Centre for Oncology and Immunology Flagship Symposium
Poster Session
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Poster Theme 1 : Liver Cancer
Poster #1:
PLK4 inhibitor, CFI-400945, suppresses liver cancer through cell cycle perturbation and eliciting anti-tumor immunity
Cerise Yuen-Ki Chan¹ ², Vincent Wai-Hin Yuen¹ ² David Kung-Chun Chiu¹, Chi-Ching Goh¹, Kelsie Thu³, David W Cescon³, Isabel Soria-Bretones³, Cheuk-Ting Law¹, Jacinth Wing-Sum Cheu¹ ², Derek Lee¹ ², Aki Pui-Wah Tse¹ ², Kel Vin Tan⁴, Misty Shuo Zhang¹ ², Bowie Po-Yee Wong¹, Chun-Ming Wong¹ ⁵, Pek-Lan Khong⁴, Irene Oi-Lin Ng¹ ⁵, Mark R Bray³, Tak W Mak² ³, Thomas Chung-Cheung Yau⁵ ⁶ *, Carmen Chak-Lui Wong¹ ² ⁵ ⁷ *
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 Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, Toronto, Canada, ⁴Department of Diagnostic Radiology, The University of Hong Kong, Hong Kong SAR, China, ⁵State Key Laboratory of Liver Research, The University of Hong Kong, Hong Kong SAR, China, ⁶Department of Medicine, The University of Hong Kong, Hong Kong SAR, China, ⁷Guangdong-Hong Kong Joint Laboratory for RNA Medicine, Sun Yat-Sen University, Guangzhou, China, *Co-corresponding authors
Prognosis of hepatocellular carcinoma (HCC) remains poor due to lack of effective therapies. Immune checkpoint inhibitors (ICIs) have delayed response and are only effective in a subset of patients. Treatments that could effectively shrink the tumors within a short period of time are idealistic to be employed together with ICIs for durable tumor suppressive effects. HCC acquires increased tolerance to aneuploidy. The rapid division of HCC cells relies on centrosome duplication. In this study, we found Polo-like kinase 4 (PLK4), a centrosome duplication regulator, represents a therapeutic vulnerability in HCC. An orally available PLK4 inhibitor, CFI-400945, potently suppressed proliferating HCC cells by perturbing centrosome duplication. CFI-400945 induced endoreplication without stopping DNA replication, causing severe aneuploidy, DNA damage, micronuclei formation, cytosolic DNA accumulation and senescence. The cytosolic DNA accumulation elicited DDX41-STING-IRF3/7-NFκ-β cytosolic DNA sensing pathway, thereby driving the transcription of senescence-associated secretory phenotypes (SASPs) which recruit immune cells. CFI-400945 was evaluated in liver specific p53/PTEN knockout mouse HCC models established by hydrodynamic tail vein injection. Tumor-infiltrated immune cells were analyzed. CFI-400945 significantly impeded HCC growth and increased infiltration of CD4+, CD8+ T cells, macrophages and natural killer cells. Combination therapy of CFI-400945 with anti-PD-1 showed tendency to improve HCC survival. Here we show that by targeting a centrosome regulator, PLK4, to activate the cytosolic DNA sensing-mediated immune response, CFI-400945 effectively restrained tumor progression through cell cycle inhibition and inducing anti-tumor immunity to achieve a durable suppressive effect even in late-staged mouse HCC.
~Published in: Hepatology, 2023 Mar 1;77(3):729-744.
Poster #2:
CFI-402257, a TTK inhibitor, effectively suppresses hepatocellular carcinoma
Cerise Yuen-Ki Chan¹ ², David Kung-Chun Chiu¹, Vincent Wai-Hin Yuen¹ ², Cheuk-Ting Law¹, Bowie Po-Yee Wong¹, Kelsie Lynn Thu³, David Ward Cescon³, Isabel Soria-Bretones³, Jacinth Wing-Sum Cheu¹ ², Derek Lee¹ ², Aki Pui-Wah Tse¹ ², Misty Shuo Zhang¹ ², Kel Vin Tan⁴, Irene Oi-Lin Ng¹ ⁵, Pek-Lan Khong⁴, Thomas Chung-Cheung Yau⁵ ⁶, Mark Robert Bray³, Tak Wah Mak¹ ² ³ *, Carmen Chak-Lui Wong¹ 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, ³The Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, Toronto, Canada, ⁴Department of Diagnostic Radiology, The University of Hong Kong, Hong Kong SAR, China, ⁵State Key Laboratory of Liver Research, The University of Hong Kong, Hong Kong SAR, China, ⁶Department of Medicine, The University of Hong Kong, Hong Kong SAR, China, *Co-corresponding authors
Deregulation of the cell cycle is a typical feature of cancer cells. Normal cells rely on the strictly coordinated spindle assembly checkpoint (SAC) to maintain the genome integrity and survive. However, cancer cells can bypass this checkpoint mechanism. In this study, we showed the clinical relevance of TTK protein kinase, a central regulator of the SAC, in hepatocellular carcinoma (HCC) and its potential as therapeutic target. Here we reported that a newly developed, orally active small molecule inhibitor targeting TTK (CFI-402257) effectively suppressed HCC growth, induced highly aneuploid HCC cells, DNA damage and micronuclei formation. For the first time, we identified CFI-402257 also induced cytosolic DNA, senescence-like response, and activated DDX41-STING cytosolic DNA sensing pathway to produce senescence-associated secretory phenotypes (SASPs) in HCC cells. These SASPs subsequently led to recruitment of different subsets of immune cells (natural killer cells, CD4+, CD8+ T cells) for tumor clearance. Our mass cytometry data illustrated the dynamic changes in the tumor-infiltrating immune populations after treatment with CFI-402257. Further, CFI-402257 improved survival in HCC-bearing mice treated with anti-PD-1, suggesting the possibility of combination treatment with immune checkpoint inhibitors in HCC patients. In summary, our study characterized CFI-402257 as a potential therapeutic for HCC, both used as a single agent and in combination therapy.
