Treatment Of Tumor-bearing Mice Research Articles

Tumor-derived nanovesicles enhance cancer synergistic chemo-immunotherapy by promoting cGAS/STING pathway activation and immunogenetic cell death

AimsCheckpoint blockade immunotherapy is a promising therapeutic modality that has revolutionized cancer treatment; however, the therapy is only effective on a fraction of patients due to the tumor environment. In tumor immunotherapy, the cGAS-STING pathway is a crucial intracellular immune response pathway. Therefore, this study aimed to develop an immunotherapy strategy based on the cGAS-STING pathway. Materials and methodsThe physicochemical properties of the nanoparticles EM@REV@DOX were characterized by TEM, DLS, and WB. Subcutaneous LLC xenograft tumors were used to determine the biodistribution, antitumor efficacy, and immune response. Blood samples and tissues of interest were harvested for hematological analysis and H&E staining. SignificanceOverall, our designed nanovesicles provide a new perspective on tumor immunotherapy by ICD and cGAS-STING pathway, promoting DCs maturation, macrophage polarization, and activating T cells, offering a meaningful strategy for accelerating the clinical development of immunotherapy. Key findingsEM@REV@DOX accumulated in the tumor site through EPR and homing targeting effect to release REV and DOX, resulting in DNA damage and finally activating the cGAS-STING pathway, thereby promoting DCs maturation, macrophage polarization, and activating T cells. Additionally, EM@REV@DOX increased the production of pro-inflammatory cytokines (e.g., TNF-α and IFN-β). As a result, EM@REV@DOX was effective in treating tumor-bearing mice and prolonged their lifespans. When combined with αPD-L1, EM@REV@DOX significantly inhibited distant tumor growth, extended the survival of mice, and prevented long-term postoperative tumor metastasis, exhibiting great potential in antitumor immunotherapy.

Abstract RF02-02: BBO-10203, a first-in-class, orally bioavailable, selective covalent small molecule that inhibits RAS-driven PI3Kalpha activity without affecting glucose metabolism

Abstract PI3Kα is the most mutated kinase and the second most mutated oncogene in human cancer. Activation of PI3Ka can be achieved by receptor tyrosine kinases such as insulin receptor and insulin-like growth factor receptor 1 and/or by directly interacting with RAS family members. Previous elegant preclinical studies have established that RAS-driven PI3Ka activation is important in tumor cells but may not be involved in cell types controlling glucose metabolism. Alpelisib, a small molecule inhibitor of the kinase activity of PI3Ka, has been approved for the treatment of ER+ PIK3CA mutant breast cancer in combination with fulvestrant after endocrine therapy in advanced or metastatic breast cancer based on an improvement in PFS versus fulvestrant alone. Inhibition of PI3Ka activity by alpelisib in normal tissues resulted in a severe (G3/4) hyperglycemia rate of 37% with frequent dose interruptions and discontinuations. Additionally, preclinical studies have demonstrated that the dysregulation of glucose homeostasis resulting from PI3Ka kinase inhibition leads to hyperinsulinemia that increases pathway flux, rendering kinase inhibitors less effective. Here, we report on a novel covalent small molecule designed to inhibit RAS-mediated activation of the AKT pathway via PI3Ka without the resultant hyperglycemia associated with direct inhibition of PI3Ka kinase activity. BBO-10203 disrupts the physical interaction between RAS and PI3Ka in tumor cells resulting in potent signaling pathway inhibition. This agent selectively binds to PI3Ka and disrupts its interaction with K-,H-, and N-RAS with low single digit nanomolar potency (~5 nM). Breaking the interaction between these two oncogenes inhibits basal pAKT cellular levels (BT-474/KYSE-410 IC50: ~5 nM) in HER2 amplified (HER2amp) and wild-type or mutant PI3Ka cell lines. Even though BBO-10203 does not inhibit the kinase activity of PI3Ka, its effects on cancer cell signaling inhibition and transcriptional regulation highly resemble those of alpelisib. BBO-10203 displays excellent drug-like properties and oral bioavailability. Single dose treatment of KYSE-410 (HER2amp/KRASG12C) tumor bearing mice with increasing doses (1-100 mg/kg, PO) of BBO-10203 results in dose and time dependent inhibition of pAKT in vivo. Maximal inhibition (~80%) is achieved at 30 mg/kg and lasts for 24 hours. Repeated dose treatment of tumor bearing mice with BBO-10203 is well tolerated and results in significant efficacy in PIK3CA mutant as well as HER2amp human xenograft models. In the KYSE-410 xenograft model, BBO-10203 daily oral dosing of 30 mg/kg results in significant tumor regressions. Importantly, treatment with BBO-10203 does not affect insulin signaling in differentiated adipocytes in vitro, nor does it impact glucose metabolism in vivo at 3-times the maximal efficacious dose level in xenograft studies. In conclusion, we have identified a novel approach to inhibit the PI3Ka signaling pathway by blocking its interaction with, and activation by RAS. This approach can achieve strong pAKT inhibition in tumor cells without changes in glucose metabolism. Clinical investigation of BBO-10203 for the treatment of both ER+/PIK3CA mutant and HER2amp breast cancer is warranted. Citation Format: Pedro Beltran, Simanshu Dhirendra, Rui Xu, Ming Chen, Daniel Czyzyk, Sofia Donovan, Siyu Feng, Cindy Feng, Lijuan Fu, Felice Lightstone, Ken Lin, Anna Maciag, Dwight Nissley, Erin Riegler, Kerstin Sinkevicius, Andrew Stephen, James Stice, David Turner, Bin Wang, Keshi Wang, Yue Yang, Cathy Zhang, Frank McCormick, Eli Wallace. BBO-10203, a first-in-class, orally bioavailable, selective covalent small molecule that inhibits RAS-driven PI3Kalpha activity without affecting glucose metabolism [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr RF02-02.

