MiR-146b-5p regulates IL-23 receptor complex expression in chronic lymphocytic leukemia cells

Abstract

Chronic lymphocytic leukemia (CLL) cells express the interleukin-23 receptor (IL-23R) chain, but the expression of the complementary IL-12Rβ1 chain requires cell stimulation via surface CD40 molecules (and not via the B-cell receptor [BCR]). This stimulation induces the expression of a heterodimeric functional IL-23R complex and the secretion of IL-23, initiating an autocrine loop that drives leukemic cell expansion. Based on the observation in 224 untreated Binet stage A patients that the cases with the lowest miR-146b-5p concentrations had the shortest time to first treatment (TTFT), we hypothesized that miR-146b-5p could negatively regulate IL-12Rβ1 side chain expression and clonal expansion. Indeed, miR-146b-5p significantly bound to the 3'-UTR region of the IL-12Rβ1 mRNA in an in vitro luciferase assay. Downregulation of miR-146b-5p with specific miRNA inhibitors in vitro led to the upregulation of the IL-12Rβ1 side chain and expression of a functional IL-23R complex similar to that observed after stimulation of the CLL cell through the surface CD40 molecules. Expression of miR-146b-5p with miRNA mimics in vitro inhibited the expression of the IL-23R complex after stimulation with CD40L. Administration of a miR-146b-5p mimic to NSG mice, successfully engrafted with CLL cells, caused tumor shrinkage, with a reduction of leukemic nodules and of IL-12Rβ1-positive CLL cells in the spleen. Our findings indicate that IL-12Rβ1 expression, a crucial checkpoint for the functioning of the IL-23 and IL-23R complex loop, is under the control of miR-146b-5p, which may represent a potential target for therapy since it contributes to the CLL pathogenesis. This trial is registered at www.clinicaltrials.gov as NCT00917540.

Conflict of interest statement

Conflict-of-interest disclosures: The authors declare no competing financial interests.

© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.

Figures

Graphical abstract

Figure 1.

Capacity of miR-146b-5p concentrations of predicting TTFT in CLL. (A) Association between miR-146b-5p concentrations and IGHV mutational status in 224 CLL cases. The numbers of CLL cases (subdivided into different quartiles according to miR-146b-5p concentrations) are indicated in each bar. (B) Kaplan-Meier curves of the 6-year TTFT probability of cases stratified according to miR-146b-5p expression. Cases with the lowest miR-146b-5p (first quartile) expression had a 47% probability of prolonged TTFT compared with that of 73%, 83%, and 79% of cases within the second, third, and fourth quartile, respectively. Statistical significance of associations between individual variables and survival was calculated using the log-rank test. (C) Kaplan-Meier curves showing TTFT probability of cases stratified in different quartiles defined by miR-146a-5p expression. Statistical significance of associations between individual variables and survival was calculated using the log-rank test. (D) Kaplan-Meier curves comparing TTFT of miR-146b-5p–low (first quartile) or miR-146b-5p higher expression (second to fourth quartiles) in IGHV-UM cases and in (E) IGHV-M cases. Statistical significance of associations between individual variables and survival was calculated using the log-rank test. (F) Cox multivariate analysis of IGVH-UM CLL cases (n = 80) showing that low miR-146b-5p expression (first quartile) maintains an independent prognostic impact in the presence of other prognostic indicators (P = .035).

Figure 2.

