EL4 Lymphoma Cells Research Articles

P2Y12 receptor-mediated cyclooxygenase 2 (COX-2) expression enhances tumor cell progression in a mouse model of lymphoma.

The pro-inflammatory enzyme cyclooxygenase 2 (COX-2) has been known to impart metastatic property to cancer cells. However, blocking of COX-2 with nonsteroidal anti-inflammatory drugs or COX-2-specific inhibitors has failed in clinical trials due to adverse effects associated with their prolonged use. We have previously shown that extracellular ATP (eATP), a major component of the tumor microenvironment, enhances COX-2 expression several-fold, both in macrophages and in various cancer cells, by acting on purinergic (P2) receptors. In this study, we show that blocking of P2 receptors significantly reduced tumor growth in a mouse model of lymphoma. Tumors were induced in mice through subcutaneous injection of syngeneic EL4 lymphoma cells. Various P2 receptor antagonists were injected within the tumors after they were palpable. The broad-spectrum P2 receptor antagonist, suramin, P2X7 receptor-specific antagonist, oATP, P2Y6 receptor-specific antagonist, MRS 2578, and P2Y12 receptor-specific antagonist, AR-C 69931, all showed significant arrest in tumor growth. Both suramin and AR-C 69931-treated tumors showed strong reduction in COX-2 expression and modulation of various metastatic markers. Disaggregated cells from AR-C 69931-treated tumors, when injected intravenously in naïve mice, did not exhibit metastasis in various tissues which was observed in mice injected with cells from saline-treated tumors. Our results show that blocking of P2 receptors is a therapeutic alternative to inhibit COX-2 expression, and thereby, arrest tumor progression and metastasis.

Melatonin mitigates vincristine-induced peripheral neuropathy by inhibiting TNF-α/astrocytes/microglial cells activation in the spinal cord of rats, while preserving vincristine's chemotherapeutic efficacy in lymphoma cells

Vincristine (VCR), an anti-tubulin chemotherapy agent, is known to cause peripheral and central nerve damage, inducing severe chemotherapy-induced peripheral neuropathy (CIPN). Although melatonin has been recently recognized for its potential anti-neuropathic effects, its efficacy in countering VCR-induced neuropathy remains unclear. This study examines the neuroprotective potential of melatonin against VCR-induced neuropathy using a rat model. Neuropathic pain was induced through 10 VCR injections (0.1 mg/kg/day i.p.), administered in two five-day cycles with a two-day break. Melatonin treatment started two days before VCR administration and continued daily throughout the experiment. Rats were assigned to five groups: control, VCR, and three melatonin-treated groups receiving VCR with melatonin (5, 10, or 20 mg/kg/day i.p.). We assessed mechanical (von-Frey and Randall-Selitto tests) and thermal (hot-plate and tail-flick tests) hyperalgesia, motor coordination (rotarod test), and sciatic nerve conduction velocity (NCV). Changes in body weight, spinal cord histopathology (H&E), and proinflammatory markers (TNF-α, IL-1β, and IL-6), reactive astrocytes (GFAP) and microglial cells (IBA-1) were also assessed, as well as spinal cord degeneration (Nissl stain) and demyelination (LFB stain and MBP). Finally, the effect of melatonin on the cytotoxic activity of VCR against EL4 lymphoma cells was assessed using an MTT assay. Our results indicated that melatonin coadministration with VCR preserved spinal cord architecture, elevated nociceptive thresholds, improved motor coordination, enhanced NCV, and maintained normal body weight gain. Melatonin also reduced inflammation, decreased reactive astrocytes and microglia, and prevented neurodegeneration and demyelination in the spinal cord. Importantly, melatonin did not affect VCR's cytotoxic activity in cancer cells.

Valrubicin-loaded immunoliposomes for specific vesicle-mediated cell death in the treatment of hematological cancers

We created valrubicin-loaded immunoliposomes (Val-ILs) using the antitumor prodrug valrubicin, a hydrophobic analog of daunorubicin. Being lipophilic, valrubicin readily incorporated Val-lLs that were loaded with specific antibodies. Val-ILs injected intravenously rapidly reached the bone marrow and spleen, indicating their potential to effectively target cancer cells in these areas. Following the transplantation of human pediatric B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), or acute myeloid leukemia (AML) in immunodeficient NSG mice, we generated patient-derived xenograft (PDX) models, which were treated with Val-ILs loaded with antibodies to target CD19, CD7 or CD33. Only a small amount of valrubicin incorporated into Val-ILs was needed to induce leukemia cell death in vivo, suggesting that this approach could be used to efficiently treat acute leukemia cells. We also demonstrated that Val-ILs could reduce the risk of contamination of CD34+ hematopoietic stem cells by acute leukemia cells during autologous peripheral blood stem cell transplantation, which is a significant advantage for clinical applications. Using EL4 lymphoma cells on immunocompetent C57BL/6 mice, we also highlighted the potential of Val-ILs to target immunosuppressive cell populations in the spleen, which could be valuable in impairing cancer cell expansion, particularly in lymphoma cases. The most efficient Val-ILs were found to be those loaded with CD11b or CD223 antibodies, which, respectively, target the myeloid-derived suppressor cells (MDSC) or the lymphocyte-activation gene 3 (LAG-3 or CD223) on T4 lymphocytes. This study provides a promising preclinical demonstration of the effectiveness and ease of preparation of Val-ILs as a novel nanoparticle technology. In the context of hematological cancers, Val-ILs have the potential to be used as a precise and effective therapy based on targeted vesicle-mediated cell death.