~Published in: Proc Natl Acad Sci USA (PNAS), 2022 Aug 9;119(32):e2119514119.
Poster #3:
Ferroptosis suppressor protein 1 inhibition promotes tumor ferroptosis and anti-tumor immune responses in liver cancer
Jacinth Wing-Sum Cheu¹ ² ^, Derek Lee^, Qidong Li² ^, Chi Ching Goh, Macus Hao-Ran Bao, Vincent Wai-Hin Yuen¹ ², Misty Shuo Zhang, Chunxue Yang, Cerise Yuen-Ki Chan¹ ², Aki Pui-Wah Tse², Irene Oi-Lin Ng² ³, Chun-Ming Wong² ³ and Carmen Chak-Lui Wong¹ ² ³ *
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, ³State Key Laboratory of Liver Research, The University of Hong Kong, Hong Kong SAR, China, ^These authors contributed equally, *Corresponding author
Ferroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation, counteracted mainly by GPX4 and FSP1 in cancer cells. Inhibition of these pathways induces ferroptosis and suppresses tumor growth. Ferroptosis is also an immunogenic cell death that elicits anti-tumor immune responses. We demonstrated that HCC cells relied on FSP1 to protect them against ferroptosis and the induction of ferroptosis by FSP1 inhibitor suppressed HCC growth. More importantly, FSP1 inhibitor promoted tumor infiltration of dendritic cells (DCs), macrophages and T cells. Single-cell RNA sequencing further revealed that FSP1 inhibitor increased DCs expressing high level of MHC molecules for antigen presentation. It also enhanced the infiltration of a macrophage subset that expressed phagocytosis gene signature. These indicated that FSP1 inhibitor treatment activates innate immunity. Despite the increase in CD8⁺ T cells upon FSP1 inhibitor treatment, these T cells were exhausted. This prompted us to explore the effect of combination treatment and we showed that FSP1 inhibitor worked synergistically with multiple immunotherapies. In sum, ferroptosis induction by FSP1 inhibition promotes innate and adaptive anti-tumor immune responses and is a promising therapeutic approach in treating HCC.
~Published in: bioRxiv. 2024; https://doi.org/10.1101/2024.04.03.588024.
Poster #4:
Cell cycle inhibitors activate hypoxia-induced DDX41-STING pathway to mediate anti-tumor immune response in liver cancer
Po Yee Wong¹ ^, Cerise Yuen Ki Chan¹ ² ^, Helen Do Gai Xue¹ ² ^, Chi Ching Goh¹, Jacinth Wing Sum Cheu¹ ², Aki Pui Wah Tse¹, Misty Shuo Zhang¹, Carmen Chak Lui Wong¹ ² ³ ⁴ ⁵ *
¹Department of Pathology, School of Clinical Medicine, the University of Hong Kong, Hong Kong SAR, China, ²Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China, ³State Key Laboratory of Liver Research, The University of Hong Kong, Hong Kong SAR, China, ⁴Guangdong-Hong Kong Joint Laboratory for RNA Medicine, Sun Yat-Sen University, Guangzhou, China, ⁵ShenZhen Hospital, The University of Hong Kong, Shenzhen, China, ^These authors contributed equally, *Corresponding author
Cancer cells are characterized by uncontrolled cell cycle progression, allowing them to proliferate fast and survive. To counteract these characteristics, cell cycle inhibitors are used to perturb their cell cycles and promote cell death. In this study, we discovered an unexpected secondary effect of cell cycle inhibitors, the immune modulation. Anti-mitotic agents such as Paclitaxel (a microtubule stabilizer), Palbociclib (a CDK4/6 inhibitor), and AZD1152 and GSK1070916 (Aurora Kinase B inhibitors) can also clear cancer cells through an alternative mechanism – activation of STING signalling.
