Templated Insertion Mediates Generation of p190 BCR/ABL Facilitating CML Heterogeneity Uniquely Detected By Optical Genome Mapping

Philadelphia chromosome (Ph) a hallmark of chronic myeloid leukemia (CML), is a result of reciprocal translocation t(9;22) generating aberrant persistently activate oncogenic tyrosine- kinase (BCR/ABL1) facilitating leukemogenesis. It was demonstrated that simultaneous breakages of two different chromosomes within BCR and ABL1 loci, serve as an oncogenic initiation of Ph⁺ leukemia. Thus, recurrency of Ph and other translocations in hematopoietic stem cells indicates that chromosomal instability (CIN) is leading pathogenetics of leukemogenesis.

Surprisingly, despite established diagnostic methods and knowledge on CML, use of Optical Genome Mapping (OGM) identified novel pathogenetic landscape of chromosomal aberrations and generation of BCR/ABL1 p190 fusion gene without formation of Ph chromosome. We found that dominant characteristics of CIN in these leukemia cells is increase of clustered aberrations at distinct breakpoints throughout a genome with proximity to microhomology-enriched segments. We detected significant accumulation of double-strand breaks (DSBs) at near-overlapping origins of replications. OGM and chromosomal analyses strongly correlate with induced distinct expression profile and activation of microhomology-mediated break induce repair (MMBIR). MMBIR utilizes microhomology for DSB repair, producing templated insertions that promote complex genomic rearrangements detected in cancer including chromosomal illegitimate recombination, integrations and fusions. Despite growing evidence on accumulation of de novo templated insertions in several cancer types, the underlying molecular and genomic mechanisms, impact on carcinogenesis and heterogeneity, particularly in hematopoietic cells are poorly understood.

Based on OGM, omics data mining and NGS, we developed sophisticated screening panels and molecular analyses to study BCR, ABL1, corresponding breakpoints loci, and near-overlapping microhomology-enriched elements, to identify and characterize this novel atypical p190 fusion gene. Our results indicate that in some CML patients', with unique CIN signature, induced mechanism of de novo templated insertions generating atypical p190 fusion gene instead of Ph chromosome formation.

It's well established that different BCR/ABL1 transcripts correspond to distinct CML clinical phenotype. Furthermore, chronic, accelerated and blast phases of CML progression evaluated and measured by expression rate of each Ph⁺ BCR/ABL1 transcript variant, resulting in prediction of therapy response, and clinical outcomes.

However, in few CML patients, standard assessments succeeded to detect fusion gene induced by templated insertion and monitor atypical transcripts. Thus, generally, standard tests were insufficient to assess expression rate of atypical p190 transcript initially and in MRD. Importantly, precise definition of the disease is challenging in patients with minimal CML. For example, preliminary screen detected templated insertions with atypical p190 BCR/ABL1 fusion genes in 6 patients. 3 patients were JAK2 V617F positive, while 2 subsequently treated for Essential Thrombocytosis. Third patient diagnosed and treated for p210 Ph⁺CML. In contrast, spontaneous recovery observed in other 3 patients, with allelic frequency of atypical p190 transcript lower than 0.035%. Thus, detection of minor clones with templated insertions, characterization of related abnormalities provide critical insights during early detection on clinical significance of atypical p190 transcript, necessity of further molecular testing and treatment evaluation. Hence, we developed robust molecular assay to measure atypical p190 transcripts to assist diagnosis and monitoring MRD. In contrast, ongoing study points on importance of MRD monitoring, detection CIN signatures, accumulation of templated insertions promoting transformation.

In conclusion, our study indicates that distinct CIN signature orchestrates unique DNA repair landscape. Lack of precise molecular assessment, and genetic identification of seemingly similar oncogenes originated from atypical fusion gene, or Ph chromosome can be providing misleading diagnostics. Furthermore, our results suggest that increased heterogeneity observed among CML patients, reflect on clonal ability to repair DNA damage, preserve genome stability and function, affecting carcinogenesis.

Ofran:Minovia Therapeutics: Membership on an entity's Board of Directors or advisory committees.