Mutations at Residues 315 and 317 in the ABL Kinase Domain Are the Main Cause of Resistance to Dasatinib in Philadelphia-Positive (Ph+) Leukemia Patients (pts).
Authors: Soverini Simona , Martinelli Giovanni , Colarossi Sabrina , Gnani Alessandra , Castagnetti Fausto , Rosti Gianantonio , Bosi Costanza , Paolini Stefania , Rondoni Michela , Piccaluga Pier Paolo , Palandri Francesca , Giannoulia Panagiota , Marzocchi Giulia , Luatti Simona , Abruzzese Elisabetta , Iacobucci Ilaria , Testoni Nicoletta , Baccarani Michele 
Published: April 2020

Abstract
Dasatinib (BMS-354825) is a second-generation BCR-ABL inhibitor active against several imatinib-insensitive BCR-ABL mutant forms. We have treated in the phase II program with dasatinib a total of forty-five Ph+ pts who were resistant to or intolerant of imatinib. At the time of writing, twenty pts have failed to respond to or relapsed on dasatinib therapy. In order to assess which pre-existent or emerging ABL kinase domain (KD) mutations are challenging for dasatinib clinical efficacy, we retrospectively analyzed ABL KD sequences before the start of treatment and every month thereafter, until dasatinib discontinuation. Mutation monitoring was done by D-HPLC, followed by sequencing in D-HPLC-positive cases. Eight pts had primary resistance to dasatinib (Table 1). In all cases, a T315I or a F317L mutation was already detectable before the onset of treatment or became detectable after one month. The mutations persisted up to the time of disease progression, which occurred at a median of 1.5 months (range, 1–4) from dasatinib start. Twelve pts had acquired resistance to dasatinib (Table 1). Relapse occurred after a median of 7.5 months (range, 3–15) from dasatinib start. Mutation analysis performed before the onset of treatment showed that five of these pts had a wild-type ABL sequence, while the remaining seven pts had evidence of various imatinib-resistant KD mutations (G250E, Y253H, E255K, D276G, M351T). At the time of relapse, however, most of the original mutant clones had disappeared, whereas mutations at residues 315 (T315I or T315A) and/or 317 (F317L or F317I) had invariably emerged in all but one pt. This pt was found to have developed a novel K356R mutation which is now under characterization. Our results indicate that residues 315 and 317 are mutation hotspots in dasatinib-resistant pts, according to the experimental observation that they are both involved in inhibitor binding. They also provide a proof of principle that novel, inhibitor-specific mutant variants (i.e., T315A, F317I, K356R) may be selected, and raise some concerns about the limitations of single-agent treatment in the long term disease control of advanced phase-CML or Ph+ ALL pts. Supported by European LeukemiaNet, AIL, AIRC, FIRB and PRIN projects.
Table 1 Pt Disease Mutation(s) before dasatinib start Best HR Best CgR Months on dasatinib Mutation(s) at relapse NE, not evaluated Primary resistance 1 CML/AP WT NR none 4 T315I 2 CML/AP T315I NR NE 1 T315I 3 CML/myBC T315I NR NE 1 T315I 4 CML/myBC F317L NR none 3 F317L 5 CML/lyBC T315I NR NE 1 T315I 6 Ph+ ALL T315I, M351T, L387M NR NE 2 T315I, M351T, L387M 7 Ph+ ALL T315I NR NE 1 T315I 8 Ph+ ALL F359V NR NE 2 T315I Acquired resistance 9 CP WT CHR minor 15 T315I 10 CML/myBC G250E NEL none 8 F317L 11 CML/lyBC Y253H CHR complete 9 CHR T315I 12 CML/lyBC WT CHR complete 4 T315I 13 CML/lyBC E255K CHR none 3 E255K, T315I 14 CML/lyBC D276G CHR complete 7 T315I 15 CML/lyBC WT CHR partial 9 F317L 16 Ph+ ALL E255K CHR NE 4 T315I 17 Ph+ ALL Y253H CHR complete 13 T315A, F317L, D276G 18 Ph+ ALL M351T CHR complete 13 M351T, F317L 19 Ph+ ALL WT CHR complete 6 F317I 20 Ph+ ALL WT CHR complete 4 K356R