Eradication of Central Nervous System Leukemia of T-Cell Origin with a Brain-Permeable LSD1 Inhibitor

Abstract

Purpose: Lysine-specific demethylase 1 (LSD1) regulates several biological processes via the bifunctional modulation of enhancer functions. Recently, we reported that LSD1 overexpression is a founder abnormality of T-cell leukemogenesis and is maintained in fully transformed T-cell acute lymphoblastic leukemia (T-ALL) cells. On the basis of this finding, we attempted to develop novel LSD1 inhibitors effective for T-ALL with central nervous system (CNS) involvement.

Experimental design: We chemically modified the prototype LSD inhibitor tranylcypromine (TCP) and screened for cytotoxicity against TCP-resistant T-ALL cell lines. In vivo efficacy of novel LSD1 inhibitors was examined in immunodeficient mice transplanted with luciferase-expressing T-ALL cell lines, which faithfully reproduce human T-ALL with CNS involvement.

Results: We found robust cytotoxicity against T-ALL cells, but not normal bone marrow progenitors, for two N-alkylated TCP derivatives, S2116 and S2157. The two compounds induced apoptosis in TCP-resistant T-ALL cells in vitro and in vivo by repressing transcription of the NOTCH3 and TAL1 genes through increased H3K9 methylation and reciprocal H3K27 deacetylation at superenhancer regions. Both S2116 and S2157 significantly retarded the growth of T-ALL cells in xenotransplanted mice and prolonged the survival of recipients as monotherapy and in combination with dexamethasone. Notably, S2157 could almost completely eradicate CNS leukemia because of its ability to efficiently pass through the blood-brain barrier.

Conclusions: These findings provide a molecular basis and rationale for the inclusion of a brain-permeable LSD1 inhibitor, S2157, in treatment strategies for T-ALL with CNS involvement.

Figure 1.. N-alkylated LSD1 inhibitors induce apoptotic cell death in T-ALL cells at clinically relevant concentrations in vitro.

(A) The IC50 values of S2101 (gray column) and OG-L002 (black column) were calculated from dose-response curves of the indicated cell lines obtained at 72 hours of culture. The mean ± S.D. (bars) of three independent experiments. The results of statistical analyses are shown in Supplementary Figure S1. (B) The IC50 values of the indicated LSD1 inhibitors were calculated from dose-response curves of T-ALL cell lines obtained at 72 hours of culture. The means ± S.D. (bars) of three independent experiments. P values were calculated by one-way ANOVA with Tukey’s multiple comparison test. (C) We cultured CD34-positive bone marrow progenitor cells from healthy volunteers with stem cell factor and thrombopoietin in the absence or presence of various concentrations of S2116 (left panel) or S2157 (right panel) for 72 hours. Jurkat cells were simultaneously treated to serve as a control. Cell viability was determined by the MTT reduction assay and expressed as a percentage of untreated controls. Each point represents the mean ± S.D. (bars) of three independent experiments. *P <0.05 by one-way ANOVA with the Bonferroni post-hoc test. (D) Left panel: T-ALL cells were cultured with various concentrations of S2116 or S2157 for 24 hours. Right panel: CEM and MOLT4 cells were cultured in the absence (control) or presence of 6 µM and 8 µM S2116, respectively, and Jurkat cells were cultured in the absence (control) or presence of 6 µM S2157 for the indicated periods. Cells were stained with annexin-V for the assessment of apoptosis by flow cytometry. The means ± S.D. (bars) of three independent experiments. *P <0.05 against untreated control by one-way ANOVA with Tukey’s multiple comparison test. See Supplementary Figure S3 for other combinations. (E) Whole cell lysates were prepared during the experiments described in panel D and Supplementary Figure S3, and subjected to immunoblotting for the indicated molecules. We performed the same set of experiments using acid-extracted histone proteins for comparison (Acid extracted). (F) Left panel: MOLT4 cells were transduced with either an empty lentiviral vector (Mock) or LSD1-overexpressing vector (LSD1) to establish stable transformants. The expression level of LSD1 was determined by immunoblotting. Right panel: We cultured each transformant with S2157 at the indicated concentrations for 72 hours. Cell viability was determined by the MTT reduction assay and expressed as a percentage of the values of untreated cells. The means ± S.D. (bars) of three independent experiments are shown. *P <0.05 by paired Student’s t test.