Our findings showed that these anti-mitotic agents induced DNA damage and accumulation of cytosolic DNA in hepatocellular carcinoma (HCC) cells. The DNA sensor DDX41 then detected these cytosolic DNA and activated the STING-TBK1-IRF3/7 pathway, leading to an increase in secretion of pro-inflammatory SASP factors in cancer cells. Further, we found that the transcription of DDX41 was regulated by the transcription factor named hypoxia-inducible factor (HIF), as suggested by the identification of HIF binding sites at the promoter region of DDX41. In HCC, where hypoxia is commonly seen in the tumor microenvironment (TME), hypoxia can upregulate the expression of DDX41 via HIF-1. Thus, hypoxic HCC cells are more susceptible to STING activation and SASP production under mitotic stress caused by cell cycle inhibitors. Within the TME, SASP facilitated immune cell infiltration into the tumor to clear cancer cells. In addition, we showed the combination of cell cycle inhibitors (Paclitaxel, Palbociclib, and AZD1152) with anti-PD-1 mAb further inhibited HCC growth in mouse models.
In conclusion, this study revealed a new immune-mediated role of cell cycle inhibitors in cancer treatment and suggested a promising direction for combining these inhibitors with the existing immunotherapy options.
Poster #5:
Precision mouse model guides precision medicine in hepatocellular carcinoma - the genetic landscape determines the immune landscape and response to immune checkpoint inhibitors
Vincent Wai-Hin Yuen¹ ³, David Kung-Chun Chiu¹, Cheuk-Ting Law¹, Jacinth Wing-Sum Cheu¹ ³, Cerise Yuen-Ki Chan¹ ³, Bowie Po-Yee Wong¹, Chi-Ching Goh¹, Misty Shuo Zhang¹, Helen Do-Gai Xue¹ ³, Aki Pui-Wah Tse¹ ³, Yan Zhang¹ ³, Henry Yee-Hin Lau¹, Derek Lee¹ ³, Rex KH Au-Yeung¹ ³, Chun-Ming Wong¹ ², Carmen Chak-Lui Wong¹ ² ³ *
¹Department of Pathology, LKS 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
Tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs) are the only two classes of FDA-approved drugs for individuals with advanced hepatocellular carcinoma (HCC). While TKIs confer only modest survival benefits, ICIs have shown remarkable outcomes in a minority of patients. This study aims to elucidate how genetic composition and specific oncogenic pathways regulate the immune composition of HCC, which directly affects response to ICIs. A collection of mouse HCCs with genotypes that closely simulate the genetic composition found in human HCCs were established using genome-editing approaches involving the delivery of transposon and CRISPR-Cas9 systems by hydrodynamic tail vein injection. Based on the CD8⁺ T cell-infiltration level, we characterized tumors with different genotypes into cold and hot tumors. Anti-PD-1 treatment had no effect in cold tumors but was greatly effective in hot tumors. As proof-of-concept, a cold tumor (Trp53ᴷᴼ/MYCᴼᴱ) and a hot tumor (Keap1ᴷᴼ/MYCᴼᴱ) were further characterized. Tumor-infiltrating CD8⁺ T cells from Keap1ᴷᴼ/MYCᴼᴱ HCCs expressed higher levels of proinflammatory chemokines and exhibited enrichment of a progenitor exhausted CD8⁺ T-cell phenotype compared to those in Trp53ᴷᴼ/MYCᴼᴱ HCCs. The TKI sorafenib sensitized Trp53ᴷᴼ/MYCᴼᴱ HCCs to anti-PD-1 treatment. Our study might help to guide the design of patient stratification systems for single or combined treatments involving anti-PD-1.
~Published in: Journal of Hepatology, 2023 Feb;78(2):376-389.
Poster Theme 2 : Gastric Cancer
Poster #6:
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 #7:
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 #8:
Divergent lineage trajectories and genetic landscapes in human gastric intestinal metaplasia organoids associated with early neoplastic progression
Sarah 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: bioRxiv. 2024; https://doi.org/10.1101/2024.04.03.588024.
Poster #9:
Human gastric intestinal metaplasia organoids display genetic profiles linked with early neoplastic growth behavior
Sarah 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: bioRxiv. 2024; https://doi.org/10.1101/2024.04.03.588024.
