The Linker Protein GAS7 Negatively Regulates Pre-B Cell Differentiation and Amplifies Proliferation and Survival Signals in Acute Lymphoblastic Leukemia

Background:Growth arrest-specific gene 7 (Gas7) functions as an adaptor for SH2- and SH3-containing proteins, in particular in cells that undergo growth arrest. Gas7 is abundantly expressed in the brain and is involved in neuronal differentiation. Interestingly, MLL-GAS7 fusion molecules resulting from the t(11;17)(q23;p13) chromosomal translocation have been reported in treatment-related acute myeloid leukemia (AML; Megonigal et al., 2000) and in a pediatric acute lymphoblastic leukemia (ALL). While the function of MLL has been extensively studied, the role of its fusion partner GAS7 in normal hematopoiesis and leukemia has not been elucidated.

Results: Studying gene expression changes during normal B cell development, we identified Gas7 as the gene with the strongest relative increase at the pre-B cell receptor checkpoint. At the transition from IL7-dependent Fraction C’ to IL7-independent small resting pre-B cells (Fraction D), GAS7 mRNA levels were upregulated by >13-fold in both human and mouse B cell progenitors. Withdrawal of IL7 cytokine signaling and Cre-mediated conditional deletion of Stat5ab recapitulated the strong increase of GAS7 expression under cell culture conditions. These finding suggest that GAS7 is part of an adaptive response of differentiating pre-B cells to attenuation of cytokine/Stat5 signaling. Consistent with this scenario, we found that Gas7^-/-pre-B cells undergo accelerated differentiation, including spontaneous Ig κ light chain gene recombination and loss of Stat5-signaling. Conversely, overexpression of GAS7, reduced responsiveness of pre-B cells to normal differentiation stimuli. These findings suggest that the linker molecule GAS7 is a negative regulator of pre-B cell differentiation.

Likewise, we found that tyrosine kinase inhibitor treatment of human Ph⁺ ALL cells resulted in a strong increased of GAS7 expression, in parallel with loss of Stat5 function. To elucidate the function of Gas7 in B cell lineage leukemia, we transformed bone marrow pre-B cells from Gas7^-/- mice with BCR-ABL1. Gas7 deficient Ph⁺ ALL cells showed decreased proliferation with reduced S phase and increased apoptosis. In agreement with effects of Stat5 on the sensitivity of Ph⁺ ALL cells against tyrosine kinase inhibitors (TKIs), Gas7 deficient Ph⁺ ALL cells showed massively increased susceptibility to Imatinib-induced apoptosis. In addition, absence of Gas7 caused loss of self-renewal capacity and failure to form colonies in methylcellulose assay. Co-immunoprecipitation experiments with flag tagged GAS7 in patient-derived Ph⁺ALL cells revealed that GAS7 physically interacts with STAT5 and retains STAT5-Y⁶⁹⁴ in an active conformation.Thereby, GAS7 can propagate even weak Stat5 activity and maintain residual cytokine or BCR-ABL1 oncogenic signaling in normal and malignant pre-B cells.

Conclusions: Here show that GAS7 functions as an important positive regulator of Stat5 downstream of cytokine receptors in normal pre-B cells and downstream of BCR-ABL1 and other oncogenes in leukemia. Owing to the GAS7-dependent reinforcement of Stat5-dependent survival and proliferation signaling, normal and leukemic pre-B cells can survive periods of reduced cytokine/oncogene signaling. These findings suggest that the interaction interface between GAS7 and Stat5 represents a potential target for small molecule scaffolds and peptides.