Figure 6.
AP-1 family members act downstream CD79a and are selectively upregulated in CNS leukemia. (A) Volcano plot showing significantly (FDR ˂0.05; log2(FC) < −1 or log2(FC) ˃ 1) regulated genes from RNA sequencing data of BCP-ALL PDX cells recovered from the CNS compared with PDX cells isolated from the spleen (SP). (B) Heat map representation of the top differentially regulated AP-1 pathway genes in CNS relative to SP (blue: downregulated; red: upregulated; samples are represented in columns whereas rows show genes. (C) Fast gene set enrichment analysis on the RNA sequencing data of CNS-isolated PDX cells vs spleen and cerebral organoid–infiltrated 697 and K2 cells vs respective noninfiltrated fraction, displaying significantly regulated gene set signatures. (D) Validation of upregulation of AP-1 genes via qRT-PCR in 7 PDX ALL samples isolated from the SP vs CNS, Mann-Whitney U test, graphs show mean with standard error; ∗P ≤ .05, ∗∗P ≤ .01, and ∗∗∗P ≤ .001. (E) The effect of shRNA-mediated knock down of CD79a (shCD79a) on the expression of AP-1 genes compared with control shRNA (shCtr) in an TCF3::PBX1+ PDX sample as determined via qRT-PCR, Mann-Whitney U test, graphs show mean with standard error. ∗P ≤ .05, ∗∗P ≤ .01, and ∗∗∗P ≤ .001. (F-G) CD79a, JUN, and other genes mRNA levels (all normalized to mRNA levels in the 697 cell line) were measured in diagnostic bone marrow (BM) samples in a selected cohort of 100 pediatric patients with BCP-ALL of mixed cytogenetics, which contained 28 patients with CNS-positive disease matched to 72 patients with CNS-negative disease of corresponding sex and age. (F) Bivariate correlation analysis between CD79a expression and JUN, FOS, and FOSB in patients with BCP-ALL diagnosed as CNS positive (CNS+) vs CNS-negative (CNS−), 2-sided t test. (G) JUN-expression levels in patients with BCP-ALL diagnosed as CNS+ vs CNS−, 2-sided t test. (H) mRNA levels of JUN were detected within patients with BCP-ALL in a cohort enriched for patients who are CNS+ and analyzed for association with the occurrence of relapse. Depicted are mRNA levels normalized to calibrator of n = 83 patients without CNS relapse vs n = 17 patients with CNS relapse, 2-tailed Mann-Whitney U test, ∗P < .05. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were used for relative quantification of the mRNA transcript. AU, arbitrary units; FC, fold change.

AP-1 family members act downstream CD79a and are selectively upregulated in CNS leukemia. (A) Volcano plot showing significantly (FDR ˂0.05; log2(FC) < −1 or log2(FC) ˃ 1) regulated genes from RNA sequencing data of BCP-ALL PDX cells recovered from the CNS compared with PDX cells isolated from the spleen (SP). (B) Heat map representation of the top differentially regulated AP-1 pathway genes in CNS relative to SP (blue: downregulated; red: upregulated; samples are represented in columns whereas rows show genes. (C) Fast gene set enrichment analysis on the RNA sequencing data of CNS-isolated PDX cells vs spleen and cerebral organoid–infiltrated 697 and K2 cells vs respective noninfiltrated fraction, displaying significantly regulated gene set signatures. (D) Validation of upregulation of AP-1 genes via qRT-PCR in 7 PDX ALL samples isolated from the SP vs CNS, Mann-Whitney U test, graphs show mean with standard error; ∗P ≤ .05, ∗∗P ≤ .01, and ∗∗∗P ≤ .001. (E) The effect of shRNA-mediated knock down of CD79a (shCD79a) on the expression of AP-1 genes compared with control shRNA (shCtr) in an TCF3::PBX1+ PDX sample as determined via qRT-PCR, Mann-Whitney U test, graphs show mean with standard error. ∗P ≤ .05, ∗∗P ≤ .01, and ∗∗∗P ≤ .001. (F-G) CD79a, JUN, and other genes mRNA levels (all normalized to mRNA levels in the 697 cell line) were measured in diagnostic bone marrow (BM) samples in a selected cohort of 100 pediatric patients with BCP-ALL of mixed cytogenetics, which contained 28 patients with CNS-positive disease matched to 72 patients with CNS-negative disease of corresponding sex and age. (F) Bivariate correlation analysis between CD79a expression and JUN, FOS, and FOSB in patients with BCP-ALL diagnosed as CNS positive (CNS+) vs CNS-negative (CNS−), 2-sided t test. (G) JUN-expression levels in patients with BCP-ALL diagnosed as CNS+ vs CNS−, 2-sided t test. (H) mRNA levels of JUN were detected within patients with BCP-ALL in a cohort enriched for patients who are CNS+ and analyzed for association with the occurrence of relapse. Depicted are mRNA levels normalized to calibrator of n = 83 patients without CNS relapse vs n = 17 patients with CNS relapse, 2-tailed Mann-Whitney U test, ∗P < .05. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were used for relative quantification of the mRNA transcript. AU, arbitrary units; FC, fold change.

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