Gefitinib targets ZAP-70-expressing chronic lymphocytic leukemia cells and inhibits B-cell receptor signaling

Chronic lymphocytic leukemia (CLL) can be divided into groups based on biomarkers of poor prognosis. The expression of the tyrosine kinase ZAP-70 (member of the Syk tyrosine kinase family) in CLL cells is associated with shorter overall survival in CLL patients. Currently, there is a lack of targeted therapies for patients with ZAP-70 expression in CLL cells. The tyrosine kinase inhibitor gefitinib has been shown to be effective at induce apoptosis in acute myeloid leukemia through inhibition of Syk. In this study, we sought to test the efficacy of gefitinib in primary human ZAP-70+ CLL cells. We demonstrate that gefitinib preferentially induces cell death in ZAP-70-expressing CLL cells with a median IC₅₀ of 4.5 μM. In addition, gefitinib decreases the viability of ZAP-70+ Jurkat T leukemia cells but fails to affect T cells from CLL patients. Western blot analysis shows gefitinib reduces both basal and B-cell receptor (BCR)-stimulated phosphorylation of Syk/ZAP-70, ERK, and Akt in ZAP-70+ CLL cells. Moreover, gefitinib inhibits the pro-survival response from BCR stimulation and decreases pro-survival proteins such as Mcl-1. Finally, ZAP-70 expression sensitizes Raji cells to gefitinib treatment. These results demonstrate that gefitinib specifically targets ZAP-70+ CLL cells and inhibits the BCR cell survival pathway leading to apoptosis. This represents the likelihood of tyrosine kinase inhibitors being effective targeted treatments for ZAP-70+ CLL cells.The clinical course of chronic lymphocytic leukemia (CLL) is highly variable, and although some patients are treated at diagnosis, others may not require therapy for years.¹ Biomarkers can help stratify these patients into indolent and aggressive disease categories. The aggressiveness of CLL is dependent on whether the leukemia cells have (60% of CLL population) or lack (40% of CLL population) mutations of the immunoglobulin variable region of the heavy chain (IgV_H). Thus, patients with early-stage disease have a median survival of 8 years if they have unmutated IgV_H (Un-IgV_H) and 24 years if they have mutated IgV_H (Mu-IgV_H) disease.² A surrogate marker for IgV_H mutational status is the expression of zeta-chain-associated protein 70 (ZAP-70); IgV_H mutated CLL cells are frequently ZAP-70 negative, whereas IgV_H unmutated cells are more typically ZAP-70 positive.³ ZAP-70 staining in CLL is not an all-or-nothing phenomenon, and to maximize the correlation with IgV_H mutational status, a ZAP-70-positive case is defined as ≥20% of the CLL cells staining for ZAP-70. Like IgV_H status, overexpression of ZAP-70 in CLL cells is associated with aggressive disease; time to treatment is 2.6 years for ZAP-70+ patients compared with 8 years for ZAP-70− patients independent of Rai stage.³ Thus, ZAP-70 is a rationale target for therapy in CLL.Although the clinical relevance of ZAP-70 in CLL is well known, its molecular function is less understood. ZAP-70 is a member of the Syk family of protein tyrosine kinases and is normally involved in signal transduction of the T-cell receptor in T cells. ZAP-70 overexpression in malignant B cells, such as CLL cells, enhances the B-cell receptor (BCR) pathway. This pathway is a key mechanism for cell survival in CLL.^4,5 Upon activation of the BCR, tyrosine kinase Lyn phosphorylates and activates Syk, leading to activation of downstream signaling pathways and upregulation of anti-apoptotic proteins, such as Mcl-1. CLL cells with both Un-IgV_H and high ZAP-70 expression show increased activation of proteins downstream of the BCR such as Akt, mitogen-activated protein kinase (MAPK), and NF-κB.^4,6,7 This suggests that alterations in the BCR signaling pathway through increased expression of the tyrosine kinase ZAP-70 are important in CLL disease progression.Gefitinib is a tyrosine kinase inhibitor known for targeting the epidermal growth factor receptor (EGFR) and is used in the treatment of non-small-cell lung cancer and other cancers of epithelial origin.⁸ The drug is well tolerated, with rash and diarrhea being the only dose-limiting toxicities. Importantly to leukemias, it is not myelosuppressive.⁹ Apart from its effects on EGFR activity, gefitinib has shown activity against >20 other kinase targets, including Lyn and Syk.^10,11 Gefitinib has been shown activity in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and acute lymphocytic leukemia (ALL), inducing both differentiation and cell death in vitro.¹² These effects are associated with inhibition of Syk phosphorylation. Thus, although gefitinib is used to treat lung cancer by inhibiting EGFR, it has potential utility in the treatment of CLL patients with high expression of Syk family members that include ZAP-70.In this study we show that gefitinib selectively induces apoptosis in ZAP-70-expressing CLL cells, both when unstimulated and BCR activated. These effects are associated in both cases with a reduction in overall tyrosine phosphorylation and specific decreases in Lyn/Lck, Syk/ZAP-70, ERK1/2, and Akt phosphorylation. These changes produce a decreased expression of Mcl-1 and blocked anti-apoptotic signaling. Forced overexpression of ZAP-70 by lentiviral infection in the Raji B-cell line increases the sensitivity of the cells to gefitinib-induced apoptosis. However, normal T cells from CLL patients, which also express ZAP-70, are not affected by gefitinib. These results suggest that tyrosine kinase inhibitors such as gefitinib are a viable treatment option for ZAP-70+ CLL patients.