Centre for Oncology and Immunology Fifth Symposium

Immunotherapy for Cancer Treatment: Breakthroughs and Innovations

Poster Session

Bioinformatics Analysis

Medicinal Chemistry

Poster Theme 1 : Liver Cancer

Poster #1:

NAD is an immunosuppressive metabolite that contributes to immunotherapy resistance

Jacinth Wing-Sum Cheu¹ ³, Kenneth Kin Leung Kwan¹ ³, Cindy Xinqi Liu¹, Grace Fu Wan Sit¹ ³, Haijing Deng¹, Wei Shen¹, Carrie Yiling Chen¹, Macus Hao-Ran Bao¹ ³, YS Chung¹ ³ ⁴, Carmen Chak-Lui Wong¹ ² ³ *

¹Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; ²State Key Laboratory of Liver Research, The University of Hong Kong, Hong Kong SAR, China; ³Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China; ⁴School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; *Corresponding author

NAD is an immunosuppressive metabolite that contributes to resistance against immunotherapy. In hepatocellular carcinoma (HCC), only a minority of patients respond to immune checkpoint blockade. To enhance therapeutic efficacy, it is crucial to understand the mechanisms of resistance and identify biomarkers that can predict patient response. Immunotherapies stimulate immune cells to secrete interferons. In the tumor microenvironment, immune cells and HCC cells are in close proximity and influence each other through their secretion profiles. Our transcriptome analysis identified NAMPT and NAD metabolism as potential contributors to immunotherapy resistance. We observed increased levels of NAMPT and serum NAD in HCC-bearing mice that were resistant to anti-PD-1 treatment. This suggests that serum NAD levels could serve as a predictor of immunotherapy response. We also found that extracellular NAD suppresses T cells. In vivo, a deficiency in NAMPT sensitized HCC to anti-PD-1 treatment. Additionally, blocking P2X7R, the NAD receptor, promoted T cell infiltration into the tumor and worked synergistically with anti-PD-1. These findings suggest a potential combination treatment strategy with anti-PD-1. Overall, our study highlights the role of NAD metabolism in immunotherapy resistance and points to new therapeutic opportunities for improving patient outcomes.

Poster #2:

CRISPR/Cas9 library screening identified CDC7 as a TTK inhibitor resistant gene in HCC treatment

Ho Wai Lau¹ ², Ou Ning Hsiu¹ ², Po Yee Wong¹ ², Yuen Ki Chan¹ ², Helen Do Gai Xue¹ ², Yifei Wang¹ ², Yan Zhang¹ ², Wei Shen¹ ², Carmen Chak-Lui Wong¹ ² *

¹Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China; ²Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; *Corresponding author

Hepatocellular carcinoma (HCC), a highly aggressive liver malignancy, is the second leading cause of cancer-related death globally. Our study revealed that the TTK inhibitor (CFI-402257) induces micronuclei and polyploidy in HCC, triggering the cytosolic DNA-sensing pathway and enhancing anti-tumor immune responses. However, resistance to cell cycle inhibitors driven by compensatory mechanisms remains a significant barrier. Using CRISPR/Cas9 screening, we identified CDC7 as a TTK inhibitor resistance gene. CDC7 phosphorylates γH2AX during DNA damage response. Dual inhibition of TTK and CDC7 exacerbates DNA damage, promoting genomic instability and suppressing tumor growth, offering a promising combination therapy for HCC.

Poster #3:

Cell cycle inhibitors activate the hypoxia-induced DDX41/STING pathway to mediate antitumor immune response in liver cancer

Po Yee Wong¹ ² ³ ^, Cerise Yuen Ki Chan¹ ² ³ ^, Helen Do Gai Xue¹ ² ³ ^, Chi Ching Goh¹ ², Jacinth Wing Sum Cheu¹ ² ³, Aki Pui Wah Tse¹ ², Misty Shuo Zhang¹ ² ³ ⁴, Yan Zhang¹ ² ³, Carmen Chak Lui Wong¹ ² ³ ⁴ ⁵ *

¹Department of Pathology, School of Clinical Medicine, The University of Hong Kong, Hong Kong SAR, China; ²State Key Laboratory of Liver Research, The University of Hong Kong, Hong Kong SAR, China; ³Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China; ⁴Department of Clinical Oncology, The University of Hong Kong-Shenzhen Hospital, Shenzhen, China; ⁵Guangdong-Hong Kong Joint Laboratory for RNA Medicine, Sun Yat-Sen University, Guangzhou, China; ^These authors contributed equally; *Corresponding author

This study demonstrates that cell cycle inhibitors possess anticancer functions beyond merely arresting the cell cycle. Specifically, these inhibitors induced DNA damage and cytosolic DNA accumulation, triggering the cytosolic DNA sensor DDX41 and activating the STING pathway. This activation led to the production of senescence-associated secretory phenotype factors (SASPs), which promote immune cell infiltration into tumors for cancer clearance. We also found that hypoxia induced the expression of DDX41 through HIF-1, rendering hypoxic hepatocellular carcinoma (HCC) cells more sensitive to these inhibitors. Combining cell cycle inhibitors with anti-PD-1 therapy significantly extended the survival of HCC-bearing mice. Our research underscores the potential of using a combination of cell cycle inhibitors and immunotherapy to achieve enhanced anticancer effects.

