Slowed Tumor Growth Research Articles

Abstract 1118: Mitigating radiation-induced lymphopenia using interleukin-15: Preclinical rationale for clinical translation

Abstract Background: Radiation therapy is a common treatment for cancer patients, with nearly 50% of them undergoing this procedure. While radiation is effective at targeting tumor cells, it also has the unintended consequence of killing lymphocytes, which are among the most sensitive cells to radiation within the erythroid, myeloid, and lymphoid lineages. This phenomenon is known as radiation-induced lymphopenia (RIL) and is considered a negative prognostic factor in various malignant solid tumors. It occurs severely in over 40% of solid tumor patients who receive radiotherapy. Some previous studies have shown promising results with IL15 therapy in increasing circulating lymphocytes in cancer patients. However, until now, no studies have investigated the impact of IL15 in the context of radiation-induced lymphopenia. Our study aims to investigate whether IL15 can boost lymphocyte numbers and decrease tumor burden following radiation treatment in a mouse model. Methods: To conduct our study, we subcutaneously injected TSA tumor cells into the flank region of mice. Once the tumors reached a palpable size, approximately 50mm3, we divided the mice into four groups: Control (Con), Irradiation (IR), Control with IL15 (Con+IL15), and Irradiation with IL15 (IR+IL15). The irradiated mouse groups received a radiation dose of 2 Gray in the thorax and 1 Gray in the spleen for five consecutive days, simulating the severe radiation-induced lymphopenia observed in cancer patients. After the radiation treatment, the IL15-treated groups received IL15 in combination with recombinant IL15 in a 1:6 ratio weekly. Throughout the experiment, we monitored the circulating lymphocytes and tumor growth. After two weeks following the last radiation dose, we euthanized the mice, collected blood, spleen, and tumor samples, and identified immune cells using flow cytometry. Results: The radiation treatment significantly increased tumor growth while decreasing the number of lymphocytes, including B cells, T cells, and NK cells, in the blood. IL15 administration led to an increase in the number of lymphocytes both in the blood and within the tumor compared to the irradiated group. We observed a trend toward reduced tumor growth in the group that received both IL15 and radiation (IR+IL15) compared to the group that received radiation alone (IR). Conclusion: IL15 effectively boosted the number of lymphocytes in both the bloodstream and tumors, resulting in slower tumor growth in the IL15-treated group compared to the irradiated group. This study is of significant importance as it demonstrates the potential of IL15 to counteract radiation-induced lymphopenia in mice with TSA tumors. Further testing of these findings is currently underway in MC38 tumor-bearing mice, a cell line known to be more radiation-sensitive. Citation Format: Priti Gupta, Min Wang, Steven Lin. Mitigating radiation-induced lymphopenia using interleukin-15: Preclinical rationale for clinical translation [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 1118.

Abstract 1369: PD-1 directly suppresses macrophage metabolism to restrict anti-tumor immunity in an obesity-cancer connection

Abstract Obesity has been established as a leading risk factor for many cancers and can drive tumor progression and metastasis. Paradoxically, obesity has in some cases been associated with better survival and improved response to immune checkpoint blockade therapies. The role of the immune system in the obesity-cancer connection and how obesity affects immunotherapy responses, however, have been unclear. Here we show that obesity enhanced PD-1 expression on macrophages to reduce phagocytosis and antigen presentation to T cells that correlated with reduced T cell expansion and function. This obesity associated immune dysfunction, however, primed for enhanced anti-tumor response to PD-1 blockade. Single cell RNAseq showed obesity remodels myeloid and T cell populations but does not impact non-immune cell populations such as fibroblasts and epithelial cells. Specifically, obesity increased the overall number of tumor-associated macrophages (TAM) while effector T cells were decreased in abundance. Interestingly, the frequency of macrophages expressing PD-1 increased, while the remaining T cells maintained similar or reduced PD-1 expression and appeared less activated. Further, T cell depletion from the tumor microenvironment, enhanced macrophage PD-1 expression and worsened PD1-associated TAM dysfunction in an obesity enhanced manor. Obesity associated cytokines and adipokines including IFN-ã, leptin and insulin induced PD-1 expression on macrophages. This expression is associated with mTOR and c-myc activation signaling pathways; as such inhibiting these signaling pathways blocked PD1 expression. Bulk RNA sequencing analysis revealed PD-1+ TAMs had an altered gene profile compared to PD-1− TAMs. PD1 expressing macrophages exhibited increased mitochondrial respiration and expression of oxidative phosphorylation, increased lipid uptake and increased cell cycling related genes. Conversely PD1- TAMs exhibited increased phagocytosis and antigen presentation. PD-1/PDL1 interaction directly regulated TAMs, as recombinant PDL-1 reduced glycolysis and phagocytosis in purified macrophages, and these effects could be reversed with blocking PD-1 antibody. Conversely, PD-1−/− TAMs had reduced lipid uptake but high rates of glycolysis, phagocytosis, and expression of MHC-II. Myeloid-specific PD-1 deficiency correlated with slower tumor growth and decreased LAG3 and increased CD69 on CD8 T cells. In addition, myeloid specific PD1 deficiency enhanced TAM antigen presentation through OVA specific CD8 T cell activation. These findings identify PD-1 as a metabolic regulator in TAM dysfunction and reveal a unique PD-1 mediated and macrophage-specific mechanism for immune tumor surveillance and checkpoint blockade. This may contribute to improved immunotherapy response in TAM-enriched tumors and obesity. Citation Format: Jackie Bader, Jeffrey Rathmell. PD-1 directly suppresses macrophage metabolism to restrict anti-tumor immunity in an obesity-cancer connection [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 1369.

