Regulation Of Antitumor Immunity Research Articles

CWF19L1 promotes T-cell cytotoxicity through the regulation of alternative splicing.

Enhancing host anti-tumor immunity is paramount for advancing cancer immunotherapy. In this study, we identify CWF19-like cell cycle control factor 1 (CWF19L1) as a novel splicing regulator that enhances T cell-mediated cytotoxicity. CWF19L1 interacts prominently with key splicing factors within the nucleus, including components of the U5 small nuclear ribonucleoprotein (snRNP) and the pre-mRNA processing factor 19 (PRPF19) complex. Deficiency of CWF19L1 disrupts alternative splicing of immune-related genes, resulting in diminished expression of cytotoxic molecules. Furthermore, CWF19L1 plays a critical role in promoting T cell-mediated anti-tumor responses by upregulating the expression of effector cytokines. Our findings unveil previously undocumented functions of CWF19L1 in alternative splicing and its involvement in the regulation of anti-tumor immunity, highlighting its potential as a therapeutic target for novel cancer immunotherapies.

Regulation of anti-tumor immunity by metal ion in the tumor microenvironment.

Metal ions play an essential role in regulating the functions of immune cells by transmitting intracellular and extracellular signals in tumor microenvironment (TME). Among these immune cells, we focused on the impact of metal ions on T cells because they can recognize and kill cancer cells and play an important role in immune-based cancer treatment. Metal ions are often used in nanomedicines for tumor immunotherapy. In this review, we discuss seven metal ions related to anti-tumor immunity, elucidate their roles in immunotherapy, and provide novel insights into tumor immunotherapy and clinical applications.

PAK1 inhibition increases TRIM21-induced PD-L1 degradation and enhances responses to anti-PD-1 therapy in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDA) is a common malignancy with a 5-year survival <10 %. Immunosuppressive tumor microenvironment (TME) plays a critical role in the progression of PDA. In recent years, programmed death-ligand 1 (PD-L1)/programmed cell death protein-1 (PD-1) blockade has emerged as a potent anti-tumor immunotherapy, while is yet to achieve significant clinical benefits for PDA patients. P21-Activated kinase 1 (PAK1) is highly upregulated in PDA and has been reported to be involved in the regulation of anti-tumor immunity. This study aims to investigate the combined effect of PAK1 inhibition and anti-PD-1 therapy on PDA and the underlying mechanisms. We have shown that PAK1 expression positively correlated with PD-L1 in PDA patients, and that inhibition of PAK1 downregulated PD-L1 expression of PDA cells. More importantly, we have demonstrated that PAK1 competed with PD-L1 in binding to tripartite motif-containing protein 21 (TRIM21), a ubiquitin E3 ligase, resulting in less ubiquitination and degradation of PD-L1. Moreover, PAK1 inhibition promoted CD8+ T cells activation and infiltration. In a murine PDA model, the combination of PAK1 inhibition and anti-PD-1 therapy showed significant anti-tumor effects compared with the control or monotherapy. Our results indicated that the combination of PAK1 inhibition and anti-PD-1 therapy would be a more effective treatment for PDA patients.

Abstract 1358: Elucidating the contribution of T cell-intrinsic VISTA to the suppression of antitumor T cell immunity

