Thus, compound mutations involving t315i and ploop mutations were. Among these mutations, the most frequent mutation, t315i mutation, is resistant to all presently registered bcrabl tyrosine. Molecular diagnostics lab t315i bcrabl mutation analysis. Characterizing of four common bcrabl kinase domain mutations. T315imutated bcrabl in chronic myeloid leukemia and. Resistant t315i clone may be present prior to initiating dasatinib, which could expand under selective pressures during treatment. The mutation burden was quantified by quantitative pcr using allele. Here we describe a typeii t315i inhibitor 2 gnf7, based upon a 3,4dihydropyrimido4,5dpyrimidin21hone scaffold which is capable of potently inhibiting wildtype and t315i bcrabl as well as other. Twelve patients displaying t315i mutations and 15 with bcrabl1 wt were examined either by direct dna sequencing or by pna directed pcr clamping displaying identical readout.
Thus, compound mutations involving t315i and ploop mutations were the major components of multiple mutations, and some lowlevel mutations with potential clinical significance were detected by subcloning. Association of t315i mutation with resistance to tyrosine. The early results from these compounds are encouraging and it is anticipated that physicians will have additional drugs at their disposal for the treatment of patients. The aim of this study was to evaluate the prevalence of this mutation in bcrablpositive cml and all patients.
Although the t315i mutation does not disturb the overall structure of the bcrabl protein, it affects the topology of the atp binding region. Detection of t315i mutation in tkiresistant patients is extremely unfavorable factor for survival, especially in the advanced phase cml, and it is a great reason for switching to ponatinib or other new potential investigated drugs if possible. A singletube allele specificpolymerase chain reaction to detect t315i resistant mutation in chronic myeloid leukemia patients. Allogeneic stem cell transplantation for patients with. Download fulltext pdf download fulltext pdf prevalence of bcrabl t315i mutation in malaysian patients with imatinibresistant chronic myeloid leukemia article pdf available december 2018. Bcrabl point mutations and tki treatment in cml patients. The second generation of bcrabl inhibitors nilotinib, dasatinib, and bosutinib developed to override imatinib resistance are not active against the t315i gatekeeper mutation. Overcoming bcrabl t315i mutation by combination of gnf2. Ponatinib is the only known tki which showed clinical efficiency in t315i positive cml patients. T315i is one of the most common acquired mutations in. Of the 9 evaluable cml patients who had the t315i mutation, all achieved a major cytogenetic response, 8 patients had a complete cytogenetic response, and 7 patients had a major molecular response. Similarly to abl kinase inhibitors akis, gnf2 failed to inhibit activity of mutated bcrabl carrying the t315i mutation.
The t315i mutation, one of the most common mutations of bcrabl. Pharmacokinetics of dasatinib for philadelphiapositive acute. The clonal evolution of two distinct t315ipositive bcr. Pharmacokinetics of dasatinib for philadelphiapositive. Pdf prevalence of bcrabl t315i mutation in malaysian. Ap24534 is an orally active multitargeted kinase inhibitor that potently inhibits bcrabl t315i. Pdf clinical features and outcomes in chronic myeloid. All patients who harbor the t315i bcrabl1 mutation or who have two or more mutations in the same bcrabl1 molecule is particularly challenging since first and secondgeneration tyrosine kinase inhibitors tkis are ineffective. Detection of t315i using digital polymerase chain reaction in. Overcoming bcrabl t315i mutation by combination of gnf2 and. In this study, we retrospectively traced small clones with the t315i mutation using dpcr at several specific time points in patients who received allogeneic hsct following dasatinibbased chemotherapy. Compound mutations involving t315i and ploop mutations. Bcrabl t315i, a mutant resistant to all approved abl kinase inhibitors, is emerging as a common pathway of failure on all three inhibitors.