Potential regulatory function of miR-146b-5p on the expression of IL-12Rβ1. (A) The inhibitory effect of a given miRNA on a target sequence (3′UTR) expression was measured in cultured HEK293 cells. These cells were transfected with either the IL-23R 3′UTR or the 3' UTRI L-12Rβ1 together with miR mimics and miR control as indicated. Both firefly luciferase and Renilla luciferase activities were measured after a 48-hour culture. Data shown are relative to the reporter vector transfected with the miR CTR mimic and represent the mean of 5 and 7 experiments, respectively, carried out in triplicate. ∗P < .05. (B) Sequence alignment of miR-146b-5p and miR-146a-5p. The seed region on miRNAs and the potential target sequence on mRNA (site type 7mer-A1, Target scan release 7.2) are indicated in green. A further base pairing of miR-146b-5p with the 3′UTR IL-12Rβ1 is indicated in red. The position coordinates are indicated for the IL-12Rβ1 transcript isoform 3′ UTR: ENSG0000096996.11: ENST00000322153.7. (C) The insert sequence of 3′ UTR clone NM_153701 (Origene, cod SC208722) (IL-12Rβ1 3' UTR-WT) and the same insert deleted of the sequence GTTCTCA complementary to the seed sequence (nt328-nt334, Origene) (IL-12Rβ1 3′ UTR-MUT). Blue, stop codon; red, cloning site; highlighted in green, the seed sequence. (D) HEK 293 cells were transfected with either the IL-12Rβ1 3′UTR-WT or with IL-12Rβ1 3′UTR-MUT with miR mimics and miR control. Both firefly luciferase and Renilla luciferase activities were measured after a 48-hour culture. Data shown are relative to the reporter vector transfected with the miR CTR mimic (reference line at 100%) and represent the mean of 4 experiments carried out in triplicate. P value is statistically significant (P < .05, t test). (E) Western blotting analysis of IL-12Rβ1 and GAPDH or β-Actin expression in purified CLL cells from 7 different cases transfected with miR-146b-5p inhibitor or miR-CTR inhibitor and cultured for 48 hours. (F) Fold change values of the IL-12Rβ1 signal normalized to that of GAPDH or β-Actin of cells transfected with miR-146b-5p inhibitor/IL-12Rβ1 normalized signal of cells transfected with miR-CTR inhibitor. Percentage (%) changes in the relative abundance of IL-12Rβ1 protein are also indicated for each CLL sample and calculated as (fold change − 1) × 100. A positive percentage indicates increased abundance relative to the control. (G) Summary of the results of the experiments in (F). Protein bands from immunoblotting were analyzed using ImageJ Analysis Software or by Alliance LD, UVITEC. Data are presented as IL-12Rβ1/GAPDH or IL-12Rβ1/β-Actin (mean ± SD). The P value of the difference between CLL cells treated with miR-146b-5p vs miR-CTR inhibitors is indicated (Wilcoxon test). ∗P < .05.

Figure 3.

Expression of the IL-12Rβ1 chain and a functional IL-23R complex following downregulation of miR-146b-5p. Purified CLL cells were transfected with miR-146b-5p inhibitor or miR CTR inhibitor, cultured for different times and analyzed for the IL-23R complex expression and for IL-23 production. (A) Time course analysis of the IL-23R complex expression after transfection with miR CTR inhibitor or miR-146b-5p inhibitor in a representative CLL case (GE1-AG114). Cells expressing the IL-23R complex were measured by flow cytometry, determining the simultaneous expression of both the IL-23R and IL-12Rβ1 chains. Double-positive cells were considered as IL-23R complex-positive. Only viable cells were gated, and of these were mainly CD19+CD5+ since cells with this phenotype that were purified before transfection (see also supplemental Figure 7). (B-D)Summary of time course expression of IL23R complex, (C) IL-12Rβ1, and (D) IL-23R side chain determined in cells from 8 CLL cases by flow cytometry before (T0) and after treatment as in (A). Data are expressed as a percentage of positive cells (mean ± SD). (E) IL-23 production in cell supernatants from 6 CLL cases treated as in (A). (F) Representative experiment on cells from GE1-AG114 CLL case to show the presence of a functional IL-23R complex. Purified CLL cells were transfected with the indicated miR inhibitors and cultured for 24 to 72 hours in the presence or absence of IL-23 (100 ng/mL), with/without IL-23-neutralizing mAbs (αIL-23p19). Viable cells (annexin-V/PI-negative cells) were determined after a 72-hour culture. (G) Summary of time course experiments on cells from 8 CLL cases treated and analyzed as in (F). Data are plotted as percent of viable cells mean ± SD, and the P value indicates the differences between the different culture conditions. (H) Determination of cell cycle phases by flow cytometry in CLL cells (GE1-AG114) transfected with the indicated miR inhibitors and cultured for 48 hours in the presence or absence of IL-23 (100 ng/mL), with/without IL-23 neutralizing mAbs (αIL-23p19) in the indicated combinations. Flow logic software was employed for the analyses. Proliferating (G2M) cells are indicated in green. (I) Summary of experiments on cells from 8 different CLL cases performed and analyzed as in (I). The values were determined after 48 hours in culture. P values are indicated (Wilcoxon test). ∗P = .04 and ∗∗P = .0078.

