Modulation Of Antitumor Immunity Research Articles

Transition Metal Oxide Nanomaterials: New Weapons to Boost Anti-Tumor Immunity Cycle.

Semiconductor nanomaterials have emerged as a significant factor in the advancement of tumor immunotherapy. This review discusses the potential of transition metal oxide (TMO) nanomaterials in the realm of anti-tumor immune modulation. These binary inorganic semiconductor compounds possess high electron mobility, extended ductility, and strong stability. Apart from being primary thermistor materials, they also serve as potent agents in enhancing the anti-tumor immunity cycle. The diverse metal oxidation states of TMOs result in a range of electronic properties, from metallicity to wide-bandgap insulating behavior. Notably, titanium oxide, manganese oxide, iron oxide, zinc oxide, and copper oxide have garnered interest due to their presence in tumor tissues and potential therapeutic implications. These nanoparticles (NPs) kickstart the tumor immunity cycle by inducing immunogenic cell death (ICD), prompting the release of ICD and tumor-associated antigens (TAAs) and working in conjunction with various therapies to trigger dendritic cell (DC) maturation, T cell response, and infiltration. Furthermore, they can alter the tumor microenvironment (TME) by reprogramming immunosuppressive tumor-associated macrophages into an inflammatory state, thereby impeding tumor growth. This review aims to bring attention to the research community regarding the diversity and significance of TMOs in the tumor immunity cycle, while also underscoring the potential and challenges associated with using TMOs in tumor immunotherapy.

Abstract PO4-19-08: Randomized phase II study of talazoparib vs talazoparib plus atezolizumab for patients with premenopausal HR+/HER2- metastatic breast cancer harboring homologous recombination deficiency scar (Young-PALETTA, KCSG BR21-09)

Abstract Background Cyclin D kinase-4/6(CDK4/6) inhibitor combined with endocrine treatment have significantly increased progression free survival(PFS) and overall survival(OS) of patients with hormone receptor(HR)-positive/HER2-negative metastatic breast cancer, and currently is a standard 1st line endocrine treatment option. Homologous recombination deficiency(HRD) including germline(g) BRCA1/2 pathogenic mutation is known to be prevalent among young premenopausal patients. Recently several retrospective data suggested that patients with germline BRCA1/2 pathogenic mutation or HRD have poorer outcome with CDK4/6 inhibitor treatment, however there is no prospective evidence. Poly ADP-ribose polymerase(PARP) inhibitors have shown significant benefit over standard chemotherapy in patients with HER2-neg metastatic breast cancer and gBRCA1/2 pathogenic mutation in two Phase 3 trials. Since preclinical data suggested that PARP inhibitors play a role in anti-tumor immune modulation, immunotherapy combined with PARP inhibitors were explored and showed promising antitumor efficacy in several single arm trials. In this trial, we investigate talazoparib and atezolizumab combination compared with talazoparib monotherapy in premenopausal women with HR+/HER2- metastatic breast cancer with HRD-scar after failure with 1st line palbociclib and endocrine treatment combination. Methods This study is a multicenter, randomized, open-label phase II trial comparing talazoparib versus talazoparib with atezolizumab in premenopausal women with HR+/HER2- metastatic breast cancer harboring HRD scar whose disease have progressed with endocrine and palbociclib combination as the 1st-line endocrine treatment. Prior one line of cytotoxic chemotherapy other than platinum for metastatic setting is allowed, but prior palliative endocrine treatment is not allowed. Eligible patients will be pre-screened with tumor tissue biopsy and peripheral blood sampling before endocrine and palbociclib treatment commencement. HRD status is determined through targeted sequencing(SOLIDaccuTestTM by NGeneBio) of tumor tissue and germline HRD genes including BRCA1/2 variant test. Whole genome sequencing and RNA sequencing are performed for biomarker analyses. Patients harboring HRD scar will receive first line endocrine treatment with palbociclib plus aromatase inhibitor with ovarian suppression. After progression with 1L endocrine treatment, patients will be randomized to either talazoparib alone or talazoparib plus atezolizumab combination. Germline BRCA1/2 pathogenic mutation is a stratification factor. Primary endpoint is a progression free survival after randomization(PFS2). Secondary objectives are a composite of PFS1 and PFS2, PFS1, overall survival, patient reported outcome, and biomarkers for treatment response. A total of 178 subjects are required to detect hazard ratio of 0.62 (median PFS2 of at least 10.5 months with talazoparib and atezolizumab combination vs 6.5 months with talazoparib alone), to achieve 80% power at a two-sided a significance level at 10%, considering drop-out rate of 5%. The study period is expected to be 72 months overall(36 months for accrual and randomization and 24 months of follow-up for primary endpoint PFS2). Citation Format: Hee Kyung Ahn, Jieun Lee, Jee Hung Kim, Kyung-Hun Lee, Jee Hyun Kim, Min Hwan Kim, Minsuk Kwon, Han Jo Kim, Kyung Hae Jung, Yeon Hee Park. Randomized phase II study of talazoparib vs talazoparib plus atezolizumab for patients with premenopausal HR+/HER2- metastatic breast cancer harboring homologous recombination deficiency scar (Young-PALETTA, KCSG BR21-09) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-19-08.

