Aberrant Splicing of the SLP65 SH2 Domain Enables Pre-B Cell Transformation and Compromises the Leukemia-Suppressive Function of the Pre-B Cell Receptor
Authors: Duy Cihangir , Klemm Lars , Nahar Rahul , van Essen Peter , Sprangers Mieke , Kim Yong-mi , Park Eugene , Martinelli Giovanni , Heisterkamp Nora , Hofmann Wolf-Karsten , Jumaa Hassan , Muschen Markus 
Published: April 2020

Abstract
The linker molecule SLP65 is critical for pre-B cell receptor-mediated differentiation signals during early B cell development. Germline mutations of the SLP65 gene in mice and humans result in a profound differentiation block at an early pre-B cell stage. Likewise, B cell lineage acute lymphoblast leukemia (ALL) cells are phenotypically arrested at an early pre-B cell stage in the vast majority of cases. In about 2–5% of cases with ALL, mutations and deletions of the SLP65 gene are found (Sprangers et al., 2006; Mullighan et al., 2007). In a comprehensive study of SLP65 function in human ALL cells, we identified numerous aberrant SLP65 splice variants, which all lack one or more exons that encode the C-terminal SLP65 SH2 domain. A splice variant lacking exon 16, which encodes the central part of the SH2 domain was particularly abundant in ALL but not in normal pre-B cells (EMBL/GenBank Accession number AM180337). The SH2 domain may have critical function because it represents the main ligand for the non-ITAM Y204 in the Igα (CD79A) signaling chain of the pre-B cell receptor (Patterson et al., 2006) and thereby connects the pre-B cell receptor to downstream signaling molecules. To examine a potential role of the SLP65 SH2 domain during the process of malignant transformation in ALL, we measured mRNA levels of full-length SLP65 vs SLP65 splice variants, in which the SH2 domain was deleted (SLP65ΔSH2) in normal pre-B cells and primary human ALL cells by quantitative RT-PCR. While normal pre-B cells express full-length SLP65 at high and SLP65ΔSH2 at very low levels, the SLP65/SLP65ΔSH2 ratio was reduced in E2A-PBX1-driven ALL and particularly low in BCR-ABL1-driven ALL. Given that SLP65ΔSH2 is overexpressed in ALL cells at the expense of full-length SLP65, we investigated biochemical properties of SLP65ΔSH2. To this end, we transduced Slp65−/− pre-B cells with either full-length Slp65, SLP65ΔSH2 or a GFP empty vector control. As opposed to full-length SLP65, SLP65ΔSH2 does not interact with Igα and does not confer tyrosine phosphorylation of BTK downstream of the pre-B cell receptor. Also Ca2+-release in response to pre-B cell receptor engagement and induction of differentiation was drastically reduced in Slp65−/− pre-B cells that were reconstituted with SLP65ΔSH2 instead of SLP65. Co-expression experiments with SLP65 showed that SLP65ΔSH2 has a dominant-negative function and impairs pre-B cell receptor signaling. We next transformed Slp65−/− pre-B cells with a retroviral BCR-ABL1 vector and tested whether SLP65 and SLP65ΔSH2 have a different impact on BCR-ABL1-mediated pre-B cell transformation and BCR-ABL1-driven leukemic growth. Reconstitution of SLP65 expression in Slp65−/− BCR-ABL1-transformed pre-B ALL cells resulted in cell death and nine days after transduction more than 90% of the SLP65-transduced cells had undergone apoptosis. Conversely, reconstitution of Slp65−/− BCR-ABL1-transformed pre-B ALL cells with SLP65ΔSH2 had no significant effect on viability of the leukemia cells. In a titration experiment, we reconstituted SLP65−/−BCR-ABL1-driven ALL cells with SLP65 and SLP65ΔSH2 at various ratios and thereby confirmed that SLP65ΔSH2 has a dominant-negative effect and compromises the leukemia-suppressive function of the pre-B cell receptor. We next tested whether aberrant splicing also interferes with the tumor suppressor function of SLP65 in vivo: To this end, we engrafted SLP65−/−BCR-ABL1- driven ALL cells that were transduced with either SLP65-GFP, SLP65ΔSH2 or GFP into sublethally irradiated NOD/SCID mice. After 24 days, mice that were engrafted with ALL cells transduced with SLP65ΔSH2 or GFP became terminally leukemic, whereas mice injected with SLP65-transduced ALL cells showed no signs of disease. At this time, all mice were sacrificed and analyzed. Whereas mice with SLP65ΔSH2- or GFP-transduced ALL cells had enlarged spleens and substantial leukemic infiltration of the bone marrow (79 and 81% leukemia cell content), leukemic infitration was reduced if SLP65-transduced ALL cells were injected (1.8% leukemia infiltration).
Whereas mutations and deletions of the SLP65 gene are rare, we conclude that aberrant splicing of the SLP65 SH2 domain represents a common mechanism in ALL cells to compromise the leukemia-suppressive function of the pre-B cell receptor.