Murine Xenograft Model Research Articles

Abstract 1356: The VISTA blocking antibody HMBD-002 promotes type-I interferon signaling and drives anti-tumor responses through macrophage reprograming and cytotoxic CD8+ T cell activation

Abstract VISTA, a predominantly myeloid immune checkpoint, promotes an immunosuppressive tumor microenvironment and has been associated with resistance to approved checkpoint therapies. While blockade of VISTA with antibodies has shown anti-tumor efficacy in preclinical models, the biological mechanisms that underlie this effect remain poorly understood. To investigate the mechanistic basis of anti-tumor responses, we performed single-cell RNA sequencing of tumor infiltrating leukocytes from the cell-derived murine colon carcinoma xenograft model, CT26, which is known to be sensitive to VISTA blockade. We used HMBD-002, a uniquely blocking but non-depleting clinical-stage IgG4 anti-VISTA antibody, to identify the key immune cells and pathways impacted by VISTA blockade in the absence of Fc-mediated cell depletion. The translatability of these findings was validated in a human PBMC mixed leukocyte reaction assay. Subsequently, targeted depletion or inhibition of the identified immune populations and pathways was performed to confirm their contribution to the anti-tumor efficacy of HMBD-002.The blockade of VISTA by HMBD-002 significantly increased CD8+ T cells in the CT26 tumors, promoting an activated cytotoxic Tc1 program without inducing exhaustion. Concurrently, a substantial increase in proinflammatory M1 tumor-associated macrophages (TAMs) was noted within treated tumors, while other immune cell populations remained unaffected. Among the signaling pathways, the type-I interferon (IFN) response exhibited a significant upregulation, particularly in TAMs, indicating a type-I IFN-driven M1 reprogramming by HMBD-002. Validation experiments further demonstrated that the depletion of TAMs or CD8+ T cells, but not CD4+ T cells, abrogated the efficacy of HMBD-002. Moreover, blocking the type-I IFN signaling effectively impeded HMBD-002-induced TAM reprogramming and subsequent anti-tumor efficacy, thus confirming the pivotal roles of CD8+ T cells, TAMs, and type-I IFN signaling in driving the anti-tumor response to VISTA blockade. In conclusion, VISTA blockade with HMBD-002 promotes an anti-tumor response by type-I IFN mediated reprogramming of TAMs and activation of cytotoxic CD8+ T cells. Citation Format: Bhushan Dharmadhikari, Olga Zharkova, Dipti Thakkar, Roberto Tirado-Magallanes, Konrad Paszkiewicz, Piers J. Ingram, Jerome D. Boyd-Kirkup. The VISTA blocking antibody HMBD-002 promotes type-I interferon signaling and drives anti-tumor responses through macrophage reprograming and cytotoxic CD8+ T cell activation [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 1356.

Abstract 6565: Improving survival of atypical teratoid/rhabdoid tumor orthotopic xenografts through the combination of PI3K inhibitor paxalisib and nucleoside analog gemcitabine

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are an aggressive infantile brain tumor with a dismal four-year event-free survival of 37%. We have previously identified high activation of mTORC1 and mTORC2 within AT/RT and shown that the highly brain penetrant PI3K inhibitor paxalisib is capable of simultaneously inhibiting mTORC1/2 by acting upstream of both complexes. In two orthotopic xenograft models, paxalisib slowed tumor growth and significantly extended survival (CHLA-06: 40 to 54 days, p=0.001; BT-12: 21 to 35 days, p=0.02). RNAseq analysis following mTOR inhibition in AT/RT cell lines showed an upregulation of genes involved in the activation of the integrated stress response (ISR) (ATF4, CHOP, PPP1R15A t-test p<0.05). Analysis of primary pediatric brain tumors through the Children’s Brain Tumor Network showed increased levels of ATF4 mRNA and eIF2α protein and mRNA in AT/RT compared to other indolent pediatric brain tumors. Increased expression of ISR components suggests this pathway may be a dependency in AT/RT. Activation of this pathway is known to be cytoprotective, however persistent over-activation drives cell death through induction of pro-apoptotic factors. Gemcitabine is a pyrimidine analog functioning to inhibit DNA synthesis and induce DNA damage; notably, recent literature has correlated DNA damage to activation of the ISR with downstream phosphorylation of eIF2α. We hypothesize that due to high baseline activation of ISR for AT/RT, paxalisib combined with gemcitabine will result in an over-activation of the ISR leading to AT/RT cell death. Western blot analysis of combination therapy compared to DMSO control showed induction of phospho-eIF2α, ATF4, and CHOP, denoting an activation of the ISR. Combination therapy in AT/RT cells induces cell death (Western blot analysis cPARP, MUSE Annexin V assay, SynergyFinder BLISS score [viability] CHLA-06: 21.50, CHLA-266: 22.60) and synergizes to decrease cell growth (SynergyFinder BLISS score [inhibition] CHLA-06: 16.80, BT-12: 14.30, CHLA-266: 13.90, BT-37: 11.40, CHLA-05: 10.40). Paxalisib/gemcitabine combination therapy in murine orthotopic xenograft models of AT/RT has slowed tumor growth (bioluminescent imaging) and led to significant extension of survival. Mice bearing BT-37 orthotopic xenografts and treated with vehicle control had a median survival of 56 days, while all of the paxalisib/gemcitabine treated mice were alive at 100 days of treatment (p<0.0001). In CHLA-06 orthotopic tumors combination therapy extended median survival from 22 days for control to 82.5 days (p<0.0001). Based on these findings, the Pacific Pediatric Neuro-Oncology Consortium (PNOC) is planning to include paxalisib/gemcitabine combination therapy in the next international clinical trial for patients with relapsed/recurrent AT/RT. Citation Format: Tyler Findlay, Kristen Malebranche, Anupa Geethadevi, Charles Eberhart, Jeffrey Rubens, Eric Raabe. Improving survival of atypical teratoid/rhabdoid tumor orthotopic xenografts through the combination of PI3K inhibitor paxalisib and nucleoside analog gemcitabine [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 6565.