Exercise may be a Novel Strategy to Reduce Vascular Toxicity of Immune Checkpoint Inhibitors in a Mouse Melanoma Model

Background: Immune checkpoint inhibitors (ICIs) are a cornerstone in advanced melanoma treatment. Unfortunately, ICI-associated cardiovascular toxicity, particularly atherosclerotic events, poses a significant challenge. Identifying interventions to mitigate the cardiovascular toxicity of ICI is critical for enhancing the quality and duration of life in cancer survivors. Given the established benefits of exercise in atherosclerosis prevention, this study explores its potential to reduce ICI-induced vascular toxicity in the context of melanoma. We hypothesized that aerobic exercise would decrease ICI's vascular toxicity in a mouse melanoma model. The primary experimental outcome was endothelial mesenchymal transition (EndMT), an early atherosclerosis indicator. Methods: C57BL/6 mice were subcutaneously injected with 0.8 x 106 BP melanoma cells ( BrafV600E/WT and Pten−/−). Upon tumor palpability, mice were assigned into four groups: IgG, Exercise, anti-PD-1, and anti-PD-1+Exercise. Mice were treated with bi-weekly administration of anti-PD-1 or IgG antibody (150 μg) for three weeks, combined with or without treadmill exercise (12 meters/minute for 45 min per day, five days/week). Body weight was monitored weekly. Following the final treatment, aortic tissues were collected and en face immunofluorescence staining for endothelial marker VE-cadherin (VE-Cad) and vimentin was performed to assess EndMT, then analyzed via confocal microscopy. Results: No significant weight differences were observed between groups. Notably, the EndMT marker vimentin expression was significantly higher in anti-PD-1 treated tumor-bearing mice compared to IgG controls (3.8% vs 0.6%, p=.03) in the atheroprotective laminar flow area. This was reduced by exercise; the anti-PD-1+Exercise group showed significantly reduced endothelial vimentin levels relative to the anti-PD-1 alone (0.8% vs 3.8%, p=.04). Disturbed flow areas did not exhibit notable differences between any group. Conclusions: Aerobic exercise may be protective against vascular toxicity in mice treated with ICIs by promoting atheroprotective flow and potentially inhibiting EndMT. Although these initial results are promising, comprehensive studies are required to ascertain the impact of exercise on preventing ICI-induced EndMT and subsequent atherosclerosis in this mouse melanoma model. Future research will aim to more deeply characterize the process of EndMT following ICI treatment and evaluate the preventive capacity of exercise against atherosclerosis in this context. MD Anderson Cancer Center Melanoma SPORE grant (NCI P50CA221703-05). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2096: A novel, orally available ferroptosis inducer targeting Fe-S assembly with significant in vivo anti-tumor efficacy

Abstract Background: Ferroptosis is a novel form of cell death driven by iron-dependent lipid peroxidation, which disrupts cellular membranes resulting in immune-activating death. A variety of tumor types, particularly those associated with enhanced lipid and/or iron content, or which experience increased oxidative stress (including drug tolerant persister cells), are sensitive to ferroptosis, rendering ferroptosis a promising therapeutic strategy for cancer. However, currently available ferroptosis inducers show limited activity in vivo, suggesting a need for novel molecular targets to validate the clinical utility of ferroptosis induction. Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of VHL resulting in constitutive activation of HIF-1α and HIF-2α, which contribute to disease progression, including by promoting lipid accumulation that sensitizes ccRCC cells to ferroptosis. Methods: We performed a high-throughput screen to identify small molecule inhibitors of HIF-2α followed by structure-activity-relationship studies for validation. The molecular target of these inhibitors was identified using the Drug Affinity Responsive Target Stability assay, and target validation was confirmed using siRNA and NMR. In vitro efficacy was determined in a panel of cancer cell lines and in vivo anti-tumor efficacy was verified using mouse models of cancer. Results: We identified a novel molecule, KD061, which decreases HIF-1/2α and induces ferroptosis in vivo through oral administration by targeting Iron Sulfur Cluster Assembly 2 (ISCA2). ISCA2 is a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex and ISCA2 inhibition either pharmacologically or using siRNA triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. ISCA2 inhibition also decreases HIF-2α protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreases HIF-1α translation. These studies reveal a novel function of ISCA2 in extra-mitochondrial Fe-S assembly that may be limiting in cancer due to the increased need for these proteins in DNA replication and maintenance. Outside of ccRCC, a 92-cell line screen reveals a variety of tumor types sensitive to ISCA2 inhibition including melanoma and clear cell ovarian cancer. Treatment of tumor-bearing mice with KD061 resulted in > 65% tumor growth inhibition, associated with significantly increased lipid peroxidation, thus confirming ferroptosis in vivo, and with decreased HIF-1/2α. The anti-tumor efficacy of KD061 exceeded that of α-PD1 and was well tolerated at the therapeutic dose. Conclusions: We describe the first orally available inducer of ferroptosis with significant anti-tumor efficacy in ccRCC and other tumor types. Citation Format: Yangsook Song Green, Maria Ferreira dos Santos, Simone Ciofi-Baffoni, Xiaohui Liu, Mei Yee Koh. A novel, orally available ferroptosis inducer targeting Fe-S assembly with significant in vivo anti-tumor efficacy [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 2096.

