Tumor Growth Inhibition Research Articles

Engineered Cellular Vesicles Displaying Glycosylated Nanobodies for Cancer Immunotherapy

AbstractImmune checkpoint blockade targeting the CD47/SIRPα axis represents an alluring avenue for cancer immunotherapy. However, the compromised efficacy and safety concerns in vivo of conventional anti‐CD47 antibodies impede their wide clinical applications. Here we introduced a single type of high‐mannose glycans into the nanobody against CD47 (HM‐nCD47) and subsequently displayed HM‐nCD47 on cellular vesicles (CVs) for enhanced cancer immunotherapy. In this platform, the CVs significantly improved the circulation time of HM‐nCD47‐CVs, the nCD47 enabled the blockade of the CD47/SIRPα axis, and the HM enhanced recognition of mannose‐binding lectin, all synergistically activating the macrophage‐mediated antitumor immunity. In both subcutaneous and metastatic murine tumor models, the HM‐nCD47‐CVs possessed significantly extended half‐lives and increased accumulation at the tumor site, resulting in a remarkable macrophage‐dependent inhibition of tumor growth, a transcriptomic remodeling of the immune response, and an increase in survival time. By integrating the chemical biology toolbox with cell membrane nanotechnology, the HM‐nCD47‐CVs represent a new immunotherapeutic platform for cancer and other diseases.

Senolytic effect of triterpenoid complex from Ganoderma lucidum on adriamycin-induced senescent human hepatocellular carcinoma cells model in vitro and in vivo.

Ganoderma lucidum (G. lucidum) is a famous medicinal mushroom that has been reported to prevent and treat a variety of diseases. Different extractions from G. lucidum have been used to manage age-related diseases, including cancer. Nevertheless, the senolytic activity of G. lucidum against senescent cancer cells has not been investigated. Although cellular senescence causes tumor growth inhibition, senescent cells promote the growth of the neighboring tumor cells through paracrine effects. Therefore, the elimination of senescent cells is a new strategy for cancer treatment. In this study, senescence was triggered in HCC cells by the chemotherapeutic agent Adriamycin (ADR), and subsequently, cells were treated with TC to assess its senolytic activity. We found for the first time that the triterpenoid complex (TC) from G. lucidum had senolytic effect, which could selectively eliminate adriamycin (ADR)-induced senescent cells (SCs) of hepatocellular carcinoma (HCC) cells via caspase-dependent and mitochondrial pathways-mediated apoptosis and reduce the levels of senescence markers, thereby inhibiting the progression of cancers caused by SCs. TC could block autophagy at the late stage in SCs, resulting in a significant activation of TC-induced apoptosis. Furthermore, TC inhibited the senescence-associated secretory phenotype (SASP) in SCs through the inhibition of NF-κB, TFEB, P38, ERK, and mTOR signaling pathways and reducing the number of SCs. Sequential administration of ADR and TC in vivo significantly reduced tumor growth and reversed the toxicity of ADR. A triterpenoid complex isolated from G. lucidum may serve as a novel senolytic agent against SCs, and its combination with chemotherapeutic agents may enhance their antitumor efficacy.

Design, Synthesis, and Biological Evaluation of Lysine-Stapled Peptide Inhibitors of p53-MDM2/MDMX Interactions with Potent Antitumor Activity In Vivo.

We introduce novel lysine-stapled peptide inhibitors targeting p53-MDM2/MDMX interactions. Leveraging the model peptides pDI (LTFEHYWAQLTS) and PMI-M3 (LTFLEYWAQLMQ) as starting points, a series of lysine-stapled analogues were designed and synthesized. Through in vitro cell assay screening, two lead compounds, SPDI-48-T1 and SPMI-48-T3, were identified for their excellent antiproliferation activity. Fluorescence polarization assays revealed that both compounds exhibited strong binding affinities against MDM2 and MDMX, ascertained by Kd values within the low micromolar spectrum. Further characterization of SPDI-48-T1 and SPMI-48-T3 demonstrated that SPDI-48-T1 possessed superior cell permeability and serum stability. Notably, SPDI-48-T1 displayed a dose-dependent suppression of tumor growth in an HCT116 xenograft mouse model. Our findings indicate that SPDI-48-T1 holds promise as a lead compound for further development as an anticancer agent by modulating p53-MDM2/MDMX interactions. Additionally, this study also proved that the lysine stapling strategy may serve as a robust approach for generating peptide ligands targeting other protein-protein interactions.

Anticancer Tetranuclear Cu(I) Complex Catalyzes a Click Reaction to Synthesize a Chemotherapeutic Agent in situ to Achieve Targeted Dual-Agent Combination Therapy for Cancer.