Poster Theme 2 : Gastric Cancer

Poster #4:

Large-scale drug sensitivity screening on cancer organoids reveal novel treatment strategies

Dessy Chan¹ ², Yin Tong¹ ², Hoi Cheong Siu¹, Sarah SK Yue¹ ², Wendy Tsui¹, Annie SY Chan¹, Tak W Mak² ³, Hans Clevers⁴, Helen HN Yan¹ ² *, Suet Yi Leung¹ ² ⁵ *

¹Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, ²Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China, ³Princess Margaret Cancer Centre, University Health Network, Toronto, Canada, ⁴The Hubrecht Institute, University Medical Centre Utrecht, Utrecht, The Netherlands, ⁵The Jockey Club Centre for Clinical Innovation and Discovery, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, *Co-corresponding authors

Gastric cancer (GC) is the 3rd leading cause of cancer death worldwide and has an exceptionally high incidence in Asia, including China and Hong Kong. Currently, other than complete surgical resection of the tumor during early-stage disease, there is no effective treatment for GC. There are a few approved chemotherapeutic agents, including 5-FU, cisplatin, epirubicin, paclitaxel, irinotecan or their combinations, along with two targeted drugs, Trastuzumab and Ramucirumab. Unfortunately, most patients are diagnosed at late stages with dismal prognosis despite these treatments. Therefore, high mortality rates highlight the urgent need for new treatment methods. With recent advances in 3D organoid culture techniques, we have successfully established a GC living biobank. Here, we show that this living biobank can serve as a valuable tool for drug sensitivity screening to guide patient treatment.

~Published in: Cell Stem Cell, 2018, Dec; 23(6):882-897.

Poster #5:

Escape from cell-cell and cell-matrix adhesion dependence underscores disease progression in gastric cancer organoid models

Yin Tong¹ ², Chung Sze Or¹, Hoi Cheong Siu¹, Siu Lun Ho¹, Simon Law³, Wai Yin Tsui¹, Annie SY Chan¹, April S Chan¹ ², Shui Wa Yun¹, Ho Sang Hui¹, Siu Tsan Yuen¹ ⁴, Suet Yi Leung¹ ² ⁵ * , Helen HN Yan¹ ² *

¹Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, ²Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China, ³Department of Surgery, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, ⁴Department of Pathology, St. Paul’s Hospital, No. 2, Eastern Hospital Road, Causeway Bay, Hong Kong SAR, China, ⁵The Jockey Club Centre for Clinical Innovation and Discovery, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, *Co-corresponding authors

Cell-cell (CC) and cell-matrix (CM) adhesions are essential for epithelial cell survival, yet dissociation-induced apoptosis is frequently circumvented in malignant cells. However, the extent of CC or CM adhesion dependence in tumor organoids and their relation to cancer progression remains unexplored. Since organoid models are more representative of a patients’ in vivo status, they can capture early tumor stages and give us a unique opportunity to assess CC independent (CCi) and CM independent (CMi) phenotypes against the patients’ clinicopathological characteristics. Here, we explored CC and CM dependence in 58 gastric cancer (GC) organoids by withdrawing either ROCK inhibitor, matrix or both to evaluate their tumorigenic potential in terms of apoptosis resistance, correlation with oncogenic driver mutations and clinical behavior. We found that almost all of the GC organoids were CMi, but only a subset were CCi or both CCi and CMi (CCi/CMi). Clinically, the CCi/CMi phenotype was associated with an infiltrative tumor edge and advanced tumor stage. Moreover, the CCi/CMi transcriptome signature was associated with poor patient survival when applied to three public GC datasets. Thus, we found that gaining cell-matrix (CMi) or cell-cell (CCi) adhesion independence or both (CMi/CCi) is a key feature that distinguishes cancer from normal organoids, and is correlated with advancing stages of tumor invasion and metastasis. This supports the importance of escaping from RHO-ROCK-mediated apoptosis as a general characteristic of cancer progression and calls for further investigation of the possible mechanisms by which such evasion is achieved, such that it can be exploited as a therapeutic strategy.

~Published in: Gut, 2023, Feb;72(2):242-255.

Poster #6:

Divergent lineage trajectories and genetic landscapes in human gastric intestinal metaplasia organoids associated with early neoplastic progression

Sarah SK Yue¹ ², Yin Tong¹ ², Wai Yin Tsui¹, Dessy Chan¹ ², Hoi Cheong Siu¹, Annie SY Chan¹, Suet Yi Leung¹ ² ³ *, Helen HN Yan¹ ² *

¹Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, ²Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China, ³The Jockey Club Centre for Clinical Innovation and Discovery, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, *Co-corresponding authors

