Abstract
Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and resistance to glucocorticoids in leukemia cells confers poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the prednisolone sensitivity of primary leukemia cells from 444 patients newly diagnosed with ALL and found significantly higher expression of CASP1 (encoding caspase 1) and its activator NLRP3 in glucocorticoid-resistant leukemia cells, resulting from significantly lower somatic methylation of the CASP1 and NLRP3 promoters. Overexpression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished the glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1-overexpressing ALL. Our findings establish a new mechanism by which the NLRP3-CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on the glucocorticoid transcriptional response suggests that this mechanism could also modify glucocorticoid effects in other diseases.
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Acknowledgements
We gratefully acknowledge the patients and their parents who participated in this research. We are appreciative of the expert technical assistance of M. Roberts, Y. Chu, Y. Wang, M.A. Payton, J. Stukenborg, S. Salehy, M. Needham, M. Chung, N. Lenchik, M. Loyd and E. Walker. We thank J. Groff and E. Stevens for figure preparation assistance and C. Simmons for assistance with manuscript preparation. We thank D. Green for his scientific advice and discussion of the manuscript. We thank C. Stewart, G. Neale, J. Morris and K. Rakestraw for their technical advice and expertise. This work was supported in part by US National Institutes of Health (NIH) National Cancer Institute grant R37CA36401 (W.E.E., M.V.R. and C.-H.P.), US NIH National Institute of General Medical Sciences Pharmacogenomics Research Network grant U01GM92666 (M.V.R. and W.E.E.), US NIH grant F32CA141762 (S.W.P.) and an American Recovery and Reinvestment Act supplement, 3R37CA036401-26S1 (W.E.E.). This work was also supported by Cancer Center Support Grant CA21765 from the National Cancer Institute and by the American Lebanese Syrian Associated Charities (ALSAC). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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S.W.P., E.J.B., D.S., L.B.R., W.E.T., P.G., R.K.S.M., M.A., A.M., J.M., D.R.C., L.T.L., Y.F., R.K.G., T.-D.K., M.V.R. and W.E.E. designed experiments. C.-H.P., S.J., M.V.R. and W.E.E. designed clinical trials. S.W.P., E.J.B., D.S., L.B.R., W.E.T., P.G., R.K.S.M., M.A., A.M., D.R.C., L.T.L., Y.F., A.Z., A.G., D.C., J.J.B. and L.H. performed experiments. S.W.P., E.J.B., D.S., L.B.R., W.E.T. and W.E.E. wrote the manuscript (reviewed by all authors). S.W.P., E.J.B., D.S., L.B.R., W.E.T., D.R.C., L.T.L., J.C.P., J.R.M., Y.F., K.R.C., G.S., M.R.W., A.M.F., C.C., W.Y., S.E.K., C.A.F., B.D., C.S., J.K.H., A.Z., A.G., D.C., J.J.B., L.H., C.G.M., M.L.d.B., R.P., S.J., T.L.D., F.L., D.B., W.L.C., C.-H.P., R.M.M., R.K.G., T.-D.K., M.V.R. and W.E.E. analyzed data.
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W.E.E., S.W.P. and E.J.B. are named as co-inventors on a pending patent application that relates to the subject matter of the article, which was filed by St. Jude Children's Research Hospital.
Integrated supplementary information
Supplementary Figure 1 Glucocorticoid-resistant leukemia cells have higher expression of CASP1 and NLRP3.
CASP1 (a–c) and NLRP3 (d–f) expression was significantly higher in glucocorticoid-resistant leukemia cells from three cohorts of newly diagnosed patients. Exact Wilcoxon Mann-Whitney rank-sum test P values are shown for a–f, with Stouffer's z-score method meta-analysis P values shown above a–f as described in the Online Methods.
Supplementary Figure 2 CASP1 and NLRP3 methylation probe locations.
DNA methylation analysis probe locations for CASP1 and NLRP3 relative to the transcription start sites of these genes. The specific base analyzed is shown in square brackets with the genomic context surrounding each site.
Supplementary Figure 3 Hypomethylation of the CASP1 and NLRP3 promoter region was associated with higher CASP1 and NLRP3 expression in leukemia cells.
In both patient cohorts for which DNA was available for DNA methylation analysis (St. Jude Protocols XV and XVI), significantly lower levels of CASP1 (a,b) and NLRP3 (d,e) methylation were found in leukemia cells with higher expression of CASP1 and NLRP3. For both CASP1 and NLRP3 methylation status, the DNA methylation site (CpG) was within 100 bp of the transcription start site (Supplementary Fig. 2). k-means clustering analysis (a triangle represents k means–identified group A, a circle represents k means–identified group B, pink and red squares represent k means–identified centers for group A and B, respectively) utilizing only CASP1 and NLRP3 methylation status significantly discriminated sensitive leukemias (blue symbols; higher methylation) from resistant leukemias (orange symbols; lower methylation) in patients from both St. Jude Protocol XV and St. Jude Protocol XVI (g,h). Exact Wilcoxon Mann-Whitney rank-sum test P values are shown for a, b, d and e, with Stouffer's z-score method meta-analysis P values shown above a, b, d and e.
