Cell Death In Jurkat Research Articles

Synthesis of Optically Pure Cyclometalated Iridium(III) Complex‐Peptide Hybrids and Their Anticancer Activity.

We report on the synthesis of optically pure cyclometalated iridium(III) complex‐peptide hybrids as amphiphilic peptide conjugates (IPH‐ACs) from Δ‐ and Λ‐Ir(tpyCO2H)3 (tpy: 2‐(4’‐tolyl)pyridine) by optical resolution via diastereomeric intermediates conjugated with the chiral alcohol, (1R,2R)‐2‐aminocyclohexanol ((R,R)‐11). Spectroscopic spectra of the optically pure IPH‐ACs that were prepared from Δ‐ and Λ‐Ir(tpyCO2H)3 are reported. It was found that both optically pure IPH‐ACs induce paraptotic cell death in Jurkat cells and the EC50 values were evaluated by MTT assays. We also performed TEM analyses of Jurkat cells treated with Λ‐13 to observe morphological changes that occurred, and cytoplasmic vacuolization, a characteristic feature of paraptosis, was observed. The intracellular uptake of IPH‐ACs by the cells measured by ICP‐MS (inductively coupled plasma‐mass spectrometry) was higher for the Λ‐forms and the EC50 values of the Λ‐forms were slightly lower than those of the Δ‐IPH‐ACs. The intrinsic cytotoxicity of the IPH‐ACs, taking into account the fact that the intracellular uptake and the intrinsic cytotoxicity is almost equal for the Δ‐ and Λ‐forms, suggests that the difference in cytotoxicity against Jurkat cells is due to the selectivity in the intracellular uptake of each stereoisomer.

Abstract 4710: The polymeric AMD3100 based drug PAMD-Ch17 induces its anti-leukemic effects in a CXCR4 independent mechanism

Abstract Background: Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are devastating blood cancers associated with 5-year survival rates of ~50% each in adults. We recently developed a new class of polymeric drugs called PAMDs based on the CXCR4 inhibitor AMD3100 (plerixafor). PAMDs have the potential to be more effective and less toxic therapeutics than the standard of care high-dose chemotherapy. We tested one variant (PAMD-Ch17), and found that unlike AMD3100, PAMD-Ch17 has several novel anti-leukemic effects. To understand the potential mechanism(s) for these new activities, we performed the following study. Methods: To test the role of CXCR4 in PAMD-Ch17’s activities, we generated CXCR4 knockout cells via Crispr/Cas9. We used PrestoBlue, 7-AAD, and Annexin V staining to assay viability and apoptosis. To identify pathways deregulated by PAMD-Ch17, we performed whole transcriptomic sequencing (RNA-Seq). To investigate if PAMD-Ch17 mediates its activities by inducing oxidative stress, we performed SeaHorse assays, and dihydroethidium (DHE), Mitosox, and Bodipy C11 staining. To investigate whether PAMD induces programmed cell death pathways, we used ferrostatin and deferoxamine to inhibit ferroptosis, necrostatin-1 against necroptosis, and Z-VAD-FMK against apoptosis. Results: We found that PAMD-Ch17 induces cell death in a dose dependent manner in human AML and ALL cell lines and, mouse primary leukemia cells, but not healthy mouse bone marrow cells. Surprisingly, PAMD-Ch17 induces equivalent levels of cell death in both wild type and CXCR4 knockout Jurkat cells, indicating that the only known target of the polymer is not required for its anti-leukemic effects. By RNA-Seq, we found altered expression of genes related to mitochondrial function, suggesting that PAMD-Ch17 could be targeting the mitochondria. To test this, we performed SeaHorse assays and found that PAMD-Ch17 induces a significant decrease in mitochondrial respiration. Consistent with this, we found that PAMD-Ch17 induces a significant increase in superoxide production, as well as lipid peroxidation. Using inhibitors of programmed cell death pathways, we found that preventing necroptosis, apoptosis, or ferroptosis caused only subtle effects on PAMD-Ch17 induced cell death, implying that the polymer’s anti-leukemic activity is likely not entirely dependent on these pathways. Conclusions: These results indicate that PAMD-Ch17 induces cell death in leukemia cells but not healthy bone marrow via a CXCR4 independent mechanism. We also found that PAMD-Ch17 induces reactive oxygen species, potentially explaining its selectivity for leukemia cells compared to healthy blood cells. Continued investigation of PAMD-Ch17 will not only further development of this new class of drugs, but also provide much needed insight into leukemia biology. Citation Format: Calvin Lam, Jogdeo Chinmay, Ekta Kapoor, Siyuan Tang, Svetlana Romanova, David Oupicky, Katherine Hyde. The polymeric AMD3100 based drug PAMD-Ch17 induces its anti-leukemic effects in a CXCR4 independent mechanism [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 4710.

