The AML microenvironment catalyzes a step-wise evolution to gilteritinib resistance
Joshi, Sunil and Nechiporuk, Tamilla and Bottomly, Daniel and Piehowski, Paul D. and Reisz, Julie A. and Pittsenbarger, Janét and Kaempf, Andy and Gosline, Sara J. C. and Wang, Yi-Ting and Hansen, Joshua R. and Gritsenko, Marina A. and Hutchinson, Chelsea and Weitz, Karl K. and Moon, Jamie and Fillmore, Thomas L. and Tsai, Chia-Feng and Schepmoes, Athena A. and Shi, Tujin and Arshad, Osama A. and McDermott, Jason E. and Babur, Ozgun and Watanabe-Smith, Kevin and Demir, Emek and D'Alessandro, Angelo and Liu, Tao and Tognon, Cristina E. and Tyner, Jeffrey W. and McWeeney, Shannon K. and Rodland, Karin D. and Druker, Brian J. and Traer, Elie, The AML Microenvironment Catalyzes a Step-Wise Evolution to Gilteritinib Resistance (January 22, 2021). Available at SSRN: https://ssrn.com/abstract=3771433 or http://dx.doi.org/10.2139/ssrn.3771433
- Organism: Homo sapiens
- Instrument: TSQ Altis
- SpikeIn:
Yes
- Keywords:
: AML, FLT3, Tumor Microenvironment, Gilteritinib, Aurora Kinase B, NRAS, Drug Resistance, Tyrosine Kinase Inhibitor
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Lab head: Tao Liu
Submitter: Yi-Ting Wang
Our study details the stepwise evolution of gilteritinib resistance in FLT3-mutated acute myeloid leukemia (AML). Early resistance is mediated by extrinsic factors from the bone marrow microenvironment, which protect residual leukemia cells. Removing supportive extrinsic ligands drives evolution of late, intrinsic resistance. Whole exome sequencing, CRISPR/Cas, metabolomics, proteomics, and pharmacologic approaches were used to mechanistically define early and late resistance. Early resistant cell lines grow more slowly, undergo metabolic reprogramming, and are dependent upon Aurora kinase B (AURKB). Late resistance cell lines are characterized by expansion of pre-existing NRAS mutant subclones and continued metabolic reprogramming. This model closely mirrors the timing and mutations of AML patients treated with gilteritinib. Pharmacological inhibition of AURKB resensitized both early resistant cell lines and primary leukemia cells from gilteritinib-treated AML patients, supporting a strategy to target early resistant AML cells with AURKB inhibitors and gilteritinib before the expansion of pre-existing resistance mutations can occur.
Stable Isotope-Labeled Peptides
Proteotypic peptides for the target proteins were selected for targeted proteomics analysis based on well-accepted criteria, and the corresponding crude heavy stable isotope-labeled peptides were synthesized with 13C/15N on C-terminal lysine or arginine (New England Peptide, Gardner, MA). The heavy peptides were dissolved individually in 15% acetonitrile (ACN) and 0.1% formic acid (FA) at a concentration of 2 mM and used for creating a peptide mixture with a final concentration of 5 pmol/μL for each peptide.
SRM Assay Development
The heavy peptides in the peptide mixture were evaluated for peptide response and fragmentation pattern using LC-SRM. For each peptide, transition settings were as follows: (1) precursor charges: 2, 3 and 4; (2) ion charges: 1, 2 and 3; (3) ion type: y; and (4) m/z window: 250-1500. Transition lists were generated with optimal collision energy values by Skyline software (Version 20.1). LC-SRM was then used to evaluate all heavy peptides for stability of peptide retention time, reliable heavy peptides identification, transition interferences, and endogenous peptide detectability. In the end, 3 transitions per peptide were selected for the final assay configuration for targeted quantitation of a total of 244 peptides of the 123 target proteins.
LC-SRM
The digested patient samples were reconstituted in 2% ACN/0.1% FA and spiked with 5 fmol/µL heavy peptides for a final concentration of 0.25 µg/µL, and 2 µL of the resulting samples were analyzed by LC-SRM using a Waters nanoACQUITY UPLC system (Waters, Milford, MA) coupled to a Thermo Scientific TSQ Altis triple quadrupole mass spectrometer (Thermo Scientific, Waltham, MA). A 100 µm i.d. × 10 cm, BEH 1.7-µm C18 capillary column (Waters) was operated at a temperature of 44 ºC. The mobile phases were (A) 0.1% FA in water and (B) 0.1% FA in ACN. The peptide samples were separated at a flow rate of 400 nL/min using a 110-min gradient profile as follows (min:%B): 7:1, 9:6, 40:13, 70:22, 80:40, 85:95, 93:50, 94:95 and 95:1. The parameters of the triple quadruple instrument were set with 0.7 fwhm Q1 and Q3 resolution, and 1.2 s cycle time. Data were acquired in time-scheduled SRM mode (retention time window: 15 min).
Data Analysis
SRM data were analyzed using the Skyline software (Version 20.1). The total peak area ratios of endogenous light peptides and their heavy isotope-labeled internal standards (i.e., L/H peak area ratios) were exported for quantitation. Peak detection and integration were carried out according to two criteria: (1) same retention time and (2) similar peak area ratios for the transitions. All data were manually inspected to ensure correct retention time, peak detection and accurate integration.
gilteritinib resistance in FLT3-mutated acute myeloid leukemia (AML)
Created on 10/19/20, 5:37 PM