Abstract 3015: HD-PTP mediates tumor suppression via modulation of anti-tumor immunity

Abstract The role of circulating extracellular vesicles in cancer biology is an area of intense research interest. Here, we reveal a tumor suppressor mechanism of HD-PTP (His Domain Protein Tyrosine Phosphatase, encoded by PTPN23), a protein associated with ESCRT (endosomal sorting complex required for transport), a complex that controls endosome sorting and exosome secretion. While loss of HD-PTP is known to be tumorigenic in mice and is frequently observed in human cancers including Triple Negative Breast Cancer (TNBC), the relevant pathways have remained obscure. We find that inhibition of HD-PTP in vivo promotes tumor progression selectively in a syngeneic, immune-competent TNBC model but not in a matched immune-deficient model. Through systematic profiling of the exosomal proteome in HD-PTP proficient and deficient cells, we identify PD-L1 as a key exosomal cargo selectively enriched due to loss of HD-PTP. Mechanistically, we show that inhibition of HD-PTP results in PD-L1 stabilization, its co-localization with the exosomal marker CD63, the enrichment of endosomal PD-L1 and subsequent increase in ExoPD-L1 release. Exosomes produced post HD-PTP loss inhibits antigen-dependent T cell activity in vitro, and HD-PTP inhibition in a syngeneic TNBC model promotes non-cell-autonomous, exosome- and PD-L1-dependent tumor progression, associated with reduced T cell infiltration and altered macrophage polarization. Consistent with these findings, low HD-PTP levels in human solid tumors are associated with decreased tumor infiltrating lymphocytes, poor response to immune checkpoint inhibition and inferior overall survival. Taken together, our studies unveil a novel tumor suppressor pathway controlling PD-L1 trafficking and ExoPD-L1 secretion, highlighting the critical roles of HD-PTP and ESCRT in anti-tumor immunity. Citation Format: Chenxu Guo, Sheng Sun, Nayana Thimmiah, Agustina Maccio, Siang Koh, Leif Ellisen. HD-PTP mediates tumor suppression via modulation of anti-tumor 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 3015.

Protein Kinase STK24 Promotes Tumor Immune Evasion via the AKT-PD-L1 Axis.

Immunotherapy targeting PD-L1 is still ineffective for a wide variety of tumors with high unpredictability. Deploying combined immunotherapy with alternative targeting is practical to overcome this therapeutic resistance. Here, the deficiency of serine-threonine kinase STK24 is observed in tumor cells causing substantial attenuation of tumor growth in murine syngeneic models, a process relying on cytotoxic CD8+ T and NK cells. Mechanistically, STK24 in tumor cells associates with and directly phosphorylates AKT at Thr21, which promotes AKT activation and subsequent PD-L1 induction. Deletion or inhibition of STK24, by contrast, blocks IFN-γ-mediated PD-L1 expression. Various murine models indicate that in vivo silencing of STK24 can significantly enhance the efficacy of the anti-PD-1 blockade strategy. Elevated STK24 levels are observed in patient specimens in multiple tumor types and inversely correlated with intratumoral infiltration of cytotoxic CD8+ T cells and with patient survival. The study collectively identifies STK24 as a critical modulator of antitumor immunity, which engages in AKT and PD-L1/PD-1 signaling and is a promising target for combined immunotherapy.

Epigenetic modulation of antitumor immunity and immunotherapy response in breast cancer: biological mechanisms and clinical implications.

Breast cancer (BC) is the most common non-skin cancer and the second leading cause of cancer death in American women. The initiation and progression of BC can proceed through the accumulation of genetic and epigenetic changes that allow transformed cells to escape the normal cell cycle checkpoint control. Unlike nucleotide mutations, epigenetic changes such as DNA methylation, histone posttranslational modifications (PTMs), nucleosome remodeling and non-coding RNAs are generally reversible and therefore potentially responsive to pharmacological intervention. Epigenetic dysregulations are critical mechanisms for impaired antitumor immunity, evasion of immune surveillance, and resistance to immunotherapy. Compared to highly immunogenic tumor types, such as melanoma or lung cancer, breast cancer has been viewed as an immunologically quiescent tumor which displays a relatively low population of tumor-infiltrating lymphocytes (TIL), low tumor mutational burden (TMB) and modest response rates to immune checkpoint inhibitors (ICI). Emerging evidence suggests that agents targeting aberrant epigenetic modifiers may augment host antitumor immunity in BC via several interrelated mechanisms such as enhancing tumor antigen presentation, activation of cytotoxic T cells, inhibition of immunosuppressive cells, boosting response to ICI, and induction of immunogenic cell death (ICD). These discoveries have established a highly promising basis for using combinatorial approaches of epigenetic drugs with immunotherapy as an innovative paradigm to improve outcomes of BC patients. In this review, we summarize the current understanding of how epigenetic processes regulate immune cell function and antitumor immunogenicity in the context of the breast tumor microenvironment. Moreover, we discuss the therapeutic potential and latest clinical trials of the combination of immune checkpoint blockers with epigenetic agents in breast cancer.

Copper homeostasis and cuproptosis in cancer immunity and therapy.