Abstract Immune checkpoint receptors (ICRs) negatively regulate antitumor immune responses. While significant advances have been made in understanding the mechanisms of well-established ICRs such as PD-1 and CTLA-4, other checkpoint proteins remain incompletely characterized and may be targeted to synergistically boost antitumor immunity. One such protein is V-domain immunoglobulin suppressor of T-cell activation (VISTA), an ICR whose expression curtails the function of several lymphoid and myeloid lineages, including tumor-reactive cytotoxic T cells. Globally blocking VISTA elicits potent T cell-mediated antitumor immunity and systemic myeloid cell-mediated inflammatory responses. Mechanistically, VISTA has been shown to inhibit T cell activation by acting as an inhibitory ligand on antigen-presenting cells. However, the impact of T cell-intrinsic VISTA on modulating tumor-reactive T cell responses has not been thoroughly investigated, and it remains unclear whether blocking T cell-specific VISTA is adequate for revitalizing T cell-mediated tumor control. In this study, we utilized VISTA conditional knockout mice models to examine the relative contribution of T cell-intrinsic VISTA versus myeloid-derived VISTA in the regulation of antitumor immunity. We present evidence that blocking VISTA on T cells alone is insufficient to induce strong antitumor immune responses, whereas blocking VISTA on myeloid cells counteracts its dominant role in orchestrating T cell suppression. Citation Format: Sachin Patnaik, Elizabeth Delaney, Cassandra Gilmour, Keman Zhang, Amin Zakeri, Hieu M. Ta, Prerana B. Parthasarathy, Tyler Alban, Timothy Chan, Li Lily Wang. Elucidating the contribution of T cell-intrinsic VISTA to the suppression of antitumor T cell 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 1358.

Identification of cytokine-induced cell communications by pan-cancer meta-analysis.

Cancer immune responses are complex cellular processes in which cytokine-receptor interactions play central roles in cancer development and response to therapy; dysregulated cytokine-receptor communication may lead to pathological processes, including cancer, autoimmune diseases, and cytokine storm; however, our knowledge regarding cytokine-mediated cell-cell communication (CCI) in different cancers remains limited. The present study presents a single-cell and pan-cancer-level transcriptomics integration of 41,900 cells across 25 cancer types. We developed a single-cell method to actively express 62 cytokine-receptor pairs to reveal stable cytokine-mediated cell communications involving 84 cytokines and receptors. The correlation between the sample-based CCI profile and the interactome analysis indicates multiple cytokine-receptor modules including TGFB1, IL16ST, IL15, and the PDGF family. Some isolated cytokine interactions, such as FN1-IL17RC, displayed diverse functions within over ten single-cell transcriptomics datasets. Further functional enrichment analysis revealed that the constructed cytokine-receptor interaction map is associated with the positive regulation of multiple immune response pathways. Using public TCGA pan-cancer mutational data, co-mutational analysis of the cytokines and receptors provided significant co-occurrence features, implying the existence of cooperative mechanisms. Analysis of 10,967 samples from 32 TCGA cancer types revealed that the 84 cytokine and receptor genes are significantly associated with clinical survival time. Interestingly, the tumor samples with mutations in any of the 84 cytokines and receptors have a substantially higher mutational burden, offering insights into antitumor immune regulation and response. Clinical cancer stage information revealed that tumor samples with mutations in any of the 84 cytokines and receptors stratify into earlier tumor stages, with unique cellular compositions and clinical outcomes. This study provides a comprehensive cytokine-receptor atlas of the cellular architecture in multiple cancers at the single-cell level.

DNASE1L3 enhances antitumor immunity and suppresses tumor progression in colon cancer.

DNASE1L3, an enzyme highly expressed in DCs, is functionally important for regulating autoimmune responses to self-DNA and chromatin. Deficiency of DNASE1L3 leads to development of autoimmune diseases in both humans and mice. However, despite the well-established causal relationship between DNASE1L3 and immunity, little is known about the involvement of DNASE1L3 in regulation of antitumor immunity, the foundation of modern antitumor immunotherapy. In this study, we identify DNASE1L3 as a potentially new regulator of antitumor immunity and a tumor suppressor in colon cancer. In humans, DNASE1L3 is downregulated in tumor-infiltrating DCs, and this downregulation is associated with poor patient prognosis and reduced tumor immune cell infiltration in many cancer types. In mice, Dnase1l3 deficiency in the tumor microenvironment enhances tumor formation and growth in several colon cancer models. Notably, the increased tumor formation and growth in Dnase1l3-deficient mice are associated with impaired antitumor immunity, as evidenced by a substantial reduction of cytotoxic T cells and a unique subset of DCs. Consistently, Dnase1l3-deficient DCs directly modulate cytotoxic T cells in vitro. To our knowledge, our study unveils a previously unknown link between DNASE1L3 and antitumor immunity and further suggests that restoration of DNASE1L3 activity may represent a potential therapeutic approach for anticancer therapy.

