Cold Tumors Research Articles

Bexmarilimab Activates Human Tumor-Associated Macrophages to Support Adaptive Immune Responses in Interferon-Poor Immune Microenvironments.

Immune checkpoint inhibitors (ICI) show substantially greater efficacy in inflamed tumors characterized by preexisting T-cell infiltration and IFN signaling than in noninflamed "cold" tumors, which often remain immunotherapy resistant. The cancer immunotherapy bexmarilimab, which inhibits the scavenger receptor Clever-1 to release macrophage immunosuppression and activate adaptive immunity, has shown treatment benefit in subsets of patients with advanced solid malignancies. However, the mechanisms that determine bexmarilimab therapy outcome in individual patients are unknown. Here we characterized bexmarilimab response in ovarian cancer ascites macrophages ex vivo using single-cell RNA sequencing and demonstrated increased IFN signaling and CXCL10 secretion following bexmarilimab treatment. We further showed that bexmarilimab was most efficacious in macrophages with low baseline IFN signaling, as chronic IFNγ priming abolished bexmarilimab-induced TNFα release. These results highlight an approach to target immunologically cold tumors and to increase the likelihood of their subsequent response to ICIs.

Genomic and Transcriptomic Profiles of Blastoid and Pleomorphic Mantle Cell Lymphoma Are Distinct from Classic Histology Mantle Cell Lymphoma

Introduction - Patients (pts) with mantle cell lymphoma (MCL) with blastoid (B-MCL) and pleomorphic (P-MCL) histology are traditionally considered as high risk; however, there is a paucity of information regarding the differences between P-MCL and B-MCL compared to classic MCL (C-MCL) histology. In this study, we present a comprehensive genomic and transcriptomic portrait of B-MCL and P-MCL. Methods - We conducted comprehensive molecular profiling using whole exome (WES) and bulk RNA sequencing (RNA seq) among the evaluable pt samples (n=260) from our center (MDA cohort) and previously reported cohorts from Bea et al ( PNAS 2013) (n=29) and Agarwal et al ( Nature Medicine 2019) (n=15) respectively. Pts with WES were (n=195) including n=151, n=29 and n=15 for MDA, Bea and Agarwal respectively while 175 pts underwent RNA sequencing (all MDA). This cohort study was conducted under an IRB approved protocol for MCL patients at our center. Patient characteristics, somatic mutation profiles, copy number abnormalities and gene expression profiles (GEP) were analysed. Lymph node, bone marrow, blood samples and any other tissue biopsies were utilized. DNA and RNA were extracted from FFPE sections from lymph nodes and non-nodal tissues. Joint WES and RNA-seq mutation calling, GEP, and transcriptomic cell deconvolution were performed using the BostonGene automated pipeline. WES and bulk RNA sequencing were performed with Illumina HiSeq4000 using a 76bp paired end configuration. Results - Among the 195 pts with WES, the distribution of tissue types was 133 nodal, 33 bone marrow, 25 blood, 4 spleen and other tissues. Mutation distribution and copy number analysis was performed for 99 C-MCL , 72 B-MCL, and 20 P- MCL pts. In the mutation profile, we found that the percentage of ATM mutations was comparable among the 3 pt groups (51%/50%/39%). The frequency of TP53 mutations was significantly lower in C-MCL (23%) compared to B-MCL (51%) and P-MCL (39%) (p < 0.001; Figure 1). NOTCH2 gain of function mutations were found in 11% of B-MCL and 4.6% of C-MCL but not detected in P-MCL. The NFkBIE Y254Sfs*13 mutation was exclusively found in B-MCL. Blastoid samples were enriched with CCND1 E36K mutations (7 of 16 CCND1 mut), while C-MCL had 2 of 16. P-MCL had a higher prevalence of SMARCA4 and DNMT3A mutations compared to B-MCL and C- MCL. We further observe that P-MCL pts exhibited higher polyploidy and aneuploidy than B-MCL and C-MCL ( p < 0.001; Figure 2), indicating that compared to C-MCL, genomic instability decreased from P-MCL to B-MCL. The most frequent arm level amplifications in P-MCL compared to B-MCL were 8q (=MYC) (38%) and 18q (=BCL2) (38%). In C-MCL and B-MCL the 3q (=BCL6) was frequently amplified (28% and 30%). MALT1, Bcl2 and CARD11 gene amplifications were highest in P-MCL. These differences were further supported when the data were compared at the gene level. Comparison of the deletion frequencies revealed that 9p (CDKN2A/B) and 17p (TP53) were frequently lost in B and P-MCL (~40%). Del8p, 6q, 10q, and 16q were frequently deleted in P-MCL compared to B-MCL. At the segmental level, mostly altered segments included del9p21.1-24.3, del13q14.2-14.3, and del17p12-13.3 that span genes like CDKN2A/B, miR15A, and TP53. Next, we performed GSEA analysis using RNA-seq data. The metabolic reprogramming pathway was upregulated in both B and P-MCL compared to C-MCL (p = 0.0096). The proportion of pts with an immune desert (D) “cold” tumor microenvironment associated with refractory disease was higher in P-MCL (50%) than in C or B-MCL. Conclusions -B and P-MCL exhibit a distinct molecular profile compared to C-MCL. P and B-MCL have the highest degree of aneuploidy and exhibit an immune cold tumor microenvironment. Further studies are ongoing to refine the molecular differences among B and P-MCL compared to C-MCL.