Abstract 5416: Purinergic P2Y6 receptor antagonists as potential anti tumor agents

Abstract Introduction: Cancer places a tremendous strain on society in both developed and economically less developed nations. It remains the most recurring and deadliest disease despite rapid advances in treatment modalities. The major contributors are the prevalence of known risk factors, urbanization, population growth and ageing. Cancer resurgence depends on the organ in which the tumor first appears, immune response, patient’s characteristics, disease stage and tumor microenvironment (TME). Among the several inflammatory molecules found within the TMEs, the levels of adenosine triphosphate (ATP) has been found to be high. In healthy tissue, the concentration of ATP in the extracellular space (eATP) is negligible and does not accumulate significantly due to the activity of ectonucleotidases. However, tissue injury, cellular stress and chemotherapy mediated cell death can increase the levels of ATP in the TME. The eATP exerts its effect mainly through purinergic P2 receptors expressed by most malignant cells. We have previously shown that eATP promoted migration and invasion of cancer cells in vitro through increased expression of the enzyme cyclooxygenase 2 (COX-2) (Sharma et al., 2021). We showed that eATP mediated COX-2 expression is through P2 receptors (Akter et al., 2021). Accordingly, we hypothesized that blocking the P2 receptors would abolish the COX-2-mediated migration of tumor cells and tested the same in an animal model. Methodology: Tumors were generated in 8-15-week-old male C57BL/6 mice (20-25 g weight) through subcutaneous injection of syngeneic EL4 lymphoma cells in the left flank of the mice. When tumors were palpable around day 10, saline or P2 receptor, antagonists were directly injected into the center of the tumor and mice were divided into three groups: saline (vehicle control), suramin (broad-spectrum P2 receptor antagonist), and MRS 2578 (P2Y6 receptor-specific antagonist). Drugs were chosen based on prior in vitro research and a vernier caliper was used to assess the growth of the tumor every day. Behavioral tests were performed on day 8 (prior to P2 receptor injections) and days 11, 14 and 17 post-tumor injection. Mice were continuously monitored for their health and were sacrificed on day 18 with deep CO2 anesthesia and cervical dislocation. Results: We found that suramin-injected tumors had a substantial reduction in tumor volume when compared to saline-injected ones. Intra-tumoral injections of P2 receptor antagonists such as MRS-2578 also resulted in similar outcomes as suramin. In tumors treated with P2 receptor antagonists, the expression of COX-2, cell cycle proteins and epithelial-to-mesenchymal (EMT) markers, which aid in metastasis, were significantly downregulated, indicating a suppressive effect. Therefore, we propose that P2 receptor-dependent anti-inflammatory drugs (PBAIDs) could be considered a potential alternative therapeutic option for preventing cancer recurrence. Citation Format: Khagendra Ghimeray, Shilpa Sharma, Ravi Shankar Akundi. Purinergic P2Y6 receptor antagonists as potential anti tumor agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5416.

Immunocytokines with target cell-restricted IL-15 activity for treatment of B cell malignancies.

Despite the advances in cancer treatment achieved, for example, by the CD20 antibody rituximab, an urgent medical need remains to optimize the capacity of such antibodies to induce antibody-dependent cellular cytotoxicity (ADCC) that determines therapeutic efficacy. The cytokine IL-15 stimulates proliferation, activation, and cytolytic capacity of NK cells, but broad clinical use is prevented by short half-life, poor accumulation at the tumor site, and severe toxicity due to unspecific immune activation. We here report modified immunocytokines consisting of Fc-optimized CD19 and CD20 antibodies fused to an IL-15 moiety comprising an L45E-E46K double mutation (MIC+ format). The E46K mutation abrogated binding to IL-15Rα, thereby enabling substitution of physiological trans-presentation by target binding and thus conditional IL-15Rβγ stimulation, whereas the L45E mutation optimized IL-15Rβγ agonism and producibility. In vitro analysis of NK activation, anti-leukemia reactivity, and toxicity using autologous and allogeneic B cells confirmed target-dependent function of MIC+ constructs. Compared with Fc-optimized CD19 and CD20 antibodies, MIC+ constructs mediated superior target cell killing and NK cell proliferation. Mouse models using luciferase-expressing human NALM-6 lymphoma cells, patient acute lymphoblastic leukemia (ALL) cells, and murine EL-4 lymphoma cells transduced with human CD19/CD20 as targets and human and murine NK cells as effectors, respectively, confirmed superior and target-dependent anti-leukemic activity. In summary, MIC+ constructs combine the benefits of Fc-optimized antibodies and IL-15 cytokine activity and mediate superior NK cell immunity with potentially reduced side effects. They thus constitute a promising new immunotherapeutic approach shown here for B cell malignancies.