Abstract 3206: Tumor-targeted nanoparticles for cancer therapy

Abstract Introduction: Untargeted cancer therapy is often toxic, limiting the maximum tolerated dose (MTD) and thus potential cure. Targeted therapy with antibody-drug conjugates (ADCs) has demonstrated greater efficacy, as with those directed at HER2 for triple negative breast cancer and other HER2 expressing tumors. However, ADC drug-antibody ratio (DAR) is limited, typically 3 to 1. In contrast, antibody-targeted nanoparticles deliver thousands of drug molecules per nanoparticle, with potentially greater anti-tumor effect. Experimental Procedures: Antibody targeted nanoparticles (TNP) were created that preferentially deliver cytotoxic molecules to tumor cells. TNPs were created with various antibodies and payloads and evaluated for their efficacy against 12 different adult and pediatric malignancies (see below), using tumor xenografts in NOG-SCID mice. Survival of control, free drug, and TNP treated animals was compared. MED was also determined. Non-tumor bearing animals treated at the same dose and schedule were evaluated for evidence of toxicity. PK/PD metrics were obtained and drug delivery to normal and tumor tissue was evaluated microscopically and by mass spectroscopy. Data: Plasma TNP drug levels persist for 48 hours, compared to free drug which is undetectable by 2 hours. Microscopically, nanoparticles accumulate in tumor tumor tissue but not normal tissue. Drug levels are typically 15 times higher in tumor tissue than normal tissue by quantitative mass spectroscopy. The minimum effective dose (MED) is reduced 25-fold for Ewing sarcoma, from 50 mg/kg to 2 mg/kg. TNPs also cross the blood-brain barrier (BBB), and were used to treat orthotopic GBM PDX xenografts, prolonging animal survival 3-fold over control or free drug treated animals. Ewing sarcoma, osteosarcoma, triple negative breast cancer, GBM, hepatoma, and adult ALL, bearing animals all showed complete tumor ablation and 100% survival when treated biweekly at 10mg/kg. Pancreatic, prostate, and ovarian Ca showed 100% survival in TNP treated mice after all control animals had died but remained tumor bearing with slow but progressive tumor growth when treated identically. Lung and colorectal Ca and melanoma bearing animals treated similarly with TNPs showed 50-80% survival at the time of control animal death. No animal in any study showed evidence of off-target toxicity. Conclusions: Tumor antigen targeted, cytotoxic molecule containing nanoparticles have been created that show broad utility in the treatment of at least a dozen different adult and pediatric cancers. Six of 12 types showed complete tumor ablation when treated at 10mg/kg biweekly injections, and the remainder showed enhanced anti-tumor effect and survival compared to free drug treatment. We conclude that use of tumor antigen targeted, small molecule cytotoxic containing nanoparticles results in dramatically enhanced tumor response and in many case, complete tumor ablation where the same free drug fails to do so. Citation Format: HyungGyoo Kang, Bryon Upton, Jon Nagy, Timothy Triche. Tumor-targeted nanoparticles for cancer therapy [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 3206.

Abstract 5387: Host-derived CCL2 drives prostate cancer bone metastasis via CCND2/STAT3 pathway

Abstract Overexpression of tumor-derived CCL2 has been shown to partially promote prostate cancer (PCa) bone metastasis, but the function of host-derived CCL2 is not fully understood. We first developed a mouse model using intracardiac injection and obtained highly metastatic PCa cell subclones through in vivo selection. The highly metastatic tumor cells were injected intracardially into CCL2 knockout (CCL2 KO) mice and wild-type (WT) controls, and the tumor cell growth and metastasis were monitored weekly by bioluminescence imaging. The osteolytic lesions were evaluated by radiography, micro-computed tomography (microCT) and immunohistochemistry (IHC) staining. The cells in the bone microenvironment were analyzed by flow cytometry. Also, RNA sequencing (RNA-seq) analysis was performed on the cells either from CCL2 KO mice or WT controls. We found that a loss of host-derived CCL2 significantly retarded tumor growth in bone and prolonged mouse survival. In addition, bone density analysis revealed a decrease in osteolytic lesion in CCL2 KO mice, compared to WT controls. Systemically, CCL2 deficiency reduced the proportion of granulocyte-myeloid-derived suppressor cell (G-MDSC) populations with immunosuppressive function and decreased the PCa conditioned medium (CM)-induced osteoclast formation. The enrichment analysis revealed that the dysregulated genes in bone metastatic cells from CCL2 KO mice were significantly enriched in several biological processes, including vitamin metabolism, monoatomic anion homeostasis, immune response, and bone growth. Additionally, we also confirmed that the CCND2/STAT3 pathway is apparently suppressed in the cells from CCL2 KO mice. This study demonstrated that targeting the host-derived CCL2 might be a novel potential anti-metastatic approach. Supported by the NSFC projects (81972766, 81972420, 82173336), as well as grants from the Science and Technology Project of Shenzhen (JCYJ20190809161811237, JCYJ20210324104214040). Key words Host-derived CCL2; Prostate cancer; Bone metastasis; osteoclast; CCND2 Citation Format: Jie Meng, Ming Chang, Siyuan Qin, Xin Huang, Weiping Liang, Haibo Tong, Jian Zhang. Host-derived CCL2 drives prostate cancer bone metastasis via CCND2/STAT3 pathway [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 5387.

