Prevent Tumor Growth Research Articles

Abstract CT276: Liberty-201: Maintenance fluoropyrimidine (FP) and bevacizumab with or without anti-lymphocyte activation gene-3 (LAG-3) antibody LBL-007 plus anti-programmed cell death protein-1 (PD-1) antibody tislelizumab (TIS) for patients (pts) with metastatic or unresectable microsatellite stable (MSS)/mismatch repair proficient (pMMR) colorectal cancer (CRC)

Abstract Background: LBL-007 is a monoclonal antibody targeting LAG-3, a receptor expressed on immune cells that negatively regulates T-cell proliferation and effector T-cell function. LAG-3 is frequently co-expressed with PD-1 on tumor-infiltrating T cells, and dual inhibition of LAG-3/PD-1 synergistically prevents tumor growth in mouse models. CRC is the second-highest cancer-related cause of death worldwide; pts diagnosed with metastatic CRC have a 5-year survival rate of 14%. Immunotherapy (IO) has only demonstrated clinical benefit in pts with microsatellite instability-high/MMR-deficient CRC, which accounts for 5% of CRC pts; however, improved therapy options for patients with MSS CRC remain limited. The combination of anti-LAG-3 + anti-PD-1 IO is currently being tested in clinical trials in pts with MSS CRC. Methods: This is a phase 1b/2, randomized, open-label study (NCT05609370). Pts with unresectable or metastatic CRC with locally or centrally confirmed pMMR or MSS status and no disease progression after induction treatment in first-line therapy are eligible. A maximum of 36 pts will be enrolled in phase 1b (safety run-in) to determine the recommended phase 2 dose. All pts will receive intravenous dual IO (LBL-007 + TIS) in combination with maintenance backbone (FP + bevacizumab). The dose of LBL-007 will be escalated from 150 mg every 3 weeks (Q3W) to 600 mg Q3W/400 mg Q2W using a Bayesian optimal interval design with informative prior. In phase 2, ~130 pts will be enrolled in the programmed death-ligand 1 (PD-L1)-positive group (tumor area positivity [TAP] ≥1%) and 60 patients in the PD-L1-negative group (TAP <1%). Pts with a PD-L1-positive status will be randomized 2:1:2 to receive dual IO + maintenance backbone (Arm A), LBL-007 + maintenance backbone (Arm B), or maintenance backbone only (Arm C). Pts with a PD-L1-negative status will be randomized 1:1 to receive dual IO + maintenance backbone (Arm D) or maintenance backbone only (Arm E). Treatment will continue until disease progression or unacceptable toxicity. The primary endpoint is safety in phase 1b and investigator-assessed progression-free survival (PFS) per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 in phase 2. Secondary endpoints include overall survival, PFS2, overall response rate, duration of response, safety, pharmacokinetics, and immunogenicity. Exploratory endpoints include potential biomarkers (including but not limited to PD-L1 and LAG-3). Citation Format: Heinz-Josef Lenz, Ying Yuan, Vivian Li, Juan Zhang, Lubna Jamal, Xiao Lin, Fuxiang Zhu, John H. Strickler. Liberty-201: Maintenance fluoropyrimidine (FP) and bevacizumab with or without anti-lymphocyte activation gene-3 (LAG-3) antibody LBL-007 plus anti-programmed cell death protein-1 (PD-1) antibody tislelizumab (TIS) for patients (pts) with metastatic or unresectable microsatellite stable (MSS)/mismatch repair proficient (pMMR) colorectal cancer (CRC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT276.

Abstract 3959: Unraveling the anti-tumor partnership of CD169+ macrophages and CD8+ T cells in a preclinical breast cancer model

Abstract In this study, we describe both the individual and the cooperative mechanisms by which CD169+ macrophages (MP) and CD8+ T cells support an anti-tumor immune response against breast cancer tumors. Breast cancer is the second leading cause of death in women of all ages each year, thus a deeper understanding of the molecular determinants that promote tumor development and suppress the anti-tumor immune response is still greatly needed. Here we use high dimensional flow cytometry, functional assays, and new mouse models to investigate the MP - T cell anti-tumor partnership. With our collaborators, we developed a preclinical mouse model of breast cancer by genetically engineering mammary stem cells to harbor three cancer driver mutations that are frequently associated in human patients: functional loss of the tumor suppressor genes p53 and mixed-lineage leukemia protein 3, and constitutive activation of the PI3 kinase. To characterize the immune cells in this tumor model, we developed a powerful 30-color high dimensional spectral flow cytometry panel to track and phenotype most known immune cell subsets. We found that CD169+ MP rapidly infiltrated and outnumbered all other populations during tumor growth, suggesting that they play an important role in shaping the tumor microenvironment. Surprisingly, although current literature describes breast cancer MP to be highly immunosuppressive, our phenotypic analysis showed only low expression of the pro-tumor MP markers TREM2 and CD206, but extremely high expression of the anti-tumor markers FOLR2 and CD64. To further investigate if these MP promoted or prevented tumor growth, we selectively depleted CD169+ MP in tumor-bearing mice and observed significantly accelerated tumor growth compared to the non-depleted control group, suggesting that these MP promoted anti-tumor responses. MP depletion also affected tumor-infiltrating CD8+ T cells, which produced lower effector functions (e.g., secretion of IFNγ, CCL3, TNF), indicating that MP may also boost anti-tumor CD8+ T cell responses. To further test if CD8+ T cells control tumor growth, we depleted either CD8+ or CD4+ T cells in tumor-bearing mice and observed that only CD8+ but not CD4+ T cell depletion accelerated tumor growth compared to the control group. MP have been documented to provide type I IFN signals to CD8+ T cells via the triggering of the STING pathway. To explore this mechanism in our model, we created a CD169creSTINGflox/KO mouse in which MP lack functional STING. Tumors in these mice grew much faster compared to those in the control group, consistent with the hypothesis that STING sensing occurs in tumor MP and promotes anti-tumor effector CD8+ T cell responses. Overall, our data provide evidence for an unexpected anti-tumor role for CD169+ MP in breast tumors, presumably through enhancing anti-tumor effector CD8+ T cell responses. Citation Format: Nicole Couturier, Marie Boutet, Kenta Nishitani, Wenjun Guo, Gregoire Lauvau. Unraveling the anti-tumor partnership of CD169+ macrophages and CD8+ T cells in a preclinical breast cancer 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 3959.