Abstract 2739: XON27, a novel and potent immunotherapy against hematologic malignancies

Abstract Background: Over the past decades, intensive research has led to the development of innovative therapies (monoclonal and bispecific antibodies, CAR T cells) that have increased the survival rate of patients with multiple myeloma (MM) and B cell lymphoma. However, despite this progress, the majority of patients relapse and become refractory due to the development of resistance. Polyclonal antibodies, with their ability to target multiple antigens, can counteract this resistance. Here we evaluate the safety and efficacy of XON27, a first-in-class glyco-humanized polyclonal antibody (GH-pAb), in MM and other blood cancers. Material and Methods: The ability of XON27 to induce antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) and apoptosis was tested in a panel of hematological malignancy cells and peripheral blood mononuclear cells (PBMC). Specific binding to human blood cancers was assessed using tissue microarrays. To study resistance mechanisms, MM cell lines were cocultured with macrophages in the presence or absence of XON27 and macrophage. To study relapse mechanisms and tumorigenicity, repeated in vitro administration of XON27 was evaluated in KMS-12-BM. The in vivo efficacy of XON27 was evaluated in KMS-12-BM NMRI nude mice model. Pharmacokinetics and safety of this drug were assessed in non-human primate after repeated IV dosing up to 60mg/kg.Results: XON27 induces potent and specific cytotoxicity in several blood malignancy cell lines including human MM cells via multiple mechanisms: CDC (EC50=35.84 ug/ml), apoptosis (IC50=100 ug/ml) and ADCP (up to 40% of cell death at 50 ug/ml). The cytotoxicity induced by XON27 in the KMS-12-BM was significantly more potent compared to daratumumab (anti-CD38) in both CDC and apoptosis assays, XON27 was able to kill up to 100% of cancer cells without affecting PBMC. XON27 showed cross-reactivity with blood cancer patient biopsies without staining healthy tissue. In addition, repeated administrations of XON27 demonstrate its ability to effectively kill tumor cells after each administration. No resistance is induced after repeated treatments. Interestingly, XON27 blocked the proliferation of residual tumor cells for up to 45 days after the second treatment at 50 ug/ml compared to the control condition, demonstrating sustainable effect of XON27 on tumorigenicity. The in vitro efficacy of XON27 was translated into in vivo efficacy in a murine MM xenograft model. It induced a significant reduction in tumor growth of up to 75%. XON27 was also characterized by a high tolerability profile and satisfactory exposure in primates. Conclusion: Although many therapeutic advances have been made in the field of blood cancers, myeloma remains an incurable disease with a real need for new innovative therapies. XON27 represents a promising and selective immunotherapy which may provide an effective and safe treatment against refractory myeloma. Citation Format: Carine Ciron, Pierre Morice, Françoise Shneiker, Odile Duvaux, Firas Bassissi. XON27, a novel and potent immunotherapy against hematologic malignancies [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 2739.

Abstract 1090: GPC2 directed CAR T cells are efficacious against fusion-positive and fusion-negative rhabdomyosarcomas

Abstract Background: Rhabdomyosarcoma (RMS) is an aggressive pediatric solid tumor with poor survival and new targeted therapies are needed. Aims: Using an integrative proteogenomic quantitative approach, we sought to identify candidate immunotherapeutic targets then validate top targets in RMS preclinical models. Methods: We first performed plasma membrane enrichment followed by mass spectrometry (MS) to define the surfaceome of 7 fusion-positive (FP) and 14 fusion-negative (FN) RMS patient-derived xenograft models. Surface proteins were scored by IMMUNOTAR, our custom pipeline which extracts and integrates quantitative features from diverse proteogenomic and annotation datasets to provide a score for each protein, using mean-average-precision for score optimization. Cell surface expression of IMMUNOTAR-prioritized proteins was validated using flow cytometry, focusing on targets with immunotherapies in development. CAR T cells targeting the lead protein were engineered and studied in RMS preclinical models. Results: A total of 847 and 882 high confidence cell surface proteins were detected in FP and FN RMS surfaceome, respectively, with 94% of proteins overlapping the phenotypes. IMMUNOTAR ranked anaplastic lymphoma kinase (ALK), a putative immunotherapeutic target in FP RMS, as the top FP target. Glypican 2 (GPC2), a differentially expressed cell surface proteoglycan identified in neuroblastoma and other neural-derived tumors, targetable with CAR T cells and antibody-drug conjugates [Bosse et al. Cancer Cell 2017], ranked as the top protein in FN and 5th protein in FP RMS. Overall, GPC2 was abundantly expressed in 16 of 21 RMS models profiled. Flow cytometry revealed expression of GPC2 comparable to neuroblastoma in 5 of 8 RMS cells lines. Additionally, both FP and FN GPC2+ RMS cell lines (FN: RH36, SMS-CTR; FP: RH41, RH30) activated GPC2 CAR T cells with either a CD28 or a 4-1BB costimulatory domain, inducing release of IFN-gamma and IL-2 upon co-incubation across different E:T ratios as quantified by ELISA. Further, GPC2 CAR T cells induced potent RMS cell cytotoxicity compared to control CD19 CAR T cells with near 100% specific cytotoxicity at 1:1 E:T ratio. In a pilot study, GPC2 CAR T cells with 4-1BB costimulatory domains induced significant tumor regression (p=0.03) and prolonged progression-free survival (p=0.01) compared to CD19 CAR T cells in an established murine RH41 flank xenograft model. Conclusion: In summary, we probed the RMS surfaceome using MS and identified candidate proteins amenable for immunotherapeutic targeting using our IMMUNOTAR algorithm. We validated GPC2 as a candidate target in FP and FN RMS and are corroborating our findings in additional pre-clinical RMS models. Demonstration of GPC2 CAR efficacy in these models could lead to inclusion of RMS in our ongoing phase 1 clinical trial (NCT05650749) in a planned expansion phase. Citation Format: Rawan Shraim, Patrick M. Schuerch, Anna Maria Giudice, Karina L. Conkrite, Amber K. Weiner, Alexander B. Radaoui, Brian Mooney, Raushan T. Kurmasheva, Peter J. Houghton, Gregg B. Morin, Yael B. Mosse, Poul H. Sorensen, Benjamin A. Garcia, Ahmet Sacan, John M. Maris, Kristopher R. Bosse, Sharon J. Diskin. GPC2 directed CAR T cells are efficacious against fusion-positive and fusion-negative rhabdomyosarcomas [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 1090.