Phorbol-12-Myristate-13-Acetate (PMA) Reactivates Replication from HIV-1 Latency and Induces Jurkat Cell Death

HIV-1 has the capability to establish latency during early infection in CD4+ cells, posing a significant challenge to the efforts aimed at curing HIV-1/AIDS. One extensively explored strategy to address this viral latency is the "shock-and-kill" approach. This involves reactivating viral replication using latency reversal agents (LRAs) to induce the death of infected cells. Regrettably, no LRAs with proven effectiveness have been identified thus far. In this study, we investigated the impact of Azidothymidine (AZT) treatment interruption and the administration of Phorbol-12-Myristate-13-Acetate (PMA), a PKC activator, as shock-and-kill approaches in vitro. We employed the susceptible Jurkat cell line and utilized a sensitive real-time PCR assay along with Western blotting analysis. Our findings revealed that AZT inhibited HIV-1 replication, and its treatment interruption led to the reactivation of viral replication. This reactivation occurred through the recruitment of host transcription factors, including NFAT, NF-κBp65, Ap-1, and Sp-1. These factors facilitated HIV production via TCR-related pathways, activation of p-TEFb pathways for transcription elongation, and upregulation of Jak/Stat pathways for viral enhancement. Furthermore, we demonstrated that PMA treatment increased the levels of these transcription factors through the activation of TCR-related signaling pathways in HIV-1 infected Jurkat cells, irrespective of the AZT treatment status. PMA also induced cell death through both extrinsic and intrinsic apoptotic signaling pathways, as well as autophagy. These results suggest that PMA effectively employs the shock-and-kill approach in HIV-1 infected Jurkat cells and highlight the potential of PKC pathway activators as promising LRAs.

Quaternary Benzophenanthridine Alkaloids Act as Smac Mimetics and Overcome Resistance to Apoptosis.

Defects in cell death signaling pathways are one of the hallmarks of cancer and can lead to resistance to conventional therapy. Natural products are promising compounds that can overcome this resistance. In the present study we studied the effect of six quaternary benzophenanthridine alkaloids (QBAs), sanguinarine, chelerythrine, sanguirubine, chelirubine, sanguilutine, and chelilutine, on Jurkat leukemia cells, WT, and cell death deficient lines derived from them, CASP3/7/6-/- and FADD-/-, and on solid tumor, human malignant melanoma, A375 cells. We demonstrated the ability of QBAs to overcome the resistance of these deficient cells and identified a novel mechanism for their action. Sanguinarine and sanguirubine completely and chelerythrine, sanguilutine, and chelilutine partially overcame the resistance of CASP3/7/6-/- and FADD-/- cells. By detection of cPARP, a marker of apoptosis, and pMLKL, a marker of necroptosis, we proved the ability of QBAs to induce both these cell deaths (bimodal cell death) with apoptosis preceding necroptosis. We identified the new mechanism of the cell death induction by QBAs, the downregulation of the apoptosis inhibitors cIAP1 and cIAP2, i.e., an effect similar to that of Smac mimetics.

Eltrombopag directly activates BAK and induces apoptosis

Small molecule direct BAK activators can potentially be used for the development of anti-cancer drugs or as tools to study BAK activation. The thrombopoietin receptor agonist eltrombopag (Eltro) inhibits BAX activation and BAX-mediated apoptosis. Here we report that, in contrast to its function as a BAX inhibitor, Eltro directly binds BAK but induces its activation in vitro. Moreover, Eltro induces or sensitizes BAK-dependent cell death in mouse embryonic fibroblasts (MEFs) and Jurkat cells. Chemical shift perturbation analysis by NMR indicates that Eltro binds to the BAK α4/α6/α7 groove to initiate BAK activation. Further molecular docking by HADDOCK suggests that several BAK residues, including R156, F157, and H164, play an important role in the interaction with Eltro. The introduction of an R156E mutation in the BAK α4/α6/α7 groove not only decreases Eltro binding and Eltro-induced BAK activation in vitro but also diminishes Eltro-induced apoptosis. Thus, our data suggest that Eltro directly induces BAK activation and BAK-dependent apoptosis, providing a starting point for the future development of more potent and selective direct BAK activators.

Caracasine, An ent-kaurane Diterpene with Proapoptotic and Pro-differentiator Activity in Human Leukaemia Cell Lines.

