A Niche-Based Cell Mutagenesis Assay Identifies ABL-Kinase Mutations Appearing in BCR-ABL T315I-Mutated Leukemic Cells Treated with Ponatinib.
Authors: Aggoune Djamel , Sorel Nathalie , Bonnet Marie Laure , Chomel Jean-Claude , Turhan Ali G 
Published: October 2019

Abstract
Abstract 2436
The occurrence of ABL-kinase (ABL-K) mutation is a major persisting concern in CML patients treated with tyrosine kinase inhibitor (TKI) therapies. Leukemic stem cell niche can protect the leukemic cells by providing survival and/or quiescence signals but also could favor the occurrence of ABL-kinase mutations. Amongst the ABL-kinase mutations, T315I is one of the most problematic as it induces resistance to all three clinically accepted TKI (Imatinib, Dasatinib, Nilotinib) and has been shown to occur at the level of stem cells (Chomel et al, Leuk lymphoma 2010). Ponatinib (Formerly AP24534) is a multi-targeted TKI which overcomes resistance to T315I mutation as well as to other ABL-kinase mutations. To model the role of the niche in the context of T315I in patients treated with Ponatinib, we designed a niche-based cell mutagenesis assay in the human hematopoietic UT7 cells engineered to express BCR-ABL (UT7-BCR-ABL native cells) and BCR-ABL with T315I (UT7-T315I), via retrovirus mediated gene transfer. Western blot analyses demonstrated that these cells express BCR-ABL and UT7 clones harboring the T315I mutation were resistant to Imatinib, Dasatinib and Nilotinib but sensitive to Ponatinib. We have treated UT7-T315I cells with N-ethyl-N-nitrosourea (ENU, 50microg/ml) for 24 hours and seeded them on previously prepared MS5 stromal cells (plate of 96 wells) in the presence of Ponatinib (30 nM final concentration). As a control, ENU-treated cells were also cultured in the absence of MS5 feeders and Ponatinib-based selection was performed in the same conditions. The same niche-based assay was also performed in the UT7-BCR-ABL native cells and selection process has taken place in the presence of Imatinib (2 microM). Cell medium was changed every week with addition of Ponatinib (UT7-T315I cells) or Imatinib (UT7-BCR-ABL native cells). At week+4, Ponatinib-resistant and IM-resistant wells cultured in the presence (MS5+) or in the absence (MS5-) of the hematopoietic niche were enumerated. In UT7-BCR-ABL native plates, the numbers of IM-resistant outgrowth was identical in MS5+ versus MS5- conditions (51/95 and 54/96, respectively). On the other hand, in UT7-T315I plates, there was a major difference in the numbers of Ponatinib-resistant T315I cells in MS5+ (96/96, 100% survival) as compared to MS5- conditions (65/96, 62 % survival). IM and Ponatinib-resistant clones were amplified and 48 clones resistant to Ponatinib (MS5+ n= 24; MS5- n= 24) and 48 clones resistant to IM (MS5+ n= 24: MS5- n=24) were screened for ABL-K mutations using a denaturing gradient gel electrophoresis assay followed by direct sequencing of the ABL-kinase domain. In the native BCR-ABL mutagenesis assay (with Imatinib), all clones were found mutated, in the presence or absence of MS5 stromal cells. Most of them were mutated in the P-loop region (E255K, G250E, Y253H). In addition, we have found 3 clones harboring the T315I mutation (1 in MS5- and 2 in MS5+ conditions). Concerning T315I clones surviving to Ponatinib, in addition to the T315I mutation, most of the resistant clones harbored an additional P-loop mutation which occrred in the absence or presence of MS5. Moreover, in two clones, two additional mutations were detected in addition to the T315I mutation. Interestingly, when Ponatinib-resistant cells were switched from MS5+ conditions to MS5- conditions, Ponatinib resistance could be abrogated in some but not all cases. In conclusion, our assay shows that the hematopoietic stem cell niche could play a crucial role in conferring resistance to Ponatinib, not only via the occurrence of novel mutations but also by providing survival signals. Preliminary results also suggest that the hematopoietic niche could facilitate the emergence of T315I mutation in cells expressing native BCR-ABL. These results could be important to study the mechanisms of the occurrence and selection of ABL-K mutations in patients treated with TKI including Ponatinib, and to develop niche-targeted therapies to overcome TKI-resistance in CML.

Disclosures:
Turhan: Novartis, Bristol Myers Squibb: Honoraria, Research Funding.