Abstract 3201: Preclinical development of a novel Romidepsin nanoparticle for the treatment of T-Cell lymphoid malignancies

Abstract The histone deacetylase inhibitors (HDACi) have long been regarded as an important class of drugs for treating T-cell lymphoid malignancies (TCL). Romidepsin (Romi), a bicyclic peptide, was approved for patients with relapsed or refractory cutaneous (CTCL) and peripheral T-cell lymphomas (PTCL). Unfortunately, the approval of Romi for relapsed/refractory PTCL was withdrawn due to a negative Phase 4 post-marketing requirement. Given its potent activity against Class I HDACs, Romi is considered one of the most effective HDAC inhibitors studied and used in patients with TCL. Herein, we report on a first-in-class nanoparticle of Romi (NanoRomi) with a superior safety and efficacy profile compared to the free drug in preclinical models of PTCL and large granular lymphocyte leukemia (LGLL). We developed a first-in-class NanoRomi using our proprietary amphiphilic di-block copolymer-based nanochemistry platform. NanoRomi was characterized in cell viability, flow cytometry, and western blotting (WB) assays to determine the concentration-effect relationships with regard to the induction of apoptosis and histone acetylation in cell lines representative of TCL and LGLL, as well as primary LGLL patient PBMC samples. In addition, we performed extensive in vivo modeling using murine xenografts of CTCL, including repeated dose toxicity, biodistribution, efficacy, and survival studies. Cytotoxicity assays demonstrated superior/comparable activity of NanoRomi (IC50: 1.1-2.3 nM) compared to free Romi (IC50: 4.8-10.6 nM) across all PTCL and LGLL cell lines studied, while in primary LGLL samples, NanoRomi exhibited superior cytotoxicity (2-fold) compared to Romi. WB analysis showed that NanoRomi induced apoptosis and increased acetylation of histone H3 and H4 in a time and concentration-dependent manner in cell lines representing CTCL, ALK+-ALCL, and LGLL. Repeat dose toxicity studies in CTCL xenografts confirmed that the overall tolerability of NanoRomi was substantially better than Romi. Tracking studies with a fluorescent dye (DiO) in the NanoRomi demonstrated preferential localization of NanoRomi in tumors of mice compared to the normal organs after 6 hours of drug administration. NanoRomi exhibited superior efficacy in the murine CTCL xenograft models, including a significant improvement in overall survival compared to Romi. Collectively, these data show that NanoRomi exhibits markedly superior pharmacokinetics, tolerability, and efficacy in models of TCL. We anticipate that the improved activity and tolerability of NanoRomi relate to the altered pharmacologic profile and superior bioavailability of the drug, which optimizes the effects of the drug for its influence on epigenetic biology. This unique platform offers the prospect of improving the tolerability and efficacy of epigenetic treatment strategies in these challenging diseases. Citation Format: Ipsita Pal, Anuradha Illendula, Andrea Joyner, Tess M. Deddens, Deepthi P. Damera, Todd E. Fox, Marya E. Dunlap-Brown, Kallesh D. Jayappa, Thomas P. Loughran, David J. Feith, Owen A. O'Connor. Preclinical development of a novel Romidepsin nanoparticle for the treatment of T-Cell lymphoid malignancies [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 3201.

Abstract 5456: Identifying the role of tissue factor in osteosarcoma metastasis

Abstract Patients with osteosarcoma who develop metastasis face very poor prognoses. Even when treated with the most effective medical and surgical interventions available, about forty percent of osteosarcoma patients will eventually succumb to lung metastasis. Despite the relevance of metastasis to clinical outcomes, we understand little about the mechanisms that drive these tumor cells into the lung and facilitate their metastatic growth. Consequently, there are no approved antimetastatic agents for osteosarcoma and outcomes have not improved in more than 40 years. Tissue factor is a tightly regulated physiologic initiator of coagulation with functions in inflammation, cytokinesis, angiogenesis, apoptosis, and growth factor secretion. These functions are divided between two separate pathways: initiation of the coagulation cascade via activation of factors VII and X, and propagation of cell signaling pathways via PAR activation. It is aberrantly expressed in many cancers and has been associated with metastatic behavior and inferior outcomes by affecting metastatic vascularity, growth, and migration. Osteosarcoma has been shown to also aberrantly express tissue factor, with descriptive studies suggesting similar effects on metastatic behavior. We hypothesized that tissue factor expressed on the surface of disseminated osteosarcoma cells activates procoagulant pathways and PAR signaling, and that both facilitate pulmonary metastasis. To test this, we first established and quantified expression of tissue factor across six osteosarcoma cell lines using Western blot, flow cytometry, and confocal microscopy in both monoculture and in coculture with human bronchial epithelial cells. PAR1 and PAR2 were also discovered to be expressed in osteosarcoma cells. RNA sequencing performed suggests that both tissue factor and PAR1 expression in osteosarcoma increase over the metastatic period, as does FVII expression by alveolar macrophages; this implies that tissue factor activation during metastasis is dynamic and dependent on host interactions. Ongoing work includes functional assays of osteosarcoma tissue factor and PARs activity as well as preclinical testing of tissue factor inhibition in a murine xenograft model of osteosarcoma. To define how tissue factor propagates metastasis, we are separately inhibiting its coagulant and cell signaling pathways after murine tail vein injection using apixaban or pathway-specific antibodies. We will also use an inducible F3 knockout to alter tumor expression of tissue factor. If we find that manipulation of tissue factor expression changes metastatic behavior across cell lines, this will suggest that tissue factor plays a crucial, mechanistic role in osteosarcoma metastasis. Varying effectiveness of coagulation or signaling pathway inhibition on metastasis will implicate tissue factor’s mechanism of metastatic propagation. Citation Format: Charles C. Treinen, Amy Gross, Maren Cam, Bryce Kerlin, Ryan Roberts. Identifying the role of tissue factor in osteosarcoma metastasis [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 5456.