Targeting DNA Damage and Repair Machinery via Delivering WEE1 Inhibitor and Platinum (IV) Prodrugs to Stimulate STING Pathway for Maximizing Chemo-Immunotherapy in Bladder Cancer.

Both cisplatin-based chemotherapy and immune checkpoint blockers (ICBs)-based immunotherapy are the first-line treatments for patients with advanced bladder cancer. Cancer cells can develop resistance to cisplatin through extensive DNA repair, while a low response rate to ICBs is mostly due to the presence of an immunosuppressive microenvironment and low PD-L1 expression. Herein, a glutathione (GSH)-responsive nanoparticle (NP2) loaded with cisplatin prodrug (Pt (IV)) and WEE1 inhibitor (MK1775) is designed. NP2 can be triggered by GSH in cancer cells, and the released MK1775 can inhibit the activity of WEE1 protein, which ultimately increases DNA damage by cisplatin. Genome-wide RNA sequencing first reveals that NP2 can inhibit DNA repair machinery by interfering with the cell cycle and significantly activate the stimulator of interferon genes pathway. Tumor growth is significantly inhibited by NP2 in vivo. As innate and adaptive immune responses are stimulated, the immunosuppressive microenvironment is modified, and the "immune cold tumor" is transformed into an "immune hot tumor". In addition, NP2 can upregulate PD-L1 expression in tumor cells, thereby increasing the response rate of PD-L1 monoclonal antibody (αPD-L1) and eliciting long-term immune responses in both primary and metastatic tumors.

Transforming Intratumor Bacteria into Immunopotentiators to Reverse Cold Tumors for Enhanced Immuno-chemodynamic Therapy of Triple-Negative Breast Cancer.

Immunotherapy of triple-negative breast cancer (TNBC) has an unsatisfactory therapeutic outcome due to an immunologically "cold" microenvironment. Fusobacterium nucleatum (F. nucleatum) was found to be colonized in triple-negative breast tumors and was responsible for the immunosuppressive tumor microenvironment and tumor metastasis. Herein, we constructed a bacteria-derived outer membrane vesicle (OMV)-coated nanoplatform that precisely targeted tumor tissues for dual killing of F. nucleatum and cancer cells, thus transforming intratumor bacteria into immunopotentiators in immunotherapy of TNBC. The as-prepared nanoparticles efficiently induced immunogenic cell death through a Fenton-like reaction, resulting in enhanced immunogenicity. Meanwhile, intratumoral F. nucleatum was killed by metronidazole, resulting in the release of pathogen-associated molecular patterns (PAMPs). PAMPs cooperated with OMVs further facilitated the maturation of dendritic cells and subsequent T-cell infiltration. As a result, the "kill two birds with one stone" strategy warmed up the cold tumor environment, maximized the antitumor immune response, and achieved efficient therapy of TNBC as well as metastasis prevention. Overall, this strategy based on a microecology distinction in tumor and normal tissue as well as microbiome-induced reversal of cold tumors provides new insight into the precise and efficient immune therapy of TNBC.

Copy number loss of KDM5D may be a predictive biomarker for ATR inhibitor treatment in male patients with pulmonary squamous cell carcinoma.

A limited number of patients with lung squamous cell carcinoma (SCC) benefit clinically from molecular targeted drugs because of a lack of targetable driver alterations. We aimed to understand the prevalence and clinical significance of lysine-specific demethylase 5D (KDM5D) copy number loss in SCC and explore its potential as a predictive biomarker for ataxia-telangiectasia and Rad3-related (ATR) inhibitor treatment. We evaluated KDM5D copy number loss in 173 surgically resected SCCs from male patients using fluorescence in situ hybridization. KDM5D copy number loss was detected in 75 of the 173 patients (43%). Genome-wide expression profiles of the transcription start sites (TSSs) were obtained from 17 SCCs, for which the cap analysis of gene expression assay was performed, revealing that upregulated genes in tumors with the KDM5D copy number loss are associated with 'cell cycle', whereas downregulated genes in tumors with KDM5D copy number loss were associated with 'immune response'. Clinicopathologically, SCCs with KDM5D copy number loss were associated with late pathological stage (p = 0.0085) and high stromal content (p = 0.0254). Multiplexed fluorescent immunohistochemistry showed that the number of tumor-infiltrating CD8+ /T-bet+ T cells was lower in SCCs with KDM5D copy number loss than in wild-type tumors. In conclusion, approximately 40% of the male patients with SCC exhibited KDM5D copy number loss. Tumors in patients who show this distinct phenotype can be 'cold tumors', which are characterized by the paucity of tumor T-cell infiltration and usually do not respond to immunotherapy. Thus, they may be candidates for trials with ATR inhibitors.