The finding of a t315i mutation in the bcrabl gene characteristic of cml is associated with clinical resistance to imatinib, nilotinib, and dasatinib. Incidence of t315i mutation in bcrablpositive cml and all. The prognosis of patients is dismal for those with a bcr. Targeted therapy of philadelphiapositive all and cml patients using imatinib im has caused significant changes in treatment course and has increased the survival of patients. The substitution of the threonine residue at position 315 of the bcrabl1 protein by isoleucine t315i places the bulky isoleucine side chain in the. Detection of t315i using digital polymerase chain reaction. Clinical activity of ponatinib in one patient with chronic. The clonal evolution of two distinct t315ipositive bcrabl1. Strategies to circumvent the t315i gatekeeper mutation in the.
Pdf molecular screening for t315i and f317l resistance. Prevalence of bcrabl t315i mutation in malaysian patients with imatinibresistant chronic myeloid leukemia. Clinical features and outcomes in chronic myeloid leukemia with t315i mutation. The proportion of patients with t315i mutation were higher than that of control group, with a significant risk association to develop resistance to tki therapy odd ratio 2. F311l, m351t, and t315i and all patients with preexisting bcrabl mutations exhibited imatinib resistance 33. We monitored the effect of gnf2 with akis on the proliferation and clonigenicity of the different baf3 cells. Mk0457, a novel kinase inhibitor, is active in patients with chronic myeloid leukemia or acute lymphocytic leukemia with the t315i bcrabl mutation. Therapeutic options against bcrabl1 t315ipositive chronic. Optimizing kinase inhibitor selection for cml patients.
The t315i rgq pcr kit enables detection of the t315i mutation in the abl gene exon 6 using realtime pcr on the rotorgene q 5plex hrm instrument. In this study, we investigated axitinib activity against ponatinibresistant cells and found that axitinib inhibited cellular growth and apoptosis in baf3 t315imutant. As expected, when the affinity between pna and template. Atp binding site codons 315 and f317, catalytic domain codons 350363 and activation loop codons 381407. In a patient with cml, we identified a pcr product corresponding to e19a2 rearrangement harboring t315i mutation. Responses were seen against each of the 15 mutations present in 1 pt at bl, including t315i, e255v, f359v, y253h. The asopcr to detect t315i mutation was performed using following sets of primers. It might represent a treatment option for patients with this mutation and complement the current clinically. All of the bcrabl mutants are inhibited by the 2nd generation inhibitors with the exception of the t315i mutant. T315i mutation occurs in atp binding site where im is found, causing resistance to treatment of patients with im. Association of t315i mutation with resistance to tyrosine kinase. Nov 27, 2012 similarly to abl kinase inhibitors akis, gnf2 failed to inhibit activity of mutated bcrabl carrying the t315i mutation.
Pna fish and pna direct pcr clamping allow to detect t315i mutation in cml patients. Baf3 cells harboring native or t315i mutated bcrabl constructs were treated with gnf2 and akis. T315i mutation exerts a dismal prognosis on adult bcrabl1. Bcrabl1 compound mutations combining key kinase domain. Ponatinib, blinatumomab, chemotherapy and transplant are the currently. Encouragingly, responses in chronic phase appeared to be remarkably durable, considering the heavily pretreated and highly resistant nature of. Incidence of t315i mutation in bcrablpositive cml and. Characteristics and outcomes of patients with chronic myeloid. T315i mutation of bcrabl1 into human philadelphia chromosomepositive leukemia cell lines by homologous recombination using the crisprcas9 system. Cleavage of bcrabl transcripts at the t315i point mutation by. Characteristics and outcomes of patients with chronic. Pdf chronic myelogenous leukemia cml is a hematological stem cell disorder caused by. This mutation confers full resistance to all avail able tki, except ponatinib, which was.
However, emergence of compound mutations in a bcrabl1 allele may confer ponatinib resistance. The gatekeeper mutation t315i is responsible for a general resistance to small molecules. It does not form a hydrogen bond with the side chain of thr 315 in native abl. Prevalence of bcrabl t315i mutation in malaysian patients with.
