Pre-B Cell Receptor Signaling Prevents Leukemic Transformation by BCR-ABL1
Authors: Trageser Daniel , Duy Cihangir , Klemm Lars , Gruber Tanja , Nahar Rahul , Park Eugene , Kim Yong-mi , Groffen John , Heisterkamp Nora , Hofmann Wolf-Karsten , Martinelli Giovanni , Williams Richard T , Jumaa Hassan , Muschen Markus 
Published: April 2020

Abstract
Pre-B cells within the bone marrow are destined to die unless they are rescued through survival signals from the pre-B cell receptor. Studying the configuration of the immunoglobulin heavy chain locus (IGHM) in sorted human bone marrow pre-B cells by single-cell PCR, we detected a functional IGHM allele consistent with the expression of a functional pre-B cell receptor in the vast majority of normal human pre-B cells. However, only in 10 of 57 cases of BCR-ABL1-transformed pre-B cell-derived acute lymphoblastic leukemia (ALL), we detected a functional IGHM allele. While normal pre-B cells respond vigorously to pre-B cell receptor engagement by Ca2+ release, the pre-B cell receptor was unresponsive even in the few cases of BCR-ABL1-driven ALL, in which we amplified a productively rearranged IGHM allele. For this reason, we studied the function of the pre-B cell receptor during early B cell development and progressive transformation in a BCR-ABL1-transgenic mouse model: Interestingly, BCR-ABL1-transgenic mice that have not yet undergone leukemic transformation show almost normal pre-B cell receptor selection. In these pre-leukemic pre-B cells, however, expression of the BCR-ABL1-transgene is very low as compared to full-blown ALL, suggesting that high levels of BCR-ABL1 expression are not compatible with normal expression of the pre-B cell receptor. Consistent with our observations in human ALL, full-blown ALL clones in BCR-ABL1-transgenic mice show defective pre-B cell receptor selection and the pre-B cell receptors expressed on few leukemic cells are not functional. Treatment of leukemic mice with the BCR-ABL1 kinase inhibitor AMN107, however, reinstated normal pre-B cell receptor selection and pre-B cell receptor function within seven days. These data suggest that the transforming signal through BCR-ABL1 and normal survival signals through the pre-B cell receptor are mutually exclusive. To test whether functional pre-B cell receptor signaling prevents transformation by BCR-ABL1, we transformed murine pre-B cells carrying a deletion of the SLP65 gene, which is required for functional pre-B cell receptor signaling. Unlike SLP65-wildtype pre-B cells, SLP65−/− pre-B cells can be transformed by BCR-ABL1 at a high efficiency. Reconstitution of SLP65 using a retroviral vector, however, induced rapid cell death of BCR-ABL1-transformed pre-B cells. We next investigated the potential impact of Slp65-reconstitution on leukemic growth of BCR-ABL1-transformed pre-B cells from SLP65−/− mice in vivo. To this end, SLP65−/− BCR-ABL1-transformed pre-B cells were labeled with firefly-luciferase and then transduced with retroviral vectors encoding SLP65/GFP or GFP alone. NOD/SCID mice were sublethally irradiated and injected with either SLP65/GFP+ or GFP+ ALL cells. Engraftment as monitored by bioluminescence imaging was delayed by more than three weeks in mice injected with SLP65/GFP+ ALL cells as compared to mice injected with GFP+ ALL cells. 36 days after injection, the first mice that were inoculated with GFP-transduced leukemia cells, became terminally ill and also the other mice in this group showed weight loss at that time. In contrast, the mice injected with SLP65-GFP-transduced ALL cells showed no signs of disease and no significant weight loss. At this time, all mice were sacrificed: Whereas mice injected with GFP-transduced ALL cells showed splenomegalia and leukemic infiltration into multiple organs, there was only mild splenic enlargement, when SLP65-reconstituted ALL cells were injected. Reconstitution of SLP65 also reduced the frequency of BCR-ABL1-transformed leukemia cells about 15-fold in the bone marrow, 5-fold in the spleen and &gt;100-fold in the peripheral blood. We conclude that deficiency of the pre-B cell receptor-related signaling molecule SLP65 not only represents a frequent feature in human ALL cells but also represents a critical requirement for BCR-ABL1-driven leukemic growth in vivo.
We conclude that pre-B cell receptor signaling renders B cell progenitor cells non-permissive to BCR-ABL1-mediated transformation. Only crippled pre-B cells with a non-functional pre-B cell receptor are susceptible to BCR-ABL1-mediated transformation.