Whole genome sequencing reveals that the Philadelphia chromosome (Ph) can evolve and gain complex chromoplectic sequence rearrangements associated with tyrosine kinase inhibitor (TKI) resistance

Abstract

Abstract Background Understanding of the heterogeneity in the formation of the Ph has evolved as technology has advanced. In the pre-TKI era, FISH revealed derivative 9 deletions that were associated with shorter survival. The poor outcomes were overcome with imatinib. Whole exome and transcriptome sequencing revealed greater complexity: inversions; novel 9;22 fusions; sequence fragmentation; and random reassembly. These were termed Ph-associated rearrangements. They occurred on the Ph and/or the derivative 9, were thought to occur at the same time as the Ph and correlated with poorer response to imatinib. Aim To use whole genome sequencing (WGS) to examine derivative chromosomes and further resolve the complexity of Ph-associated rearrangements in patients at diagnosis and pan-TKI resistant cell lines. Methods WGS was performed for 7 chronic phase CML patients at diagnosis and 7 matched sensitive/resistant Ph-positive cell lines. Resistance was induced by exposure to increasing concentrations of imatinib. Data was analyzed using Oncoanalyzer. Derivative chromosome structure was resolved using the LINX algorithm, which clusters structural variants that are highly unlikely to occur independently. Rearrangements were validated using orthogonal methods, including Optical Genome Mapping and SNP Array. Six of 7 patients had pre-characterized Ph-associated fusions identified using RNAseq. Results For most patients, a balanced reciprocal Ph translocation involves 2 rearrangements: 1 that generates the Ph and BCR::ABL1; and 1 reciprocal derivative 9 with ABL1::BCR. These structures were resolved for 1 patient without pre-characterized Ph-associated rearrangements and for 5 of 7 parental cell lines. A distinct class of complex structural rearrangement called chromoplexy was identified involving the Ph translocation in 11 samples. These Ph-associated rearrangements had the highest number of linked structural variants within the individual genomes of 10 of the 11 samples. Chromoplexy involves the formation of extensive chained rearrangements affecting multiple chromosomes that create an intricate series of inter/intra-chromosomal fusions, deletions, duplications and inversions. Many DNA strands were broken and ligated in new configurations in the affected samples. Multiple genes on the derivative chromosomes were disrupted, including recurrent cancer drivers. For 3 of 7 matched sensitive/resistant cell lines, Ph-associated rearrangements were acquired in the resistant lines. The most complex events occurred in the LAMA84 resistant line. The parallel sensitive line had the standard 2 rearrangements. However, the resistant line gained 17 rearrangements with highly amplified Ph sequences, inversions and novel genomic fusions. Remarkably, small fragments from 6 chromosomes (2, 4, 10, 13, 17, X) were inserted upstream and downstream of BCR::ABL1, and disrupted the NUP214 driver gene. The K562 sensitive line displayed 24 rearrangements with non-contiguous amplified Ph regions of different copy number. Events included NOTCH1 deletion, 9;22 NUP214 fusion and an adjacent 9;13 fusion. Six fragments from chromosome 13 were incorporated into the Ph. The K562 resistant line displayed further evolution with 31 Ph rearrangements. The AR230 sensitive line displayed Ph chromoplexy with 9 rearrangements. The resistant line evolved with 19 additional Ph rearrangements. A fragment from chromosome 21 was inserted upstream of BCR::ABL1 and disrupted the MAPK1 driver gene. The LZTR1 tumor suppressor was deleted. All other cell lines maintained the standard Ph configuration. Ph-associated chromoplexy was evident in 6 patient samples: average 10 rearrangements. The patient with the highest number (15) progressed to blast phase at 4 months of imatinib. Four Ph-associated subclones were detected at diagnosis with non-contiguous amplified regions, consistent with the evolved cell lines. Furthermore, the LZTR1 tumor suppressor gene was deleted, as detected in AR230. The blast phase sample was unavailable. Deep molecular response was achieved in 1 patient on imatinib and 4 on frontline or switch to nilotinib/dasatinib. Conclusion We have demonstrated that Ph chromoplectic rearrangement can be present at diagnosis but can also be initiated during leukemic progression and disrupt multiple genes, including cancer drivers. The Ph may be prone to extensive DNA breakage. Ph chromoplexy could constitute a new mechanism that contributes to acquired TKI resistance.