A typeii kinase inhibitor capable of inhibiting the t315i. At the time the mutation was detected, 8 patients were in the chronic phase cp, 7 in the accelerated phase. Association of t315i mutation with resistance to tyrosine kinase inhibitor therapy in patients with cml attended the oncologyhematology center in alnajaf city of iraq. The q252h t315i, t315i m351t, t315i f359v, and t315i h396r mutants exhibited marginal ponatinib sensitivity ic 50. Dynamics of bcrabl kinase domain mutations in chronic. Apr 16, 2009 the gatekeeper mutation t315i confers resistance against small molecules by increasing or restoring the ablkinase activity accompanied by aberrant transphosphorylation of endogenous bcr, even in. Ponatinib therapy in recurrent philadelphia chromosome. The t315i mutation occurs in approximately 15% of imatinibresistant patients with a abl kinase domain mutation and may be more frequently detected in patients with advanced cml and ph. By contrast, t315i was the only mutation recovered in mutagenesis screening assays when high doses of nilotinib were used, and in phase ii studies of dasatinib for patients with cml after imatinib failure, t315i and f317li were the mutations more frequently encountered. The gatekeeper mutation t315i confers resistance against small. Allogeneic stem cell transplantation sct is currently the only curative treatment option for chronic myeloid leukemia cml patients with bcrabl t315i mutations. For monitoring of the levels of the thr to ile mutation at codon 315 t315i of the bcrabl kinase seen in cml patients who have developed resistance to imatinib or other kinase inhibitors.
The product contained different mutation sites encoding abl amino acids 207 to 414. T315i mutation of bcrabl1 into human philadelphia chromosome. The t315i substitution is one of the most common mutations found in imatinibresistant patients and the clinical importance of this mutation is expected to grow because it is predicted to represent the major mechanism of resistance to secondgeneration inhibitors, such as dasatinib and nilotinib 6 8. In this study, we investigated axitinib activity against ponatinibresistant cells and found that axitinib inhibited cellular growth and apoptosis in baf3 t315i mutant. Despite the remarkable success of imatinib against bcrabl, development of secondary resistance, most often due to point mutations in the bcrabl tyrosine kinase tk domain, is quite common. Join our community just now to flow with the file mutations and make our shared file collection even more complete and exciting. Pdf the development of imatinib mesylate im, the first generation specific tyrosine kinase inhibitor tki of bcrabl, has had a major impact in. However, axitinib, a vascular endothelial growth factor receptor inhibitor, is effective against this mutation. T315i bcrabl mutation analysis quantitative indication. Compound mutations involving t315i and ploop mutations are. Pdf the bcrabl mutations t315i and y253h do not confer. Antileukemic activity of axitinib against cells harboring.
Detection of bcrabl t315i mutation by peptide nucleic acid. The kit provides all necessary reagents, optimized for rapid and sensitive detection of a low percentage of mutant dna in a background of wildtype genomic dna. Therefore, detection of t315i mutationin cml patients are clinically useful in the selection of. We assessed the prognostic value of small clones with the t315i mutation at specific time points using the novel technology digital polymerase chain reaction pcr, which is more sensitive than direct sequencing. Chronic myeloid leukemia harboring t315i and f317l. At the second step, specific pcr was performed for common mutations y253h, t315i, and e255k and m351t. The key structural feature of the molecule is a carboncarbon triple bond linkage that makes productive hydrophobic contact with the side chain of. In one t315i positive patient with more than 1 log increase of bcrablabl level, the mutation probably expanded as a result of selective pressure exerted by im 4.
Sequencing of the bcrabl gene in relapsed patients revealed a set of mutants that mediate drug resistance. T315i is one of the most common acquired mutations in this domain, which occurs in atp binding site and inhibits the formation of hydrogen bond with im. Characterizing of four common bcrabl kinase domain. Aurora a kinase inhibitor aki603 induces cellular senescence. Bcrabl tyrosinekinase inhibitors tki are the firstline therapy for most patients with chronic myelogenous leukemia cml. At the time of mutational analysis, during dasatinib treatment, the t315i clone was 100% and the quantification of bcr. Pdf the bcrabl mutations t315i and y253h do not confer a. The t315i is a unique mutation because of its resistance to all approved bcrabl inhibitors, prior to ponatinib. The cml patients with t315i mutation have the median of pfs only 11. Several 3rd generation inhibitors such as ap24534, vx680 mk0457, pha739358, ppya, xl228, sgx70393, fty720 and tg1011 are being developed to target the t315i mutation. More than 90% of cml cases are caused by a chromosomal abnormality that results in the formation of a socalled philadelphia chromosome. Clinical significance of t315i abl kinase domain mutation. Abl t315i mutation, which is resistant to tkis including second.