Kaurane-type diterpenoids, obtained from various natural sources, have shown many biological activities, including anti-inflammatory and antitumor effects. Caracasine, an ent-kaurane diterpenoid isolated from the flowers of Croton micans, was shown to induce apoptosis in leukaemia cell lines. The present study aimed to ascertain the compound's mechanism of cell death induction using two leukaemia cell lines, Jurkat E6.1 (T cell) and HL-60 (promyeloblast cells). Cell death in Jurkat and HL60 cells were evaluated by flow cytometry for apoptosis with annexin-V/PI, mitochondrial membrane potential disturbance, changes in cell cycle, CD95 expression, caspase activation, Nuclear Factor kappa B inhibition, and differentiation into a neutrophil-like cell (dHL60). Caracasine (10 μM) increased the G0/G1 phase in Jurkat and arrested the cell cycle in the S phase in HL60. Caracasine increased CD95 expression (p<0.01 in Jurkat and p<0.05 in HL60) and caspase-8 activation (p<0.001 in Jurkat and p<0.05 in HL60). Caspase-9 was activated in both cell lines (p<0.001) along with the decline in mitochondrial Δψm (p<0.05 in Jurkat and p<0.001 in HL60). In HL60 cells, the kaurane induced neutrophil differentiation was assessed by CD40 expression and reactive oxygen species production. In Jurkat cells, caracasine inhibited the NF-κB pathway in cells pretreated with PHA to activate the NF-κB pathway, suggesting a possible role in inflammatory diseases. Caracasine induced apoptosis through the intrinsic and extrinsic pathways in both cell lines were evaluated which could be the leading structure for new anti-leukemic and anti-inflammatory drugs.

Discovery of highly potent HDAC8 PROTACs with anti-tumor activity

Various diseases are deeply associated with aberrations in HDAC8 functions. These aberrations can be assigned to either structural functions or catalytic functions of HDAC8. Therefore, development of HDAC8 degradation inducers might be more promising than HDAC8 inhibitors. We employed the proteolysis targeting chimera (PROTAC) strategy to develop a selective and potent HDAC8 degradation inducer CT-4 with single-digit nanomolar DC50 values and over 95% Dmax in both triple-negative breast cancer MDA-MB-231 cells and T-cell leukemia cells. Notably, CT-4 demonstrated potent anti-migration activity and limited anti-proliferative activity in MDA-MB-231 cells. In contrast, CT-4 effectively induced apototic cell death in Jurkat cells, as assessed by a caspase 3/7 activity assay and flow cytometry. Our findings suggest that the development of HDAC8 degradation inducers holds great potential for the treatment of HDAC8-related diseases.

A new inhibitor of human peptide deformylase suppresses cell proliferation and induces cell apoptosis and autophagy in cancers.

Human peptide deformylase (hsPDF) has been found overexpressed in many cancer cells and its inhibitors exhibit antitumor activity. Studies were performed to validate that hsPDF is a good antitumor target. The inhibitory effect of PDF64 on hsPDF enzymatic activity was measured and confirmed by computation analysis. Antiproliferation activity was determined and in-vivo antitumor activity were analyzed in HCT116 and HL60 nude mice xenografts. Mitochondrial membrane potential (MMP), cell apoptosis, and autophagic cell death were analyzed by flow cytometry. ATP level was quantified using an ATP assay kit. Protein expression and kinase phosphorylation were determined by western blotting. A new hsPDF inhibitor PDF64 was identified. It showed evident antiproliferation activity in 10 cancer cells and significantly suppressed tumor growth in HCT116 and HL60 xenografts. It induced an obvious decrease in MMP and caused apparent cell apoptosis and autophagy in HCT116 and Jurkat cells. PDF64 treatment also led to an evident decrease in cellular ATP levels in these cells. Moreover, PDF64 downregulated c-Myc expression and had some effects on extracellular regulated protein kinases (ERK) and protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) pathways. PDF64 exhibited good antitumor effects both in vivo and in vitro . It caused cell apoptosis and autophagic death in HCT116 and Jurkat cells. The effects may be mediated by inhibiting c-Myc expression and ERK or PI3K-Akt-mTOR pathway. Therefore, PDF64 may be a promising reagent for antitumor drug development, which further supports that hsPDF is a good antitumor drug target.

