BACH2 Mediates Early B Cell Differentiation and Oncogene-Induced Senescence in Acute Lymphoblastic Leukemia
Authors: Swaminathan Srividya , Huang Chuanxin , Titz Bjorn , Buchner Maike , Geng Huimin , Graeber Thomas G , Willman Cheryl L , Igarashi Kazuhiko , Melnick Ari , Muschen Markus 
Published: October 2019

Abstract
Abstract 562FN2

Background:
The BACH2 (BTB and CNC homology, basic leucine zipper transcription factor 2) transcription factor is required for class-switch recombination and somatic hypermutation of immunoglobulin genes during affinity maturation of mature germinal center B cells. Interestingly, 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).

Results:
Bach2 mRNA levels are significantly lower in Ph+ ALL (n=72) compared to normal human bone marrow pre-B cells (n=10). We next studied 49 samples 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. Consistent with these findings, an independent study (Children's Oncology Group; NCT00005603) demonstrated that BACH2 mRNA levels in childhood ALL samples at diagnosis negatively correlated with early minimal residual disease (MRD) findings on day 29 (n=207; p<0.0001). Compared to normal pre-B cells (n=5), CpG islands in the BACH2 promoter were hypermethylated in Ph+ ALL cells (n=70). A detailed sequence analysis of the BACH2 coding region in 10 primary cases of Ph+ ALL revealed 7 unique point mutations including 5 amino acid changes in the BACH2 BTB domain. These findings suggest that BACH2 is affected by somatic mutations in a fraction of cases of Ph+ ALL.
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. An Affymetrix GeneChip analysis revealed that many of the genes that are differentially expressed between Bach2+/+ and Bach2−/− ALL cells are shared with a common gene expression signature reflecting TKI-treatment and inducible deletion of Myc or Stat5a/Stat5b. Interestingly, Bach2−/− normal pre-B cells lack the ability to upregulate expression of Rag1 and Rag2. The two Rag enzymes are required for Vk-Jk gene recombination and as a consequence, Bach2−/− pre-B cells fail to differentiate into k light chain expressing B cells. 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 Imatinib-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).
While BCR-ABL1-transformed pre-B ALL cells already express Myc at high levels, forced overexpression of Myc through a retroviral vector results in oncogene-induced senescence (OIS; senescence-associated b-galactosidase+) and subsequent apoptosis (Annexin V+). Whereas Bach2+/+ leukemia cells are non-permissive to forced Myc expression and die within four days following OIS, 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.

Conclusions:
These findings collectively identify Bach2 as a barrier mechanism against malignant transformation of pre-B cells. Bach2 is required for induction of Arf and p53 expression in the context of OIS. BACH2 is often hypermethylated at its promoter or somatically mutated in regions encoding its BTB domain. Consistent with these findings, lack of Bach2 mRNA expression is predictive of positive MRD at day 29 and associated with relapse of childhood ALL.

Disclosures:
No relevant conflicts of interest to declare.