Abstract 2465: A novel agonist of CD137 as a single agent shows cancer immunoprevention efficacy against various tumor types that is not dependent on the trained immunity

Abstract Introduction: We recently demonstrated that pretreatment of mice with SA-4-1BBL, a novel oligomeric form of CD137 agonist, as a single agent prevents the development of various tumor types in mice. The tumor immunoprevention efficacy is long-lasting (>14 weeks) and contingent upon CD4+ immune cells, NK cells, and IFN-γ, but not CD8+ T and B cells. Trained immunity mediated by previously activated myeloid cells has been shown to have efficacy against cancer. The objective of this study was twofold: i) assess the role of trained immunity and ii) elucidate the mechanistic underlying of cancer immunoprevention. Study Design: Wild-type C57BL/6 mice and various transgenic (CD4+ OT-II.Rag+/+, CD4+ OT-II.Rag−/−, CD8+ OT-I/Rag2−/- KO, and Rag1−/−) mice on C57BL/6 background were pretreated subcutaneously with SA-4-1BBL or an agonistic Ab that does not have immunoprevention efficacy (3H3) twice two weeks apart. Mice were challenged two weeks later with a cervical cancer cell line (TC-1) and monitored for tumor development. A group of mice was also treated with monophosphoryl lipid A (MPLA), an agonist of TLR4 involved in trained immunity, using the above scheme. Mice treated with saline served as the control throughout all studies. Deep immunophenotyping was conducted to assess immune correlates of cancer immunoprevention. Results: Pretreatment with SA-4-1BBL prevented tumor growth in wild-type mice (> 60%), whereas all MPLA treated mice developed tumor. SA-4-1BBL did not show tumor immunoprevention efficacy in mice lacking adaptive immune cells (Rag1−/- KO), OT-II cells on Rag−/- or Rag+/+ background, and OT-I on Rag2−/− background. Deep immunophenotyping revealed a significant increase in the absolute number of CD4+ T central memory and CD4+ T stem cell-like memory cells, NK cells, NKT cells, and various myeloid cells in mice treated with SA-4-1BBL as compared with those treated with the 3H3 Ab or saline treated controls. Conclusions: These results demonstrate that cancer immunoprevention efficacy of SA-4-1BBL requires a diverse T cell receptor repertoire as OT-II mice on Rag+/+ background, restricted to a TCRβ chain, but have all other myeloid and NK cells, do not show efficacy. Furthermore, treatment with MPLA involved in trained immunity has no efficacy. Altogether, these observations do not implicate trained immunity as a mechanism of cancer immunoprevention. Elucidation of the mechanistic basis of SA-4-1BBL immunoprevention may have significant clinical implications. Citation Format: Feyza Nur Arguc, Mohammad Tarique, Vahap Ulker, Esma S. Yolcu, Haval Shirwan. A novel agonist of CD137 as a single agent shows cancer immunoprevention efficacy against various tumor types that is not dependent on the trained 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 2465.

Abstract 3341: Targeting CXCR4 via the small molecule inhibitor NMX1 as a therapeutic strategy to treat high-risk malignancies

Abstract Introduction: Cytokines are secreted by various cells of the immune system, endothelium, and stroma that play a critical role in cell survival. The activation of C-X-C chemokine receptor type 4 (CXCR4) by its ligand SDF-1 leads to signaling by the AKT and mTOR pathways. CXCR4 is highly expressed in several types of cancer and contributes to tumor growth, metastasis, and resistance to therapy. Interleukin 11 (IL-11) is a cytokine that often drives disease progression, as well as contributing to tumor immune evasion. NMX1 (NovoMedix) is a novel oral small molecule that inhibits mTOR signaling, CXCR4 expression, IL-11 secretion, and activates AMPK. In animal models of breast cancer, NMX1 effectively prevented tumor growth and metastasis, while also protecting from doxorubicin-induced cardiotoxicity. In this study, we investigated the anticancer activity of NMX1 in preclinical models of high-risk pediatric and adult malignancies. Methods: Cell viability assays using alamar blue were performed in a panel of pediatric and adult cancer cells, including leukemia, brain, breast, lung, neuroblastoma, and sarcoma. Cells were treated with increasing concentrations of NMX1 ranging from 250 nM to 16 µM for 96 hours, followed by determination of half maximal inhibitory concentrations (IC50). To validate target modulation, the level of IL-11 and CXCR4 was determined by ELISA and immunoblot, respectively. Protein kinase activity was assessed by phosphorylation levels. Induction of apoptosis was confirmed by caspase-mediated PARP cleavage. To identify effective drug combinations, NMX1-treated cells were screened with a comprehensive drug library. Synergy was evaluated in the top candidates by dose-response matrices and SynergyFinder. Safety and tolerability was tested in animal models. Results: NMX1 treatment induced cytotoxicity at micromolar concentrations in diverse types of cancer, with IC50 values ranging from 0.2-8.7 µM and 0.4-4.4 µM for leukemia and solid tumor cells, respectively. NMX1 induced cancer cell death by PARP cleavage and apoptosis. Mechanistically, CXCR4 expression, mTOR signaling, and IL-11 secretion were decreased upon treatment with NMX1, validating effective target modulation. The drug combination screen identified several candidates in a tumor type-dependent manner. Synergistic effects were observed using NMX1 combined with clinically relevant concentrations of rucaparib, linsitinib, and crizotinib, which target PARP, IGFR, and c-MET, respectively. Conclusions: Our study shows antitumor activity by NMX1 through inhibition of CXCR4, IL-11, and mTOR signaling, providing an effective therapeutic strategy for high-risk and refractory malignancies. Preclinical data on feasible drug combinations and biological correlates of activity support development of early phase clinical studies involving NMX1. Citation Format: Patrick Sipila, Son Tran, Chunfen Zhang, Laura G. Corral, Kyle W. Chan, Cathy A. Swindlehurst, Aru Narendran. Targeting CXCR4 via the small molecule inhibitor NMX1 as a therapeutic strategy to treat high-risk 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 3341.

