Abstract

Background: Resistance to imatinib in patients with chronic myeloid leukemia (CML) is commonly associated with reactivation of BCR-ABL due to kinase domain mutations or increased expression. Activation of SRC family kinases has been shown in isolated patients without evidence of BCR-ABL reactivation. We hypothesized that activating mutations of the RAS pathway (one of the major downstream mediators of BCR-ABL) or of leukemia-associated tyrosine kinases other than BCR-ABL may cause BCR-ABL-independent resistance.

Patients and Methods: CML patients with imatinib resistance were initially screened for BCR-ABL mutations. Two sets of nested primers were used to specifically amplify BCR-ABL. Sequence analysis covered the entire 5′ end of ABL including the Cap, SH3, SH2 and kinase domains. Bone marrow samples from patients without BCR-ABL mutations (N=17) were screened for mutations of NRAS, KRAS and FLT3 by direct DNA sequencing. Analysis of PTPN11 and PDGFR is ongoing and will be presented.

Results: We detected a KRAS exon 3 mutation (T58I) in 1/17 patients (5.8%) and confirmed this mutation by 2-directional sequencing of 3 independently amplified PCR products. No mutations in NRAS or FLT3 were observed. Analysis of archived samples revealed that T58I was absent while the patient was responding to 800mg of imatinib and achieved a major cytogenetic response (MCyR), became detectable before relapse and remained detectable (at approximately 30%) in 4 subsequent samples collected over a period of 18 months (Table 1), while the patient achieved a second MCyR on dasatinib. The T58I allele became undetectable prior to relapse on dasatinib. None of the samples tested were positive for BCR-ABL kinase mutations. T58I is an activating mutation of KRAS that has been detected in patients with Noonan syndrome who developed JMML (Schubbert et. al, Nature Genetics, 2006, 38:331). We tested the functional relevance of T58I in the context of imatinib resistance in 32D cells co-expressing T58I and BCR-ABL, with KRAS G12D and native KRAS as controls. Sensitivity to imatinib was reduced (G12D>T58I>native), with 15% 32D cells expressing KRAS T58I retaining viability at 2.5 μM over 3 Days. These data demonstrate that T58I confers partial imatinib resistance, although it does not induce complete growth factor independence.

Conclusion: Activating KRAS mutations may contribute to resistance or disease persistence in CML patients treated ABL kinase inhibitors.

Table 1:

Follow-up summary of patient with KRAS T58I mutation

Sample dateTreatment prior to cytogenetic evaluation (mg/day)Cytogenetics (Ph+ metaphases)FISH (Bcr-Abl)%KRAS T58I Allele%Bcr-Abl kinase domain
Feb-02 Imatinib 400 11/17 41.5 WT 
Dec-02 Imatinib 400 20/20 78 WT 
Dec-03 Imatinib 600–800 +Arsenic trioxide 5/25 31.5 WT 
Oct-04 Imatinib 800+Arsenic trioxide 7/20 14.5 31.8 WT 
Jun-05 Imatinib 800 17/20 62.5 ND WT 
Oct-05 Dasatinib 140 ND 23 28.8 WT 
Dec-05 Dasatinib 100 6/20 24.5 ND WT 
Mar-06 Dasatinib 80 1/1 13.5 37.4 WT 
Apr-06 Dasatinib 80 1/12 10 ND WT 
Sep-06 Dasatinib 80 3/20 24 34.9 WT 
Dec-06 Dasatinib 80 1/3 29 33.1 WT 
Feb-07 Dasatinib 80 4/20 51 WT 
May-07 Dasatinib 80 19/20 60.5 WT 
Sample dateTreatment prior to cytogenetic evaluation (mg/day)Cytogenetics (Ph+ metaphases)FISH (Bcr-Abl)%KRAS T58I Allele%Bcr-Abl kinase domain
Feb-02 Imatinib 400 11/17 41.5 WT 
Dec-02 Imatinib 400 20/20 78 WT 
Dec-03 Imatinib 600–800 +Arsenic trioxide 5/25 31.5 WT 
Oct-04 Imatinib 800+Arsenic trioxide 7/20 14.5 31.8 WT 
Jun-05 Imatinib 800 17/20 62.5 ND WT 
Oct-05 Dasatinib 140 ND 23 28.8 WT 
Dec-05 Dasatinib 100 6/20 24.5 ND WT 
Mar-06 Dasatinib 80 1/1 13.5 37.4 WT 
Apr-06 Dasatinib 80 1/12 10 ND WT 
Sep-06 Dasatinib 80 3/20 24 34.9 WT 
Dec-06 Dasatinib 80 1/3 29 33.1 WT 
Feb-07 Dasatinib 80 4/20 51 WT 
May-07 Dasatinib 80 19/20 60.5 WT 

Author notes

Disclosure: No relevant conflicts of interest to declare.