To develop next-generation metal-based drugs and dual-drug combination therapy for cancer, we proposed to develop a copper (Cu) complex that exerts anticancer function by integrating chemotherapy, immunotherapy and catalyzes a click reaction for the in situ synthesis of a chemotherapeutic agent, thereby achieving targeted dual-agent combination therapy. We designed and synthesized a tetranuclear Cu(I) complex (Cu4) with remarkable cytotoxicity and notable catalytic ability for the in situ synthesis of a chemotherapeutic agent via Cu(I)-catalyzed azide-alkyne 1,3-cycloaddition (CuAAC). We also constructed an apoferritin (AFt)-Cu4 nanoparticles (NPs) delivery system. AFt-Cu4 NPs not only showed an enhanced performance of tumor growth inhibition, but also improved the targeting ability and reduced the systemic toxicity of Cu4 in vivo. Importantly, the anticancer effect was enhanced by combining the AFt-Cu4 NPs with the resveratrol analogue obtained from the CuAAC reaction in situ. Finally, we revealed the anticancer mechanism of the Cu4/AFt-Cu4 NPs, which involves both cuproptosis and cuproptosis-induced systemic immune response.

Anticancer Tetranuclear Cu(I) Complex Catalyzes a Click Reaction to Synthesize a Chemotherapeutic Agent in situ to Achieve Targeted Dual‐Agent Combination Therapy for Cancer

To develop next‐generation metal‐based drugs and dual‐drug combination therapy for cancer, we proposed to develop a copper (Cu) complex that exerts anticancer function by integrating chemotherapy, immunotherapy and catalyzes a click reaction for the in situ synthesis of a chemotherapeutic agent, thereby achieving targeted dual‐agent combination therapy. We designed and synthesized a tetranuclear Cu(I) complex (Cu4) with remarkable cytotoxicity and notable catalytic ability for the in situ synthesis of a chemotherapeutic agent via Cu(I)‐catalyzed azide‐alkyne 1,3‐cycloaddition (CuAAC). We also constructed an apoferritin (AFt)‐Cu4 nanoparticles (NPs) delivery system. AFt‐Cu4 NPs not only showed an enhanced performance of tumor growth inhibition, but also improved the targeting ability and reduced the systemic toxicity of Cu4 in vivo. Importantly, the anticancer effect was enhanced by combining the AFt‐Cu4 NPs with the resveratrol analogue obtained from the CuAAC reaction in situ. Finally, we revealed the anticancer mechanism of the Cu4/AFt‐Cu4 NPs, which involves both cuproptosis and cuproptosis‐induced systemic immune response.

Relationship of Signaling Pathways between RKIP Expression and the Inhibition of EMT-Inducing Transcription Factors SNAIL1/2, TWIST1/2 and ZEB1/2.

Untreated primary carcinomas often lead to progression, invasion and metastasis, a process that involves the epithelial-to-mesenchymal transition (EMT). Several transcription factors (TFs) mediate the development of EMT, including SNAIL1/SNAIL2, TWIST1/TWIST2 and ZEB1/ZEB2, which are overexpressed in various carcinomas along with the under expression of the metastasis suppressor Raf Kinase Inhibitor Protein (RKIP). Overexpression of RKIP inhibits EMT and the above associated TFs. We, therefore, hypothesized that there are inhibitory cross-talk signaling pathways between RKIP and these TFs. Accordingly, we analyzed the various properties and biomarkers associated with the epithelial and mesenchymal tissues and the various molecular signaling pathways that trigger the EMT phenotype such as the TGF-β, the RTK and the Wnt pathways. We also presented the various functions and the transcriptional, post-transcriptional and epigenetic regulations for the expression of each of the EMT TFs. Likewise, we describe the transcriptional, post-transcriptional and epigenetic regulations of RKIP expression. Various signaling pathways mediated by RKIP, including the Raf/MEK/ERK pathway, inhibit the TFs associated with EMT and the stabilization of epithelial E-Cadherin expression. The inverse relationship between RKIP and the TF expressions and the cross-talks were further analyzed by bioinformatic analysis. High mRNA levels of RKIP correlated negatively with those of SNAIL1, SNAIL2, TWIST1, TWIST2, ZEB1, and ZEB2 in several but not all carcinomas. However, in these carcinomas, high levels of RKIP were associated with good prognosis, whereas high levels of the above transcription factors were associated with poor prognosis. Based on the inverse relationship between RKIP and EMT TFs, it is postulated that the expression level of RKIP in various carcinomas is clinically relevant as both a prognostic and diagnostic biomarker. In addition, targeting RKIP induction by agonists, gene therapy and immunotherapy will result not only in the inhibition of EMT and metastases in carcinomas, but also in the inhibition of tumor growth and reversal of resistance to various therapeutic strategies. However, such targeting strategies must be better investigated as a result of tumor heterogeneities and inherent resistance and should be better adapted as personalized medicine.

Modeling serum M-protein response for early detection of biochemical relapse in myeloma patients treated with bortezomib, lenalidomide and dexamethasone.

