Lipidomic Analysis of Flt3-ITD AML Cells Treated with Intensive Chemotherapy and Independent of Concomitant Sorafenib Display a Profile of Increased Susceptibility to Ferroptosis

Introduction

The randomised trial of (pre-allogeneic transplant) 2:1 sorafenib to placebo plus intensive chemotherapy in Flt3-ITD AML (Blood, 2023 Dec 7) did not meet the primary end-point of improved event-free survival (EFS) after a median 49 months of follow-up. To investigate changes in the lipidome of AML, we studied up to 810 lipid species in this cohort of AML patients, in paired samples from at diagnosis to post-induction chemotherapy, and a smaller number at relapse.

Methods

From the 98 AML patients recruited to the above trial, bone marrow (BM) and peripheral blood (PB) cells and plasma samples were available for lipidome analyses from 47 patients. Post sample protein extraction and quality control (QC), we had 15 BM cells; 11 BM plasma; 29 PB cells and 28 PB plasma samples paired from at diagnosis to post-induction chemotherapy timepoints. There were combined BM and PB cells and plasma from 4 patients at relapse for analysis. All QC viable BM and PB cell and plasma samples together with 3 separate QC samples underwent lipid extraction for liquid chromatography-tandem mass spectrometry (LC-MS) analysis using an Agilent 1290 Infinity I/II LC machine. Following separation by LC, lipids were assessed via Agilent 6495C Triple Quadrupole Mass Spectrometer for abundance quantification. Relative lipid abundances were categorised into lipid classes and species.

Two-way ANOVAs was performed to analyse the potential effect of Sorafenib on AML lipid abundances. Repeated Measures (RM) ANOVAs were run to identify differences in lipid abundances across clinical timepoints. Student t-tests were performed on differences of lipid species of interest. All p values underwent Benjamini-Hochberg (BH) corrections to decrease the false discovery rate. Significance was set at p ≤ 0.05 for all analyses.

Results

The initial analysis was aimed to identify the effect of the FLT 3 inhibitor, Sorafenib on lipid abundances in Flt3-ITD AML cases. The lipidome of all 4 sample cohorts (BM and PB cells and BM and PB plasma) showed no statistical differences in lipid abundance across Sorafenib and placebo groups, paired from at diagnosis to post-induction timepoints.

Subsequently, all lipid analyses were grouped as one cellular sample i.e. BM cells or PB cells for each respective timepoint of AML at diagnosis, post-induction or at relapse. Comparative lipid analyses of AML BM cells at diagnosis to post induction chemotherapy showed an increase in total phospholipids, in particular phosphatidylcholine (PC, p=1.21E-03), phosphatidyl ethanolamine (PE, p=2.54E-03) and phospholipids of ³4 PUFA (polyunsaturated fatty acids, p=4.62E-11) composition whilst phospholipids of MUFA (monounsaturated fatty acids) composition were reduced (p=2.54E-09) in AML cells at diagnosis. These same statistically significant differences in lipid class and species were seen in AML PB cell sample analyses at diagnosis in comparison to post induction timepoints.

Deeper analyses of PUFA species based on carbon:double bond numbers shows PUFA 20:4 (p=2.64E-10) and 22:6 (p=3.87E-03) species were significantly higher in Flt3-ITD AML cells at diagnosis compared to post-induction chemotherapy. Further, these PUFA species, for example, PUFA 20:4 amount (p=9.91E-14) re-increases in relapsed AML cells in comparison to post-induction cells. These lipid species measurements allowed formulation of an estimated cellular (phospholipid) peroxidation index (CPI) based on relative hydrogen atom transfer rate constants of fatty acids. The CPI in both AML BM (p=2.33E-12) and PB (p=1.06E-12) cells was higher at diagnosis compared to post-chemotherapy, and also, re-increased in AML relapsed cells compared to post-induction i.e. remission samples.

Conclusions

PUFA phospholipids are sensitive to lipid peroxidation, a critical process in iron-dependent ferroptosis. In contrast MUFA phospholipids are relatively resistant to lipid peroxidation and hence, ferroptosis. The CPI is an indirect read-out of ferroptosis sensitivity. These lipidome results suggest Flt3-ITD AML cells, both at diagnosis and at relapse have high levels of lipid peroxidation and hence, may be sensitive to ferroptosis.