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. 2024 Jul 1;109(7):2073-2084.
doi: 10.3324/haematol.2023.283494.

B-cell precursor acute lymphoblastic leukemia elicits an interferon-α/β response in bone marrow-derived mesenchymal stroma

Mandy W E Smeets  1 , Elisabeth M P Steeghs  2 , Jan Orsel  3 , Femke Stalpers  3 , Myrthe M P Vermeeren  3 , Christina H J Veltman  3 , Lotte Slenders  3 , Stefan Nierkens  4 , Cesca Van de Ven  3 , Monique L Den Boer  5
Affiliations

B-cell precursor acute lymphoblastic leukemia elicits an interferon-α/β response in bone marrow-derived mesenchymal stroma

Mandy W E Smeets et al. Haematologica. .

Abstract

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) can hijack the normal bone marrow microenvironment to create a leukemic niche which facilitates blast cell survival and promotes drug resistance. Bone marrow-derived mesenchymal stromal cells (MSC) mimic this protective environment in ex vivo co-cultures with leukemic cells obtained from children with newly diagnosed BCP-ALL. We examined the potential mechanisms of this protection by RNA sequencing of flow-sorted MSC after co-culture with BCP-ALL cells. Leukemic cells induced an interferon (IFN)-related gene signature in MSC, which was partially dependent on direct cell-cell signaling. The signature was selectively induced by BCP-ALL cells, most profoundly by ETV6-RUNX1-positive ALL cells, as co-culture of MSC with healthy immune cells did not provoke a similar IFN signature. Leukemic cells and MSC both secreted IFNα and IFNβ, but not IFNγ. In line, the IFN gene signature was sensitive to blockade of IFNα/β signaling, but less to that of IFNγ. The viability of leukemic cells and level of resistance to three chemotherapeutic agents was not affected by interference with IFN signaling using selective IFNα/β inhibitors or silencing of IFN-related genes. Taken together, our data suggest that the leukemia-induced expression of IFNα/β-related genes by MSC does not support survival of BCP-ALL cells but may serve a different role in the pathobiology of BCP-ALL.

