The PAX5-JAK2 translocation acts as dual-hit mutation that promotes aggressive B-cell leukemia via nuclear STAT5 activation (2022)
Figure 3

Figure 3. The loss of wild-type Pax5 accelerates B-ALL development in Pax5Jak2/+ mice.

(A) RNA-seq expression profile at the Pax5 locus in Pax5Jak2/+ B-ALL cells (black) and Pax5+/+ pro-B cells (grey). The exon-intron structure and the two alternative promoters at exons 1A and 1B of Pax5 are shown below.

(B) Immunoblot analysis of nuclear extracts isolated from Pax5+/+ (WT) pro-B cells and Pax5Jak2/+ lymph node tumor cells. The wild-type Pax5 and Pax5-Jak2 proteins were detected with an antibody raised again the N-terminal (N-term) paired domain of Pax5. The size of marker proteins is indicated in kilodaltons (kDa) to the left.

(C) Flow-cytometric analysis of Pax5 protein expression by intracellular Pax5 staining of lymph node B220+CD19+ B cells from a Pax5+/+ mouse (grey filled) and B220lowCD19+ B-ALL cells from a 10-week-old Pax5Jak2/+ tumor mouse (black). The full-length Pax5 protein was detected with an antibody that recognizes C-terminal Pax5 sequences that are absent in the Pax5-Jak2 protein.

(D) Flow-cytometric analysis of bone marrow cells from 4-week-old Pax5ihCd2/+ and Pax5Jak2/ihCd2 mice. The expression of human (h) CD2 from the Pax5ihCd2 allele is shown for CD19+B220+ (grey) and CD19+B220low (black) B cells.

(E) Loss of the wild-type Pax5 allele in CD19+B220low B cells from the bone marrow of 6-week-old Pax5Jak2/+ mice. Genomic DNA isolated from flow cytometry-sorted CD19+B220+ (+) and CD19+B220low (low) B cells was analyzed by PCR with primers amplifying exon 4 of the wild-type Pax5 allele and unique sequences of the Pax5Jak2 allele (Table EV1), as shown in the schematic diagram of the two Pax5 alleles. The positions of the PCR fragments corresponding to the two Pax5 alleles are indicated to the right of the agarose gel.

(F) Copy number analysis of the Pax5Jak2 allele by quantitative PCR analysis with primers amplifying unique sequences across the Pax5 exon 5-Jak2 exon 19 junction of the Pax5Jak2 allele. The PCR analysis was performed with genomic DNA isolated from lymph node (LN) Pax5Jak2/+ B-ALL cells (3 experiments), from flow cytometry-sorted CD19+B220+ and CD19+B220low B cells from the bone marrow (BM) of 6-week-old Pax5Jak2/+ mice (2 experiments each) and from CD19+B220+ B cells from Pax5+/+ bone marrow (2 experiments). The PCR data were normalized to the product amplified from a control region located 35 Mb upstream of Pax5, and the ratio obtained for B220+ B cells from Pax5Jak2/+ bone marrow was set to 1 copy number of the Pax5Jak2 allele.

(G) Kaplan-Meier survival analysis of Cd79a-Cre Ikzf1neo/+ Pax5LSL-Jak2/+ (black) and Cd79a-Cre Pax5LSL-Jak2/+ (grey) mice. Statistical analysis of the survival curves was performed with the log-rank (Mantel-Cox) test; ****P < 0.0001. n, number of mice analyzed.

(H) Flow-cytometric analysis of B220 and CD19 expression in B-ALL tumor cells from the lymph node of a Cd79a-Cre Ikzf1neo/+ Pax5LSL-Jak2/+ mouse (black; left). Pax5 expression in these B-ALL tumor cells (black line) and control Pax5+/+ lymph node B cells (grey filled) was determined by intracellular Pax5 staining (right).