:Immunomedics, Inc.: Employment. Cardillo:Immunomedics, Inc.: Employment.

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Evaluation of the Anti-Tumor Activity of MLN4924, A Novel NEDD8 Activating Enzyme Inhibitor, in Pre-Clinical Models of Rituximab Chemotherapy-Sensitive or -Resistant B-Cell Lymphoma.

Blood ◽

10.1182/blood.v120.21.2761.2761 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2761-2761

Author(s):

Natalie M Czuczman ◽

Matthew J. Barth ◽

Richa Dwar ◽

Cory Mavis ◽

Pavel Klener ◽

...

Keyword(s):

Cell Cycle ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Single Agent ◽

B Cell Lymphoma ◽

Lymphoma Cells ◽

Clinical Models ◽

Chemotherapy Agents ◽

Anti Tumor Activity

Abstract Abstract 2761 Clinical outcome of patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) remains poor with currently available therapies. Recently, the ubiquitin-proteasome system (UPS) appears to play an important role in the development of resistance in MCL and some subtypes of DLBCL. Targeting UPS represents a rational approach in an attempt to eradicate drug-resistant lymphoma clones. MLN4924 is a novel, potent and selective inhibitor of the NEDD8-activating enzyme (NAE) that is necessary for the modification of cullin-RING ubiquitin ligases. We evaluated the anti-tumor activity of MLN4924 against a panel of rituximab-sensitive (RSCL) or rituximab/chemotherapy–resistant (RCRCL) DLBCL and Burkitt lymphoma cell lines, cytarabine-sensitive or -resistant (AraCR) MCL cell lines, and primary tumor cells freshly isolated from lymphoma patients (n=13). Lymphoma cells were exposed to escalating doses of MLN4924 alone or in combination with selected chemotherapy agents for up to 72 hrs. Changes in the cell viability or ATP content were determined by alamar Blue reduction or CellTiterGlo assays, respectively. Induction of apoptosis and changes in the levels of NFkB and UPS regulatory proteins were analyzed by Western blotting. Cell cycle alterations were determined by propidium iodide staining and NFkB activity was quantified by flow cytometry using the Imagestream technology. MLN4924 demonstrated time- and dose-dependent anti-lymphoma activity in all cell lines tested. The IC50 in RSCLs were Raji=400nM, RL=1uM and U2932=>3uM. All RCRCLs were less responsive to MLN4924 as a single agent with IC50 concentrations 4–10× those of their respective sensitive parental cell lines. The MCL cell lines Mino, MinoAraCR, Z-138, HBL-2 and HBL-2AraCR were most sensitive to MLN4924 anti-tumor effects (IC50=250nM) with no significant difference between cytarabine-sensitive and -resistant cell lines; while the MCL cell lines Rec-1, Rec-1AraCR, Jeko-1 and Jeko-1AraCR were less sensitive (IC50=500–1000nM). A variable degree of anti-tumor activity was also observed in primary lymphoma cells. In addition to single-agent activity, MLN4924 plus selected anti-lymphoma chemotherapy agents (bortezomib, bendamustine and cytarabine) demonstrated synergy in cytarabine-sensitive and (to a lesser degree) cytarabine-resistant MCL cell lines. Combinations with additional chemotherapeutic agents (doxorubicin and vincristine) resulted in additive effects. Exposure of MCL cells to MLN4924 resulted in G1 cell cycle arrest. In vitro exposure of the more sensitive MCL cell lines Mino and MinoAraCR to MLN4924 resulted in an increase in p-IkBα and down-regulation of both total and nuclear NFkB. The less sensitive cell lines Rec-1 and Rec-1AraCR demonstrated little to no change in NFkB activation following exposure to MLN4924. Additional studies are ongoing to further define the molecular mechanisms of the anti-tumor activity observed following NAE inhibition by MLN4924 in these pre-clinical models and to further evaluate the activity of MLN4924 in in vivo SCID mouse models of B-cell lymphoma. Our data suggests that MLN4924, a novel NAE inhibitor, is active against B-cell lymphomas, particularly MCL, and is a promising agent warranting further investigation in relapsed/refractory aggressive B-cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

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Effects of Proteasome Inhibition in the Anti-Tumor Activity of Venetoclax in Mantle Cell Lymphoma Pre-Clinical Models