Copper is an essential nutrient for maintaining enzyme activity and transcription factor function. Excess copper results in the aggregation of lipoylated dihydrolipoamide S-acetyltransferase (DLAT), which correlates to the mitochondrial tricarboxylic acid (TCA) cycle, resulting in proteotoxic stress and eliciting a novel cell death modality: cuproptosis. Cuproptosis exerts an indispensable role in cancer progression, which is considered a promising strategy for cancer therapy. Cancer immunotherapy has gained extensive attention owing to breakthroughs in immune checkpoint blockade; furthermore, cuproptosis is strongly connected to the modulation of antitumor immunity. Thus, a thorough recognition concerning the mechanisms involved in the modulation of copper metabolism and cuproptosis may facilitate improvement in cancer management. This review outlines the cellular and molecular mechanisms and characteristics of cuproptosis and the links of the novel regulated cell death modality with human cancers. We also review the current knowledge on the complex effects of cuproptosis on antitumor immunity and immune response. Furthermore, potential agents that elicit cuproptosis pathways are summarized. Lastly, we discuss the influence of cuproptosis induction on the tumor microenvironment as well as the challenges of adding cuproptosis regulators to therapeutic strategies beyond traditional therapy.

Potential role of intratumor bacteria outside the gastrointestinal tract: More than passengers.

Tumor-associated bacteria and gut microbiota have gained significant attention in recent years due to their potential role in cancer development and therapeutic response. This review aims to discuss the contributions of intratumor bacteria outside the gastrointestinal tract, in addition to exploring the mechanisms, functions, and implications of these bacteria in cancer therapy. We reviewed current literature on intratumor bacteria and their impact on tumorigenesis, progression, metastasis, drug resistance, and anti-tumor immune modulation. Additionally, we examined techniques used to detect intratumor bacteria, precautions necessary when handling low microbial biomass tumor samples, and the recent progress in bacterial manipulation for tumor treatment. Research indicates that each type of cancer uniquely interacts with its microbiome, and bacteria can be detected even in non-gastrointestinal tumors with low bacterial abundance. Intracellular bacteria have the potential to regulate tumor cells' biological behavior and contribute to critical aspects of tumor development. Furthermore, bacterial-based anti-tumor therapies have shown promising results in cancer treatment. Understanding the complex interactions between intratumor bacteria and tumor cells could lead to the development of more precise cancer treatment strategies. Further research into non-gastrointestinal tumor-associated bacteria is needed to identify new therapeutic approaches and expand our knowledge of the microbiota's role in cancer biology.

Comprehensive profiling of clock genes expression in hepatocellular carcinoma (HCC).

4108 Background: The circadian clock mechanism controls the physiological homeostasis of the liver and plays a key role in hepatocarcinogenesis. Recent evidence unveiled core clock proteins as novel therapeutic targets in cancer. Our group showed that clock regulators BMAL1 and CLOCK can promote proliferation of liver cancer cells by modulating the cell cycle checkpoint kinase Wee1. Here we further evaluated the molecular landscape of clock pathway alterations in HCC leveraging multi-platform profiling of patient tumor samples. Methods: 780 HCC tested at Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) were analyzed. Clock gene Score (CS) was determined using expression of core clock genes Z scores (positives of CLOCK, ARNTL, RORA/B/C and negatives of repressors CRY1/2, PER1/2/3, REVERBA/B) stratified by quartiles. xCell was used to quantify cell infiltration in the tumor microenvironment (TME). Significance was determined as P-values and adjusted for multiple testing ( q) of < .05. Gene expression profiles were analyzed for transcriptional signatures predictive of response to immunotherapy including the T cell inflamed score (TIS) and IFG score. Real world survival was obtained from insurance claims data and Kaplan-Meier estimates were calculated for comparison. Results: CS was higher in metastatic sites than primary tumors (median transcripts per million [TPM]: 0.81 vs 0.37, P < .05). No significant differences in patient age and sex were observed between CS Q1 (lowest) and Q4 (highest) cohorts, although a trend towards a higher frequency of males was observed in Q4 (76% vs 68%, Q4 vs Q1, P = .07). CS was positively associated with telomerase subunit TERT mutations (64% vs 52%, Q4 vs Q1, P = .04) and negatively correlated with FGF3 copy number amplification (2% vs 6%, P = .04) and WEE1 gene expression (median TPM: 15 vs 28, q < .05). No dMMR/MSI-H tumors were observed in our series and there were no significant associations with tumor mutational burden and PD-L1 protein expression. Expression of immune related genes was lower in tumors with high CS, including IDO1, CD80, PD-L1, LAG3, CD86, TIM3, PD-1 and PD-L2 (fold change: 0.57-0.67 q < .05). NK cell infiltration in the TME and the TIS score were also significantly lower in CS-high HCC ( q < .05). Individually, lower CLOCK and CRY1 tumor mRNA expression were associated with longer OS (Q1 vs Q4: CLOCK HR 0.71, 95%CI [0.51-0.98], P = .04 and CRY1 HR 0.70 [0.51-0.95], P = .02, respectively). Conclusions: This is the most extensive profiling study to investigate the expression of clock genes in HCC. Our data show that clock genes expression impacts patient survival and is associated with alterations in immune-related gene expression and TIS score which suggest a role in the modulation of anti-tumor immunity. These results support the clock pathway role as a oncogenic driver and its potential as a therapeutic target in HCC.

Characterization of MCL-1 in patients with colorectal cancer (CRC): Expression, molecular profiles, and outcomes.