Multiple myeloma (MM) treatment guidelines recommend waiting for formal progression criteria (FPC) to be met before proceeding to the next line of therapy. As predicting progression may allow early switching to next-line therapy while the disease burden is relatively low, we evaluated the predictive accuracy of a mathematical model to anticipate relapse 180 days before the FPC is met. A subset of 470/1143 patients from the IA16 dataset who were initially treated with VRd (Velcade (bortezomib), Revlimid (lenalidomide), and dexamethasone) in the CoMMpass study (NCT01454297) were randomly split 2:1 into training and testing sets. A model of M-protein dynamics was developed using the training set and used to predict relapse probability in patients in the testing set given their response histories up to 12 or more months of treatment. The predictive accuracy of this model and M-protein "velocity" were assessed via receiver operating characteristics (ROC) analysis. The final model was a two-population tumor growth inhibition model with additive drug effect and transit delay compartments for cell killing. The ROC area under the curve value of relapse prediction 180 days ahead of observed relapse by FPC was 0.828 using at least 360 days of response data, which was superior to the M-protein velocity ROC score of 0.706 under the same conditions. The model can predict future relapse from early M-protein responses and can be used in a future clinical trial to test whether early switching to second-line therapy results in better outcomes in MM.

The identification of potent dual-target monopolar spindle 1 (MPS1) and histone deacetylase 8 (HDAC8) inhibitors through pharmacophore modeling, molecular docking, molecular dynamics simulations, and biological evaluation.

Overexpression of monopolar spindle 1 (MPS1) and histone deacetylase 8 (HDAC8) is associated with the proliferation of liver cancer cells, so simultaneous inhibition of both MPS1 and HDAC8 could offer a promising therapeutic approach for the treatment of liver cancer. Dual-targeted MPS1/HDAC8 inhibitors have not been reported. A combined approach of pharmacophore modeling and molecular docking was used to identify potent dual-target inhibitors of MPS1 and HDAC8. Enzyme inhibition assays were performed to evaluate the optimal compound with the strongest inhibitory activity against MPS1 and HDAC8. The selectivity of MPH-5 for MPS1 and HDAC8 was assessed on a panel of 68 kinases and other histone deacetylases. Subsequently, molecular dynamics (MD) simulation verified the binding stability of the optimal compound to MPS1 and HDAC8. Ultimately, in vitro cellular assays and in vivo antitumor assays evaluated the antitumor efficacy of the most promising compound for the treatment of hepatocellular carcinoma. Six dual-target compounds (MPHs 1-6) of both MPS1 and HDAC8 were identified from the database using a combined virtual screening protocol. Notably, MPH-5 showed nanomolar inhibitory effect on both MPS1 (IC50 = 4.52 ± 0.21nM) and HDAC8 (IC50 = 6.07 ± 0.37nM). MD simulation indicated that MPH-5 stably binds to both MPS1 and HDAC8. Importantly, cellular assays revealed that MPH-5 exhibited significant antiproliferative activity against human liver cancer cells, especially HepG2 cells. Moreover, MPH-5 exhibited low toxicity and high efficacy against tumor cells, and it overcomes drug resistance to some extent. In addition, MPH-5 may exert its antitumor effects by downregulating MPS1-driven phosphorylation of histone H3 and upregulating HDAC8-mediated K62 acetylation of PKM2. Furthermore, MPH-5 showed potent inhibition of HepG2 xenograft tumor growth in mice with no apparent toxicity and presented favorable pharmacokinetics. The study suggests that MPH-5 is a potent, selective, high-efficacy, and low-toxicity antitumor candidate for the treatment of hepatocellular carcinoma.

Potent combination benefit of the AKT inhibitor capivasertib and the BCL-2 inhibitor venetoclax in diffuse large B cell lymphoma.

The therapeutic potential of targeting PI3K/AKT/PTEN signalling in B-cell malignancies remains attractive. Whilst PI3K-α/δ inhibitors demonstrate clinical benefit in certain B-cell lymphomas, PI3K signalling inhibitors have been inadequate in relapsed/refractory diffuse large B-cell lymphoma (DLBCL) in part, due to treatment related toxicities. Clinically, AKT inhibitors exhibit a differentiated tolerability profile offering an alternative approach for treating patients with B-cell malignancies. To explore how AKT inhibition complements other potential therapeutics in the treatment of DLBCL patients, an in vitro combination screen was conducted across a panel of DLCBL cell lines. The AKT inhibitor, capivasertib, in combination with the BCL-2 inhibitor, venetoclax, produced notable therapeutic benefit in preclinical models of DLBCL. Capivasertib and venetoclax rapidly induced caspase and PARP cleavage in GCB-DLBCL PTEN wildtype cell lines and those harbouring PTEN mutations or reduced PTEN protein, driving prolonged tumour growth inhibition in DLBCL cell line and patient derived xenograft lymphoma models. The addition of the rituximab further deepened the durability of capivasertib and venetoclax responses in a RCHOP refractory DLBCL in vivo models. These findings provide preclinical evidence for the rational treatment combination of AKT and BCL-2 inhibitors using capivasertib and venetoclax respectively alongside anti-CD20 antibody supplementation for treatment of patients with DLBCL.