Anticancer effect of metformin alone and in combination with 2-deoxy-D-glucose on mouse T cell lymphoma EL4 cells

Metformin is a treatment used widely for non-insulin-dependent diabetes mellitus with few side effects and acts by inhibiting hepatic gluconeogenesis and glucose absorption from the gastrointestinal tract. Lymphoma is one of the most common hematological malignancies in dogs. Chemotherapy is used mainly on lymphoma, but further research on developing anticancer drugs for lymphoma is needed because of its severe side effects. This study examined the anticancer effects of metformin alone and in combination with 2-deoxy-D-glucose (2-DG), a glucose analog, on EL4 cells (mouse T-cell lymphoma). Metformin reduced the metabolic activity of EL4 cells and showed an additive effect when combined with 2-DG. In addition, cell death was confirmed using a trypan blue exclusion test, Hochest 33342/propidium iodide (PI) staining, and Annexin V/PI staining. An analysis of the cell cycle and mitochondria membrane potential (MMP) to investigate the mechanism of action showed that metformin stopped the G2/M phase of EL4 cells, and metformin + 2-DG decreased MMP. Metformin exhibited anticancer effects as a G2/M phase arrest mechanism in EL4 cells and showed additive effects when combined with 2-DG via MMP reduction. Unlike cytotoxic chemotherapeutic anticancer drugs, metformin and 2-DG are related to cellular glucose metabolism and have little toxicity. Therefore, metformin and 2-DG can be an alternative to reduce the toxicity caused by chemotherapeutic anticancer drugs. Nevertheless, research is needed to verify the in vivo efficacy of metformin and 2-DG before they can be used in lymphoma treatments.

Abstract LB324: BB3008, a potent and selective small molecular HPK1 inhibitor effective in multiple syngeneic tumor models

Abstract Introduction: Hematopoietic progenitor kinase 1 (HPK1) is a serine/threonine kinase in the MAP4K family predominantly expressed in immune cells, and has been identified as a negative regulator of T cell and B cell receptor signaling and dendritic cell function. Inhibition of the kinase activity of HPK1 results in the activation of exhausted T cell in tumor microenvironment and enhanced anti-tumor immune response. Observation of HPK1 overexpression in various tumor tissues further indicates the kinase as a novel target for immuno-oncology therapy. Here we report BB3008 as a small molecular HPK1 inhibitor. Methods: BB3008 was developed through structure-based drug design, and optimized by SAR analysis and medicinal chemistry iteration. Biochemical assays and cell-base functional assays were applied in compound evaluation. Pharmacokinetic (PK) and preliminary toxicity studies were performed with mice and dogs. Syngeneic tumor models in mice were conducted to demonstrate the in vivo efficacy of BB3008 as a single agent and in combination with mouse PD-1 antibody. Results: BB3008 shows sub-nanomolar HPK1 potency, with good selectivity against kinases in the MAP4K family and other TCR signaling-related kinases, such as FYN and ZAP70. Inhibition of HPK1 by BB3008 strongly suppresses the phosphorylation at Ser376 of the downstream biomarker protein SLP-76, with IC50 at 30 nM. With the stimulation of CD3/CD28, boost of IL-2 production was observed in Jurkat cells, purified human T cells, and human peripheral blood mononuclear cells upon treatment of BB3008 at sub-micromolar concentration. BB3008 also enhanced the killing activity of T cell against EL4 lymphoma cell in vitro in a concentration-dependent manner. PK profile of BB3008 showed good bioavailability (>80%) with a nearly linear dose-dependent exposure. The compound had no significant suppression on various normal cell lines proliferation, and had no hERG liability. In preliminary safety evaluation, no abnormal clinical symptoms and serum/blood tests were observed in mice and dogs dosed up to 1000mpk and 150mpk for 14 days, respectively. BB3008 showed significant tumor-growth inhibition rates in CT26, Hepa 1-6 and 4T1 mouse models as single agent, and in MC-38 mouse model in combination with mouse PD-1 antibody. Conclusion: As a potent, selective and oral available small molecule HPK1 inhibitor, BB3008 shows good safety tolerance in preclinical animals, and promising efficacy in multiple solid tumor models, both as single agent or in combination with immune checkpoint blockade. The IND-enabling study of BB3008 is in progress. Citation Format: Gongping Duan, Min Li, Xingmin Zhang. BB3008, a potent and selective small molecular HPK1 inhibitor effective in multiple syngeneic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB324.

