PRDM16 isoforms differentially regulate normal and leukemic hematopoiesis and inflammatory gene signature

Abstract

PRDM16 is a transcriptional coregulator involved in translocations in acute myeloblastic leukemia (AML), myelodysplastic syndromes, and T acute lymphoblastic leukemia that is highly expressed in and required for the maintenance of hematopoietic stem cells (HSCs), and can be aberrantly expressed in AML. Prdm16 is expressed as full-length (fPrdm16) and short (sPrdm16) isoforms, the latter lacking the N-terminal PR domain. The role of both isoforms in normal and malignant hematopoiesis is unclear. We show here that fPrdm16 was critical for HSC maintenance, induced multiple genes involved in GTPase signaling, and repressed inflammation, while sPrdm16 supported B cell development biased toward marginal zone B cells and induced an inflammatory signature. In a mouse model of human MLL-AF9 leukemia, fPrdm16 extended latency, while sPrdm16 shortened latency and induced a strong inflammatory signature, including several cytokines and chemokines that are associated with myelodysplasia and with a worse prognosis in human AML. Finally, in human NPM1-mutant and in MLL-translocated AML, high expression of PRDM16, which negatively impacts outcome, was associated with inflammatory gene expression, thus corroborating the mouse data. Our observations demonstrate distinct roles for Prdm16 isoforms in normal HSCs and AML, and identify sPrdm16 as one of the drivers of prognostically adverse inflammation in leukemia.

Keywords: Adult stem cells; Bone marrow; Hematology; Leukemias; Stem cells.

Figure 1. Prdm16 supports normal HSC function.

(A and B) Frequency (n = 9) (A) and absolute number (n = 6) (B) of HSCs (Lin^–cKit⁺Sca1⁺Flt3^–CD48^–CD150⁺) in BM of adult Vav-Cre^–/– Prdm16^fl/fl (WT) and Vav-Cre^+/– Prdm16^fl/fl (KO) mice. (C) Peripheral blood (PB) donor chimerism of WT or KO BM HSCs in competitive transplants with CD45.1 BM 16 weeks after transplantation (n = 16–18 mice, 3 independent transplants). (D) Percentage lymphoid (CD19⁺ or CD3⁺) and myeloid (Gr1⁺ or Mac1⁺) donor cells from C (n = 16–18 mice). (E) Change in donor/competitor ratio (log₁₀ scale) in primary competitive transplantation recipients and secondary recipients (n = 8 mice, 2 independent transplants). (F) Limiting-dilution assay of WT versus KO BM HSCs. (G) PB donor chimerism of WT or KO FL HSCs 16 weeks after competitive transplantation (n = 8–10 mice, 2 independent transplants). (H) Percentage of donor lymphoid or myeloid donor cells from G (n = 8–10 mice). Mean ± SEM. NS, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test.

Figure 2. Increased respiration in adult Prdm16-deficient HSCs.

(A) GO pathways significantly up- or downregulated in Prdm16-deficient HSCs. Values expressed as –log₁₀ of the P value, determined by PANTHER analysis. (B) HSC frequency in PB of Vav-Cre^–/– Prdm16^fl/fl (WT) and Vav-Cre^+/– Prdm16^fl/fl (KO) mice (n = 3). (C) Fraction of genes upregulated (red) in Prdm16-deficient HSCs among all genes and the 5 respiratory complexes. (D) Extracellular metabolic flux analysis of WT and KO BM HSCs (n = 3 experiments in duplicate, 5 mice per experiment). Rot, rotenone; Ant, antimycin; Oligo, oligomycin. (E and F) Basal oxygen consumption rate (OCR) (E) and respiratory ATP production (F) measured from D (n = 3). (G) ROS measured by the percentage of CellROX Deep Red–positive WT or KO BM HSCs (n = 3). Mean ± SEM. NS, P > 0.05; *P < 0.05; ***P < 0.001; Student’s t test, and χ² test for observed vs. expected values.