3085 Background: Myeloid cell leukemia 1 (MCL-1) is a member of the BCL-2 protein family and is anti-apoptotic/pro-survival in function. Dysregulation of MCL-1 expression has been reported in several solid tumors, including lung and breast cancer. In CRC, MCL-1 has been associated with resistance to chemotherapeutic drugs and multi-kinase inhibitor regorafenib. Our study aimed to characterize the molecular features associated with MCL-1 gene expression in CRC. Methods: 28,576 CRC samples were analyzed by Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES). MCL-1 expression was stratified by quartiles where top quartile transcripts per million (TPM) were considered high (Q4) and bottom quartile low (Q1). Cell infiltration (CI) in the tumor microenvironment (TME) was estimated by RNA deconvolution analysis using QuantiSEQ. Interferon-gamma and T-cell inflamed signatures were also calculated from RNA data. X2 and Fisher-Exact tests were used, and statistical significance was determined as a P-value adjusted for multiple comparisons ( q < 0.05). Real world survival was obtained from insurance claims data and Kaplan-Meier estimates were calculated for molecularly defined patients. Results: MCL-1 expression was significantly increased in rectal tumors compared to other primary tumor sites (median TPM [mTPM] 20.8 vs 18.9 vs 18.7, in rectal vs left-sided vs right-sided, respectively, q < 0.0001). MCL-1 high tumors were associated with increased rate of PD-L1 IHC levels (Q1 vs Q4: 4.2% vs 9.7% [all CRC]; 3.7% vs 8.9% [pMMR/MSS], q < 0.0001), while MCL-1-low showed marginally increased rates of TMB-high (8.0% vs 7.0% [all CRC]; 3.3% vs 2.6% [pMMR/MSS], P < 0.05). MCL-1-high expression was associated with a higher T-cell inflamed signature and IFN score ( q < 0.0001). MCL-1 high was associated with higher mutation rates of CDKN2A, BRCA2, KRAS and GNAS, while lower mutation rates of TP53, PIK3CA, PTEN, BRAF, APC, FBXW7, AMER1, SOX9, and SMAD2 and copy number amplifications in several genes ( q < 0.05). MCL-1 high TME had higher CI of M1 and M2 macrophages, monocytes, B cells, NK cells, and T-reg infiltration, while dendritic cells and CD4+ T-cell were lower ( q < 0.05). Patients with high MCL-1 expressing CRC had longer survival (Q4 vs Q1: 33.5 vs 24.8 months, HR 0.72, 95% CI 0.67-0.77, P < 0.0001) and longer time-on-treatment with regorafenib although with limited clinical benefit (Q4 vs Q1: 2.03 vs 1.06 months, HR: 0.67, 95% CI: 0.50-0.89, P = 0.005). Conclusions: Our data show a strong correlation between distinct immune biomarkers, TME cell infiltration and MCL-1 expression in CRC. Furthermore, increased tumor MCL-1 expression improved patient prognosis and treatment outcomes. These findings suggest a key clinical role for MCL-1 as an important modulator of anti-tumor immunity and TME and a potential biomarker in CRC.

Abstract 2892: A SREBF2-dependent genetic program drives a novel immunotolerant dendritic cell population that supports cancer progression

Abstract The development of immune responses to cancers are crucially dependent upon effective antigen cross-presentation by local dendritic cell (DC) populations. Indeed, DC-mediated T cell stimulation is essential for generating responses to anti-PD-1 immunotherapy. Tumors have been shown to hijack DC function in a process broadly known as tolerization. Despite the significant implications of this process on the modulation of anti-tumor immunity, the underlying mechanisms of this process remain unclear. We have identified a unique population of DCs in transgenic models of both melanoma and non-small cell lung cancer that arise within the tumor microenvironment and migrate to the draining lymph node (LN) tissues during the course of disease progression. RNAseq and flow cytometry studies revealed this DC population to exhibit a unique CD63 surface marker, enabling us to determine this subset to drive regulatory T cell (Treg) and TH2 expansion while suppressing conventional DC-mediated activation of CD8+ T cell responses, even at rare numbers. Additional transcriptional and metabolic studies showed these CD63+ DCs to be a subset of the previously identified 'mature DCs enriched in immunoregulatory molecules' (mregDC) population and to exhibit both enhanced activity of the mevalonate biosynthetic pathway as well as increased lipid stores and dependence on fatty acid oxidation. Inhibitor studies confirmed that mevalonate biosynthesis drives CD63+ mregDC-mediated immune tolerance while scATACseq studies have identified open regulatory elements in CD63+ mregDCs associated with key rate-limiting genes associated with the mevalonate pathway. Additional lineage analysis revealed the CD63+ mregDC population to evolve primarily from type 2 conventional DCs in vivo. Both DC-specific genetic deletion of the SREBF2 master transcription factor as well as SREBP2 pharmacologic inhibition suppresses melanoma progression while enhancing tumor CD8+ T cell infiltration. Adoptive transfer of CD63+ mregDCs enhances melanoma metastases and promotes the accumulation of Tregs in draining LN tissues. scRNAseq studies as well as spatial transcriptomic analysis has confirmed this same DC population in the sentinel LN tissues of melanoma patients. We propose that this novel DC subset represents a key target for augmenting anti-tumor immune responses and suppressing metastatic progression. Ongoing work is focused on dissecting the regulatory pathways dictating the transcriptional program of CD63+ mregDCs and developing a CD11c-CD63 bispecific depletion antibody for further pre-clinical testing. Citation Format: Michael P. Plebanek, Yue Xue, Y-Van Nguyen, Nicholas C. DeVito, Xueying Wang, Georgia Beasley, Nagendra Yarla, Bala Thievanthiran, Brent A. Hanks. A SREBF2-dependent genetic program drives a novel immunotolerant dendritic cell population that supports cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2892.