Identification of the relationship between single-cell N6-methyladenosine regulators and the infiltrating immune cells in esophageal carcinoma

BackgroundN6-methyladenosine (m6A) RNA methylation plays a crucial role in important genomic processes in a variety of malignancies. However, the characterization of m6A with infiltrating immune cells in the tumor microenvironment (TME) in esophageal squamous carcinoma (ESCC) remains unknown. MethodsThe single-cell transcriptome data from five ESCC patients in our hospital were analyzed, and TME clusters associated with prognosis and immune checkpoint genes were investigated. Cell isolation and qPCR were conducted to validate the gene characterization in different cells. ResultsAccording to distinct biological processes and marker genes, macrophages, T cells, and B cells clustered into three to four different subgroups. In addition, we demonstrated that m6A RNA methylation regulators were strongly related to the clinical and biological features of ESCC. Analysis of transcriptome data revealed that m6A-mediated TME cell subsets had high predictive value and showed a close relationship with immune checkpoint genes. The validation results from qPCR demonstrated the characteristics of essential genes. CellChat analysis revealed that RNA from TME cells m6A methylation-associated cell subtypes had substantial and diversified interactions with cancer cells. Further investigation revealed that MIF- (CD74+CXCR4) and MIF- (CD74+CD44) ligand-receptor pairings facilitated communication between m6A-associated subtypes of TME cells and cancer cells. ConclusionOverall, our study demonstrated for the first time the function of m6A methylation-mediated intercellular communication in the microenvironment of tumors in controlling tumor development and anti-tumor immune regulation in ESCC.

Programmed death-ligand 1 and p53 as a biomarker in predicting oropharyngeal squamous cell carcinoma

Introduction. Currently, the pathogenetic role of the human papillomavirus (HPV) in carcinogenesis is well studied, that most cases of oropharyngeal squamous cell carcinoma are associated with the persistence of this infection. In addition to HPV infection, the involvement of programmed death-ligand 1 (PD-L1) and p53 proteins in the initiation and progression of oropharyngeal squamous cell carcinoma provides their formation as additional biomarkers in predicting the disease.Aim. To study the prognostic role of PD-L1 and p53 expression in oropharyngeal squamous cell carcinoma depending on HPV status.Materials and methods. The study included 62 patients with oropharyngeal squamous cell carcinoma T1–4N0–3M0 (Tumor, Nodus and Metastasis, (TNM), 7th edition) treated in 2015–2020 at the Republican Specialized Scientific and Practical Medical Center of Oncology and Radiology and in the Tashkent and Samarkand city branches. All patients underwent immunohistochemical analysis for the presence of p16INK4a, PD-L1 and p53 protein expression in formalin-fixed and paraffin-embedded tumor tissue samples.Results. Based on the results of the assessment, it was found that positive HPV status, low and medium levels of PD-L1 expression and positive regulation of wild-type p53 are associated with favorable outcomes in patients with oropharyngeal squamous cell carcinoma. Negative HPV status, high and very high levels of PD-L1 expression, loss of wild type p53 function with upregulation of p53 mutant type leads to worse disease outcomes.Conclusion. Thus, the assessment of the expression of the onco-suppressor protein p53 helps to determine the biology of cancer cells in patients with oropharyngeal squamous cell carcinoma and suggest sensitivity to ongoing therapy, and by studying the regulation of the PD-L1 protein, antitumor immune regulation can be suggested in these patients.

Kindlin-1 regulates IL-6 secretion and modulates the immune environment in breast cancer models.