Figure 2.. LSD1 inhibitors modify the gene expression program in favor of cell death in T-ALL cells.

(A) MOLT4 cells were cultured with either vehicle alone (0.1% DMSO) or 12 µM S2157 for 24 hours. RNA samples were subjected to microarray analysis for 32,078 genes including microRNAs. Red lines correspond to 2-fold changes (P <0.05 with an FDR threshold of 0.05). Some genes related to T-ALL biology are annotated. (B) Left panel: The expression of the annotated genes in panel A was examined by semiquantitative RT-PCR with suboptimal amplification cycles (40 cycles) in S2157-treated MOLT4 cells (see Supplementary Table S1 for primer sequences). Right panel: The signal intensities of bands in the left panel were quantified and are shown as relative values to those of GAPDH. (C) CEM and MOLT4 cells were cultured with 6 µM S2116, and Jurkat cells were cultured with 8 µM S2157 for the indicated periods. The expression level of NOTCH3 (left panel) and TAL1 (right panel) was determined by RQ-PCR, normalized to that of GAPDH, and quantified by the 2^−∆∆Ct method with the T-0 values set at 1.0. The means ± S.D. (bars) of three independent experiments. *P <0.05 vs. T-0 control by one-way ANOVA with Tukey’s multiple comparison test. See Supplementary Figure S7 for other combinations. (D) Left panel: T-ALL cells were cultured with various concentrations of S2116 or S2157 for 24 hours. Right panel: CEM and MOLT4 cells were cultured with 6 µM S2116, and Jurkat cells were cultured with 8 µM S2157 for the indicated periods. Whole cell lysates were subjected to immunoblotting for Notch3, TAL1 and GAPDH expression. See Supplementary Figure S8 for other combinations. (E) Using ChIP assays, we analyzed the status of chromatin modifications at super-enhancer regions of the NOTCH3 and TAL1 genes in MOLT4 cells treated with either S2116 or S2157 at 12 µM for 24 hours. Representative data of 40 cycles are shown. Input indicates that PCR was performed with genomic DNA. (F) Purified chromatin was isolated from MOLT4 cells treated with either 0.1% DMSO (vehicle) or 12 µM S2157 for 24 hours, followed by immunoprecipitation with anti-histone H3K27ac antibody. Genomic DNA fragments were purified from the precipitants and subjected to high-throughput sequencing. Left panel: The status of H3K27 acetylation surrounding transcription-start sites (TSS) in the entire genome. Red; vehicle-treated control, Blue; S2157-treated MOLT4 cells, Green; Pooled input. Right panel: ChIP seq tracks of normalized tags showing enrichment of histone H3K27 acetylation (purple) in the enhancer regions of the NOTCH3 (upper panel) and TAL1 (lower panel) genes visualized on the UCSC genome browser. Arrowheads indicate the regions examined in ChIP assays (panel E) and ChIP-qPCR (Supplementary Figure S11).

Figure 3.. Notch3 and TAL1 are bona fide targets of N-alkylated LSD1 inhibitors.

(A) MOLT4 cells were transduced with lentiviral vectors encoding shRNA against LSD1 (sh-LSD1 #1, #2 and #3) or an ineffective control (sh-Control). Whole cell lysates were prepared after 72 hours to examine the expression of the indicated molecules with immunoblotting. (B) Upper panel: Immunoblot analyses of Notch3 and TAL1 in lentivirus-transduced stable MOLT4 transformants. Lower panel: We cultured selected clones with S2116 at the indicated concentrations for 72 hours. Cell viability was determined by the MTT reduction assay and expressed as a percentage of the values for corresponding untreated cells. The means ± S.D. (bars) of three independent experiments are shown. *P <0.05 against Mock-transfected cells by paired Student’s t test. (C) We cultured a Mock-transformant and the NOTCH3-stable transformant #2 with 8 µM S2116 for the indicated periods. Whole cell lysates were subjected to immunoblot analyses for the expression of some T-cell oncogene products. (D) Upper panel: Wright-Giemsa staining of primary leukemic cells (> 90% purity) from T-ALL patients. Lower panel: Cells were cultured with vehicle (0.1% DMSO), 1 µM S2116 (left panel) or 1 µM S2157 (right panel) for 48 hours, and subjected to the MTT reduction assay for relative cell numbers (%Control) and RQ-PCR for TAL1 and NOTCH3 mRNA expression. Broken lines indicate the values for corresponding DMSO-treated cells.