Synthesis of targeted doxorubicin-loaded gold nanorod −mesoporous manganese dioxide core–shell nanostructure for ferroptosis, chemo-photothermal therapy in vitro and in vivo

In the current study, a core–shell inorganic nanostructure comprising a gold nanorod core and −mesoporous manganese dioxide shell was synthesized. Then, the mesoporous manganese dioxide shell was loaded with doxorubicin (DOX) and then coated with pluronic F127 and pluronic F127-folic acid conjugate (1.5:1 wt ratio of pluronic F127: pluronic F127-folic acid conjugate) to prepare targeted final platform. In this design, mesoporous manganese dioxide acted as a reservoir for DOX loading, anti-hypoxia, and MRI contrast agent, while the gold nanorod core acted as a photothermal and CT scan imaging agent. DOX was encapsulated in the mesoporous manganese dioxide shell with a loading capacity and loading efficiency of 19.8 % ± 0.2 and 99.0 % ± 0.9, respectively. The in vitro release experiment showed the impact of glutathione (GSH), mildly acidic pH, and laser irradiating toward accelerated stimuli-responsive DOX release. The ·OH production of the prepared platform was verified by methylene blue (MB) decomposition reaction. Furthermore, thermal imaging exhibited the ability of the prepared platform to convert the NIR irradiation to heat. In vitro cytotoxicity tests on the folate receptor-positive 4 T1 cell line revealed the remarkable cytotoxicity of the targeted formulation compared to the nontargeted formulation (statistically significant). The MTT experiment demonstrated that exposure to laser 808 irradiation enhanced cytotoxicity of the targeted formulation (p < 0.0001). The production of ROS in 4 T1 cells following treatment with the targeted formulation was demonstrated by the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. Furthermore, in vivo investigations by implementing subcutaneous 4 T1 tumorized female BABL/c mice indicated that the prepared platform was an effective system in suppressing tumor growth by combining chemotherapy with PTT (photothermal therapy). Additionally, simultanous PTT and anti-hypoxic activity of this system showed potent tumor growth suppression impact. The percent of tumor size reduction in mice treated with FA-F127-DOX@Au-MnO2 + 808 nm laser compared to the control group was 99.7 %. The results of the biodistribution investigation showed tumor accumulation and modified pharmacokinetics of the targeted system. Lastly, 6 and 24 h post-intravenous injection, CT-scan and MR imagings capability of the prepared platform was verified in preclinical stage. The prepared multipurpose system introduces great opportunity to provide multiple treatment strategy along with multimodal imaging capability in a single platform for breast cancer treatment.

Abstract B012: Randomized phase II trial of two different nutritional approaches for patients receiving treatment for their advanced pancreatic cancer: initial results of safety and feasibility of the medically supervised ketogenic diet

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by stromal fibrosis, hypoxia, and nutritional deprivation. Nutritional metabolic interventions may fundamentally change the tumor microenvironment and improve outcomes. A ketogenic diet (KD) (lower carbohydrate, moderate protein, higher fat) can significantly reduce glucose and insulin and stimulate ketogenesis. Triplet chemotherapy with gemcitabine/cisplatin/nab-paclitaxel has been associated with a median OS 16.4 months, PFS 10.1 months in patients with metastatic PDAC in the first-line setting. Recently, KD plus triplet chemotherapy was shown to inhibit murine pancreatic KPC tumor growth and prolong animal survival over chemotherapy alone (Yang et al, Med, 2022). Tumor growth inhibition was associated with glucose depletion, altered TCA substrate use and NADH elevation. Methods: In this Phase II trial, patients with treatment-naive metastatic PDAC were randomized to triplet chemotherapy with a medically supervised ketogenic diet (MSKD) or continuing their usual diet (non-MSKD). The MSKD is supported by Virta Health, who offers tracking of daily ketone (beta-hydroxybutyrate, BHB) and glucose levels, a web-based application, education, and communication with a remote care team to ensure sustained nutritional ketosis (BHB 0.5–3.0mM). Patients with BMI &amp;lt; 18, type 1 DM or history of DKA were excluded. Primary endpoint was PFS. Secondary endpoints: DCR (PR+CR+SD ≥ 9 weeks), change in CA 19-9 (CA125, CEA if not CA 19-9 expressers), average fasting insulin levels, HbA1c, body weight, gut microbiome, serum metabolites, QOL (QLQ- C30). Results: A total of 36 patients with untreated metastatic PDAC were enrolled. Among 32 evaluable patients (median age 65.9 years; 53% male), 16 were randomized to each arm. Both groups had similar baseline weight, HgbA1c, insulin and BHB levels (all p&amp;gt;0.05). Nine patients had type 2 DM; 6 required insulin. In the MSKD group, 15/16 achieved nutritional ketosis at any point during the study, with mean BHB of 0.57 mM (95% CI 0.40–0.73) and median proportion of days in ketosis of 39.4% (range 0-95.8%) by daily ketone tracking. Lab data showed significantly higher mean ± SD maximum change in BHB in the MSKD (0.5 ± 0.9 mmol/L) versus non-MSKD (–0.2 ± 0.3 mol/L) group (p = 0.018). There were no significant differences between groups in maximum change in weight, HbA1C, or insulin (all p &amp;gt; 0.05). The most common MSKD-related adverse events were Gr 1-2 fatigue (n=4), constipation (n=3), weight loss (n=3), and none stopped MSKD due to adverse events.Conclusion: A medically supervised ketogenic diet is feasible in patients with metastatic PDAC receiving triplet chemotherapy, with median proportion of days in ketosis of 39.4% and an acceptable safety profile. The MSKD is associated with comparable changes in insulin and HgbA1c levels compared to the non-MSKD diet, and sustained ketosis does not result in significantly greater weight loss. MSKD warrants further prospective exploration and analyses of the primary endpoint and correlative studies are ongoing. Citation Format: Gayle S Jameson, Erkut Borazanci, Diana L Hanna, Caroline GP Roberts, Meredith S Pelster, Richard C Frank, Angela T Alistar, Julia E Wiedmeier-Nutor, Sandra D Algaze, Brandon Fell, Sarah J Hallberg, Denise J Roe, Betsy C Wertheim, Derek Cridebring, Joshua D Rabinowitz, Stephen Gately, Daniel D Von Hoff, Drew W Rasco. Randomized phase II trial of two different nutritional approaches for patients receiving treatment for their advanced pancreatic cancer: initial results of safety and feasibility of the medically supervised ketogenic diet [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B012.