Abstract 3445: Immune cells infiltration and activation are higher in breast cancers resistant to antiestrogen therapy

Abstract We investigated the tumor microenvironment (TME) in estrogen receptor positive (ER+) primary breast cancers from stage I-III patients treated with estrogen deprivation (ED, induced with letrozole) for 10-21 days. We used primary breast cancer biopsies, collected from 198 patients, to assess tumor-infiltrating lymphocytes (TILs), transcriptomic profiles, and TME composition. Diagnostic biopsy and surgical on-treatment tumor specimens were collected; Ki67, ERα, PR, and HER2 expression were measured using automated quantitative analysis (AQUA). Tumors were categorized as estrogen deprivation-sensitive (ED-S) or -resistant (ED-R) based on the natural log (ln) of the Ki67 score (ln ≤1.0 or ≤2.7% Ki67+ cells vs. ln ≥2.0 or ≥7.4%, respectively) in the on-treatment (surgical) biopsy. Scoring of stromal (s)TILs, using hematoxylin and eosin staining of on-treatment specimens, revealed that sTILs were elevated in ED-R compared to ED-S tumors (p<0.0001). We next constructed tissue microarrays (TMAs) of on-treatment tumor sections and performed cyclic immunofluorescence (CyCIF) with 38 antibodies, including markers of hormone receptors, immune cells, signaling pathways, proliferation, DAPI (nuclei), and others. We then segmented single cells from each tumor core and designated them as immune, cancer, or stromal cells based on expression of a set of markers. Briefly, cells that stained strongly for CD45 (leukocyte marker), and/or CD4 (T lymphocyte marker), or CD68 (macrophage marker) were considered immune cells. CD45/CD4/CD68-negative cells were categorized as tumor, if E-cadherin and/or cytokeratin-positive, or stromal cells, if -negative. To investigate the TME composition, we next assessed cell specific spatial enrichment by quantifying the expression of immune markers in the area immediately adjacent to each tumor cell. Relative to ED-S, ED-R samples exhibited higher CD45+ cells (p<0.0001) and higher CD8+ T cells (p=0.0329), while CD4+ T cells showed no difference. Conversely, immune-suppressive T-reg (CD4+FOXP3+) cells were enriched in the ED-S samples (p=0.0004). In addition, PD1, a marker for T cell exhaustion, was higher in the ED-S group (p=0.0004), as were CD68+ cells (p<0.0001). To confirm and expand upon these findings, RNA-sequencing was performed on the on-treatment tumor sections. Gene set enrichment analysis of hallmark gene signatures revealed that immune-related gene sets, such as “IFNα response”, “IFNɣ response”, “allograft rejection”, and “inflammatory response” were enriched in ED-R vs. ED-S tumors. In summary, our study shows that activated anti-tumor immune cells are enriched in the TME of ER+ breast tumors resistant to estrogen deprivation, whereas ED-sensitive tumors show a more immunosuppressed or immune cold milieu. These data suggest a potential causal link between antiestrogen treatment and modulation of antitumor immunity in ER+ breast cancers. Citation Format: Fabiana Napolitano, Dhivya R. Sudhan, Yunguan Wang, Paula I. González-Ericsson, Luigi Formisano, Kyung-Min Lee, Chang-Ching Lin, María Rosario Chica-Parrado, Hongli Ma, Nathaniel Evans, Alberto Servetto, Saurabh Mendiratta, Spencer Barnes, Yisheng V. Fang, Justin M. Balko, Gordon B. Mills, Marilyne Labrie, Ariella B. Hanker, Carlos L. Arteaga. Immune cells infiltration and activation are higher in breast cancers resistant to antiestrogen therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3445.

Abstract 4616: Automated multiplex immunofluorescence enables single cell analysis of tumor stroma

Abstract Background: The tumor microenvironment (TME) consists of malignant cells and supporting non-malignant cellular and non-cellular components that form the tumor stroma. The tumor stroma plays an important role in tumor progression and has emerged as a modulator of anti-tumor immunity (Salmon et al., Nat Rev Cancer 2019) and responses to therapy (Hirata and Sahai, Cold Spring Harb Perspect Med 2017). As such, several therapeutic approaches have recently been developed to target stromal cells as anti-cancer treatments (Valkenburg et al., Nat Rev Clin Oncol 2018, Bejarano et al., Cancer Discov 2021). In addition, the composition of the TME has been recognized as a prognostic factor for survival in cancer patients (Pagès et al., Oncogene 2011). Current protein-based approaches to characterize and better understand the cell composition of the tumor stroma face many limitations such as reagent availability and lengthy protocols. In this study, we identified a list of 22 markers to characterize non-tumoral immune cells, fibroblasts and endothelial cells in the TME, in a single tissue slide. We propose an approach that overcomes reagent incompatibility and opens new avenues of research of tumor stroma. Method: Multiorgan Tumor Microarray (TMA) was interrogated with a sequential immunofluorescence (seqIF™) panel encompassing protein markers enabling characterization of TME. A 22-plex panel was created based on expanding an already established 13-plex panel (CD3, CD4, CD8, CD11c, CD20, CD45, CD56, CD68, aSMA, FoxP3, Ki67, PD1, PD-L1) by adding 9 additional antibodies (CD11b, CD14, CD31, CD47, CK, FAP, LaminB1, SIRPα, Vimentin). Hyperplex immunofluorescent staining was performed using automated staining-imaging COMET™ platform generating ome-tiff images containing 25 layers: DAPI, 2 autofluorescent and 22 marker channels. Postprocessing of images was done with HORIZON™ image analysis software. Results: We established a panel of 22 markers that can be analyzed simultaneously on a single tissue slide despite limited variability in primary antibody species. Using seqIF™ protocol allowed the study of colocalization and co-expression of markers not compatible to study simultaneously in the traditional immunofluorescence approach. Our hyperplex data revealed distinct composition of the stromal compartment between different tumor types, highlighting high heterogeneity in the tissue composition and stromal architecture. Conclusion: COMET™ platform enabled studying in detail TME components and highlighted heterogeneity of tumor stroma across different tissue types. SeqIF™ protocol lifted the limitation imposed by same species antibodies and allowed simultaneous interrogation of markers’ expression preserving their spatial relationship. Citation Format: François Rivest, Victor de Gautard, Vytautas Navikas, Nadine Nelson, Bastian Nicolai, Joanna Kowal, Saska Brajkovic. Automated multiplex immunofluorescence enables single cell analysis of tumor stroma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4616.