Synergetic regulation of cancer cells and exhausted T cells to fight cold tumors with a fluorinated EGCG-based nanocomplex.

Immune therapy that targets PD-L1 (programmed cell death-ligand 1) is attractive to augment immune response by breaking the programmed cell death-1 (PD-1)/PD-L1 axis. However, T cell exhaustion associated with insufficient T cells infiltration may diminish the efficacy of cancer therapy. Here, we report a novel delivery system of FEGCG/FPEI@siTOX composed of fluorinated EGCG (FEGCG) and fluorinated polyethyleneimine (FPEI) for delivery of small interfering RNA anti-TOX (thymus high mobility group box protein, TOX) to treat tumor and metastasis. In this way, the reduction in PD-L1 expression by FEGCG can promote T-cell function, while inhibition of TOX expression with siTOX can alleviate T-cell exhaustion. FPEI are designed to deliver siRNA with high efficiency and low toxicity compared to classical PEI. Integrating FEGCG, FPEI and siTOX into such a novel system resulted in excellent anti-tumor and antimetastatic effects. It is a promising delivery system and potential strategy for the treatment of "cold" tumors.

DDEL-12. A CONFORMABLE IMPLANTABLE DEVICE - MICROMESH - FOR THE SUSTAINED DELIVERY OF CHEMOTHERAPEUTIC MOLECULES AND IMMUNE CHECK POINT INHIBITORS AGAINST HIGH-GRADE GLIOMAS

Abstract High-grade gliomas, like glioblastoma (GBM), continue to have a poor prognosis with therapeutic options limited to maximal safe resection and adjuvant radiochemotherapy. Effective treatment is hampered by the blood-brain barrier, limiting the intratumor permeation of systemically administered agents; and biological heterogeneity of malignant cells, requiring complex therapeutic regimens. A conformable, compartmentalized polymeric implant – microMESH – has been engineered to overcome these obstacles by realizing a localized, sustained release of drugs, small inhibitors, antibodies, and nanomedicines. In the present configuration, microMESH comprises a water-soluble polyvinyl alcohol microlayer, loaded with the immune checkpoint aCD47, intercalated with a microfabricated network of poly lactic-co-glycolic acid, carrying docetaxel (DTXL). Upon deposition over the tumor margins, microMESH adheres and conforms to the local tissue releasing thereof its therapeutic cargo for several weeks, circumventing possible systemic toxicity. Gel permeation chromatography, nuclear magnetic resonance spectroscopy, weight and pH variations were measured to assess the microMESH biodegradation over time. microMESH biocompatibility and cytotoxicity were assessed on cancer cell monolayers and spheroids. The preclinical performance of microMESH was evaluated in two syngeneic mouse models of high-grade gliomas obtained via the intracranial inoculation of murine cancer cells – CT-2A, cold tumor; GL261, hot tumor. Flow cytometry was used to map the immune component in the tumor and assess the expression of immune checkpoints. Bioluminescence imaging was used to monitor tumor proliferation longitudinally. microMESH biodegraded in ~2 months with no documented toxicity. Preliminary experiments with an aCD47/DTXL–microMESH applied over the unresected margins of an orthotopic CT-2A model documented a median overall survival of 33.5 days against 18 days for untreated mice, 22 days for intravenously injected DTXL, 24 days for intraperitoneally administered aCD47 (statistically not significant difference among the control groups). microMESH allowed the sustained delivery of complex combination therapies directly at the tumor bed demonstrating superior efficacy over systemic therapies.