Abstract C025: A novel pan-RAS/β-catenin inhibitor, ADT-030, produces tumor regression in PDX models of pancreatic cancer

Abstract BACKGROUND: The absence of symptoms often leads to late diagnosis of pancreatic ductal adenocarcinoma (PDAC) limiting treatment options. Tumor cell proliferation and invasion/metastasis are usually driven by KRAS mutations and activation of Wnt/β-catenin pathway components. The recent approval of KRASG12C-specific inhibitors for lung cancer has validated RAS as a druggable target but has had limited application for PDAC given the rarity of this codon in PDAC. In addition, no Wnt/β-catenin inhibitors have been FDA approved. Thus, drugs which inhibit these important targets are urgently needed for PDAC and other cancers. Here, we describe treatment results with ADT-030, a novel dual pan-RAS/β-catenin inhibitor in murine models of PDAC and lung cancer. METHODS: In vitro assays of growth inhibition, colony formation, apoptosis, and cell cycle, along with western blotting for RAS signaling were performed to assess potency and selectivity of ADT-030. Murine 2838c3-Luc KRASG12D PDAC cells were implanted orthotopically into the pancreas of C57BL/6J mice and cells from two PDAC patient-derived xenograft (PDX) models harboring KRASG12D or KRASG12C mutations were implanted subcutaneously (SC) in NSG mice to evaluate the antitumor activity of ADT-030. ADT-030 was administered once daily by gavage 5x/week for 4 weeks. RESULTS: ADT-030 potently inhibited the growth of PDAC cells in vitro harboring the most prevalent KRASG12D mutation as well as those with KRASG12C. Annexin V assay revealed the capacity of ADT-030 to trigger apoptosis in KRAS mutant cells with minimal impact on BxPC3 RASWT cells. Cell cycle analysis using propidium iodide staining revealed that ADT-030 arrested cells in the G2/M phase. Other experiments showed that ADT-030 simultaneously suppressed both MAPK/AKT signaling and β-catenin transcriptional by inhibiting PDE10 and activating cGMP/PKG signaling. ADT-030 caused tumor regression in SC PDAC PDX mouse tumor models harboring either KRASG12D or KRASG12C mutations without discernable toxicity. Inhibition of orthotopic PDAC tumor growth by ADT-030 was dose-dependent, causing up to 80% reduction in tumor volume following a 4-week treatment period. In addition, ADT-030 significantly extended survival and yielded a high percentage of cures after stopping treatment in a murine model of KRAS- mutant lung cancer. CONCLUSION: ADT-030, novel dual pan-RAS/β-catenin inhibitor, showed strong and durable antitumor activity in clinically relevant and aggressive murine models of PDAC and lung cancer. ADT-030 is a promising development candidate for treatment of PDAC and other RAS-driven cancers, particularly given the complex KRAS mutation landscape of many cancers. Citation Format: Dhana Sekhar Reddy Bandi, Ganji P Nagaraju, Jeremy B Foote, Adam B Keeton, Xi Chen, Kristy L Berry, Yulia Y Maxuitenko, Upender Manne, Donald J Buchsbaum, Gary A Piazza, Bassel F El-Rayes. A novel pan-RAS/β-catenin inhibitor, ADT-030, produces tumor regression in PDX models of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C025.

Abstract A052: Silencing MICAL2 Expression in Pancreatic Cancer Cells rewires the tumor microenvironment through the IL1-a/p38 MAP kinase/STAT-3 axis and Sensitizes Tumors to Immune Checkpoint Blockade therapy