Poster #10:
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 #11:
Pathogenetic role of RNA splicing and NRAS mutation combination in chronic myelomonocytic leukaemia
Yanan Chen¹ ², Chun-fung Sin¹ ³, Lichuan Zheng¹ ², Koon-Chuen Chan¹ ², Dandan Wang¹ ², Lingge Tu¹ ², Fangfang He¹ ², Wan Hei Jeffrey Hui², Hoi Ki Leung², Leslie Kar-Fai Chan², Sze Pui Tsui³, Hoi-yi Chan³, Alvin Chun Hang Ma⁴ ⁵, Anskar YH Leung¹ ² ⁵ * and Xuan Sun¹ ² ⁵ *
¹Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China, ²Department of Medicine and ³Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, ⁴Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong SAR, China, ⁵ZeBlast Technology Limited, Hong Kong Science Park, Hong Kong SAR, China, *Co-corresponding authors
Transgenic expression of SRSF2ᴾ⁹⁵ᴴ and NRASᴳ¹²ᴰ in zebrafish induced myelodysplasia, monocytosis, and leukaemia transformation that resembled human chronic myelomonocytic leukaemia (CMML) and acute myeloid leukaemia (AML). Furthermore, leukaemic phenotypes were accentuated upon serial transplantations. Transcriptomic analysis revealed global alterations in splicing in SRSF2ᴾ⁹⁵ᴴ mutants and an inflammatory phenotype associated with the activation of the cGAS-STING pathway, triggered by DNA damage and R-loop formation. Targeting R-loop formation using an RNA polymerase III inhibitor effectively reduced cytosolic R-loop levels, cGAS activation, expression of cytokines and immune-related genes, and alleviated leukaemia phenotypes in SRSF2ᴾ⁹⁵ᴴ mutant zebrafish. Thus, our SRSF2ᴾ⁹⁵ᴴ zebrafish model has shed light on the pathogenesis of human myeloid diseases and provided a platform for identifying therapeutic agents based on the mitigation of the immune and inflammatory response to SRSF2 mutation.
~Published in: Blood, 2022; 140 (Supplement 1): 11491.
Poster #12:
Downregulation of BRCA2 sensitizes FLT3-ITD AML to PARP inhibitor and induced activation of cGAS-STING pathway
Nelson K.L. Ng¹ ² ^, Lam Ng¹ ² ^, Michael K.C. Chan¹ ² ^, Lichuan Zheng¹ ² ^, Wing Lam¹ ², Stephen S. Y. Lam¹, Michael S.Y. Huen³, Eric C.W. So⁴ * and Anskar Y.H. Leung¹ ² *
¹Department of 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, ⁴Leukaemia and Stem Cell Biology Group, Department of Haematological Medicine, Rayne Institute, King’s College, London, UK, ^These authors contributed equally, *Co-corresponding authors
Internal tandem duplication (ITD) of FMS-like Tyrosine Kinase 3 (FLT3) is one of the most common mutations in acute myeloid leukaemia (AML). FLT3-ITD AML has been demonstrated to be susceptible to DNA repair inhibition. In this study we examined the therapeutic effects and underlying mechanisms of poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) in this AML subtype. We found that FLT3-ITD AML showed down-regulation of BRCA2 via its transcription repressor SNAI1 and a decrease in homologous recombination. PARPi treatment led to an increase in interferon signaling, a decrease in double-strand break (DSB) repair, activation of cGAS-STING signaling and enhancement of macrophage functions. Furthermore, PARPi upregulated AXL expression, and both AXL inhibition and knockout amplified the anti-leukemia effects of PARPi. Taken together, our findings reveal a novel FLT3-ITD/SNAI1/BRCA2 axis in AML, a non-cell autonomous effect of PARPi via cGASSTING activation, and identify AXL as a potential therapeutic target, providing valuable insights for the development of innovative therapeutic strategies for FLT3-ITD AML.
Poster #13:
Identifying mediators of intrinsic and acquired resistance to decitabine in TP53-mutant AML
Vivian Wang¹, Nelson KL Ng² ³, Anskar YH Leung² ³, Steven Chan¹ ² ⁴ *
¹Department of Medical Biophysics, University of Toronto, Canada, ²Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China, ³Department of Medicine, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, ⁴Princess Margaret Cancer Centre, University Health Network, Toronto, Canada
TP53-mutated AML (TP53Mut AML), representing ~5-15% of de novo AML cases, is an aggressive subtype that is associated with poor outcomes and chemoresistance. In a 2016 clinical trial studying the efficacy of a 10-day decitabine course for AML, 100% of TP53Mut AML patients (21/21) demonstrated a response, while only 41% of TP53-wildtype patients (32/78) responded. These findings suggest that decitabine might be an effective treatment for TP53Mut AML. However, the rapid selection of resistant subclones led to non-durable responses. In this study, we conducted genome-wide CRISPR knockout screens to identify genes that, upon inactivation, could overcome resistance to decitabine in paired decitabine-sensitive and decitabine-resistant TP53-mutated AML cell lines.
