Pre-B Cell Receptor Signaling Distinguishes E2A-PBX1 From Other Subtypes of Acute Lymphoblastic Leukemia

The E2A-PBX1 [t(1;19)(q23;p13)] fusion is found in ≂f4% of cases of childhood ALL and involves a rearrangement of the TCF3 gene (encoding the E2A transcription factor). TCF3 (E2A) is not only a critical regulator of B cell lineage commitment and early B cell development (Müschen et al., 2002; Sigvardsson et al., 2002), it also cooperates with LEF1 to activate canonical WNT/β-catenin signaling (Hovanes et al., 2001; Merrill et al., 2001).

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, which strongly suggests that E2A-PBX1 leukemia clones are selected for active pre-B cell receptor signaling. 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) or suppresses (BCR-ABL1, MLL-AF4, Hyperdiploid, likely other subtypes) leukemic growth. In a proof-of-concept experiment, we super-transformed E2A-PBX1-induced ALL cells (active pre-B cell receptor signaling) and MLL-AF4-ALL cells lacking pre-B cell receptor function with the BCR-ABL1 oncogene. The BCR-ABL1 oncogene was chosen, because it is only found in leukemia cells that lack pre-B cell receptor function. Whereas growth of pre-B cell receptor-negative MLL-AF4 ALL cells was strongly accelerated by BCR-ABL1-transformation, pre-B cell receptor-positive E2A-PBX1 ALL cells were rapidly eliminated within 9 days after BCR-ABL1-transduction. Interestingly, incubation of E2A-PBX1 ALL cells survived BCR-ABL1-transduction only in the presence of 10 μ mol/l of the BCR-ABL1 kinase inhibitor Imatinib.

To identify factors that distinguish E2A-PBX1 and other ALL subtypes (BCR-ABL1, MLL-AF4, Hyperdiploid) that may explain the divergent role of pre-B cell receptor signaling in these groups, we performed a comparative gene expression including a meta-analysis of published microarray data and quantitative RT-PCR. In this analysis, E2A-PBX1 ALL cells were distinguished by high expression levels of pre-B cell receptor-related signaling molecules (e.g. BLNK, SYK, BTK). The most prominent gene expression differences involve canonical WNT/β-catenin signaling. As opposed to other ALL subtypes, E2A-PBX1 ALL cells express the β-catenin cofactors TCF3 and LEF1 at >5-fold higher levels and WNT16 at >12-fold higher levels compared to BCR-ABL1, MLL-AF4, Hyperdiploid and TEL-AML1-driven ALL subtypes.

Constitutive activation of the canonical WNT/β-catenin signaling pathway in E2A-PBX1 ALL cells may explain the distinct role of pre-B cell receptor signaling in this ALL subset: Since pre-B cell receptor signaling via BTK negatively regulates WNT/β-catenin-dependent survival and self-renewal signaling (James et al., 2009), the level of constitutive WNT/β-catenin-signaling may determine permissiveness of ALL cells to pre-B cell receptor function.

No relevant conflicts of interest to declare.