Carbon Nanomaterials: Emerging Roles in Immuno-Oncology.

Cancer immunotherapy has made breakthrough progress in cancer treatment. However, only a subset of patients benefits from immunotherapy. Given their unique structure, composition, and interactions with the immune system, carbon nanomaterials have recently attracted tremendous interest in their roles as modulators of antitumor immunity. Here, we focused on the latest advances in the immunological effects of carbon nanomaterials. We also reviewed the current preclinical applications of these materials in cancer therapy. Finally, we discussed the challenges to be overcome before the full potential of carbon nanomaterials can be utilized in cancer therapies to ultimately improve patient outcomes.

The role of AMPK in cancer metabolism and its impact on the immunomodulation of the tumor microenvironment.

Adenosine monophosphate-activated protein kinase (AMPK) is a key metabolic sensor that is pivotal for the maintenance of cellular energy homeostasis. AMPK contributes to diverse metabolic and physiological effects besides its fundamental role in glucose and lipid metabolism. Aberrancy in AMPK signaling is one of the determining factors which lead to the development of chronic diseases such as obesity, inflammation, diabetes, and cancer. The activation of AMPK and its downstream signaling cascades orchestrate dynamic changes in the tumor cellular bioenergetics. It is well documented that AMPK possesses a suppressor role in the context of tumor development and progression by modulating the inflammatory and metabolic pathways. In addition, AMPK plays a central role in potentiating the phenotypic and functional reprogramming of various classes of immune cells which reside in the tumor microenvironment (TME). Furthermore, AMPK-mediated inflammatory responses facilitate the recruitment of certain types of immune cells to the TME, which impedes the development, progression, and metastasis of cancer. Thus, AMPK appears to play an important role in the regulation of anti-tumor immune response by regulating the metabolic plasticity of various immune cells. AMPK effectuates the metabolic modulation of anti-tumor immunity via nutrient regulation in the TME and by virtue of its molecular crosstalk with major immune checkpoints. Several studies including that from our lab emphasize on the role of AMPK in regulating the anticancer effects of several phytochemicals, which are potential anticancer drug candidates. The scope of this review encompasses the significance of the AMPK signaling in cancer metabolism and its influence on the key drivers of immune responses within the TME, with a special emphasis on the potential use of phytochemicals to target AMPK and combat cancer by modulating the tumor metabolism.

The Species of Gut Bacteria Associated with Antitumor Immunity in Cancer Therapy.

Both preclinical and clinical studies have demonstrated that the modulation of gut microbiota could be a promising strategy for enhancing antitumor immune responses and reducing resistance to immunotherapy in cancer. Various mechanisms, including activation of pattern recognition receptors, gut commensals-produced metabolites and antigen mimicry, have been revealed. Different gut microbiota modulation strategies have been raised, such as fecal microbiota transplantation, probiotics, and dietary selection. However, the identification of gut bacteria species that are either favorable or unfavorable for cancer therapy remains a major challenge. Herein, we summarized the findings related to gut microbiota species observed in the modulation of antitumor immunity. We also discussed the different mechanisms underlying different gut bacteria's functions and the potential applications of these bacteria to cancer immunotherapy in the future.

Ablation of the endoplasmic reticulum stress kinase PERK induces paraptosis and type I interferon to promote anti-tumor Tcell responses.

Activation of unfolded protein responses (UPRs) in cancer cells undergoing endoplasmic reticulum (ER) stress promotes survival. However, how UPR in tumor cells impacts anti-tumor immune responses remains poorly described. Here, we investigate the role of the UPR mediator pancreatic ER kinase (PKR)-like ER kinase (PERK) in cancer cells in the modulation of anti-tumor immunity. Deletion of PERK in cancer cells or pharmacological inhibition of PERK in melanoma-bearing mice incites robust activation of anti-tumor Tcell immunity and attenuates tumor growth. PERK elimination in ER-stressed malignant cells triggers SEC61β-induced paraptosis, thereby promoting immunogenic cell death (ICD) and systemic anti-tumor responses. ICD induction in PERK-ablated tumors stimulates type I interferon production in dendritic cells (DCs), which primes CCR2-dependent tumor trafficking of common-monocytic precursors and their intra-tumor commitment into monocytic-lineage inflammatory Ly6C+CD103+ DCs. These findings identify how tumor cell-derived PERK promotes immune evasion and highlight the potential of PERK-targeting therapies in cancer immunotherapy.