Blood ◽

10.1182/blood-2018-99-116510 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2942-2942

Author(s):

Shalin K. Kothari ◽

Cory Mavis ◽

Juan Gu ◽

Francisco J. Hernandez-Ilizaliturri

Keyword(s):

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Scid Mice ◽

Parp Cleavage ◽

Synergistic Activity ◽

Clinical Models ◽

Anti Tumor Activity

Abstract Background: At the molecular level, mantle cell lymphoma (MCL) is characterized by the deregulation of Bcl-2 family members (Mcl-1, BIM) and cell cycle (cyclin D1) regulatory proteins. Perhaps related to this, the clinical outcome of MCL continues to be poor specially for those patients with disease progression after high dose chemotherapy and autologous stem cell rescue and/or BTK inhibitors, stressing the need to develop novel therapeutic strategies or optimize current available options. Venetoclax (V), a highly selective Bcl-2 inhibitor, has shown modest activity against relapsed/refractory MCL. Over-expression of Mcl-1 has been postulated to be a mechanism of resistance to V limiting its anti-tumor activity in subtypes of lymphoma including MCL. The lethality by proteasome inhibitors (PIs) has been associated with changes in the Bcl-2 family members (Bax, Noxa, Mcl-1 and Bcl-XL) in lymphoma pre-clinical models, making them ideal agents to combine with V. To this end, we studied the anti-tumor activity of combining PIs with V in MCL pre-clinical models. Materials and Methods: A panel cytarabine sensitive (Rec-1, Jeko, Granta, HBL-2, Z-138 and Mino) and resistant (araC) cell lines (Jeko araC, HBL-2 araC, and Mino araC) were exposed to V, Bortezomib (BTZ), carfilzomib (CFZ), or ixazomib (IXZ) for 24, 48 and 72 hours. Cell viability was calculated measuring the ATP content. IC50 drug concentrations were calculated for each agent. Subsequently, MCL cell lines were exposed to escalating doses of V (0.001uM-5uM) and CFZ (1.5625nM-50nM), BTZ (3.125nM-100nM) or IXZ (3.125nM-100nM). In addition, primary tumor cells isolated from B-cell lymphoma patients (N=21) including MCL patients were exposed to V +/- BTZ or CFZ for 48 hrs. Cell viability was determined by Cell Titerglo. Coefficient of synergy were calculated using CalcuSyn software program. Induction of apoptosis was detected by Annexin V/Propidium iodine staining and PARP cleavage. Changes in Bcl-2 and cell cycle regulatory proteins were evaluated by Western blotting in HBL-2 cells. For in vivo experiments, 6-8 weeks old severe combined immunodeficiency (SCID) mice were inoculated with 10x106 HBL-2 cells via tail vein injection (IV). Subsequently, SCID mice were treated with V (100mg/kg/dose via gastric lavage on days 3-7, 10-14 and 17-21) or IXZ (6mg/kg/dose IV days 3, 6, 10, 13, 17 and 20) or combination of both agents. A group of untreated animals was used as a control. Differences in survival were evaluated between treatment groups. Results: In vitro exposure of MCL cell lines to either V, BTZ, CFZ, and IXZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining both V with CFZ or IXZ at known sub-therapeutic and therapeutic doses of individual agents measured by ATP content and apoptosis potential. Anti-tumor activity was observed in cytarabine sensitive and resistant cell lines. Similar findings were observed in primary tumor cells isolated from B-cell lymphoma patients. In vitro exposure of MCL cell lines with the lowest IC50 (HBL-2) to V and PIs (BTZ, CFZ, or IXZ) resulted in the upregulation of Noxa, BIM, Mcl-1 cleavage form (pro-apoptotic) and downregulation of Bcl-XL leading to PARP cleavage and apoptosis. In vivo treatment of MCL bearing SCID mice with V resulted in significant anti-tumor activity when compared to single agent IXZ treated or control animals. Of interest, MCL bearing SCID animals treated with V and IXZ exhibited a better disease control and the survival was longer than SCID animals treated with V or IXZ single agent (P<0.05). Conclusion: Our data suggests that V exhibits strong synergistic activity with PIs, especially with CFZ (in vitro) or IXZ (in vitro and in vivo). Together, our data supports the evaluation of V in combination with readily available novel PIs (IXZ or CFZ) in relapsed/refractory MCL. (Supported by LRF grant 555463, an NIH grant R01 CA136907-01A1 and a grant from The Roswell Park Cancer Institute Alliance Foundation) Disclosures No relevant conflicts of interest to declare.