Poster #14:
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 SY 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 leukemia (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 leukemic burden and the naïve T cell population and increased the 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 #15:
Combination of QUIZartinib and Omacetaxine Mepesuccinate (QUIZOM) remodels proteostasis and triggers inflammatory response in FLT3-ITD AML
Li-Chuan Zheng¹ ² ^, Kelvin KW Wong³ ^, Stephen SY Lam¹ ^, Garret MK Leung¹, Chenqinyao Li¹, Kui-Wa Tong¹ ², Wing Lam¹ ², Xiao-yuan Zeng¹, Koon-Chuen Chan¹ ², Natalie Nok-Man Chan¹ ², Ka-Lam Ng¹ ², Chee-Chean Dang¹, Tsz-Ho Kwok¹, Sze-Pui Tsui⁴, Rakesh Sharma³, Jason WH Wong⁵, Anskar YH Leung¹ ² * Cheuk-Him Man¹ ² *
¹Department of Medicine, 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, ³Proteomics and Metabolomics Core, Centre for PanorOmic Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, ⁴Department of Pathology, Queen Mary Hospital, Hong Kong SAR, China, ⁵School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, ^These authors contributed equally, *Co-corresponding authors
In this study, we investigated the vulnerability of FLT3-ITD AML and identified its dependency on protein translation. We found that a combination therapy of Quizartinib and Omacetaxine Mepesuccinate (QUIZOM) disrupted proteostasis by suppressing of HSP70 chaperones and induced an inflammatory response via cGAS-STING pathway activation. To confirm our observations we conducted a phase 2 clinical trial by treating 40 FLT3-ITD AML patients with QUIZOM and we achieved a high composite complete remission rate of 82.5%. Single cell RNA-seq analysis of clinical trial samples confirmed T cell activation and expansion of a terminally differentiated effector memory T cell subset upon remission. Clinical resistance to QUIZOM was associated with the presence of leukemic stem cells expressing high levels of HSPA1B and JUN, while the additional of c-Jun inhibitor mitigated this resistance and further enhanced QUIZOM’s anti-leukemic effect. In conclusion, QUIZOM effectively suppressed FLT3-ITD and induced immune clearance of leukaemia cells via cGAS-STING pathway activation.
Poster Theme 4 : Immuno-oncology
Poster #16:
Characterizing the spatial single cell landscape and clonal dynamics of tumor-infiltrating T lymphocytes during pancreatic cancer development
Matthew Bianca¹, Teresa Ciudad¹, Sara Lamorte¹, Rene Quevedo¹, Tracy McGaha¹ ² *
¹Princess Margaret Cancer Centre, University Health Network, Toronto, Canada, ²Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China, *Corresponding author
Pancreatic Ductal Adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates. The disease is preceded by inflammatory pancreatic intraepithelial neoplasia (PanIN) or intraductal papillary mucinous neoplasia (IPMN). Early lesions represent an “immunologically-hot” tumor microenvironment (TME) where T cells possess a high cytotoxic capacity to kill malignant cells, whereas late-stage disease contains dysfunctional T cells lacking antitumor characteristics. Recent studies have confirmed that PDAC has the potential to respond to immunotherapy. The spatial interactions of neoantigen-reactive lymphocytes throughout PDAC progression have yet to be explored and hold unexploited therapeutic potential. In this study, we aim to utilize single-cell and spatial sequencing technologies to interrogate the phenotypic evolution and clonal dynamics of T cells across disease progression within clinical tissue specimens
Poster #17:
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
¹Centre for Oncology and Immunology, Hong Kong Science ParkHong 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 comprise 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 #18:
Determining the influence of SMAD4 loss on the tumor immune microenvironment of pancreatic ductal adenocarcinoma
Robbie Jin¹ ², M. Teresa Ciudad¹, Rene Quevedo¹, Sara Lamorte¹, Zhe Qi Liu¹ ², Xin Zhang¹, Matthew Bianca¹ ², Tracy L. McGaha¹ ² ³ *
¹Princess Margaret Cancer Centre, University Health Network, Toronto, Canada, ²Department of Immunology, Temerty Faculty of Medicine, University of Toronto, Toronto, Canada, ³Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China, *Corresponding author
Pancreatic cancer has a dismal five-year survival rate of 10%, beckoning the need to a deeper understand of its underlying tumor biology. Mutations in KRAS, TP53, and SMAD4 are frequent in pancreatic cancer and are believed to be key drivers of disease pathophysiology in pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic cancer type. KRAS and TP53 mutations have been reported to influence the tumor immune microenvironment (TIME), promoting pro-tumorigenic capabilities of the tumor microenvironment’s myeloid populations. In this study, we aim to investigate the role of cancer cell SMAD4 mutations in shaping the PDAC TIME. Findings from this study could identify novel mechanisms leveraged by cancer cells to subvert anti-tumor immunity which could then be targeted for developing future therapeutics.
