Deng Pan’s research while affiliated with Tsinghua University and other places

The formation of immune synapses (ISs) between cytotoxic T cells and tumor cells is crucial for effective tumor elimination. However, the role of ISs in immune evasion and resistance to immune checkpoint blockades (ICBs) remains unclear. We demonstrate that ICAM-1, a key IS molecule activating LFA-1 signaling in T and natural killer (NK) cells, is often expressed at low levels in cancers. The absence of ICAM-1 leads to significant resistance to T and NK cell-mediated anti-tumor immunity. Using a CRISPR screen, we show that ICAM-1 is epigenetically regulated by the DNA methylation pathway involving UHRF1 and DNMT1. Furthermore, we engineer an antibody-based therapeutic agent, “LFA-1 engager,” to enhance T cell-mediated anti-tumor immunity by reconstituting LFA-1 signaling. Treatment with LFA-1 engagers substantially enhances immune-mediated cytotoxicity, potentiates anti-tumor immunity, and synergizes with ICB in mouse models of ICAM-1-deficient tumors. Our data provide promising therapeutic strategies for re-engaging immune stimulatory signals in cancer immunotherapy.

Bone metastasis is a lethal consequence of breast cancer. Here we used single-cell transcriptomics to investigate the molecular mechanisms underlying bone metastasis colonization—the rate-limiting step in the metastatic cascade. We identified that lymphotoxin-β (LTβ) is highly expressed in tumour cells within the bone microenvironment and this expression is associated with poor bone metastasis-free survival. LTβ promotes tumour cell colonization and outgrowth in multiple breast cancer models. Mechanistically, tumour-derived LTβ activates osteoblasts through nuclear factor-κB2 signalling to secrete CCL2/5, which facilitates tumour cell adhesion to osteoblasts and accelerates osteoclastogenesis, leading to bone metastasis progression. Blocking LTβ signalling with a decoy receptor significantly suppressed bone metastasis in vivo, whereas clinical sample analysis revealed significantly higher LTβ expression in bone metastases than in primary tumours. Our findings highlight LTβ as a bone niche-induced factor that promotes tumour cell colonization and osteolytic outgrowth and underscore its potential as a therapeutic target for patients with bone metastatic disease.

Bone metastatic relapse is a lethal consequence of breast cancer, occurring years after initial diagnosis. By analyzing single-cell transcriptomes of bone-seeding tumor cells and in vivo barcoded cDNA library screening, LTβ (lymphotoxin-β) is identified as a key factor highly expressed in early-stage bone metastatic cells, associated with poor bone metastasis-free survival, and capable of promoting dormancy reactivation in multiple breast cancer models. Mechanistically, tumor-derived LTβ activates NF-κB2 signaling in osteoblasts to express CCL2/5, facilitating tumor cell seeding and accelerating osteoclastogenesis. Both processes contribute to the reactivation of dormancy and metastatic outgrowth. Blocking LTβ signaling with a decoy receptor significantly suppressed bone colonization and metastatic progression, whereas clinical sample analysis revealed significantly higher LTβ expression in bone metastases than in primary tumors. Our findings highlight LTβ as a bone niche-induced factor that promotes tumor cell seeding and dormancy reactivation, underscoring its potential as a therapeutic target for preventing bone metastatic relapse in patients with breast cancer.

Metabolic reprogramming toward glycolysis is a hallmark of cancer malignancy. The molecular mechanisms by which the tumor glycolysis pathway promotes immune evasion remain to be elucidated. Here, by performing genome-wide CRISPR screens in murine tumor cells co-cultured with cytotoxic T cells (CTLs), we identified that deficiency of two important glycolysis enzymes, Glut1 (glucose transporter 1) and Gpi1 (glucose-6-phosphate isomerase 1), resulted in enhanced killing of tumor cells by CTLs. Mechanistically, Glut1 inactivation causes metabolic rewiring toward oxidative phosphorylation, which generates an excessive amount of reactive oxygen species (ROS). Accumulated ROS potentiate tumor cell death mediated by tumor necrosis factor alpha (TNF-α) in a caspase-8- and Fadd-dependent manner. Genetic and pharmacological inactivation of Glut1 sensitizes tumors to anti-tumor immunity and synergizes with anti-PD-1 therapy through the TNF-α pathway. The mechanistic interplay between tumor-intrinsic glycolysis and TNF-α-induced killing provides new therapeutic strategies to enhance anti-tumor immunity.