Figure 3.

Expression of the IL-12Rβ1 chain and a functional IL-23R complex following downregulation of miR-146b-5p. Purified CLL cells were transfected with miR-146b-5p inhibitor or miR CTR inhibitor, cultured for different times and analyzed for the IL-23R complex expression and for IL-23 production. (A) Time course analysis of the IL-23R complex expression after transfection with miR CTR inhibitor or miR-146b-5p inhibitor in a representative CLL case (GE1-AG114). Cells expressing the IL-23R complex were measured by flow cytometry, determining the simultaneous expression of both the IL-23R and IL-12Rβ1 chains. Double-positive cells were considered as IL-23R complex-positive. Only viable cells were gated, and of these were mainly CD19+CD5+ since cells with this phenotype that were purified before transfection (see also supplemental Figure 7). (B-D)Summary of time course expression of IL23R complex, (C) IL-12Rβ1, and (D) IL-23R side chain determined in cells from 8 CLL cases by flow cytometry before (T0) and after treatment as in (A). Data are expressed as a percentage of positive cells (mean ± SD). (E) IL-23 production in cell supernatants from 6 CLL cases treated as in (A). (F) Representative experiment on cells from GE1-AG114 CLL case to show the presence of a functional IL-23R complex. Purified CLL cells were transfected with the indicated miR inhibitors and cultured for 24 to 72 hours in the presence or absence of IL-23 (100 ng/mL), with/without IL-23-neutralizing mAbs (αIL-23p19). Viable cells (annexin-V/PI-negative cells) were determined after a 72-hour culture. (G) Summary of time course experiments on cells from 8 CLL cases treated and analyzed as in (F). Data are plotted as percent of viable cells mean ± SD, and the P value indicates the differences between the different culture conditions. (H) Determination of cell cycle phases by flow cytometry in CLL cells (GE1-AG114) transfected with the indicated miR inhibitors and cultured for 48 hours in the presence or absence of IL-23 (100 ng/mL), with/without IL-23 neutralizing mAbs (αIL-23p19) in the indicated combinations. Flow logic software was employed for the analyses. Proliferating (G2M) cells are indicated in green. (I) Summary of experiments on cells from 8 different CLL cases performed and analyzed as in (I). The values were determined after 48 hours in culture. P values are indicated (Wilcoxon test). ∗P = .04 and ∗∗P = .0078.

Figure 4.