Supplementary Figure 4 Germline versus somatic DNA methylation status.
In a subset of patients (n = 55) enrolled on St. Jude Protocol XVI, both germline and somatic DNA methylation was analyzed. (a,b) DNA methylation in these patients, with lines connecting paired samples. Orange symbols show prednisolone-resistant patients, blue symbols show prednisolone-sensitive patients and gray symbols show patients with intermediate resistance as defined in Fig. 1c. Paired t-test P values are shown for a and b.
Supplementary Figure 5 CASP1 increases resistance to glucocorticoids.
Enforced expression of CASP1 in a human B-lineage leukemia cell line (697; harboring an E2A-PBX1 translocation) increased resistance to prednisolone after activation of the NALP3 inflammasome (by addition of LPS and ATP). 697 cells were transduced with a lentivirus containing full-length CASP1 and genes encoding puromycin N-acetyltransferase or puromycin N-acetyltransferase alone (control). Cells were selected with puromycin, and their sensitivity to prednisolone was measured using the MTT assay, in the presence (+) or absence (–) of inflammasome activation (LPS and ATP). LC50 values for control and CASP1-expressing cells in the presence of LPS and ATP were 0.73 ± 0.16 µM and 1.9 ± 0.73 µM prednisolone, respectively (P = 0.01).
Supplementary Figure 6 CrmA expression increases glucocorticoid receptor levels and restores glucocorticoid sensitivity in primary glucocorticoid-resistant leukemia cells.
Primary leukemia cells isolated from a patient with glucocorticoid-resistant ALL and high levels of CASP1 expression were transduced with lentivirus either expressing RFP plus the CASP1-inhibitory protein CrmA (CrmA) or RFP alone. (a) The sensitivity (LC50) of these cells to dexamethasone was determined by MTT assay, revealing that overexpression of CrmA reversed glucocorticoid resistance (LC50 = 0.14 μM (95% confidence interval = 0.1426 × 10–2 to 0.2773 μM versus >10 μM; P < 1 × 10–14)). (b) Western blot analysis demonstrates restoration of glucocorticoid receptor protein expression.
Supplementary Figure 7 Endogenous CASP1 protein levels in glucocorticoid-resistant primary ALLs are comparable to CASP1 protein level in the NALM-6 leukemia cells expressing recombinant CASP1.
CASP1 protein levels by western blot in primary leukemia cells from two glucocorticoid-resistant patients and NALM-6 cells expressing recombinant CASP1 are shown. Lane 1 is lysate from NALM-6 ALL cells transduced with the control (empty) lentivirus and has undetectable CASP1, lane 2 depicts lysate from CASP1-transduced NALM-6 leukemia cells (in which CASP1 was not activated with LPS and ATP), and lanes 3 and 4 are lysates from primary leukemia cells isolated from patients with glucocorticoid-resistant ALL (LC50 = 1,387 and 206.4 μM, respectively). Recombinant CASP1 has a higher molecular weight than endogenous CASP1, owing to the Myc-DDK protein tag.
Supplementary Figure 8 Glucocorticoid-resistant cell lines derived from ALL patient xenografts and NCI60 cell lines often show increased expression of CASP1.
Publically available data from xenografts generated from glucocorticoid-resistant and -sensitive primary leukemia samples (Online Methods) were analyzed for CASP1 expression. (a) Xenografts that were resistant to dexamethasone showed higher CASP1 expression than those that were responsive to dexamethasone, including mRNA expression data from two glucocorticoid-resistant ALL xenografts established from newly diagnosed patients at St. Jude Children’s Research Hospital. (b) In a separate analysis, the REH and Nalm6 cell lines and ALL cells in the NCI60 leukemia panel were combined to compare CASP1 expression in glucocorticoid-sensitive and -resistant ALLs. Similar to the xenograft data (a), cell lines that were resistant to prednisolone had higher expression of CASP1. Combining these data revealed a highly significant difference in CASP1 expression levels in glucocorticoid-sensitive and glucocoritcoid-resistant cell lines and xenografts (Wilcoxon rank-sum test, P = 8.9 × 10–7).
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Supplementary Figures 1–8 and Supplementary Tables 1 and 5. (PDF 466 kb)
Supplementary Table 2
Upregulated genes. (XLS 60 kb)
Supplementary Table 3
Downregulated genes. (XLS 67 kb)
Supplementary Table 4
Random unchanged genes. (XLS 488 kb)
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Paugh, S., Bonten, E., Savic, D. et al. NALP3 inflammasome upregulation and CASP1 cleavage of the glucocorticoid receptor cause glucocorticoid resistance in leukemia cells. Nat Genet 47, 607–614 (2015). https://doi.org/10.1038/ng.3283
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DOI: https://doi.org/10.1038/ng.3283
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