The adhesion protein Kindlin-1 is over-expressed in breast cancer where it is associated with metastasis-free survival; however, the mechanisms involved are poorly understood. Here, we report that Kindlin-1 promotes anti-tumor immune evasion in mouse models of breast cancer. Deletion of Kindlin-1 in Met-1 mammary tumor cells led to tumor regression following injection into immunocompetent hosts. This was associated with a reduction in tumor infiltrating Tregs. Similar changes in T cell populations were seen following depletion of Kindlin-1 in the polyomavirus middle T antigen (PyV MT)-driven mouse model of spontaneous mammary tumorigenesis. There was a significant increase in IL-6 secretion from Met-1 cells when Kindlin-1 was depleted and conditioned media from Kindlin-1-depleted cells led to a decrease in the ability of Tregs to suppress the proliferation of CD8+ T cells, which was dependent on IL-6. In addition, deletion of tumor-derived IL-6 in the Kindlin-1-depleted tumors reversed the reduction of tumor-infiltrating Tregs. Overall, these data identify a novel function for Kindlin-1 in regulation of anti-tumor immunity, and that Kindlin-1 dependent cytokine secretion can impact the tumor immune environment.

HDAC3 Inhibition Promotes Antitumor Immunity by Enhancing CXCL10-Mediated Chemotaxis and Recruiting of Immune Cells.

It is generally believed that histone deacetylase (HDAC) inhibitors, which represent a new class of anticancer agents, exert their antitumor activity by directly causing cell-cycle arrest and apoptosis of tumor cells. However, in this study, we demonstrated that class I HDAC inhibitors, such as Entinostat and Panobinostat, effectively suppressed tumor growth in immunocompetent but not immunodeficient mice. Further studies with Hdac1, 2, or 3 knockout tumor cells indicated that tumor-specific inactivation of HDAC3 suppressed tumor growth by activating antitumor immunity. Specifically, we found that HDAC3 could directly bind to promotor regions and inhibit the expression of CXCL9, 10, and 11 chemokines. Hdac3-deficient tumor cells expressed high levels of these chemokines, which suppressed tumor growth in immunocompetent mice by recruiting CXCR3+ T cells into the tumor microenvironment (TME). Furthermore, the inverse correlation between HDAC3 and CXCL10 expression in hepatocellular carcinoma tumor tissues also suggested HDAC3 might be involved in antitumor immune regulation and patient survival. Thus, our studies have illustrated that HDAC3 inhibition suppresses tumor growth by enhancing immune cell infiltration into the TME. This antitumor mechanism may be helpful in guiding HDAC3 inhibitor-based treatment.

The Role of miR-155 in Antitumor Immunity.

MicroRNAs belong to a group of short non-coding RNA molecules that are involved in the regulation of gene expression at multiple levels. Their function was described two decades ago, and, since then, microRNAs have become a rapidly developing field of research. Their participation in the regulation of cellular processes, such as proliferation, apoptosis, cell growth, and migration, made microRNAs attractive for cancer research. Moreover, as a single microRNA can simultaneously target multiple molecules, microRNAs offer a unique advantage in regulating multiple cellular processes in different cell types. Many of these cell types are tumor cells and the cells of the immune system. One of the most studied microRNAs in the context of cancer and the immune system is miR-155. MiR-155 plays a role in modulating innate and adaptive immune mechanisms in distinct immune cell types. As such, miR-155 can be part of the communication between the tumor and immune cells and thus impact the process of tumor immunoediting. Several studies have already revealed its effect on antitumor immune responses, and the targeting of this molecule is increasingly implemented in cancer immunotherapy. In this review, we discuss the current knowledge of miR-155 in the regulation of antitumor immunity and the shaping of the tumor microenvironment, and the plausible implementation of miR-155 targeting in cancer therapy.

Abstract 2101: RNF2 ablation reprograms the tumor-immune microenvironment and stimulates durable NK and CD4+ T cell-dependent anti-tumor immunity