Gastric intestinal metaplasia (IM) represents a critical pre-cancerous stage, characterized by a morphological spectrum that is currently inadequately represented by human cell line models. Understanding the progression of IM along the cancer spectrum is pivotal in early detection, prevention and treatment of gastric cancer. However, the lack of suitable cell models for IM has hindered our understanding of its pathogenesis and neoplastic progression. To fill this gap, we have generated a large cohort of human gastric IM organoids (IMOs) and performed comprehensive multi-omics profiling and functional characterization. We also performed single-cell RNA sequencing to characterize stem/progenitor populations and their lineage trajectories. Our analysis showed that lineage plasticity in IM goes beyond gastric or intestinal fates, and demonstrated a reversion to a fetal phenotype. We observed that IMO cells spanned a spectrum from hybrid gastric/intestinal to advanced intestinal differentiation, and uncovered lineage trajectories that connected different cycling and quiescent stem and progenitors, highlighting their differences in the gastric to IM transition. Lastly, we found that cell populations in gastric IM and cancer tissues were highly similar to those derived from IMOs and exhibited a fetal signature. Our findings provide crucial insights into the lineage trajectories and genetic landscapes of IM, demonstrating that our IM organoid models are ideal for studying early gastric neoplastic transformation and chemoprevention. This research advances our understanding of IM pathogenesis and its neoplastic progression, potentially paving the way for the future development of targeted therapeutic strategies and improved patient outcomes.

~Published in: Gut. 2024; https://doi.org/10.1136/gutjnl-2024-332594.

Poster #7:

Human gastric intestinal metaplasia organoids display genetic profiles linked with early neoplastic growth behavior

Sarah SK Yue¹ ², Yin Tong¹ ², Frank Lai¹ ², Wai Yin Tsui¹, Annie SY Chan¹, Hoi Cheong Siu¹, Suet Yi Leung¹ ² ³ *, Helen HN Yan¹ ² *

¹Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, ²Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China, ³The Jockey Club Centre for Clinical Innovation and Discovery, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, *Co-corresponding authors

Gastric intestinal metaplasia (IM) is a pre-cancerous stage of gastric cancer (GC) characterized by the transformation of gastric cells into a more intestinal-like cell type. The prevalence of IM and GC varies globally, with East Asia, particularly China, exhibiting high incidence rates. However, traditional methods for classifying IM have limited success in predicting GC risk, leading to the development of molecular studies and multi-omics approaches to better identify high-risk patients. In this study, we established a gastric IM organoid cohort derived from 47 GC patients, and conducted comprehensive molecular profiling, including genomic, epigenomic, and transcriptomic analyses. We also performed single-cell RNA sequencing to characterize stem/progenitor populations. This IM cohort represents an expansion of our previously established gastric and colon cancer organoid biobanks. Thus, our biobank, encompassing genomic, transcriptomic, and clinico-pathological data, is an invaluable resource for understanding IM heterogeneity, stemness, lineage plasticity, differentiation trajectories, and signaling pathway deregulation. This information has the potential to guide the development of novel strategies for GC prevention and provide insights into the neoplastic progression of IM.

~Published in: Gut. 2024; https://doi.org/10.1136/gutjnl-2024-332594.

Poster #8:

An orthotopic organoid transplantation model for understanding the role of ARHGAP fusions in conferring cell-cell and cell-matrix adhesion independence in gastric tumor cells

Priscilla SW Cheng¹ ², Sarah SK Yue¹ ², Yin Tong¹ ², Hoi Cheong Siu¹, Wai Yin Tsui¹, Dessy Chan¹ ², Stephanie KY Ma³, Annie SY Chan¹, April S Chan¹ ², Shui Wa Yun¹, Ho Sang Hui¹, Siu Tsan Yuen¹ ⁴, Suet Yi Leung¹ ² ⁵ *, Helen HN Yan¹ ² *

¹Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, ²Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China, ³School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, ⁴Department of Pathology, St. Paul’s Hospital, Hong Kong SAR, China, ⁵The Jockey Club Centre for Clinical Innovation and Discovery, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, *Co-corresponding authors

ARHGAP fusion events are frequently observed in diffuse-type gastric cancer, but the mechanism by which they promote carcinogenesis is not well studied. As part of our ongoing effort to expand our gastric organoid biobank, we established 9 tumor organoid lines from 6 gastric cancer patients carrying three distinct combinations of ARHGAP fusions, including CLDN18-ARHGAP6 (n=3), CLDN18-ARHGAP26 (n=5) and CTNND1-ARHGAP26 (n=1). We used an inducible Cas9 system to perform ARHGAP fusion specific KO in these organoids, followed by ROCK inhibitor withdrawal and spheroid formation assays. Furthermore, we developed an orthotopic xenograft model to introduce luciferase labelled tumor organoids with ARHGAP fusion or ARHGAP fusion KO to the stomachs of NGS mice. We found that ARHGAP fusion organoids were cell-cell (CC) and cell-matrix (CM) independent, but knockout of ARHGAP fusion partially restored both CC and CM adhesions, with enhanced RHO and apoptotic pathway activity in tumour organoids. Moreover, the ARHGAP fusion KO organoids became re-sensitised to CC and CM dissociation-induced apoptosis. ARHGAP fusion knockdown also inhibited xenograft formation in vivo, suggesting that the tumor promoting ability of ARHGAP fusion is strongly associated with cell-cell and cell-matrix adhesion independence. This represents the first cell models of ARHGAP fusion gastric cancer organoids that are shown to be CC/CM independent, with their knockdown leading to reversion back to CC/CM dependent states and continuous reliance on ROCK inhibitor for growth. Along with inhibition of orthotopic xenograft formation, our study confirms the importance of ARHGAP fusion in RHO-ROCK pathway inactivation for mediating the CC/CM independent phenotype.