Abstract 7539: First-in-class anti-PVR mAb NTX1088 advancing through Phase I: Safe and potent in restoring DNAM1 expression to enhance antitumor immunity

Abstract NTX1088, a first-in-class anti-PVR (CD155) mAb, is currently evaluated in a Phase 1, open-label, multi-center study (NCT05378425). The trial initiated at MD Anderson Cancer Center, and was expanded into additional sites, including City of Hope, Ochsner Health, Sheba Medical Center and Hadassah Medical Center. Study objectives are safety, dose-finding, and efficacy with focus on pharmacokinetics, pharmacodynamics, and biomarker discovery. NTX1088 is investigated as a single agent and in combination with the anti-PD1 mAb, pembrolizumab (Keytruda) in patients with advanced solid malignancies. Ongoing dose escalation demonstrates a safe profile across ascending dose levels. NTX1088 is a high affinity humanized IgG4-S228P mAb that binds PVR and blocks all known interacting receptors at a single nM EC50. PVR, is a highly upregulated membrane protein on numerous tumor cells. PVR expression has been associated with worse patient outcome, due to its central role in immune suppression. PVR’s impact is mediated through interaction with the key stimulatory receptor, DNAM1 (CD226), on T and NK cells, leading to internalization and degradation of DNAM1. PVR also interacts with the inhibitory immune checkpoint receptors, TIGIT, CD96 and KIR2DL5A. Blocking PVR by NTX1088 has multiple immune-stimulating roles, through restoration of DNAM1 immune activation function, while simultaneously neutralizing TIGIT, CD96 and KIR2DL5A inhibitory signals in immune cells. Importantly, DNAM1 downmodulation was recently identified as a key resistance mechanism to approved immune checkpoint inhibitors, and its restoration by NTX1088 is a novel MoA, not demonstrated previously by other therapies.In vitro, as a monotherapy, NTX1088 significantly increased immune cell activation, superior to TIGIT, CD112R, and PD1 antibody blockade, leading to a greater immune-mediated tumor cell killing, IFNγ secretion, and CD137 induction. Importantly, only NTX1088 was able to restore DNAM1 to the surface of immune cells. Synergy was obtained when NTX1088 was combined with PD1 blockers, or with the anti-CD112R mAb, NTX2R13.Syngeneic models of PVRK.O, demonstrated a complete immune-mediated tumor regression. Numerous humanized murine xenograft models were investigated whereby NTX1088 exhibited robust tumor growth inhibition as a standalone and in combination with PD1 blockade.In conclusion, PVR blockade by NTX1088 has a remarkable pre-clinical efficacy, suggesting a potential clinical breakthrough, based on its ability to simultaneously overcome multiple tumor escape mechanisms. First-in-human (FIH) trial is currently ongoing and biomarker analysis is showing initial mechanistic proof of concept for NTX1088 mode of action, including the restoration of DNAM1 surface expression on patients’ immune cells. This is the first case of surface DNAM1 elevation in clinical settings. Citation Format: Anas Atieh, Akram Obeidat, Alon Vitenshtein, Guy Cinamon, Keren Paz, Tihana Roviš, Paola Brilc, Ofer Mandelboim, Stipan Jonjic, Pini Tsukerman. First-in-class anti-PVR mAb NTX1088 advancing through Phase I: Safe and potent in restoring DNAM1 expression to enhance antitumor immunity [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 7539.

Abstract 665: MEN1703/SEL24 exhibits promising antitumoral activity in preclinical models of myelofibrosis both as single agent and combined with ruxolitinib

Abstract Myelofibrosis (MF) is a pathological chronic bone marrow (BM) condition characterized by abnormal growth of erythroid and megakaryocytic precursors and deposition of fibrotic tissue. The JAK/STAT pathway plays an important role in MF (Cardoso 2015), and multiple JAK1/2 inhibitors (JAKis) are approved for MF treatment. Despite initially limiting disease symptoms, JAKis often fail due to persistent JAK2 activation and survival of MF-driving cells, hence new treatment strategies are highly needed. PIM kinases also contribute to MF pathogenesis. PIM1 is highly overexpressed in granulocytes, peripheral blood mononuclear cells (PBMCs), and BM of patients with MF. PIM1/2 expression is dependent upon JAK2 signaling in myeloproliferative neoplasms (MPNs). Hence, PIM inhibitors represent potential therapeutic targets for MPNs and have been evaluated in several preclinical and clinical studies (Rampal 2021). MEN1703/SEL24 (MEN) is an oral, first-in-class, dual PIM/FLT3 kinase inhibitor in development for hematologic malignancies. This study aims to investigate the efficacy of MEN alone and in combination with the JAKi ruxolitinib (RUX) in preclinical MF models and to elucidate the underlying signaling pathways. MF cell lines (JAK2V617F and JAK2 wild type) were used in MTS assays to assess the in vitro cytotoxicity of MEN alone and in combination with RUX, compared with the PIM inhibitor TP-3654 (Dutta 2022). Cell cycle distribution and signaling pathways were analyzed via propidium iodide/FACS analysis and immunocapillary electrophoresis, respectively, upon treatment with the single agents and MEN + RUX. In vivo efficacy was assessed in a murine xenograft model treated with MEN +/- RUX. In vitro studies showed single agent MEN activity with an IC50 ranging from 0.3 to 2.3 µM. Moreover, a synergistic drug interaction was found with MEN + RUX in all tested cell lines (Combination Index [CI] range 0.41-0.80). MEN presented cytotoxic and cytostatic capacity, inducing apoptosis and arresting a proportion of cells in G2/M phase. This effect was maintained in combination with RUX. Signaling pathway analysis showed that MEN inhibits phosphorylation of STAT proteins and decreases Mcl1 protein expression. These effects were enhanced with MEN + RUX. Compared with TP-3654, MEN was more effective both as a single agent (IC50 2-3 fold lower than TP-3654) and in combination with RUX (mean CI of 0.77-0.80 compared to 0.79-0.98 with TP-3654). MEN showed anti-tumoral efficacy in MF preclinical models, moderately higher than TP-3654, exhibiting in vitro activity at clinically relevant concentrations. Importantly, MEN combined with the standard of care RUX was synergistic, and molecular analyses confirmed the role of PIM downstream target inhibition. Our results support the therapeutic potential and relevance of MEN in MF treatment strategies. Citation Format: Maria Luisa Iannitto, Diego Bisignano, Sonia Zicari, Francesco Belli, Alessio Fiascarelli, Simone Talucci, Giuseppe Merlino, Vittoria Nicolis di Robilant, Clelia Irrissuto, Mario Bigioni, Alessandro Bressan, Daniela Bellarosa, Monica Binaschi. MEN1703/SEL24 exhibits promising antitumoral activity in preclinical models of myelofibrosis both as single agent and combined with ruxolitinib [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 665.

