Analysis of BCR-ABL1 Tyrosine Kinase Domain Mutations In Primitive Chronic Myeloid Leukemia Cells Identifies a Unique Mutator Phenotype.
Authors: Jiang Xiaoyan , Grant Hannah , Stebbing Justin , Foroni Letizia , Craddock Charles , Griffiths Mike , Clark Richard E. , O'Brien Stephen , Khorashad Jamshid S. , Gerrard Gareth , Wang Lihui , Irving Julie , Wang Meng , Karran Loraine , Dyer Martin JS , Forrest Donna L. , Page Karen , Eaves Connie J. , Woolfson Adrian 
Published: October 2019

Abstract
Abstract 3397
The tyrosine kinase inhibitor (TKI), imatinib mesylate (IM), induces remissions in most chronic phase chronic myeloid leukaemia (CML-CP) patients, but emergence of drug resistance attributable to critical mutations in the BCR-ABL1 TK domain remains a significant clinical problem. We previously demonstrated that primitive (CD34+) CML-CP cells are both intrinsically insensitive to IM and genetically unstable. BCR-ABL1 TK domain mutations are, furthermore, readily detectable in CD34+ CML-CP cells even prior to IM treatment. However, the mechanisms by which they arise are unknown. In this study, we characterized the spectrum of mutations at each of the 3 codon positions in the BCR-ABL1 TK domain of IM-naïve CD34+ cells and used a mathematical model to compare these with those found in the same region of DNA in IM-resistant cells. A total of 460 TK mutations were identified in cells from 15 IM-naïve and from 316 IM-resistant patients. The mutations were non-randomly distributed across the 3 codon positions, with very few mutations at position 3 (1 of 136 mutations, 0.7%, in IM-naïve patients and 31 of 329, 9%, in IM-resistant patients). The remainder showed no bias in their distribution across codon positions 1 and 2 in IM-naïve patient cells but, in IM-resistant cells, a significant positive bias was observed at codon position 2 (p=0.042). Conversely, BCR-ABL1 TK domain sequences from IM-naïve patient cells had significantly more transitions relative to unselected regions of the genome across all codon positions, a significant positive A-to-G mutational bias (A>G, 1.12×10−10) and an under-representation of C-to-T mutations (C>T, 5.34×10−5) when these mutations were compared with unselected regions of the genome. In addition, we observed a T-to-C mutational hotspot (T>C, 1.23×10−4) at codon position 2. IM-naïve cells exhibited a transitional bias at position 2 and overall (1.99×10−4 and 6.19×10−5, respectively; at position 1, P=0.052). We observed a similar pattern of TK codon position mutations at position 1 and overall in sequences derived from IM-resistant cells. Interestingly, in IM-resistant cells, A-to-T (A>T) transversions were over-represented at position 2 and overall (1.41×10−11 and 3.77×10−5). These cells also had a profound transversional bias (1.45×10−4) at codon position 3, suggesting that positive selection occurs at a position where transversions are typically non-synonymous. However, a skewed transition:transversion ratio and transition and transversion frequency at TK codon positions 1 and 2 was observed in both IM-naïve and IM-selected cells with mutations distributed across these 2 codon positions in a significantly uneven fashion. In IM-resistant cells, the frequency of C-to-T mutations (C>T, 3.82×10−11) indicated a bias affecting codon position 2 more often than position 1, and A-to-T transversions occurred significantly more frequently at position 2 compared to position 1 (A>T, 7.45×10−9). This was also the case for T-to-C transitions in TK sequences of IM-naïve cells (T>C, 1.13×10−4). Frequencies of these TK mutations in IM-naïve and IM-resistant cells were also higher than in unselected regions of the genome, providing further evidence that the TK domain mutations in CML-CP cells have a distinct mutational profile. For example, the clinically observed M244V and D276G mutations result from A-to-G transitions and the F359L mutation arises from a T-to-C transition, both of which would be predicted by the activity of a CML-CP mutator. However, the most clinically important mutation, T315I, that confers resistance to most currently available TKIs, is generated by a C-to-T transition, suggesting a high mutational rate generating mutational escape around the principal mutator pattern and profound selection thereafter. Notably, the CML-CP mutational signature is distinct from that of the activation-induced cytidine deaminase (AID)-induced hypermutation reported in CML blast crisis, both in targeting specific codon positions and in the overall mutational pattern. This strongly favors the hypothesis that the genomic instability of primitive CML-CP cells is generated by a different, specific mutational process. The results also suggest that monitoring BCR-ABL mutational hotspots may be clinically useful in anticipating TKI resistance.

Disclosures:
No relevant conflicts of interest to declare.