~Published in: Gut, 2023, Feb;72(2):242-255

Poster Theme 3 : AML

Poster #9:

Activation of cGAS-STING pathway by hypomethylating agent in TP53 mutated MDS/AML

Nelson Ka-Lam Ng¹ ² ^, Lam Ng¹ ² ^, Stephen Lam¹ ^, Xingliang Liu³, Koon C. Chan¹ ², Lichuan Zheng¹ ², Chunxiao Zhang¹, Rachel L. S. Tse³, Wing Lam¹ ², Natalie Nok-Man Chan¹ ², Cheuk Him Man¹ ², Arthur K.L. Cheung⁴, Ho-Wan Ip⁵, Chun H. Au⁶, Edmond Ma⁷, Chi T. Ng⁸, Ying Ni⁹, Run S. Li⁹, Guang Sheng Ling³, Suet Yi Leung² ⁴ ⁵ ¹⁰, Asif Javed³ ¹⁰ *, Anskar Y. H. Leung¹ ² ¹⁰ *

Presenter: Kerwin Cheung¹ ²

¹Department of Medicine, School of Clinical Medicine, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; ²Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China; ³School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; ⁴Centre for PanorOmic Sciences, The University of Hong Kong, Hong Kong SAR, China; ⁵Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; ⁶Hong Kong Genome Institute, Hong Kong SAR, China; ⁷Department of Pathology, Hong Kong Sanatorium and Hospital, Hong Kong SAR, China; ⁸Clinical Trials Centre, The University of Hong Kong, Hong Kong SAR, China; ⁹Department of Infectious Diseases and Public Health, City University of Hong Kong, Hong Kong SAR, China; ¹⁰The Jockey Club Centre for Clinical Innovation and Discovery, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; ^These authors contributed equally; *Co-corresponding author

We examined the therapeutic mechanisms of decitabine in TP53-mutant (TP53m) acute myeloid leukemia (AML) using cellular studies, transcriptomic analysis, nanopore sequencing and a xenotransplantation model. This research included AML cell lines, primary AML samples with mutant and wildtype TP53, and an isogenic system with introduced TP53 mutations. In a Phase II clinical trial (NCT03080766), we treated 34 patients with complex or monosomy karyotype MDS/AML, carrying either wildtype or mutant TP53, with a 10-day course of decitabine, followed by nanopore sequencing and single-cell transcriptome analysis. We found that decitabine preferentially affected TP53m AML cells, increasing senescence, apoptosis, cytosolic double-stranded RNA, and DNA breaks. Transcriptomic analysis revealed increased expression of endogenous retroviruses, transposable elements, and genes related to inflammation and immune activation. These results were further validated in TP53 wildtype cell lines with knocked-in TP53 missense variants. Activation of the cGAS-STING and RIG-1-MAVS pathways was confirmed, and macrophage activation was demonstrated in co-culture studies. Moreover, anti-CD47 antibody enhanced decitabine's anti-leukemic effects in vivo. Single-cell transcriptome analysis of 47 samples from 17 patients revealed positive enrichment in inflammatory and immune response gene sets in TP53m leukemic and immune cells among responders. At relapse, leukemic cells up-regulated MYC signaling and heat shock response, while T-cells showed an exhaustion signature. Our study highlights the interplay between leukemic and immune cells in TP53m patients undergoing decitabine treatment, suggesting that hypomethylating agents induce both intrinsic anti-leukemia effects and immune activation through the RIG-1-MAVS and cGAS-STING pathways. This model offers insights for future clinical trial designs for this disease subtype.

Poster #10:

Inhibition of Polo-like Kinase 4 activates immune response via cGAS-STING pathway in TP53-mutated AML

Wing Lam¹, Chee-Chean Dang¹, Cheuk-Him Man¹, Xiao-yuan Zeng¹, Li-Chuan Zheng¹, Ka-Lam Ng¹, Koon-Chuen Chan¹, Tsz-Ho Kwok¹, Timothy Chi-Chun Ng¹, Wing-Yan Leung¹, Michael Shing-Yan Huen², Carmen Chak-Lui Wong³ ⁴ ⁵, Chi Wai Eric So⁶, Zhixun Dou⁷ ⁸, Mark Robert Bray⁹, Tak Wah Mak³ ⁵ ⁹, Anskar YH Leung¹ ⁵ *

¹Department of Medicine, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; ²School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; ³Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; ⁴State Key Laboratory of Liver Research, The University of Hong Kong, Hong Kong SAR, China; ⁵Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China; ⁶Department of Haematological Medicine, Leukemia and Stem Cell Biology Team, King’s College London, London, UK; ⁷Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; ⁸Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA; ⁹Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada; *Corresponding author

Acute myeloid leukemia (AML) with TP53 mutation portends an extremely poor prognosis. Based on in-silico analyses in TP53-mutated AML, we identified polo-like kinase 4 (PLK4) as a novel therapeutic target for this subtype. PLK4 inhibition activated the cGAS-STING pathway, leading to secretion of cytokines which activate the immune response of macrophages and T cells against TP53-mutated AML. Combination of PLK4 inhibition with macrophage checkpoint inhibitor (anti-CD47 antibody) synergistically reduced leukemic burden and prolonged animal survival in vivo. The study has shed light on a novel therapeutic strategy for TP53-mutated AML.