Tumor heterogeneity is a major barrier to cancer therapy, including immunotherapy. Activated T cells can efficiently kill tumor cells following recognition of MHC class I (MHC-I)–bound peptides, but this selection pressure favors outgrowth of MHC-I–deficient tumor cells. We performed a genome-scale screen to discover alternative pathways for T cell–mediated killing of MHC-I–deficient tumor cells. Autophagy and TNF signaling emerged as top pathways, and inactivation of Rnf31 (TNF signaling) and Atg5 (autophagy) sensitized MHC-I–deficient tumor cells to apoptosis by T cell–derived cytokines. Mechanistic studies demonstrated that inhibition of autophagy amplified proapoptotic effects of cytokines in tumor cells. Antigens from apoptotic MHC-I–deficient tumor cells were efficiently cross-presented by dendritic cells, resulting in heightened tumor infiltration by IFNγ-and TNFα-producing T cells. Tumors with a substantial population of MHC-I–deficient cancer cells could be controlled by T cells when both pathways were targeted using genetic or pharmacologic approaches. Significance Tumor heterogeneity is a major barrier to immunotherapy. We show that MHC-I–deficient tumor cells are forced into apoptosis by T cell–derived cytokines when TNF signaling and autophagy pathways are targeted. This approach enables T cell–mediated elimination of tumors with a substantial population of resistant, MHC-I–deficient tumor cells.

Theoretically a broad spectrum of cancers, including those with low tumor burdens, could respond to NK cell therapy and NK cells might combat resistance to T cell-based therapies. Allogenic NK cells have also demonstrated a good safety profile in clinical trials. These promising features have led to increasing efforts to advance NK-based immunotherapies in recent years. However, efficacy of NK therapy remains limited. Strategies to augment NK efficacy are therefore much needed. To augment the efficacy of adoptive NK cells, most current studies revolve around two focal points: optimizing the source of NK cells and improving their functionality and persistence in vivo. In the current study, we took a different approach by studying how to make cancer cells more vulnerable to NK-mediated killing. It remains unclear to what extent mitochondrial apoptosis is required for NK-mediated killing. We found that primary human NK cells robustly induce mitochondrial apoptosis. Moreover, mitochondrial apoptosis is essential for efficient NK killing, especially at physiologically relevant low E:T ratios (Effector:Target ratios). It is traditionally believed that cytotoxic cell-cancer cell contacts are binary live/death events. We found that NK engagement is often sub-lethal and push cancer cells towards apoptotic threshold (i.e. priming cancer cells for apoptosis), making them more susceptible for killing by subsequent NK contacts. Upregulation of anti-apoptotic proteins has been widely implicated in cancer resistance to chemo- and targeted therapies. We found that overexpression of these proteins such as BCL-2, BCL-XL, and MCL-1 reduced mitochondrial priming for apoptosis and made cancer cells less susceptible to NK killing. We reasoned that additional agents that increase cancer cell mitochondrial priming for apoptosis (e.g., BH3 mimetics) might augment NK-induced killing, as long as NK cells can tolerate these agents. While primary resting NK cells are sensitive to BCL-2, BCL-XL, and MCL-1 inhibitors, unexpectedly, pre-activation with IL-2 conferred resistance of NK cells to these inhibitors. NK cells and BH3 mimetics synergized in both priming and killing cancer cells in vitro. BH3 profiling could also predict the ideal BH3 mimetics to be combined with NK cells for different tumor models. Using liquid and solid tumor xenograft models, we demonstrated that BH3 mimetics synergized with NK cells in suppressing tumor growth and prolonging mouse survival. In summary, we propose a rational strategy to sensitize cancer cells to NK cellular therapy. Moreover, we elucidate the mechanism underlying the apoptotic signaling that is the scientific basis for this strategy. Our results could potentially enable basic, pre-clinical, and clinical studies investigating the combined effects of BH3 mimetics with NK cells in cancers. Citation Format: Rongqing Aaron Pan, Jeremy Ryan, Deng Pan, Kai Wucherpfennig, Anthony Letai. Augmenting NK cell based immunotherapy by targeting mitochondrial apoptosis [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 4174.

