Qi-Jing Li's research while affiliated with Institute of Molecular and Cell Biology and other places

SifiNet is a robust and accurate computational pipeline for identifying distinct gene sets, extracting and annotating cellular subpopulations, and elucidating intrinsic relationships among these subpopulations. Uniquely, SifiNet bypasses the cell clustering stage, commonly integrated into other cellular annotation pipelines, thereby circumventing potential inaccuracies in clustering that may compromise subsequent analyses. Consequently, SifiNet has demonstrated superior performance in multiple experimental datasets compared with other state-of-the-art methods. SifiNet can analyze both single-cell RNA and ATAC sequencing data, thereby rendering comprehensive multi-omic cellular profiles. It is conveniently available as an open-source R package.

Melanoma cells, deriving from neuroectodermal melanocytes, may exploit the nervous system’s immune privilege for growth. Here we show that nerve growth factor (NGF) has both melanoma cell intrinsic and extrinsic immunosuppressive functions. Autocrine NGF engages tropomyosin receptor kinase A (TrkA) on melanoma cells to desensitize interferon γ signaling, leading to T and natural killer cell exclusion. In effector T cells that upregulate surface TrkA expression upon T cell receptor activation, paracrine NGF dampens T cell receptor signaling and effector function. Inhibiting NGF, either through genetic modification or with the tropomyosin receptor kinase inhibitor larotrectinib, renders melanomas susceptible to immune checkpoint blockade therapy and fosters long-term immunity by activating memory T cells with low affinity. These results identify the NGF–TrkA axis as an important suppressor of anti-tumor immunity and suggest larotrectinib might be repurposed for immune sensitization. Moreover, by enlisting low-affinity T cells, anti-NGF reduces acquired resistance to immune checkpoint blockade and prevents melanoma recurrence.

Necroptosis is an inflammatory form of cell suicide that critically depends on the kinase activity of Receptor Interacting Protein Kinase 3 (RIPK3). Previous studies showed that immunization with necroptotic cells conferred protection against subsequent tumor challenge. Since RIPK3 can also promote apoptosis and NF-κB-dependent inflammation, it remains difficult to determine the contribution of necroptosis-associated release of damage-associated molecular patterns (DAMPs) in anti-tumor immunity. Here, we describe a system that allows us to selectively induce RIPK3-dependent necroptosis or apoptosis with minimal NF-κB-dependent inflammatory cytokine expression. In a syngeneic tumor challenge model, immunization with necroptotic cells conferred superior protection against subsequent tumor challenge. Surprisingly, this protective effect required CD4 ⁺ T cells rather than CD8 ⁺ T cells and is dependent on host type I interferon signaling. Our results provide evidence that death-dependent type I interferon production following necroptosis is sufficient to elicit protective anti-tumor immunity.

The essential branched‐chain amino acids (BCAAs) leucine, isoleucine, and valine play critical roles in protein synthesis and energy metabolism. Despite their widespread use as nutritional supplements, BCAAs’ full effects on mammalian physiology remain uncertain due to the complexities of BCAA metabolic regulation. Here a novel mechanism linking intrinsic alterations in BCAA metabolism is identified to cellular senescence and the senescence‐associated secretory phenotype (SASP), both of which contribute to organismal aging and inflammation‐related diseases. Altered BCAA metabolism driving the SASP is mediated by robust activation of the BCAA transporters Solute Carrier Family 6 Members 14 and 15 as well as downregulation of the catabolic enzyme BCAA transaminase 1 during onset of cellular senescence, leading to highly elevated intracellular BCAA levels in senescent cells. This, in turn, activates the mammalian target of rapamycin complex 1 (mTORC1) to establish the full SASP program. Transgenic Drosophila models further indicate that orthologous BCAA regulators are involved in the induction of cellular senescence and age‐related phenotypes in flies, suggesting evolutionary conservation of this metabolic pathway during aging. Finally, experimentally blocking BCAA accumulation attenuates the inflammatory response in a mouse senescence model, highlighting the therapeutic potential of modulating BCAA metabolism for the treatment of age‐related and inflammatory diseases.

N6-methyladenosine (m6A) RNA modification plays important roles in the governance of gene expression and is temporally regulated in different cell states. In contrast to global m6A profiling in bulk sequencing, single-cell technologies for analyzing m6A heterogeneity are not extensively established. Here, we developed single-nucleus m6A-CUT&Tag (sn-m6A-CT) for simultaneous profiling of m6A methylomes and transcriptomes within a single nucleus using mouse embryonic stem cells (mESCs). m6A-CT is capable of enriching m6A-marked RNA molecules in situ, without isolating RNAs from cells. We adapted m6A-CT to the droplet-based single-cell omics platform and demonstrated high-throughput performance in analyzing nuclei isolated from thousands of cells from various cell types. We show that sn-m6A-CT profiling is sufficient to determine cell identity and allows the generation of cell-type-specific m6A methylome landscapes from heterogeneous populations. These indicate that sn-m6A-CT provides additional dimensions to multimodal datasets and insights into epitranscriptomic landscape in defining cell fate identity and states.