Abstract 3327: L-pampo™, a TLR2/3 agonist, enhances anti-tumor immune responses and synergistically improves the anti-tumor efficacy of immune checkpoint inhibitors in cold tumors

Abstract Introduction: Immunotherapy is a promising therapeutic approach for a wide range of cancers. However, some tumors called "cold tumors" lack infiltrating T-cells and are not responsive to immune checkpoint inhibitors (ICIs), leading to worse clinical outcomes. This study demonstrates that L-pampo, a novel TLR2/3 agonist, promotes an anti-tumor immune response in cold tumors. Also, combining L-pampo with ICIs can improve the anti-tumor efficacy more safely than standard-of-care (SoC) chemotherapies. Method: We utilized syngeneic tumor models using CT26 colon cancer and 4T1 triple-negative breast cancer (TNBC). L-pampo was administered alone or in combination with ICIs such as anti-PD-1 (aPD-1) and anti-CTLA-4 (aCTLA-4) antibodies to tumor-bearing mice (n=8/group). Tumor size was measured every three days, and the survival rate was monitored until day 60. On day 21 or 27 after transplantation, we analyzed the tumor weight and tumor-infiltrating immune cells. Furthermore, we assessed the respective anti-tumor efficacies and safety of L-pampo and SoC chemotherapies (FOLFOX and paclitaxel). Result: In the CT26 colon cancer model, L-pampo effectively reduced the tumor size compared to the vehicle group (83.7%). The combination of L-pampo and ICI reduced tumor size by 86.1% (with aCTLA4) and 90.5% (with aPD-1), while the ICI alone did not effectively inhibit tumor growth (aPD-1; 17.3%, aCTLA-4; 23.6%). Notably, the triple concurrent treatment with L-pampo, aPD-1 and aCTLA-4 reduced tumor size by 96.6% and induced completed tumor regression in 1 out of 8. In the 4T1 TNBC model, L-pampo reduced tumor size by 64.2% compared to the vehicle treatment, and combined treatment with L-pampo and a-PD-1 suppressed tumor growth compared to aPD-1 alone (9.6% vs. 61.5%). Additionally, L-pampo improved median overall survival in each tumor model (CT26; 25.5 vs. 35.5, 4T1; 29.5 vs. 53.5 days). To compare the anti-tumor efficacy of L-pampo and conventional cytotoxic chemotherapy, we treated tumor-bearing mice with FOLFOX or paclitaxel as SoC regimen, respectively. Compared with each chemotherapy, L-pampo revealed comparable antitumor efficacies (Tumor growth inhibition CT26; 75.6% vs. 70.6%, 4T1; 65.5% vs. 58.8%). Interestingly, L-pampo inhibited tumor growth without significant adverse effects. In contrast, conventional chemotherapies induced acute hematological side effects such as leukopenia/neutropenia/thrombocytopenia and rapid weight loss. Furthermore, L-pampo increased the active cytotoxic T-cells infiltration through antigen-presenting cells stimulation within the tumors. Conclusion: This study suggests that L-pampo, a novel TLR2/3 agonist, can be a potent anti-tumor agent, improving the anti-tumor efficacy of ICIs in cold tumors with enhanced anti-tumor immunity and minimal toxicities than conventional chemotherapies. Citation Format: Guentae Kim, Yoonki Heo, Hye Jung Kim, Sejung Park, Byung Cheol Ahn, Won Suk Lee, Chan Kim, Hong Jae Chon, Eunyoung Chun, Jung Sun Yum. L-pampo™, a TLR2/3 agonist, enhances anti-tumor immune responses and synergistically improves the anti-tumor efficacy of immune checkpoint inhibitors in cold 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 3327.

Abstract B084: Triple checkpoint blockade, but not anti-PD1 alone, enhances the efficacy of engineered adoptive T cell therapy in advanced ovarian cancer

Abstract Background: Over 20,000 women are diagnosed with ovarian cancer annually, and more than half will die within 5 years. This rate has changed little in the last 30 years, highlighting the need for therapy innovation. Although immunotherapy has revolutionized cancer treatment, efforts to harness endogenous patient immune responses have yielded limited therapeutic activity in ovarian cancer patients. T cells engineered to express a T cell receptor (TCR) targeting proteins uniquely overexpressed in tumors have the potential to control tumor growth without toxicity. Mesothelin (Msln) is over-expressed in ovarian cancer, contributes to the malignant and invasive phenotype, and has limited expression in healthy cells, making it a candidate immunotherapy target in these tumors. Methods: The ID8VEGF mouse cell line was used to evaluate if T cells engineered to express a mouse Msln-specific high-affinity T cell receptor (TCRMsln) can kill ovarian cancer. Tumor-bearing mice were treated with TCRMsln T cells plus anti-PD-1, anti-Tim-3 or anti-Lag-3 checkpoint-blocking antibodies alone or in combination, ultimately targeting up to three inhibitory receptors simultaneously. Single-cell RNA-sequencing (scRNAseq) was used to profile the impact of combination checkpoint blockade on engineered T cells and the tumor microenvironment (TME). Results: In a disseminated ID8 tumor model, adoptively transferred TCRMsln T cells preferentially accumulated in established tumors, delayed ovarian tumor growth, and prolonged mouse survival. However, elements in the TME limited engineered T cell persistence and cytolytic function. Triple checkpoint blockade, but not single- or double-agent treatment, dramatically increased antitumor function by intratumoral TCRMsln T cells. scRNAseq of tumor-infiltrating cells revealed distinct transcriptome changes in engineered and endogenous T cells and myeloid-derived cells. Engineered T cells, when combined with triple checkpoint blockade, increased expression of genes associated with effector and memory gene signatures, including proliferation and metabolic function, and reduced expression of genes associated with exhaustion. Moreover, combining adoptive immunotherapy with triple checkpoint blockade significantly prolonged survival in the cohort of treated tumor-bearing mice, relative to mice that received TCRMsln T cells alone or with anti-PD1 or double-agent treatments. Conclusions: Inhibitory receptor/ligand interactions within the TME can dramatically reduce T cell function, suggesting tumor cells may upregulate the ligands for PD-1, Tim-3 and Lag-3 for protection from tumor-infiltrating lymphocytes. In an advanced ovarian cancer model, triple checkpoint blockade significantly improved engineered T cell function and outcomes in mice in a setting where single checkpoint blockade had no significant activity. These results suggest that disrupting multiple inhibitory pathways simultaneously, which can be more safely pursed in a cell intrinsic form through genetic engineering, may be necessary for improved efficacy in patients. Citation Format: Kristin G. Anderson, Yapeng Su, Madison G. Burnett, Breanna M. Bates, Magdalia L. Rodgers Suarez, Susan L. Ruskin, Valentin Voillet, Raphael Gottardo, Philip D. Greenberg. Triple checkpoint blockade, but not anti-PD1 alone, enhances the efficacy of engineered adoptive T cell therapy in advanced ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B084.