Polycationic peptide R7-G-Aβ25-35 selectively induces cell death in leukemia Jurkat T cells through speedy mitochondrial depolarization, and CASPASE-3 -independent mechanism

BackgroundAcute lymphoblastic leukemia (ALL) is still incurable hematologic neoplasia in an important percentage of patients. Therefore, new therapeutic approaches need to be developed. MethodsTo evaluate the cellular effect of cell-penetrating peptides (C-PP) on leukemia cells, Jurkat cells -a model of ALL were exposed to increasing concentration (50–500 μM) Aβ25–35, R7-G-Aβ25-35 and Aβ25–35-G-R7 peptide for 24 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry (FC), and fluorescent microscopy (FM) analysis were used to assess metabolic viability, cell cycle and proliferation, mitochondria functionality, oxidative stress, and cell death markers. ResultsWe report for the first time that the R7-G-Aβ25-35, but not Aβ25–35 peptide, induced selective cell death in Jurkat cells more efficiently than the Aβ25–35-G-R7 peptide. Indeed, R7-G-Aβ25-35 (200 μM) altered the metabolic activity (−25%), arrested the cell cycle in the G2/M-phase (15%), and induced a significant reduction of cellular proliferation (i.e., −74% reduction of Ki-67 nuclei reactivity). Moreover, R7-G-Aβ25-35 induced the dissipation of mitochondrial membrane potential (ΔΨm, 51%) and produced an important amount of reactive oxygen species (ROS, 75% at 8 h) in Jurkat cells. The exposure of cells to antioxidant/cytoprotectant N-acetylcysteine (NAC) did not prevent R7-G-Aβ25-35 from a loss of ΔΨm in Jurkat cells. The peptide was also unable to activate the executer CASPASE-3, thereby preserving the integrity of the cellular DNA corroborated by the fact that the caspase-3 inhibitor NSCI was unable to protect cells from R7-G-Aβ25-35 -induced cell damage. Further analysis showed that the R7-G-Aβ25-35 peptide is specifically localized at the outer mitochondria membrane (OMM) according to colocalization with the protein translocase TOMM20. Additionally, the cytotoxic effect of the poly-R7 peptide resembles the toxic action of the uncoupler FCCP, mitocan oligomycin, and rotenone in Jurkat cells. Importantly, the R7-G-Aβ25-35 peptide was innocuous to menstrual mesenchymal stromal cells (MenSC) –normal non-leukemia proliferative cells. ConclusionOur findings demonstrated that the cationic Aβ peptide possesses specific anti-leukemia activity against Jurkat cells through oxidative stress (OS)- and CASPASE-3-independent mechanism but fast mitochondria depolarization.

The fungal Clitocybe nebularis lectin binds distinct cell surface glycoprotein receptors to induce cell death selectively in Jurkat cells.

Clitocybe nebularis lectin (CNL) is a GalNAcβ1-4GlcNAc-binding lectin that exhibits an antiproliferative effect exclusively on the Jurkat leukemic T cell line by provoking homotypic aggregation and dose-dependent cell death. Cell death of Jurkat cells exhibited typical features of early apoptosis, but lacked the activation of initiating and executing caspases. None of the features of CNL-induced cell death were effectively blocked with the pan-caspase inhibitor or different cysteine peptidase inhibitors. Furthermore, CNL binding induced Jurkat cells to release the endogenous damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1). A plant lectin with similar glycan-binding specificity, Wisteria floribunda agglutinin (WFA) showed less selective toxicity and induced cell death in Jurkat, Tall-104, and Hut-87 cell lines. HMGB1 release was also detected when Jurkat cells were treated with WFA. We identified the CD45 and CD43 cell surface glycoproteins on Jurkat cells as the main targets for CNL binding. However, the blockade of CD45 phosphatase activity failed to block either CNL-induced homotypic agglutination or cell death. Overall, our results indicate that CNL triggers atypical cell death selectively on Jurkat cells, suggesting the potential applicability of CNL in novel strategies for treating and/or detecting acute T cell leukemia.

Antagonistic Antibody Targeting TNFR2 Inhibits Regulatory T Cell Function to Promote Anti-Tumor Activity.

