A sequencing study led by the University of California, San Francisco, in collaboration with Pacific Biosciences, has taken advantage of the PacBio RS's long reads to validate the disputed potential of internal tandem duplication mutations in the FLT3 gene as a therapeutic target in acute myeloid leukemia.
The team published results of the study in a letter in Nature this week. Sequencing samples from eight patients who showed response but then relapsed in a phase II trial of Ambit Bioscience's investigational drug AC220 (quizartinib), the researchers identified secondary mutations in residues of the FLT3-ITD kinase domain that conferred increased resistance to the FLT3 inhibitor. This, they reported, supports FLT3-ITD as a driver lesion and valid drug target in human AML.
The group also suggested in its report that the resistance-conferring kinase domain mutations in FLT3-ITD identified during the study could be high-value targets for future drug development.
Neil Shah, leader of the research team and a professor of hematology and oncology at UCSF, said that the study showed that future AML patients might benefit from a combination of multiple inhibitors, guided by their particular resistance-mutation profile — potentially as measured by sequencing technologies like the PacBio system.
Shah told Clinical Sequencing News this week that the group was "quite pleased by the fact that [the PacBio RS] detected a greater complexity of the mutational landscape at the time of relapse."
The platform "certainly has the potential to be adapted for use in clinical testing," he said. "What we were encouraged by is its potential to be applicable to a growing number of malignancies that are activated by a particular mutation and treated with a tyrosine kinase inhibitor that then develop drug-resistant mutations some distance away."
While he acknowledged that there is still much to learn about "the heterogeneity of tumors at the time of diagnosis, to say nothing of the time of acquired resistance," he noted that a sequencing-based approach "could, very easily, in the not-too-distant future have a substantial impact on clinical decision making provided that we have a number of effective next-gen therapeutics from which to choose."
Shah's lab is now working to identify such compounds and bring them to the clinic.
"We think that … if we had the ability to recapture inhibition of these drug-resistant mutant forms with perhaps another type of FLT3 inhibitor, we might once again regain remission in some or most of these patients," he said.
The recent validation study, meantime, was intended to answer open questions about FLT3-ITD's basic validity as a drug target so that such work could move forward, Shah said.
Mutations in FLT3 show up in about 20 percent of AML patients and have been known for years to be associated with poor prognosis. "The reason we got interested in the issue," he said, "is because despite this provocative evidence, the first several attempts to develop and test clinical inhibitors of FLT3 really failed to achieve anything that was very impressive, so that led to a lot of skepticism about whether or not it was a valid therapeutic target."
Nevertheless, "whether or not this first generation of drugs was really very effective or not was never completely known," he said.
In the study, Shah and his colleagues decided to use the clinical activity of a new drug, AC220, as a way to define whether FLT3-ITD is a valid target for inhibitory therapy.
Shah said Ambit Biosciences developed AC220 around the time most other pharmaceutical companies and academic investigators were moving away from FLT3 inhibitors.
"Lo and behold, in the original phase I study [of AC220] there were a substantial proportion of patients who had deep bone marrow remissions with some cases lasting well over a couple of years," Shah said.
"We thought if we analyzed patients [from the phase II trial,] that we would see some patients relapsing and that would enable us to really ascertain whether response and relapses were associated with FLT3 inhibition, [and subsequent] reactivation."
To test this, the group first identified AC220 resistance-conferring mutations using an in vitro saturation mutagenesis assay, finding three residues in the kinase domain of FLT3-ITD that conferred high degrees of AC220 resistance in cell proliferation and biochemical assays.
The researchers then evaluated eight pairs of pre-treatment and relapse samples from patients in the trial who initially showed reduction of bone marrow blasts to less than five percent, but then relapsed despite ongoing treatment. Subcloning and sequencing of individual FLT3-ITD alleles revealed that mutations in two of the three residues identified as critical in the in vitro experiments were present at the time of relapse but not detected pre-treatment in all eight patients.
For a more precise look, the group then used the PacBio RS to confirm and expand the results. The platform allowed the team to generate "hundreds of reads" from individual patient samples spanning the ITD region and kinase domain "with an average read length greater than one kilobase," the authors wrote.
The results were consistent with those found using the subcloning method, the team reported, but revealed in more detail the genetic complexity of evolving drug resistance in the disease.
"We basically found that one or more of the mutations we found in the lab screen evolved at the time AC220-resistant disease developed in all eight patients," Shah said. "That, in our minds, validates FLT3-ITD as a therapeutic target."
Shah highlighted the PacBio technology's applicability to the task of revealing resistance-conferring mutations "on the backbone of FLT3 activation."
"These ITD mutations typically occur [in a] domain which is several hundred nucleotides or almost a kilobase away from where some of these drug resistance mutations we found in the lab existed," he said.
"Moreover, some, such as D835 or Y842, have been previously described in the absence of an ITD mutation," said Shah. "So the onus was on us to really demonstrate these are occurring in the context of the FLT3-ITD mutation."
"The only technology we were aware of that would afford such long reads and the degree of sensitivity, short of our lab-based subcloning and sequencing method … was the PacBio RS platform," he said.
Shah said one limiting aspect of the group's approach was the focused scope of the sequencing method. "You have to have an idea of where you are looking for this platform to be used to its greatest potential," he said. Because the group only interrogated the one gene, there could still be other complementary mechanisms of resistance, as has been described with other kinase inhibitors. Further studies to identify these will be required, the authors wrote in the Nature letter.
Moving forward, Shah said his team is also interested in applying the same approach to other disease settings.
"What we're doing right now is looking at samples of patients treated with BCR-Abl kinase inhibitors [for other forms of leukemia] to get an idea of the degree of heterogeneity that exists in patients that failed more than one kinase inhibitor in a sequential manner.
Obviously we've had BCR-Abl kinase inhibitors for a while and there are patients that fail sequentially one, two, even three [drugs.] We've not yet had an opportunity to appreciate the degree of heterogeneity present," he said.
Shah's lab is working to discover compounds that can target AC220-resistant FLT3 mutations and has "identified several promising candidates," according to a UCSF statement.
In addition, Catherine Smith, who works in Shah's lab and is the first author of the Nature letter, is leading a clinical trial testing Plexxicon's FLT3 inhibitor PLX3397 in patients positive for FLT3-ITD mutations.
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