Abstract Expanding the utility of immune-based cancer treatments is a clinical challenge due to tumor-intrinsic factors that suppress the immune response. Here we report the identification of tumoral Ring Finger Protein 2 (RNF2), the core subunit of the Polycomb Repressor Complex 1 (PRC1), as a negative regulator of antitumor immunity in various human cancers, including breast cancer. In syngeneic murine models of triple negative breast cancer, we found that deleting genes encoding PRC1 subunits Rnf2 or BMI1 proto-oncogene, polycomb ring finger (Bmi1), or the downstream effector of Rnf2, Remodeling and Spacing Factor 1 (Rsf1), was sufficient by itself to induce durable tumor rejection and establish immune memory by enhancing infiltration and activation of NK and CD4+ T-cells, but not CD8+ T-cells, into the tumor and enabled their cooperativity. These findings uncover an epigenetic reprogramming of the tumor-immune microenvironment which fosters durable antitumor immunity and memory. Citation Format: Zhuo Zhang, Lin Luo, Chuan Xing, Yu Chen, Peng Xu, Mao Li, Ling Zeng, Chao Li, Sadashib Ghosh, Deborah Della Manna, Tim Townes, William J. Britt, Narendra Wajapeyee, Barry P. Sleckman, Zechen Chong, Jianmei Wu Leavenworth, Eddy Yang. RNF2 ablation reprograms the tumor-immune microenvironment and stimulates durable NK and CD4+ T cell-dependent anti-tumor immunity [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 2101.

Associations of different immune checkpoints-expressing CD4+ Treg/ T cell subsets with disease-free survival in colorectal cancer patients

There are different subsets of T regulatory cells (Tregs), orchestrating critical roles in the regulation of anti-tumor immunity in colorectal cancer (CRC). In this study, we report that a high frequency of circulating CD4+FoxP3+ Tregs was associated with poorer disease-free survival (DFS), while their higher frequencies in tumor-infiltrating CD4+ Tregs was associated with better DFS. We further investigated such associations with four Tregs/T cells expressing or lacking FoxP3 and Helios (FoxP3±Helios±). For the first time, we report that a high frequency of circulating CD4+FoxP3+Helios+ Tregs was associated with poorer DFS, while a high frequency of tumor-infiltrating CD4+FoxP3−Helios− T cells was associated with poorer DFS. In the four FoxP3±Helios± T cell subsets expressing any of the immune checkpoints (ICs) investigated, we found that a high frequency of CD4+FoxP3+Helios−PD-1+ Tregs in circulation was associated with worse DFS. We also found that high frequencies of FoxP3+Helios+CTLA-4+ Tregs, FoxP3+Helios−CTLA-4+ Tregs, and FoxP3−Helios+CTLA-4+ CD4+ T cells in circulation were associated with worse DFS. In contrast, high frequencies of CD4+TIM-3+ T cells, FoxP3+Helios+TIM-3+ Tregs, and FoxP3−Helios+TIM-3+ CD4+ T cells in circulation were associated with longer DFS. Our data show that certain CD4+ Treg/T cell subsets could serve as independent predictive biomarkers in CRC patients. Identification of the exact subpopulations contributing to clinical outcomes is critical for prognoses and therapeutic targeting.

LINC00892 Is an lncRNA Induced by T Cell Activation and Expressed by Follicular Lymphoma-Resident T Helper Cells.

Successful immunotherapy in both solid tumors and in hematological malignancies relies on the ability of T lymphocytes to infiltrate the cancer tissue and mount an immune response against the tumor. Biomarkers able to discern the amount and the types of T lymphocytes infiltrating a given tumor therefore have high diagnostic and prognostic value. Given that lncRNAs are known to have a highly cell-type-specific expression pattern, we searched for lncRNAs specifically expressed by activated T cells and at the same time in a kind of lymphoma, follicular lymphoma, where the microenvironment is known to play a critical role in the regulation of antitumor immunity. We focused on a non-coding transcript, annotated as LINC00892, which reaches extremely high expression levels following cell activation in Jurkat cells. Interestingly LINC00892 has an expression pattern resembling that of genes involved in T cell memory. Accordingly, LINC00892 is mostly expressed by the effector memory and helper CD4+ T cell sub-types but not by naïve T cells. In situ analyses of LINC00892 expression in normal lymph nodes and in follicular lymphoma biopsies show that its expression is limited to CD4+ PD1hi T cells, with a subcellular localization within the germinal center matching that of follicular helper T cells. Our analysis therefore suggests that the previously uncharacterized lncRNA LINC00892 could be a useful biomarker for the detection of CD4+ memory T cells in both normal and tumor tissues.

