Dosing-specific modes of resistance to quizartinib in individual acute myeloid leukemia cells exposed by ResolveOME™ combined genomics and transcriptomics chemistry

S.D. Velivela1, I. Salas-González1, V.J. Weigman1, J.A.A. West1, G.L. Harton1, J.S. Zawistowski1

1BioSkryb Genomics, Inc., Durham, NC.

Dose and dose scheduling influence mechanisms of resistance to targeted therapeutics in clinical trials at both genomic and transcriptomic levels. To ascertain interplay between these levels, we created a model of quizartinib resistance in an acute myeloid leukemia (AML) cell line harboring an internal tandem duplication (ITD) mutation in FLT3 with continual near-IC50 (2nM) dosing, and a second dose-escalation model where an initial 200 pM dose was increased by 100 pM at weekly intervals until the growth rate of the cells was near that of the treatment-naïve parental cells. We hypothesized that in utilizing single cell analysis between models we would see distinct, single-nucleotide and copy number changes, in conjunction with transcriptional adaptation and therefore employed ResolveOME to concomitantly assess DNA and RNA modulation. Copy number variation (CNV) analysis with BaseJumper™ showed that treatment-naïve parental single cells harbored Chr.5, 6 & 13 trisomies and pentasomy of Chr.8, consistent with karyotypic analysis. In the continual dosing model, resistance was correlated with gain of Chr. 19q and loss of Chr.5 trisomy to 2n level, whereby these modulations where heterogenous in single cells. By contrast, the dose-escalation model revealed Chr. 1q gain and Chr. 2p loss and ploidy reduction of Chr. 8 to 2n level in a fraction of the individual cells. At the single nucleotide variation (SNV) level, we identified a secondary mutation N841K in the drug target FLT3, present in all resistant cells of the continual dosing model and previously found in AML patients. We identified FLT3 N841K only in a subset of dose-escalation single cells, and, intriguingly, the single cells that harbor this missense mutation lack the CNV specific to that model. This suggests differential mechanisms of resistance among single cells of a given treatment model, whereby N841K may be sufficient to drive resistance in isolation, but in other cells copy number alterations acquired during dosage increases were a primary mode of resistance. In addition to these divergent CNV paradigms we have defined AXL pathway bypass of FLT3 signaling inhibition via GAS6 upregulation in resistant cells of the continual-dosing model and contrast the magnitude of this adaptation between single dose-escalation single cells. These data highlight distinct and heterogeneous modes of single-cell drug resistance depending on the nature and duration of the dosing and spotlight the necessity of joint genomic and transcriptomic information to comprehensively elucidate drug resistance mechanisms.