Haploinsufficiency of Ebf1 Collaborates with BCR-ABL1 to Decrease the Latency of Acute Lymphoblastic Leukemia.

In previously published genome-wide copy number analysis of leukemic samples from 242 pediatric acute lymphoblastic leukemia (ALL) patients, we reported that mutations in genes regulating B lymphoid development are the most common lesion in B-progenitor ALL, and these include PAX5, IKZF1, and EBF1. Mono-allelic deletion of EBF1 was observed in 8/200 B-progenitor leukemia samples, including a BCR-ABL1 ALL. EBF1 encodes a transcription factor that is required for the development of B cells, and with E2A regulates the expression of B-lineage specific genes. Mice null for Ebf1 arrest B cell development at the pro-B cell stage, whereas Ebf1^+/− mice have a 50% reduction in the number of immature and mature B cells but a normal number of pro-B cells. Importantly, neither haploinsufficiency nor the complete loss of Ebf1 results in the development of leukemia in mice. To examine the role of genetic alterations targeting B-lymphoid differentiation in the pathogenesis in BCR-ABL1 ALL, we transduced Ebf1^+/+ and Ebf1^+/− bone marrow cells with MSCV-GFP-IRES-p185 BCR-ABL1 retrovirus and transplanted the resultant cells into lethally irradiated wild-type C57BL6 recipient mice. Mice transplanted with BCR-ABL1 Ebf1^+/− cells developed B lineage ALLs at a shorter latency than observed with BCR-ABL1 Ebf1^+/+ cells (median overall survival of 17 days in Ebf1^+/− vs 42 days in Ebf1^+/+, P<0.0001). All leukemias had a B220⁺Cd19⁺Bp1⁺ pre-B cell immunophenotype; however, the leukemias that developed from the Ebf1^+/− cells aberrantly expressed high levels of the stem cell marker Sca1 (mean fluorescence level for Sca1 of 69.6 in Ebf1^+/− (n=22) vs 16.8 in Ebf1^+/+ (n=14), p<0.0001). To begin to understand how a decrease in the copy number of Ebf1 may contribute to leukemogenesis, we examined early B cell development in bone marrow (BM) cells from two week-old C57BL6 Ebf1^+/− and Ebf1^+/+ mice. Our analysis confirmed previous reports indicating a 2-fold reduction of B220⁺CD43⁻ B cells in Ebf1^+/− compared to Ebf1^+/+ mice. Interestingly, however, we also detected an approximately 6-fold increase in a transitional population of B220^loIL-7R⁺cKit^lo Pre-pro B cells that also expressed Sca1 (2194 mean number of Ebf1^+/− cells per 100,000 BM cells (n=10) vs 372 mean number of Ebf1^+/+ cells per 100,000 BM cells (n=8), p<0.0001), an observation that raises the possibility that Ebf1 haploinsufficiency expands the pool of cells that are susceptible to transformation by BCR-ABL expression. It will be important to examine whether the accelerated tumorigenesis resulting from Ebf1 haploinsufficiency is a consequence of a subtle shift in differentiation, or some alternative mechanism of oncogenic cooperativity. Studies are underway to directly assess the role of B220^loIL-7R⁺cKit^lo Sca1⁺ cells in BCR-ABL1 driven ALL.