Glucocorticoid receptor mediated sensitization of colon cancer to photodynamic therapy induced cell death

Photodynamic therapy (PDT) is a clinically approved, non-invasive alternate cancer therapy. A synthetic glucocorticoid (GC), dexamethasone (Dex) has previously been demonstrated to sensitize cancer cells to chemotherapy. However, to the best of our knowledge, the sensitization effect of GCs on PDT has not yet been investigated. We hypothesized that glucocorticoid receptor (GR) targeting can selectively make cancer cells more sensitive to PDT treatment, as PDT induces hypoxia wherein GR-activity gets enhanced. In addition, Dex was reported to act against the PDT-induced cell survival pathways like HIF-1α, NRF2, NF-κB, STAT3 etc. Thus, both the treatments can complement each other and may result in increasing the effectiveness of combination therapy. Hence, in this study, we developed liposomal formulations of our previously reported PDT agent P-Nap, either alone (D1P-Nap) or in combination with Dex (D1XP-Nap) to elucidate the sensitization effect. Interestingly, our RT-PCR results in hypoxic conditions showed down-regulation of HIF-1α and over expression of GR-activated genes for glucose-6-phosphatase (G6Pase) and PEPCK enzymes, indicating prominent GR-transactivation. We also observed higher phototoxicity in CT26.WT cells treated with D1XP-Nap PDT under hypoxic conditions as compared to normoxic conditions. These effects were reversed when cells were pre-treated with RU486, a competitive inhibitor of GCs. Moreover, our in vivo findings of subcutaneous tumor model of Balb/C mice for colon cancer revealed a significant decrease in tumor volume as well as considerable enhancement in the survivability of PDT treated tumor-bearing mice when Dex was present in the formulation. A high Bax/Bcl-xL ratio, high p53 expression, enhanced E-cadherin expression and down-regulation of pro-tumorigenic transcription factors NF-κB and c-Myc were found in tumor lysates from mice treated with D1XP-Nap under PDT, indicating GR-mediated sensitization of the tumor to PDT-induced cell death and enhancement of life-span for tumor bearing mice.

HDAC6 inhibitor ACY-1215 enhances STAT1 acetylation to block PD-L1 for colorectal cancer immunotherapy

The search for effective combination therapy with immune checkpoint inhibitors (ICI) has become important for cancer patients who do not respond to the ICI well. Histone deacetylases (HDACs) inhibitors have attracted wide attention as anti-tumor agents. ACY-1215 is a selective inhibitor of HDAC6, which can inhibit the growth of a variety of tumor. We previously revealed that HDAC family is highly expressed in colorectal cancer specimens and mouse models. In this study, ACY-1215 was combined with anti-PD1 to treat tumor-bearing mice associated with colorectal cancer. ACY-1215 combined with anti-PD1 effectively inhibited the colorectal tumor growth. The expression of PD-L1 in tumor of mice were inhibited by ACY-1215 and anti-PD1 combination treatment, whereas some biomarkers reflecting T cell activation were upregulated. In a co-culture system of T cells and tumor cells, ACY-1215 helped T cells to kill tumor cells. Mechanically, HDAC6 enhanced the acetylation of STAT1 and inhibited the phosphorylation of STAT1, thus preventing STAT1 from entering the nucleus to activate PD-L1 transcription. This study reveals a novel regulatory mechanism of HDAC6 on non-histone substrates, especially on protein acetylation. HDAC6 inhibitors may be of great significance in tumor immunotherapy and related combination strategies.

Short Antiangiogenic MMP-2 Peptide-Decorated Conjugated Linoleic Acid-Coated SPIONs for Targeted Paclitaxel Delivery in an A549 Cell Xenograft Mouse Tumor Model.