TMIC-78. ENDOTHELIAL REGULATION OF THE IMMUNOSUPPRESSIVE PHENOTYPE OF TUMOR-ASSOCIATED MICROGLIA IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is considered a non-immunogenic “cold tumor” with an immunosuppressive microenvironment that blocks T-cell infiltration and function, limiting the efficacy of targeted immunotherapy. Tumor-associated microglia and macrophages (TAM) constitute approximately 30-50% of the non-neoplastic immune cells, and they are closely associated with blood vessels in GBM. However, the precise mechanism by which TAM acquire the immunosuppressive phenotype remains poorly understood. In this study, we show that tumor endothelial cells (TEC) from primary and recurrent GBM express inflammatory cytokines and chemokines that influence the function and polarization of microglia. We demonstrate that conditioned media (CM) of TEC promotes the immunosuppressive phenotype of normal microglia and patient-derived TAM. RNA-sequencing of TAM treated with TEC-CM shows upregulation of immunosuppressive cytokines, as well as T-cell co-inhibitory molecules such as PD-L1. Culturing CD8 T-cells in CM of TAM-polarized by TEC inhibits their cytotoxic function and cytokine production, and induces expression of PD-1 and CTLA-4. Furthermore, gene set enrichment analysis uncovers alterations in calcium signaling-related pathways in TAM polarized by TEC-CM. Blocking calcium signaling with small molecule inhibitors in microglia reverses the immunosuppressive phenotype induced by TEC. To determine whether the interactions between GBM, endothelial cells and TAM promotes immunosuppression in vivo, we utilize the humanized immune system mouse model (huNOG-EXL) that harbor human lymphocytes and monocyte populations. In preliminary studies, we found that co-transplantation of GBM tumor cells with TAM and TEC significantly promotes tumor growth compared with GBM cells alone. Single-cell mass cytometry analysis of peripheral blood mononuclear cells (PBMC) pre- and post-transplant, and scRNA-sequencing and spatial-transcriptomic studies of these co-transplanted mice will potentially reveal key insights into local- and peripheral immune responses regulated by tumor endothelial-microglia interactions. Together, our findings highlight a key role for endothelial-mediated calcium signaling in regulation of the immunosuppressive phenotype of microglia in GBM.

TMIC-85. TGFB2 AND IFNGR2 MRNA EXPRESSION LEVELS IN THE TUMOR MICROENVIRONMENT (TME) PROGNOSITICALLY IMPACT OS IN PEDIATRIC DIPG PATIENTS

Abstract DIPG patients expressed significantly higher levels of TGFB2 and IFNGR2 than control pons samples and the expression profile indicated higher levels of TGFB2 than TGFB1 and TGFB3 in DIPG patients suggesting TGFB2 mRNA level is preferentially increased in DIPG patients. TGFB2 is a prognostic indicator (HR for patients in the TGFB2high group of DIPG patients = 2.88 (1.12-7.39); P = 0.028) for poor overall survival (OS) independent of IFNGR2 levels, the age of the patient, and the significant interaction effect observed between IFNGR2 and TGFB2. Predictive modeling using the Cox regression parameters showed that the context of low expression of IFNGR2, TGFB2low resulted in more favorable OS times compared to TGFB2high whereby the median OS time of 11 months for the TGFB2low group of patients was greater than the upper 95% confidence limit for the TGFB2high group of patients (Median = 7 months; Upper 95% Confidence interval = 10 months). At higher levels of IFNGR2 expression, the TGFB2high group of patients exhibited a median OS time of 15 months that was within the (95% confidence interval for the TGFB2low group of patients (Median = 13 months; 95% CI = 10-17 months). Worse survival outcomes comparing DIPG patients with high versus low TGFB2 levels in the context of low levels of IFNGR2 levels suggest that abrogating the TGFB2 mRNA expression in the immunologically cold tumor microenvironment indicated by low levels of IFNGR2 expression can potentially treat pediatric DIPG patients. Furthermore, DIPG patients with low levels of JAK1 or STAT1 mRNA expression in combination with TGFB2high also exhibited poor OS outcomes suggesting that the inclusion of (Interferon-gamma) IFNγ to stimulate and activate JAK1 and STAT1 in anti-tumor immune cells present the brainstem tumor microenvironment can enhance the effect of TGFB2 blockade.

IMMU-42. PP2A-INHIBITION INDUCES STING-TYPE I-MEDIATED IMMUNOLOGIC CELL DEATH IN GBM

Abstract Glioblastoma (GBM) is one of the most lethal forms of brain cancer, and is known to be an aggressive, immunologically cold tumor, resulting in a lack of T-cell infiltration and killing. Thus increasing immune-mediated killing of GBM is a promising therapeutic method. Our group has found that Protein Phosphatase 2A (PP2A), a ubiquitous serine/threonine phosphatase that has been shown to enhance tumor immunogenicity in glioblastoma, does so by inducing immunogenic cell death. We used LB-100, a small molecule inhibitor of the catalytic subunit of PP2A, as a promising drug-therapy. CRISPR-KO of PP2A was generated in murine glioma lines GL261 and SB28. Markers of immunologic cell death, such as calreticulin, gamma-H2AX, and HMGB1 were assessed. In vitro, western blotting of SB28 and GL261 showed significantly increased expression of gamma-H2AX in PP2A-KO compared to WT. Flow-cytometry staining of SB28 and GL261 revealed a dramatic increase in calreticulin expression in PP2A-KO compared to WT. An antibody-based fluorescence assay for HMGB1 also showed dramatic increase in PP2A-KO compared to WT. All experiments were repeated by treating LB-100 for 24 hour to SB28 and GL261 WT and PP2A-KO cells, and revealed a dose-dependent increase in expression of immunogenic cell death markers. In vivo, B6 mice were injected intracranially with SB28 WT or PP2A-deficient tumor and monitored till endpoint. At the endpoint, tumors were harvested, cryosectioned, and stained for calreticulin and gamma-H2AX. Mice injected with PP2A-KO tumor survived longer than WT, and had the highest expression of both calreticulin gamma-H2AX when compared to WT tissue. In conclusion, PP2A-deficiency and inhibition via LB-100 is demonstrated to induce immunogenic cell death in tumors, thus being a promising target for immunotherapy.

