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4612 Exploration of the Specific Mechanism of EGR2 Hotspot Mutation Mediating the Upregulation of Galectin-1 Expression in the Progression of CLL Following Treatment with BTK Inhibitor

Program: Oral and Poster Abstracts
Session: 641. Chronic Lymphocytic Leukemia: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Lymphoid Leukemias, Fundamental Science, Apoptosis, CLL, CHIP, Genomics, Clinical Research, Diseases, Immunology, Real-world evidence, Lymphoid Malignancies, Biological Processes
Monday, December 9, 2024, 6:00 PM-8:00 PM

Xiao Lu, MD1*, Yeqin Sha, MD2*, Xinyu Song, MD3*, Luomengjia Dai, MD2*, Ziyuan Zhou, MD2*, Hanning Tang, MD2*, Yi Miao, MD1*, Shuchao Qin, MD PHD1*, Yi Xia, MD, PhD2*, Tonglu Qiu, MD1*, Lei Fan4*, Jianyong Li, MD5 and Huayuan Zhu6*

1Lymphoma Center, Department of Hematology, Jiangsu Province Hospital, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China
2Lymphoma Center, Department of Hematology, Jiangsu Province Hospital, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China
3School of Basic Medical Sciences of Nanjing Medical University, Nanjing, Jiangsu Province, China
4Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, China
5First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, China
6Pukou CLL Center, Pukou Division of Jiangsu Province Hospital, Nanjing, Jiangsu, China

Objective

To investigate the clinical and biological characteristics, mutation landscape of CLL/SLL patients harboring EGR2 mutation and its potential role in the progression of CLL treated with BTK inhibitors.

Methods

We retrospectively investigated the clinical and biological characteristics, mutation landscape of 353 CLL/SLL patients who received targeted next-generation sequencing (NGS) examination in the Department of Hematology, the First Affiliated Hospital of Nanjing Medical University. We explored the potential role of EGR2 hotspot mutation in the progression of CLL following treatment with BTK inhibitor using targeted NGS, scRNA-seq, flow cytometry, and cell co-culture, Western blotting, immunofluorescence staining CCK-8 and other multi-omics methods. Kaplan–Meier analysis was performed to construct survival curves and the analysis of variance was used to determine differences between groups. A significance level of p<0.05 was applied.

Results

353 patients with CLL/SLL in the Department of Hematology, the First Affiliated Hospital of Nanjing Medical University were included in this study and 204 of them were treated with BTK inhibitors (ibrutinib, zanubrutinib, orelabrutinib and LOXO-305). The median follow-up time for patients who had the TTFT over months was 90.3 months, and for patients treated with BTK inhibitor was 21.9 months.

EGR2 mutations were detected in 11.0% (39/353) of all patients. All EGR2 mutations were heterozygous somatic mutations and within the DNA-binding sites of the zinc-finger domains, predominantly affected codons E356K (74.4%, 29/39), H384N (12.8%, 5/39) and D411(12.8%, 5/39). Furthermore, it was associated with higher frequency of unmutated IGHV genes (p<0.001), Del (11q) (p<0.05), ATM mutation(p<0.01) and mutually exclusive with Del (13q) (p<0.001) and trisomy 12 (p<0.001). 62.9% (22/35) of patients at baseline (19 of whom had EGR2 p.E356K) had clonal mutations (adjusted VAF>45%). Patients with EGR2 mutations showed younger (median age: 46 vs 57 years, p<0.001), higher frequency of complex karyotype(CK≥3) (38.5% vs 14.4%, p<0.001), bulky lymphadenopathy (>5cm) (25.6% vs 8.3%,p=0.002), shorter TTFT (median, 12.9 vs 31.1 months, p<0.001), inferior PFS(median, 22.3 vs 54.3 months, p<0.001) and higher risk of developing Richter transformation(27.8% vs 2.2%, p<0.001) under BTK inhibitor, as compared with patients without EGR2 mutations.

We further investigated the potential role of EGR2 p.E356K. Primary CLL cells from EGR2 p.E356K mutaed patients and EGR2 p.E356K overexpressing MEC-1 cells presented with higher proliferation and more anti-apoptotic, even with treatment of different concentrations zanubrutinib and venetoclax (p<0.001). In addition, scRNA-seq data found that PI3K-AKT pathway, anti-apoptotic pathway and Galectins family member LGALS1were aberrational activated in EGR2 p.E356K overexpressing MEC-1 cells, which was verified by Western blotting.

Furthermore, scRNA-seq also showed a different T cell subset in patients with EGR2 p.E356K mutation, manifested as enrichment of naïve T, regulatory T and exhausting T cells, indicating a more suppressed T cell immune microenvironment. The interaction of the LGALS1-CD45 pathway significantly increased in patients with EGR2 p.E356K, which was more profoundly in samples collected post progression on BTKi. Further bioinformatics analysis indicated that EGR2 molecules might have a direct transcriptional regulatory effect on the expression of LGALS1 and we verified this by luciferase assay. OTX008, a Galectin-1 inhibitor, alone or combined with zanubrutinib or venetoclax, could significantly suppress the proliferation and induce cell apoptosis in EGR2 p.E356K overexpressing MEC-1 cells, partly by downregulating the activation of PI3K-AKT pathway.

Conclusions

CLL patients harboring the EGR2 p.E356K mutation exhibit enrichment of poor prognostic characteristics. The CLL EGR2 p.E356K mutation facilitated the expression of Galectin-1, promoting the proliferation and anti-apoptosis of CLL cells, and leading to a suppressed T cell immune microenvironment.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH