The Pre-B Cell Receptor Suppresses Leukemogenesis by Censoring MYC Expression.
Authors: Nahar Rahul , Trageser Daniel , Klemm Lars , Duy Cihangir , van Essen Peter , Kim Yong-mi , Heisterkamp Nora , Martinelli Giovanni , Hofmann Wolf-Karsten , Jack Hans-Martin , Jumaa Hassan , Muschen Markus 
Published: April 2020

Pre-B cells in human bone marrow are destined to die unless they are rescued through survival signals from a successfully assembled pre-B cell receptor. Congenital defects in pre-B cell receptor-related signaling molecules cause a severe differentiation block at an early pre-B cell stage. Likewise, B cell lineage acute lymphoblastic leukemia (ALL) cells are arrested at an early pre-B cell stage in the vast majority of cases. Given that the pre-B cell receptor drives both proliferation and differentiation of normal B cell precursors, we test here the hypothesis that pre-B cell receptor signaling represents a critical target for malignant transformation towards ALL.

Studying 148 cases of pre-B cell-derived human ALL, we found that pre-B cell receptor expression and function is linked to specific cytogenetic subgroups: ALL cells carrying an E2A-PBX1-gene rearrangement are –like normal pre-B cells- highly selected for the expression of a functional pre-B cell receptor. In all 8 ALL cases with E2A-PBX1 fusion, engagement of the pre-B cell receptor resulted in a strong Ca2+ signal. In striking contrast, ALL cells carrying BCR-ABL1- or MLL-AF4 fusion genes and ALL cells with hyperdiploid karyotype lack expression of a functional pre-B cell receptor in virtually all cases. Only 10 of 57 cases with BCR-ABL1, 0 of 7 cases with MLL-AF4 and 1 of 30 cases with hyperdiploid karyotype a productively rearranged μ-heavy chain locus encoding the central component of the pre-B cell receptor, was found. Even in the few BCR-ABL1 ALL cases, in which a productively rearranged μ-chain was amplified, no pre-B cell receptor was expressed. Based on these findings, we hypothesize that ALL can be subdivided into two groups based on whether pre-B cell receptor signaling enables (E2A-PBX1; Type I) or suppresses (BCR-ABL1, MLL-AF4, Hyperdiploid; Type II) leukemic growth. In a proof-of-concept experiment, we super-transformed E2A-PBX1-induced Type I ALL cells (active pre-B cell receptor signaling) and MLL-AF4-induced Type II ALL cells (lack of pre-B cell receptor expression) with the BCR-ABL1 oncogene. Whereas growth of pre-B cell receptor-negative Type II ALL cells was accelerated by BCR-ABL1-transformation, pre-B cell receptor-positive Type I ALL cells rapidly eliminated by apoptosis within 9 days after BCR-ABL1-transduction.

To identify factors that distinguish Type I (E2A-PBX1) and Type II (BCR-ABL1, MLLAF4, Hyperdiploid) ALL and that may explain the divergent role of pre-B cell receptor signaling in these groups, we performed a comparative gene expression including a metaanalysis of published microarray data and quantitative RT-PCR.

Compared to E2A-PBX1 Type I ALL, MYC mRNA levels are on average 4-fold, 6-fold and 2.5-fold higher in BCR-ABL1, MLL-AF4 and Hyperdiploid ALL cells, respectively. To test whether high expression levels of MYC render leukemia cells non-permissive to pre-B cell receptor expression, we studied bone marrow B cell precursors from Rag2−/− mice that carry a tetracycline-inducible μ-chain transgene (Hess et al., 2001) as cell culture model for inducible pre-B cell receptor expression. When expression of the pre-B cell receptor was induced in normal IL7-dependent B cell precursors, the cells were induced to first proliferate and subsequently differentiate, hence mirroring normal stages of early B cell development. We then transformed Rag2−/− Tet-μ-chain B cell precursors by retroviral transduction with MYC. MYC-transformed cells rapidly outcompeted untransduced normal IL7-dependent B cell precursors in cell culture. Induction of pre-B cell receptor expression, however, completely reversed growth kinetics and within a few days, normal untransduced pre-B cell receptor-positive cells had a substantial growth advantage over MYC-transduced pre-B cell receptor-positive cells that were progressively lost in cell culture. Interestingly, this growth pattern was reversible by subsequent ablation of pre-B cell receptor expression: After Tet-mediated ablation of pre-B cell receptor expression, the initial growth kinetics were restored and MYC-transduced pre-B cell receptor-negative cells regained a substantial growth advantage.

These findings demonstrate that different levels of MYC expression determine permissiveness of ALL cells for pre-B cell receptor signaling. Hence, the pre-B cell receptor suppresses outgrowth of Type II leukemia by censoring high levels of MYC expression.