Abstract 2719: Papiliximab, a bispecific nanobody targeting CD47 and PDL1 retards tumor growth without hemolysis

Abstract Cancer cells are known to express immune checkpoint proteins, enabling them to evade immune surveillance. In response, anti-cancer therapies targeting these proteins have been developed. While immune checkpoint inhibitors targeting PD-L1 have been clinically approved, their effectiveness as standalone therapies is limited, with only a 20% response rate. To improve responsiveness, bispecific monoclonal antibodies (MoAbs) targeting both CD47 and PD-L1 have been developed. These bispecific antibodies activate both the innate (by blocking CD47) and adaptive (by blocking PD-L1) immune systems simultaneously. However, conventional antibodies targeting CD47 have been dropped during development due to the risk of hemolysis. In this study, we developed a bispecific single-domain antibody (nanobody, Nb) that targets both CD47 and PD-L1 with a minimal hemagglutination risk. Using phage display libraries from alpacas immunized with recombinant antigens, we screened for an anti-CD47 Nb and an anti-PD-L1 Nb. The affinities (Kd) of these Nbs for their antigens were 7.0 × 10−9 and 9.7 × 10−9, respectively. To maximize affinity for both antigens, a bispecific antibody (Papiliximab) was designed to tandemly array anti-CD47 and PD-L1 Nbs with the Fc region of IgG4. Papiliximab has a lower affinity for RBC (up to 3 μM) than conventional anti-CD47 MoAbs reported earlier. Papiliximab successfully inhibited interactions between CD47/SIRP-α and PD-L1/PD-1, with IC50 values of 7.09 nM and 2.67 nM, respectively. Papiliximab induced the expression of IFN-γ by inhibiting the PD-L1/PD-1 interaction in the PBMC-based MLR (Mixed Lymphocyte reaction) assay. In addition, Papiliximab demonstrated the induction of a phagocytosis effect through CD47 blocking and its IgG4 Fc domain. Therefore, Papiliximab is a multifunctional hybrid bispecific antibody that modulates both innate and acquired immunity. Papiliximab exhibited no cross-reactivity with mouse antigens, but did with cynomolgus antigens. We tested its efficacy on the growth of human B cell lymphoma (Raji cells) and human breast cancer (MDA-MB-231 cells) in humanized NSG mice. Papiliximab was more effective in inhibiting tumor growth than mono-specific Abs and Nbs for PD-L1 or CD47, and their combination. These observations suggest that Papiliximab might be able to improve clinical remission more effectively than the combination of monotherapies without the risk of anemia associated with anti-CD47 MoAbs. We are now working to elucidate the mechanisms behind Papiliximab's superior efficacy and safety compared to the combination of monotherapies. Citation Format: Bum Seo Baek, Min Geun Kim, Jong Hyun Kim, In Young Jang, Sung Min Kim, Jeong Hwan Kim, Sang Beum Lee, Hyoung Tae Kim, Seung-Yong Seong. Papiliximab, a bispecific nanobody targeting CD47 and PDL1 retards tumor growth without hemolysis [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 2719.

Abstract 5830: A first-of-its-kind model that reconstitutes targeted drug-induced cellular transdifferentiation

Abstract Introduction: Lung tumors treated with targeted therapies ultimately relapse due to intra-genic resistance mutations, bypass pathway activation, or cellular lineage changes. Newer precision therapies have higher selectivity and potency against both resistant and activating gene mutations, resulting in a significant increase in incidence of cellular transdifferentiation. The most common form of transdifferentiation is the change from lung adenocarcinomas (LUADs) to small cell lung cancer (t-SCLC) upon drug treatment which remains very poorly understood. Methods: We have combined patient derived xenografts (PDX) and ex vivo lines with multi-omic characterization, high-content image-based morphometry and fluorescence tracking, and single-cell barcode tracing to better understand the process of lung tumor transdifferentiation. Results: We have developed a first-of-its-kind t-SCLC model that reconstitutes ex vivo LUAD to SCLC transdifferentiation. Genomic and transcriptomic analyses of our NSCLC PDX collection identified tumors with mutation in TP53 and RB1, two genes that are altered in virtually all de novo SCLCs. PDX sample CBX336 was identified as having mutations in both TP53 and RB1, in addition to expressing the KRASG12C oncogene. Upon treatment with Sotorasib, CBX336 tumors initially demonstrated slow tumor growth, followed by a dramatic increase in the growth after prolonged drug exposure. Our experimental and computational data suggest a highly coordinated procession toward transdifferentiation with the parallel upregulation of the neuroendocrine transcription factor NEUROD1 across numerous epithelial cellular clones. Conclusions: Our new experimental system allows characterization at the single-cell level. Ongoing work includes the integration of new resources to study lineage reprogramming, and advancing new therapeutic strategies. Citation Format: Priyanka Gopal, Mohamed Abazeed. A first-of-its-kind model that reconstitutes targeted drug-induced cellular transdifferentiation [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 5830.