M244v t315i was the only t315i inclusive compound mutant in the panel predicted to be sensitive to ponatinib and rebastinib at clinically achievable. Imatinib mesylate, a selective inhibitor of bcrabl, has been successful in chronic myeloid leukemia clinical trials, but shortlived remissions are usually observed in blast crisis patients. Pdf inhibitors of abl and the ablt315i mutation boris. Strategies to circumvent the t315i gatekeeper mutation in. Clinical features and outcomes in chronic myeloid leukemia. To overcome imatinib resistance, more potent tyrosine kinase inhibitors tkis such as nilotinib, dasatinib, and bosutinib have been developed with demonstrable activity against most of the bcrabl kinase domain mutations seen in patients treated with imatinib, with the notable exception of the t315i mutation. Ponatinib, blinatumomab, chemotherapy and transplant are the. Mutation specific restriction enzymes rsai, ddei, mnli and ncoi were applied for unveiling the proportion of mutated and unmutated alleles.
Chronic myeloid leukemia harboring t315i and f317l mutations. The t315i mutant form of bcrabl lacks a threonine residue, which provides a. Detection of bcrabl t315i mutation by peptide nucleic. This abnormality was discovered by peter nowell in 1960 and is a consequence of fusion between the abelson tyrosine kinase gene at. The gatekeeper mutation t315i confers resistance against small molecules by increasing or restoring the ablkinase activity accompanied by aberrant transphosphorylation of. Panel a and b show t315i positive cells from a patient with 60 % of mutated cells positive cells, panel c and d show t315i positive cells from a patient with 45 % of mutated cells. We found that clinically reported bcrabl1 compound mutants center on 12 key.
The early stage of chronic myeloid leukemia is triggered by the tyrosine kinase bcrabl. Bad prognosis and resistance to tkis in such cases boosted development of novel drugs. Pdf inhibitors of abl and the ablt315i mutation glenn. Ponatinib ap24534, iclusig is a multitargeted tki optimized using structurebased drug design to bind to the inactive conformation of abl and abl t315i. However, emergence of the t315i mutation has been found to be a main cause of failure after dasatinibcontaining treatments. The bcrabl fusion transcript encodes the bcrabl tyrosine kinase tk, which causes chronic myelogenous leukemia cml. T315i affects a contact residue for most available tyrosine kinase inhibitors tkis, thus patients with chronic myeloid leukemia cml harboring this mutation are resistant to these agents. However, how dna mutation occurs, particularly the t315i mutation. The highly conserved threonine 315 residue is called the gatekeeper because it is located near the abl catalytic domain controlling the access to a hydrophobic pocket of the enzymatic active site. Pdf a singletube allele specificpolymerase chain reaction to. It is caused by a single cytosine to thymine c t base pair substitution at position 944 of the abl gene codon 315 of the abl protein sequence resulting in amino acid threonine being substituted by isoleucine at that. The bcrabl mutations t315i and y253h do not confer a growth advantage in the absence of imatinib.
In other words, growth competition between mutant clones can lead to a change in major mutations by varying kinase activity from an addon mutation within the clone 6,30, and the presence of the t315i mutation appears to play an important role in the selection and deselection processes of compound mutant clones 24,31,32. Acquired mutations in tyrosine kinase domain of bcrabl protein are a mechanism for development of resistance. Thus, the usefulness of crisprcas9 system for functional analysis of somatic mutations in cancers was demonstrated. Pdf inhibitors of abl and the ablt315i mutation researchgate. The results and pcr conditions are summarized in fig.
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