Phase II INTERACT-ION study: ezabenlimab (BI 754091) and mDCF (docetaxel, cisplatin, and 5-fluorouracil) followed by chemoradiotherapy in patients with Stage III squamous cell anal carcinoma.

BackgroundChemoradiotherapy alone is the standard treatment for locally advanced squamous cell anal carcinoma (SCAC). However, up to 50% of patients will experience recurrence; thus, there is a need for new treatments to improve outcomes. Modified docetaxel, cisplatin and 5-fluorouracil (mDCF) is a treatment option for first-line metastatic SCAC, having shown efficacy in the Epitopes-HPV01 and -02 trials (NCT01845779 and NCT02402842). mDCF treatment also plays a role in the modulation of anti-tumor immunity, suggesting it may be a good combination partner for immunotherapy in patients with SCAC. Anti-programmed death protein-1 (PD-1) immunotherapy has been shown to be effective in metastatic SCAC. We therefore designed the INTERACT-ION study to assess the combination of mDCF with ezabenlimab (BI 754091), an anti-PD-1 antibody, followed by chemoradiotherapy, in patients with Stage III SCAC.MethodsINTERACT-ION is a pivotal, open-label, single-arm phase II study in patients with treatment-naïve Stage III SCAC. Patients will receive induction treatment with mDCF (docetaxel 40 mg/m2 and cisplatin 40 mg/m2 on Day 1, 5-fluorouracil 1200 mg/m2/day for 2 days) every 2 weeks for 4 cycles and ezabenlimab (240 mg given intravenously) every 3 weeks for 3 cycles. In the absence of disease progression at 2 months, two additional cycles of mDCF and one additional cycle of ezabenlimab will be administered. Patients with radiological objective response, pathological complete/near-complete response and biological complete response will then receive an involved-node radiotherapy with intensity-modulated radiation therapy and concurrent chemotherapy, followed by ezabenlimab alone for seven cycles. All other patients will receive standard chemoradiotherapy. The primary endpoint is the clinical complete response rate 10 months after the first cycle of mDCF plus ezabenlimab. Major secondary endpoints are major pathological response and biological complete response after induction treatment. An extensive ancillary biomarker study in tumor tissue and peripheral blood will also be conducted.DiscussionThe addition of immunotherapy to chemotherapy is an area of active interest in metastatic anal cancer. This pivotal study will evaluate this combination in the locally advanced setting. Ancillary biomarker studies will contribute to the understanding of predictors of response or resistance to treatment.Clinical Trial Registration https://clinicaltrials.gov/ct2/show/NCT04719988, identifier NCT04719988.

ScRNA-seq reveals ATPIF1 activity in control of T cell antitumor activity

ABSTRACT ATP synthase inhibitory factor 1 (ATPIF1) is a mitochondrial protein with an activity in inhibition of F1Fo-ATP synthase. ATPIF1 activity remains unknown in the control of immune activity of T cells. In this study, we identified ATPIF1 activity in the induction of CD8+ T cell function in tumor models through genetic approaches. ATPIF1 gene inactivation impaired the immune activities of CD8+ T cells leading to quick tumor growth (B16 melanoma and Lewis lung cancer) in ATPIF1-KO mice. The KO T cells exhibited a reduced activity in proliferation and IFN-γ secretion with metabolic reprogramming of increased glycolysis and decreased oxidative phosphorylation (OXPHOS) after activation. T cell exhaustion was increased in the tumor infiltrating leukocytes (TILs) of KO mice as revealed by the single-cell RNA sequencing (scRNA-seq) and confirmed by flow cytometry. In contrast, ATPIF1 overexpression in T cells increased expression of IFN-γ and Granzyme B, subset of central memory T cells in CAR-T cells, and survival rate of NALM-6 tumor-bearing mice. These data demonstrate that ATPIF1 deficiency led to tumor immune deficiency through induction of T cell exhaustion. ATPIF1 overexpression enhanced the T cell tumor immunity. Therefore, ATPIF1 is a potential molecular target in the modulation of antitumor immunity of CD8+ T cells in cancer immunotherapy. Induction of ATPIF1 activity may promote CAR-T activity in cancer therapy.

Abstract 4166: Small-molecule inhibition of glycogen synthase kinase-3 (GSK-3) increases the efficacy of anti-PD-L1 therapy in a murine model of microsatellite stable colorectal cancer (CRC); Therapeutic response correlates with T cell ratios and serum cytokine profiles in mice