Poster #11:

HPK1 inhibition potentiated the therapeutic efficacy of venetoclax in AML by synergistically enhancing T-cell response

Xiao-Yuan Zeng¹ ² , Kwui-Wa Tong¹ ² , Koon-Chuen Chan¹ ² , Li-Chuan Zheng¹ ² , Stephen S.Y. Lam¹, Mark- Robert Bray³, Tak-Wah Mak² ³ ⁴, Cheuk-Him Man¹ ² *, Anskar Y.H. Leung¹ ² *

¹Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; ²Center for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China; ³The Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, Toronto, Canada; ⁴Department of Pathology, The University of Hong Kong, Hong Kong SAR, China; *Co-corresponding authors

Haematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T cell signaling. Venetoclax, a BCL2 inhibitor, has become a standard of care for unfit or elderly patients with acute myeloid leukaemia (AML). We hypothesize that HPK1 inhibitor can synergize with venetoclax and improve treatment outcome in AML by co-activating the cell-death pathway and T cell response. In vitro, HPK1 inhibitor promoted human T-cell function. In an in vivo model in which MLL-AF9 AML was transplanted into non-irradiated recipient mice, HPK1 inhibitor and venetoclax combination significantly decreased leukaemic burden and the naïve T cell population and increased effector memory T cell subset and cytokine secretion in the cytotoxic T cell populations. Thus, the regimen may be effective in AML and should be tested in clinical trials.

Poster Theme 4 : Immuno-oncology

Poster #12:

Tumor-specific cholinergic CD4+ T lymphocytes guide immunosurveillance of hepatocellular carcinoma

Chunxing Zheng¹ ², Bryan E. Snow², Yin Tong¹ ³, Suet Yi Leung¹ ³, Tak W. Mak¹ ² ³ *

Presenter: Enyu Huang¹ ³ ⁴

¹Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China; ²Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada; ³Department of Pathology, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong SAR, China; *Corresponding author

Choline acetyltransferase (ChAT) positive T cells have been shown to relay the adrenergic signals from splenic nerves and suppress inflammation via acetylcholine. However, their roles in hepatocellular carcinoma (HCC) regulation is unclear. In this research, we showed ChAT⁺ T cells undergo clonal expansion during oncogenesis of HCC, and they are comprised of regulatory T cells and dysfunctional PD-1⁺ T cells. The cholinergic activity intrinsic in T cells keeps CD25⁺ T regulatory cells and PD-1+ T cells in check and maintain immunosurveillance in liver cancer. Our results unveil a previously unappreciated role for cholinergic T cells in liver cancer immunobiology.

~Published in: Nature Cancer, 2023 Oct;4(10):1437-1454.

Poster #13:

Defining the epigenetic and somatic status of molecules facilitating immune evasion and T cell-based immunotherapy resistance in diffuse large B-cell lymphoma (DLBCL)

Jamie Lam¹ ² ³ ⁴, Michael St Paul², Liam Hendrikse², Ping Luo², Hanae Ida², Yoshihiro Iwamoto², Thorsten Berger², Drew Wakeham², Anskar YH Leung¹ ³ *, Tak-Wah Mak¹ ² ⁴ ⁵ *

¹Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China; ²Princess Margaret Cancer Centre, University Health Network, Toronto, Canada; ³Department of Medicine, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; ⁴Department of Medical Biophysics, University of Toronto, Toronto, Canada; ⁵Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; *Co-corresponding authors

The loss of antigen presentation-related proteins enables tumor cells to evade immunosurveillance and anti-tumor responses, resulting in patients developing resistance to immunotherapy. Over 50% of diffuse large B-cell lymphoma (DLBCL) cells have lost surface expression of HLA class I molecules or β2M, potentially due to selection pressure imposed by immunosurveillance mechanisms. This project aims to advance the design of T cell-based therapies against DLBCL by identifying alterations in antigen presentation proteins, defining HLA-bound peptides recognized by T cells, and studying gene expression changes in other lymphocytes in the tumor microenvironment that may contribute to resistance to immunosurveillance.