Abstract 3339: TTFields for the management of spinal metastases in in vitro and in vivo models

Abstract Spine metastases often affect cancer patients, leading to significant morbidity and mortality. Their prevalence has increased over time due to the progressive improvements in cancer management which led to a longer survival of these individuals. Radiation therapy is the mainstay treatment for local control of these lesions, with surgery reserved for selected cases (e.g., spinal instability, neurological deficits secondary to neural compression requiring urgent decompression, separation surgery). Chemotherapy addresses the diffusely metastatic spread. However, when spinal metastases progress despite using all the appropriate available treatment, there follows a progressive decline in function and further treatment options are limited, highlighting the need for development of novel therapies. Tumor treating fields (TTFields) has demonstrated to impair tumor cell replication and are influenced by surrounding tissue, such as bone (presents non-conductive properties). Our goal was to investigate the effects of TTFields in in vitro and in vivo spinal metastasis models. We hypothesize that the deposition of TTFields is increased in bone, and this would ultimately result in vertebral metastasis growth suppression. A computational model using a simulated spine resection cavity with metallic hardware was used to investigate the TTFields intensity in various tissues. For in vitro studies, luciferase tagged KRIB osteosarcoma and A549 lung adenocarcinoma cell lines were cultured in demineralized human bone graft. These were treated with TTFields and cell viability was quantified. In vivo assays were performed using a previous validated murine orthotopic tumor model. The effects of TTFields in these animals were monitored with imaging (bioluminescence and MRI) and clinical findings (progressive neurological deficits milestones that corresponds to the degree of spinal cord compression). At the end of the study, tissue was collected for histologic analysis. Sham stimulation was used for control groups. Computational simulation demonstrated an elevated TTFields intensity in the tumor-bone interface of the resected vertebral body. In all experimental models, TTFields significantly suppressed tumor cell growth. In vivo models, this effect on cell proliferation prevented tumor growth into the spinal canal, delaying, or even preventing, neurological deficits. TTFields inhibited tumor growth in three-dimensional cultures and orthotopic murine models of spinal metastases, preventing functional loss. The bony environment surrounding the tumor demonstrated to enhance the intensity of electric fields on a computational simulation, which stands as an advantage with the use of TTFields for vertebral tumors. These results provide solid evidence supporting future clinical trials investigating the effects of this novel and promising technology for the treatment of spine metastases. Citation Format: Romulo Augusto Andrade de Almeida, Daniel Ledbetter, Xizi Wu, Ariel Naveh, Chirag Patel, Queena Gonzalez, Thomas H. Beckham, Robert Y. North, Laurence Rhines, Christopher A. Alvarez-Breckenridge, Claudio E. Tatsui. TTFields for the management of spinal metastases in in vitro and in vivo models [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 3339.

Abstract 4103: Novel mRNA-encoded HPV vaccine with APC maturation signal and endolysosomal trafficking domain demonstrates superior T cell-mediated immunogenicity and efficacious tumor clearance in vivo

Abstract Human Papillomavirus (HPV), particularly HPV16, is a known causative agent of cancers in various anatomical sites, notably the anogenital and oropharyngeal regions. Despite the availability of prophylactic HPV vaccines for almost two decades, vaccination rates among young females, who are at a heightened risk for cervical intraepithelial neoplasia (CIN) and subsequent invasive cancer, remain suboptimal. The current standard of care for cervical cancer, involving invasive surgery and chemotherapy-based treatments, underscores the pressing need for safer and more effective therapeutics to enhance patient survival and quality of life. In this context, while several HPV16-E6/E7 targeted therapeutic vaccines are in development and clinical trials, their clinical efficacy has been limited due to inadequate antigen presentation and suboptimal immunogenicity. Addressing this, our team has developed a novel, highly immunogenic mRNA therapeutic cancer vaccine, engineered to activate a robust T cell-mediated adaptive immune response and exhibit strong anti-tumor potential. Our innovative approach includes the design of an HPV16-E6/E7 mRNA vaccine (ABO2101) that incorporates a unique antigen-expressing cassette. This cassette features endolysosomal trafficking domains and antigen presenting cell (APC) activation stimulators, enhancing both Class I and Class II antigen presentation and APC maturation, thereby amplifying antigen-specific T cell responses. In both murine and human-derived experimental models, ABO2101 has demonstrated a significantly more potent adaptive T cell response against HPV16 antigens compared to other HPV16 mRNA therapeutic vaccines. In antitumor efficacy evaluations using TC-1 tumor-bearing mice, ABO2101 single-dose administration successfully eradicated both early-stage (50 mm³) and advanced (500 mm³) tumors. This was accompanied by a marked increase in HPV16-specific CD8+ cytotoxic T lymphocytes within the tumor microenvironment. Moreover, in prophylactic and adjuvant therapy models, ABO2101 prevented tumor growth across various dosages, indicating its robust preventative capabilities. Further, the combination of ABO2101 with immune checkpoint inhibitors, including PD-1 and CTLA-4 antibodies, displayed a synergistic effect in both immune-oncology sensitive and resistant animal models, suggesting a potential for combinational clinical therapy. In conclusion, our development of ABO2101, a mRNA-based HPV16-E6/E7 therapeutic cancer vaccine inclusive of APC maturation signals, represents a groundbreaking advancement in HPV-associated cancer treatment, as supported by our compelling preclinical data. Citation Format: Liang Du, Hongya Han, Jingshu Ma, Zhenxing Yang, Jinjuan Mao, Jijun Yuan, Bo Ying. Novel mRNA-encoded HPV vaccine with APC maturation signal and endolysosomal trafficking domain demonstrates superior T cell-mediated immunogenicity and efficacious tumor clearance in vivo [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 4103.