Infiltration of regulatory T cells (Tregs) in the tumor microenvironment suppresses anti-tumor immune response, and promotes tumor progression. Tumor necrosis factor receptor‐2 (TNFR2), which is highly expressed on Tregs, activates Tregs through nuclear factor kappa B (NF-κB) pathway. Moreover, TNFR2+ Tregs have been shown to be most suppressive among all Tregs populations in tumor. Due to the unique expression pattern and function of TNFR2 on Tregs, a TNFR2 blocking antibody is expected to compromise Tregs function, relieve Tregs-mediated immunosuppression, and hence to enhance anti-tumor immune response. AN3025 is an antagonistic anti-human TNFR2 (hTNFR2) antibody that is currently under preclinical development. This study investigates the immunomodulatory and anti-tumor activity of AN3025. AN3025 was generated through rabbit immunization with extracellular domain of human TNFR2 and subsequent humanization by complementarity-determining regions (CDRs) grafting. AN3025 binds to the extracellular domain of both human and cynomolgus with sub-nanomolar affinity and specificity, but not mouse or rat TNFR2. AN3025 inhibited tumor necrosis factor alpha (TNFα) induced cell death of hTNFR2-overexpressing Jurkat cells by competing with TNFα for binding to hTNFR2. In the Tregs/T effector co-culture assay, AN3025 increased T effector proliferation and enhanced interferon gamma (IFNγ) production. As a monotherapy, AN3025 significantly inhibited MC38 tumor growth in TNFR2 humanized mouse model. Subsequent flow cytometry (FACS) and immunohistochemistry (IHC) analysis revealed that administration of AN3025 led to decreased Tregs population, increased CD4+ and CD8+ T cell numbers in the tumor. The anti-tumor activity of AN3025 was dependent on the existence of CD4+ and CD8+ T cells, as depletion of CD4+ and CD8+ T cells abolished the anti-tumor activity of AN3025. In addition, AN3025 in combination with anti-PD-1 antibody demonstrated stronger in-vivo anti-tumor activity. The potent anti-tumor efficacy of AN3025, either as a monotherapy or in combination with anti-PD-1 antibody, supports its further clinical development for the treatment of various human tumors.

Cyclometalated Iridium(III) Complex-Cationic Peptide Hybrids Trigger Paraptosis in Cancer Cells via an Intracellular Ca2+ Overload from the Endoplasmic Reticulum and a Decrease in Mitochondrial Membrane Potential.

In our previous paper, we reported that amphiphilic Ir complex–peptide hybrids (IPHs) containing basic peptides such as KK(K)GG (K: lysine, G: glycine) (e.g., ASb-2) exhibited potent anticancer activity against Jurkat cells, with the dead cells showing a strong green emission. Our initial mechanistic studies of this cell death suggest that IPHs would bind to the calcium (Ca2+)–calmodulin (CaM) complex and induce an overload of intracellular Ca2+, resulting in the induction of non-apoptotic programmed cell death. In this work, we conduct a detailed mechanistic study of cell death induced by ASb-2, a typical example of IPHs, and describe how ASb-2 induces paraptotic programmed cell death in a manner similar to that of celastrol, a naturally occurring triterpenoid that is known to function as a paraptosis inducer in cancer cells. It is suggested that ASb-2 (50 µM) induces ER stress and decreases the mitochondrial membrane potential (ΔΨm), thus triggering intracellular signaling pathways and resulting in cytoplasmic vacuolization in Jurkat cells (which is a typical phenomenon of paraptosis), while the change in ΔΨm values is negligibly induced by celastrol and curcumin. Other experimental data imply that both ASb-2 and celastrol induce paraptotic cell death in Jurkat cells, but this induction occurs via different signaling pathways.

Roles for ADAM17 in TNF-R1 Mediated Cell Death and Survival in Human U937 and Jurkat Cells.

Signaling via death receptor family members such as TNF-R1 mediates pleiotropic biological outcomes ranging from inflammation and proliferation to cell death. Pro-survival signaling is mediated via TNF-R1 complex I at the cellular plasma membrane. Cell death induction requires complex IIa/b or necrosome formation, which occurs in the cytoplasm. In many cell types, full apoptotic or necroptotic cell death induction requires the internalization of TNF-R1 and receptosome formation to properly relay the signal inside the cell. We interrogated the role of the enzyme A disintegrin and metalloprotease 17 (ADAM17)/TACE (TNF-α converting enzyme) in death receptor signaling in human hematopoietic cells, using pharmacological inhibition and genetic ablation. We show that in U937 and Jurkat cells the absence of ADAM17 does not abrogate, but rather increases TNF mediated cell death. Likewise, cell death triggered via DR3 is enhanced in U937 cells lacking ADAM17. We identified ADAM17 as the key molecule that fine-tunes death receptor signaling. A better understanding of cell fate decisions made via the receptors of the TNF-R1 superfamily may enable us, in the future, to more efficiently treat infectious and inflammatory diseases or cancer.

Naegleria fowleri Induces Jurkat T Cell Death via O-deGlcNAcylation.