TMIC-24. FERROPTOSIS INDUCES IMMUNOGENIC CELL DEATH AND SYNERGIZES WITH CD47 BLOCKADE TO REPROGRAM THE GLIOMA MICROENVIRONMENT

Abstract Immunotherapy has thus far proven ineffective in glioblastoma, in part due to the immune cold tumor microenvironment. Here we demonstrate that ferroptosis specifically targets a quiescent, immunosuppressive astrocyte-like glioma population and can therefore be used as a novel inducer of immunogenic cell death to repolarize the immune microenvironment by promoting tumor cell phagocytosis and antigen presentation. First, we characterized the immunogenic effects of ferroptosis by using a glioma cell-myeloid cell co-culture system and patient-derived organotypic slice cultures and performed phagocytosis assays, flow-cytometry, immunohistochemistry, cytokine arrays and single cell RNAseq. GPX4-dependent ferroptosis induced translocation of the pro-phagocytic eat-me signal calreticulin in both murine glioma cells and human tissue samples. Ferroptotic-driven tumor cell death reprogramed local myeloid populations to promote glioma phagocytosis, MHCII-driven antigen presentation, and paracrine cytokine signaling involved in T-cell recruitment. Local delivery of RSL3, a GPX4 inhibitor, in a murine glioma model polarized myeloid cells to a pro-phagocytic phenotype and significantly increased intra-tumoral levels of CD4+ and CD8+ T-cells. Astrocytic-like tumor cells upregulate CD47 as a mechanism to evade phagocytosis. Therefore, we combined ferroptosis with an anti-CD47 monoclonal antibody (aCD47) to enhance reprogramming of the immune cold microenvironment. In an orthotopic murine glioma model, adding aCD47 to RSL3 led to significantly increased tumor phagocytosis, antigen presentation and prolonged survival. Thus, we demonstrate that local delivery of ferroptosis-inducing agents in conjunction with CD47 blockade is an effective multi-modality therapeutic regimen capable of reversing the immunosuppressive glioma microenvironment.

IMMU-08. EOSINOPHILS ARE IMPORTANT MODULATORS OF AN IMMUNE-RESPONSIVE TUMOR-MICROENVIRONMENT IN GLIOBLASTOMA

Abstract Eosinophils, bone marrow-derived granulocytes involved in allergic asthma and autoimmunity, have attracted increasing attention due to their assumed role in effective responses to immune checkpoint blockade in some extracranial tumors. However, the role of this rare type of immune cells in immunologically cold tumors such as glioblastoma (GBM) has not been studied so far. When quantifying immune infiltrates and levels of 30 immune-relevant cytokines in 60 matched pairs of human primary and recurrent GBM, strongest correlations (r= 0.71-0.93) were observed for the eosinophil-associated cytokines Eotaxin and Il-5 as well as for TNFa, IFNg and Il2 which are important cytokines for cytotoxic T cell responses. A combined score of these cytokines (Eo score) turned out to be an independent prognostic marker for improved overall survival of GBM patients. Furthermore, multicolor immunofluorescent staining revealed that Eo score high GBM are significantly higher infiltrated by effector T cells and show a significantly lower infiltration with anti-inflammatory M2-like tumor-associated macrophages / microglia cells (M2-TAM) in recurrent GBM. Flowcytometry analysis of GBM-derived single cell suspensions confirmed a positive correlation of higher eosinophil numbers with higher numbers of tumor-infiltrating T cells, while an inverse relation between eosinophils and monocytes could be demonstrated in the TCGA data set. Spatial transcriptomic data of human GBM confirmed that areas with a higher eosinophil infiltration showed higher numbers of T cells and pro-inflammatory M1-TAM, lower numbers of M2-TAM and a remarkable vessel normalization. Finally, in a newly developed patient-derived GBM organoid model we could corroborate the functional impact of eosinophils on the tumor-microenvironment. An increase of eosinophil numbers in these patient avatars resulted in a significantly decreased number of M2-TAM. Altogether, tumor-infiltrating eosinophils seem to be a novel type of immune-modulator in GBM and may enhance immune responses by shaping the tumor niche from an immunosuppressive to an immunepermissive microenvironment.