Differential cytotoxic effects of fenbendazole on mouse lymphoma EL-4 cells and spleen cells

Fenbendazole (FBZ) is a benzimidazole anthelmintic widely used to treat parasitic infections. The anticancer effect of FBZ has been recently highlighted leading to its consideration as a potential anticancer agent. Although previous studies have demonstrated the effect of FBZ on cancer cells, there is a paucity of studies on the effect of FBZ on lymphoma cells and normal immune cells. Herein, we investigated the effects of FBZ on a mouse lymphoma cell line, EL-4 cells, and spleen cells, using vincristine as a positive control. The cellular metabolic activity of EL-4 cells was decreased by FBZ, but that of the spleen cells was not decreased. Moreover, FBZ reduced the mitochondrial membrane potential and induced reactive oxygen species production in EL-4 cells, but not in spleen cells. FBZ induced G2/M phase arrest and increased the sub G0/G1 phase ratio, indicating apoptosis. Furthermore, compared to the control cells, the reactivity of spleen cells pretreated with FBZ to lipopolysaccharide was maintained. In summary, FBZ is cytotoxic to EL-4 cells, but not to spleen cells. This study provides experimental evidence that FBZ exerts an anticancer effect, and less cytotoxic effects and functional damage to normal spleen cells.

Effect of pulsed magnetic field in murine T lymphoma EL4 cells

Maintenance of homoeostasis in human body is a very important indicator in all cell activities. When exposed to a disease, various immune cells are activated due to the inflammatory response, and particularly T cells play a role in inducing apoptosis of mutated cells such as tumor cells. When the activity of T cells is very low, infection by external invasion is easy, and on the contrary, excessive activation leads to chronic inflammation caused by autoimmune diseases. Many clinical studies related to pulsed magnetic field (PMF) demonstrated its efficacy in reducing pain, improving blood circulation, as well as blood’s acid-base balance. Therefore, our study has tried to investigate the influence of PMF on the regulation of acid-base homeostasis in EL4 T lymphoma cell. In addition, we have tried to explain the role of PMF on immune cell activity by measuring the level of pro-inflammatory cytokine, TNF-α in culture supernatants. EL4 cells were cultured in a DMEM medium supplemented with 10% FBS and 1% penicillin in an incubator at 37 °C and 5% CO2 condition. Our PMF stimulator has the maximum strength of 4700 G at a transition time of 222 μs with pulse intervals of 1 Hz. The homoeostasis in pH was improved as PMF strength increases. Cell viability decreased by 32% after PMF stimulation of 4700 G. It was observed that the concentration of TNF-α, a cytokine related to inflammation, also decreased as the strength of PMF increased. These results suggest that PMF stimulation improves the anti-inflammatory effect, therefore, it is thought to affect the immune system by balancing the activation and suppression of immune cells. For clinical use, our study might suggest non-invasive PMF can be developed as a medical devices modulating immune system, although it is necessary to optimize the PMF conditions such as pulse shape, duration, or repetition rate.

Impact of Epichaperome Inhibitor PU-H71 on Anti-Tumor T Cell Responses and Its Implications for Immune and Cellular Therapy

The epichaperome is a tightly integrated network of proteins supporting critical roles in the tumor-associated microenvironment and cancer cell survival. The epichaperome inhibitor PU-H71 is under development in clinical trials. A patient with an unclassified myeloproliferative neoplasm harboring a novel PML-SYK fusion, who progressed to acute myeloid leukemia despite allogeneic stem cell transplantation was successfully treated using PU-H71 (Sugita et al. 2021, NPJ Precis Oncol). We reported changes in donor immune cells after PU-H71 treatment, suggesting that epichaperome inhibition was modulating antitumor immune responses and improving the efficacy of cellular immunotherapy. To investigate this hypothesis, we analyzed the effects of PU-H71 on antitumor immune responses, GvHD and CAR-T cells. We tested the effects of PU-H71 on C57BL/6 mice immune responses to ovalbumine (OVA) immunization. OVA-specific T- and B-cell responses were evaluated by ELISPOT and ELISA, respectively. When administered at the time of priming, PU-H71 significantly increased the number of IFNγ-secreting T cells as compared to controls (median 126 vs 42 per million of cells, p=0.0002; n=8), without an impact on T cell phenotype or anti-OVA antibody levels. In contrast, PU-H71 had no effect when administered once the OVA-specific T cell response was ongoing. Then, we tested the effect of PU-H71 on antitumor immune responses in OVA-primed mice challenged with E.G7 (OVA+) or parental EL4 lymphoma cell lines (OVAneg). PU-H71 improved in vivo anti-tumor immune responses in OVA-primed mice challenged with E.G7 but not EL4 cells (Mean survival 17.5 vs 14 days, p=0.0003, n=8 per group). Since PU-H71 augmented early but not established T cell responses, we next evaluated whether it would have an effect on a GvHD murine model. Lethally-irradiated B6C3F1 mice received bone marrow and splenocytes from C57BL/6 mice and were treated with PU-H71 or vehicle at the first signs of GvHD (D11). GvHD clinical scores and histological analysis (colon, small intestine, liver and skin) were assessed. PU-H71 did not aggravate GvHD at both clinical and histological levels. Therefore, PU-H71 augments antigen-specific mouse T cell responses by acting during priming. To better understand its effect on human T cells and its potential adjuvant effect during cellular therapy, we next evaluated in vitro and in vivo CAR-T cells phenotype and function in the presence of PU-H71. T cells were purified from healthy donor PBMCs and were transduced with a CD19.4-1Bb.3z lentiviral vector to obtain CART19 cells. First, we found that epichaperome indeed forms in CART19 cells upon activation with anti-CD3/CD28 beads vs control. In vitro cytotoxicity of CAR-T cells toward NALM6 B-ALL cells was significantly improved (55.39 vs 74.66%, p=0.0356) by PU-H71. Finally, the therapeutic effect of the combination of PU-H71 and CART19 was evaluated in a B-ALL NSG mouse model using NALM6-BLIV cells, with bioluminescence imaging and flow cytometry (CART19 and leukemic cells) monitoring. Results suggest that PU-H71 administration increases CART19 numbers in the bone marrow and improves their in vivo antitumor efficacy against NALM6. Together, our results show that PU-H71 augments antigen-specific T cell responses during priming including CAR-T cell cytotoxicity, with little effect on ongoing immune responses including GvHD. Our data suggest that PU-H71 improves the in vivo efficiency of CAR-T cells in a B-ALL mouse model. In addition to its previously demonstrated direct anti-tumor effect, PU-H71 also had an adjuvant effect on anti-tumor T cell responses while not aggravating GvHD, which supports its clinical evaluation in the post-transplant setting or in combination with immune therapies like CAR-T cells.

