Session: 621. Lymphomas: Translational – Molecular and Genetic: Poster III
Hematology Disease Topics & Pathways:
Research, Adult, Lymphoid Leukemias, Elderly, Translational Research, Bioinformatics, LGL, Diseases, Lymphoid Malignancies, Computational biology, Biological Processes, Technology and Procedures, Molecular biology, Study Population, Human, Omics technologies
Methods: We generated a PBMC CITE-seq dataset obtained from four healthy donors and six NK-LGLL patients stratified into three subtypes based on mutations in TET2 (n=2), STAT3 (n=2), or CCL22 (n=2). Cell types were identified using cell surface marker proteins and known cell type-specific gene markers, and these identifications were used for all downstream analyses.
Results: Malignant NK cells were clearly identified by the higher expression of CD16 cell surface marker and exhibited upregulation of granzymes (B, H, M, A), perforin, and NK cytotoxicity and activation genes compared to other cell types. However, inhibitory receptors (PD1, TIM3) and several exhaustion markers were also upregulated, suggesting negative regulation of these chronically activated cells. Non-leukemic NK cells in patients were more cytotoxic and exhausted than those from healthy controls, based on higher module scores calculated from gene expression of relevant markers, which mirrors findings of the non-leukemic compartment in T-cell LGLL patients. Restricting comparisons to patient cells, we found that malignant cells downregulated cytokine-cytokine receptor interaction genes compared to CD56bright (e.g., TNFRSF11A, IL6R, TNFRSF18, CCR7) and CD56dim (e.g., TNFRSF4, CXCR2, TNFRSF18) normal NK cells. Among the most downregulated genes in malignant cells were several members of the TNF superfamily with a role in signaling pathways that modulate immune response.
We also compared features of the three molecular subtypes. Malignant NK cells in TET2 mutated samples exhibited increased expression of CD57, decreased CD56 and CD94 expression, and several down regulated genes involved in cell adhesion (IL32, IL7R and TIGIT). In CCL22 mutated samples, malignant cells exhibited increased CD57, CD56, and CD94 expression and upregulated genes involved in leukocyte cell-cell adhesion and chemotaxis, consistent with findings that CCL22 mutations lead to biased G-protein-coupled receptor signaling and dysregulation of microenvironmental crosstalk. Malignant cells in STAT3 mutated samples showed the lowest expression of CD57, CD56, and CD94 and upregulated genes involved in cell killing and leukocyte activation. Consistent with these findings, STAT3 mutated malignant cells were the most cytotoxic and exhausted, followed by malignant cells in TET2, then CCL22 mutated patients.
Hierarchical clustering of cell type-specific regulons identified CD8+ TEMRA cells as the closest normal cell type to malignant NK cells. The TBX21 regulon was specific to both malignant NK and healthy CD8+ TEMRA cells. Several target genes of TBX21 associated with immune regulation and cytotoxicity were upregulated in malignant cells compared to CD8+ TEMRA cells, including IL32, LAG3, S1PR5, CXCR1, and NKG7. Ligand-receptor (LR) analysis determined that malignant NK cells interact primarily with monocytes and dendritic cells through MHC-II, CD99, and MIF pathways. We also found enhanced cell-cell communication in CCL22 mutated samples, with malignant NK cells predominantly interacting with non-malignant CD56bright NK cells through CCL, CD99, and MHC-I signaling pathways.
Conclusions: We showed that malignant NK cells exhibited unique gene expression profiles with high cytotoxicity but also signs of negative regulation and exhaustion. Non-leukemic NK cells in patients were more cytotoxic and exhausted compared to healthy individuals. We identified mutation-specific gene expression profiles in malignant cells and found CD8+ TEMRA cells as the closest normal cell type. This initial analysis identified several unique features of malignant NK cells that warrant further investigation.
Disclosures: Loughran: Dren Bio, Inc.: Consultancy, Current equity holder in private company, Current holder of stock options in a privately-held company; Recludix Pharma, Inc.: Consultancy, Current equity holder in private company, Current holder of stock options in a privately-held company; Prime Genomics: Consultancy, Current equity holder in private company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Kymera Therapeutics, Inc.: Consultancy, Current equity holder in private company, Current holder of stock options in a privately-held company; Keystone Nano, Inc.: Consultancy, Current equity holder in private company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Nouveau Biosciences LLC: Consultancy, Current equity holder in private company, Current holder of stock options in a privately-held company; CanceRX Foundation, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Flagship Pioneering: Consultancy, Current equity holder in private company, Current holder of stock options in a privately-held company. Feith: AstraZeneca: Research Funding; Dren Bio: Consultancy, Current holder of stock options in a privately-held company, Research Funding; Recludix Pharma: Research Funding; Kymera Therapeutics: Honoraria, Research Funding. Ratan: AstraZeneca: Research Funding; MedGenome: Consultancy.
See more of: Oral and Poster Abstracts