New sequencing studies in acute myeloid leukemia (AML) patients have identified specific mutations in the FLT3 gene that are associated with resistance to treatment with FLT3 inhibitor drugs, and could point the way to the development of more effective, personalized AML therapeutics.
The work, by a University of California, San Francisco (UCSF)-led team, used Pacific Biosciences’ single molecule real-time (SMRT) sequencing platform to look more closely at the sequences of activating internal tandem duplication (ITD) mutations in the FLT3 (FLT3-ITD) alleles carried by patients who had relapsed after treatment with the investigational FLT3 inhibitor AC220 (quizartinib; Ambit Biosciences).
Their results identified point mutations within the kinase domain of FLT3-ITD that were evident at the point of treatment relapse but weren’t present before AC220 therapy, and which led to one of three alterations in the FLT3 structure that conferred substantial resistance to AC220. Reporting in Nature, Neil P. Shah, Ph.D., and colleagues conclude that their findings “validate FLT3-ITD as a therapeutic target in human AML.” Their published paper is titled “Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia.”
FLT3-ITD mutations occur in approximately 20% of AML patients and are associated with poor prognosis. However, previous work has been unable to determine definitively whether such mutations play a causal in AML development and progression, or whether they are passenger mutations that occur alongside the true genetic drivers. Indeed, the researchers note, the lack of “convincing clinical evidence” with early FLT3 inhibitors indicated that FLT3-ITD probably represents a passenger lesion. And while studies with the broad-spectrum kinase inhibitor sorafenib have "anecdotally" achieved bone marrow remission in FLT3-ITD+ AML patients, whether the drug’s mechanism of action is mediated through FLT3 inhibition is still unclear.
In fact to date, they add, “perhaps the most compelling evidence so far that FLT3-ITD could represent a driver mutation in AML was the identification of a secondary FLT3 kinase domain mutation that conferred moderate resistance to the multikinase inhibitor PKC412 in a single FLT3-ITD+ patient who relapsed after an initial bone marrow response.”
To try and provide more definitive data on whether FLT3-ITD mutations in AML are drivers or passengers in disease progression, the UCSF researchers sequenced the FLT3-ITD genes in eight patients who had relapsed after treatment with AC220 in a Phase II clinical trial. They looked for the presence of drug-resistance mutations in the kinase domains of FLT3-ITD in samples taken both before treatment and after relapse.
In every case, subcloning and sequencing of individual FLT3-ITD alleles revealed the presence of mutations at the time of relapse that weren’t detected prior to drug treatment. Importantly, the mutations were all found at two of three critical residues that the team had already identified as AC220 resistance-conferring mutations using an in vitro assay.
The researchers then applied the Pacific Biosciences SMRT sequencing platform to more accurately sequence the ITD region and kinase domain of the FLT3-ITD alleles in each patient’s sample. They claim the technique effectively provides sequencing reads of sufficient length to allow focused interrogation of FLT3-ITD alleles, and enabled them to obtain from individual patient samples hundreds of reads spanning the ITD region and kinase domain with an average read length of greater than 1 kilobase (kb).
Using this technique the presence of resistance-conferring kinase domain mutations were identified in the FLT3-ITD alleles of all eight patient samples.
Moreover, all the mutations identified at relapse were present at a frequency significantly higher than that observed in a normal controls, and were not detected before treatment. And the substitutions that conferred a high degree of resistance to AC220 in vitro were also associated with an equivalent level of resistance to sorafenib in cell-based growth and biochemical assays.
By marrying the obtained sequence data with known structure of the FLT3 kinase domain, the researchers postulated that the mutations lead to conformational changes that obstruct the binding of AC220 and sorafenib. Alternatively, they note, the mechanism of AC220 resistance may relate to increased kinase activity that impacts on downstream effector activation.
Whatever then mechanisms, the investigators say their findings demonstrate that FLT3-ITD can represent a driver lesion and valid therapeutic target in human AML. “AC220-resistant FLT3 kinase domain mutants represent high-value targets for future FLT3 inhibitor development efforts.”