Fc gamma receptor is not required for in vivo processing of radio- and drug-conjugates of the dead tumor cell-targeting monoclonal antibody, APOMAB®

The Fc region of a monoclonal antibody (mAb) can play a crucial role in its biodistribution and therapeutic activity. The chimeric mAb, chDAB4 (APOMAB®), which binds to dead tumor cells after DNA-damaging anticancer treatment, has been studied pre-clinically in both diagnostic and therapeutic applications in cancer. Given that macrophages contribute to the tumor accumulation of chDAB4 and its potency as an antibody drug conjugate in vivo, we next wanted to determine whether the Fc region of the chDAB4 mAb also contributed. We found that, regardless of prior labeling with chDAB4, dead EL4 lymphoma or Lewis Lung (LL2) tumor cells were phagocytosed equally by wild-type or Fcγ knock-down macrophage cell lines. A similar result was seen with bone marrow-derived macrophages from wild-type, Fcγ knock-out (KO) and NOTAM mice that express Fcγ but lack immunoreceptor tyrosine-based activation motif (ITAM) signaling. Among EL4 tumor-bearing wild-type, Fcγ KO or NOTAM mice, no differences were observed in post-chemotherapy uptake of 89Zr-labeled chDAB4. Similarly, no differences were observed between LL2 tumor-bearing wild-type and Fcγ KO mice in post-chemotherapy uptake of 89Zr-chDAB4. Also, the post-chemotherapy activity of a chDAB4-antibody drug conjugate (ADC) directed against LL2 tumors did not differ among tumor-bearing wild-type, Fcγ KO and NOTAM mice, nor did the proportions and characteristics of the LL2 tumor immune cell infiltrates differ significantly among these mice. In conclusion, Fc-FcγR interactions are not essential for the diagnostic or therapeutic applications of chDAB4 conjugates because the tumor-associated macrophages, which engulf the chDAB4-labelled dead cells, respond to endogenous ‘eat me’ signals rather than depend on functional FcγR expression for phagocytosis.

Targeting the Cytosolic Branched Chain Aminotransferase (BCATc) for T Cell Driven Anti‐Lymphoma Immunotherapy

The lymphoma microenvironment is a dynamic network between lymphoma cells and non‐malignant components that may promote tumor growth and consequently drug resistance. Progress in T cell metabolism has demonstrated that T cells experience metabolic disadvantage in the tumor microenvironment, which commonly manifests in T cell exhaustion and jeopardizes their potential to destroy cancer cells. We identified the cytosolic branched chain aminotransferase (BCATc) as a novel metabolic checkpoint of T cell activation. BCATc catalyzes the first step in the degradation of the essential amino acid leucine, which is a well‐known activator of complex 1 of the mammalian target of rapamycin (mTORC1). In T cells, BCATc is induced upon T cell activation and is a part of a negative feedback loop regulation of mTORC1. Based on extensive earlier research, we proposed that BCATc exerts immunosuppressive properties in the tumor microenvironment and we hypothesized that deleting BCATc from T cells renders them effective in eradicating lymphoma tumors.By using our newly designed mouse model with BCATc deleted from CD4+ and CD8+ T cells (T‐BCATcKO mice), we performed tumor studies where male and female mice were subcutaneously injected with 2.5x105 murine EL‐4 lymphoma cells [n=8] or phosphate‐buffered saline [n=4‐6]. As controls, mice expressing the floxed BCATc gene (T‐BCATcfl/flmice, n=9) were used. Tumor growth, body weight, and food intake were monitored for up to 15 days followed by collection of tumor tissues and organs. In addition, CD4+ T cells were isolated from spleens of T‐BCATcfl/fl and T‐BCATcKO mice, activated with a plate‐bound anti‐CD3 and soluble anti‐CD28 and left untreated or treated with 5 mM gabapentin (a competitive inhibitor of BCATc) or 10 mM N‐acetyl‐leucine amide (NALA, a structural analog of leucine) for 24‐72 hours to measure changes in the secretion of interferon gamma (IFNγ) from T helper 1 (Th1) cells or the expression of Foxp3, a master activator of regulatory T cells (CD4+CD25+ T reg cells).Deletion of BCATc from T cells or inhibition of the BCATc activity by gabapentin caused a significant increase in the secretion of IFNγ from Th1 cells after 24‐72 hours of activation, while CD4+CD25+ T reg cells expressed reduced levels of Foxp3 in response to gabapentin. In contrast, NALA blocked the IFNγ secretion and caused an increase in the expression of Foxp3. Following tumor inoculation, the T‐BCATcKO mice demonstrated a significant delay in tumor appearance and growth (~50 % reduction in tumor volumes) and the final tumor masses were reduced by 25% compared to control mice. None of the animals differed in body, organ weights or food intake and deletion of BCATc from T cells did not lead to systemic overactivation of the immune system. This study revealed that deleting BCATc from T cells may offer an advantage to better fight lymphoma and identified BCATc as a new therapeutic target to improve the efficiency of checkpoint inhibitors and help increase the low rates of remission seen in lymphoma patients.

HIF activation enhances Fc\u03b3RIIb expression on mononuclear phagocytes impeding tumor targeting antibody immunotherapy

BackgroundHypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (FcγR) and impaired by the single inhibitory FcγR, FcγRIIb.MethodsWe applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription.ResultsWe report that TAMs are FcγRIIbbright relative to healthy tissue counterparts and under hypoxic conditions, mononuclear phagocytes markedly upregulate FcγRIIb. This enhanced FcγRIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human FcγRIIb+/+ transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of FcγRIIb can partially restore phagocytic function in human monocytes.ConclusionOur findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of FcγRIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies.

Synergistic anticancer activity of disulfiram/copper against mouse lymphoma cells

Disulfiram (DSF) is a marketed drug to treat patients with alcohol dependence by inhibiting aldehyde dehydrogenase. Over the last few decades, DSF has been shown to have anticancer effects through different mechanisms. Moreover, this effect can be elevated when used with copper (Cu). Subsequent studies have been conducted on various cancers, but few on lymphoma. This study investigated the anticancer effects of DSF on lymphoma and how this effect changed when treated with Cu. DSF synergistically decreased the metabolic activity of EL4 lymphoma cells when combined with Cu. At 1 µM of DSF alone, the metabolic activity of EL4 cells decreased by 49% compared to the control, whereas it decreased by 87% with a DSF + CuCl2 treatment. Rhodamine 123 and 2',7'–dichlorofluorescein diacetate staining showed that DSF induced the reduction of the mitochondrial membrane potential and promoted the production of reactive oxygen species. In particular, the combined treatment of DSF + Cu induced cell death based on multiple assays, including annexin V-fluorescein isothiocyanate/propidium iodide staining. Overall, DSF has anticancer effects on lymphoma cells and exhibits synergistic effects when combined with Cu. This study provides some valuable information to broaden the use of DSF in clinics and basic research.

TSLP up-regulates inflammatory responses through induction of autophagy in T cells.

Thymic stromal lymphopoietin (TSLP), a type I cytokine belonging to the IL-2 cytokine family, promotes Th2-mediated inflammatory responses. The aim of this study is to investigate whether TSLP increases inflammatory responses via induction of autophagy using a murine T cell lymphoma cell line, EL4 cells, and lipopolysaccharide (LPS)-injected mice. TSLP increased expression levels of autophagy-related factors, such as Beclin-1, LC3-II, p62, Atg5, and lysosome associated membrane protein 1/2, whereas these factors increased by TSLP disappeared by neutralization of TSLP in EL4 cells. TSLP activated JAK1/JAK2/STAT5/JNK/PI3K, while the blockade of JAK1/JAK2/STAT5/JNK/PI3K signaling pathways reduced the expression levels of Beclin-1, LC3-II, and p62 in TSLP-stimulated EL4 cells. In addition, TSLP simultaneously increased levels of inflammatory cytokines via induction of autophagy by activation of JAK1/JAK2/STAT5/JNK/PI3K signaling pathways. In an LPS-induced acute liver injury (ALI) mouse model, exogenous TSLP increased expression levels of Beclin-1 and LC3-II, whereas functional deficiency of TSLP by TSLP siRNA resulted in lower expression of Beclin-1, LC3-II, and inflammatory cytokines, impairing their ability to form autophagosomes in ALI mice. Thus, our findings show a new role of TSLP between autophagy and inflammatory responses. In conclusion, regulating TSLP-induced autophagy may be a potential therapeutic strategy for inflammatory responses.