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HMPL-523, a Novel SYK Inhibitor Showed Anti-Tumor Activities In Vitro and In Vivo

Blood ◽

10.1182/blood.v128.22.3970.3970 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3970-3970

Author(s):

Na Yang ◽

Wei Deng ◽

Qiaoling Sun ◽

Junqing Liang ◽

Linfang Wang ◽

...

Keyword(s):

Tyrosine Kinase ◽

Cell Viability ◽

B Cell ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Syk Inhibitor ◽

Anti Tumor Activity

Abstract Introduction: Spleen Tyrosine Kinase (SYK), a non-receptor type of tyrosine kinase, is a member of Syk/ZAP70 tyrosine kinase family. It plays a pivotal role in the regulation of B-cell receptor (BCR) signal pathway, which regulates proliferation, differentiation and survival of B lymphocytes. The abnormal activation of BCR singling is closely related to transformation and development of B cell lymphoma. Targeting BCR downstream molecules, such as Bruton' tyrosine kinase (BTK) and phosphoinositide-3-kinase δ (PI3Kδ) has emerged as new therapeutic approaches and inhibitors of BTK and PI3Kδ were approved recently by FDA for treatment of some subtypes of B-cell malignancies. Currently, a couple small molecular inhibitors against SYK, another BCR downstream molecule, are under the early clinical development and showed initial efficacy in B cell lymphomas. HMPL-523, discovered and currently being developed in Phase I clinical trial by Hutchison MediPharma, is a novel, highly potent and selective SYK inhibitor (IC50: 0.025 μM). The anti-tumor activity of HMPL-523 was evaluated in this study. Methods: Inhibitory effects of HMPL-523 on cell viability were investigated in a panel of B cell lymphoma cell lines with SYK/BCR dysregulation by CellTiter-Glo luminescent or CCK-8 assay. The effect of HMPL-523 on SYK signaling pathway was detected by western blot. Annexin-V- positive and PI-negative population was recognized as apoptotic cells by FACS. Nude mice bearing B cell lymphoma xenograft tumors with SYK/BCR dysregulation were used to determine anti-tumor activity of HMPL-523 in vivo. Result: HMPL-523 blocked phosphorylation of BLNK, downstream protein of Syk, in human mantle cell line REC-1 and human plasma cell line ARH-7777 with IC50 of 0.105 µM and 0.173 μM, respectively. HMPL-523 also inhibited cell viability of Ba/F3 Tel-Syk with IC50 of 0.033 μM. Furthermore, inhibitory effects of HMPL-523 on cell viability were evaluated in a panel of B -cell lymphoma cell lines with SYK/BCR deregulation. Results showed that HMPL- 523 potently inhibited cell survival with IC50s from 0.4 to 2 μM. Consistent with the effect on cell viability, HMPL-523 increased the apoptotic rate of REC-1 cells. Moreover, HMPL-523 showed the synergistic activities on killing human diffused large B cell lymphoma (DLBCL) in combination with other drugs such as BTK inhibitor, PI3Kδ inhibitors and Bcl2 family inhibitor. The detailed mechanisms underlying the synergism are still under investigation. Anti-tumor activity of HMPL-523 was determined in Syk dependent xenograft models. Daily oral administration of 100 mg/kg HMPL-523 showed potent anti-tumor activity in B cell lymphoma REC-1 (TGI: 59%). Conclusion:HMPL-523 is a highly potent SYK inhibitor with good activity against B-cell lymphoma in pre-clinical in vitro and in vivo models, supporting further clinical research for HMPL-523 as either single agent or combination drug with other agents to treat B-cell malignancies e.g. DLBCL Disclosures Yang: Hutchison MediPharma Ltd: Employment, Research Funding. Deng:Hutchison MediPharma Ltd: Employment, Research Funding. Sun:Hutchison MediPharma Ltd: Employment, Research Funding. Liang:Hutchison MediPharma Ltd: Employment, Research Funding. Wang:Hutchison MediPharma Ltd: Employment, Research Funding. Fan:Hutchison MediPharma Ltd: Employment, Research Funding. Tang:Hutchison MediPharma Ltd: Employment, Research Funding. Yu:Hutchison MediPharma Ltd: Employment, Research Funding. Sun:Hutchison MediPharma Ltd: Employment, Equity Ownership. Zhou:Hutchison MediPharma Ltd: Employment, Research Funding. Dai:Hutchison MediPharma Ltd: Employment, Research Funding. Qing:Hutchison MediPharma Ltd: Employment, Research Funding. Su:Hutchison MediPharma Ltd: Employment, Research Funding. Ren:Hutchison MediPharma Ltd: Employment, Research Funding.