Interest in harnessing natural killer (NK) cells for cancer immunotherapy is rapidly growing. However, efficacy of NK cell-based immunotherapy remains limited in most trials. Strategies to augment the killing efficacy of NK cells are thus much needed. In the current study, we found that mitochondrial apoptosis (mtApoptosis) pathway is essential for efficient NK killing, especially at physiologically relevant effector-to-target ratios. Furthermore, NK cells can prime cancer cells for mtApoptosis and mitochondrial priming status affects cancer-cell susceptibility to NK-mediated killing. Interestingly, pre-activating NK cells confers on them resistance to BH3 mimetics. Combining BH3 mimetics with NK cells synergistically kills cancer cells in vitro and suppresses tumor growth in vivo. The ideal BH3 mimetic to use in such an approach can be predicted by BH3 profiling. We herein report a rational and precision strategy to augment NK-based immunotherapy, which may be adaptable to T cell-based immunotherapies as well.

Immune checkpoint inhibitor (ICI) therapy is generating remarkable responses in individuals with cancer, but only a small portion of individuals with breast cancer respond well. Here we report that tumor-derived Jagged1 is a key regulator of the tumor immune microenvironment. Jagged1 promotes tumorigenesis in multiple spontaneous mammary tumor models. Through Jagged1-induced Notch activation, tumor cells increase expression and secretion of multiple cytokines to help recruit macrophages into the tumor microenvironment. Educated macrophages crosstalk with tumor-infiltrating T cells to inhibit T cell proliferation and tumoricidal activity. In individuals with triple-negative breast cancer, a high expression level of Jagged1 correlates with increased macrophage infiltration and decreased T cell activity. Co-administration of an ICI PD-1 antibody with a Notch inhibitor significantly inhibits tumor growth in breast cancer models. Our findings establish a distinct signaling cascade by which Jagged1 promotes adaptive immune evasion of tumor cells and provide several possible therapeutic targets.

Purpose: Immune checkpoint blockade (ICB) has shown remarkable efficacy, but in only a minority of cancer patients, suggesting the need to develop additional treatment strategies. Aberrant glycosylation in tumors, resulting from the dysregulated expression of key enzymes in glycan biosynthesis, modulates the immune response. However, the role of glycan biosynthesis enzymes in anti-tumor immunity is poorly understood. We aimed to study the immunomodulatory effects of these enzymes. Experimental design: We integrated transcriptional profiles of treatment-naïve human tumors and functional CRISPR screens to identify glycometabolism genes with immunomodulatory effects. We further validated our findings using in vitro co-culture and in vivo syngeneic tumor growth assays. Results: We identified MAN2A1, encoding an enzyme in N-glycan maturation, as a key immunomodulatory gene. Analyses of public immune checkpoint blockade trial data also suggested a synergy between MAN2A1 inhibition and anti-PD-L1 treatment. Loss of Man2a1 in cancer cells increased their sensitivity to T cell-mediated killing. Man2a1 knockout enhanced response to anti-PD-L1 treatment and facilitated higher cytotoxic T cell infiltration in tumors under anti-PD-L1 treatment. Furthermore, a pharmacological inhibitor of MAN2A1, swainsonine, synergized with anti-PD-L1 in syngeneic melanoma and lung cancer models, whereas each treatment alone had little effect. Conclusions: Man2a1 loss renders cancer cells more susceptible to T cell-mediated killing. Swainsonine synergizes with anti-PD-L1 in suppressing tumor growth. In light of the limited efficacy of anti-PD-L1 and failed phase II clinical trial on swainsonine, our study reveals a potential therapy combining the two to overcome tumor immune evasion.