Niche-based screening identifies small-molecule inhibitors of leukemia stem cells

Abstract

Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those compounds that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on target, via inhibition of HMG-CoA reductase. These results illustrate the power of merging physiologically relevant models with high-throughput screening.

Figure 1. A High-Throughput System for Probing Primary, Stem-enriched Leukemia Cells Within a Stromal Niche

(a) LSCe cells generate cobblestone area-forming cell (CAFC (arrows)) and non-CAFC morphologies when plated with bone marrow stroma (primary BMSCs or OP9 cells), at the 6-day assay endpoint. Qualitative images are shown as a visual introduction to the devised assay. The red channel has been pseudocolored to accommodate colorblindness. Scale bars represent 50 μM. (b) A representative example of the “Number of neighbors” rule, one of 50 computational rules that together identify CAFCs. A raw image of LSCe cells in co-culture (left, dsRed channel) is converted to a heatmap (middle) reflecting the number of adjacent cells (red = many, blue = few), identifying cell “objects “(right; see Methods) that are part of a cobblestone area (orange = CAFC, blue = non-CAFC). Qualitative images are shown as a conceptual introduction to the computational method. Scale bars represent 50 μM. (c) Schematic of the small-molecule co-culture screen protocol. (d) The co-culture screen data for 14,718 compounds (blue) added at ~5 μM, in duplicate. Values were normalized to the means of both neutral (DMSO carrier, yellow, set at 0% effect) and positive (10 μM XK469, a topoisomerase IIβ inhibitor, red, set at −100% effect) controls. (e) Schematic summarizing the filtering steps used to define high priority compounds. The number of compounds prioritized at each step is shown.

Figure 2. Prioritized Screening Hits Display Selective Toxicity to LSCe Cells Relative to HSPCs in Co-culture

The names and structures of 15 prioritized small-molecule screening hits are shown, with EC₅₀ values (from dose-response curves across 8 concentrations) for both LSCe cells (as percent cobblestone area effect; 2 replicates per concentration) and HSPCs (as total cells per well; 6 replicates per concentration) co-cultured under identical conditions on primary BMSC stromal cells. Celastrol, piperlongumine, and 2-methoxy estradiol were previously identified, validating the screening approach.

Figure 3. Novel Small Molecule BRD7116 Selectively Targets LSCe Cells by Cell-Autonomous and Cell-Non-Autonomous Mechanisms

(a) Dose-response effects of bis-aryl sulfone hit BRD7116 on LSCe cells (red, in duplicate) and normal HSPCs (blue, 6 replicates per dose) co-cultured on primary BMSC stroma, shown overlaid. BRD7116 was applied to the co-cultures in both cases. (b) The effects of treating only the stromal monolayer prior to co-culture are compared to the effects of treating both the stroma and LSCe cells together in co-culture. The data shown is from the stromal pretreatment secondary screen in which OP9 stromal cells were treated with BRD7116 for three days prior to LSCe cell plating (green). The LSCe co-culture retest data with OP9s (black) is also shown. Both screens were performed in duplicate. (c) The effects of treating only the stromal monolayer with BRD7116 for three days prior to the plating of admixed LSCe cells and HSPCs on primary BMSC stroma (as mean +/− SEM of quadruplicate replicates). These two hematopoietic populations are quantified as percent of total hematopoietic cells under each stromal pretreatment condition. * p < 0.05; n.s. = not significant, for effects relative to DMSO (0 μM BRD7116) controls. (d) Compared to DMSO control, gene expression changes present in LSCe cells after 6 hours of direct treatment with 5 μM BRD7116 are significantly enriched for an AML differentiation gene signature, as defined by all-trans retinoic acid (ATRA) treatment of human AML cells, by Gene Set Enrichment Analysis (GSEA).

Figure 4. Lovastatin Selectively Inhibits Murine and Human Leukemia Cells in Co-culture

(a) Lovastatin displays leukemia-selective activity (red dose-response curve, in duplicate) compared to HSPCs (blue, 6 replicates per dose) in co-culture with BMSCs. (b) The effects of lovastatin on CAFC activity of primary human CD34⁺ cells isolated from either normal or leukemic patient samples. Co-cultures were treated for 6 days, and then rinsed. The fraction of replicate co-cultures containing cobblestone areas at the 5-week assay endpoint (2 weeks for FLT3-ITD sample) is shown (n ≥ 6). The clinical characteristics of the AML samples are as follows, including whether each AML case is primary (P) or directly related to previous cytotoxic therapy, as therapy-related (TR) leukemia is associated with a worse prognosis than de novo disease. AML 1: FLT3-ITD⁺ (P), AML 2: NPM1 mutation (P), AML 3: CBFB-MYH11 fusion (P), AML 4: trisomy 11, del(17p) (TR), AML5: CEPBα double mutation (P), AML 6: MLL-AF9 fusion (TR). (c) Representative images of admixed LSCe cells and HSPCs co-cultured with uncolored primary BMSCs at 5 days of exposure to lovastatin. The red channel has been pseudocolored to accommodate colorblindness. Scale bars represent 50 μM. (d) Quantification of the effect (mean +/− SEM of quadruplicate replicates) is also shown and is representative of two independent experiments. * p < 0.001 for LSCe cells relative to DMSO (0 μM lovastatin) controls.

Figure 5. Sensitivity of LSCe Cells to HMGCR Inhibition

(a) The addition of 2 mM mevalonate to co-cultures of LSCe cells with OP9 stroma rescued the anti-leukemia effect of 1 μM lovastatin, with no stromal toxicity observed. Data are mean +/− SEM of triplicate replicates, and are representative of two independent experiments. P-values shown are relative to DMSO control. * p < 0.001, n.s. = not significant. (b) Schematic summarizing genetic and pharmacologic findings for the mevalonate pathway in LSCe cells in vitro (co-culture experiments) and in vivo (RNAi pooled screen). (c) Limiting dilution analysis of varying numbers of LSCe cells pretreated in co-culture with BMSCs for 24 hours with DMSO control or 5 μM lovastatin prior to syngeneic transplantation into mice (n ≥ 3 as shown). (d) Ex vivo treatment for 48 hours with 5 μM lovastatin impaired the ability of LSCe cells co-cultured with BMSCs to induce leukemia upon transplantation into mice relative to DMSO treatment (n = 6). See Supplementary Figure 5 for effects on admixed normal HSPCs.