Abstract 1671: Exploring the mechanism of PP2A regulated cell death via macropinocytosis in pancreatic cancer

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death in the United States. KRAS is an oncogene mutated in 90% of PDAC patients. Oncogenic KRAS leads to aberrant cell proliferation and survival. KRAS is considered hard to target because resistance mechanisms to KRAS inhibitors are common highlighting the need to understand alternative strategies. PDAC tumor microenvironment evolves with cancer cell progression leading to vascular remodeling and blood vessel collapse. This makes the tumors nutrient deplete. To circumvent this, cells employ KRAS-dependent macropinocytosis- a nutrient scavenging pathway. Macropinocytosis is the process by which cells take up extracellular material by membrane ruffling to form vesicles which then fuse with lysosomes to release nutrients. Targeting KRAS-driven macropinocytosis can be crucial to limiting nutrients to the cell to prevent tumor growth. Protein Phosphatase 2A (PP2A), a heterotrimeric Serine/Threonine phosphatase, is a downstream regulator of KRAS. Its activity is repressed by mutated KRAS. We have seen that the activation of PP2A with small molecular activator, DT061, prevents macropinosomes from fusing with lysosomes which leads to cell death. But how DT061 regulated PP2A activity affects the macropinocytosis pathway is still unknown. The purpose of this study is to understand the mechanism of cell death brough upon by the activation of PP2A with DT061. Membrane ruffling is critical for the initiation of macropinocytosis. Rac is a GTPase usually involved with membrane ruffling. To show that Rac is needed for DT061 mediated macropinocytosis, we used EHT 1864, a Rac family inhibitor. Cells were treated with EHT 1864 before being treated with DT061 and stained with crystal violet. The inhibition of Rac prevented DT061 driven cell death, indicating that Rac is crucial to DT061 driven cell death. High molecular weight TMR-Dextran can only be taken up by the cells through macropinocytosis. Cells were treated with TMR-Dextran and EHT 1864. Cells with Rac inhibition showed lower uptake of TMR-Dextran than cells that were treated with DT061. Together these findings indicate to us that Rac plays a crucial role in the initiation of macropinocytosis and the regulation of cell death caused by the activation of PP2A. Understanding the pathway of DT061 mediated cell death can be vital to developing this concept as a therapeutic method of treatment for PDAC patients who currently have the lowest 5-year survival rate (12%) of all cancers. Citation Format: Indiraa Doraivel, Garima Baral, Claire M. Pfeffer, Brittany L. Allen-Petersen. Exploring the mechanism of PP2A regulated cell death via macropinocytosis in pancreatic cancer [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 1671.

Abstract 641: SCD1 upregulation due to STK11/KEAP1 loss upregulates SLC7A11 and causes deregulation of fatty acid metabolism leading to ferroptosis evasion

Abstract Introduction: Non-small cell lung cancer (NSCLC) with co-occurring loss-of-function (LOF) mutations in serine/threonine kinase 11 (STK11) and Kelch-like ECH-associated protein1 (KEAP1) (SK) are notably aggressive and unresponsive to chemo and immunotherapy, in current care standards. Novel therapeutic strategies are in demand to improve the outcomes for SK co-mutant NSCLC patients. We reported earlier that SK LOF mutations enhance cell proliferation and tumor growth in xenograft lung cancer models. We also found the ferroptosis evasion genes are enriched, and targeting ferroptosis regulators is a therapeutic opportunity in SK co-mutant NSCLC. Methods: We used CRISPR/Cas9 gene editing to create stable knockouts of STK11 (SKO), KEAP1 (KKO) or both (DKO), non-targeting control (NTC) in two NSCLC lines. We performed an in vitro druggable genome library CRISPR screening in the same NSCLC lines. We performed kinase profiling and RNA-seq in the cell lines to depict the transcriptomic and proteomic landscape, followed by western blotting and qRT-PCR validation. We also performed BODIPY/C11 assay and lipidomics to explore the ferroptosis and metabolic landscape of SK co-mutant NSCLC models. Results: Stearoyl CoA desaturase-1 (SCD1) was the top CRISPR screen hit, a critical modulator of fatty acid metabolism and ferroptosis evasion. SCD1 expression is significantly higher in NSCLC tissue than adjacent normal tissue (p<0.0001), and high SCD1 expression correlates with poorer prognosis in patients with NSCLC (p=0.027). GSEA from the bulk RNA sequencing in SK co-mutant cells pre-and post-SCD1 inhibitor treatment showed SCD1 inhibition leads to upregulation of the glutathione and glutamic acid pathways compared to NTC cells. Moreover, SK co-mutant cells have a significantly higher expression of SLC7A11, enabling cystine uptake and subsequent conversion to cysteine, essential for maintaining redox balance and cell survival. Hence, SK co-mutant cells are more resistant to cysteine depletion than NTC cells. SCD1 inhibition causes a decrease in SLC7A11 expression exclusively in DKO cells. Eventually, SCD1 inhibition leads to global metabolomic changes in SK co-mutant cells, including key pathways involved in lipid and glucose metabolism. Finally, genetic and pharmacological inhibition of SCD1 prevents tumor growth and sensitizes DKO cells to ferroptosis inducers like erastin. Conclusions: Our findings highlight the importance of the SCD1-SLC7A11 axis in regulating ferroptosis in SK co-mutant models. Current data impact our understanding of ferroptosis in NSCLC and its ability to target SCD1 or ferroptosis in cancer treatment. Compounds such as inhibitors of SLC7A11 (Erastin/IKE) are safe to use with acceptable toxicity and established doses. Hence, our study will facilitate the translation of ferroptosis inducers or SCD1 inhibitors in clinical trials. Citation Format: Utsav Sen, Charles Coleman, Dan Hasson, Triparna Sen. SCD1 upregulation due to STK11/KEAP1 loss upregulates SLC7A11 and causes deregulation of fatty acid metabolism leading to ferroptosis evasion [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 641.