EPCO-35. IDENTIFICATION OF DIVERSE IMMUNE PHENOTYPES IN NF-1 GLIOMA BY SINGLE NUCLEI GENE EXPRESSION ANALYSIS

Abstract Neurofibromatosis type 1 (NF-1) is the most common cancer predisposition syndrome in which 15-20% of affected individuals develop glioma. Large scale DNA and RNA bulk profiling showed the high molecular complexity of NF-1 glioma with the tumor cellular ecosystem constituted by multiple malignant clones and heterogenous immune microenvironment. However, the composition and function of infiltrating cells was mostly hidden in the bulk tumor mass, and the extended granularity of NF-1 glioma immune microenvironment remained still unexplored. Here, to address this challenge, we collected glioma samples from 53 NF-1 patients including 27 high-grade and 26 low-grade tumors, and we analyzed their gene expression at the highest resolution through single nuclei RNA sequencing. A total of 288,372 single cells have been classified by integrating multiple computational approaches (including genomic copy number inference, gene signature enrichment, pathway-based deconvolution and integrative non-negative matrix factorization). The non-tumor cell compartment (124,082 cells, 43%) has been dissected for the deep characterization of each cell type that populate the microenvironment of NF-1 glioma. We unraveled the complexity of the immune infiltration and identified different subpopulations exhibiting specific immune functions within myeloid and lymphoid components. Different glioma niches have been revealed by comparing the relative composition of the immune microenvironment across the tumors. Recruitment and activation of cytotoxic CD8+ T cells and natural killers by an active crosstalk with dendritic and pro-inflammatory myeloid cells defined an immune-supportive phenotype that can mediate a potential anti-tumor response in low-grade NF-1 glioma. Regulatory T cell infiltration and effector T cell exhaustion, instead, induced immune suppression in another tumor niche. Conversely, the complete absence of lymphocytes characterized a large set of cold tumors, mostly high-grade glioma. The elucidation of the complex immune interplay in the different tumor niches provides novel insights for the application of immunotherapies in NF-1 glioma patients.

CTIM-41. ONCOLYTIC IMMUNOTHERAPY LINKS IMMUNOACTIVATION TO SUBJECT SURVIVAL IN PHASE I TRIAL OF RECURRENT GLIOBLASTOMA

Abstract A “first-in-human” phase 1 trial of intralesional injection of CAN-3110, an immunotherapeutic oncolytic based on herpes simplex virus 1 (HSV1), was conducted in 41 subjects with recurrent high-grade glioma (rHGG). The impact of CAN-3110 on the tumor immune microenvironment was assessed using immunohistochemistry, multiplexed immunofluorescence, TCR-beta DNA sequencing, and bulk RNA sequencing of paired pre- and post-treatment samples. These analyses were combined with patient features to identify potential biomarkers of therapy response in rHGG. In IDH wild type recurrent glioblastoma (IDHwt rGBM) patients, post-treatment survival times were positively associated with positive pre-treatment HSV1 serology after controlling for additional covariates such as tumor volume, age, gender, CAN-3110 dose, etc. (HR = 0.16, p < 0.001, n = 32, CoxPH). Positive HSV1 serology was also significantly associated with clearance of CAN-3110 from injected tumors. Interestingly, increased intratumoral CD8 and CD4 T cell numbers following treatment were positively associated with prolonged survival in HSV1 seropositive patients (p = 0.017 and 0.026, respectively), but not when analyses were conducted including HSV1 seronegative patients. Likewise, post-treatment immune expression signatures (such as for antitumor cytokines, T cell traffic, etc.) were more strongly associated with prolonged survival for HSV1 seropositive patients than for seronegative patients, and most assessed immune signatures only became associated with survival after CAN-3110 treatment. Extended survival was also associated with increased TCR-beta diversity following therapy in both the tumor and in PBMCs. Combined with a lack of observed dose-limiting toxicities and a median post-treatment survival of 14.2 months (95% CI: 9.5-15.7) in IDHwt rGBM patients with positive HSV1 serology, these data provide evidence that intralesional CAN-3110 treatment can instigate immune activation in a traditionally immunologically cold tumor and that this immune activation may influence survival time—particularly when patients have had prior exposure to HSV1. (clinicaltrials.gov NCT03152318).

Fabrication of An Immunostimulatory Supramolecular Nanomedicine for Potent Cancer Chemoimmunotherapy.

Chemoimmunotherapy can boost strong antitumor immune responses by triggering immunogenic cell death (ICD), which highlights a promising prospect in clinical applications. However, current chemoimmunotherapy shows limited efficacy due to the low delivery efficiency and insufficient immunogenicity of available chemotherapeutic drugs. A supramolecular polymeric nanomedicine (Pt-Tu@NP) is herein reported using cucurbit[7]uril-based host-guest recognition and noncovalent self-assembly. Pt-Tu@NPs have excellent biodistribution and strongly evoke the endoplasmic reticulum stress-mediated ICD of tumor cells, triggering potent antitumor immune responses by promoting dendritic cell (DC) maturation and cytotoxic T cell infiltration. The coordinated butyrate promotes a positive feedback regulation between DCs and CD8+ T cells. Pt-Tu@NPs stimulate immune cold tumors into hot ones, working in synergy with an immune checkpoint blockade to effectively suppress tumor growth and metastasis, which suggests a promising approach for cancer chemoimmunotherapy.

Cooperative Networks between MYC and XPO1 Associated with Decreased T-Cell Presence and a Depleted Tumor Microenvironment May be Addressed By the Synergistic Combination of AZD4573 and Selinexor

Background: Genomically-targeted Diffuse Large B-cell (DLBCL) treatments for refractory or relapsing patients remain a clinical need alongside the growing success of Chimeric Antigen T-cells (CAR-T), which have ushered in durable responses in 40-70% of patients. However, recent studies have identified factors indicative of an inferior CAR-T response. Recent tumor Ecotype studies from Kotlov and Steen reveal that a Depleted or Cold tumor microenvironment (TME) is associated with poor CAR-T response. Notable features of these environments include a decreased presence of native T-cells alongside oncogenic alterations associated with their exhaustion, namely MYC and XPO1. Few precision treatments exist given the difficulty targeting MYC, but two avenues have shown recent success: inhibition of the downstream oncogene CDK9 via AZD4573 and upstream inhibition of a MYC propagator, the nuclear exportin XPO1 via Selinexor. Herein, we report the first observations of synergy between AZD4573 and Selinexor within DLBCL cell lines and data supporting that these genes may serve as accessible markers for inferior tumor immune microenvironments. Methods: We combined progression vs. response results from four previous CAR-T genomics studies, noting MYC status (Jaeger 2020, Jain 2021, Sworder 2021, and Shouval 2022). We next analyzed a 418-patient de-novo DLBCL patient cohort (Xu-Monette 2020) and 48-patient TCGA cohort after GEDIT immune deconvolution for differential expression and component enrichment between patients expressing high levels of MYC and XPO1 (+1 Standard Deviation) compared to normal counterparts. Significantly expressed genes were evaluated via gene set enrichment analyses. We next assayed AZD4573 and Selinexor vs. 7 DLBCL cell lines, 5 of which harbor MYC alterations, across a series of 9 concentrations (78nM-20µM) across 3-9 replicates. We followed these results by combining treatments in the MYC-altered Ly3, DHL6, and DHL4 cell lines, analyzing results using the BLISS synergy model. Results: Integrative analyses support that MYC alterations are associated with significantly reduced CAR-T outcomes. Of the 93 MYC-altered patients within the total 246-patient cohort, 65.59% of them experienced progression during CAR-T treatment compared to just 50.33% of WT CAR-T patients (p = 0.0242). Differential gene expression analyses between de-novo patients expressing high MYC or XPO1 revealed losses in Cytokine Signaling pathways (FDR = 6.96E-18) and gains in Cell Cycle modulators (FDR = 2.525E-20). Importantly, CD8A expression was significantly lower in these patients (FDR < 0.0001) alongside other key reductions of CDKN1A and TNFAIP3. After immune deconvolution, both High-MYC and High-XPO1 patients displayed significant losses of CD8 T-cell association, respectively (FDR = 0.0005, FDR = 0.0396). Notably, High-MYC patients without a CD8 T-cell presence experienced an inferior survival prognosis compared to High-MYC counterparts with a CD8 presence (p = 0.0272). High MYC or XPO1 patients that experienced a failure event before 24-months (EFS24) had significantly lower activated CD4 T-cell presence compared to normal counterparts as well (FDR = 0.0095, FDR = 0.0069). Next, AZD4573 and Selinexor assays vs. DLBCL cell lines demonstrated dose and MYC-dependent cell inhibition, with ED50 values ranging between 58.1 nM to 8.07 µM. Lastly, the molecules were combined vs. Ly3, DHL6 and DHL4 cell lines, resulting in synergistic reduction of cell viability and BLISS synergy scores of 33.76, and 11.86, and 5.04, respectively. Conclusions: Our results support that the cooperation between MYC and XPO1 is associated with T-cell reductions characteristic of a Depleted or Cold TME, a key issue for CAR-T success. We applied the targeted therapies AZD4573 and Selinexor vs. cell line models to address this pathway, with both displaying anti-tumor effects as single agents. Most importantly, we observed synergistic activity in the MYC-harboring Ly3 and DHL6 cell lines, and in the WT DHL4 cell line when these molecules were combined. These results are supported by integrative analyses that highlight the importance of targeting aberrant MYC signaling via CDK9 and XPO1 inhibition, revealing a potential avenue to address inferior response rates faced by patients harboring MYC-positive, Depleted tumor microenvironments that would otherwise face an inferior CAR-T prognosis.

Hydrophobic ion pairing and microfluidic nanoprecipitation enable efficient nanoformulation of a small molecule indolamine 2, 3-dioxygenaseinhibitor immunotherapeutic.