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Figures

Figure 1.
Figure 1.
B-cell precursor acute lymphoblastic leukemia cells induce an interferon-related gene signature in mesenchymal stromal cells. (A) Diagram of the experimental setup. RNA sequencing was performed on sorted viable mesenchymal stromal cells (MSC) and acute lymphoblastic leukemia (ALL) fractions after mono-culture and after MSC/ALL co-culture; for the gating strategy see Online Supplementary Figure S1. Secreted interferon levels were measured in equal volumes of supernatant of mono-cultures and MSC/ALL co-cultures (Figure 6A). (B) IFI6 mRNA levels expressed in paired samples of MSC sorted after mono-culture (squares; MSC#1-3 indicated in red, blue, and green, respectively.) and MSC sorted after co-culture with BCP-ALL cells from 15 individual patients (#1, ETV6-RUNX1-like; #2-6, B-other; #7, high hyperdiploid; #8-15, ETV6-RUNX1; circles). Boxplots represent the interquartile range, the median is depicted by a line. IFN: interferon; FDR: P value for the false discovery rate; FPKM: fragments per kilobase million; HD: high hyperdiploid.
Figure 2.
Figure 2.
ETV6-RUNX1 B-cell precursor acute lymphoblastic leukemia cells most potently induce an interferon-related gene signature in mesenchymal stromal cells. (A) mRNA expression levels in mesenchymal stromal cells (MSC) of IFI6, MX1, OAS3 and IFI27 upon mono-culture (gray bars) and co-culture (white bars) summarized according to B-cell precursor acute lymphoblastic leukemia (BCP-ALL) subtype (N=8 for ETV6-RUNX1, N=1 for high hyperdiploid, N=6 for B-other). Each dot represents the average expression of interferon (IFN)-related genes in three MSC per sort. The mean ± standard error of mean is indicated. (B) Heatmap of expression levels of IFN-related genes in MSC sorted after mono-culture (- BCP-ALL) and those after co-culture with 15 different ALL cases (+ BCP-ALL). The heatmap is grouped by BCP-ALL subtype: high hyperdiploid (HD) and B-other ALL cases (#1-7) or ETV6-RUNX1 ALL cases (#8-15). Blue, low expression. Red, high expression. *P<0.05, **P<0.01, ***P<0.001. MSC: mesenchymal stromal cells; FPKM: fragments per kilobase million; Mono: mono-culture; Co: co-culture; HD: high hyperdiploid; BCP-ALL: B-cell precursor acute lymphoblastic leukemia.
Figure 3.
Figure 3.
Normal cord blood, with the exception of monocyte-containing preparations, has a limited effect on expression levels of interferon-related genes in mesenchymal stromal cells. IFI6, ISG15, IFIT1, MX1, CXCL10, IFI44L, IFI27, IFITM1, and OAS3 gene expression levels measured by reverse transcriptase quantitative polymerase chain reaction in mesenchymal stromal cells (MSC) (MSC#2) sorted after co-culture with mononuclear cells from five different cord blood samples: cord blood with relatively (A) low (N=2) or (B) high (N=2) percentages of monocytes (>20%), or (C) an unknown cellular composition (N=1). Bars are means of triplicate measurements ± standard error of mean for two independent co-culture experiments. The dashed line (---) indicates imRNA expression levels of MSC after mono-culture, set at 100%. P<0.05 MSC: mesenchymal stromal cells.
Figure 4.
Figure 4.
Direct cell-cell contact contributes to induction of Interferon-related gene expression in mesenchymal stromal cells. (A) Schematic overview of the experimental setup of direct co-culture and transwell co-culture of REH cells with mesenchymal stromal cells (MSC) (MSC#2) using 0.4 µm pore-size transwells. (B) Percentage of DiI-positive MSC after direct (circles, white bars) and transwell (squares, gray bars) co-culture with REH leukemic cells for 40 hours. Dots represent averaged duplicate measurements; bars represent means ± standard error of mean (SEM) for three independent experiments. (C) Interferon-related gene expression levels, measured by reverse transcriptase quantitative polymerase chain reaction, in MSC sorted after direct (circles, white bars) or transwell (squares, gray bars) co-culture with REH cells for 40 hours. Dots represent averaged triplicate measurements; bars represent means ± SEM for four independent experiments. The dashed line (---) indicates mRNA expression levels of MSC after mono-culture, set at 100%. *P<0.05, **P<0.01, ****P<0.0001. MSC: mesenchymal stromal cells.
Figure 5.
Figure 5.