The pathogenic free-living amoeba Naegleria fowleri causes primary amoebic meningoencephalitis, a fatal infection, by penetrating the nasal mucosa and migrating to the brain via the olfactory nerves. N. fowleri can induce host cell death via lytic necrosis. Similar to phosphorylation, O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation) is involved in various cell-signaling processes, including apoptosis and proliferation, with O-GlcNAc addition and removal regulated by O-GlcNAc transferase and O-GlcNAcase (OGA), respectively. However, the detailed mechanism of host cell death induced by N. fowleri is unknown. In this study, we investigated whether N. fowleri can induce the modulation of O-GlcNAcylated proteins during cell death in Jurkat T cells. Co-incubation with live N. fowleri trophozoites increased DNA fragmentation. In addition, incubation with N. fowleri induced a dramatic reduction in O-GlcNAcylated protein levels in 30 min. Moreover, pretreatment of Jurkat T cells with the OGA inhibitor PUGNAc prevented N. fowleri–induced O-deGlcNAcylation and DNA fragmentation. These results suggest that O-deGlcNAcylation is an important signaling process that occurs during Jurkat T cell death induced by N. fowleri.

Abstract 1451: AN3025: A novel anti-human TNFR2 antibody that exhibits immune activation and strong anti-tumor activity in vivo

Abstract Background: The unique biology and expression pattern of tumor necrosis factor receptor-2 (TNFR2) make it an attractive therapeutic target for immuno-oncology. TNFR2 highly expresses on a subset of regulatory T cells (Tregs) and MDSCs within tumor microenvironment that can activate these cells through nuclear factor kappa B (NF-kB) pathway. TNFR2+ Treg has been shown to be most suppressive among all Treg populations in tumor. On the other hand, TNFR2 is also abundantly expressed on the surface of many human tumors. TNFR2 blocking antibody is expected to relieve TNFR2-mediated immunosuppression and inhibit TNFR2-expressing tumor cell survival. AN3025 is a novel humanized IgG1 (variant) anti-hTNFR2 antibody under preclinical development. The immunomodulatory and anti-tumor activity of AN3025 were evaluated both in vitro and in vivo. Materials and methods: AN3025 was generated through rabbit immunization followed by phage display, then humanized by CDRs grafting. The binding affinity and specificity were studied by ELISA and FACS. The ability of AN3025 to mitigate TNF/TNFR2 signaling pathway was characterized using TNFR2 overexpressing Jurkat cell line in vitro. The in vivo anti-tumor activity was evaluated in TNFR2 humanized mouse model bearing MC38 tumor. The tumor samples from control and AN3025 treated mice were taken for further FACS and RNA seq analysis. Results: AN3025 binds to the extracellular domain of human TNFR2 with sub-nanomolar affinity and specificity. It cross-reacts with cynomolgus TNFR2 with similar affinity, but not with mouse or rat TNFR2. Mechanistically, AN3025 partially competes with TNFα for binding to TNFR2 receptor and inhibits TNFα induced hTNFR2 overexpressing Jurkat cell death, however it lacks of agonist activity towards TNFR2 even in the presence of hFC crosslinking. In vivo AN3025 significantly inhibits MC38 tumor growth as a monotherapy in hTNFR2 mouse model, while no impact on body weight. Subsequent FACS analysis suggests decreased Tregs% in AN3025 treated tumor. RNA seq suggests immune activation such as increased IFN-gamma and Granzyme expression. In addition, AN3025 enhances anti-tumor efficacy of mPD-1 antibody in a combination study. Conclusions: AN3025 is a novel anti-hTNFR2 antibody and demonstrates immunomodulatory activity and potent anti-tumor efficacy in vivo, supporting its clinical development for the treatment of human cancers. Citation Format: Yonglin Chen, Manxue Jia, Sherry Xu, Yanhui Zhao, Eric Chan, Meng Zhang, Emma Chen, Yi Zhang. AN3025: A novel anti-human TNFR2 antibody that exhibits immune activation and strong anti-tumor activity in vivo [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 1451.

Synthesis and Anticancer Properties of Bis‐ and Mono(cationic peptide) Hybrids of Cyclometalated Iridium(III) Complexes: Effect of the Number of Peptide Units on Anticancer Activity