Abstract Introduction: PDAC is marked by a fibroinflammatory stroma and thrives on interactions between cancer cells and their microenvironment, including CAFs and immune cells, driving disease progression and treatment resistance. Using ChIP-seq on resected human PDAC samples, we identified MICAL2 as a super-enhancer-associated gene and its role in tumor progression. MICAL2 is a flavin monooxygenase that promotes actin depolymerization and SRF transcription. Here, we evaluated how tumor cell-expressed MICAL2 rewires the PDAC microenvironment through the IL1-a/p38 MAP kinase/STAT3 axis. Methods: KPC-Shcontrol (ShCNT) and MICAL2 (M2KD) were orthotopically injected to assess tumor growth. Sc-RNA seq, immunophenotyping, and immunohistochemistry were performed on ShCNT and M2KD tumors. Atomic force microscopy was done to assess tissue stiffness. CD8+ specific antibodies were used for T-cell depletion. Adoptive transfer of CD8+ T cells enriched from M2KD tumors was performed by tail vein injection. Anti-PD1 and anti-IL-1a antibodies were injected IP. RNA-Seq analysis was performed on human PDAC cells (AsPC-1 ShCNT and M2KD) and validation of IL-1α and IL-1R expression was done in human and mouse pancreas control, KD, OE cancer cell lines, and PSCs by q-PCR. We exposed PSCs to conditioned media from cancer cells with and without MICAL2 and checked the expression of genes related to inflammation and fibrosis by qPCR. Results: Orthotopic injections of KPC-M2KD cells led to decreased tumor growth compared to ShCNT (0.26 gm vs. 1 gm, p = 0.008). Sc-RNA and immunohistochemistry revealed reduced PDPN+ and a-SMA+ CAFs in M2KD tumors. We also observed less collagen and fibronectin deposition in M2KD tumors resulting in reduced tissue stiffness as measured by atomic force microscopy. Flow cytometry and immunofluorescence showed increased intertumoral infiltration of cytotoxic and effector CD8+T cells in M2KD tumors. CD8+T cell depletion reversed growth inhibition in M2KD tumors. Adoptive transfer of CD8+T cells enriched from M2KD tumors impeded control tumor growth. RNA-seq revealed decreased IL1-α expression in M2KD cells (p&amp;lt;0.05). IL1R expression was reduced on PSCs when cocultured with M2KD vs. control cell media. PSCs co-cultured with M2KD cancer cells also showed significant downregulation of genes including IL-6, Cxcl1, and LIF changes that were rescued by adding recombinant IL-1α in M2KD cells. Western blot showed MICAL2 regulates the phosphorylation of p38 MAP kinase which in turn regulates the expression of IL1-α and STAT3 activation confirmed by treating dox-inducible ShCNT and ShM2KD cancer cells with p38 inhibitor. Treating M2KD tumors with immune checkpoint blockade PD-1 alone significantly reduced tumor size and 50% of M2KD tumor-bearing mice had complete tumor regression after treatment with PD-1 and neutralizing IL-1a antibodies. Conclusion: MICAL2 mediates tumor-stromal crosstalk through the IL1-a/p38/STAT3 axis and creates an immunosuppressive environment in PDAC. MICAL2 may be a potential immunomodulatory target for pancreatic cancer therapy. Citation Format: Bharti Garg, Evangeline Mose, Edgar Esparaza, Jay Patel, Rithika Medari, Alexei Martsinkovskiy, Carrie Bishop, Gissele Gonzalez, Adam Engler, Parag Katira, Herve Tiriac, Andrew M Lowy. Silencing MICAL2 Expression in Pancreatic Cancer Cells rewires the tumor microenvironment through the IL1-a/p38 MAP kinase/STAT-3 axis and Sensitizes Tumors to Immune Checkpoint Blockade therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A052.

Binary proton therapy of Ehrlich carcinoma using targeted gold nanoparticles

Proton therapy can treat tumors located in radiation-sensitive tissues. This article demonstrates the possibility of enhancing the proton therapy with targeted gold nanoparticles that selectively recognize tumor cells. Au-PEG nanoparticles at concentrations above 25 mg/L and 4 Gy proton dose caused complete death of EMT6/P cells in vitro. Binary proton therapy using targeted Au-PEG-FA nanoparticles caused an 80% tumor growth inhibition effect in vivo. The use of targeted gold nanoparticles is promising for enhancing the proton irradiation effect on tumor cells and requires further research to increase the therapeutic index of the approach.

Abstract B058: Rewiring CD8+ T cell responses to PD-1 immune checkpoint blockade in PDAC via the inhibitory receptor PSGL-1