Silencing of interferon-related genes in mesenchymal stromal cells does not affect the viability of primary B-cell precursor acute lymphoblastic leukemia cells. Fold-change in percentage of viable leukemic cells after 72 hours of co-culture with transduced mesenchymal stromal cells (MSC) (MSC#2). A non-silencing control - short-hairpin luciferase (NSC-shLuc) is used as reference. (A) ETV6-RUNX1 (N=3), and (B) B-other (N=2) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells. Five shRNA per gene were tested for knockdown efficiency; only shRNA resulting in >60% knockdown (shIFI6 #4, shISG15 #4, shMX1 #3/5, shIFI44L #1/3, and shIFI27 #2, see Online Supplementary Methods) were used. Bars represent means ± standard error of mean of duplicate measurements for three independent experiments. A dashed line (---) indicates that viability of ALL cells is equal to the viability of ALL cells co-cultured with NSC-shLuc-MSC (fold-change = 1; gray bars). ALL: acute lymphoblastic leukemia; NIC: non-infected control; NSC-shLuc: non-silencing control - short-hairpin-luciferase; sh: short hairpin.
Figure 6.
Figure 6.
Interferon-α secretion and IFNAR1 expression decreased in mesenchymal stromal cells after co-culture with primary B-cell precursor acute lymphoblastic leukemia cells. (A) Interferon-alpha (IFNα) concentration in supernatants collected after 40 hours from 15 mesenchymal stromal cells (MSC) (MSC#1-3) and B-cell precursor acute lymphoblastic leukemia (BCP-ALL) (ALL#1-15) mono- and co-cultures. IFNα concentration is corrected for medium background level. Squares and circles indicate IFNα secretion level in the sum of MSC and BCP-ALL mono-cultures and their co-cultures, respectively. The limit of detection is indicated by a solid line. The range of detection for IFNα is 0.5-2,503 pg/mL. (B) IFNAR1 and (C) IFNAR2 mRNA levels expressed in paired samples of MSC sorted after mono-culture (squares; MSC#1-3 indicated in red, blue, and green, respectively.) and MSC sorted after co-culture with BCP-ALL cells from 15 individual patients (#1-15; circles). Boxplots represent the interquartile range, the median is depicted by a line. IFN: interferon; MSC: mesenchymal stromal cells; BCP-ALL: B-cell precursor acute lymphoblastic leukemia; HD: high hyperdiploid; FDR: P-value false discovery rate; FPKM: fragments per kilobase million.
Figure 7.
Figure 7.
Interferon-mediated response in mesenchymal stromal cells does not affect primary ETV6-RUNX1 B-cell precursor acute lymphoblastic leukemia viability. (A) mRNA expression levels of IFI6 in mesenchymal stromal cells (MSC) (MSC#2) after 40 hours (left graph) and 120 hours (right graph) exposure to recombinant human interferon (IFN)α (3.0 U/mL; striped bars), IFNβ (60 pg/mL; gray bars) and inhibitors thereof (i-IFNa/p; 0.9, 1.8 and/or 3.6 ng/mL) (open diamonds). Unstimulated MSC served as the control (white bars; filled circles). The dashed line (---) indicates baseline mRNA expression levels in mono-cultures of MSC set at 100%. (B) Log10-transformed IFN-related gene expression levels, measured by reverse transcriptase quantitative polymerase chain reaction, in MSC exposed to primary ETV6-RUNX1-positive B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells for 40 hours with and without inhibitors of IFNa/p (1.8 ng/mL; open diamonds) or IFNy (3 ng/mL; open squares). Bars represent means ± standard error of mean (SEM) of triplicate measurements for three ETV6-RUNX1-positive samples (ALL#9-11). (C) Fold change in viability of ETV6-RUNX1 BCP-ALL cells after 40 hours (left graph) and 120 hours (right graph) of mono-culture with i-IFNa/p (1.8 ng/mL), MSC co-culture without i-IFNa/p, or MSC co-culture with i-IFNa/p (1.8 ng/mL), normalized to mono-culture without i-IFNa/p. Bars represent means ± SEM of triplicate measurements for three independent experiments. *P<0.05, **P<0.01, ***P<0.001. MSC: mesenchymal stromal cells; i-IFNa/p: inhibitors of interferon α/β; i-IFNy: inhibitor of interferon y; BCP-ALL: B-cell precursor acute lymphoblastic leukemia; mono: mono-culture; co: co-culture.
Figure 8.
Figure 8.
The interferon-mediated response in mesenchymal stromal cells does not affect the sensitivity of primary ETV6RUNX1 B-cell precursor acute lymphoblastic leukemia cells to drugs. Fold change in percentages of viable ETV6-RUNX1 B-cell precursor acute lymphoblastic leukemia cells in 120-hour mono-culture or mesenchymal stromal cell co-culture upon exposure to (A) L-asparaginase, (B) daunorubicin, or (C) prednisolone (concentration as determined in Online Supplementary Figure S2) and/or inhibitors of interferon α/β (1.8 ng/mL), normalized to values in mono-culture without drug exposure or interferon inhibitors. Bars represent means ± standard error of mean of triplicate measurements for three independent experiments. i-IFNα/β: inhibitors of interferon α/β; BCP-ALL: B-cell precursor acute lymphoblastic leukemia; MSC: mesenchymal stromal cells; ASP: L-asparaginase; DNR: daunorubicin; PRED: prednisolone.
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Funding: This study was funded by the Pediatric Oncology Foundation Rotterdam (SKOCR) and the Oncode Institute in Utrecht.