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Antibody-Targeted Chemotherapy with Immunoconjugates of Calicheamicin: Differential Anti-Tumor Activity of Conjugated Calicheamicin Targeted to B-Cell Lymphoma Via B-Cell Lineage Specific Molecules CD19, CD20 and CD22.

Blood ◽

10.1182/blood.v104.11.2490.2490 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2490-2490 ◽

Cited By ~ 1

Author(s):

John F. DiJoseph ◽

Douglas C. Armellino ◽

Maureen M. Dougher ◽

Arthur Kunz ◽

Erwin R. Boghaert ◽

...

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Targeted Chemotherapy ◽

Phase I Clinical Trials ◽

Anti Cd20 ◽

Anti Tumor Activity ◽

Acid Labile

Abstract Antibody-targeted chemotherapy using tumor-targeted immunoconjugates of the cytotoxic agent, calicheamicin, is a clinically validated strategy for the treatment of acute myeloid leukemia. Calicheamicin is a potent cytotoxic natural product that binds DNA in the minor groove and causes double strand DNA breaks. B lymphoid lineage-specific antigens CD19, CD20, and CD22 have been studied extensively as potential targets for therapeutic applications of immunotoxins. In order to determine which one of these three antigens is most suitable for antibody-targeted calicheamicin therapy, we conjugated monoclonal antibodies, BU12 (murine anti-CD19 mAb), rituximab (chimeric anti-CD20 mAb), and m5/44 (murine anti-CD22 mAb) to a hindered disulfide derivative of N-acetyl gamma calicheamicin and evaluated the anti-tumor activity of these conjugates against three human B-cell lymphoma lines (BCL), Ramos, Raji and RL. Each of these three mAb bound to their respective antigens on the surface of BCL and was modulated, indicative of their potential internalization. Immunoconjugates of these mAbs, prepared by covalently linking calicheamicin via either acid-labile or acid-resistant linkers, caused a potent inhibition of BCL growth in vitro (IC50s ranged from 7 pM for the acid-labile linked m544 up to 6.8 nM for the acid-resistant linked anti-CD20 conjugates of calicheamicin). Immunoconjugates with acid-labile linkers were more potent than their counterparts with the acid-stable linker and conjugates targeted to either CD19 or CD22 were more potent than those targeted to CD20 in inhibiting BCL growth in vitro. In contrast, unconjugated mAb to CD19 or CD22 had no effect on BCL growth in vitro whereas anti-CD20 mAb, at concentrations >1 μg/ml, had an inhibitory effect of 30% on in vitro BCL growth. When examined for their effects on the growth of established subcutaneous BCL xenografts in nude mice, calicheamicin conjugated to anti-CD22 was by far the most efficacious conjugate against each of the three BCL xenografts studied. Calicheamicin conjugated to rituximab caused significant inhibition of BCL growth but was less effective than the conjugates of anti-CD22 or anti-CD19 mAb. Interestingly, anti-CD19 conjugates of calicheamicin, while effective in vitro against both Raji and Ramos BCL and effective against Raji BCL xenografts, had no effect on the growth of Ramos BCL xenografts in vivo. The reasons underlying the lack of anti-tumor activity of CD19-targeted calicheamicin conjugate against Ramos xenografts in vivo remain unknown. Based on a number of factors including the potent and consistent anti-tumor activity of the anti-CD22-conjugated calicheamicin, CD22 was selected as the molecular target for further development. A calicheamicin conjugate containing an acid-labile linker of humanized anti-CD22 mAb, CMC-544, is currently being evaluated in phase I clinical trials in non-Hodgkin’s B-cell lymphoma.

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