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, poorly immunogenic cancer and non-responsive to all currently available treatments, including immune checkpoint blockade (ICB) therapies. As survival rates remain low (13%), there is an urgent need for new therapeutic options. A hallmark of PDAC is the limited infiltration of anti-tumor cytotoxic CD8+ T cells. Analysis of single-cell RNA-sequencing data of PDAC tumors from treatment naïve patients reveals high expression of P-selectin glycoprotein ligand 1 (PSGL-1) on tumor-infiltrating CD8+ T cells. PSGL-1 is an intrinsic negative regulator of CD8+ T cells that promotes the development of functional exhaustion. We therefore hypothesized that PSGL-1-mediated immune suppression is a critical barrier to effective anti-tumor immunity in PDAC. To test this, we used preclinical models of orthotopic injection PDAC tumor cells into pancreata, recapitulating patient responses to PDAC with limited T cell infiltrates and uncontrolled tumor growth in C57BL/6 (wildtype) control mice and compared anti-tumor immune responses in PSGL-1KO (C57BL/6 background) mice. At 28 days post-injection of KPC.4662 PDAC tumor cells, tumor burden in PSGL-1KO mice was reduced by &amp;gt;50% compared to controls, with 45% of PSGL-1KO animals exhibiting &amp;gt;70% reduction. Total immune infiltration (CD45+) was 2-fold greater in tumors from PSGL-1KO mice, with significant increases in infiltrating CD4+ and CD8+ T cells. Detailed analyses revealed that infiltrating CD8+ T cells from PSGL-1KO mice were less terminally differentiated towards exhaustion. PSGL-1KO PD-1+ CD8+ T cells expressed less TOX expression and fewer cells co-expressed CD38 and CD101 while more expressed CX3CR1 compared to controls. Intratumoral PSGL-1KO CD8+ T cells also retained greater functionality as well as indicated by inflammatory cytokine production (IFNg, TNFa) and Granzyme B expression. Moreover, PSGL-1KO mice showed protection against metastatic disease to the lungs. We find that protection by PSGL-1KO mice is T cell dependent as transient depletion of CD4+ and CD8+ T cells prior to tumor cell implantation results in the loss of PDAC tumor growth inhibition by PSGL-1KO mice. Less-differentiated, proliferation-competent precursors of exhausted CD8+ T cells (TPEX) are essential to PD-1 ICB responsiveness in patients and preclinical models. Given that PSGL-1-deficiency limits terminal differentiation of CD8+ T cells in PDAC, we hypothesized that PSGL-1-deficiency would promote responsiveness to PD-1 ICB, which is ineffective as a monotherapy in PDAC patients and mouse models. While therapeutic treatment with anti-PD-1 had no effect on the tumor growth in the control animals, PD-1 ICB resulted in tumor ablation in 85% of PSGL-1KO animals, demonstrating a profound synergistic effect of PD-1 inhibition in the absence of PSGL-1. From these studies, we conclude that PSGL- 1 is a critical inhibitor anti-tumor T cell immunity in PDAC and represents a novel ICB target with high translational potential for promoting immune responses to PDAC tumors. Citation Format: Jennifer L Hope, Yijuan Zhang, Hannah A. F. Hetrick, Evelyn S. Sanchez Hernandez, Gabriele Romano, Sreeja Roy, Michelle Lin, Ashley B Palete, Swetha Maganti, Dennis C Otero, Katelyn T Byrne, Cosimo Commisso, Linda M Bradley. Rewiring CD8T cell responses to PD-1 immune checkpoint blockade in PDAC via the inhibitory receptor PSGL-1 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B058.

X-ray triggered scintillator versatile nanocatalytic platform for synergistic photodynamic/chemodynamic therapy

Nanocatalysts with photodynamic therapy (PDT) and chemodynamic therapy (CDT) are excellent for tumor therapy. However, it is still challenging to achieve complete tumor eradication due to the drawbacks of limited penetration depth of intratumoural tissues, hypoxia and complexity of the tumor microenvironment (TME). Herein, we fabricated an integrated multifunctional nanoreactor (LuAG:Tb/Ce-RB@ZIF-8-Au2Pt-HA, LRZAPH) combining scintillating nanoparticles (SCNPs, LuAG:Tb/Ce), a metal-organic framework (ZIF-8), and bimetallic Au2Pt for X-ray-triggered PDT and dual noble-metal nanozyme catalyzed CDT. Such a nanoreactor not only significantly enhanced the PDT effect under X-ray irradiation through full resonance energy transfer from LuAG:Tb/Ce scintillator to Ross Bengal (RB), but also facilitated the reactive oxygen species (ROS) and oxygen (O2) production through the excellent peroxidase-like (POD-like) and catalase-like (CAT-like) catalytic properties of Au2Pt nanozymes. O2 also alleviates hypoxia in intratumoural tissues during coordinated PDT. In addition, the dissociation behavior of ZIF-8 with pH-responsive and targeted of hyaluronic acid (HA) in acidic TME significantly enhanced the therapeutic efficacy of LRZAPH nanocatalysts. Significantly, the high tumor growth inhibition rate of 93 % was revealed due to radiotherapy (RT)/PDT/CDT synergetic therapy in vivo, which minimized the toxic and side effects of conventional clinical radiotherapy/chemotherapy on human. The synergistic effect of LRZAPH nanocatalysts on PDT and catalytically induced CDT is expected to provide new pathways for effective treatment of deep tumors.

Anti-4-1BB×PDL1 Bispecific Antibody Reinvigorates Tumor-Specific Exhausted CD8+ T Cells and Enhances the Efficacy of Anti-PD1 Blockade.