Abstract 4291: Bai1 inhibition promotes ferroptosis of breast cancer cells to prevent tumor growth in human xenograft tumor models

Abstract The adhesion G-protein coupled- receptor 1 ADGRB1/Bai1 was identified as a phosphatidylserine receptor that cooperates with ELMO/Dock180/Rac to promote maximal engulfment of apoptotic cells. This function of Bai1 was also shown to promote myoblasts fusion. Cell-cell fusion has been demonstrated to occur in normal physiological functions as well as in diseases. Studies have shown in cancer that cell-cell fusion increases tumor heterogeneity, drug resistance, and metastasis as hybrids acquire new properties that allow them to migrate and proliferate at new sites. Activation of ELMO-Dock signaling pathway has been associated with breast cancer metastasis. Moreover, ELMO1 has been identified as a modifier of breast cancer risk for BRCA mutation carriers. Rac1 was also shown to be overexpressed in proliferative breast disease, preinvasive and invasive breast carcinoma as well as lymph node metastases. Our recent studies showed that breast cancer cell fusion with bone marrow-derived mesenchymal stem cells was modulated by hypoxic conditions and promoted by apoptosis. Our hypothesis is that hypoxia stress induced apoptosis activates the phosphatidylserine receptor Bai1, and it signals through the ELMO/DOCK180/Rac to promote breast cancer cell fusion-driven tumor heterogeneity and metastasis. In this study, we determined the ability of Bai1 depleted breast cancer cells to induce tumor growth in immunocompromised mice. We used Bai1 sgRNA to inhibit Bai1 expression in the breast cancer cell MDA-MB-231. These cells (1x 106) together with bone marrow-derived mesenchymal stem cells (MSCs) (1x 106) were injected into the fat pad of the breast of 15 NOD SCID female mice. Control mice (15) received the same cells with no inhibition of Bai1. The mice were monitored for eight weeks. We found that Bai1 inhibition significantly reduced tumor growth in these mice in the course of time (P<0.05). The average tumor weight was also significantly reduced when Bai1 was inhibited (P<0.001). RNA sequencing of tumors generated from these mice revealed a significant increase in the expression of genes driving ferroptosis and a reduction in critical oncogenes in the experiment group compared to the control group. These findings will be helpful in the development of new classes of drugs effective in breast cancer treatment. Grant Support The NIH/NIMHD grant 5U54MD015929-03, the Society for Investigative Dermatology (SID) Freinkel Diversity Fellowship, and NSF Award Notice for Award ID 2142465 (awarded to FNK). Citation Format: Felicite Kamdem Noubissi, Oluwatowin Odubanjo, Hung-Chung Huang, Brenda Ogle, Paul Tchounwou. Bai1 inhibition promotes ferroptosis of breast cancer cells to prevent tumor growth in human xenograft tumor models [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 4291.

A self-assembled, genetically engineered, irradiated tumor cell debris vaccine.

Vaccine-based therapeutics for cancers face several challenges including lack of immunogenicity and tumor escape pathways for single antigen targets. It has been reported that radiotherapy has an in situ vaccine effect that provides tumor antigens following irradiation, helping to activate antigen-presenting cells (APCs). Herein, a new vaccine approach is developed by combining genetically engineered irradiated tumor cell debris (RTD) and hyaluronic acid (HA), termed HA@RTD. A cancer cell line is developed that overexpresses granulocyte-macrophage colony-stimulating factor (GM-CSF). A hydrogel was developed by covalent conjugation of HA with RTD proteins that acted as a potent vaccine system, the effects which were probed with T cell receptor sequencing. The engineered vaccine activated antitumor immunity responses and prevented tumor growth in mice even with a single immunization. HA@RTD vaccine efficacy was also assessed in therapeutic settings with established tumors and in combination with immune checkpoint blockade.

Ferroptosis Inducers as Promising Radiosensitizer Agents in Cancer Radiotherapy.

Radiotherapy (RT) failure has historically been mostly attributed to radioresistance. Ferroptosis is a type of controlled cell death that depends on iron and is caused by polyunsaturated fatty acid peroxidative damage. Utilizing a ferroptosis inducer may be a successful tactic for preventing tumor growth and radiotherapy-induced cell death. A regulated form of cell death known as ferroptosis is caused by the peroxidation of phospholipids containing polyunsaturated fatty acids in an iron-dependent manner (PUFA-PLs). The ferroptosis pathway has a number of important regulators. By regulating the formation of PUFA-PLs, the important lipid metabolism enzyme ACSL4 promotes ferroptosis, whereas SLC7A11 and (glutathione peroxidase 4) GPX4 prevent ferroptosis. In addition to introducing the ferroptosis inducer chemicals that have recently been demonstrated to have a radiosensitizer effect, this review highlights the function and methods by which ferroptosis contributes to RT-induced cell death and tumor suppression in vitro and in vivo.