The design of targeted antiangiogenic nanovectors for the delivery of anticancer drugs presents a viable approach for effective management of nonsmall-cell lung carcinoma (NSCLC). Herein, we report on the fabrication of a targeted delivery nanosystem for paclitaxel (PTX) functionalized with a short antimatrix metalloproteinase 2 (MMP-2) CTT peptide for selective MMP-2 targeting and effective antitumor activity in NSCLC. The fabrication of the targeted nanosystem (CLA-coated PTX-SPIONs@CTT) involved coating of superparamagnetic iron-oxide nanoparticles (SPIONs) with conjugated linoleic acid (CLA) via chemisorption, onto which PTX was adsorbed, and subsequent surface functionalization with carboxylic acid groups for conjugation of the CTT peptide. CLA-coated PTX SPIONs@CTT had a mean particle size of 99.4 nm and a PTX loading efficiency of ∼98.5%. The nanosystem exhibited a site-specific in vitro PTX release and a marked antiproliferative action on lung adenocarcinoma cells. The CTT-functionalized nanosystem significantly inhibited MMP-2 secretion by almost 70% from endothelial cells, indicating specific anti-MMP-2 activity. Treatment of tumor-bearing mice with subcutaneous injection of the CTT-functionalized nanosystem resulted in 69.7% tumor inhibition rate, and the administration of the nanosystem subcutaneously prolonged the half-life of PTX and circulation time in vivo. As such, CLA-coated PTX-SPIONs@CTT presents with potential for application as a targeted nanomedicine in NSCLC management.

Cells of the Osteoblast Lineage Cross-Talk with Myeloid-Derived Suppressor Cells to Promote Multiple Myeloma Growth

Multiple myeloma (MM) is a plasma cell malignancy that thrives in the bone marrow (BM) and is highly influenced by the BM tumor microenvironment. Osteolytic bone disease is a hallmark of MM and is partly due to osteoblast (OB) inhibition. MM cells suppress osteoblast maturation and function via inhibition of the critical Runt-related transcription factor 2 (Runx2) in immature OBs. However, immature cells of the osteoblast lineage (e.g. pre-osteoblasts) remain present and even increased in the MM bone microenvironment and their contribution to the progression of MM has not been well understood. We previously developed a syngeneic mouse model of MM in which Runx2 is specifically deleted in the immature OBs of C57BL6/KaLwRij mice (OB-Runx2 -/- mice), and demonstrated that OB-Runx2 deficiency creates a highly inflammatory BM microenvironment that is responsible for MM cell bone-homing to new bone sites. We therefore hypothesize that pre-osteoblasts play a role as promoters of MM cell survival and proliferation. To determine the direct effect of pre-osteoblasts on MM cells, we established co-cultures of pre-osteoblasts with MM cells. 5TGM1 murine MM cells were cocultured with MC3T3 murine pre-osteoblasts and newborn mouse calvarial pre-osteoblasts respectively. Within 48hrs of co-culture, each of pre-osteoblast populations slightly stimulated proliferation of 5TGM1 MM cells. These data suggest that pre-osteoblasts do not directly affect myeloma growth in vitro. Therefore, we next focus our studies on the role of pre-osteoblasts in MM progression in vivo. Firstly, we used OB-Runx2 deficient mouse model to assess the impact of pre-osteoblast accumulation on MM progression in vivo. 2x10 5 5TGM1-Luciferase MM cells were injected into the right tibia of OB-Runx2 -/- mice and control mice. Bioluminescence imaging and serum IgG2bκ (a soluble marker of 5TGM1 MM cells) ELISA demonstrated that, compared with controls, OB-Runx2 -/- mice had a much larger tumor burden. Following, to more thoroughly evaluate the role of pre-osteoblasts in early stage of MM cell engraftment in bones, we used MC3T3 murine pre-osteoblasts to modulate pre-osteoblast populations at the sites of MM engraftment. 2x10 5 5TGM1-Luciferase MM cells were injected directly into the right tibia of NSG mice with or without equal numbers of MC3T3 pre-osteoblasts. Tumors grew significantly larger when co-injected with 5TGM1 MM cells and MC3T3 pre-osteoblasts than injected with 5TGM1 MM cells alone, which was confirmed by luciferin imaging and serum IgG2bκ ELISA. These data suggest that pre-osteoblasts provide critical support for MM progression in vivo. Given that pre-osteoblasts have no direct effect on the growth of MM cells in vitro, we hypothesized that the pre-osteoblastic niche may regulate MM cells via other intermediary cells in BM. We previously showed that OB-Runx2 deficiency induces an immunosuppressive BM microenvironment that is marked by an increase of myeloid-derived suppressor cells (MDSCs). To investigate whether pre-osteoblasts support MM cell survival and proliferation through the functional crosstalk with MDSCs, 5TGM1 MM cells were co-cultured with MC3T3 pre-osteoblasts and mouse calvarial pre-osteoblasts respectively in the presence or absence of MDSCs. Interestingly, pre-osteoblasts significantly promoted the proliferation of MM cells under the existence of MDSCs. To extend these findings in vivo, we next determined whether MDSC depletion can alleviate the promoting effect of pre-osteoblasts on MM progression. Gemcitabine (GEM) is an FDA-approved anti-cancer agent and MDSC inhibitor used in the treatment of solid tumors but not commonly used in MM therapy. We again injected 5TGM1-Luciferase MM cells into the right tibia of NSG mice with or without co-injection of MC3T3 pre-osteoblasts. We treated tumor-bearing mice with PBS or GEM (i.p. 30 mg/kg/week) for 4 weeks. Bioluminescence imaging and serum IgG2bκ ELISA demonstrated that GEM could overcome the proliferative effect of pre-osteoblasts on MM cells in mouse model, and this effect was independent of NK, B and T cells. Taken together, using in vivo and in vitro models, we show that pre-osteoblasts have a positive regulatory impact on MM cells through communication with MDSCs. Ongoing mechanistic studies aim to delineate how pre-osteoblasts modulate the functions of MDSCs to promote MM progression.

