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4132 Activating STAT3 Mutations Drive CD38 Expression in T-Large Granular Lymphocytic Leukemia

Program: Oral and Poster Abstracts
Session: 603. Lymphoid Oncogenesis: Basic: Poster III
Hematology Disease Topics & Pathways:
Lymphoid Leukemias, Research, Translational Research, LGL, Diseases, Lymphoid Malignancies, Biological Processes, Gene editing, Technology and Procedures, Pathogenesis
Monday, December 9, 2024, 6:00 PM-8:00 PM

Giulia Calabretto1,2,3*, Elena Buson3*, Zachary Hutchins1*, Colleen Isabelle4, Sydnee Scozzafava1*, Valentina Trimarco2*, Barbara Mariotti5*, Rachel Burzinski1*, Amy Boles1*, Elisa Rampazzo2,3*, Antonella Teramo2,3*, Sabrina Manni2,3*, Monica Facco2,3*, Gregorio Barilà6*, Flavia Bazzoni5*, Gianpietro Semenzato3*, Livio Trentin2, Jonathan E. Brammer7, Renato Zambello2,3* and Anjali Mishra1

1Thomas Jefferson University, Sydney Kimmel Cancer Center, Philadelphia, PA
2Department of Medicine, Hematology and Clinical Immunology section, Padua University School of Medicine, Padova, Italy
3Veneto Institute of Molecular Medicine (VIMM), Padova, Italy
4Thomas Jefferson University, Philadelphia, PA
5Department of Medicine, Division of General Pathology, University of Verona, Verona, Italy
6Hematology Unit, Ospedale San Bortolo, Vicenza, Italy
7Division of Hematology, The Ohio State University, Columbus, OH

The tumor-specific expression of CD38 is a crucial prognostic marker and a key therapeutic target in various hematologic malignancies. However, the regulatory mechanisms of CD38 expression in malignant cells are not well understood. This study identifies a cohort of T-cell large granular lymphocytic leukemia (T-LGLL) patients where CD38 expression correlates with STAT3 mutations. T-LGLL, a rare and incurable disease, involves the clonal proliferation of mature cytotoxic T-cells. STAT3 gain-of-function mutations (STAT3MUT), found in 40-70% of patients, result in constitutively active STAT3 proteins with pro-oncogenic properties. Our flow cytometry analysis reveals that CD38 expression in gated T-large granular lymphocytes (T-LGLs) from STAT3MUT T-LGLL patients (18/45, 40%) is significantly higher compared to STAT3 wild-type (STAT3WT) T-LGLL (n=27) and healthy donors (HD, n=10). Specifically, mutated patients showed a markedly increased percentage of CD38+ cells (STAT3MUT: 69±22%; STAT3WT: 12±15%; HD: 21±8%, p<0.001) and an elevated CD38 mean fluorescence intensity (MFI) (STAT3MUT: 839±440; STAT3WT: 179±227; HD: 289±116, p<0.001). Notably, T-LGLL patients which exhibited higher CD38 expression also experienced a severe disease progression compared to the indolent clinical course observed in CD38dim/- cases. To investigate the role of STAT3 in regulating CD38 expression, we performed chromatin immunoprecipitation (ChIP) sequencing, which revealed increased STAT3 binding at the CD38 gene locus in STAT3MUT T-LGLL patients compared to those with STAT3WT (n=12 and 7, respectively). In addition, ChIP-qPCR demonstrated an increased H3K27Acetylation, a marker of active transcription, at the CD38 promoter in STAT3MUT T-LGLL patient [STAT3MUT (n=5): 0,9±0,4; STAT3WT (n=10): 0,4±0,2; p<0.01)]. These findings were further substantiated at the transcript level, showing a significant 5-fold increase (p<0.01) of CD38 mRNA level in STAT3MUT T-LGLL patients. Our results indicate that STAT3 directly influences CD38 transcription, providing a mechanistic link between aberrant STAT3 activation and elevated CD38 expression in T-LGLL patients. To further evaluate the role of STAT3 in regulating CD38 expression, we utilized the MOTN-1 human T-LGLL cell line with STAT3WT and generated an isogenic cell line model harboring one of the most frequently observed STAT3 mutation (D661Y) found in patients. This was achieved using a CRISPR/Cas9-mediated homology-directed repair editing strategy. The resulting cell line exhibited constitutively active STAT3 and displayed a higher CD38 mRNA level and MFI compared to the WT counterpart, underscoring the impact of the STAT3 mutation on CD38 expression. Both cell lines were transduced with a firefly luciferase–GFP lentivirus for efficient in vivo tracking and then transplanted into immunodeficient Rag−/−γc−/− mouse models, representing either STAT3WT or STAT3MUT. This setup was used to assess the potential of targeting T-LGLL with the anti-CD38 mAb daratumumab. After confirming cell engraftment via bioluminescence imaging, mice were randomized into vehicle and daratumumab treatment groups. The T-LGLL cell burden resulted lower in daratumumab-treated mice compared to vehicle-treated controls, as measured by IVIS imaging. Notably, the tumor reduction in STAT3WT T-LGLL was less pronounced (1.8-fold decrease, n=4 each, p=0.23) compared to the significant reduction observed in mutant T-LGLL (2.64-fold decrease, n=4 each, p=0.04). This indicates the effectiveness of daratumumab in inhibiting tumor growth in STAT3 mutant T-LGLL model. In summary, our work provides new insight on the oncogenic role of STAT3 in regulating CD38 expression in T-LGLL and suggests that immune-targeting of CD38 could serve as a promising new therapeutic strategy for T-LGLL patients.

Disclosures: Brammer: Incyte: Other: Trial Support, Research Funding; Secura Bio, INc.: Consultancy. Mishra: TEVA: Research Funding; ONO: Research Funding.

*signifies non-member of ASH