AbstractCyclometalated iridium (Ir) complexes having Ir(tpy)3 (tpy=2‐(4’‐tolyl)pyridine) scaffold exhibit promising potential for the diagnosis of cancer, as phosphorescent probes for cellular imaging and in the treatment of cancer, due to their high stability, tunable photophysical properties, and biological activities. We previously reported on the synthesis of some cationic amphiphilic Ir(III) complex‐peptide hybrids (IPHs) such as 2 and 3 that contain KK(K)GG peptide units (K: lysine, G: glycine) that are attached via alkyl chain (C2∼C16) linkers and induce paraptosis‐like cell death in Jurkat (T‐lymphocyte leukemia) cancer cells through multiple cell death pathways. Herein, we report on the design and synthesis of two IPHs, 4 and 5, which possess two and one KKKGG sequences, respectively, and an examination of the effect of the number of peptide units on their anticancer activity. Our evaluations of the anticancer properties of 3–5 suggested that 3 and 4 are more cytotoxic than 5 against Jurkat cells, indicating a positive relationship between the number of H2N‐KKKGG units in 3–5 and their anticancer activity. In addition, a parallel relationship was observed between the cytotoxicity of 3–5 and Ca2+ overload in mitochondria, suggesting the direct transfer of Ca2+ from the endoplasmic reticulum to mitochondria.

Epigenetic priming by EHMT1/EHMT2 in acute lymphoblastic leukemia induces TP53 and TP73 overexpression and promotes cell death

Euchromatic histone-lysine N-methyltransferase 1 (EHMT1) and EHMT2 are upregulated in various human cancers, and their deregulation is associated with tumor development and progression. In this paper, we investigated the expression level of EHMT1/EHMT2 in acute lymphoblastic leukemia (ALL) and whether the modulation of these enzymes could have any cellular or molecular impact on ALL cells. For this, we used UNC0646 as a priming strategy to target EHMT1/EHMT2 and investigated its effect on proliferation and cell viability of Jurkat cells by MTT assay. Then, considering the IC50 and IC75, cellular death was determined by Annexin V/PI staining using flow cytometry. Finally, we investigated by RT-PCR the molecular bases that could be involved in the observed effects. Interestingly, accessing the International Microarray Innovations in Leukemia (MILE) study group, we detected that both EHMT1 and EHMT2 are overexpressed in ALL. More important, we determined that inhibition of EHMT1/EHMT2 significantly decreased Jurkat cell viability in a dose-dependent manner. Accordingly, we observed that inhibition of EHMT1/EHMT2 promoted Jurkat cell death, which was accompanied by increased expression of P53, TP73, BAX, and MDM4. These results clearly indicate that inhibition of EHMT1/EHMT2 induces pro-apoptotic gene expression in ALL and promotes cell death. More importantly, the modulation of these histone methyltransferases may be a promising epigenetic target for ALL treatment.

Abstract 4515: The anti-CD47 antibody SRF231 increases anti-tumor activity of standard of care chemotherapy in platinum-resistant PDX models of ovarian cancer

Abstract Background: CD47 is a type I integral membrane protein expressed on multiple human tumors, including ovarian cancer, and modulates cell processes such as cell migration, adhesion, T-cell function, and cell death via interaction with multiple ligands. Interaction of CD47 with SIRPα expressed on myeloid cells results in an inhibitory “don't eat me” signal that prevents phagocytosis of CD47-expressing cancer cells. Enhancement of the anti-tumor activity of chemotherapy has also been reported with CD47 antagonists. We investigated the effects of combining SRF231, an investigational fully human IgG4 antibody, with chemotherapy in models of human ovarian cancer. Methods: Expression of CD47 in 8 established platinum-resistant PDX models of ovarian cancer was measured by immunohistochemistry with the anti-CD47 antibody SP279. SRF231-mediated phagocytosis of ovarian cancer cell lines was assessed using a macrophage coculture system. Cell death from combining SRF231 with either doxorubicin or platinum in vitro was assessed by Annexin V assay. The activity of SRF231 combined with doxorubicin in vivo was compared to isotype control, SRF231, or doxorubicin monotherapy in an ovarian cancer subcutaneous xenograft model, OVCAR3. Additionally, the activity of SRF231 combined with platinum was compared to isotype control, SRF231, or platinum monotherapy in two luciferase-expressing intraperitoneal PDX ovarian cancer models, as measured by bioluminescent imaging. Results: CD47 expression was high across all 8 PDX models. SRF231 induced phagocytosis of ovarian cancer cell lines by human monocyte-derived macrophages. SRF231 potentiated doxorubicin- or oxaliplatin-mediated cell death in Jurkat cells in Annexin V assays. In the OVCAR3 xenograft model, the combination of SRF231 and doxorubicin resulted in significant tumor growth inhibition and improved survival compared to isotype control, SRF231 monotherapy, or doxorubicin monotherapy. In the PDX model DF216, which demonstrates no evidence of monotherapy response to SRF231, combination of SRF231 and carboplatin resulted in tumor regression with significantly enhanced anti-tumor activity compared to carboplatin or SRF231 monotherapy, as well as to isotype control. Similarly, in the PDX model DF86, where SRF231 demonstrates modest monotherapy activity, combination SRF231 and carboplatin resulted in tumor regression and significant increase in anti-tumor activity compared to carboplatin or SRF231 monotherapies. Conclusion: Anti-CD47 directed therapy with SRF231, a fully human antibody, demonstrated the ability to significantly increase the anti-tumor activity of standard chemotherapies in xenograft and platinum-resistant PDX models of ovarian cancer. Further exploration of combining anti-CD47 and platinum regimens in ovarian cancer is warranted. Citation Format: Joyce F. Liu, Kshama A. Doshi, Benjamin H. Lee, Marisa O. Peluso, Li Zhang, Shan Zhou, Qing Zeng, Stephen Wang, Paul T. Kirschmeier, Cam A. Tran, Cloud Paweletz, Matthew Rausch, Alison M. Paterson, Prafulla C. Gokhale, Ursula A. Matulonis. The anti-CD47 antibody SRF231 increases anti-tumor activity of standard of care chemotherapy in platinum-resistant PDX models of ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4515.