Downregulation of miR-146b-5p expression by CLL cells cocultured with CD40L-TC. (A) Comparison of IL-23R complex expression by cells from a representative CLL case (GE1-AG114) transfected with the indicated miR inhibitors or cocultured with CD40L-TC or mock control cells for 48 hours and analyzed by flow cytometry. (B) Summary of data on IL-23R complex expression (determined by flow cytometry) after a 48-hour exposure of purified CLL cells to miR-146b-5p inhibitor or CD40L-TC as in (A). CLL cells were from cases GE1-AG114, GE1-DM210, GC0015, SV1-SA, SR1-ME1077, MG0482, VF0384, and CM18. P values of the difference between CLL cells treated with miR-146b-5p vs miR CTR inhibitors or exposed to CD40L-TC vs mock cells are indicated (Wilcoxon test). ∗∗P = .0078. (C) Time course experiments to determine cell viability, cell activation, and miR-146b-5p expression following coculture with CD40L-TC. The top panels show CLL cells morphology based on their side scatter-A and forward scatter-A features before (T0) and after CD40L engagement in the representative CLL case GE1-CC190. The gate (live gate) indicates viable cells. Lower panels summarize data of the experiments in 3 different CLL cases (GE1-GA191, GE1-CC190, and SR1-ME1077). Cell viability was measured by flow cytometry by excluding annexin-V/PI-positive cells (left panel). Expression of the CD80 activation marker by purified CLL cells was evaluated by flow cytometry and is indicated as the percentage of positive cells in the viable cell gate (middle panel). The lower right panel shows miRNA expression by RT-qPCR. Data are expressed as ΔCT of miR-146b-5p vs miR-93. Data are plotted as mean ± SD. (D) RT-qPCR analysis of miR-146b 5p (CLL cases PF0024, HG0135, SR0112, CA0058, RD0468, and LG0337) and IL-12Rβ1 mRNA (CLL cases PF0024, HG0135, CA0058, RD0468, MA0151, and AR0090) expression by CLL cells transfected with miR-146b-5p inhibitors and subsequently cultured with CD40L-TC for 48 hours. miR-146b-5p expression was calculated as fold change compared with values observed in CLL cells transfected with miR-CTR inhibitors normalized to RNU44 and U6 small nuclear RNA(left panel). IL-12Rβ1 mRNA expression was calculated as fold change using CLL cells transfected with miR CTR inhibitor cells as calibrator normalized vs POL2RA gene mRNA (right panel). Data are plotted as mean ± SD. (E) IL-12Rβ1 chain expression by CLL cells from cases PF0024, HG0135, SR0112, CA0058, DF0319, RD0468, MA0151, AR0090, PD0164, and SR1-ME1077 transfected with the indicated miR inhibitors and cultured for 72 hours in the presence of CD40L-TC. Cells were analyzed by flow cytometry, and data are expressed as a percentage of positive cells. (F) IL-23R complex expression by CLL cells of the same cases analyzed in (E). Data are expressed as a percentage of positive cells (mean ± SD). P values of the difference between stimulated CLL cells and control samples are indicated (Wilcoxon test). ∗P < .05. (G) Flow cytometric analysis of IL-21 receptor expression by CLL cells treated as in (E) in a representative case DF0319. (H) Comparison of IL-23R complex or IL-21R expression by CLL cells from 5 cases (HG0135, SR0112, DF0319, RD0468, and PD0164) treated with the indicated miR inhibitors and cultured with CD40L-TC cells. Data are expressed as a percentage of positive cells (mean ± SD).

Figure 4.

