Heterogeneous resistance to quizartinib in acute myeloid leukemia revealed by single-cell analysis

Abstract

Genomic studies have revealed significant branching heterogeneity in cancer. Studies of resistance to tyrosine kinase inhibitor therapy have not fully reflected this heterogeneity because resistance in individual patients has been ascribed to largely mutually exclusive on-target or off-target mechanisms in which tumors either retain dependency on the target oncogene or subvert it through a parallel pathway. Using targeted sequencing from single cells and colonies from patient samples, we demonstrate tremendous clonal diversity in the majority of acute myeloid leukemia (AML) patients with activating FLT3 internal tandem duplication mutations at the time of acquired resistance to the FLT3 inhibitor quizartinib. These findings establish that clinical resistance to quizartinib is highly complex and reflects the underlying clonal heterogeneity of AML.

Figure 1.

D835 mutations in native FLT3 cause resistance to quizartinib. (A) Normalized cell viability of Ba/F3 populations stably expressing FLT3-mutant isoforms after 48 hours in various concentrations of quizartinib (error bars represent standard deviation of triplicates from the same experiment). (B) Western blot analysis using the indicated antibodies performed on lysates from interleukin-3–independent Ba/F3 populations expressing the FLT3-mutant isoforms indicated. Cells were exposed to quizartinib at the noted concentrations for 90 minutes.

Figure 2.

Genetic heterogeneity revealed by single-cell sorting of samples from patients relapsed on quizartinib. (A) Schematic of experimental setup for single-cell sorting followed by FLT3 mutation genotyping. Percent of cells with indicated FLT3 genotype in patients (B) 1005-007, (C) 1005-009, and (D) 1009-003. Each circle represents a particular FLT3 genotype. The relative size of circles is representative of the relative proportion of the overall population with that genotype.

Figure 3.

Genetic heterogeneity in colonies grown from a patient relapsed on quizartinib. (A) Schematic of experimental setup for genotyping of colonies grown from bone marrow of a patient relapsed after a response to quizartinib. (B) Number of colonies after 14 days in methylcellulose in dimethyl sulfoxide (DMSO) and 20 nM quizartinib. Error bars represent standard error of triplicate platings. (C) FLT3 genotype of single colonies plucked from methylcellulose after 14 days of growth in DMSO or 20 nM quizartinib. Each circle represents a particular FLT3 genotype. The relative size of circles is representative of the relative proportion of the overall population with that genotype. Number of colonies with each genotype is shown for DMSO (left) and quizartinib 20 nM (right).

Figure 4.

Models for evolution of polyclonal FLT3-dependent and non-FLT3–dependent resistance after TKI treatment. Schematic representation illustrates proportion of FLT3 mutant and native FLT3 leukemia cell population at treatment time points. (A) At diagnosis, FLT3-ITD⁺ cells represent a portion of bulk tumor. Debulking after chemotherapy results in Remission #1, but is followed by emergence of chemotherapy-resistant FLT3-dependent leukemia cell populations at the time of Relapse #1. FLT3 TKI treatment induces Remission #2, but at the time of Relapse #2, multiple drug-resistant clones evolve, including FLT3 cells containing ITD and native FLT3 cells. (B) At diagnosis, FLT3-ITD⁺ cells represent a portion of bulk tumor. FLT3 D835V and D835Y mutations arise on native FLT3 alleles in separate leukemic stem cells, and these clones persist through chemotherapy. Debulking after chemotherapy results in Remission #1 but is followed by the emergence of chemotherapy-resistant FLT3-dependent leukemia cell populations at the time of Relapse #1. FLT3 TKI treatment induces Remission #2, but at the time of Relapse #2, multiple drug-resistant clones arise, including FLT3-ITD–containing clones (D835-mutant and off-target resistant), expansion of TKI-resistant native FLT3 D835–mutant clones, and off-target–resistant native FLT3 clones.