Abstract 1789: Machine learning-based method to analyze metabolic fluxes of patient tumors

Abstract Personalized oncology must overcome the complex challenge of distinct cancer phenotypes in each patient, formed in part in response to unique metabolic crosstalk among cells in the tumor microenvironment which can be a determining factor in therapeutic response. Although isotope tracing sheds light on differences of metabolite enrichments in tumor and normal tissues, quantifying the metabolic fluxes in patients remains elusive. The conventional metabolic flux analysis tools are unable to estimate patient metabolic fluxes because we are limited to a single time point enrichment datapoint from each patient’s in-vivo measurement at isotopic non-steady state. We overcame this challenge by generating training data from the limited patient data and implementing a machine learning model to predict fluxes. To generate training data, we first simulated constrained random fluxes and enrichment patterns at different time points using patient circulating metabolites enrichment via an isotopic non-stationary metabolic flux analysis framework. Using this data, we trained a convolutional neural network to predict flux ratios. To validate our model, we compared predicted flux ratios to the experimental data from mouse models for two different treatments. Our recent findings from a glioblastoma (GBM) mouse model study using conventional metabolic flux analysis revealed higher de novo guanosine monophosphate (GMP) synthesis in GBM than normal cortex. These tumors can be sensitive to mycophenolate mofetil (MMF) which inhibits inosine monophosphate dehydrogenase, but only if the major source of GMP production is de novo synthesis and not salvage. Our machine learning model can discriminate between sources of GMP synthesis in patients, thus enabling prediction of potential responders. Furthermore, we previously showed that serine uptake is higher in GBM than normal cortex, and a serine- and glycine-restricted diet slows tumor growth in mice. While both circulating serine and microenvironment-derived serine could account for serine uptake in GBM, we hypothesized that the circulating serine dependence would better predict response to dietary serine depletion. Hence, we combined patient scRNA-seq and metabolomics data to feed our neural network framework to estimate microenvironmental and circulating serine uptake in six patients with brain cancer. Thus, we have shown that our model can identify patients who will benefit from MMF treatment or a serine- and glycine-restricted diet, enabling administration of highly effective personalized treatments for GBM patients. Citation Format: Baharan Meghdadi, Anjali Mittal, Andrew J. Scott, Sravya Palavalasa, Daniel R. Wahl, Deepak Nagrath. Machine learning-based method to analyze metabolic fluxes of patient 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 1789.

Abstract 4010: Assessing impacts of tumor burden and CAR-T cell dosage on the toxicity and efficacy profile of CD19 CAR-T therapy in a PBMC-humanized mouse model

Abstract Chimeric antigen receptor T-cell (CAR-T) therapy is a significant advancement in treating hematological malignancies, yet it faces challenges due to its variable therapeutic responses and the risk of severe toxicities. This study delves into the effects of tumor burden and CAR-T cell doses on the toxicity profile of the therapy, employing a PBMC-humanized mouse model engrafted with luciferase-labeled Raji B cell lymphoma (Raji-luc). By exploring high vs. low tumor burden and high vs. low CAR-T dose scenarios, we aim to gain insights into the dynamic relationship between these factors and CAR-T-induced toxicity and efficacy. In the first experiment, we established PBMC humanized mouse models with a high tumor burden and a low tumor burden. 10 days (high burden) or 7 days (low burden) after the Raji-luc inoculation, mice were engrafted with 10M human PBMCs and dosed with CD19 autologous CAR-Ts. In mice with low tumor burden, CAR-T treatment demonstrated significant efficacy, as evidenced by IVIS imaging, while the high tumor burden model exhibited reduced efficacy. CAR-T treatment in Raji-bearing mice did not induce weight loss in either high or low burden models. CD19 CAR-T cells effectively reduced the target cell population in peripheral blood, with greater expansion observed in the higher tumor burden model. We also evaluated human cytokine levels post-CAR-T treatment, revealing higher cytokine induction in the high tumor burden model, peaking at 2 days post-treatment. Secondly, in a CAR-T dose-response study, we treated PBMC-humanized Raji-bearing mice with 10M, 15M, and 20M CAR-T cells. Higher CAR-T cell doses (15M and 20M) resulted in observable toxicity measured by body weight loss, while the 10M dose did not. All CAR-T doses effectively slowed tumor growth and induced significant CAR-T cell expansion. Selected human cytokines, such as IL-5 and RANTES, demonstrated a dose-response correlation with CAR-T treatment. Additionally, IL-6 and IL-10 were significantly correlated with tumor burden rather than CAR-T doses. The differential cytokine responses observed in our study provide valuable insights into the utility of the PBMC-humanized mouse model for investigating the biological responses associated with CD19 CAR-T therapy. Our findings underscore the utility of the PBMC-humanized mouse model in assessing variability in toxicity and cytokine responses to CAR-T therapy. This model offers valuable insights into the factors influencing CAR-T treatment outcomes and provides a platform for planning more precise treatment and enhancing the safety and efficacy of CAR-T therapy. Citation Format: Won Lee, Destanie Rose, James G. Keck, Jiwon Yang. Assessing impacts of tumor burden and CAR-T cell dosage on the toxicity and efficacy profile of CD19 CAR-T therapy in a PBMC-humanized mouse 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 4010.

Abstract 5531: Loss of csf2(gm-csf) with cisplatin treatment decreases metastasis and improves survival in a hpv positive head and neck cancer murine model