Abstract 2825: Experimental hyperthermic intraperitoneal chemotherapy in mice: Establishment of a novel ovarian cancer xenograft model

Abstract Introduction: The peritoneum is a common metastatic site for several abdominal cancers, including ovarian and colorectal cancer. Peritoneal metastasis is associated with poor prognosis and poor response to therapy. A potentially curative treatment is cytoreductive surgery (CRS) to remove all visible tumor tissue, followed by perfusion with hyperthermic intraperitoneal chemotherapy (HIPEC) to eliminate small residual tumors and free-floating tumor cells. Hyperthermia can enhance the cytotoxic effect of the drug, inhibit DNA repair and activate the immune system. At the current time, there is no standardized protocol for performing HIPEC, and there is a large variation regarding key parameters, such as choice of drug, drug concentration, treatment duration, carrier solution, volume and temperature. The impact of these parameters is unknown, and in vivo models can be a helpful tool to better understand some of these parameters to improve this treatment strategy. Methods: HIPEC was established in a murine xenograft model of ovarian cancer to evaluate the response to intraperitoneal perfusion of cisplatin and mitomycin C for 30 min at 41.5°C (HIPEC) and 37°C (NIPEC) compared to no treatment or intraperitoneal perfusion with saline at 41.5°C. The luciferase transfected B76 ovarian cancer cell line was injected intraperitoneally and treatment was performed on day 4 when the tumors were < 2 mm. Treatment efficacy was assessed by weekly luminescence measurement, tumor weight at an early time point (day 17), and overall survival. Results: Intraperitoneal perfusion with cisplatin or mitomycin C significantly inhibited tumor growth as assessed by luminescence, tumor weight on day 17, as well as increased overall survival of the mice compared to control treatment from 25-27 days to 36 days for cisplatin and 37 days for mitomycin C. No significant differences in tumor growth or survival were observed by the addition of hyperthermia. However, a slight reduction of tumor weight at day 17 was observed in the HIPEC groups. Conclusion: We have established a functional closed HIPEC model in mice bearing peritoneal metastases from the B76 ovarian cancer cell line which accurately mimics many features of the procedure in patients. Intraperitoneal perfusion of both cisplatin and mitomycin C efficaciously inhibited tumor growth and improved overall survival of the mice. Addition of hyperthermia caused an additional modest, temporary growth inhibition which did not translate into improved long-term outcome. Citation Format: Karianne Giller Fleten, Yvonne Andersson, Emil Løvstakken, Theodor M. Herud, Stein Waagene, Kjersti Flatmark. Experimental hyperthermic intraperitoneal chemotherapy in mice: Establishment of a novel ovarian cancer xenograft model [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 2825.

Abstract 5118: Neuroblastoma extracellular vesicles present GPC2 and activate GPC2 CAR T cells in an antigen-dependent manner

Abstract Glypican 2 (GPC2) chimeric antigen receptor (CAR) T cells are safe and efficacious in neuroblastoma and are currently being tested in a first-in-human Phase 1 clinical trial at the Children’s Hospital of Philadelphia. Tumor-derived extracellular vesicles (TEVs) are nanosized vesicles secreted by cancer cells, often enriched with glypicans. However, the presence of GPC2 on neuroblastoma TEVs and any interaction with GPC2 CAR T cells has not been explored. High-levels of GPC2 were found on TEVs isolated from neuroblastoma preclinical models [7 cell lines, circulating EVs from 6 patient-derived xenograft (PDX)-bearing mice] and from the peripheral blood of 16 neuroblastoma patients, but not on circulating EVs from 7 non-tumor bearing mice or 12 healthy human donors. The level of circulating GPC2+ EVs isolated from the peripheral blood of PDX-bearing mice positively correlated with PDX size (R=0.96; P<0.0001) and parent PDX GPC2 expression. To evaluate how GPC2 on EVs modulates CAR T cell functionality, we generated EVs with varied levels of GPC2 (Hi/Lo/Neg) and co-incubated each EV subset with GPC2 CAR T cells. Here using immunofluorescence and flow assays we found that EVs bind GPC2 CAR T cells proportional to the amount of GPC2 on their surface. Further, GPC2Hi EV-CAR T cell synapses induced potent T cell activation shown by CD69/Granzyme B expression and release of IL-2/IFN-γ. GPC2Hi EV pre-incubated CAR T cells also displayed enhanced target neuroblastoma cell cytotoxicity, inducing 4-fold more specific cytotoxicity than CAR T cells incubated with control GPC2Neg EVs. In vivo we observed that injection of GPC2Hi EVs into GPC2Lo neuroblastoma SK-N-AS xenografts genetically altered to have significantly decreased endogenous EV production (via RAB27A knock-out) greatly enhanced the efficacy of co-infused GPC2 CAR T cells (p<0.05). Similarly, in an isogenic SK-N-AS-GPC2 xenograft murine model treated with a limiting number of GPC2 CAR T cells concurrently with the EV-inhibiting drug GW4869, GPC2 CAR efficacy was dependent on the presence of GPC2Hi EVs. Here, pharmacologic EV inhibition in mice decreased GPC2 CAR efficacy which could be rescued by intratumoral injection of GPC2Hi EVs. In both in vivo models, the presence of intratumoral GPC2Hi EVs resulted in a significantly enhanced tumor infiltration of activated (CD69+/CD25+) GPC2 CAR T cells (p<0.05). Taken together, GPC2Hi EVs are selectively secreted from neuroblastomas and bind and activate GPC2 CAR T cells enhancing their anti-tumor cytotoxicity. To further capitalize on these findings, we engineered GPC2Hi EVs to have an extended circulation half-life through albumin binding or tumor targeting via GD2 binding that are currently being tested in vivo and results will be reported. EVs offer a versatile platform for CAR antigen presentation and should be further validated as a strategy to enhance CAR T cell efficacy for solid tumors. Citation Format: Anna M. Giudice, Stephanie Matlaga, Guillem Pascual-Pasto, Patrick M. Schuerch, Geoffrey Rouin, Brendan McIntyre, Vincent P. Zecchino, Kristopher R. Bosse. Neuroblastoma extracellular vesicles present GPC2 and activate GPC2 CAR T cells in an antigen-dependent manner [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 5118.