2052 Background: Diffuse intrinsic pontine glioma (DIPG) harboring H3.3-K27M mutation is a malignant pediatric brain tumor with a >90% mortality rate within two-year of diagnosis. Current therapeutic options for DIPG are limited. Aiming to improve therapeutic outcomes, we herein report the preliminary findings of a phase I trial studying a neoantigen peptide vaccine targeting H3.3-K27M. Methods: ENACTING is an open-label, single center, two-armed phase 1 trial to assess the safety and T cell immunity of a neoantigen peptide vaccine against H3.3-K27M. Patients aged ≥ 5 years old with newly diagnosed DIPG were consented and screened. HLA-A*02+/H3.3-K27M+ patients were enrolled to a two-arm study: Arm A consists of subjects receiving open debulking surgery, and Arm B consists of subjects without surgery eligibilities who received stereotactic biopsy. All patients subsequently received conformal radiotherapy and neoantigen vaccine treatment designed to elicit both CD4+ and CD8+ T cell immune response. Vaccine was administered intramuscularly in combination with polyinosinic-polycytidylic acid-poly-L-Iysine carboxymethylcellulose (Poly-ICLC). The primary objective is to evaluate the safety (AEs graded by CTCAE v4.03) and survival outcomes. Secondary objectives include maximum tolerated dose (MTD) and immunological responses. Results: As of Jan 2023, 11 patients have been treated, with 7 in Arm A and 4 in Arm B. No grade 3-4 treatment-related adverse events have been observed, with fever (81.9%) and injection site pain (54.5%) being the most common AEs. Among 10 efficacy-assessable patients, median progression-free survival (mPFS) and median overall survival (mOS) were 11.4 months (95% CI: 5.8~14.7) and 15.4 months (95% CI: 7.53~not reached), respectively. One-year OS rate was 66.7% (95% CI: 42~100%). One patient was assessed as complete response (CR). T cell responses against neoantigen were detected and H3.3-K27M mutation-specific CD4 ⁺ and CD8 ⁺ TCR clones were validated. Conclusions: The H3.3-K27M neoantigen vaccine was well tolerated. Initial results from this ongoing study suggest that, compared with other current therapies against DIPG, H3.3-K27M peptide vaccination may provide superior patient survival outcomes. Clinical trial information: NCT04749641 . [Table: see text]

6047 Background: Nasopharyngeal carcinoma (NPC) refraction and metastasis is common, but therapeutics are limited. As adoptive immunotherapy has emerged as effective against other cancers, engineered T cells bearing a transgenic Epstein-Barr virus (EBV)-specific TCR (TCR-T) represent a viable approach to treat EBV-associated NPC. Given PD-1/PD-L1 induced T cell hypofunction, we herein report preliminary findings of a phase I trial of EBV-targeting TCR-T cells armored with secreted PD-1 blockade for patients who failed in two or more lines of standard therapies. Methods: Patients with advanced NPCs were consented and screened for EBV serotype and HLA haplotype. EBV+/HLA-A*02+ patients were enrolled in a rapid titration setting to escalate the single infusion dose from 5x10 ⁶ to 1.0x10 ⁷ and 5.0x10 ⁷ /kg TCR-T cells. After safety evaluation, the 5.0X10 ⁷ /kg cohort was expanded for further investigation of combining with IL-2 administration. Fludarabine/cyclophosphamide were administered prior to TCR-T cell transfer as pre-conditioning. Patient monitoring and peripheral blood analysis occurred weekly over the first month and then monthly until disease progression or patient withdrawal. The primary objective was to determine safety and a recommended phase 2 dose (RP2D), while the secondary objective was investigator assessed ORR (RECIST v1.1). Results: One patient per TCR-T dose was treated, and, as no dose-limiting toxicity (DLT) has been observed, dose level 3 (5.0x10 ⁷ /kg) was expanded with IL-2 administration upon TCR-T cell infusion. As of January 2023, six patients have been treated. One patient (16.7%) exhibited grade 1 CRS (n=6). No grade 4 treatment-related adverse events (TRAEs) have been observed, with leukopenia and fever being the most common AEs. Two patients (33.3%) were assessed as partial response (PR), with one reaching a response duration of 9 months to date. Three patients (50%) were assessed as stable disease (SD). For all patients, pharmacokinetic analysis revealed that levels of TCR-T cells in peripheral blood peak between 3- and 14-days post-infusion, with a maximum duration of 180 days. Conclusions: EBV-targeting TCR-T cells armored with PD-1 blockade are well tolerated. Initial results from this ongoing study indicate that EBV proteins may be safe and effective TCR-T targets to achieve superior outcomes in advanced EBV-positive NPC patients. Clinical trial information: NCT04139057 . [Table: see text]

Single-cell sequencing has provided a means of quantifying cellular omic phenotypes. Identifying cell-type-specific feature genes is a crucial aspect of understanding cellular heterogeneity. Over the past decade, many methods have been developed to identify feature genes; however, these methods either depend on dubious cell clustering or fail to provide subpopulation-specific markers. We introduce SifiNet, a robust and accurate approach for identifying marker gene sets based on gene co-expression network topology. The identified gene sets facilitate the calculation of cellular gene set enrichment scores and cell annotation, and can reveal potential transitional relationships between cell subpopulations. SifiNet outperforms state-of-the-art methods in marker gene set identification and cell-type annotation accuracy. It is applicable to both single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) data. We have applied SifiNet to various experimental studies, successfully identifying novel gene markers, annotating cells with complex heterogeneity, and uncovering intriguing cell developmental trajectories.

