FDA-approved ferumoxytol displays anti-leukaemia efficacy against cells with low ferroportin levels

Abstract

Acute myeloid leukaemia is a fatal disease for most patients. We have found that ferumoxytol (Feraheme), an FDA-approved iron oxide nanoparticle for iron deficiency treatment, demonstrates an anti-leukaemia effect in vitro and in vivo. Using leukaemia cell lines and primary acute myeloid leukaemia patient samples, we show that low expression of the iron exporter ferroportin results in a susceptibility of these cells via an increase in intracellular iron from ferumoxytol. The reactive oxygen species produced by free ferrous iron lead to increased oxidative stress and cell death. Ferumoxytol treatment results in a significant reduction of disease burden in a murine leukaemia model and patient-derived xenotransplants bearing leukaemia cells with low ferroportin expression. Our findings show how a clinical nanoparticle previously considered largely biologically inert could be rapidly incorporated into clinical trials for patients with leukaemia with low ferroportin levels.

Conflict of interest statement

Competing interests: J.G., C.K., H. Z., and M.G. have filed pending US patent application 15/759,161 and pending EU application 16845094.8 around the therapeutic use of Ferumoxytol. C.K. is currently an employee of Alnylam Pharmaceuticals.

Figures

Figure 1.. Reduced expression of FPN is a feature of leukaemia cell lines and primary AML samples.

SLC40A1 (FPN) transcript expression relative to normal bone marrow CD34+ progenitor cells (nBM CD34+) in (A) leukaemia cell lines (n=19 cell lines) and (B) primary AML samples (n=31 AML samples), blasts and CD34+ are shown. (C) Representative example of the flow cytometry analysis for FPN expression in cell lines, n = 20 cell lines measured and reported in Supplementary Table 1.(D) Percent expression of FPN using flow cytometry in cell lines (n=19 cell lines), and (E) primary AML samples (n=31 AML samples) in the indicated populations. Human AML cell lines stratified by FPN expression relative nBM CD34+ cells, are represented as low (blue) and high (red) to evaluate (F) iron content at 24h (n=3 cell lines for FPN-low and FPN-high) determined by ICP-MS and (G) cell survival, represented by cell counts at 24h and 48 h after exposure to ferumoxytol (FH) or PBS (n=3 per group and timepoint). Green and pink dot represents KCL22 and MV411 respectively through panels g-i. (H) Levels of gene expression for HMOX1, GCLC, and SLC7A11 relative to untreated samples (n=3 technical replicates per cell line, n=3 cell lines (shown with SEM) for FPN-low and FPN-high lines (n=6 cell lines total). (I) Cytosolic and mitochondrial staining for ROS using H2DCFDA and MitoSox, respectively reveal elevated ROS levels in FPN-low lines (n=2 independent measurements per cell line (n=3 cell lines per FPN low or FPN high group) per timepoint). SEM determined based on cell lines in each FPN low or high group as biological replicates). Each symbol represents a sample, bar represents the mean and the error bar represents the SEM. An ordinary one-way ANOVA was used for Fig. 1e. A two-tailed unpaired t-test was used for Figs. 1f-h. Flow experiments were performed one time with corresponding controls (unstained, isotype, or FMO). RT-PCR data was also performed once with each cell line and gene in triplicate.

Figure 2.. In vivo treatment of mice having blast crisis CML with ferumoxytol reduces leukaemia burden and improves overall survival.

(a) Schematic representation for the experimental plan to evaluate anti-leukaemia activity in vivo using the murine blast crisis CML model (bcCML). (b) Representative flow cytometry panel showing leukemic blasts (red boxes) in peripheral blood (PB), bone marrow (BM), and spleen (SPL) after the treatment with saline (blue, n=7 mice), Ara-C (red, n=4 mice), ferumoxytol 3 mg/kg (green, n=3 mice), ferumoxytol (FH) 6 mg/kg (purple, n=6 mice) flow analysis was performed once on the samples with controls (unstained, isotype, or FMO) present and samples run in triplicate. Scatter plots for the percent leukemic blasts for all animals from the indicated cohorts in PB (c), BM (d), and SPL (e). Each symbol represents an individual mouse. Bar represents the mean; error bar represents the SEM. (f) Spleen index (spleen weight normalized to body weight). (g) Schematic representation for the experimental plan to evaluate the effect of ferumoxytol in overall survival. (h) Percent leukemic blasts in PB at the indicated time points. Each point represents an individual surviving mouse with the average represented as the solid line. Day 7: saline n=10 mice, ferumoxytol n=10 mice; day 14: saline n=7 mice, ferumoxytol n=10 mice; day 21: saline n=3 mice, ferumoxytol n=8 mice). Experiment performed one time. (i) Kaplan-Meier survival plot for mice treated with saline or ferumoxytol (n=10 mice per group). Data are shown as mean ± SEM. Unpaired two-tailed t-tests were used to compare between saline and ferumoxytol 6mg kg−1 in Fig. 2c-f. Experiment performed twice and replicate curve reported in Supplementary Fig. 13. Multiple t-tests for 7, 14, and 21 days assuming different SDs were used for Fig. 2h with a false discovery rate of 5% using a two-stage set-up method. Comparison of survival curves in Fig. 2i was performed using a log-rank Mantel-Cox test.

Figure 3.. Ferumoxytol treatment targets leukaemia cells from patient derived xenografts (PDX) from primary AML samples with low FPN without harming normal cells.

(a) Schematic representation for the experimental plan to evaluate anti-leukaemia activity in vivo using PDX mice from primary AML samples with low or high FPN, xenografts were also established using normal CD34+ cells from cord blood (CB). (b) Percent human cell engraftment relative to saline-treated control for mice treated with 5 mg/kg ferumoxytol (FH) for 4 weeks. FPN (SLC40A1) low samples (blue, n=4) AML33, AML9 (n=5), and AML1 (n=8), FPN (SLC40A1) high sample (red, n=5) AML4 and CB (grey, n=3) are shown. Each symbol represents an individual mouse, bars represent the mean, error bar represents the SEM. (c) Fold change expression in transcriptional levels of HMOX1 relative to normal bone marrow CD34+ after treatment with ferumoxytol (n=6 mice AML9, n=5 mice AML 33). Data are shown as mean ± SEM and analysis was done using an ordinary one-way ANOVA for Fig. 3b while a two-tailed unpaired t-test was used in Fig. 3c. AML-PDX experiment performed once due to sample availability.

Figure 4.. Oxidative Ferrotherapy through low ferroportin expression in leukaemia cells.

Normal cells (left) maintain a sufficiently high expression of FPN that allow cells to digest and transport iron without damaging effects of ROS from Fenton chemistry. Leukemic cells, including LSCs, in contrast that contain abnormally low expression of FPN (middle) will allow the accumulation of iron and cellular stress due to increased ROS. Upon addition of ferumoxytol (FH) (Right), AML cells low in FPN are increasingly stressed, overcoming antioxidant production, resulting in cell death.