Abstract Granulocyte macrophages colony stimulating factor (GM-CSF) also known as CSF2 is a popular cytokine responsible for homeostasis. According to TCGA databank CSF2 expression upregulated in human HPV+ (Human Papilloma Virus) Head and Neck Small Cell Carcinoma (HNSCC) compared to normal tissue. The mechanism of CSF2 in tumor progression and chemo resistance has not been well studied. Previously, our lab generated a murine model of HPV+ HNSCC that is partially responsive to cisplatin and radiation (like human disease). The role of our study is to evaluate the importance of Csf2 with cisplatin treatment (CT) in HPV + HNSCC in the murine model. Using CRISPR-Cas9 technology we knocked out the expression of the Csf2 ligand in our parental mEERL (murine E6 E7 Ras Luciferase) tumor cell line and implanted both the parental mEERL tumor and the Csf2 KO mEERL tumor in the oral cavity of C57BL6/J mice. When tumors reached a palpable size, we administered Cisplatin (20mg/m2, 3 doses total). We observed slow tumor growth in absence of Csf2 without treatment. On the other hand, we observed tumor regression in both tumor models with cisplatin treatment. Post treatment, mice with Csf2 KO tumors had a significantly prolonged rate of survival. To evaluate the role of Csf2 in the Tumor Microenvironment (TME), we collected the tumors 96 hours post CT and characterized the TME using multicolored flow cytometry. The results showed that post treatment mice with Csf2 KO tumors increased the number of the Natural Killer cell population and a surge of M-MDSC (Ly6Chi) population. The synergistic effect of knocking out Csf2 and cisplatin treatment in our tumor model appears to be driven by chemokine and cytokine changes in the TME. Simultaneously, we observed significantly lower levels of lung metastasis in Csf2 KO tumor bearing mice compared to those with parental mEERL tumors. Whereas we have seen increased metastasis in ipsilateral lymph node of Csf2 KO mice. This metastasis data mimics human disease where many patients have regional lymph node involvement, but few develop distant metastasis. These results indicate that GM-CSF (CSF2) in the TME could be an excellent target that would cause less metastasis and improved overall survival. Further investigations are needed to understand the underlying mechanism of immune response in the absence of CSF2 in the TME. Citation Format: Payal Ghosh, Craig Welbon, William C. Spanos. Loss of csf2(gm-csf) with cisplatin treatment decreases metastasis and improves survival in a hpv positive head and neck cancer murine 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 5531.

Abstract 1418: Priming of the glioblastoma tumour microenvironment via copper chelation to enhance the efficacy of immunotherapies

Abstract Glioblastoma, a highly aggressive brain tumor with a dismal prognosis necessitates novel therapeutic approaches as current standard treatments yield limited success. Despite the transformative impact of immune checkpoint blockade in various malignancies, its efficacy in glioblastoma remains elusive. Similarly, chimeric antigen receptor T (CAR-T) cell therapy, while successful in hematological malignancies, faces challenges in solid tumors. This study addresses the immunosuppressive barriers hindering the effectiveness of immunotherapies in glioblastoma. Highlighting copper's pivotal role in glioblastoma, our research group demonstrated its elevation and influence on PD-L1 expression. Given the success of copper chelators in treating Wilson's Disease, in which neurological manifestations are alleviated following treatment, we hypothesized that copper chelation therapy could be a promising strategy for glioblastoma, especially when combined with immunotherapies. Flow cytometry analysis revealed a significant increase in the tumor-associated antigen GD2 following treatment with the copper chelator trientine (TETA), suggesting its potential synergy with GD2-targeted immunotherapies. In an immunocompetent murine glioblastoma model, combination therapy with GD2-targeted CAR-T cells and TETA effectively slowed tumor growth, demonstrating enhanced efficacy. Additionally, TETA as a monotherapy significantly improved survival compared to the control. The combination of TETA with anti-PD1 antibody therapy also exhibited promising results in inhibiting tumor growth and subsequently prolonging survival, with some mice demonstrating complete tumor regression. Importantly, TETA decreased copper levels in the brains of the mice, indicating the drug can act on the brain, a crucial therapeutic hurdle that is hampering the efficacy of novel therapies in glioblastoma. Further exploration of immune cell populations using OPAL Multiplex immunohistochemistry following TETA, anti-PD1, and GD2-targeted CAR-T cell treatments will underscore the potential of copper chelation in reshaping the tumor microenvironment. Our findings suggest that copper chelation, with agents like TETA, can be repurposed as a viable anticancer therapy when combined with immunotherapies. This study provides a compelling rationale for further pre-clinical validation, offering a potential breakthrough in the quest for effective glioblastoma treatments. Citation Format: Tyler Shai-Hee, Toni Rose Jue, Riccardo Cazzoli, Jourdin R. Rouaen, Filip Michniewicz, Jessica Bell, Tessa Gargett, Michael P. Brown, Orazio Vittorio. Priming of the glioblastoma tumour microenvironment via copper chelation to enhance the efficacy of immunotherapies [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 1418.

Abstract 6027: 89Zr-matuzumab and 225Ac-matuzumab as a theranostic for epidermal growth factor receptor-positive KRAS wild-type colorectal and breast cancer xenografts