Figure 3. Hematopoietic phenotype of fPrdm16-deficient mice.

(A) HSC frequency and absolute number (Lin^–cKit⁺Sca⁺Mac1⁺CD48^–CD150⁺) in FL of Δ47-fPrdm16^–/– (KO), Δ47-fPrdm16^+/– (HET), and WT littermate mice (n = 34 mice). (B) Analysis of Δ13-fPrdm16^–/– mice, performed as in A (n = 28 mice). (C) PB donor chimerism 16 weeks after competitive transplantation of WT or KO FL HSCs (n = 12–14 mice, 3 independent experiments). (D) BM donor chimerism in recipient mice from C 16 weeks after transplantation (n = 6 mice). (E) Percentage lymphoid (CD19⁺ or CD3⁺) donor cells in PB from C (n = 12–14). (F) Percentage of B cells (CD19⁺) among lymphoid cells in E. (G) GO pathways significantly up- or downregulated in Δ47-fPrdm16^–/– FL HSCs. Values expressed as –log₁₀ of the P value, determined by PANTHER analysis. Mean ± SEM. NS, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; 1-way ANOVA for multiple comparisons or Student’s t test for single comparisons.

Figure 4. sPrdm16 supports development of an LSK^– B cell progenitor.

(A) Donor chimerism in BM, thymus, and spleen 16 weeks after competitive transplantation of Δ47-fPrdm16^–/– FL HSCs (n = 6). (B) Representative flow cytometry plots showing gating of Lin^–Sca1⁺Kit^– (LSK^– cells) in BM of recipients of Δ47-fPrdm16^–/– (KO) and WT littermate FL cells (n = 6). (C) Donor LSK^– frequency in recipients of WT and KO FL cells (n = 6). (D) Ratio of marginal zone (CD21^hiCD23^lo) to follicular B cells (CD23^hiCD21^lo) (MZ/FO) among donor splenic B cells (CD19⁺) in recipients of WT and KO FL cells (n = 3). (E) Sca1 mean fluorescence intensity (MFI) of donor MZ cells (n = 3). (F) Relative CD150 MFI of FL HSCs (n = 54 mice). (G) LSK^– frequency (n = 73 mice) in FL from WT, Δ47-fPrdm16^+/– (HET), and KO FL expressed as a percentage relative to litter average. (H) Prdm16 mRNA copies per cell in HSC and LSK^– populations from 8-week-old WT mice (n = 3–4 mice, in triplicate). Mean ± SEM. NS, P > 0.05; *P < 0.05; **P < 0.01; 1-way ANOVA for multiple comparisons or Student’s t test for single comparisons.

Figure 5. sPrdm16 expression in HSCs shortens latency of MLL-AF9 leukemia.

(A) Survival of lethally irradiated mice transplanted with BM HSC-derived MLL-AF9 cells from Vav-Cre^–/– Prdm16^fl/fl (WT) and Vav-Cre^+/– Prdm16^fl/fl (KO) mice. (B) Colony-forming assays of MLL-AF9 cells from A (n = 4 independent assays in duplicate). (C) Survival of lethally irradiated mice transplanted with Prdm16^+/+ (WT), Prdm16^+/– (HET), or Prdm16^–/– (KO) FL HSC-derived MLL-AF9 cells. (D) Colony-forming assays of MLL-AF9 cells from C (n = 4 independent assays in duplicate). (E) Survival of lethally irradiated mice transplanted with MLL-AF9 cells generated from BM Lin^–Sca1^–Kit⁺ cells from Vav-Cre^–/– Prdm16^fl/fl (WT) and Vav-Cre^+/– Prdm16^fl/fl (KO) mice. (F) Survival of lethally irradiated mice transplanted with FL HSC-derived MLL-AF9 cells from Δ47-fPrdm16^–/– (KO) or WT littermate mice. (G) Expression of Prdm16 relative to HSC controls in stem and progenitor cells and in MLL-AF9 leukemic cells (n = 3, in triplicate). MPP, multipotent progenitor. (H) GO pathways significantly up- or downregulated in KO relative to WT MLL-AF9 cells from A. Values expressed as –log₁₀ of the P value, determined by PANTHER analysis. Mean ± SEM. NS, P > 0.05; 1-way ANOVA for multiple comparisons, Gehan-Breslow-Wilcoxon test for comparison of survival curves; n = 13–15 recipients from 3 independently derived MLL-AF9 lines for each of the survival experiments in A, C, E, and F.

