Aging-associated inflammation promotes selection for adaptive oncogenic events in B cell progenitors - PubMed (original) (raw)

. 2015 Dec;125(12):4666-80.

doi: 10.1172/JCI83024. Epub 2015 Nov 9.

Matias Casás-Selves, Jihye Kim, Vadym Zaberezhnyy, Leila Aghili, Ashley E Daniel, Linda Jimenez, Tania Azam, Eoin N McNamee, Eric T Clambey, Jelena Klawitter, Natalie J Serkova, Aik Choon Tan, Charles A Dinarello, James DeGregori

Aging-associated inflammation promotes selection for adaptive oncogenic events in B cell progenitors

Curtis J Henry et al. J Clin Invest. 2015 Dec.

Abstract

The incidence of cancer is higher in the elderly; however, many of the underlying mechanisms for this association remain unexplored. Here, we have shown that B cell progenitors in old mice exhibit marked signaling, gene expression, and metabolic defects. Moreover, B cell progenitors that developed from hematopoietic stem cells (HSCs) transferred from young mice into aged animals exhibited similar fitness defects. We further demonstrated that ectopic expression of the oncogenes BCR-ABL, NRAS(V12), or Myc restored B cell progenitor fitness, leading to selection for oncogenically initiated cells and leukemogenesis specifically in the context of an aged hematopoietic system. Aging was associated with increased inflammation in the BM microenvironment, and induction of inflammation in young mice phenocopied aging-associated B lymphopoiesis. Conversely, a reduction of inflammation in aged mice via transgenic expression of α-1-antitrypsin or IL-37 preserved the function of B cell progenitors and prevented NRAS(V12)-mediated oncogenesis. We conclude that chronic inflammatory microenvironments in old age lead to reductions in the fitness of B cell progenitor populations. This reduced progenitor pool fitness engenders selection for cells harboring oncogenic mutations, in part due to their ability to correct aging-associated functional defects. Thus, modulation of inflammation--a common feature of aging--has the potential to limit aging-associated oncogenesis.

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Figures

Figure 8

Figure 8. Reducing inflammation in aged IL-37tg mice abrogates NRASV12-mediated oncogenesis.

Young and old (2-month-old and 20-month-old, respectively) littermate (LC) or IL-37tg mice were transplanted with young _NRASV12_-expressing cells, as in Figure 7A. Three months after transplantation, BM was analyzed by flow cytometry for (A) the frequency of _NRASV12_-expressing cells in B progenitor cell populations and (B) activation of STAT5 and ERK in pro–B cells expressing NRASV12 (red-outlined gray boxes) or not expressing the oncogene. (C) mRNA levels of Hprt, Gmps, and Myc in sorted pro–B cells were determined by qPCR (note: _NRASV12_-initiated cells only expanded in the old littermate control mice). (D) Model for how aging and aging-associated inflammation regulate progenitor cell fitness and oncogenesis. Values in A–C represent the mean ± SEM, with more than 5 mice per group. *P < 0.05 and #P < 0.001, by Student’s t test relative to young controls.

Figure 7

Figure 7. Reducing inflammation in aged AATtg mice suppresses selection for oncogenic _NRASV12_–expressing B progenitors.

(A) Experimental overview. (B and C) Frequencies of young, vector-expressing (CFP+) cells (B) or young, _NRASV12_-expressing (GFP+) cells (C) in the peripheral blood of recipient mice were monitored for 2 months after transplantation. (D) After 2 months, mice were sacrificed, and the frequencies of _NRASV12_-expressing pre–B cell progenitors in the BM of recipient mice were determined by flow cytometry. LD, limit of detection. (E) Recipient pro–B cells (CD45.2+) were analyzed by flow cytometry for STAT5 and ERK activation. (F) STAT5 activation in donor pro–B cells (CD45.1+) expressing vector (CFP+) or NRASV12 (GFP+) was analyzed using flow cytometry. (G and H) Expression levels of the indicated genes in sorted donor (CD45.1+) pro–B cells expressing vector (V) or NRASV12 (RAS) were determined by qPCR . Values in B–H represent the mean ± SEM, with more than 5 mice per group. (B–E and G) #P < 0.001, by Student’s t test relative to young controls. (F and H) *P < 0.05, **P < 0.01, and #P < 0.001, by 1-way ANOVA. Ctrl, control.