Abstract Background: About 80% of colorectal cancer (CRC) and 50 % of Triple-Negative breast cancer (TNBC) patients overexpress epidermal growth factor receptor (EGFR). Mutations in the KRAS oncogene (present in 40% of mCRC) lead to constitutive over-activation of EGFR and drive de novo resistance to anti-EGFR drugs. Here, we propose to target KRAS-mutant/BRAF mutant EGFR positive mCRC and TNBC using 225Ac-labeled matuzumab, a humanized anti-EGFR monoclonal antibody. Methods: p- -SCN-macropa was used to conjugate matuzumab for radiolabeling with 225Ac and SCN-deferoxamine was used to conjugate matuzumab for labelling with 89Zr. The radioimmunoconjugates were characterized by flow cytometry, HPLC and iTLC. in vitro cytotoxicity was evaluated in EGFR-positive mCRC, TNBC cell lines and 3D spheroids with different levels of EGFR density. Tumor growth was monitored using digital caliper. Mice were treated with either 10 MBq of 89Zr-matuzumab for imaging or three doses of 13 KBq/dose administered of 225Ac-matuzumab 10 days apart. In vivo study endpoint was tumor volume greater than or equal to 1500 mm3. Results: Flow cytometry showed about 95% binding to the cells in all EGFR-positive colorectal cell lines (DLD-1, SW620, SNU-C2B, HT-29) and breast cancer cell lines (MDA-MB-468, MDA-MB-231). in vitro studies showed enhanced cytotoxicity of 225Ac-matuzumab compared with matuzumab. IC50 in the MDA-MB-468 cell line for 225Ac-matuzumab (1.86 ± nM) was 35 times more effective than matuzumab (65.25± 7 nM). Similar trends were observed in the other KRAS-mutant mCRC cell lines and breast cancer cell lines. In 3D spheroid models, the IC50 of 225Ac-matuzumab (3.0 ± 2 nM) in MDA-MB-468, was 20.79 times more effective than matuzumab (62.3 ± 1.38 nM). Similar trends were observed for all the other spheroid models as well. High uptake of 89Zr-matuzumab was observed in both mCRC and TNBC xenografts. 225Ac-matuzumab slowed tumor growth rate in MDA-MB-468 and MDA-MB-231 tumors models compared with control antibody or non-treated controls for 30 days. This study is still ongoing and will last for 2 more months. Conclusion: 225Ac-matuzumab shows very promising outcomes in KRAS-mutant mCRC models and breast cancer models and warrants further investigation. Citation Format: Florence Anjong Tikum, Humphrey Fonge, Fabrice Njotu, Hanan Babeker, Jessica K. Pougoue, Nikita Henning, Alireza Doroudi. 89Zr-matuzumab and 225Ac-matuzumab as a theranostic for epidermal growth factor receptor-positive KRAS wild-type colorectal and breast cancer xenografts [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 6027.

Combined KRAS-MAPK pathway inhibitors and HER2-directed drug conjugate is efficacious in pancreatic cancer

Targeting the mitogen-activated protein kinase (MAPK) cascade in pancreatic ductal adenocarcinoma (PDAC) remains clinically unsuccessful. We aim to develop a MAPK inhibitor-based therapeutic combination with strong preclinical efficacy. Utilizing a reverse-phase protein array, we observe rapid phospho-activation of human epidermal growth factor receptor 2 (HER2) in PDAC cells upon pharmacological MAPK inhibition. Mechanistically, MAPK inhibitors lead to swift proteasomal degradation of dual-specificity phosphatase 6 (DUSP6). The carboxy terminus of HER2, containing a TEY motif also present in extracellular signal-regulated kinase 1/2 (ERK1/2), facilitates binding with DUSP6, enhancing its phosphatase activity to dephosphorylate HER2. In the presence of MAPK inhibitors, DUSP6 dissociates from the protective effect of the RING E3 ligase tripartite motif containing 21, resulting in its degradation. In PDAC patient-derived xenograft (PDX) models, combining ERK and HER inhibitors slows tumour growth and requires cytotoxic chemotherapy to achieve tumour regression. Alternatively, MAPK inhibitors with trastuzumab deruxtecan, an anti-HER2 antibody conjugated with cytotoxic chemotherapy, lead to sustained tumour regression in most tested PDXs without causing noticeable toxicity. Additionally, KRAS inhibitors also activate HER2, supporting testing the combination of KRAS inhibitors and trastuzumab deruxtecan in PDAC. This study identifies a rational and promising therapeutic combination for clinical testing in PDAC patients.

MCL1 inhibition targets Myeloid Derived Suppressors Cells, promotes antitumor immunity and enhances the efficacy of immune checkpoint blockade

Immune checkpoint inhibitors (ICIs) are now the first-line treatment for patients with advanced melanoma. Despite promising clinical results, many patients fail to respond to these therapies. BH3 mimetics, a novel class of small molecule inhibitors that bind and inhibit anti-apoptotic members of the BCL2 family proteins such as BCL2 or MCL1, have been very successful in treating hematologic malignancies. However, there are limited studies on the immunomodulatory role of the BH3 mimetics. Several factors contribute to ICI resistance including myeloid-derived suppressor cells (MDSCs) that exert immunosuppressive effects through direct and indirect inhibition of antitumor immunity. Thus, targeting MDSCs to enhance antitumor immunity has the potential to enhance the efficacy of ICIs. In this study, we show that the MCL1 inhibitor S64315 reduces melanoma tumor growth in an immune cell-dependent manner in mice. Specifically, S64315 enhances antitumor immunity by reducing MDSC frequency and by promoting the activity of CD8+T cells. Additionally, human MDSCs are 10 times more sensitive to S64315 than cutaneous melanoma lines. Further, we found that a higher expression of MCL1 is associated with poor survival for patients treated with anti-PD-1. Finally, combining S64315 and anti-PD-1 significantly slowed tumor growth compared to either agent alone. Together, this proof-of-concept study demonstrates the potential of combining an MCL1 inhibitor with anti-PD-1 in the treatment of melanoma. It justifies the further development of next generation MCL1 inhibitors to improve efficacy of ICIs in treating malignant melanoma.