Downregulation of miR-146b-5p expression by CLL cells cocultured with CD40L-TC. (A) Comparison of IL-23R complex expression by cells from a representative CLL case (GE1-AG114) transfected with the indicated miR inhibitors or cocultured with CD40L-TC or mock control cells for 48 hours and analyzed by flow cytometry. (B) Summary of data on IL-23R complex expression (determined by flow cytometry) after a 48-hour exposure of purified CLL cells to miR-146b-5p inhibitor or CD40L-TC as in (A). CLL cells were from cases GE1-AG114, GE1-DM210, GC0015, SV1-SA, SR1-ME1077, MG0482, VF0384, and CM18. P values of the difference between CLL cells treated with miR-146b-5p vs miR CTR inhibitors or exposed to CD40L-TC vs mock cells are indicated (Wilcoxon test). ∗∗P = .0078. (C) Time course experiments to determine cell viability, cell activation, and miR-146b-5p expression following coculture with CD40L-TC. The top panels show CLL cells morphology based on their side scatter-A and forward scatter-A features before (T0) and after CD40L engagement in the representative CLL case GE1-CC190. The gate (live gate) indicates viable cells. Lower panels summarize data of the experiments in 3 different CLL cases (GE1-GA191, GE1-CC190, and SR1-ME1077). Cell viability was measured by flow cytometry by excluding annexin-V/PI-positive cells (left panel). Expression of the CD80 activation marker by purified CLL cells was evaluated by flow cytometry and is indicated as the percentage of positive cells in the viable cell gate (middle panel). The lower right panel shows miRNA expression by RT-qPCR. Data are expressed as ΔCT of miR-146b-5p vs miR-93. Data are plotted as mean ± SD. (D) RT-qPCR analysis of miR-146b 5p (CLL cases PF0024, HG0135, SR0112, CA0058, RD0468, and LG0337) and IL-12Rβ1 mRNA (CLL cases PF0024, HG0135, CA0058, RD0468, MA0151, and AR0090) expression by CLL cells transfected with miR-146b-5p inhibitors and subsequently cultured with CD40L-TC for 48 hours. miR-146b-5p expression was calculated as fold change compared with values observed in CLL cells transfected with miR-CTR inhibitors normalized to RNU44 and U6 small nuclear RNA(left panel). IL-12Rβ1 mRNA expression was calculated as fold change using CLL cells transfected with miR CTR inhibitor cells as calibrator normalized vs POL2RA gene mRNA (right panel). Data are plotted as mean ± SD. (E) IL-12Rβ1 chain expression by CLL cells from cases PF0024, HG0135, SR0112, CA0058, DF0319, RD0468, MA0151, AR0090, PD0164, and SR1-ME1077 transfected with the indicated miR inhibitors and cultured for 72 hours in the presence of CD40L-TC. Cells were analyzed by flow cytometry, and data are expressed as a percentage of positive cells. (F) IL-23R complex expression by CLL cells of the same cases analyzed in (E). Data are expressed as a percentage of positive cells (mean ± SD). P values of the difference between stimulated CLL cells and control samples are indicated (Wilcoxon test). ∗P < .05. (G) Flow cytometric analysis of IL-21 receptor expression by CLL cells treated as in (E) in a representative case DF0319. (H) Comparison of IL-23R complex or IL-21R expression by CLL cells from 5 cases (HG0135, SR0112, DF0319, RD0468, and PD0164) treated with the indicated miR inhibitors and cultured with CD40L-TC cells. Data are expressed as a percentage of positive cells (mean ± SD).

Figure 5.

Influence of miR-146b-5p concentrations on the expression of the IL-12Rβ1 chain by CLL cells cocultured with CD40L-TC. (A) Purified CLL cells were transfected with the indicated miRNA mimics and cultured for 48 hours with CD40L-TC and IL-12Rβ1 mRNA concentrations determined by RT-qPCR. The cases studied were PF0024, DF0319, SR0112, CA0058, RD0468, MA0151, AR0090, LG0337, PD0164, and HG0135. Data are plotted as mean ± SD. (B) Evaluation of IL-23R and IL-12Rβ1 chain expression by flow cytometry in 1 representative CLL case (SR1-ME1077) cultured with CD40L-TC for 72 hours. Only viable cells were gated, and double-positive cells were considered IL-23R complex-positive. (C) IL-23R complex expression by CLL cells (cases PF0024, HG0135, SR0112, CA0058, DF0319, RD0468, MA0151, AR0090, PD0164, and SR1-ME1077) transfected with the indicated miRNAs and cultured with CD40L-TC for 72 hours. Data are expressed as a percentage of double (IL-12Rβ1+IL-23R+) positive cells (mean ± SD). (D) IL-12Rβ1 side chain, (E) IL-23R side chain, and (F) IL-23 side chain-only expression by the CLL cells from the same cases analyzed in (C). Data are expressed as a percentage of positive cells (mean ± SD). (G) Ki67 expression was analyzed by flow cytometry in CLL cells cultured with CD40L-TC for 72 hours following transfection of the indicated miRNA mimics. Data from a representative case (CLL RD0468) are shown. (H) Multiparametric flow cytometry test to detect cells expressing both IL-23R complex and Ki67 in the same representative experiment as in (E). The cells were stained for Ki67 and the IL-12Rβ1 and IL-23R chains of the IL-23 receptor complex. Ki67+ cells gated in (E) are highlighted by the green dots, whereas the whole cell population is indicated by gray dots. (I) Summary of Ki67+ cells determinations obtained on 11 different CLL cases (PF0024, HG0135, SR0112, CA0058, DF0319, RD0468, MA0151, AR0090, PD0164, LG0337, and SR1-ME1077) after a 72-hour culture with CD40L-TC following transfection of the indicated miRNA mimics. Data are expressed as mean ± SD. P values of the difference between treated and control cells are indicated (Wilcoxon test). ∗P < .05.