Figure 6

Figure 6. Reducing inflammation prevents declines in aging-associated B progenitor fitness.

BM from young (2-month-old) and old (20-month-old) littermate and AATtg and IL-37tg mice was analyzed for the frequency of B cell progenitor populations (A and D). Activation of STAT5, STAT3, ERK, and STAT1 in pro–B cells was determined by flow cytometry (B and E). mRNA expression of genes involved in cell-cycle regulation and inflammation in sorted pro–B cells was determined by qPCR (C and F). Values represent the mean ± SEM of 2 independent experiments, with more than 5 mice per group. *P < 0.05 and #P < 0.001, by Student’s t test relative to young controls for each experiment.

Figure 5

Figure 5. Proinflammatory cytokines are reduced in old antiinflammatory transgenic mice.

ELISAs for TNF-α, IL-6, and IL-1β were performed on BM aspirates and serum collected from young (2-month-old) and old (20-month-old) littermates, old AATtg mice (A and B), and old IL-37tg mice (C–E). Values represent the mean ± SEM of 2 independent experiments (6 mice total). *P < 0.05, **P < 0.01, and #P < 0.001, by Student’s t test relative to young controls.

Figure 3

Figure 3. Increased inflammation in the BM with age coincides with decreased expression of genes regulating cell-cycle progression in B progenitors.

(A) PCA of the microarray data was generated using the Partek Genomics Suite. (B) Gene expression profiles from the microarray analysis performed on B cell progenitors isolated from young and old mice were analyzed by GSEA for the expression of genes regulated by E2F and MYC and of those involved in inflammatory processes. (C and D) BM aspirates were collected from young (Y; 2-month-old), middle-aged (M; 14-month-old), and old (O; 24-month-old) mice, and IL-6 (C) and TNF-α (D) levels were determined using ELISA. Values represent the mean ± SEM of 2 independent experiments, with more than 6 mice per age group. (E) Network analysis of the microarray data was performed using IPA software, which identified TNF-α as an important cytokine that increases in aged B progenitors. (F) Heatmap showing a subset of the inflammatory genes shown in B. (G) Pro–B cell progenitors from young (2-month-old) and old (24-month-old) mice were sorted, and expression levels of inflammatory genes (Tnfa, Ifnz, and Ifi27) or of those regulated by inflammation (e.g., Muc5b) were determined by qPCR. (H) Expression levels of TNF-α in sorted young or old pro–B cells expressing vector or oncogenic BCR-ABL, NRASV12, or Myc (all GFP+) were determined using qPCR. Values in G and H represent the mean ± SEM of 4 independent experiments, with more than 10 mice per age group. #P < 0.001, by Student’s t test relative to young controls. ND, not determined.

Figure 4

Figure 4. Induction of inflammation promotes functional decline in B progenitors.

(A–C) Young mice were injected every 4 days for 2 weeks with PBS (vehicle), αIL-7–neutralizing Abs, LPS, or recombinant TNF-α. After 2 weeks of treatment (3 injections total), the mice were sacrificed and the percentage of B progenitors in the BM determined using flow cytometry. Expression levels of B lineage–specification genes (B) and purine synthesis genes (C) were determined using qPCR. Values in A–C represent the mean ± SEM for 5 mice per treatment group. (D–G) Serum TNF-α levels in 5-month-old TNF-αΔARE mice and their littermate controls were measured by ELISA (D), frequencies of B cell (B220+) and myeloid (MAC1+) progenitor cells in BM were determined by flow cytometry (E and F), and mRNA levels of purine synthesis genes in pro–B cells were determined by qPCR (G). Values represent the mean ± SEM of 3 independent experiments, with more than 9 mice per group. *P < 0.05, **P < 0.01, and #P < 0.001, by Student’s t test relative to PBS-injected mice or littermate controls. LC, littermate control.

Figure 2

Figure 2. Oncogenic mutations correct aging-associated functional defects in B progenitors, leading to increased leukemogenesis.