Poster #19:
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;³3Department 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 #20:
Characterizing the role of acetylcholine in pancreatic cancer liver metastases
Xiaotong Long¹ ², Linlin Zhao³, Christine Chio⁴, Tak Mak¹ ² ³ ⁵ *
¹Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada, ²Princess Margaret Cancer Centre, University Health Network, Toronto, Canada, ³Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China, ⁴Institute for Cancer Genetics, Department of Genetics and Development, Columbia University Irving Medical Center, New York, NY 10032, USA, ⁵Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, *Corresponding author
Pancreatic ductal carcinoma (PDAC) is a leading cause of cancer-associated death worldwide. The liver is the most common metastatic site for PDAC metastasis, with a 5-year survival rate of approximately 3%. Previous studies demonstrated that acetylcholine signaling protects against PDAC, and subdiaphragmatic denervation promoted PDAC liver metastases. Our lab revealed that cholinergic CD4⁺ T cells (which express choline acetyltransferase) play a protective role in hepatocellular carcinoma by maintaining immunosurveillance. Yet, the role of cholinergic immune cells in PDAC liver metastases remains unclear, and the impact on immune composition within the hepatic metastatic environment remains to be investigated. In this study, we aim to examine the effect of acetylcholine on cancer cells, and to investigate the role of cholinergic immune cells in PDAC and its metastases.
Poster #21:
Unraveling the landscape of cholinergic CD4+ T lymphocytes in hepatocellular carcinoma
Chunxing Zheng¹ ², Bryan E. Snow¹, Yin Tong² ³, Suet Yi Leung² ³, Tak W. Mak¹ ² ³ ⁴ *
Presenter: Nathan Ng²
¹Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada, ²Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China, ³Department of Pathology, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong SAR, China, ⁴Departments of Immunology and Medical Biophysics, University of Toronto, Toronto, Ontario, Canada, *Corresponding author
Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and the third leading cause of cancer-related deaths worldwide¹. CD4⁺ T cells play a vital role in antitumor immunity by differentiating into subsets such as regulatory T cells (Tregs) and effector T cells, which modulate immune responses within the tumor microenvironment². Isolating CD4⁺ T cells typically involves harvesting lymphoid tissues, mechanical dissociation, and magnetic-activated cell sorting (MACS) using CD4-specific antibodies³.
Recent research has highlighted the importance of cholinergic signaling in immune regulation, mediated by T cells expressing choline acetyltransferase (ChAT). ChAT-expressing T cells influence inflammatory responses and various immune functions⁴. Recent work demonstrated that T cells produce acetylcholine to modulate immune responses and inflammation, linking the nervous and immune systems⁵. This finding is significant for understanding immune regulation in cancer and other inflammatory diseases.
In this study, we investigated the role of cholinergic CD4⁺ T cells in HCC immunosurveillance by utilizing genetic and molecular techniques, including CRISPR/Cas9-mediated gene editing and single-cell RNA sequencing⁶. We aimed to elucidate how ChAT-expressing T cells contribute to antitumor immunity and the potential therapeutic implications of targeting this pathway in liver cancer.
~Published in: Nature Cancer, 2023 Oct;4(10):1437-1454.
Poster #22:
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 *
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
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 #23:
Development of 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 *, ^These authors contributed equally, *Co-corresponding authors
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 using droplet microfluidics for studying the cell-cell fusion process. We illustrate the efficacy of our system by examining the case of syncytia formation, a cell-cell fusion phenomenon triggered by the SARS-CoV-2 spike protein, which is commonly observed in patients who develop severe chronic respiratory symptoms. Our system effectively annotates the syncytium-forming potential of spike protein variants and identifies mutations that may cause serious pathological consequences. These identified mutations are of potential concern and warrant continued monitoring. Our high-throughput platform offers broad potential for studying cancer-related cell-cell fusion phenotypes.
~Published in: Nature Biomedical Engineering, 2024 Mar;8(3):291-309.
Poster #24:
Top variants identifications in mutagenesis screens via low-N sampling
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 *
¹Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China, ²Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China, ³School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China, ⁴Department of Statistics and Actuarial Science, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
Screening mutagenesis libraries is routine practice in protein engineering, but it often involves significant experimental resources spent on cloning and characterizing non-functional variants, which constitute the majority of the library. Therefore, we aimed to develop a more efficient strategy to maximize the identification of top-performing variants with minimal experimental effort across the vast combinatorial mutational landscape. Towards this goal, we have developed a streamlined machine learning-based approach that outperforms other state-of-the-art methods. Our strategy integrates zero-shot prediction with multi-round sampling and machine learning to prioritize the experimental characterization of the most promising variants in the library. We found that four rounds of low-N pick-and-validate sampling of 12 variants for machine learning yielded the best accuracy of up to 92.6% in selecting the true top 1% variants in combinatorial mutant libraries. Alternatively, two rounds of 24 variants was also found to be effective. Thus, we have successfully demonstrated our strategy by identifying high-performance protein variants from diverse families, including the CRISPR-based genome editors, supporting the potential for our approach to be generally applied for solving protein engineering tasks.
