Type: Oral
Session: 621. Lymphomas: Translational – Molecular and Genetic: Molecular Profiling and Prognostic Biomarkers in Hodgkin and Non-Hodgkin Lymphomas
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Hodgkin lymphoma, Translational Research, Lymphomas, Diseases, Lymphoid Malignancies
Materials and methods: We performed targeted and whole exome sequencing of Hodgkin and Reed-Sternberg (HRS) cells (n=116) purified using laser capture microdissection and flow cytometry (FCM)-based cell sorting. For in vitro functional studies, we retrovirally expressed CSF2RB WT or mutants observed in CHL tumors into two CHL-derived cell lines (L428, U-HO1). Using these isogenic models, we characterized the full spectrum of domain-specific mutations and elucidated the role of CSF2RB alterations in oncogenic signaling. Moreover, to investigate spatial tumor microenvironment architecture, we performed imaging mass cytometry (IMC) on the exome/targeted sequencing cohort using a customized antibody panel.
Results: In 26/107 cases (24.3%), we identified recurrent CSF2RB mutations, which clustered in the cytoplasmic domain (exon 14). Mutations were categorized as missense (n=2), multiple-hit (n=3), and truncating (n=21) mutations. Functional characterization in the L428 model showed that STAT5 was hyperphosphorylated in cells expressing all truncating mutants observed in cases with CHL (p.S723*, p.E788*, p.Lys838*, p.Q853*, p.Q867*, and p.Gln885*) compared to WT cells in an IL-5-dependent manner. Focusing on the functional consequences of the most commonly observed hotspot, the CSF2RB-p.E788* frameshift mutation, we generated cells with a CSF2RB heterozygous genotype by 1:1 transfection of WT and mutant alleles (WT/p.E788*) in U-HO1. With IL-3 or IL-5 (but not GM-CSF) stimulation, we validated that pSTAT5 was markedly higher in WT/p.E788* compared to WT cells. Differential gene expression analysis by RNA-seq revealed up-regulation of common IL-2 signaling pathway molecules and cytokine gene expression signatures, including genes such as IL-13 and CCL17 (TARC) in p.E788* cells compared to WT cells in the L428 model. These findings were validated in the U-HO1 model with IL-3 or IL-5 stimulation. In accord with the gene expression results, we detected higher amounts of IL-13 and TARC by ELISA in the supernatant from cells expressing all truncating mutants compared with WT cells in the L428 model. Furthermore, these gain-of-function phenotypes could be reversed by a selective JAK2 inhibitor (pacritinib), suggesting deregulated cytokine-dependent JAK2-STAT5 signaling leads to increases in IL-13 and CCL17 protein expression. Next, we performed an in-vitro transwell T‐cell migration assay using peripheral blood mononuclear cells from healthy donors and found that supernatant from p.E788* cells significantly enhanced migration of CCR4+ T cells, which contains FOXP3+ Tregs, compared with supernatant from WT cells. Enhanced migration was completely blocked by an anti‐CCR4 antagonist. Finally, spatial analysis using IMC data confirmed the close proximity between CCL17+ HRS cells and CCR4+ T cells in primary CHL samples. In particular, we observed a significant enrichment of FOXP3+ Tregs in close proximity to HRS cells in the CSF2RB-mutation enriched molecular cluster.
Conclusion: CSF2RB mutations contribute to deregulated, cytokine-dependent, oncogenic signaling, leading to downstream IL-13 related cell-autonomous phenotypes and TARC related tumor microenvironment composition and functions. We predict these phenotypes will pinpoint therapeutically targetable vulnerabilities, and may provide precision therapeutic approaches based on genetic subtype definitions in CHL.
Disclosures: Takata: Abbvie: Honoraria; Jansen Pharmaceutical K.K: Honoraria; AstraZeneca: Honoraria. Savage: Bristol Myers Squibb: Consultancy, Research Funding; Seagen: Consultancy, Honoraria, Research Funding; Regeneron: Other: DSMC; AbbVie: Consultancy. Scott: Roche: Consultancy, Honoraria; Genmab: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; AstraZenenca: Consultancy, Honoraria; Veracyte: Consultancy, Honoraria; Roche/Genentech: Research Funding; Nanostring: Patents & Royalties: use of gene expresssion to subtype aggressive lymphoma. Steidl: Bristol Myers Squibb: Research Funding; Epizyme: Research Funding; EISAI: Consultancy; Seattle Genetics: Consultancy; AbbVie: Consultancy; Bayer: Consultancy; Trillium Therapeutics Inc: Research Funding.