(A) Ba/F3 cells expressing vector (V) or oncogenes (BCR-ABL, NRASV12, Myc) were grown in various concentrations of IL-3 for 24 hours, and STAT5 activation in these cells was determined by flow cytometry. (B) Ba/F3 cells were grown overnight in media containing or lacking IL-3, and expression levels of Myc and Hprt in these cells were determined by qPCR. Values in A and B represent the mean ± SEM of 3 independent experiments (9 total samples). (C–F) c-KIT+ BM cells were isolated from young (2-month-old) or old (24-month-old) mice, retrovirally transduced to express vector or oncogenic BCR-ABL, _NRAS_V12,, or Myc (each with coexpressed GFP), and transplanted into sublethally irradiated young BALB/c mice. Three weeks after transplantation, mice were sacrificed, and STAT5 activity (C) and mRNA levels of Hprt, Myc, and Ebf (D–F) were determined in vector-expressing or oncogene-expressing pro–B cell progenitors. Values in C–F represent the mean ± SEM for more than 5 mice per group. (G and H) Young mice were lethally irradiated and transplanted with either 2 × 106 young or old whole BM cells. Four days later, mice reconstituted with young or old BM cells were transplanted, respectively, with young or old c-KIT+ cells expressing oncogenic NRASV12 or Myc. Leukemia-free survival is plotted using Kaplan-Meier graphs. Most mice developed B220+CD43+ pro–B cell–like acute lymphoblastic leukemia (ALL) (87% and 80% for _NRAS_- and _Myc_-driven leukemias, respectively, on the old backgrounds). (I) Young or old mice were treated with busulfan and transplanted with young or old c-KIT+ cells expressing oncogenic NRASV12. Leukemia-free survival is plotted using Kaplan-Meier graphs. Values in G and H represent the mean ± SEM of 2 independent experiments, with more than 15 mice per group in total, and values in I represent 5 mice per treatment group. (A–F) **P < 0.01 and #P < 0.001, by Student’s t test . In C–F, oncogene-bearing samples were compared with vector-expressing controls (old to old). (G–I) **P < 0.01 and #P < 0.001, by Cox proportional hazards test. BMT, BM transplantation.

Figure 1

Figure 1. Impaired metabolism, nucleotide anabolism, and cell cycling accompany aging B lymphopoiesis.

(A) Nucleotide synthesis GSEA sets were derived from microarray analysis of B progenitors isolated from young and old BALB/c mice. Y1, young mouse #1; O1, old mouse #1. (B) Hprt, Gmps, and Myc expression in sorted young and old pro–B cells was determined using qPCR. Values represent the mean ± SEM of 4 independent experiments (8 donor mice/age group). (C) Young BALB/c mice were injected with 1× PBS or IL-7–neutralizing Abs (αIL-7) every 4 days for 2 weeks, and Pax5 and Hprt expression was determined by qPCR in B220-purified B progenitors. Values represent the mean ± SEM of 3 independent experiments (6 mice/age group). (D) B progenitors were isolated from young and old BALB/c mice, and NMR metabolomics was performed. Values represent the mean ± SEM of 2 independent experiments (8 donor mice/age group). (E) ATP and NADH levels were determined in B progenitors isolated from young and old BALB/c mice. Values represent the mean ± SEM of 3 independent experiments (9 donor mice/age group). (F) Samples used in D were analyzed by mass spectrometry for relative nucleotide levels.(G) Young and old BALB/c mice were treated with 1× PBS (Veh.), or young mice were treated with IL-7–neutralizing Abs as described in C, and the energy balance of purine nucleotides in B220+ cells was determined by mass spectrometry. Values represent the mean ± SEM of 3 independent experiments (4 donor mice/group). (H–J) C57BL/6 mice were injected with EdU, BM was harvested 2 hours later, and cell-cycle analysis in pro–B cells was performed. (H) B220+/MAC1+ (B/M) cell ratio. (I) Representative cell-cycle profiles. (J) Normalized x-mean MFI of EdU+ cell populations for pro–B cells. Statistical analyses in H and J are relative to the levels observed in 5-month-old mice and represent the mean ± SEM of 3 independent experiments (4 donor mice/age group). *P < 0.05, **P < 0.01, and #P < 0.001, by Student’s t test relative to young controls for each experiment. Y, young; O, old.

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