~Published in: Cell Systems, 2024 Feb 21;15(2):193-203.
Poster #25:
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 *
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
Selecting and engineering optimal base editors for precise genome editing applications is pivotal for achieving maximum efficiency and minimal undesired edits. We present a platform for creating and analyzing a library of base editor variants, enabling large-scale evaluation of their editing performance. Our comparison provides quantitative measures of each variant’s efficiency, purity, motif preference, and bias for generating single and multiple base conversions. Additionally, we revealed undesired higher indel rates and noncanonical base conversions in some existing editors. As the number of editor variants continues to expand, our platform addresses the need for an unbiased, scalable method to benchmark editing outcomes, accelerating the development of next-generation genome editor tools.
~Published in: Cell Systems, 2023 May 17;14(5):392-403.
Poster #26:
Size-exclusion-based genome-wide CRISPR screen identifies determinants for cell-cell fusion
Bei Wang¹ ²,Charles W.F. Chan¹ ², Xiner Huang³, Gigi C.G. Choi¹ ², Hin Chu³, Alan S.L. Wong¹ ²,*
¹Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China, ²Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Hong Kong SAR, China, ³State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Hong Kong SAR, China
Cell-cell fusion occurs in various pathological processes, such as fusion between tumor and normal somatic cells and enveloped viruses-induced cell fusion. Investigating the host factors crucial for these cell fusion events could offer potential strategies for combating associated diseases. In this study, we developed a size-exclusion-based selection strategy and coupled it with a genome-wide CRISPR knockout screen to systematically identify host factors essential for SARS-CoV-2 spike-mediated syncytium formation. In addition to the known receptor ACE2, we identified and validated two core regulators of clathrin-mediated endocytosis (CME), AP2M1 and FCHO2, as essential for spike-mediated syncytia formation. Furthermore, we confirmed that two FDA-approved CME-inhibiting drugs, chlorpromazine and fluvoxamine, can effectively inhibit SARS-CoV-2-induced syncytia formation and alleviate disease severity, as demonstrated through in vitro and hamster models.
~Published in: Nature Biomedical Engineering, 2024 Mar;8(3):291-309.
Poster Theme 6 : Bioinformatics Analysis
Poster #27:
Integrative analysis of long- and short-read RNA sequencing enables quantification of a cancer specific transposable element driven transcript
Sojung Lee¹ ², Jayne A Barbour², Yee M Tam², Haocheng Yang², Yuanhua Huang² ³, Jason WH Wong¹ ² *
¹Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China, ²School of Biomedical Sciences, The University of Hong Kong, Hong Kong SAR, China, ³Department of Statistics and Actuarial Science, The University of Hong Kong, Hong Kong SAR, China, *Corresponding author
With an ability to compromise genome integrity, endogenous transposable elements (TEs) have significant associations with human diseases. Short-read sequencing has been used to study the expression of TEs; however, the highly repetitive nature of these elements makes multimapping a critical issue. Here we implement LocusMasterTE, an improved quantification method by integrating long-read sequencing. Introducing computed transcript per million (TPM) counts from long-read sequencing as prior distribution during Expectation-Maximization (EM) model in short-read TE quantification, multi-mapped reads are re-assigned to correct expression values. Based on simulated short reads, LocusMasterTE outperforms current quantitative approaches and is significantly favourable in capturing newly inserted TEs. We also verified that TEs quantified by LocusMasterTE clearly related to euchromatins and heterochromatin in cell line samples. With LocusMasterTE, we anticipate that more accurate quantification can be performed, allowing novel functions of TEs to be uncovered.
~Published in: bioRxiv, 2023; https://doi.org/10.1101/2023.03.21.533716.