Inhibition of MER proto-oncogene tyrosine kinase by an antisense oligonucleotide enhances treatment efficacy of immunoradiotherapy

BackgroundThe combination of radiotherapy and immunotherapy (immunoradiotherapy) has been increasingly used for treating a wide range of cancers. However, some tumors are resistant to immunoradiotherapy. We have previously shown that MER proto-oncogene tyrosine kinase (MerTK) expressed on macrophages mediates resistance to immunoradiotherapy. We therefore sought to develop therapeutics that can mitigate the negative impact of MerTK. We designed and developed a MerTK specific antisense oligonucleotide (ASO) and characterized its effects on eliciting an anti-tumor immune response in mice.Methods344SQR cells were injected into the right legs on day 0 and the left legs on day 4 of 8-12 weeks old female 129sv/ev mice to establish primary and secondary tumors, respectively. Radiation at a dose of 12 Gy was given to the primary tumors on days 8, 9, and 10. Mice received either anti-PD-1, anti-CTLA-4 or/and MerTK ASO starting from day 1 post tumor implantation. The composition of the tumor microenvironment and the level of MerTK on macrophages in the tumor were evaluted by flow cytometry. The expression of immune-related genes was investigated with NanoString. Lastly, the impact of MerTK ASO on the structure of the eye was histologically evaluated.ResultsRemarkably, the addition of MerTK ASO to XRT+anti-PD1 and XRT+anti-CTLA4 profoundly slowed the growth of both primary and secondary tumors and significantly extended survival. The ASO significantly reduced the expression of MerTK in tumor-associated macrophages (TAMs), reprograming their phenotype from M2 to M1. In addition, MerTK ASO increased the percentage of Granzyme B+ CD8+ T cells in the secondary tumors when combined with XRT+anti-CTLA4. NanoString results demonstrated that the MerTK ASO favorably modulated immune-related genes for promoting antitumor immune response in secondary tumors. Importantly, histological analysis of eye tissues demonstrated that unlike small molecules, the MerTK ASO did not produce any detectable pathology in the eyes.ConclusionsThe MerTK ASO can significantly downregulate the expression of MerTK on TAMs, thereby promoting antitumor immune response. The combination of MerTK ASO with immunoradiotherapy can safely and significantly slow tumor growth and improve survival.Graphical

Deletion of MyD88 in T Cells Improves Antitumor Activity in Melanoma

Cytotoxic CD8+ T cells are central to the antitumor immune response by releasing cytotoxic granules that kill tumor cells. They are activated by antigen-presenting cells, which become activated by DAMPs (damage associated molecular patterns) through MyD88. However, the suppressive tumor microenvironment promotes T-cell tolerance to tumor antigens, in part by enhancing the activity of immune checkpoint molecules that prevent CD8+ T-cell activation and cytotoxicity. MyD88 limits CD4+ T-cell activation during cardiac adaptation to stress. A similar mechanism is hypothesized to exist in CD8+ T cells that could be modulated to improve antitumor immunity. Herein, adoptive transfer of MyD88-/- CD8+ T cells in melanoma-bearing T-cell-deficient mice resulted in slower tumor growth, greater intratumoral T-cell accumulation, and higher melanoma cell death compared with transfer of wild-type CD8+ T cells. These findings were also observed in T-cell-specific MyD88-/- mice compared with wild-type littermates implanted with melanoma. Mechanistically, deletion of MyD88 enhanced CD8+ T-cell activation and survival, and T-cell receptor induced degranulation of cytotoxic molecules, overall improving the killing of melanoma cells. This enhanced cytotoxicity was retained in mice bearing tumors expressing the specific antigen for which cytotoxic T-cells were restricted. This study's results demonstrate a conserved mechanism for MyD88 in modulating CD8+ T-cell activation and represent a novel target in improving cancer immunotherapy.

In Vivo Stimulation of Therapeutic Antigen-Specific T Cells in an Artificial Lymph Node Matrix.

T cells are critical mediators of antigen-specific immune responses and are common targets for immunotherapy. Biomaterial scaffolds have previously been used to stimulate antigen-presenting cells to elicit antigen-specific immune responses; however, structural and molecular features that directly stimulate and expand naïve, endogenous, tumor-specific T cells in vivo have not been defined. Here, an artificial lymph node (aLN) matrix is created, which consists of an extracellular matrix hydrogel conjugated with peptide-loaded-MHC complex (Signal 1), the co-stimulatory signal anti-CD28 (Signal 2), and a tethered IL-2 (Signal 3), that can bypass challenges faced by other approaches to activate T cells in situ such as vaccines. This dynamic immune-stimulating platform enables direct, in vivo antigen-specific CD8+ T cell stimulation, as well as recruitment and coordination of host immune cells, providing an immuno-stimulatory microenvironment for antigen-specific T cell activation and expansion. Co-injecting the aLN with naïve, wild-type CD8+ T cells results in robust activation and expansion of tumor-targeted T cells that kill target cells and slow tumor growth in several distal tumor models. The aLN platform induces potent in vivo antigen-specific CD8+ T cell stimulation without the need for ex vivo priming or expansion and enables in situ manipulation of antigen-specific responses for immunotherapies.

Hypoxia-related carbonic anhydrase 9 induces serpinB9 expression in cancer cells and apoptosis in T cells via acidosis.

Hypoxia is a common feature of solid tumors. However, the impact of hypoxia on immune cells within tumor environments remains underexplored. Carbonic anhydrase 9 (CA9) is a hypoxia-responsive tumor-associated enzyme. We previously noted that regardless of human CA9 (hCA9) expression, hCA9-expressing mouse renal cell carcinoma RENCA (RENCA/hCA9) presented as a "cold" tumor in syngeneic aged mice. This study delves into the mechanisms behind this observation. Gene microarray analyses showed that RENCA/hCA9 cells exhibited elevated mouse serpinB9, an inhibitor of granzyme B, relative to RENCA cells. Corroborating this, RENCA/hCA9 cells displayed heightened resistance to antigen-specific cytotoxic T cells compared with RENCA cells. Notably, siRNA-mediated serpinB9 knockdown reclaimed this sensitivity. Invivo tests showed that serpinB9 inhibitor administration slowed RENCA tumor growth, but this effect was reduced in RENCA/hCA9 tumors, even with adjunctive immune checkpoint blockade therapy. Further, inducing hypoxia or introducing the mouse CA9 gene upregulated serpinB9 expression, and siRNA-mediated knockdown of the mouse CA9 gene inhibited the hypoxia-induced induction of serpinB9 in the original RENCA cells. Supernatants from RENCA/hCA9 cultures had lower pH than those from RENCA, suggesting acidosis. This acidity enhanced serpinB9 expression and T cell apoptosis. Moreover, coculturing with RENCA/hCA9 cells more actively prompted T cell apoptosis than with RENCA cells. Collectively, these findings suggest hypoxia-associated CA9 not only boosts serpinB9 in cancer cells but also synergistically intensifies T cell apoptosis via acidosis, characterizing RENCA/hCA9 tumors as "cold."

