Ibrutinib immunomodulation via CTLA4 downregulation in CLL

Chronic lymphocytic leukemia (CLL) is the most common lymphoid malignancy in the United States. It causes significant morbidity and mortality, largely through its suppressive effects on the immune system. Our group has previously demonstrated that CLL cells express CTLA4, an immune checkpoint molecule normally found on activated T cells. CTLA4 is brought to the leukemic cell surface after interaction with a CLL-reactive activated T cell and functions to decrease CD80 expression on neighboring cells, thereby decreasing T cell activation. Previous data indicated a decrease in CTLA4 expression on T cells in patients treated with ibrutinib or acalabrutinib, two irreversible Bruton’s Tyrosine Kinase (BTK) inhibitors. Here, we expand our understanding of CTLA4 regulation in CLL and demonstrate that ibrutinib treatment directly modulates CTLA4 expression on CLL cells while indirectly modulating T cell CTLA4 expression. In CLL cells, we demonstrate that in vitro treatment with ibrutinib suppresses CTLA4 at each stage of its expression - mRNA, total protein, and surface trafficking (mean reductions: mRNA 53%, p>.0001, n=18; total protein 36%, p=.044, n=10; surface protein 35%, p=.0006, n=10). In contrast, treatment with acalabrutinib (a BTK-specific inhibitor) decreases mRNA (mean reduction 44%, p=.006, n=14) while sparing protein expression and trafficking. This means that CTLA4 surface protein (and therefore CTLA4 function) is regulated by non-BTK “off-target” effects of ibrutinib, while transcription is at least partly BTK-dependent. Comparing patient samples obtained before treatment versus after five months on ibrutinib, we demonstrate that CTLA4 suppression does occur with in vivo treatment (mean reduction: 54% total, p=.047; 40% surface, p=.034; n=5). In contrast to its in vivo effects and direct suppression on CLL cells, in vitro ibrutinib treatment failed to suppress T cell expression of CTLA4. This indicates that the T cell CTLA4 modulation seen in treated patients is due to indirect effects and points to different regulatory mechanisms in T versus CLL cells. Previous published data indicates that IFNγ levels in CLL patients decrease with ibrutinib treatment – here, we show that IFNγ can stimulate CTLA4 expression on CLL cells (qPCR mean increase 80%, p=.012, n=10). This indicates a second mechanism of CTLA4 reduction in patients receiving this treatment, via loss of IFNγ stimulation. Overall, we demonstrate a new mechanism of immunomodulation by ibrutinib – decreased CTLA4 on CLL cells. We demonstrate a different regulatory mechanism driving CTLA4 expression in CLL cells vs. T cells, and show that the decrease in T cell CTLA4 expression during ibrutinib treatment occurs indirectly. While recent efforts have developed more selective BTK inhibitors in an effort to decrease medication side effects, these results indicate that off-target immunomodulatory effects may be an underappreciated benefit from ibrutinib treatment. Citation Format: Max Yano, Priscilla Do, Xiaokui Mo, Natarajan Muthusamy, John C. Byrd. Ibrutinib immunomodulation via CTLA4 downregulation in CLL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3959.