Blockade of programmed cell death-1 (PD-1) is a transformative immunotherapy. However, only a fraction of patients benefit, and there is a critical need for broad-spectrum checkpoint inhibition approaches that both enhance the recruitment of cytotoxic immune cells in cold tumors and target resistance pathways. Indoleamine 2, 3-dioxygenase (IDO) small molecule inhibitors are promising but suboptimal tumor bioavailability and dose-limiting toxicity have limited therapeutic benefits in clinical trials. This study reports on a nanoformulation of the IDO inhibitor navoximod within polymeric nanoparticles prepared using a high-throughput microfluidic mixing device. Hydrophobic ion pairing addresses the challenging physicochemical properties of navoximod, yielding remarkably high loading (>10%). The nanoformulation efficiently inhibits IDO and, in synergy with PD-1 antibodies improves the anti-cancer cytotoxicity of T-cells, in vitro and in vivo. This study provides new insight into the IDO and PD-1 inhibitors synergy and validates hydrophobic ion pairing as a simple and clinically scalable formulation approach.

Extracellular vesicles hybrid plasmid-loaded lipid nanovesicles for synergistic cancer immunotherapy

Combination immunotherapy of cancer vaccines with immune checkpoint inhibitors (ICIs) represents a promising therapeutic strategy for immunosuppressed and cold tumors. However, this strategy still faces challenges, including the limited therapeutic efficacy of cancer vaccines and immune-related adverse events associated with systematic delivery of ICIs. Herein, we demonstrate the antitumor immune response induced by outer membrane vesicle from Akkermansia muciniphila (Akk-OMV), which exhibites a favorable safety profile, highlighting the potential application as a natural and biocompatible self-adjuvanting vesicle. Utilizing tumor cell-derived exosome as an antigen source and Akk-OMV as a natural adjuvant, we construct a cancer vaccine formulation of extracellular vesicles hybrid lipid nanovesicles (Lipo@HEV) for enhanced prophylactic and therapeutic vaccination by promoting dendritic cell (DC) maturation in lymph node and activating cytotoxic T cell (CTL) response. The Lipo@HEV is further loaded with plasmid to enable gene therapy-mediated PD-L1 blockade upon peritumoral injection. Meanwhile, it penetrates into lymph node to initiate DC maturation and CTL activation, synergistically inhibiting the established tumor. The fabrication of extracellular vesicles hybrid plasmid-loaded lipid nanovesicles reveals a promising gene therapy-guided and vesicle-based hybrid system for therapeutic cancer vaccination and synergistic immunotherapy strategy.

Chemical Biology Perspectives on STING Agonists as Tumor Immunotherapy.

Stimulator of interferon genes (STING) is a crucial adaptor protein in the innate immune response. STING activation triggers cytokine secretion, including type I interferon and initiates T cell-mediated adaptive immunity. The activated immune system converts "cold tumors" into "hot tumors" that are highly responsive to T cells by recruiting them to the tumor microenvironment, ultimately leading to potent and long-lasting antitumor effects. Unlike most immune checkpoint inhibitors, STING agonists represent a groundbreaking class of innate immune agonists that hold great potential for effectively targeting various cancer populations and are poised to become a blockbuster in tumor immunotherapy. This review will focus on the correlation between the STING signaling pathway and tumor immunity, as well as explore the impact of STING activation on other biological processes. Ultimately, we will summarize the development and optimization of STING agonists from a medicinal chemistry perspective, evaluate their potential in cancer therapy, and identify possible challenges for future advancement.

Immunogenicity Threshold in Allogeneic Cells Impacts CTL Response to Nondominant Congenic Antigens.

Transplantation and cancer expose the immune system to neoantigens, including immunogenic (dominant and subdominant) and nonimmunogenic Ags with varying quantities and affinities of immunodominant peptides. Conceptually, immunity is believed to mainly target dominant Ags when subdominant or nondominant Ags are linked within the same cell due to T cell interference. This phenomenon is called immunodominance. However, our previous study in mice showed that linked nonimmunogenic Ags (OVA and GFP) containing immunodominant peptides mount immunity irrespective of the MHC-matched allogeneic cell's immunogenicity. Consequently, we further explored 1) under what circumstances does the congenic marker CD45.1 provoke immunity in CD45.2 mice, and 2) whether linking two dominant or subdominant Ags can instigate an immune response. Our observations showed that CD45.1 (or CD45.2), when connected to low-immunogenic cell types is presented as an immunogen, which contrasts with its outcome when linked to high-immunogenic cell types. Moreover, we found that both dominant and subdominant Ags are presented as immunogens when linked in environments with lower immunogenic thresholds. These findings challenge the existing perception that immunity is predominantly elicited against dominant Ags when linked to subdominant or nondominant Ags. This study takes a fundamental step toward understanding the nuanced relationship between immunogenic and nonimmunogenic Ags, potentially opening new avenues for comprehending cancer immunoediting and enhancing the conversion of cold tumors with low immunogenicity into responsive hot tumors.