Poster #14:

Transcriptional landscape and TCR expansion of cholinergic CD4+ T cells in hepatocellular carcinoma

Chunxing Zheng¹ ², Bryan E. Snow², Yin Tong¹ ³, Suet Yi Leung¹ ³, Tak W. Mak¹ ² ³ *

Presenter: Guanghao Liang¹

Cholinergic nerves play a role in tumor progression and dissemination. However, unlike other visceral tissues, cholinergic innervation in the hepatic parenchyma is poorly detected, and it remains unclear whether cholinergic signaling regulates liver cancer. In this study, we used a mouse multi-hit model of hepatocellular carcinoma (HCC) and single-cell genomic technologies to characterize the transcriptional landscape and TCR repertoire of cholinergic CD4⁺ T cells. We identified two distinct populations of CD4⁺ T cells expressing choline acetyltransferase (ChAT): regulatory T cells and dysfunctional PD-1⁺ T cells. By combining TCR repertoire analysis with experimental validation, we found that tumor antigens drove the clonal expansion of these cholinergic T cells in HCC.

~Published in: Nature Cancer, 2023 Oct;4(10):1437-1454.

Poster #15:

Artesunate suppresses Th17 response via inhibiting IRF4-mediated glycolysis and ameliorates Sjögren's syndrome

Fan Xiao¹ ², Chong Deng¹, Enyu Huang¹ , Yuan Tang¹ and Liwei Lu¹ ² *

Primary Sjögren’s syndrome (pSS) is a chronic autoimmune disease characterized by secretory dysfunction and inflammation in salivary glands (SG) and lacrimal glands. Increasing evidence indicates that glycolysis controls effector functions of Th17 cells that are important during pSS development. However, it remains unclear whether targeting Th17 glycolysis could be an effective therapeutic strategy for treating pSS. In this study, we found that artesunate (ART), an important derivative of artemisinin and a first-line antimalarial agent, effectively ameliorated SG tissue inflammation accompanied by profoundly suppressed Th17 responses in both experimental Sjögren’s syndrome (ESS) mice and humanized SS mice. Notably, ART treatment markedly suppressed glycolysis in Th17 cells. Mechanistically, ART inhibited glycolysis via promoting the proteasomal degradation of IRF4 in Th17 cells. Together, our findings have demonstrated that ART suppresses Th17 responses via inhibiting IRF4-mediated glycolysis and may serve as a promising therapeutic candidate for the treatment of pSS patients.

~Published in: Signal Transduction and Targeted Therapy, 2022 Aug 29;7(1):274.

Poster Theme 5 : CRISPR technologies

Poster #16:

Development of a high-throughput functional genomics profiling system for studying cell-cell fusion

Charles W. F. Chan¹ ² ^, Bei Wang¹ ² ^, Lang Nan³ ⁴ ^, Xiner Huang⁵ ^, Tianjiao Mao³ ⁴, Hoi Yee Chu¹ ², Cuiting Luo⁵, Hin Chu⁵ ⁶ ⁷ *, Gigi C.G. Choi¹ ² ³ *, Ho Cheung Shum³ ⁴ *, Alan S.L. Wong¹ ² *

¹Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Hong Kong SAR, China; ²Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China; ³Department of Mechanical Engineering, The University of Hong Kong, Hong Kong SAR, China; ⁴Advanced Biomedical Instrumentation Centre, Hong Kong Science Park, Hong Kong SAR, China; ⁵State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Hong Kong SAR, China; ⁶Centre for Virology, Vaccinology and Therapeutics, Hong Kong Science Park, Hong Kong SAR, China; ⁷Department of Infectious Disease and Microbiology, The University of Hong Kong-Shenzhen Hospital, Shenzhen, China; ^These authors contributed equally; *Co-corresponding authors

We have developed a high-throughput functional genomics profiling system for studying the cell-cell fusion process using droplet microfluidics. Using a case in which the Spike protein of the SARS-CoV-2 causes syncytia, a cell-cell fusion phenomenon that is commonly observed in patients who develop severe chronic respiratory symptoms, we demonstrate the feasibility of our system to annotate the syncytium-forming potential of Spike protein variants and inform mutations that could cause serious pathological consequences. They are the potential mutations of concern and should be tracked. Our high-throughput platform holds broad potential for studying cancer-related cell-cell fusion phenotypes.

~Published in: Nature Biomedical Engineering, 2024 Mar;8(3):291-309.

Poster #17:

Effective low-N sampling strategy that identifies top protein variants in complex combinatorial mutagenesis settings

Hoi Yee Chu¹ ², John H.C. Fong¹, Dawn G.L. Thean¹, Peng Zhou¹ ², Frederic K.C. Fung¹ ², Yuanhua Huang³ ⁴, Alan S.L. Wong¹ ² *

We developed a strategy to obtain the greatest number of best-performing protein variants with the least amount of experimental effort for mutagenesis screens to alleviate the experimental resources spent on cloning and testing non-functional variants. Our strategy uses zero-shot prediction and machine learning to guide multi-round sampling of top variants in the library. We found that four rounds of low-N pick-and-validate sampling with 12 variants for machine learning yielded up to 92.6% accuracy in selecting the true top 1% variants mutant libraries, while two rounds of 24 variants identify top variants with higher sequence diversity. Our proposed strategy outperforms other state-of-the-art methods in terms of efficiency and accuracy and can be generalized for a range of protein-function inference, including Cas9 and APOBEC protein in the CRISPR genome editor toolkit.

~Published in: Cell Systems, 2024 Feb 21;15(2):193-203.