Increase in serum choline levels predicts for improved progression-free survival (PFS) in patients with advanced cancers receiving pembrolizumab

BackgroundRecent studies have demonstrated that T cells can induce vasodilation in a choline-acetyltransferase dependent manner, leading to an increase in T cell migration to infected tissues in response to viral...

Correlations between Circulating and Tumor-Infiltrating CD4+ T Cell Subsets with Immune Checkpoints in Colorectal Cancer.

T regulatory cells (Tregs) play different roles in the regulation of anti-tumor immunity in colorectal cancer (CRC), depending on the presence of different Treg subsets. We investigated correlations between different CD4+ Treg/T cell subsets in CRC patients with immune checkpoint-expressing CD4+ T cells. Positive correlations were observed between levels of different immune checkpoint-expressing CD4+ T cells, including PD-1, TIM-3, LAG-3, and CTLA-4 with FoxP3+ Tregs, Helios+ T cells, FoxP3+Helios+ Tregs, and FoxP3+Helios− Tregs in the tumor microenvironment (TME). However, negative correlations were observed between levels of these immune checkpoint-expressing CD4+ T with FoxP3−Helios− T cells in the TME. These correlations in the TME highlight the role of cancer cells in the upregulation of IC-expressing Tregs. Additionally, positive correlations were observed between levels of FoxP3+ Tregs, Helios+ T cells, FoxP3+Helios+ Tregs, and FoxP3+Helios− Tregs and levels of CD4+CTLA-4+ T cells and CD4+PD-1+ T cells in peripheral blood mononuclear cells (PBMCs) and normal tissue-infiltrating lymphocytes (NILs). These observations suggest that CTLA-4 and PD-1 expressions on CD4+ T cell subsets are not induced only by the TME. This is the first study to investigate the correlations of different FoxP3+/−Helios+/− T cell subsets with immune checkpoint-expressing CD4+ T cells in CRC patients. Our data demonstrated strong correlations between FoxP3+/Helios+/− Tregs but not FoxP3−Helios+/− non-Tregs and multiple immune checkpoints, especially in the TME, providing a rationale for targeting these cells with highly immunosuppressive characteristics. Understanding the correlations between different immune checkpoints and Treg/T cell subsets in cancer patients could improve our knowledge of the underlying mechanisms of Treg-mediated immunosuppression in cancer.

The SETDB1–TRIM28 Complex Suppresses Antitumor Immunity

The tumor immune microenvironment is influenced by the epigenetic landscape of the tumor. Here, we have identified the SETDB1-TRIM28 complex as a critical suppressor of antitumor immunity. An epigenetic CRISPR-Cas9 screen of 1,218 chromatin regulators identified TRIM28 as a suppressor of PD-L1 expression. We then revealed that expression of the SETDB1-TRIM28 complex negatively correlated with infiltration of effector CD8+ T cells. Inhibition of SETDB1-TRIM28 simultaneously upregulated PD-L1 and activated the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) innate immune response pathway to increase infiltration of CD8+ T cells. Mechanistically, SETDB1-TRIM28 inhibition led to micronuclei formation in the cytoplasm, which is known to activate the cGAS-STING pathway. Thus, SETDB1-TRIM28 inhibition bridges innate and adaptive immunity. Indeed, SETDB1 knockout enhanced the antitumor effects of immune checkpoint blockade with anti-PD-L1 in a mouse model of ovarian cancer in a cGAS-dependent manner. Our findings establish the SETDB1-TRIM28 complex as a regulator of antitumor immunity and demonstrate that its loss activates cGAS-STING innate immunity to boost the antitumor effects of immune checkpoint blockade.