Small Molecule Inhibitors Targeting Nuclear Factor κB Activation Markedly Reduce Expression of Interleukin-2, but Not Interferon-γ, Induced by Phorbol Esters and Calcium Ionophores.

The T-box transcription factor Eomesodermin (Eomes) promotes the expression of interferon-γ (IFN-γ). We recently reported that the small molecule inhibitors, TPCA-1 and IKK-16, which target nuclear factor κB (NF-κB) activation, moderately reduced Eomes-dependent IFN-γ expression in mouse lymphoma BW5147 cells stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). In the present study, we investigated the direct effects of NF-κB on IFN-γ expression in mouse lymphoma EL4 cells and primary effector T cells. Eomes strongly promoted IFN-γ expression and the binding of RelA and NFATc2 to the IFN-γ promoter when EL4 cells were stimulated with PMA and IM. Neither TPCA-1 nor IKK-16 reduced IFN-γ expression; however, they markedly decreased interleukin (IL)-2 expression in Eomes-transfected EL4 cells. Moreover, TPCA-1 markedly inhibited the binding of RelA, but not that of Eomes or NFATc2 to the IFN-γ promoter. In effector CD4+ and CD8+ T cells activated with anti-CD3 and anti-CD28 antibodies, IFN-γ expression induced by PMA and A23187 was not markedly decreased by TPCA-1 or IKK-16 under conditions where IL-2 expression was markedly reduced. Therefore, the present results revealed that NF-κB is dispensable for IFN-γ expression induced by PMA and calcium ionophores in EL4 cells expressing Eomes and primary effector T cells.

Doxorubicin Paradoxically Ameliorates Tumor-Induced Inflammation in Young Mice.

Doxorubicin (DOX) is one of the most widely used chemo-therapeutic agents in pediatric oncology. DOX elicits an inflammatory response in multiple organs, which contributes to DOX-induced adverse effects. Cancer itself causes inflammation leading to multiple pathologic conditions. The current study investigated the inflammatory response to DOX and tumors using an EL4-lymphoma, immunocompetent, juvenile mouse model. Four-week old male C57BL/6N mice were injected subcutaneously with EL4 lymphoma cells (5 × 104 cells/mouse) in the flank region, while tumor-free mice were injected with vehicle. Three days following tumor implantation, both tumor-free and tumor-bearing mice were injected intraperitoneally with either DOX (4 mg/kg/week) or saline for 3 weeks. One week after the last DOX injection, the mice were euthanized and the hearts, livers, kidneys, and serum were harvested. Gene expression and serum concentration of inflammatory markers were quantified using real-time PCR and ELISA, respectively. DOX treatment significantly suppressed tumor growth in tumor-bearing mice and caused significant cardiac atrophy in tumor-free and tumor-bearing mice. EL4 tumors elicited a strong inflammatory response in the heart, liver, and kidney. Strikingly, DOX treatment ameliorated tumor-induced inflammation paradoxical to the effect of DOX in tumor-free mice, demonstrating a widely divergent effect of DOX treatment in tumor-free versus tumor-bearing mice.

Abstract 2377: microRNA expression profile in urinary exosomes is dependent on non-invasive lymphoma induction in mice

Abstract Lymphoma accounts for approximately 4% of cancers in the United States, with an estimated 20,910 number of deaths per year. The standard method of diagnosis and staging of lymphoma involves surgical biopsy of the tumor - a procedure that has many negative associated risks. Exosomes are extracellular vesicles secreted in biological fluids that can serve as “liquid biomarkers”. Identification of unique cancer-related biomarkers in urinary exosomes may provide a novel non-invasive and cost-effective tool for lymphoma diagnosis. Analyses of biomarkers obtained from urinary exosomes have been used to evaluate urological cancers, however, these methods have not yet been translated to non-urological pathologies, such as lymphomas. The objective for this study was to determine the profile of microRNAs (miRNAs) expressed in urinary exosomes of mice challenged with lymphoma and compare it to miRNAs identified in urinary exosomes of control mice. Male and female C57BL/6 mice, (tumor (+), n=12), were injected with either 2.5x105 mouse EL-4 lymphoma cells or phosphate-buffered saline (tumor (-), n=12). Tumor growth was monitored for 20 days. Urine was collected for 48 hours starting on day 17 and serum, tumor tissues, and organs were collected on day 20. Extraction of urinary exosomes was followed by total RNA isolation and RT-qPCR for which a set of PCR arrays consisting of 709 mouse-specific miRNA primers was used. Fold changes in miRNA expression were quantified using the ΔΔCt method. Mice developed tumors by day 13 with initial tumor appearance around day seven. There were no statistically significant differences between final tumor mass, body weight, or food and water intake in tumor (+) versus tumor (-) mice. RT-qPCR arrays of miRNAs extracted from urinary exosomes revealed 464 miRNAs that were differentially expressed between tumor (+) and tumor (-). 215 miRNAs were up-regulated, while 249 miRNAs were down-regulated in tumor (+) mice. These results will be compared to miRNAs from serum and tumor tissues to identify tumor-specific miRNAs that can be used for potential application in the clinical setting. Citation Format: Brittany Wilson, Rebekah Betar, Alexander Martin, Zackaria Niazi, Michael Boyer, Lori Winter, Victor Babich, Francesca Di Sole, Elitsa Ananieva. microRNA expression profile in urinary exosomes is dependent on non-invasive lymphoma induction in mice [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 2377.

