Combined epigenetic and metabolic treatments overcome differentiation blockade in acute myeloid leukemia

Abstract

A hallmark of acute myeloid leukemia (AML) is the inability of self-renewing malignant cells to mature into a non-dividing terminally differentiated state. This differentiation block has been linked to dysregulation of multiple cellular processes, including transcriptional, chromatin, and metabolic regulation. The transcription factor HOXA9 and the histone demethylase LSD1 are examples of such regulators that promote differentiation blockade in AML. To identify metabolic targets that interact with LSD1 inhibition to promote myeloid maturation, we screened a small molecule library to identify druggable substrates. We found that differentiation caused by LSD1 inhibition is enhanced by combined perturbation of purine nucleotide salvage and de novo lipogenesis pathways, and identified multiple lines of evidence to support the specificity of these pathways and suggest a potential basis of how perturbation of these pathways may interact synergistically to promote myeloid differentiation. In sum, these findings suggest potential drug combination strategies in the treatment of AML.

Keywords: molecular biology; stem cell research; systems biology.

Graphical abstract

Figure 1

Small molecule screen identifies two compounds that enhance LSD1i-mediated differentiation of ER-HOXA9 cells (A) ER-HOXA9 cells were treated with GSK-LSD1 (LSD1i) combined with individual drugs from a compound library to determine which combinations induced differentiation to greater level than LSD1i treatment alone. Insert shows merged fluorescent images of ER-HOXA9 cells in non-differentiated and differentiated states following 50nM LSD1i treatment (White bar = 10 microns). Differentiated cells activate the Lyz2-GFP reporter, which provides a readout for differentiation. (B) Compound screen measuring differentiation of ER-HOXA9 cells treated with combinations of LSD1i with lipid (left) and nucleic acid (right) metabolism drugs for five days. Alternating bands of color represent a different drug, where individual dots (average of two replicates) represent a specific dose of that drug. Drug doses that significantly reduced viability (i.e. ≤ 10% of total cells were live) were assigned 0% differentiation. Horizontal line at 19% differentiation, which is the average efficiency observed with the cytarabine positive control, designated the significance threshold. Two candidates, mercaptopurine (6MP) and cerulenin (CER) are highlighted. See Table S2 for full compound screen information. (C) Chemical structures of LSD1i, 6MP, and CER. See also Figures S1 and S2 and Table S1.

Figure 2

6MP and CER suppress proliferation and enhance differentiation in combination with LSD1i (A) Time course measurement of proliferation (top) and differentiation (bottom) status of ER-HOXA9 cells treated with 200nM LSD1i, 5μM 6MP, and 5μM CER for one to five days as single or triple combinations. Proliferation was measured as live cell numbers relative to initial cell numbers at day 0 (approximately 2500 live cells), where for example a value of 25 indicates 25 × 2500 = 62,500 live cells. Differentiation was measured as percentage of cells positive for Lyz2-GFP. Bars represent average of three to four technical replicates, and error bars represent standard deviation. See Table S6 for data. (B) ER-HOXA9 cells treated with LMC combination (200nM LSD1i, 5μM 6MP, and 5μM CER) display morphologies consistent with mature murine leukocytes. Images were acquired at 120× magnification (White bar = 10 microns). Cells treated with vehicle or drugs were classified as immature or mature cells in blinded manner. (C) Genes related to neutrophil granule formation (Target) were quantified for expression changes in ER-HOXA9 cells treated with vehicle or LMC combination (200nM LSD1i, 5μM 6MP, and 5μM CER) for two to four days. Values represent average of two replicates and normalized to the specified housekeeping reference genes (Ref). (D) Differentiation status of ER-HOXA9 cells after treatment with multiple doses of LSD1i and 6MP (top) or with LSD1i and CER (bottom) as double drug combinations for four days. Bars represent average of five to six technical replicates. See also Figures S3–S8 and Tables S3–S7 and S18.

Figure 3

Metabolic profiling and suppression of the differentiation response to drugs (A) ER-HOXA9 cells were treated with combinations of 200nM LSD1i, 5μM 6MP, and 5μM CER and suppressed with vehicle (white), 10μM HYP (light gray) or 1mM HCY (dark gray) for four days. Differentiation for Panels A-C was assessed by Lyz2-GFP expression. Bars and vertical lines represent average and standard deviation, respectively, of eight technical replicates. Asterisks in Panels A-C represent significant differences (∗p_adj < 0.01) as determined by Games-Howell test. See Table S12 for data for Panels A-C. (B) ER-HOXA9 cells treated with vehicle, 200nM LSD1i, 2.5 μM 6MP, and double combination of both drugs were co-incubated with vehicle or 5μM ribavirin (RBV) for four days. Bar and vertical lines represent average and standard deviation of six technical replicates. (C) ER-HOXA9 cells treated with vehicle, 200nM LSD1i, 5μM CER, or double combination of both drugs were co-incubated with or without 60μM (HCY) for three days. Bar and vertical lines represent average and standard deviation of eight technical replicates. (D) Principal component analysis of the relative abundances of polar metabolites extracted from ER-HOXA9 cells treated with vehicle (VEH), triple drug combination (LMC, at 200nM LSD1i, 5μM 6MP, and 5μM CER), or estradiol withdrawal (E2M). The heatmap depicts the natural log fold change of the abundance of a given metabolite over the average abundance of that metabolite across all samples (red and blue represent enriched or depleted over average). Histogram represents distribution of fold changes across all metabolites and treatments. See Table S13 for data. (E) Relative abundances of select polar metabolites extracted from ER-HOXA9 cells treated with vehicle (white), drugs (light gray), or estradiol withdrawal (dark gray). Bars and vertical lines represent mean and standard deviation of three replicates. (F) Relative abundances of select acylcarnitine and monoglyceride species from ER-HOXA9 cells treated with vehicle (white), drugs (light gray), or estradiol withdrawal (dark gray). Bars and vertical lines represent mean and standard deviation of three replicates. See also Figures S9–S16 and Table S8. Liquid chromatography-mass spectrometry data of LSD1i, 6MP, and CER drugs, related to Figure 3, Table S9. Flow cytometry data of ER-HOXA9 cells treated with drugs in different orders, related to Figure 3, Table S10. Raw and normalized flow cytometry data, and associated statistical analyses, of ER-HOXA9 cells treated with various concentrations of TCP, 6MP, and CER for four days, related to Figure 3, Table S11. Flow cytometry data of ER-HOXA9 cells treated with various concentrations of LSD1i and 6-thioguanine (first tab), nucleosides (second tab), and ACC inhibitors (third tab) for four days, related to Figure 3, Table S12. Flow cytometry data of ER-HOXA9 suppression studies, related to Figure 3, Table S13. Liquid chromatography-mass spectrometry data of polar (first tab) and non-polar (second tab) metabolites extracted from ER-HOXA9 cells treated with vehicle (Veh), triple drug combination (LMC, at 200nM LSD1i, 5μM 6MP, 5μM CER), and estradiol withdrawal (E2m), related to Figure 3, Table S14. Genomics data of ER-HOXA9 cells, related to Figure 3.