To overcome the limited efficacy of immune checkpoint blockade, there is a need to find novel cancer immunotherapeutic strategies for the optimal treatment of cancer. The novel anti-4-1BB×PDL1 bispecific antibody-ABL503 (also known as TJ-L14B)-was designed to simultaneously target PDL1 and 4-1BB and demonstrated strong antitumor T-cell responses without considerable toxicity. In this study, we investigated the mechanisms by which the combination of ABL503 and anti-PD1 blockade affected the reinvigoration of exhausted tumor-infiltrating CD8+ T cells (CD8+ TIL) and antitumor efficacy. Single-cell suspensions of hepatocellular carcinoma and ovarian cancer tissues from treatment-naïve patients were used for immunophenotyping of CD8+ TILs and in vitro functional assays. Humanized hPD1/hPDL1/h4-1BB triple-knock-in mice were used to evaluate the effects of ABL503 and anti-PD1 blockade in vivo. We observed that ABL503 successfully restored the functions of 4-1BB+ exhausted CD8+ TILs, which were enriched for tumor-specific T cells but unresponsive to anti-PD1 blockade. Importantly, compared with anti-PD1 blockade alone, the combination of ABL503 and anti-PD1 blockade further enhanced the functional restoration of human CD8+ TILs in vitro. Consistently, the combination of ABL503 with anti-PD1 in vivo significantly alleviated tumor growth and induced enhanced infiltration and activation of CD8+ TILs. ABL503, a PDL1 and 4-1BB dual-targeting bispecific antibody, elicits pronounced additive tumor growth inhibition, with increased infiltration and functionality of exhausted CD8+ T cells, which in turn enhances the anticancer effects of anti-PD1 blockade. These promising findings suggest that ABL503 (TJ-L14B) in combination with PD1 inhibitors will likely further enhance therapeutic benefit in clinical trials. See related commentary by Molero-Glez et al., p. 3971.

Design, Synthesis, and Biological Evaluation of 5-Amino-4-fluoro-1H-benzo[d]imidazole-6-carboxamide Derivatives as Novel and Potential MEK/RAF Complex Inhibitors Based on the "Clamp" Strategy.

Herein, we described the rational drug design and synthesis of a series of 5-amino-4-fluoro-1H-benzo[d]imidazole-6-carboxamide derivatives that inhibit MEK and RAF kinases. The detailed screening cascades revealed that 16b was a preferred compound, which might act like a "clamp" to stabilize the MEK/RAF complex, thereby effectively inhibiting MEK1, BRAF, and BRAFV600E with IC50 values of 28, 3, and 3 nM, respectively. 16b possessed an excellent selectivity over other 312 human-related kinases at 1 μM. In vitro, 16b showed potent antiproliferative activities against MIA PaCa-2 (G12C KRAS), HCT116 (G13D KRAS), and C26 (G12D KRAS) cells with IC50 values of 0.011, 0.079, and 0.096 μM, respectively. CoIP experiments demonstrated that 16b could induce MEK/RAF complex formation. Most importantly, in the C26 syngeneic colorectal and HCT116 mice xenograft tumor models, 16b demonstrated tumor growth inhibition of 70 and 93%, respectively, suggesting that 16b may be a promising MEK/RAF complex inhibitor and worthy of further development.

SRBD1 Regulates the Cell Cycle, Apoptosis, and M2 Macrophage Polarization via the RPL11-MDM2-p53 Pathway in Glioma.

Low expression of certain ribosomal proteins leads to the inactivation of p53, which is mediated mainly by RPL5 or RPL11 (ribosomal protein L11). It is also unknown what mechanisms drive aberrant ribosomal proteins expression in tumor. SRBD1 (S1 RNA-binding domain 1), as a highly conserved RNA-binding protein, is lowly expressed in glioma tissues and correlated with glioma prognosis. In this study, we observed that SRBD1 was closely related to p53 signaling. The upregulation of SRBD1 elevated p53 levels, thereby activating the p53 signaling pathway. As an RNA bind protein, SRBD1 could bind to the 5'-UTR of target genes and regulate RNA translation. We further conducted RNA immunoprecipitation using anti-SRDB1 antibody and noticed 29 hub RNA, including RPL11. RPL11 could inhibit MDM2-mediated p53 ubiquitination. SRBD1 upregulation promoted RPL11 binding to MDM2 via elevating RPL11 protein levels, which in turn activated the p53 signaling. Disrupting the p53 signaling blocked SRBD1-induced glioma suppression. In mouse xenograft model, SRBD1 ectopic expression was effective in reducing the total M2 tumor-associated macrophages (TAMs) density and suppressed glioma tumor growth. In summary, these data show that SRBD1 has a critical role in inhibition of glioma tumor growth and M2 macrophage polarization, and targeting RPL11-MDM2-p53 signaling may be an effective strategy to improve therapy and survival for glioma patients.

An endogenous oxygen self-supplied nanoplatform with GSH-depleted and NIR-II triggered electron-hole separation for enhanced photocatalytic anti-tumor therapy.

The use of artificial enzymes and light energy in photocatalytic therapy, a developing drug-free therapeutic approach, can treat malignant tumors in vivo. However, the relatively deficient oxygen concentration in the tumor microenvironment (TME) restrains their further tumor treatment capability. Herein, a novel nanoplatform with Cu7S4@Au nanocatalyst coated by MnO2 was successfully designed. After 1064 nm light irradiation, the designed nanocatalyst can promote the separation of light generated electron-hole pairs, resulting in ROS generation and tumor cell apoptosis. The MnO2 shelled nanoplatform can function as a TME-responsive oxygen self-supplied producer to improve photocatalyst treatment and GSH depletion. In summary, the designed novel nanoplatform shows efficient inhibition of tumor growth via GSH depletion and synergistic photocatalytic therapy, which is of great significance for improving the clinical tumor treatment effect.