Thermo-sensitive PLGA-PEG-PLGA hydrogel for sustained release of EGF to inhibit cervical cancer recurrence

Overexpression of epidermal growth factor receptor (EGFR) in cancer is a key cause of recurrence of cervical cancer (CC). Although the EGF-EGFR pathway has been studied for decades, preventing tumor growth and recurrence caused by peripheral EGF remains a great challenge. In this work, a strategy is proposed to reduce the stimulation of high concentration EGF on tumor growth by using a thermo-sensitive hydrogel. The hydrogel is a triblock copolymer composed of polyethylene glycol (PEG) and poly (lactide glycolide) (PLGA). Based on the excellent temperature sensitivity, carrier capacity, swelling property and biocompatibility, the hydrogel can absorb the liquid around the tumor by injection and release EGF continuously at low concentration. The inhibitory effect of hydrogel on tumor growth is fully confirmed by an implanted tumor mouse model with human cervical cancer cell lines (HeLa) using triple-immunodeficient NCG mice. Compared with free EGF, the EGF-loaded hydrogel can hardly induce surface plasmon resonance (SPR) response, which proves that hydrogel can effectively weaken cytoskeleton rearrangement and inhibit cell migration by continuously releasing low concentration EGF. In addition, the EGF-loaded hydrogel can reduce cell proliferation by delaying the progress of cell cycle progression. Taken together, the hydrogel can effectively protect tumor microenvironment from the stimulation of high concentration EGF, delay cancer cellular processes and tumor growth, and thus providing an approach for inhibiting tumor recurrence of CC.

Rapamycin inhibits B16 melanoma cell viability invitro and invivo by inducing autophagy and inhibiting the mTOR/p70‑S6k pathway.

Rapamycin is an immunosuppressant that has been shown to prevent tumor growth following organ transplantation. However, its exact mode of antitumor action remains unknown. The present study used the B16-F10 (B16) murine melanoma model to explore the antitumor mechanism of rapamycin, and it was revealed that rapamycin reduced B16 cell viability in vitro and in vivo. In addition, in vitro and in vivo, the results of western blotting showed that rapamycin reduced Bcl2 expression, and enhanced the protein expression levels of cleaved caspase 3 and Bax, indicating that it can induce the apoptosis of B16 melanoma cells. Furthermore, the results of cell cycle analysis and western blotting showed that rapamycin induced B16 cell cycle arrest in the G1 phase, based on the reduction in the protein expression levels of CDK1, cyclin D1 and CDK4, as well as the increase in the percentage of cells in G1 phase. Rapamycin also significantly increased the number of autophagosomes in B16 melanoma cells, as determined by transmission electron microscopy. Furthermore, the results of RT-qPCR and western blotting showed that rapamycin upregulated the protein expression levels of microtubule-associated protein light chain 3 (LC3) and Beclin-1, while downregulating the expression of p62 in vitro and in vivo, thus indicating that rapamycin could trigger cellular autophagy. The present study revealed that rapamycin in combination with chloroquine (CQ) further increased LC3 expression compared with that in the CQ group, suggesting that rapamycin induced an increase in autophagy in B16 cells. Furthermore, the results of western blotting showed that rapamycin blocked the phosphorylation of p70 ribosomal S6 kinase (p70-S6k) and mammalian target of rapamycin (mTOR) proteins in vitro and in vivo, thus suggesting that rapamycin may exert its antitumor effect by inhibiting the phosphorylation of the mTOR/p70-S6k pathway. In conclusion, rapamycin may inhibit tumor growth by inducing cellular G1 phase arrest and apoptosis. In addition, rapamycin may exert its antitumor effects by inducing the autophagy of B16 melanoma cells in vitro and in vivo, and the mTOR/p70-S6k signaling pathway may be involved in this process.

Co(II), Mn(II), and Fe(III) complexes of water‐soluble hydrazone bearing 2‐nicotinoylhydrazineylidene moiety: Preparation, characterization, cyclic voltammetry, computational and biological studies

A water‐soluble hydrazone ligand bearing 2‐nicotinoylhydrazineylidene moiety, sodium 4‐hydroxy‐3‐([2‐nicotinoylhydrazineylidene]methyl) benzenesulfonate (NaH2NH), and its complexes with Co(II), Fe(III), and Mn(II) have been synthesized and characterized. The structure of the ligand and its complexes were analyzed using various spectroscopic techniques such as FT‐IR, 1H‐ and 13C‐NMR, EI‐mass, powder X‐ray diffraction, UV–visible spectra, and magnetic measurements. The infrared spectra indicated that the ligand behaves as a negatively charged bidentate ligand (ON) through the deprotonation of the enolic (OH) group and the azomethine (C=N) group in the [Co (NaHNH)2(H2O)4Cl2].3H2O complex. In the [Fe (NaNH)2(H2O)Cl].2H2O complex, the ligand behaves as bi‐negative tetradentate (O2N2) through the protonation of the enolic oxygen and the nitrogen atom of the azomethine group in two nicotinic hydrazone molecules. Another coordination type was observed for [Mn (NaNH)(H2O)3] complex in which NaH2NH acts as bi‐negative tridentate ONO through the nitrogen atom of the azomethine group, the protonated of enolic and phenolic groups. The fluorescence spectrum of the NaH2NH was found to be quenched upon addition of these Co(II) and Mn(II) ions. The antimicrobial results indicated that the Mn(II) complex exhibited the highest antibacterial potency against all targeted organisms. The results of antioxidants demonstrated that the complexes exhibited higher activity compared to the free ligand, indicating an enhancement of antioxidant activity through complexation. The tested substances established strong nuclease‐like activity at a high concentration of 7.5 mM, leading to the complete degradation of DNA as observed in agarose gel electrophoresis. This suggests that these compounds have the potential to serve as antitumor agents in vivo by inhibiting DNA replication in cancer cells and preventing tumor growth. The interaction of the isolated compounds with 1c14 and 6vj3 targets was discussed using molecular docking.