Cellular senescence is a stress-induced, stable cell cycle arrest phenotype which generates a pro-inflammatory microenvironment, leading to chronic inflammation and age-associated diseases. Determining the fundamental molecular pathways driving senescence instead of apoptosis could enable the identification of senolytic agents to restore tissue homeostasis. Here, we identify thrombomodulin (THBD) signaling as a key molecular determinant of the senescent cell fate. Although normally restricted to endothelial cells, THBD is rapidly upregulated and maintained throughout all phases of the senescence program in aged mammalian tissues and in senescent cell models. Mechanistically, THBD activates a proteolytic feed-forward signaling pathway by stabilizing a multi-protein complex in early endosomes, thus forming a molecular basis for the irreversibility of the senescence program and ensuring senescent cell viability. Therapeutically, THBD signaling depletion or inhibition using vorapaxar, an FDA-approved drug, effectively ablates senescent cells and restores tissue homeostasis in liver fibrosis models. Collectively, these results uncover proteolytic THBD signaling as a conserved pro-survival pathway essential for senescent cell viability, thus providing a pharmacologically exploitable senolytic target for senescence-associated diseases.

Background: Diffuse intrinsic pontine glioma (DIPG) harboring H3.3-K27M mutation is a malignant pediatric brain tumor with a >90% mortality rate within two years of diagnosis. Aiming to improve therapeutic outcomes, we herein describe a neoantigen peptide vaccine against H3.3-K27M which effectively triggers both CD8+ and CD4+ T cell responses. Methods: A neoantigen vaccine was designed to trigger T cell immunity against DIPGs harboring the H3.3-K27M mutation. ENACTING (NCT04749641) was then initiated as an open-label, single center, two-armed phase 1 trial to assess T cell immune responses and vaccine safety. The vaccine was administered intramuscularly with poly-ICLC adjuvant until tumor progression or untolerated toxicity. PBMCs before and after each vaccine treatment were collected for TCR repertoire analysis and immune response assessment. Results: As of November 2022, 10 patients have been treated. No grade 3-4 treatment-related adverse events have been observed, with fever (80%) and injection site pain (60%) being the most common AEs. On a per patient basis, vaccines induce a landscape change of TCR repertoire in patients’ PBMC after 4-6 times of dosing, indicating multiple dosing is required to trigger extensive T cell responses. T cell responses against neoantigens were detected and H3.3-K27M mutation-specific CD4+ and two CD8+ clones were validated. Among 9 efficacy-assessable patients, the one-year overall survival rate was 71.4%. The mPFS has reached 11.7 months and increasing. One patient reached complete response. As this trial remains ongoing, subgroup analysis will be reported in the future. Conclusion: The H3.3-K27M neoantigen vaccine was well tolerated and elicited mutation-specific CD4+ and CD8+ T cell responses in patients. Initial results from this ongoing study suggest that, compared with other current immunotherapies against DIPG, H3.3-K27M peptide vaccination may provide superior patient outcomes, for both life qualities and survival outcomes. Table 1. Clinical efficacy and adverse events Efficacy Complete response (CR) 1 (11.1%) Partial response (PR) 0 (0) Stable disease (SD) 8 (88.9%) Progressive disease (PD) 0 (0) Disease control rate (DCR) 100% 12-month overall survival 71.4% Median progression-free survival (mPFS) 11.7 months (95% CI, 7.0-NR) Median overall survival (mOS) 15.7 months (95% CI, 10.0-NR) Treatment-Related Adverse Events All grades Grade 3 Grade 4 Fever 9 (90.0%) 0 0 Injection site pain 6 (60.0%) 0 0 Bloating 1 (10.0%) 0 0 Abdominal pain 1 (10.0%) 0 0 Vomiting 1 (10.0%) 0 0 Increased blood LDH 1 (10.0%) 0 0 Proteinuria 1 (10.0%) 0 0 Hypocalcemia 1 (10.0%) 0 0 Citation Format: Yang Zhang, Nan Ji, Gang Chen, Haiyang Wu, Yi Wang, Xiao’ou Li, Wei Xu, Ling Peng, Tian Li, Yi Wang, Li-Feng Zhang, Shengjun Sun, Xiaobing Zhao, Si Li, Peter Alexander, Liwei Zhang, Qi-Jing Li. H3.3-K27M neoantigen vaccine elicits CD4+ and CD8+ T cells immunity and improved prognosis against diffuse intrinsic pontine glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT086.