Figure 4

6MP incorporates into nucleic acids (A) LC-MS analysis of deoxythioguanosine (dTG) and other deoxynucleotides processed from ER-HOXA9 cells treated with vehicle, single drug (5μM 6MP), double drug combination (200nM LSD1i and 5μM 6MP), and triple drug combination (200nM LSD1i, 5μM 6MP, and 5μM CER) for four days. Levels of dTG were normalized to deoxyguanosine. To access variability across conditions, levels of deoxyadenosine were compared to deoxyguanosine. Bar and vertical lines represent average and standard deviation of three technical replicates. See Table S16 for data. (B) LC-MS analysis of thioguanosine (TG) and other nucleotides processed from ER-HOXA9 cells treated with vehicle, single drug (5μM 6MP), double drug combination (200nM LSD1i and 5μM 6MP), and triple drug combination (200nM LSD1i, 5μM 6MP, and 5μM CER) for four days. Levels of TG were normalized to guanosine. See Figure S18 for biological replicate. Bar and vertical lines represent average and standard deviation of three technical replicates, and asterisks represent significant differences (∗∗∗p_adj < 0.0005) as determined by t test. (C) HPLC elution profiles of RNA-derived nucleosides from ER-HOXA9 cells treated with vehicle or 5μM 6MP. A water-only sample served as a negative control and thioguanosine standard served as a positive control. Note the similar retention times between the standard and the highlighted peak in the 6MP-treated sample. (D) Heavy hypoxanthine (HYP) was used as a tracer for purine salvage in total RNA. Black circles represent the ¹³C carbon isotopes. ER-HOXA9 cells were incubated without heavy HYP as a negative control, or with 5μM heavy HYP combined with various drug combinations for four days. Heavy guanosine and adenosine were normalized to the respective light nucleosides to determine their relative abundance. To assess variability across conditions, levels of total adenosine were compared to total guanosine (heavy and light). Bar and vertical lines represent average and standard deviation of three replicates. (E) Pulse-chase experiment of ¹³C₅-HYP labeling of guanosine and adenosine in total RNA, where cells were incubated with 5μM ¹³C₅-HYP for 2 days, followed by 5μM ¹²C₅-HYP for 2 more days. Heavy guanosine and adenosine were normalized to the respective light nucleosides to determine their relative abundance, and the change in abundance before and after chase was determined. Bar and vertical lines represent average and standard deviation of three replicates. See also Figures S17 and S18 and Tables 15 and S16.

Figure 5

Optimal dosage schemes of LSD1i and 6MP administration to reduce tumor cell numbers (A) Schematic model of tumor growth and differentiation. We consider 2 types of cells: non-differentiated and differentiated cells, that have their own birth rates termed α₁ and α₂ respectively and death rates termed δ₁ and δ₁, respectively. The parameter α₂ was set to a low value to represent the lack of self-renewal of differentiated cells. We assume that a cell can differentiate through a mitosis-independent process at a rate of γ. (B) Change in rates of growth, differentiation, and death rates described in Panel A, where 0.5mg/kg LSD1i and 5mg/kg 6MP dose are given every twenty-four hours. (C) Contour plot depicting the percentage of differentiated cells after 2 weeks of treatment (left) and the log₂ fold change in total cell numbers after 2 weeks of the group treated with various doses of LSD1i and 6MP administered every twenty-four hours compared to the non-treated group (right). Color of lines represents levels of differentiation or fold change. (D) Contour plot comparing two different drug schedules: treatment with 6MP daily and LSD1i every 48 hr or treatment with LSD1i daily and 6MP every 48 hr at the doses specified along the axes after one week (left) or two weeks (right) of treatment. The blue area represents doses where the former schedule would be more effective at decreasing the number of cells, whereas the red area represents doses where the latter schedule would be more effective. Color of lines represent the relative reduction ratio of the former schedule over the latter, where positive values indicate the former outperforms the latter. See also Figures S19–S22 and Table S17.