Figure 6.

Effects of in vivo treatment with miRNA mimics on CLL cells engrafted in NSG mice. Mice were injected with 50 × 106 CLL cells prestimulated with autologous activated T cells. After 4 to 6 weeks, blood samples were evaluated for the presence of circulating leukemic cells by flow cytometry, and after CLL cell engraftment was achieved, the mice were treated with 3 doses of miR-146b-5p or miR CTR mimic. Mice were sacrificed after 3 days from the last inoculum, and cell suspensions from the spleen, bone marrow (BM), and peripheral blood (PBL) were analyzed by flow cytometry for the percentage of CD19+CD5+ CLL cells or CD19−CD5+ T cells over the total of human CD45+ cells. (A) Flow cytometry analysis of 2 representative mice injected with CLL GE1-PM129 and treated with miR CTR mimic (NSG 13) or miR-146b-5p (NSG 5). (B) Summary of the flow cytometry analyses of 14 mice injected with CLL cells from 2 different cases (GE1-PM129 and GE1-RO148) and treated as in (A). Percentages of CD19+CD5+ CLL cells or (C) CD19−CD5+ T cells are shown. (D) Apoptotic neoplastic cells and T cells in the spleen from a miR-146b-5p (NSG 16) and a miR CTR (NSG 13) mimic-treated mouse. Apoptotic cells were detected by flow cytometry as annexin-V–positive cells by gating CD45+CD19+CD5+ CLL cells (solid red histogram profiles) or CD45+CD19−CD5+ T cells (solid blue histogram profiles). (E) Summary of the flow cytometry tests carried out in 14 mice treated as in (D). Percentages of apoptotic CLL and T cells are expressed as mean ± SD. (F) High-power magnification (200×) images of longitudinal sections of paraffin-embedded mouse spleen stained with αCD20-Ab or αCD3-Ab showing the infiltrating foci present in mice treated with miR CTR (NSG 13) or miR-146b-5p mimics (NSG 16).

Figure 7.

Treatment with miR-146b-5p mimic inhibits CLL growth, proliferation, and IL-12Rβ1 expression in the NSG mouse model. (A) Low magnification image (top) of longitudinal sections of paraffin-embedded mouse spleen (CLL GE1-PM129) stained for CD20 (left) or CD3 (right). The spleen from a mouse treated with miR CTR mimic (NSG13, top) or with miR-146b-5p mimic (NSG16, bottom) is represented. (B) Higher magnification (40×) of the areas of spleen highlighted by the boxes in (A) showing a decrease of neoplastic B cells (and not of T cells) in the follicular infiltrates after treatment with miR-146b-5p compared with control samples. (C) IHC analysis of Ki67+ cycling cells at 100× and 200× magnification in spleens of the same mice treated as indicated in (A). Decreased proliferating cells in the spleen infiltrate of mice treated with miR-146b-5p mimic (lower panels) compared with control samples (upper panels) is evident. (D) High magnification image (400×) of longitudinal sections of the spleens of the same mice as in (C) double-stained for CD20 and Ki67. (E) Double-marker immunofluorescence (IF) and confocal microscopy (high-magnification image 400×) analysis of IL-12Rβ1 chain and CD20 (upper panels) or CD3 chain (lower panels) and DAPI of the spleens of miR-CTR (NSG 14, left) or miR-146b-5p mimic-treated mice (NSG 5, right). (F) Double-marker IF and confocal microscopy (high-magnification 200×) analysis of IL-12Rβ1 chain and IL-23 or DAPI of a representative of the spleens of miR-CTR (NSG 14, upper panel) or miR-146b-5p mimic-treated mice (NSG 5, lower panel). IF microphotographs are representative of analyses of ≥5 low-power magnification (100×) or 10 high-power magnification (200× and 400×) microscopic fields performed on each mouse tissue sample.