BACH2 (BTB and CNC homology 1, basic leucine zipper transcription factor 2) is required for class-switch recombination and somatic hypermutation of immunoglobulin genes during affinity maturation of mature germinal center B cells. We and others found that BACH2 is strongly upregulated in BCR-ABL1-transformed acute lymphoblastic leukemia (Ph+ ALL) cells upon treatment with tyrosine kinase inhibitors (TKI).
Bach2 mRNA levels are significantly lower in Ph+ ALL (n=72) compared to normal human bone marrow pre-B cells (n=10). Studying gene expression in a clinical trial for children with high risk ALL (Children's Oncology Group, P9906; n=207), we found in a multivariate analysis that high Bach2 levels at the time of diagnosis represents an independent predictor of favorable clinical outcome (negative MRD at day and higher overall and relapse-free survival; p<0.0001). We next studied 49 sample pairs from patients with childhood ALL at diagnosis and relapse. In 44 of these sample pairs, the relapse sample showed drastically reduced mRNA levels of Bach2 (p=0.019), suggesting that loss of BACH2 expression is associated with relapse of childhood ALL. A comparison of the methylation status of BACH2 promoter of normal pre-B cells (n=5), with Ph+ ALL cells (n=70) revealed that CpG islands in the BACH2 promoter were heavily hypermethylated in the leukemia samples. These findings are also consistent with genomic analyses on patient derived samples and the identification of small deletions at 6q15 in 4 of 11 cases of childhood ALL cases that all span the BACH2 locus.
To study the role of Bach2 in pre-B ALL in a genetic experiment, we transformed pre-B cells from Bach2−/− mice with BCR-ABL1. We observed that Bach2−/− normal pre-B cells lack the ability to counterselect pre-B cell clones that failed to undergo successful V(D)J rearrangement. In the absence of Bach2, a significant number of B cells survive even though they failed to rearrange immunoglobulin heavy chain genes. Besides this unexpected role in early B cell differentiation, quantitative RT-PCR and Western blot confirmed that BACH2 is also required for expression of the tumor suppressors Cdkn2a (Arf), p53 and Btg2. Consistent with extremely low protein levels of Arf and p53 in Bach2−/− leukemia cells, Bach2−/− ALL cells are more resistant to TKI-treatment, more actively proliferating (increased S-phase; p=0.02) and exhibit a ∼90-fold increased ability to form colonies in methyl cellulose (p=0.001). Studying Cre-mediated inducible deletion of p53 in p53-fl/fl leukemia cells, we found that Bach2-induced tumor suppression is largely dependent on p53 function.
Forced overexpression of Myc results in oncogene-induced senescence (OIS) followed by apoptosis. Whereas Bach2+/+ leukemia cells are non-permissive to forced Myc expression and die within four days after Myc induction, Bach2−/− ALL cells tolerate forced expression of Myc and evade OIS and subsequent cell death. Similarly, overexpression of Myc alone fails to transform Bach2+/+ pre-B cells. By contrast, retroviral overexpression of Myc results in rapid transformation and growth factor-independence of Bach2−/− pre-B cells. Bach2−/− Myc-high pre-B cells cause fatal leukemia in 100% of recipient mice within 22 days, whereas all mice that received Bach2+/+ Myc-high pre-B cells survived without signs of disease until day 67, when all mice were sacrificed and analyzed for MRD by flow cytometry and PCR. No evidence of MRD was detected in most mice injected with Bach2+/+ Myc-high pre-B cells. Three mice had positive MRD PCR findings, however, at 4 log orders below findings in mice injected with Bach2−/−Myc-high pre-B cells.
Our findings identify Bach2 as a novel tumor suppressor upstream of p53 in pre-B ALL. Bach2 is a regulator of negative selection during normal pre-B cell differentiation but also limits excessive proliferation of pre-B cell clones by induction of oncogene-induced senescence and activation of p53. In addition, our multivariate analyses identify high expression levels of Bach2 as powerful predictor of favorable clinical outcome in children, which may be useful in future approaches for risk stratification.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.