Abstract 690: Investigation of BH-3 mimetics as radiosensitizing agents in glioblastoma

Abstract Glioblastoma (GBM) is a highly aggressive primary brain tumor. Treatment consists of maximal safe tumor resection, chemotherapy (temozolomide) and radiotherapy; however, this aggressive treatment offers only a modest improvement in outcomes, with average life expectancy of approximately 12 months. Consequently, novel, effective and easily translatable therapies are urgently required. While radiotherapy is an effective treatment for many solid tumors, GBM displays an innate radio-resistance that contributes to treatment failure. Therefore, there is a need to identify chemotherapeutics that can be used as radiosensitizers to increase efficacy. Notably, a key pathway involved in therapeutic resistance is apoptotic evasion, which is driven by upregulation of pro-survival BCL-2 proteins that inhibit the pro-apoptotic proteins necessary for the apoptotic response. Recent advances in drug development have produced a range of BH3 mimetics - novel chemotherapeutics with potential to reinstate apoptosis by targeting specific pro-survival molecules. We hypothesized that the radiation sensitivity of GBM cells would be enhanced by using BH3 mimetics to inhibit pro-survival molecules and release the apoptotic block. A panel of BH3 mimetics was tested in combination with radiotherapy across three patient-derived GBM cell lines (G7, E2 and R15), using a 3D clonogenic culture system that recapitulates in vivo growth patterns and responses. We found that the BCL-XL inhibitor, A1331852, significantly radiosensitized all 3 GBM lines. In addition, Western blot analysis revealed A1331852 to induce PARP1 cleavage in G7 cells when used as a single agent. Subsequent in vivo studies were conducted in our well characterized orthotopic G7 xenograft murine model of GBM. Immunohistochemical studies showed that the pro-survival proteins MCL-1 and BCL-XL were upregulated in irradiated tumors. Furthermore, a pharmacokinetic study in the same orthotopic murine model, allowing for drug delivery evaluation, and optimized treatment scheduling. Importantly, A1331852 was found to be blood brain barrier penetrant at 25 mg/kg, with increased drug detected in tumor vs normal brain tissue. From these data, a survival study investigating A1331852 as a radiosensitizing agent was carried out in the same G7 GBM orthotopic xenograft model. Kaplan-Meier analysis showed an increase in survival of mice treated with A133852 plus radiotherapy compared with the radiotherapy only group (p = 0.0309). Overall, we have demonstrated that dependence on BCL-2 family proteins is a vulnerability that can be targeted to improve radiotherapy response in GBM. Furthermore, we have identified a promising BH3 mimetic, A1331852, to use in combination with RT and have completed a proof-of-concept in vivo study that confirms the potential of BH3 mimetics to improve outcomes for patients with GBM. Citation Format: Louise R. Dutton, Clara Mullen, Anna L. Koessinger, Mark Jackson, Katrina Stevenson, Anthony J. Chalmers, Stephen Tait, Kirsteen J. Campbell, Joanna L. Birch. Investigation of BH-3 mimetics as radiosensitizing agents in glioblastoma [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 690.

Abstract 7127: An ATM inhibitor: ZN-B-2262 in combination with radiation/ADCs containing topoisomerase inhibitors for the treatment of solid tumors

Abstract Background: ZN-B-2262 is a best-in-class, IND-approved, small molecule serine/threonine kinase receptor inhibitor targeting Ataxia Telangiectasia Mutated (ATM), which plays a key role in DNA damage response (DDR) associated with double-strand breaks (DSB). Radiotherapy (RT) and topoisomerase inhibition can directly cause DSB. ZN-B-2262 is expected to enhance the therapeutic effect of RT, or antibody drug conjugates (ADCs) containing topoisomerase inhibitors by suppressing DSB DNA repair and blocking checkpoint controls. Methods: The inhibition of isolated recombinant ATM, ATR, mTOR, DNA-PK, PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ enzymes was evaluated using In-Cell-Western assay to determine the effect of ZN-B-2262 on ATM enzyme activity by detecting phosp-KAP1 (Ser824) expression. The inhibition of phospho-ATM (Ser1981) was evaluated by ATM assay on HT29 cells using a High-Content Imaging System (HCS). Therapeutic efficacy, as measured by tumor growth inhibition (TGI), was evaluated using ZN-B-2262 in combination with DS-1062 (Trop2 ADC), irinotecan or RT in MDA-MB-468 triple negative breast cancer (TNBC), SW620 colorectal cancer, and Fadu head and neck squamous cell carcinoma (HNSCC) subcutaneous xenograft murine models, respectively. In addition, ZN-B-2262 was tested for Human Aldehyde Oxidase (AO) activity. Safety margins were determined based on the ratio of unbound AUC obtained from 28-day GLP animal toxicology studies and predicted human PK at efficacious dose. Results: ZN-B-2262 exhibited strong inhibition against ATM with the IC50 at 4.4 nM. No inhibition effect was observed on other IKK family enzymes with IC50 value >10 uM. ZN-B-2262 showed high inhibition activity on phosp-KAP1 with IC50 of 13.25 nM and on phospho-ATM with IC50 of 21.5 nM. In the MDA-MB-468 xenograft efficacy study, ZN-B-2262 combined with a clinically relevant dose of DS-1062 showed significantly superior tumor inhibition effects compared to DS-1062 alone. In the SW620 xenograft efficacy study, in combination with irinotecan, ZN-B-2262 showed synergistic tumor inhibition effects. In the Fadu xenograft efficacy study, ZN-B-2262 demonstrated synergistic anti-tumor activity when combined with RT. Sustained and enhanced anti-tumor efficacy was observed post withdrawal of ZN-B-2262 combined with RT, which was not observed after stopping treatment of RT alone. Based on 28-day GLP tox in rat and dog, the predicted human safety margin is 25- to 50-fold. Conclusion: ZN-B-2262, in combination with ADCs containing topoisomerase inhibitors, (e.g., Dato-Dxd), or RT, is expected to be a best-in-class (possibly first-in-class) compound, for the treatment of solid tumors. In particular, ZN-B-2262 should demonstrate improved PK and minimal inter-patient PK variability compared to current ATM clinical candidates since ZN-B-2262 is not an AO substrate. Citation Format: Ding Zhou, Zheng Wang, Yan (Pita) Liu, Tingting Fu, Ziqiang (Zack) Cheng. An ATM inhibitor: ZN-B-2262 in combination with radiation/ADCs containing topoisomerase inhibitors for the treatment of solid tumors [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 7127.