Abstract B029: Combinations of metformin and BH3-mimetic drugs synergistically induce immunogenic cell death in breast cancer cells

Abstract There is a compelling medical need for novel and safe treatment options for patients with advanced and difficult to treat breast cancer. Our previous findings have demonstrated that a combination of metformin and navitoclax (BCL-2/BCL-XL inhibitor) successfully prevents tumor growth in patient derived xenograft and syngrafted mouse models of aggressive MYC-driven breast cancer. This outcome is due to the synthetic lethal action of these drugs in tumors with elevated MYC expression. Here, we explored the combinations of metformin with all clinically applicable anti-apoptotic BCL-2 family protein inhibitors (BH3-mimetics) in a panel of 15 triple-negative breast cancer cell lines with distinct endogenous MYC levels, as well as inducible MYC models. We used cytotoxicity assays and analyzed the data with SynergyFinder, an interactive analysis and visualization tool, to calculate the degree of combination synergy utilizing Zero interaction potency reference model. Through these cytotoxicity assays, we defined which metformin and BH3-mimetic drug combination induces most synergistic cell death in a MYC-synthetic lethal manner. Additionally, we used DAMP reporter cell lines and peripheral blood mononuclear cell-based dendritic cell activation assays to evaluate the immunogenic potential of combination-induced cell death. We show that targeting MCL-1 or BCL-2/XL with BH3-mimetics MIK-665 and navitoclax, respectively, and combining them with metformin, induces synergistic cell death in MYC-high cancer cells. We also show that cell death caused by these drug combinations promotes maturation of dendritic cells. In conclusion, targeting MYC-high tumors with synthetic lethal strategies, including BH3-mimetics and metformin, may offer new opportunities to combine with immunotherapies for the treatment of breast cancer. Citation Format: Mariel Savelius, Linda Id, Johanna Anttila, Ruixian Liu, Ilida Suleymanova, Jani Saarela, Juha Klefström. Combinations of metformin and BH3-mimetic drugs synergistically induce immunogenic cell death in breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B029.

Abstract B047: Epigenetic reprogramming by CBP/P300 bromodomain inhibition of triple-negative breast cancer and the immune microenvironment

Abstract Triple-negative breast cancer (TNBC) is a biologically heterogeneous and clinically important breast cancer subtype because if considered a distinct disease, TNBC would rank as the 5th leading cause of cancer deaths in women. TNBC patients resistant to standard-of-care therapies have poor survival. Tumor-associated immune cells such as neutrophils and macrophages can display a pro-tumoral phenotype and contribute to therapy resistance. Epigenetic enzymes have been shown to reprogram both the tumor cells and tumor-associated immune cells. Thus, targeting epigenetic enzymes is a potential strategy to improve the response of TNBC to standard-of-care therapies. Previously, our laboratory performed an epigenetic drug screen and identified several epigenetic inhibitors. We selected IACS-70654, a novel selective p300/CBP BRD inhibitor, to be the primary focus of this study. We determined the effects of IACS-70654 in our unique syngeneic TNBC mouse models. In a neutrophil-enriched luminal-like TNBC model 2208L, IACS-70654 as a single agent initially caused tumor regression and then durably prevented tumor growth, which is not usually observed for single agents in these aggressive TNBC models. IACS-70654 also substantially decreased infiltrated neutrophils. In the peripheral blood and bone marrow of 2208L tumor-bearing mice, IACS-70654 reduced neutrophils to the same level as non-tumor-bearing mice. Based on this finding, we treated 2208L tumors with IACS-70654 in combination with immune checkpoint blockade anti-PD1 and docetaxel. After 27 days, all treatments besides anti-PD1 were stopped, and the combination-treated tumors grew slower than the ones only treated with IACS-70654. More importantly, in tumors treated with IACS-70654 in combination with anti-PD1, there was a substantial increase in CD8+ T cell infiltration. In addition, B cell infiltration was induced in the tumor stroma, indicating that IACS-70654 stimulated immune response in the tumor microenvironment. In contrast, IACS-70654 failed to significantly reduce the growth of T12, a Claudin-low macrophage-enriched model. Therefore, we hypothesized that the infiltrated immune profiles, specifically myeloid cell infiltration, might partially account for this differential response. Accordingly, we studied the effects of IACS-70654 in two additional models, basal-like T6 and luminal-like 2250L tumors, which also have relatively high neutrophil infiltration. However, one of the principal differences in immune profiles between the two models and 2208L is that they also are infiltrated with twice as many macrophages. The response of infiltrated immune cells to IACS-70654 in those two models differed from those of 2208L tumors. IACS-70654 reduced monocytic myeloid-derived suppressor cells and pro-tumor macrophages. These results illustrate the immune heterogeneity among our tumor models. We are currently performing ChIP-seq and single-cell ATAC-seq to elucidate potential mechanisms and pathways employed by IACS-70654 in both tumor and tumor-associated immune cells. Citation Format: Xueying Yuan, Michael Soth, Na Zhao, Philip Jones, Jeffrey Rosen. Epigenetic reprogramming by CBP/P300 bromodomain inhibition of triple-negative breast cancer and the immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B047.