Poster #18:

Parallel engineering and profiling of a cytosine base editor system

John H.C. Fong¹, Hoi Yee Chu¹ ², Peng Zhou¹ ², Alan S.L. Wong¹ ² *

Selecting and engineering the most suitable base editors for installing specific base conversions with maximal efficiency and minimal undesired edits are pivotal for precise genome editing applications. We present here a platform for creating and analyzing a library of base editor variants to evaluate their editing performance at scale. Our comparison provides quantitative measures of each variant’s efficiency, purity, motif preference, and bias in generating single and multiple base conversions. We also uncover undesired higher indel rates and noncanonical base conversions in some existing editors. With the growing number of editor variants, our platform addresses the need for an unbiased, scalable method to benchmark editing outcomes and accelerate next-generation genome editor engineering.

~Published in: Cell Systems, 2023 May 17;14(5):392-403.

Poster Theme 6 : Bioinformatics Analysis

Poster #19:

ERCC2 mutations alter the genomic distribution pattern of somatic mutations and are independently prognostic in bladder cancer

Jayne A. Barbour¹, Tong Ou², Haocheng Yang¹, Hu Fang¹ ³, Noel C. Yue¹, Xiaoqiang Zhu¹, Michelle W. Wong-Brown⁴ ⁵, Yuen T. Wong⁶, Nikola A. Bowden⁴ ⁵, Song Wu² ⁷ *, Jason W. H. Wong¹ ⁸ ⁹ *

¹School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; ²Urology Institute of Shenzhen University, The Third Affiliated Hospital of Shenzhen University, Shenzhen University, Shenzhen, China; ³Institute of Biomedical Data, South China Hospital, Medical School, Shenzhen University, Shenzhen, China; ⁴Centre for Drug Repurposing and Medicines Research, University of Newcastle, NSW, Australia; ⁵Hunter Medical Research Institute, Newcastle, NSW, Australia; ⁶Adult Cancer Program, Lowy Cancer Research Centre, UNSW Sydney, NSW, Australia; ⁷Department of Urology, South China Hospital, Medical School, Shenzhen University, Shenzhen, China; ⁸Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China, ⁹Centre for PanorOmic Sciences, The University of Hong Kong, Hong Kong SAR, China; *Co-corresponding authors

Excision repair cross-complementation group 2 (ERCC2) encodes the DNA helicase xeroderma pigmentosum group D, which functions in transcription and DNA repair. Point mutations in ERCC2 are putative drivers in around 10% of bladder cancers (BLCAs) and a positive biomarker for cisplatin therapy response. We first demonstrated that mutant ERCC2 is an independent predictor of prognosis in BLCA. We then examined its impact on the somatic mutational landscape using a cohort of ERCC2 wild-type (n = 343) and mutant (n = 39) BLCA whole genomes. The genome-wide distribution of somatic mutations is significantly altered in ERCC2 mutants, especially with respect to epigenetics. We leverage these alterations to develop a machine learning model for predicting pathogenic ERCC2 mutations.

Poster #20:

A cell type-specific cryptic promoter drives ERVK-7 overexpression in lung cancer

Sojung Lee¹ ², Yin Yee Ho¹, Suyu Hao¹, Yingqi Ouyang¹, U Ling Liew¹, Ashish Goyal³, Stephen Li¹, Jayne A. Barbour¹, Mu He¹, Yuanhua Huang¹ ⁴ ⁵, Jason W. H. Wong¹ ² *

¹School of Biomedical Sciences, The University of Hong Kong, Hong Kong SAR, China; ²Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China; ³Cancer Epigenomics, German Cancer Research Center (DKFZ), Heidelberg, Germany; ⁴Center for Translational Stem Cell Biology, Hong Kong Science and Technology Park, Hong Kong SAR, China; ⁵Department of Statistics and Actuarial Science, The University of Hong Kong, Hong Kong SAR, China; *Corresponding author

Human endogenous retroviruses have been implicated to play important roles in health and disease, with recent evidence linking ERVK-7 envelope glycoprotein to immune checkpoint blockade response and prognosis in lung adenocarcinoma (LUAD) patients. However, the mechanisms underlying ERVK-7 regulation remain unclear. We show that the 5' long terminal repeat (LTR) of ERVK-7 is methylated and non-functional, requiring alternative upstream promoters to drive its expression via two novel transcripts, ERVK-7.long and ERVK-7.short. ERVK-7.long is the primary driver of ERVK-7 overexpression in LUAD, with the transcript predominantly active in malignant cells. Epigenetic and single-cell RNA analyses reveal that ERVK-7.long activation is predetermined by cellular origin, with tumor necrosis factor-alpha (TNF-α) contributing to its upregulation. Higher levels of ERVK-7 observed in LUAD compared with lung squamous cell carcinoma (LUSC) can be further explained by increased interferon signaling, the key regulator of ERVK-7.short, in LUAD. Our findings uncover the mechanistic basis of ERVK-7 upregulation in LUAD and potential avenues for its use as a therapeutic or diagnostic target in non-small cell lung cancer.

~Published in: Genome Medicine, 2022 Oct 31;14(1):124.