Mannosylated STING Agonist Drugamers for Dendritic Cell-Mediated Cancer Immunotherapy.

The Stimulator of Interferon Genes (STING) pathway is a promising target for cancer immunotherapy. Despite recent advances, therapies targeting the STING pathway are often limited by routes of administration, suboptimal STING activation, or off-target toxicity. Here, we report a dendritic cell (DC)-targeted polymeric prodrug platform (polySTING) that is designed to optimize intracellular delivery of a diamidobenzimidazole (diABZI) small-molecule STING agonist while minimizing off-target toxicity after parenteral administration. PolySTING incorporates mannose targeting ligands as a comonomer, which facilitates its uptake in CD206+/mannose receptor+ professional antigen-presenting cells (APCs) in the tumor microenvironment (TME). The STING agonist is conjugated through a cathepsin B-cleavable valine-alanine (VA) linker for selective intracellular drug release after receptor-mediated endocytosis. When administered intravenously in tumor-bearing mice, polySTING selectively targeted CD206+/mannose receptor+ APCs in the TME, resulting in increased cross-presenting CD8+ DCs, infiltrating CD8+ T cells in the TME as well as maturation across multiple DC subtypes in the tumor-draining lymph node (TDLN). Systemic administration of polySTING slowed tumor growth in a B16-F10 murine melanoma model as well as a 4T1 murine breast cancer model with an acceptable safety profile. Thus, we demonstrate that polySTING delivers STING agonists to professional APCs after systemic administration, generating efficacious DC-driven antitumor immunity with minimal side effects. This new polymeric prodrug platform may offer new opportunities for combining efficient targeted STING agonist delivery with other selective tumor therapeutic strategies.

Hsa_circ_0003356 suppresses gastric cancer progression via miR-556-5p/FKBP5 axis

BackgroundCircRNAs are implicated in the tumorigenesis of various human cancers. This study aims to explore how circ_0003356 contributes to the development of gastric cancer (GC). MethodsCirc_0003356 expression was analyzed in GSE184882 dataset and validated in our cohort of GC patients and human GC cell lines. The correlations between circ_0003356 levels and prognostic parameters were analyzed. The contribution of circ_0003356 in GC cell malignant behaviors such as cell survival, apoptosis and invasion were investigated by circ_0003356 overexpression in GC cell lines. The downstream targets of circ_0003356 were predicted and verified in vitro and in vivo. The in vivo function of circ_0003356 was studied as well in a xenograft mouse model. ResultsCirc_0003356 expressed at a low level in human GC tissues and cells, which was closely associated with poor outcome of GC patients. Circ_0003356 overexpression induced GC cell apoptosis while depressed the growing, migration and invasive abilities through miR-556-5p/FKBP5 axis. In vivo model showed retarded tumor growth when circ_0003356-overexpressed cells were inoculated. ConclusionCirc_0003356 is identified as a potential biomarker of the prognosis of human gastric cancer, and circ_0003356/miR-556-5p/FKBP5 axis could be a promising target in gastric cancer treatment.

Oral administration of Manuka honey induces IFNγ-dependent resistance to tumor growth that correlates with beneficial modulation of gut microbiota composition.

To investigate the potential of Manuka honey (MH) as an immunomodulatory agent in colorectal cancer (CRC) and dissect the underlying molecular and cellular mechanisms. MH was administered orally over a 4 week-period. The effect of MH treatment on microbiota composition was studied using 16S rRNA sequencing of fecal pellets collected before and after treatment. Pretreated mice were implanted with CRC cells and followed for tumor growth. Tumors and lymphoid organs were analyzed by flow cytometry (FACS), immunohistochemistry and qRT-PCR. Efficacy of MH was also assessed in a therapeutic setting, with oral treatment initiated after tumor implantation. We utilized IFNγ-deficient mice to determine the importance of interferon signaling in MH-induced immunomodulation. Pretreatment with MH enhanced anti-tumor responses leading to suppression of tumor growth. Evidence for enhanced tumor immunogenicity included upregulated MHC class-II on intratumoral macrophages, enhanced MHC class-I expression on tumor cells and increased infiltration of effector T cells into the tumor microenvironment. Importantly, oral MH was also effective in retarding tumor growth when given therapeutically. Transcriptomic analysis of tumor tissue highlighted changes in the expression of various chemokines and inflammatory cytokines that drive the observed changes in tumor immunogenicity. The immunomodulatory capacity of MH was abrogated in IFNγ-deficient mice. Finally, bacterial 16S rRNA sequencing demonstrated that oral MH treatment induced unique changes in gut microbiota that may well underlie the IFN-dependent enhancement in tumor immunogenicity. Our findings highlight the immunostimulatory properties of MH and demonstrate its potential utilization in cancer prevention and treatment.