Image-based RNA interference screening reveals an individual dependence of acute lymphoblastic leukemia on stromal cysteine support

Abstract

Interactions with the bone marrow microenvironment are essential for leukemia survival and disease progression. We developed an imaging-based RNAi platform to identify protective cues from bone marrow derived mesenchymal stromal cells (MSC) that promote survival of primary acute lymphoblastic leukemia (ALL) cells. Using a candidate gene approach, we detected distinct responses of individual ALL cases to RNA interference with stromal targets. The strongest effects were observed when interfering with solute carrier family 3 member 2 (SLC3A2) expression, which forms the cystine transporter xc- when associated with SLC7A11. Import of cystine and metabolism to cysteine by stromal cells provides the limiting substrate to generate and maintain glutathione in ALL. This metabolic interaction reduces oxidative stress in ALL cells that depend on stromal xc-. Indeed, cysteine depletion using cysteine dioxygenase resulted in leukemia cell death. Thus, functional evaluation of intercellular interactions between leukemia cells and their microenvironment identifies a selective dependency of ALL cells on stromal metabolism for a relevant subgroup of cases, providing new opportunities to develop more personalized approaches to leukemia treatment.

Figure 1. Bone marrow derived mesenchymal stromal cells (MSC) provide different pro-survival cues to support precursor B-cell ALL

(A) ALL cell viability in monoculture or in co-culture with MSC after 6 days was measured by flow cytometry using anti-CD45 and 7-AAD stainings. (B) 22 BCP-ALL samples were tested for their survival in co-culture with MSC or in mono-culture after 6 days. The percentage of viable cells at day 6 was normalized to the input viable cell numbers. SR, standard risk; MR, medium risk; HR, high risk; VHR, very high risk; MNR, morphological non-responder, relapsed ALL cases. (C) Comparison of ALL cell viability in co-culture with MSC (MSC+ALL), separated via a transwell (MSC/ALL) or in mono-culture (ALL) after 3 days. Viability was assessed by FACS and performed at least in duplicates and normalized to the viable cell numbers of input.

Figure 2. Live cell imaging based RNAi screening platform to examine ALL cell viability in co-cultures

(A) Workflow of imaging-based assessment of ALL cell viability after RNA interference in MSC. (B) Living cells in the co-culture were stained using CyQUANT in a 384 well format. After automated microscopy which acquired 9 images per well covering more than 50% of each well and subsequent image segmentation using CellProfiler, the number of living ALL cells was assessed with the machine learning software Advanced Cell Classifier (ACC). (C) Correlation of the viability assessment of ALL cells by ACC and by flow cytometry. In total, 5 experiments were run in parallel for both methods. To get the full range of possible ALL cell viabilities, conditions with negative control siRNAs and positive control siRNAs (killing the MSC) as well as incubation with chemotherapeutic agents (dexamethasone, daunorubicin and vincristine) in different concentrations and seeding different numbers of cells were used.

Figure 3. The stromal cystine transporter x_c⁻ is essential for maintaining the viability of a subset of ALL cases

(A) Identification of patient specific signals from MSC supporting ALL cell viability by RNAi interference. In the first screen, the effect of RNA interference with 110 genes in MSC on the viability of three ALL cases was assessed. 20 genes were found to significantly decrease ALL cell viability after RNA interference in MSC in at least 2 of the three cases. In the second screen, the effect of RNA interference with these 20 genes in MSC was assessed for 10 ALL cases. Depicted is the mean survival for each patient sample, normalized to the mean survival in the scrambled controls using the following color code: blue, decrease; red, increase. Neg Ctrl, scramble siRNA; Pos Ctrl, toxic siRNA that kills MSC. (B) ALL cell viability was assessed by flow cytometry for 10 patient samples after RNA interference with SLC3A2 at MSC level. The 3 most sensitive patients (white bars) and the 3 most resistant patients (black bars) were used for further experiments. (C) ALL cell viability was assessed by flow cytometry for 2 SLC3A2-dependent and 2 SLC3A2-independent cases after RNA interference with SLC3A2 in MSC using 4 individual siRNA sequences. (D) Cell viability assessed by flow cytometry after 6 days of co-culture with MSC in cystine depleted RPMI medium was decreased in 3 SLC3A2-dependent compared to 3 SLC3A2-independent samples. The supplementation of cystine rescued cell viability in a dose dependent manner. (E) Cell viability assessed by flow cytometry for 27 precursor B-ALL cases after RNA interference with SLC7A11 in MSC using 2 individual siRNA sequences identified a subset of ALL cases that depends on stromal SLC7A11.

Figure 4. Stromal cysteine supply is required to control oxidative stress in ALL samples that are dependent on stromal SLC3A2/SLC7A11

Comparison of the GSH (A) and ROS levels (B) of patient samples that are dependent (n = 3) and independent (n = 3) on stromal SLC3A2 assessed after RNA interference with SLC3A2 in MSC after 3 days of co-culture. (C) Sensitivity to H₂O₂ was decreased in the 3 SLC3A2 dependent samples but not in the 3 independent samples after RNA interference with stromal SLC3A2. (D) The stromal support function lost after RNA interference with SLC3A2 is rescued and (E) ROS levels are restored to control levels by the addition of cysteine (100 μM), GSH (5 mM) or NAC (5 mM), but not cystine (100 μM). ALL cell viability and ROS levels (DCF-DA) were assessed by flow cytometry after 6 days of co-culture. (F and G) GSH and ROS levels were assessed by flow cytometry after incubation with SSZ for 72 hours in the stromal x_c⁻-dependent ALL sample SR-13. (H) SSZ decreased ALL cell viability of 3 stromal x_c⁻ dependent ALL samples (blue curve) while 3 independent samples (red curve) remained largely unaffected after 3 days incubation with SSZ in coculture. (I) Recombinant human cysteine dioxygenase (CDO1) decreased the viability of x_c⁻ dependent ALL samples. In contrast, x_c⁻ independent ALL was not affected by CDO1 treatment. Histograms in A, B, C, D, E, H and I show the mean ± SD of values normalized to the respective negative controls (*P < 0.05; **P < 0.01; ***P < 0.001, Mann-Whitney U-test).

Figure 5. Model depicting the dependence of leukemia cells on cystine metabolism of stromal cells

The stromal cystine transporter composed of SLC3A2 and SLC7A11 is required for the import of cystine in MSC. Reduction of cystine to cysteine and subsequent supply to leukemia cells are critical steps for GSH synthesis in leukemia cells. GSH is an important antioxidant to maintain the redox state in leukemia cells that generate more ROS than non-malignant cells. The experimental evidence is summarized in the discussion.