Deng Pan’s research while affiliated with Dana-Farber Cancer Institute and other places

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.

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.

Resistance to cytotoxic T cells is frequently mediated by loss of MHC class I expression or IFNγ signaling in tumor cells, such as mutations of B2M or JAK1 genes. NK cells could potentially target such resistant tumors, but suitable NK cell-based strategies remain to be developed. We hypothesized that such tumors could be targeted by NK cells if sufficient activating signals were provided. Human tumors frequently express the MICA and MICB ligands of the activating NKG2D receptor, but proteolytic shedding of MICA/B represents an important immune evasion mechanism in many human cancers. We showed that B2M- and JAK1-deficient metastases were targeted by NK cells following treatment with a monoclonal antibody (mAb) that blocks MICA/B shedding. We also demonstrated that the FDA-approved HDAC inhibitor panobinostat and a MICA/B antibody acted synergistically to enhance MICA/B surface expression on tumor cells. The HDAC inhibitor enhanced MICA/B gene expression, whereas the MICA/B antibody stabilized the synthesized protein on the cell surface. The combination of panobinostat and the MICA/B antibody reduced the number of pulmonary metastases formed by a human melanoma cell line in NSG mice reconstituted with human NK cells. NK cell-mediated immunity induced by a mAb specific for MICA/B, therefore, provides an opportunity to target tumors with mutations that render them resistant to cytotoxic T cells.

NK cells contribute to protective antitumor immunity, but little is known about the functional states of NK cells in human solid tumors. To address this issue, we performed single-cell RNA-seq analysis of NK cells isolated from human melanoma metastases, including lesions from patients who had progressed following checkpoint blockade. This analysis identified major differences in the transcriptional programs of tumor-infiltrating compared with circulating NK cells. Tumor-infiltrating NK cells represented 7 clusters with distinct gene expression programs indicative of significant functional specialization, including cytotoxicity and chemokine synthesis programs. In particular, NK cells from 3 clusters expressed high levels of XCL1 and XCL2, which encode 2 chemokines known to recruit XCR1+ cross-presenting DCs into tumors. In contrast, NK cells from 2 other clusters showed a higher level of expression of cytotoxicity genes. These data reveal key features of NK cells in human tumors and identify NK cell populations with specialized gene expression programs.

Many human cancers are resistant to immunotherapy for reasons that are poorly understood. We used a genome-scale CRISPR/Cas9 screen to identify mechanisms of tumor cell resistance to killing by cytotoxic T-cells, the central effectors of antitumor immunity. Inactivation of >100 genes sensitized mouse B16F10 melanoma cells to killing by T-cells, including Pbrm1, Arid2 and Brd7, which encode components of the PBAF form of the SWI/SNF chromatin remodeling complex. Loss of PBAF function increased tumor cell sensitivity to interferon-γ, resulting in enhanced secretion of chemokines that recruit effector T-cells. Treatment-resistant tumors became responsive to immunotherapy when Pbrm1 was inactivated. In many human cancers, expression of PBRM1 and ARID2 inversely correlated with expression of T-cell cytotoxicity genes, and Pbrm1-deficient murine melanomas were more strongly infiltrated by cytotoxic T-cells. Citation Format: Deng Pan, Aya Kobayashi, Peng Jiang, Guo-Cheng Yuan, X. Shirley Liu, John Doench, Xintao Qiu, Prakash Rao, Henry Long, Myles A. Brown, Kai W. Wucherpfennig, Lucas Ferrari de Andrade, Rong En Tay, Adrienne M. Luoma, Daphne Tsoucas, Klothilda Lim. Systematic discovery of immune regulatory mechanisms in tumor cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A146.