Extensive RAG-Mediated Rearrangements and Mutations in BCR-ABL1 and BCR - ABL1 -like Adult Acute Lymphoblastic Leukemia

BCR-ABL1 and BCR-ABL1 -like acute lymphoblastic leukemia (ALL) are two major pre-B cell acute leukemia subtypes characterized by genetic alterations affecting lymphoid-specific transcription factors. Studies examining the chain of genetic events necessary to develop leukemia established that the BCR-ABL1 fusion gene and kinase-activating BCR-ABL1 -like lesions are initiating events, however, insufficient for leukemia development. Secondary genetic events targeting B cell development genes are therefore an essential requirement for overt ALL. A recent study (Papaemmanuil et al , Nat. Genet., 2014) revealed that illegitimate RAG-mediated recombination is the predominant mutational mechanism establishing these secondary genetic events in ETV6-RUNX1 ALL. Of note, ETV6 - RUNX1 ALL is mainly restricted to pediatric cases and it remains unanswered whether this mutational process also plays a prominent role in adult ALL pathogenesis. We carried out a detailed genomic characterization to determine whether aberrant RAG activity is also a prominent mutational driver in certain adult B cell ALL (B-ALL) subtypes. Diagnostic material of 53 unselected B-ALL cases and matched remission specimens were characterized using DNA mapping arrays to discern copy number alterations (CNAs). We observed multiple BCR-ABL1 / BCR-ABL1 -like patients with abundant genetic lesions and selected 5 cases for targeted sequencing of CNA boundaries to determine whether these lesions were driven by RAG-mediated recombination. Whole genome sequencing (WGS) for a single BCR-ABL1 -like patient was used to asses this mutational mechanism genome-wide. In total 64 structural variants (SVs) could be analyzed at base-pair level. De novo motif detection on breakpoint sequences revealed the prominence of the heptamer CACAGTG (E-value=5.68x10 -91 ), a constituent of the recombination signal sequence (RSS), present in 121 out of 128 breakpoints (94.5%). RSS detection revealed that 58 out of 64 SVs (90.6%) had a cryptic RSS (cRSS) on one or both sides of the lesion. Incorporation of non-templated sequences was observed for 54 out of the 64 (84.4%) SVs. Superimposition of breakpoints on chromatin marks revealed a strong enrichment for active promoters and enhancers (p -16 ). WGS data revealed cRSS motifs and incorporation of non-templated sequences for 23 out of 26 SVs (88.5%). Integrative analysis of all 6 cases confirmed 125 unique SV breakpoints strongly enriched for the active chromatin marks H3K4me3 and H3K27ac. STAT5 binding, a postulated regulator of V(D)J recombination, is similarly enriched at the breakpoints. Promiscuous binding of RAG1 and RAG2 was previously noted in human thymocytes and murine pre-B cells (Teng et al , Cell, 2015). Strikingly, the breakpoints are frequently bound by RAG2 in human thymocytes. In total 66 out of 125 breakpoints could be translated to the murine genome and revealed a strong enrichment of RAG1 and RAG2 binding at homologous positions in murine pre-B cells. Exhaustive mutation detection revealed complex somatic mutations within cRSS motifs, which are rare V(D)J recombination products introduced by erroneous cleavage and error-prone repair (open-and-shut joints). Strikingly, 4 out of 6 BCR-ABL1 / BCR-ABL1 -like cases had mutations in the BTLA promoter-situated cRSS, frequently in combination with a RAG-mediated deletion of the other allele (Figure 1). Genomic screening in 142 B-ALL patients confirmed 8 additional cases with BTLA promoter mutations, predominantly (6 out of 8) belonging to the BCR - ABL1 / BCR - ABL1- like subgroups. We provide strong evidence that aberrant RAG activity plays a pivotal role in the development of BCR - ABL1/BCR - ABL1 -like adult ALL. We demonstrate that breakpoints are strongly enriched for RAG binding implying a predisposition for illegitimate V(D)J recombination. Importantly, we report on a novel mutational mechanism introducing mutations in cRSS motifs through open-and-shut joints, frequently resulting in the biallelic inactivation of BTLA . Proliferation and V(D)J recombination during pre-B cell development is orchestrated by the interplay of IL7R and pre-BCR signalling. Strikingly, most kinase-activating lesions constitutively activate these signalling cascades and could enact, in concert with BTLA inactivation, constant proliferation, pro-survival and V(D)J recombination-initiating signals with disastrous consequences. Disclosures No relevant conflicts of interest to declare.