Dual-specificity tyrosine-regulated kinase 2 exerts anti-tumor effects by induction of G1 arrest in lung adenocarcinoma

ObjectivesLung cancer is a leading cause of cancer-related mortality and remains one of the most poorly prognosed disease worldwide. Therefore, it is necessary to identify novel molecular markers with potential therapeutic effects. Recent findings have suggested that dual-specificity tyrosine-regulated kinase 2 (DYRK2) plays a tumor suppressive role in colorectal, breast, and hepatic cancers; however, its effect and mechanism in lung cancer remain poorly understood. Therefore, this study aimed to investigate the tumor-suppressive role and molecular mechanism of DYRK2 in lung adenocarcinoma (LUAD) by in vitro experiments and xenograft models. Materials and methodsThe evaluation of DYRK2 expression was carried out using lung cancer cell lines and normal bronchial epithelial cells. Overexpression of DYRK2 was induced by an adenovirus vector, and cell proliferation was assessed through MTS assay and Colony Formation Assay. Cell cycle analysis was performed using flow cytometry. Additionally, proliferative capacity was evaluated in a xenograft model by subcutaneously implanting A549 cells into SCID mice (C·B17/Icr-scidjcl-scid/scid). ResultsImmunoblotting assays showed that DYRK2 was downregulated in most LUAD cell lines. DYRK2 overexpression using adenovirus vectors significantly suppressed cell proliferation compared with that in the control group. Additionally, DYRK2 overexpression suppressed tumor growth in a murine subcutaneous xenograft model. Mechanistically, DYRK2 overexpression inhibited the proliferation of LUAD cells via p21-mediated G1 arrest, which was contingent on p53. ConclusionTaken together, these findings suggest that DYRK2 may serve as potential prognostic biomarker and therapeutic target for LUAD.

Antigen Presenting Cell Mimetic Lipid Nanoparticles for Rapid mRNA CAR T Cell Cancer Immunotherapy.

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable clinical success in the treatment of hematological malignancies. However, producing these bespoke cancer-killing cells is a complicated ex vivo process involving leukapheresis, artificial T cell activation, and CAR construct introduction. The activation step requires the engagement of CD3/TCR and CD28 and is vital for T cell transfection and differentiation. Though antigen-presenting cells (APCs) facilitate activation in vivo, ex vivo activation relies on antibodies against CD3 and CD28 conjugated to magnetic beads. While effective, this artificial activation adds to the complexity of CAR T cell production as the beads must be removed prior to clinical implementation. To overcome this challenge, this work develops activating lipid nanoparticles (aLNPs) that mimic APCs to combine the activation of magnetic beads and the transfection capabilities of LNPs. It is shown that aLNPs enable one-step activation and transfection of primary human T cells with the resulting mRNA CAR T cells reducing tumor burden in a murine xenograft model, validating aLNPs as a promising platform for the rapid production of mRNA CAR T cells.

TREM1/DAP12 based novel multiple chain CAR-T cells targeting DLL3 show robust anti-tumour efficacy for small cell lung cancer.

Small cell lung cancer (SCLC), recognized as the most aggressive subtype of lung cancer, presents an extremely poor prognosis. Currently, patients with small cell lung cancer face a significant dearth of effective alternative treatment options once they experience recurrence and progression after first-line therapy. Despite the promising efficacy of immunotherapy, particularly immune checkpoint inhibitors in non-small cell lung cancer (NSCLC) and various other tumours, its impact on significantly enhancing the prognosis of SCLC patients remains elusive. DLL3 has emerged as a compelling target for targeted therapy in SCLC due to its high expression on the membranes of SCLC and other neuroendocrine carcinoma cells, with minimal to no expression in normal cells. Our previous work led to the development of a novel multiple chain chimeric antigen receptor (CAR) leveraging the TREM1 receptor and DAP12, which efficiently activated T cells and conferred potent cell cytotoxicity. In this study, we have developed a DLL3-TREM1/DAP12 CAR-T (DLL3-DT CAR-T) therapy, demonstrating comparable anti-tumour efficacy against SCLC cells in vitro. In murine xenograft and patient-derived xenograft models, DLL3-DT CAR-T cells exhibited a more robust tumour eradication efficiency than second-generation DLL3-BBZ CAR-T cells. Furthermore, we observed elevated memory phenotypes, induced durable responses, and activation under antigen-presenting cells in DLL3-DT CAR-T cells. Collectively, these findings suggest that DLL3-DT CAR-T cells may offer a novel and potentially effective therapeutic strategy for treating DLL3-expressing SCLC and other solid tumours.

Potential of targeting signal-transducing adaptor protein-2 in cancer therapeutic applications.