Ethyl 2,2-difluoro-2-(2-oxo-2H-chromen-3-yl) acetate attenuates the malignant biological behaviors of non-small cell lung cancer via suppressing EGFR/PI3K/AKT/mTOR signaling pathway.

The goal of the study is to examine the impact on the malignant biological behaviors of non-small cell lung cancer (NSCLC) of a novel coumarin derivative, ethyl 2,2-difluoro-2-(2-oxo-2H-chromen-3-yl) acetate (C2F). It also aims to define its underlying mechanism. NSCLC cell lines and xenograft nude mice model were conducted to explore the anti-NSCLC effects of C2F in vitro and in vivo. Then, network pharmacology analysis and molecular docking were applied to estimate the possible targets of C2F in NSCLC. Finally, the underlying mechanism of C2F against NSCLC cellular proliferation and tumor development was confirmed using inhibitors or activators of the PI3K/AKT signaling pathway. Our results showed that C2F was able to inhibit proliferation, migration, and invasion of NSCLC cell lines, induce cell cycle arrest and apoptosis in vitro, and prevent tumor growth in vivo. In addition, the estimated glomerular filtration rate and its downstream pathway (PI3K/AKT/mTOR) were found to be critical for the anti-NSCLC activity of C2F. C2F inhibits malignant biological behaviors of NSCLC by suppressing EGFR/PI3K/AKT/mTOR signaling pathway.

Polymeric nanocapsules loaded with poly(I:C) and resiquimod to reprogram tumor-associated macrophages for the treatment of solid tumors.

In the tumor microenvironment (TME), tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer. Toll-like receptors (TLRs) ligands, such as poly(I:C) or resiquimod (R848) are able to reprogram TAMs towards M1-like antitumor effector cells. The objective of our work has been to develop and evaluate polymeric nanocapsules (NCs) loaded with poly(I:C)+R848, to improve drug stability and systemic toxicity, and evaluate their targeting and therapeutic activity towards TAMs in the TME of solid tumors. NCs were developed by the solvent displacement and layer-by-layer methodologies and characterized by dynamic light scattering and nanoparticle tracking analysis. Hyaluronic acid (HA) was chemically functionalized with mannose for the coating of the NCs to target TAMs. NCs loaded with TLR ligands were evaluated in vitro for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry, using primary human monocyte-derived macrophages. For in vivo experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry and multispectral immunophenotyping. We have developed polymeric NCs loaded with poly(I:C)+R848. Among a series of 5 lead prototypes, protamine-NCs were selected based on their physicochemical properties (size, charge, stability) and in vitro characterization, showing good biocompatibility on primary macrophages and ability to stimulate their production of T-cell attracting chemokines (CXCL10, CCL5) and to induce M1-like macrophages cytotoxicity towards tumor cells. In mouse tumor models, the intratumoral injection of poly(I:C)+R848-protamine-NCs significantly prevented tumor growth and lung metastasis. In an orthotopic murine lung cancer model, the intravenous administration of poly(I:C)+R848-prot-NCs, coated with an additional layer of HA-mannose to improve TAM-targeting, resulted in good antitumoral efficacy with no apparent systemic toxicity. While no significant alterations were observed in T cell numbers (CD8, CD4 or Treg), TAM-reprogramming in treated mice was confirmed by the relative decrease of interstitial versus alveolar macrophages, having higher CD86 expression but lower CD206 and Arg1 expression in the same cells, in treated mice. Mannose-HA-protamine-NCs loaded with poly(I:C)+R848 successfully reprogram TAMs in vivo, and reduce tumor progression and metastasis spread in mouse tumors.

Improved immunotherapy techniques using nano technology to treat targeted cancer

Immunotherapy, a promising cancer treatment method, faces challenges such as off-target effects and limited efficacy. Researchers are utilizing nanotechnology to enhance the accuracy and effectiveness of immunotherapy. In a clinical trial, nanoparticles were used to directly carry immunomodulatory drugs to tumor locations, enhancing cytotoxic T cell activation and preventing tumor growth. Patients receiving nanoparticle-enhanced immunotherapy experienced longer progression-free survival times and higher response rates than those receiving standard immunotherapy alone. This study demonstrates how nanotechnology is revolutionizing immunotherapy approaches for focused cancer treatment.

Arsenic Trioxide Induces Retinoic Acid-Related Orphan Receptor Beta and Blocks the WNT Pathway to Inhibit Stemness in Glioblastoma.

Glioblastoma (GBM) is a malignant primary brain tumor and an essential contributor to morbidity and mortality globally. Arsenic trioxide (ATO) exerts specific roles in preventing tumor growth. This study investigated the role of ATO in GBM cell behaviors and stemness. The effects of ATO on the malignant behavior of GBM cells, tumor stemness, and epithelial-mesenchymal transition (EMT) factors in mouse tumor tissues were explored. Targets of ATO in GBM were predicted using multiple databases. Subsequently, the expression of retinoic acid-related orphan receptor beta (RORB), WNT-1, β-Catenin, and c-Myc expression were examined in GBM cells before and after ATO treatment.ATO inhibited the malignant behavior of GBM cells in vitro and slowed down the GBM growth in vivo by inhibiting the stemness. The inhibitory effect of ATO on GBM was achieved by promoting RORB levels and strengthening the antagonism to β-Catenin to inhibit Wnt signaling, thus inhibiting tumor growth. Collectively, ATO induced RORB levels in GBM cells and strengthened the antagonistic effect on β-Catenin, thus inhibiting WNT signaling and tumor growth.