Analysis of microRNAs in Urinary Exosomes of Tumor‐bearing Mice as a Non‐invasive Tool for Lymphoma Diagnosis

Lymphoma consists of a subgroup of immunological cancers, which arise from malignant B and T lymphocytes. Comprising approximately 4% of cancers in the United States, lymphoma accounts for up to 19,940 deaths per year. Surgical biopsy is the primary method for lymphoma staging and diagnosis. Unfortunately, there are many risks associated with this procedure, including cosmetic disfigurement, seeding of malignant cells into previously unaffected tissue, and expense incurred by patients. Identification of unique cancer-related biomarkers in urinary exosomes may provide a novel non-invasive and cost-effective tool for lymphoma diagnosis. Although analyses of biomarkers obtained from urinary exosomes have been used to evaluate urological cancers, these methods have not yet been translated to non-urological pathologies, such as lymphomas. The objective for this study was to analyze the profile of microRNAs (miRNAs) obtained from urinary exosomes of mice bearing lymphoma tumors and compare it to miRNAs identified in urinary exosomes of tumor free (-) control mice. Male and female C57BL/6 mice were injected with either 2.5x105 mouse EL-4 lymphoma cells [tumor (+) mice, n=6] or phosphate-buffered saline [tumor (-) mice, n=6]. Tumor growth was monitored for up to 20 days with collection of serum, tumor tissues, and organs at the end of this period. Urine was collected for 48 hours beginning on day 17. Extraction of urinary exosomes was followed by total RNA isolation and RT-qPCR using a set of PCR arrays consisting of 709 mouse-specific miRNA primers. Fold changes in miRNA expression were quantified using the ∆∆Ct method. Mice developed tumors by day 13 with initial tumor appearance around day seven. There were no statistically significant differences between final tumor mass, body weights, or food and water intake in tumor (+) versus tumor (-) mice. RT-qPCR arrays of miRNAs extracted from urinary exosomes revealed 464 differentially expressed miRNAs between tumor (+) and tumor (-) mice. Specifically, the expression of two miRNAs, significantly increased in urinary exosomes of tumor (+) mice compared to tumor (-) mice, was also increased in the lymphoma tissue of origin. One miRNA with a significantly decreased expression in the urinary exosomes of tumor (+) mice was found to have a decreased expression in the lymphoma tissue of origin. Expression of miRNAs in mouse tissue was analyzed by RT-qPCR. These results revealed that mice challenged with lymphoma tumors are releasing tumor-specific miRNAs in their urine. These findings are in support of future clinical studies on the use of miRNA analysis in urinary exosomes for non-invasive diagnostics of lymphoma patients.

PD-1 antibody and ruxolitinib enhances graft-versus-lymphoma effect without increasing acute graft-versus-host disease in mice.

Boosting T cell immune response posttransplant with checkpoint inhibitors increases graft-versus-lymphoma (GVL) effects at the cost of increasing acute graft-versus-host disease (aGVHD). A combined targeted therapy is needed to decrease checkpoint inhibitors-induced aGVHD without impairing GVL. We studied whether this competition could be avoided by giving concurrent anti-PD-1 antibody and ruxolitinib in allotransplant mouse models in which recipients were challenged with A20 or EL4 lymphoma cells. Given alone the PD-1 antibody increased GVL but did not improve survival of recipients challenged with A20 cells because of increased deaths from aGVHD. Adding ruxolitinib decreased levels of effector T cells and related cytokines. Tbx21- T cells had higher PD-1 levels compared with Tbx21+ T cells. Ruxolitinib increased PD-1 levels on donor T cells by suppressing Tbx21 expression. Ruxolitinib increased apoptosis of T cells which was reversed by the PD-1 antibody. PD-1 antibody preserved expression of granzyme B and cytotoxicity of T cells which were decreased by ruxolitinib. The net result of combined therapy was increased GVL, no increase in aGVHD and increased survival. The combined therapy improved survival of recipients challenged by A20 cells which expressed high level of PD-L1, but not EL4 cells which do not express PD-L1.