Cannabinoid CP55940 selectively induces apoptosis in Jurkat cells and in ex vivo T-cell acute lymphoblastic leukemia through H2O2 signaling mechanism

T-cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous malignant hematological disorder arising from T-cell progenitors. This study was aimed to evaluate the cytotoxic effect of CP55940 on human peripheral blood lymphocytes (PBL) and on T-ALL cells (Jurkat). PBL and Jurkat cells were treated with CP55940 (0−20 μM), and morphological changes in the cell nucleus/ DNA, mitochondrial membrane potential (ΔΨm), and intracellular reactive oxygen species levels were determined by fluorescence microscopy and flow cytometry. Cellular apoptosis markers were also evaluated by western blotting, pharmacological inhibition and immunofluorescence. CP55940 induced apoptotic cell death in Jurkat cells, but not in PBL, in a dose–response manner with increasing fragmentation of DNA, arrest of cell cycle and damage of ΔΨm. CP55940 increased dichlorofluorescein fluorescence (DCF) intensity, increased DJ-1 Cys106- sulfonate, a marker of intracellular stress, induced the up-regulation of p53 and phosphorylation of transcription factor c-JUN. It increased the expression of BAX and PUMA, up-regulated mitochondrial proteins PINK1 and Parkin, and activated CASPASE-3. Antioxidant NAC, pifithrin-α, and SP600125 blocked CP55940 deleterious effect on Jurkat cells. However, the potent and highly specific cannabinoid CB1 and CB2 receptor inverse agonist SR141716 and SR144528 were unable to blunt CP55940-induced apoptosis in Jurkat cells. Conclusively CP55940 provokes cell death in Jurkat through CBR-independent mechanism. Interestingly, CP55940 was also cytotoxic to ex vivo T-ALL cells from chemotherapy-resistant pediatric patients. In conclusion, CP55940 selectively induces apoptosis in Jurkat cells through a H2O2-mediated signaling pathway. Our findings support the use of cannabinoids as a potential treatment for T-ALL cells.

New 1Z,5Z-diene macrodiolides: Catalytic synthesis, anticancer activity, induction of mitochondrial apoptosis, and effect on the cell cycle.

An original scheme was developed for the synthesis of previously undescribed unsaturated macrodiolides containing a 1Z,5Z-diene moiety in 44-80% yields and with high stereoselectivity (>95%) based on the intermolecular esterification of α,ω-diols with α,ω-alka-nZ,(n+4)Z-dienedicarboxylic acids (1,12-dodeca-4Z,8Z-dienedicarboxylic acid, 1,14-tetradeca-5Z,9Z-dienedicarboxylic acid, 1,18-octadeca-7Z,11Z-dienedicarboxylic acid) catalyzed by hafnium triflate [Hf(OTf)4]. The unsaturated dicarboxylic acids were prepared via homo-cyclomagnesiation of tetrahydropyran ethers of O-containing 1,2-dienes with EtMgBr in the presence of Mg metal and the Cp2TiCl2 catalyst (10mol.%) and the subsequent Jones oxidation of pyran ethers formed after the acid hydrolysis of magnesacyclopentanes. The thus prepared macrodiolides exhibit high cytotoxic activity in vitro against Jurkat, K562, U937, Hek293 and HeLa cancer cell lines. It was found that induction of the programmed cell death in Jurkat cells by macrodiolides corresponds to the mitochondrial apoptosis pathway. Also, it was shown that the prepared macrodiolides efficiently suppress phosphorylation of Akt and p38 kinases and CREB transcription factor in cancer cells.