Adaptor proteins play essential roles in various intracellular signaling pathways. Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein that possesses pleckstrin homology (PH) and Src homology 2 (SH2) domains, as well as a YXXQ signal transducer and activator of transcription 3 (STAT3)-binding motif in its C-terminal region. STAP-2 is also a substrate of breast tumor kinase (BRK). STAP-2/BRK expression is deregulated in breast cancers and enhances STAT3-dependent cell proliferation. In prostate cancer cells, STAP-2 interacts with and stabilizes epidermal growth factor receptor (EGFR) after stimulation, resulting in the upregulation of EGFR signaling, which contributes to cancer-cell proliferation and tumor progression. Therefore, inhibition of the interaction between STAP-2 and BRK/EGFR may be a possible therapeutic strategy for these cancers. For this purpose, peptides that interfere with STAP-2/BRK/EGFR binding may have great potential. Indeed, the identified peptide inhibitor successfully suppressed the STAP-2/EGFR protein interaction, EGFR stabilization, and cancer-cell growth. Furthermore, the peptide inhibitor suppressed tumor formation in human prostate- and lung-cancer cell lines in a murine xenograft model. This review focuses on the inhibitory peptide as a promising candidate for the treatment of prostate and lung cancers.

Cytolethal Distending Toxin Modulates Cell Differentiation and Elicits Epithelial to Mesenchymal Transition.

The bacterial genotoxin, cytolethal distending toxin (CDT), causes DNA damage in host cells, a risk factor for carcinogenesis. Previous studies have shown that CDT induces phenotypes reminiscent of epithelial to mesenchymal transition (EMT), a process involved in cancer initiation and progression. We investigated different steps of EMT in response to Helicobacter hepaticus CDT and its active CdtB subunit using in vivo and in vitro models. Most of the steps of the EMT process were induced by CDT/CdtB and observed throughout the study in murine and epithelial cell culture models. CdtB induced cell-cell junction disassembly, causing individualization of cells and acquisition of a spindle-like morphology. The key transcriptional regulators of EMT (SNAIL and ZEB1) and some EMT markers were upregulated at both RNA and protein levels in response to CDT/CdtB. CdtB increased the expression and proteolytic activity of matrix metalloproteinases, as well as cell migration. A range of these results were confirmed in Helicobacter hepaticus-infected and xenograft murine models. In addition, colibactin, a genotoxic metabolite produced by Escherichia coli, induced EMT-like effects in cell culture. Overall, these data show that infection with genotoxin-producing bacteria elicits EMT process activation, supporting their role in tumorigenesis.

Radiolabeling Diaminosarcophagine with Cyclotron-Produced Cobalt-55 and [55Co]Co-NT-Sarcage as a Proof of Concept in a Murine Xenograft Model.

Cobalt-sarcophagine complexes exhibit high kinetic inertness under various stringent conditions, but there is limited literature on radiolabeling and in vivo positron emission tomography (PET) imaging using no carrier added 55Co. To fill this gap, this study first investigates the radiolabeling of DiAmSar (DSar) with 55Co, followed by stability evaluation in human serum and EDTA, pharmacokinetics in mice, and a direct comparison with [55Co]CoCl2 to assess differences in pharmacokinetics. Furthermore, the radiolabeling process was successfully used to generate the NTSR1-targeted PET agent [55Co]Co-NT-Sarcage (a DSar-functionalized SR142948 derivative) and administered to HT29 tumor xenografted mice. The [55Co]Co-DSar complex can be formed at 37 °C with purity and stability suitable for preclinical in vivo radiopharmaceutical applications, and [55Co]Co-NT-Sarcage demonstrated prominent tumor uptake with a low background signal. In a direct comparison with [64Cu]Cu-NT-Sarcage, [55Co]Co-NT-Sarcage achieved a higher tumor-to-liver ratio but with overall similar biodistribution profile. These results demonstrate that Sar would be a promising chelator for constructing Co-based radiopharmaceuticals including 55Co for PET and 58mCo for therapeutic applications.

CircSEPT9 promotes breast cancer progression by regulating PTBP3 expression via sponging miR-625-5p.

Breast cancer (BC) is a common malignancy which threatens the health of women. Circular RNAs (circRNAs) are critical factors in multiple cancers, including BC. The aim of this experiment was to investigate the molecular mechanisms of circRNA Septin 9 (circSEPT9) in the progression of BC. CircSEPT9, microRNA-625-5p (miR-625-5p) and polypyrimidine tract-binding protein 3 (PTBP3) levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was performed to detect the protein levels of PTBP3, E-cadherin and vimentin. Cell counting kit-8 assay (CCK8) and thymidine analog 5-ethynyl-2'-deoxyuridine (EDU) was utilized for proliferation examination. Flow cytometry was conducted to measure apoptosis. Transwell assay and wound healing assay to investigate the migration of BC cells. Glucose uptake and lactate production were determined by specific kits. Additionally, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were utilized to verify the interaction. A murine xenograft model was established to investigate the function of circSEPT9 in BC in vivo. Overexpression of circSEPT9 was found in BC tissues and cells. Silencing circSEPT9 impeded BC cell proliferation, migration, epithelial-mesenchymal transition (EMT) and glycolytic metabolism but facilitated cell apoptosis in vitro. Meanwhile, circSEPT9 knockdown constrained tumor growth in vivo. MiR-625-5p was targeted by circSEPT9. The influence of silencing circSEPT9 on BC cell function was regained by miR-625-5p inhibitor. Furthermore, miR-625-5p regulated BC cell malignant phenotypes via downregulating PTBP3. circSEPT9 contributed to the malignant progression of BC by up-regulating PTBP3 via sponging miR-625-5p.

Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner.

Our findings unveiled that iNKT cells recognize and kill CD1d-negative target tumors via the anti-iNKT TCR mAb bound to CD32 at the tumor site, thereby bridging iNKT cells and CD1d-negative tumors. These findings shed light on the therapeutic potential of anti-iNKT TCR mAbs in NKT cell-based immunotherapy to treat CD1d-negative CD32+ cancers.