Immune Checkpoint Blockade Promotes Thrombosis Via T-Cell and Neutrophil Activation, and Tumor-Cell Associated Tissue Factor (TF) in a Murine Model of Colorectal Cancer

Background: Immune checkpoint blockade (ICB) is a paradigm shift in cancer therapy. Despite clinical benefit, various adverse events are associated with ICB using immune checkpoint inhibitors (ICI). Venous thrombosis is an emerging clinical problem in cancer patients receiving ICI. Little is known about how ICI promotes thrombosis in cancer. To define the underlying mechanisms of ICI-associated thrombosis (IAT), we assessed the ICI impact on blood hypercoagulability and tumor TF expression using a mouse thrombosis model of colorectal cancer. Methods: We developed a CT26 mouse thrombosis model with ICI (aPD-1+aCTLA4) administration. Systemic hypercoagulability was assessed by measurement of circulating nucleosomes (Nu.Q H3.1 Immunoassay), neutrophil extracellular traps (NETs) (CitH3 immunofluorescence), circulating neutrophil-platelet aggregates (immunophenotyping), and levels of thrombin-antithrombin (TAT) complexes in ICI-treated mice. Tumor-associated TF and TF activity in circulating extracellular vesicles (EV) were assessed by western blotting and Factor Xa generation assays. To gain mechanistic insights into TF induction by ICI, tumor cytokines were also profiled (proteome profiler array). The role of cancer cell-derived TF in IAT was determined using mice implanted with CRISPR/Cas9-mediated TF knockout (TFKO) CT26 tumor cells. Results: ICI treated tumor-bearing mice developed larger thrombi than control mice and demonstrated elevated levels of circulating nucleosomes (114 vs 82 ng/ml), NET (15.8 vs 6.8%), platelet-neutrophil aggregates (44.8 vs 22.3%), and TAT complexes (12.6 vs 7.9 ng/ml) compared to IgG-treated mice. Tumor TF expression was increased in tumor extracts. Cytokine profiling suggested enhanced production of IFNɣ (2-fold) and TNFα (5-fold), as well as CXCL11 (6.8-fold), an effector T cell-recruiting chemokine. IFNɣ-increased TF expression in cultured CT26 cells, suggesting that T-cell derived IFNɣ may play a role in enhancing tumor TF expression in vivo. Circulating EV TF antigen and procoagulant activity were increased by ICI. CRISPR/Cas9-mediated TF KO in CT26 cells resulted in complete loss of TF expression, and mice bearing these cells showed reduced tumor and EV-associated TF (Fig. A), associated with a significant decrease in thrombus formation after ICI treatment (24.2 vs 20.5 mg, Fig. B). There was no significant difference in growth of wild-type and TF KO CT26 tumors. Conclusions: Our findings provide insights into cellular and molecular mechanisms associated with ICI-induced thrombosis, and suggest a role for neutrophil and granulocyte activation, and tumor-associated TF. Additional studies are ongoing to further define these pathways in the murine model and in patients.

Combination therapy of HIFα inhibitors and Treg depletion strengthen the anti-tumor immunity in mice.

Hypoxia-inducible factor 1 alpha (HIF1α), under hypoxic conditions, is known to play an oxygen sensor stabilizing role by exerting context- and cell-dependent stimulatory and inhibitory functions in immune cells. Nevertheless, how HIF1α regulates T cell differentiation and functions in tumor settings has not been elucidated. Herein, we demonstrated that T-cell-specific deletion of HIF1α improves the inflammatory potential and memory phenotype of CD8+ T cells. We validated that T cell-specific HIF1α ablation reduced the B16 melanomas development with the indication of ameliorated antitumor immune response with enhanced IFN-γ+ CD8+ T cells despite the increase in the Foxp3+ regulatory T-cell population. This was further verified by treating tumor-bearing mice with a HIF1α inhibitor. Results indicated that HIF1α inhibitor also recapitulates HIF1α ablation effects by declining tumor growth and enhancing the memory and inflammatory potential of CD8+ T cells. Furthermore, a combination of Treg inhibitor with HIF1α inhibitor can substantially reduce tumor size. Collectively, these findings highlight the notable roles of HIF1α in distinct CD8+ T-cell subsets. This study suggests the significant implications for enhancing the potential of T cell-based antitumor immunity by combining HIF1α and Tregs inhibitors.

Personalized neoantigen viro-immunotherapy platform for triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) corresponds to approximately 20% of all breast tumors, with a high propensity for metastasis and a poor prognosis. Because TNBC displays a high mutational load compared...

Inducible nitric oxide synthase (iNOS)-activated Cxcr2 signaling in myeloid cells promotes TGFβ-dependent squamous cell carcinoma lung metastasis

Transforming growth factor beta (TGFβ) activity is linked to metastasis in many cancer types, but whether TGFβ activity is necessary for squamous cell carcinoma (SCC) lung metastasis has not been studied. Here we used a lung metastatic SCC model derived from keratin 15 (K15). KrasG12D.Smad4−/− SCC and human SCC specimens to identify metastasis drivers and test therapeutic interventions. We demonstrated that a TGFβ receptor (TGFβR) inhibitor reduced lung metastasis in mouse SCC correlating with reduced CD11b+/Ly6G+ myeloid cells positive for inducible nitric oxide synthase (iNOS). Further, TGFβ activity and iNOS were higher in primary human oral SCCs with metastasis than SCCs without metastasis. Consistently, either depleting myeloid cells with anti-Gr1 antibody or inhibiting iNOS with L-N6-(1-iminoethyl)-l-lysine (L-NIL) reduced SCC lung metastasis. L-NIL treated tumor-bearing mice exhibited reductions in tumor-infiltrating myeloid cells and in plasma Cxcl5 levels, and attenuated primary tumor growth with increased apoptosis and decreased proliferation. Blocking Cxcl5 with an antagonist of its receptor Cxcr2, SB225002, also reduced SCC lung metastasis.