Abstract P241: A highly potent HPK1 inhibitor augments immune cell activation and anti-tumor immunity in a syngeneic tumor mouse model

Abstract Background: HPK1 (MAP4K1) is a serine/threonine Ste20-related protein kinase that belongs to the mitogen-activated protein kinase (MAPK) family. HPK1 is mainly expressed in hematopoietic cells and serves as a negative regulator of anti-tumor immunity through modulating the activation of lymphocytes and dendritic cells. Upon TCR activation, HPK1 phosphorylates the adaptor protein SLP76 at Ser376 to destabilize the SLP76 microclusters, which leads to the attenuation of the TCR signaling. The reported anti-tumor efficacy data from HPK1 knockout and kinase-dead knock-in mouse models support HPK1 as a novel intracellular I/O target. Methods: Regor CARD platform (Computer Accelerated Rational Discovery) was deployed to identify potent and selective inhibitors of HPK1. Biochemical assays and primary human pan T cell-based cellular assays were utilized to support the structure-activity relationship (SAR) analysis and inhibitor optimization. In vitro and in vivo target engagement studies were conducted in Jurkat T cells and mouse splenocytes, respectively. In vivo efficacy study data were generated using CT-26 syngeneic tumor mouse model. Results: Highly potent HPK1 inhibitors with good selectivity against the liable immune kinases were identified. RGT-197, one of the lead inhibitors demonstrated a significant increase of IL-2 secretion in primary human pan T cells upon TCR activation. It inhibited TCR-induced phosphorylation of SLP76 at Ser-376 in vitro and in vivo. Anti-tumor efficacy was observed in CT-26 tumor-bearing mice by oral dosing of RGT-197 as a single agent and in combination with an anti-PD1-monoclonal antibody. Furthermore, RGT-197 increased the level of cytokines important for anti-tumor immunity in vivo. Conclusion: RGT-197, a potent and selective HPK1 inhibitor, provides a potential opportunity as a small molecule I/O agent to boost anti-tumor immunity either as monotherapy or in combination with immune checkpoint inhibitors. Citation Format: Hao Liu, Lei Wu, Song Feng, Wei Huang, Huijuan Li, Jing Lin, Yangyang Liu, Liufeng Mei, Baoqi Ren, Julie Xie, Lili Yao, Wenge Zhong. A highly potent HPK1 inhibitor augments immune cell activation and anti-tumor immunity in a syngeneic tumor mouse model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P241.

A Novel Lair-1 Antibody Selectively Targets Acute Myeloid Leukemia (AML) Stem Cells

