Activating STAT3 Mutations Drive CD38 Expression in T-Large Granular Lymphocytic Leukemia
Authors: Calabretto Giulia , Buson Elena , Hutchins Zachary , Isabelle Colleen , Scozzafava Sydnee , Trimarco Valentina , Mariotti Barbara , Burzinski Rachel , Boles Amy , Rampazzo Elisa , Teramo Antonella , Manni Sabrina , Facco Monica , Barilà Gregorio , Bazzoni Flavia , Semenzato Gianpietro , Trentin Livio , Brammer Jonathan E. , Zambello Renato , Mishra Anjali 
Published: December 2024

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&lt;0.001) and an elevated CD38 mean fluorescence intensity (MFI) (STAT3MUT: 839±440; STAT3WT: 179±227; HD: 289±116, p&lt;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&lt;0.01)]. These findings were further substantiated at the transcript level, showing a significant 5-fold increase (p&lt;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.