LACK OF SMARCB1 EXPRESSION CHARACTERIZES A SUBSET OF PERIPHERAL T‐CELL LYMPHOMAS ENRICHED IN CHILDREN AND YOUNG ADULTS

Introduction: Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is a heterogeneous group of malignancies with poor outcome. Because patients respond poorly to current treatment regimens, identification of new therapeutic strategies is required. In a mouse tumor model, conditional deletion of Smarcb1 encoding a member of the SWI/SNF chromatin remodeling complex causes the majority of mice to develop mature T-cell lymphomas. The human counterpart, SMARCB1, is a bona fide tumor suppressor gene associated with the development of for example, rhabdoid tumors and schwannomas, but has also been shown to be involved in lymphocyte development. Methods: SMARCB1 (INI1) gene and protein expression was assessed in 315 patients with diverse mature T cell lymphomas by array-based mRNA profiling, immunohistochemistry and/or Western Blot. The DNA methylome was analyzed in SMARCB1-negative pediatric PTCL-NOS patients (n = 5) and murine T cell lymphomas of the Cd4-cre::Smarcb1fl/fl model (n = 5) using the Infinium MethylationEPIC and Mouse Methylation BeadChip, respectively. Murine Smarcb1-deficient T-cell lymphomas were further studied using droplet-based single cell RNA sequencing (Chromium, 10X Genomics). Results: SMARCB1 expression was heterogeneous between and within the 315 different human mature T-cell lymphoma entities. Loss of SMARCB1 expression in PTCL-NOS patients correlated with young age (Wald test, p value = 0.011). Molecular characterization and DNA methylation analysis revealed that loss of SMARCB1 expression in human PTCL largely occurs via somatic mutation and/or epigenetic silencing, whereas in contrast to rhabdoid tumors, germline SMARCB1 mutations were not observed. Comparison of the DNA methylome of human and murine PTCL-NOSSmarcb1- showed similar DNA methylation profiles, with hypermethylation of T-cell-related genes and hypomethylation of genes involved in myeloid development. Increase of myeloid cell populations was confirmed in murine tumors by scRNA-seq analyses, which further revealed an immunosuppressive and pro-inflammatory tumor microenvironment (TME). Treatment of tumor-bearing mice with SAHA, a pan-HDACi, triggered remodeling of the TME, promoting replenishment of lymphoid compartments and reversion of the exhaustion phenotype. Conclusions: Here we describe SMARCB1-negative PTCL-NOS as a potential new molecular subtype of PTCL-NOS significantly enriched in young patients. A strong concordance between naturally occurring SMARCB1-deficient PTCL in humans and in the targeted mouse model were found regarding epigenetic features. Our results provide a rationale for further investigation of potential HDACi combination therapies in SMARCB1-negative PTCL-NOS. Keywords: aggressive T-cell non-Hodgkin lymphoma, genomics, epigenomics, and other-omics No conflicts of interests pertinent to the abstract.

A highly selective humanized DDR1 mAb reverses immune exclusion by disrupting collagen fiber alignment in breast cancer

BackgroundImmune exclusion (IE) where tumors deter the infiltration of immune cells into the tumor microenvironment has emerged as a key mechanism underlying immunotherapy resistance. We recently reported a novel role...

CD4 depletion enhances the efficacy of Super2 IL-33 armored CAR T cell against solid tumors and leads memory formation of both endogenous and CAR T cells

Abstract Chimeric antigen receptor (CAR) T cells exhibit tremendous efficacy in patients with relapsed and recurrent liquid cancers. However, CAR T cells fail to control solid tumors because of their inability to infiltrate the suppressive tumor environment and their poor persistence in vivo. The development of 4 thgeneration armored CAR T cells that employ direct tumor killing and release cytokines that can modulate the immune response to enhance tissue/tumor infiltration may improve CAR T cell persistence. Our lab has developed Super2 and IL-33 (S233) armored CAR T cells that significantly delayed B16F10 melanoma tumor growth and enhanced tumor infiltration of CARs and endogenous immune cells. Combination therapy of S233 CAR T Cells with anti-CTLA4 or anti-CD4 further enhanced this phenotype. S233 CAR T cells and anti-CD4 combination therapy promoted Melanoma-associated vitiligo (MAV), which correlates with development of tumor protective Tissue Resident Memory (TRM) cells. To assess whether CAR T cells differentiate into memory T cells, we treated tumor-bearing mice with S233 CAR T Cells with or without anti-CD4, resected their residual tumors and tracked memory T cell responses. We found that S233 CAR T cells persisted longer in the tumor draining lymph node and developed accelerated MAV when combined with anti-CD4. Both CAR T cells and endogenous T cells differentiated into central and effector memory but only endogenous T cells in the CD4 depleted group became TRM cells. While these findings provide insights on strategies to enhance long-term persistence of CAR T cells, additional studies are needed to induce CAR TRM cells and assess the durability of anti-tumor responses. NIH grant T32-AI007363 NIH grant P30-CA023108

Examining methotrexate’s safety and efficacy in combination with immune checkpoint inhibitors to control immune related inflammatory arthritis in cancer

Abstract Immune checkpoint inhibitors (ICIs) are associated with high rates of toxic side effects known as immune related adverse events such as inflammatory arthritis (ir-RA). ir-RA is empirically treated with drugs for rheumatoid arthritis such as methotrexate (MTX) and hydroxychloroquine which was recently shown to reduce the anti-tumor benefit of ICIs in mouse models of cancer. We have set out to determine the safety of MTX use in combination with ICIs, its effect on the anti-tumoral immune response and its efficacy in treating ir-RA in a novel arthritis-B16 PD-L1 tumor mouse model. We observed that MTX did not diminish the anti-tumor benefit of ICIs in our melanoma model and significantly altered the intra-tumoral CD8 +T cell population by promoting the formation of central memory cells and diminishing PD1 expression. We have identified that MTX acts on neuropilin-1 (NRP1), a receptor identified as a CD8 +T cell exhaustion marker promoting terminal exhaustion and limiting memory cell formation in tumors. We have shown that MTX treatment in vitro reduces the expression of NRP1 on stimulated CD8 +T cells. We have also advanced in the development of a novel arthritis-tumor model showing that MTX can control and diminish inflammation in the delayed type hypersensitivity arthritis (DTHA) and reduced the frequency of Th1 and Th17 cells in the popliteal lymph nodes of mice, with Th17 cells known to have a crucial role in ir-RA. Together these results show that MTX is safe for use in combination with ICIs in our tumor model and is effective in our DTHA model that mimics the immune profile of ir-RA. With the development of our combined arthritis-tumor model we will be able to assess MTX’s efficacy in controlling arthritis in ICIs treated tumor bearing mice.

Abstract LB036: IRF8 regulates tumor cell sensitivity to intrinsic ferroptosis by repressing p53

Abstract Tumor cell resistance to ferroptosis, which is a recently discovered CTL induced cell death pathway, remains incompletely understood. We report here that interferon regulatory factor 8 (IRF8) functions as a regulator of tumor cell intrinsic ferroptosis. Genome-wide gene expression profiling identified ferroptosis pathway as an IRF8-regulated pathway in tumor cells. IRF8.KO tumor cells are resistant to intrinsic ferroptosis induction and also exhibit decreased ferroptosis in response to tumor specific CTL killing. Loss of IRF8 increases p53 express in tumor cells and knocking out Tp53 in IRF8.KO cells restored tumor cell sensitivity to intrinsic ferroptosis induction. We were able to show that treating tumor-bearing mice with IRF8-encoding plasmid NTC9385R-mIRF8 encapsulated in a DOTAP-cholesterol lipid nanoparticle significantly reduced tumor growth and increased the percentage of dead cells in the tumor. Our data therefore determine that IRF8 represses p53 expression to maintain tumor cell sensitivity to intrinsic ferroptosis. Citation Format: Dakota Poschel, Mercy Kehinde-Ige, John Klement, Dafeng Yang, Alyssa Merting, Natasha Savage, Huidong Shi, Kebin Liu. IRF8 regulates tumor cell sensitivity to intrinsic ferroptosis by repressing p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB036.

Abstract LB337: Inhibition of the E3 ligase CBL-B enhances the effector function and proliferation of natural killer cells

Abstract The E3 ubiquitin ligase CBL-B is a master regulator of effector T cells through its downstream regulation of T-cell receptor and co-stimulatory stimulation, but it has also been shown to play a negative feedback role in NK cells. CBL-B depletion enhances the production of IFNγ and the cytotoxicity of activated NK cells. Similar to the effect of depleting CD8 T cells in mice, depletion of NK cells abolishes the tumor growth delay observed in Cbl-b-/- mice. With emerging data supporting the potential role of exogenous NK cells in anti-tumor immunity, inhibition of CBL-B represents a promising approach to enhance both adaptive and innate immunity for cancer immunotherapy. Using one of our small molecules, allosteric CBL-B inhibitors, HOT-A, we demonstrated that CBL-B inhibition enhanced NK activation, IFNγ production and granzyme B secretion in response to the various stimulation conditions, including cytokines (such as IL-15 and IL12 + IL-18) and stimulatory receptors (such as NKp30 and NKG2D + CD244). NK cell proliferation was also enhanced in the presence of HOT-A. Single cell analysis further suggested that HOT-A increased NK polyfunctional cytokine expression during IL-12 + IL-18 activation. In a coculture system of NK cells and K562 target cells, HOT-A dose-dependently promoted primary human NK cell activation, cytokine production and cytotoxicity in the K562 cells. In the in vivo CT-26 tumor model, granzyme B+ NK cells in the TIL were significantly increased in the HOT-A treated tumor bearing mice and NK function gene signature was also increased by Nanostring analysis, suggesting an enhanced NK function in the tumor microenvironment. Taken together, the pre-clinical data presented here demonstrate that our CBL-B inhibitor enhances NK cell function in vitro and in vivo, suggesting that its enhancement of anti-tumor immunity may be driven through both effector T cell and NK cell-driven biology. Citation Format: Yilin Qi, Jun Kuai, Yingzhi Bi, David Greco, Samira Jaeger, Ken Carson, Hadi Danaee, Timothy Reilly, Geraldine Harriman, Fang Wang. Inhibition of the E3 ligase CBL-B enhances the effector function and proliferation of natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB337.