Poster #28:
Ganciclovir-induced mutations are present in a diverse spectrum of post-transplant malignancies
Hu Fang¹ ² ^, Helen HN Yan³ ⁴ ^, Rebecca A Bilardi⁵ ⁶ ^, Christoffer Flensburg⁵ ⁶, Haocheng Yang¹, Jayne A Barbour¹, Hoicheong Siu³, Michelle Turski⁷, Edward Chew⁵, Zhen Xu⁵ ⁶, Siu T Lam⁸, Rakesh Sharma⁸, Mengya Xu¹, Junshi Li¹, Ho W Ip⁹, Carol YM Cheung¹⁰, Michael SY Huen¹, E Alejandro Sweet-Cordero⁷, Ian J Majewski⁵ ⁶ *, Suet Y Leung³ ⁴ ⁸ ¹¹ *, Jason WH Wong¹ ⁴ ⁸ ¹¹ *
¹School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, ²Department of Research & Development, South China Hospital, Health Science Center, Shenzhen University, China, ³Department of Pathology, School of Clinical Medicine, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, ⁴Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China, ⁵The Walter and Eliza Hall Institute of Medical Research, ⁶University of Melbourne, Department of Medical Biology, ⁷Division of Hematology/Oncology, Department of Pediatrics, University of California San Francisco, ⁸Centre for PanorOmic Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China, ⁹Department of Pathology and ¹⁰Department of Medicine, Queen Mary Hospital, Hong Kong SAR, China, ¹¹The 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 authors
Ganciclovir (GCV) is an antiviral widely used in solid organ and allogeneic hematopoietic stem cell transplant patients for prophylaxis and treatment of cytomegalovirus. Although GCV has been shown to be mutagenic, its contribution to cancer incidence and other factors that influence its mutagenicity is unknown. By analysing cancer genomes in a cohort of 121,771 patients, we identified 22 patients of diverse cancer types showing evidence of GCV-associated mutagenesis. All patients with detailed clinical history available had previously undergone organ transplantation, and were prescribed GCV and the immunosuppressant, mycophenolate. In vitro testing in cell line and organoid models showed that mycophenolate, when used in combination with GCV, increased the misincorporation of GCV in genomic DNA and GCV-induced DNA mutations. Our study provides strong evidence that GCV contributes to carcinogenesis in a broad range of tissues.
~Published in: Genome Medicine, 2022 Oct 31;14(1):124.
Poster #29:
Investigating anchorage independent growth of colorectal adenoma organoids using multiomics profiling
Jonathan W.T. Tse¹ ², 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, *Corresponding author
In this study, we examined the anchorage independent growth of colorectal adenoma organoids, a critical factor in tumor metastasis. Using multiomics profiling, we investigated the potential drivers of this phenomenon. We utilized colony and spheroid formation assays to mimic anchorage independence, with successful formation used as a proxy for this trait. The hierarchical structure of the samples, derived from the same pooled organoids of an individual, provided insight into the evolutionary pressures contributing to anchorage independence. We performed comprehensive multiomics analysis, combining whole genome sequencing, whole exome sequencing, RNA-sequencing, and DNA-methylation data. To avoid false positive associations due to relatedness, a kinship matrix, based on standardized genotypes, was incorporated to capture unobserved factors. Various pipelines and tools were utilized to identify chromosomal aberrations that might be contributing to the evolutionary pressures leading to anchorage independence. Our study aims to broaden the understanding of metastasis drivers, identify potential biomarkers for targeted therapy, and improve prognosis for patients with metastasis by investigating the development of anchorage independence in benign tumors.
Poster Theme 7 : Medicinal Chemistry
Poster #30:
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, P. R. 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 faster reaction kinetics than conventional probes and enabled identification of more and unique reactive cysteine as compared to the reported cysteine profiling experiments. We successfully utilized NAIA to identified covalent ligand which can modulate cell cycle, implying the applicability of NAIA in covalent drug screening.
~Published in: Nature Communications, 2023 Jun 15;14(1):3564.
Poster #31:
Development of superoxide-responsive probes for chemoproteomic investigation of superoxide-mediated signaling
Hinyuk Lai¹ ² and Clive Yik-Sham Chung¹ 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
Superoxide is a reactive oxygen species (ROS) with complex roles in biological systems. It can contribute to the development of serious diseases, from aging to cancers and neurodegenerative disorders. However, it can also serve as a signaling molecule for important life processes. Monitoring superoxide levels and identifying proteins regulated by superoxide are crucial to enhancing our understanding of this growing field of redox biology and signaling. Given the high reactivity and very short lifetime of superoxide compared to other ROS in biological systems, proteins redox-modified by superoxide should be in close proximity to where superoxide is generated endogenously, i.e. the superoxide hotspots. This inspires us to develop superoxide-specific quinone methide-based precursors, QMP-SOs, for proximity labeling of proteins within/near superoxide hotspots to image superoxide and profile proteins associated with superoxide biology by chemoproteomics. QMP-SOs specifically react with superoxide to generate electrophilic quinone methide intermediate, which subsequently reacts with nucleophilic amino acids to induce a covalent tag on proteins, as revealed by LC-MS and shotgun proteomics experiments. The alkyne handle on the covalent tag enables installation of fluorophore onto the tagged proteins for fluorescence imaging of superoxide in cells under oxidative stress. Through establishing a chemoproteomics platform, QMP-SO-TMT, we identify DJ-1 and DLDH as proteins associated with superoxide biology in liver cancer cells treated with menadione. This work should provide insights into the crosstalk between essential cellular events and superoxide redox biology, as well as the design principles of quinone methide-based probes to study redox biology through proximity labeling and chemoproteomics.
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