Poster #21:

Investigating anchorage independent growth of colorectal adenoma organoids using multiomics profiling

Jonathan W.T. Tse¹ ², Hoi Cheong Siu³, Yin Tong¹ ³, Suet Yi Leung¹ ³, Helen H.N. Yan¹ ³, David J.H. Shih¹ ² *

¹Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China; ²School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; ³Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; *Corresponding author

Anchorage-independent growth, a hallmark of tumor cell malignancy, enables cells to survive and proliferate without attachment to the extracellular matrix, facilitating metastasis. This study aims to elucidate the phenotypic and evolutionary factors contributing to this process in colorectal adenoma organoids. We employ multiomics profiling on hierarchically-structured organoid samples cultured under two distinct assays that simulate anchorage independence. To address the hierarchical structure of the data and potential confounding due to relatedness, we utilize a mixed-effects model incorporating a kinship matrix. Our findings have the potential to identify novel cancer biomarkers, which could enhance immunotherapy strategies and improve prognostic outcomes for patients with metastatic colorectal cancer.

Poster Theme 7 : Medicinal Chemistry

Poster #22:

Lead identification by N-Acryloylindole-alkyne (NAIA) for covalent targeting of Rac1 to modulate cell cycle

Tin-yan Koo¹ ², Hinyuk Lai¹ ², Daniel K. Nomura³ and Clive Yik-Sham Chung¹ ² ³ ⁴ *

¹Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China; ²School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; ³Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA; ⁴Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; *Corresponding author

Activity-based protein profiling (ABPP) allows identification of reactive residues on protein targets for investigating protein modifications and discovering covalent ligands. Cysteine is one of the amino acids being targeted with significant biological roles in life. Yet, conventional cysteine reactive probes are far from perfect for proteome-wide profiling of cysteines. We reported N-acryloylindole-alkyne (NAIA) as a promising cysteine-reactive probe with improved performance and compatibility with cell-based experiments. NAIA demonstrated superior properties than both conventional and newly developed probes in terms of reaction kinetics, selectivity and adduct stability. This enabled identification of more and unique reactive cysteine, as compared to the reported cysteine profiling experiments. We successfully utilized NAIA to identify covalent ligands that can modulate the cell cycle through Rac1, suggesting the applicability of NAIA in covalent drug screening.

~Published in: Nature Communications, 2023 Jun 15;14(1):3564.

Poster #23:

Chemoproteomics assisted discovery of a small covalent RhoA inhibitor

Tin-Yan Koo¹ ², Jason Ying Ki Li¹ ², Nga-Sze Lee¹ ², Jintian Chen³, Hillary Yui-Yan Yip¹ ², Ianto Bosheng Huang¹ ⁴, Kai-Yu Ng¹ ⁴, Helen H. N. Yan² ⁵, Suet Yi Leung² ⁵ ⁶, Stephanie Ma¹ ⁴ ⁷ ⁸, Jingying Zhou³, Clive Yik-Sham Chung¹ ² ⁵ *

¹School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; ²Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China; ³School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong SAR, China; ⁴Laboratory for Synthetic Chemistry and Chemical Biology, Hong Kong Science Park, Hong Kong SAR, China; ⁵Department of Pathology, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; ⁶The Jockey Club Centre for Clinical Innovation and Discovery, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; ⁷The University of Hong Kong – Shenzhen Hospital, Hong Kong SAR, China; ⁸State Key Laboratory of Liver Research, The University of Hong Kong, Hong Kong SAR, China; *Corresponding author

RhoA is a key contributor to cancer development but has not yet been targeted clinically. Research has shown that levels and functions of RhoA are elevated in colorectal cancer (CRC), indicating it could be a promising treatment target for CRC. Through activity-based protein profiling (ABPP) and mass spectrometry (MS) studies, we have discovered a covalent inhibitor for RhoA, CL16. Targeting RhoA Cys16 with CL16 hinders GTP binding of RhoA and suppresses its function in CRC cells. This leads to cell cycle arrest. Importantly, CL16 inhibits tumour growth in mouse models of CRC, while also promoting the infiltration of T cells into the tumour microenvironment. Our study demonstrates the development of a potent RhoA covalent inhibitor for CRC treatment.

Poster #24:

Targeted protein degradation of Aryl Hydrocarbon Receptor (AHR) through autophagy pathway

Wong Clara Shania¹ ², Jacinth Wing-Sum Cheu¹ ³, Carmen Chak-Lui Wong¹ ³ *, Clive Yik-Sham Chung¹ ² ³ *

¹Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China; ²School of Biomedical Science, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; ³Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; *Co-corresponding author

Liver cancer remains a leading cause of cancer deaths globally, with limited treatment options. Aryl hydrocarbon receptor (AHR) has emerged as a key driver for cancer development, such as liver cancer, in which its aberrant expression and increased activity promotes tumour growth and immune escape. We developed an AHR degrader harnessing autophagy-mediated protein degradation to selectively degrade AHR. In vitro and cellular studies demonstrated the ability of the AHR degrader to kills hepatocellular carcinoma (HCC) cells while also restoring the immune system, demonstrating the potential of AHR degraders as a cancer therapy.