Figure 6. Distinct roles of Prdm16 isoforms in MLL-AF9 leukemia.

(A) Colony-forming assays of Prdm16-deficient Vav-Cre^+/– Prdm16^fl/fl (KO) MLL-AF9 cells expressing empty vector, fPrdm16, or sPrdm16 (n = 3 independent assays in duplicate). (B) Survival of lethally irradiated mice transplanted with MLL-AF9 cells expressing empty vector, fPrdm16, sPrdm16, or both (n = 14–15 recipients from 3 independent experiments). (C) Percentage of MLL-AF9 cells in BM of recipient mice 24 hours after transplantation (n = 9 recipients from 3 transplants). (D) Percentage Ki67⁺ cells among MLL-AF9 cells of leukemic mice (n = 4 recipients). (E) GO pathways significantly upregulated in sPrdm16- or fPrdm16-expressing MLL-AF9 cells isolated from leukemic mice. Values expressed as –log₁₀ of the P value, determined by PANTHER analysis. (F) Basal oxygen consumption rate (OCR). (G) Spare respiratory capacity (SRC) (P[EV/fPrdm16] = 0.07, P[fPrdm16/sPrdm16] = 0.08). (H) Respiratory ATP production. (I) Proton leak in MLL-AF9 cells from leukemic mice. n = 4 recipients (F–I). (J) ROS measured by MFI of CellROX Deep Red in MLL-AF9 cells (n = 4). Mean ± SEM. NS, P > 0.05; *P < 0.05; **P < 0.01; 1-way ANOVA for multiple comparisons, Gehan-Breslow-Wilcoxon test for comparison of survival curves.

Figure 7. PRDM16 is associated with an inflammatory signature in a subset of human AML.

(A) Correlation between PRDM16 RPKM and overall survival in all 179 human AML samples from the Cancer Genome Atlas (CGA) (n = 179). (B) PRDM16 RPKM for all samples from A, ranked by RPKM, illustrating Q1/Q2 (PRDM16^lo) and Q4 (PRDM16^hi). (C) Principal component (PC) analysis of cohorts described in B. (D) PRDM16 RPKM compared within 4 mutually exclusive groups from the CGA AML cohort: NPM1 mutated, NPM1wt HOXA9/B4 double-positive (HOXA/B), HOXA9 or HOXB4 single-positive (One HOX), and HOXA9/HOXB4 double-negative (No HOX) (n = 179). (E and F) Correlation between PRDM16 RPKM and overall survival among NPM1-mutated AML samples (n = 47) (E) and MLL-rearranged AML samples (F) in the CGA (n = 21). (G and H) Principal component analysis of NPM1-mutated (G) and MLL-rearranged (H) AML cases from the CGA, comparing PRDM16^hi and PRDM16^lo cohorts. (I and J) Representative list of GO pathways upregulated in PRDM16^hi or PRDM16^lo cohorts of NPM1-mutated (I) or MLL-rearranged (J) AML cases in the CGA. Values expressed as –log₁₀ of the P value, determined by PANTHER analysis. (K) χ² analysis of observed versus expected number of dysregulated MDS-related genes in common with genes from our RNA-Seq analysis in Figure 5H and Figure 6E. Data represent mean ± SEM. NS, P > 0.05; *P < 0.05; **P < 0.01; Pearson’s test for linear correlations, 1-way ANOVA for multiple comparisons, χ² test for comparing observed vs. expected values.