Figure 4.. The N-alkylated LSD1 inhibitor S2157 eradicates CNS leukemia in murine xenotransplanted models.

(A) We injected 5 × 10⁶ luciferase-expressing MOLT4 cells into NOD/SCID mice via a tail vein and started treatments at day 10 after transplantation with either vehicle alone (0.1% DMSO) or 50 mg/kg S2157 twice a week for 3 weeks (vertical arrows in panel B). Tumor-derived luciferase activity was measured ex vivo by the IVIS Imaging System after D-luciferin injection on the indicated days. (B) Quantitative data of in vivo bioluminescence imaging as photon units (photons/s). *P <0.05 vs. the vehicle-treated group determined by one-way ANOVA with Tukey’s multiple comparison test (n=3). (C) Brains were resected from each mice on day 29 and subjected to a whole-mount luciferase assay (Day 29 Brain) and histopathological examination. Representative hematoxylin-eosin staining of bregma + 2 mm and + 6 mm sections is shown. Arrows indicate apoptotic bodies in the bregma + 6 mm panel. (D) Day 29 brain was subjected to an in situ TUNEL assay to detect apoptoic cells (green). Transplanted MOLT4 cells were identified by human CD3 expression (red) and DAPI staining (blue). (E) Immunohistochemical staining of brain tissues with specific antibodies recognizing Notch3, TAL1 and cleaved caspase-9 (green). Transplanted MOLT4 cells were identified by human CD3 expression (red) and DAPI staining (blue). Only merged images are shown. Data shown are representative of multiple independent samples collected on day 29. (F) We injected 1 × 10⁷ luciferase-expressing Jurkat cells into NOD/SCID mice via a tail vein and started treatments at day 20 after transplantation with vehicle alone (0.1% DMSO), 12.5 mg/kg OG-L002, or 50 mg/kg S2157 twice a week for 3 weeks. Left panel: Representative photographs of mice on day 56. Right panel: Quantitative data of tumor-derived luciferase activity on day 56 (photons/s). The means ± S.D. (bars) of three independent experiments are shown. P values were calculated by a paired Student’s t test.

Figure 5.. The LSD1 inhibitors exhibit synergistic effects with conventional anti-leukemic agents in vitro and in murine xenotransplanted models.

(A) MOLT4 cells were cultured with four anti-leukemia drugs in the absence or presence of S2157 for 72 hours to obtain dose-response curves for each combination. The combination index plots were generated by the CompuSyn software according to the method of Chou and Talalay. A combination index (CI) <1.0 indicates synergism of the two drugs. (B) The means ± S.D. (bars) of CI between the indicated drugs and either S2116 or S2157 from three independent experiments are shown. (C) We injected 5 × 10⁶ luciferase-expressing MOLT4 cells into NOD/SCID mice via a tail vein and randomized them into four treatment groups at day 10 after transplantation: the vehicle alone (0.1% DMSO, intraperitoneally, 5 times a week; n=3), dexamethasone alone (10 mg/kg, intraperitoneally, 5 times a week; n=3), S2157 alone (50 mg/kg, intraperitoneally, twice a week; n=4) and the combination of S2157 and dexamethasone (n=5). Kaplan-Meier survival curves were generated and statistically analyzed by the log-rank test using the Prism software (GraphPad Software, La Jolla, CA). P-values and hazard ratios (95% confidential interval) between the vehicle-treated group (Control) and the S2157-alone (green) or combined-treatment group (red) are shown.