Extensive research has led to recent approval of novel therapies such as mylotarg, venetoclax, glasdegib and CC486, and small molecule inhibitors against actionable mutations such as ivosidenib (IDH1), enasidenib (IDH2), gliteritinib and midostaurin (FLT3) in AML. However, the mainstay of treatment in AML remains unchanged since the 1970s. There is a significant unmet need for AML patients that fail to respond to or relapse after standard-of-care (SOC) treatments including allogeneic stem cell transplantation and targeting actionable mutations. In addition, a large fraction of SOC patients invariably relapse due to persistence of chemotherapy-resistant leukemia stem cells (LSCs) or immune evasion. Therefore, identification of unique therapies that preferentially target elusive LSCs and promote immune responses to AML to prevent relapse are highly sought after. Unlike, targeting acute lymphoblastic leukemia (ALL) with CD19 or CD22 with various modalities, when developing AML therapies, it is of paramount importance to differentiate LSCs from hematopoietic stem cells (HSCs) to lessen or abolish unavoidable cytopenias.Leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) is an immune checkpoint receptor on T cells and myeloid cells that delimits immune cell activation through binding to endogenous collagen ligands. In addition, LAIR-1 is universally expressed on AML blasts and may sustain AML survival signals. We demonstrated using multi-color flow cytometry that LAIR-1 is highly expressed in AML blasts (n=9 of 9) and that LAIR-1 expression in LSCs (markers: CD34 +CD38 -CD90 -CD45RA +/- or CD34 -CD117 +CD244 +/-) is high compared with negligible expression of LAIR-1 in HSCs (markers: CD34 +CD38 -CD90 +CD99 -) (n=3) (Figure 1). Based on these findings, we hypothesized that a LAIR-1 monoclonal antibody (mAb) would disrupt LAIR-1 mediated survival signaling and preferentially target LAIR-1 expressing AML LSCs and blast cells but not HSCs. To test this, we developed a novel LAIR-1 targeting mAb with a functional human IgG 1 isotype that blocks LAIR-1 binding to its ligands (including collagens, complement component C1q, MBL and SP-D)To characterize the anti-leukemic effect of the LAIR-1 mAb we performed an in vitro antibody dependent cell cytotoxicity (ADCC) assay with LAIR-1 expressing AML cells (MOLT4 and MV-4-11). Compared with isotype control, the LAIR-1 mAb significantly increased leukemia cell death (MV411 = 17% above isotype, and MOLT4 = 29.24% above isotype at 15 µg/ml), suggesting that the LAIR-1 mAb confers ADCC activity against LAIR1 + AML cells (Figure 2). To elucidate if the LAIR-1 mAb has a direct signaling effect on LAIR-1 + AML cells, a colony forming unit assay using primary AML cells was carried out. Interestingly, the LAIR-1 mAb inhibited colony formation by AML CD34 + cells (40-60% decreased compared with isotype control, N=4), but not normal CD34 + cells. These data suggests that our LAIR-1 mAb stimulated LAIR-1 signaling that inhibits LSC self-renewal. We then tested the in vivo anti-leukemia effect of the mAb in cell line derived xenograft (CDX) models (immune deficient mice transplanted with MV-4-11 expressing luciferase). In vivo bioluminescence imaging indicated that the LAIR-1 mAb significantly inhibited in vivo AML growth (91% reduction of total flux)(Figure 3). A significant increase in cell death was observed in the presence of the mAb in the blood (47%), spleen (89.4%) and bone marrow (27.6%). Similar to the anti-leukemic effect in CDX AML models, the LAIR-1 mAb significantly suppressed in vivo growth of AML patient derived xenografts (5 different primary AML donors) (10-90% human CD33 + AML cells in isotype control treatment vs 0.5-5% CD33 + AML cells in anti-LAIR-1 treatment, N=3) (Figure 4), while minimally impacting normal immune cells.Taken together, our studies suggest that the LAIR-1 mAb we generated is a novel AML immunomedicine that preferentially eradicates AML LSCs and blasts while preserving healthy HSCs through disruption of AML survival signals and clearance of AML through ADCP and ADCC. Additional studies are currently evaluating if this novel LAIR-1 mAb has other mechanisms of action that contribute to overall in vivo activity, including reduction of AML niche implantation, regulation of bone marrow homing and regulation of anti-tumor immunity. [Display omitted] DisclosuresTian: NextCure: Ended employment in the past 24 months. Paucarmayta: NextCure: Current Employment. Lovewell: NextCure: Current Employment. Maloveste: NextCure: Current Employment. Copeland: NextCure: Current Employment. O'Neill: NextCure: Current Employment. Patel: NextCure: Current Employment. Liu: NextCure: Current Employment, Current holder of stock options in a privately-held company. Myint: NextCure: Current Employment, Current holder of stock options in a privately-held company. Langermann: NextCure: Current Employment, Current holder of stock options in a privately-held company. Flies: NextCure: Current Employment, Current holder of stock options in a privately-held company. Kim: Nextcure: Research Funding.

A single-cell and spatially resolved atlas of human breast cancers.

Breast cancers are complex cellular ecosystems where heterotypic interactions play central roles in disease progression and response to therapy. However, our knowledge of their cellular composition and organization is limited. Here we present a single-cell and spatially resolved transcriptomics analysis of human breast cancers. We developed a single-cell method of intrinsic subtype classification (SCSubtype) to reveal recurrent neoplastic cell heterogeneity. Immunophenotyping using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) provides high-resolution immune profiles, including new PD-L1/PD-L2+ macrophage populations associated with clinical outcome. Mesenchymal cells displayed diverse functions and cell-surface protein expression through differentiation within three major lineages. Stromal-immune niches were spatially organized in tumors, offering insights into antitumor immune regulation. Using single-cell signatures, we deconvoluted large breast cancer cohorts to stratify them into nine clusters, termed 'ecotypes', with unique cellular compositions and clinical outcomes. This study provides a comprehensive transcriptional atlas of the cellular architecture of breast cancer.