Abstract Immune checkpoint blockade (ICB) with αPD-1/PD-L1 has impressive efficacy in microsatellite unstable (MSI+) CRC but no significant activity in the 85% of CRC cases that are microsatellite stable (MSS). Our group previously demonstrated that 9-ING-41, a small-molecule inhibitor of GSK-3 in clinical development, stimulates both NK and T cell activity. This project characterizes the effects of 9-ING-41 in combination with ICB in vivo. In a syngeneic murine colon carcinoma BALB/c model using MSS cell line CT-26, we compared isotype, 9-ING-41 (70 mg/kg 2x/wk), αPD-1 (10 mg/kg 2x/wk), αPD-L1 (10 mg/kg 2x/wk), 9-ING-41 + αPD-1, and 9-ING-41 + αPD-L1 treatment groups. The median overall survival was 19 days (d) with 9-ING-41 (hazard ratio for death (HR) 0.12; 95% confidence interval (CI), 0.03-0.54; p=0.008), 16 d for 9-ING-41 + isotype (HR 0.40; 95% CI, 0.08-1.9; p=0.14), 20.5 d for αPD-1 (HR 0.15; 95% CI, 0.03-0.67; p=0.0185), 18 d for αPD-L1 (HR 0.21; 95% CI, 0.05-0.89; p=0.0559), 17 d for 9-ING-41 + αPD-1 (HR 0.37; 95% CI, 0.09-0.1.5; p=0.29), and 45.5 d for 9-ING-41 + αPD-L1 (HR 0.08; 95% CI, 0.02-0.36; p=0.0019), compared to 16 d for the isotype control group. Tumor response rates observed were 33.3% in the 9-ING-41 + αPD-L1 group, 16.6% in the αPD-1 group, and a 0% for all other treatment groups. We hypothesize that the 9-ING-41-mediated upregulation of PD-L1 in CRC cells (HCT-116, HT-29) observed via both flow cytometry (FC) and western blot analysis may contribute to the increased efficacy of combination therapy with αPD-L1, as compared to αPD-1. Significant differences between responders and non-responders in intratumoral and splenic natural killer (NK) and T cell subsets 14-days post-treatment initiation were shown by multi-color FC. Compared to non-responders, regardless of treatment group, responders had lower percentages of splenic CD4+ (p=0.0145) and CD8+ T cells (p=0.0001), increased percentages of splenic CD69+ activated T cells (p=0.0070) and FOXP3+ regulatory T cells (p=0.001), and increased percentages of tumor-infiltrating CD3+ (p=0.0006) and CD4+ T cells (p<0.0001). Responders had lower splenic CD8+/Treg (p=0.0007) and CD4+/Treg (p=0.001) ratios and higher intra-tumoral CD8+/Treg (p=0.0032) and CD4+/Treg (p=0.0001) ratios. Murine serum cytokine profiling showed that responders had lower concentrations of tumorigenic cytokines (BAFF, CCL7, CCL12, VEGF, VEGFR2, CCL21) and higher concentrations of immunomodulatory cytokines (CCL4, TWEAK, GM-CSF, CCL22, IL-12p70) compared to non-responders. These results demonstrate that small-molecule inhibition of GSK-3 with 9-ING-41 may increase the anti-tumor effects of ICB and improve response in patients with MSS CRC via modulation of anti-tumor immunity and cytokine signaling. Citation Format: Kelsey E. Huntington, Anna Louie, Lanlan Zhou, Benedito A. Carneiro, Wafik El-Deiry. Small-molecule inhibition of glycogen synthase kinase-3 (GSK-3) increases the efficacy of anti-PD-L1 therapy in a murine model of microsatellite stable colorectal cancer (CRC); Therapeutic response correlates with T cell ratios and serum cytokine profiles in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4166.

Abstract 1380: Setdb1-loss reactivates ERV expression and interferon signaling to induce immune-mediated melanoma clearance

Abstract While immunotherapies have been revolutionary for the treatment of many cancers including melanoma, most patients do not respond, or develop resistance to these therapies. Several epigenetic modulators have been identified as potential targets for augmenting anti-tumor immunity, however the full breadth of their role is not well-understood. To identify modulators of anti-tumor immunity in melanoma, we performed a whole-genome CRISPR screen using the YUMMER-G model and identified members of the HUSH complex and its associated methyltransferase, Setdb1, as drop-out hits. To validate these findings, we used CRISPR-Cas9 to knockout Setdb1 individually and observed that Setdb1-/- tumors are cleared by their host mice in a CD8+ T-cell dependent manner. To determine the source of immunogenicity in Setdb1-/- cells, we performed RNA-sequencing, cytokine profiling, and evaluated antigen presentation by flow cytometry. These assays revealed increased MHC-I levels, elevated secretion of Ifn-, and induction of an interferon-stimulated gene (ISG) expression program, consistent with the observed immunogenicity. We further determined that treating Setdb1-/- cells with IFNAR-blocking antibodies restores MHC-I levels to wild-type levels, suggestive of a tumor-intrinsic interferon loop. We hypothesized that type-I interferon signaling is generated through sensing of expressed endogenous retroviral elements (ERVs), and consistently observe upregulation of ERVs in Setdb1-/- cells. Previous studies have described the potential for ERV-derived antigens to act as substrates for anti-tumor activity by CD8+ T cells. We identified T cells in the tumor microenvironment of Setdb1-/- tumors that bind tetramer carrying the P15E peptide derived from ERV envelope. Together, these findings suggest that tumors lacking expression of Setdb1-/- upregulate ERV transcripts, driving expression of type-I interferons which generate an inflamed tumor microenvironment and potentiate efficient tumor clearance of Setdb1-/- tumors. Overall, this study emphasizes a critical role for regulators of ERV expression and type-I interferon expression, and suggests their potential as therapeutic targets for augmenting anti-cancer immune responses. Citation Format: Meaghan K. McGeary, William Damsky, Drew Daniels, Goran Micevic, Eric Song, Hua Jane Lou, Clotilde Calderwood, Sateja Paradkar, Akiko Iwasaki, David Calderwood, Benjamin Turk, Marcus Bosenberg. Setdb1-loss reactivates ERV expression and interferon signaling to induce immune-mediated melanoma clearance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1380.