Advances in Understanding of Metabolism of B-Cell Lymphoma: Implications for Therapy

There have been significant recent advances in the understanding of the role of metabolism in normal and malignant B-cell biology. Previous research has focused on the role of MYC and mammalian target of rapamycin (mTOR) and how these interact with B-cell receptor signaling and hypoxia to regulate glycolysis, glutaminolysis, oxidative phosphorylation (OXPHOS) and related metabolic pathways in germinal centers. Many of the commonest forms of lymphoma arise from germinal center B-cells, reflecting the physiological attenuation of normal DNA damage checkpoints to facilitate somatic hypermutation of the immunoglobulin genes. As a result, these lymphomas can inherit the metabolic state of their cell-of-origin. There is increasing interest in the potential of targeting metabolic pathways for anti-cancer therapy. Some metabolic inhibitors such as methotrexate have been used to treat lymphoma for decades, with several new agents being recently licensed such as inhibitors of phosphoinositide-3-kinase. Several other inhibitors are in development including those blocking mTOR, glutaminase, OXPHOS and monocarboxylate transporters. In addition, recent work has highlighted the importance of the interaction between diet and cancer, with particular focus on dietary modifications that restrict carbohydrates and specific amino acids. This article will review the current state of this field and discuss future developments

Prep1 (pKnox1) Regulates Mouse Embryonic HSC Cycling and Self-Renewal Affecting the Stat1-Sca1 IFN-Dependent Pathway

<div><p>A hypomorphic <i>Prep1</i> mutation results in embryonic lethality at late gestation with a pleiotropic embryonic phenotype that includes defects in all hematopoietic lineages. Reduced functionality of the hematopoietic stem cells (HSCs) compartment might be responsible for the hematopoietic phenotype observed at mid-gestation. In this paper we demonstrate that Prep1 regulates the number of HSCs in fetal livers (FLs), their clonogenic potential and their ability to <i>de novo</i> generate the hematopoietic system in ablated hosts. Furthermore, we show that Prep1 controls the self-renewal ability of the FL HSC compartment as demonstrated by serial transplantation experiments. The premature exhaustion of Prep1 mutant HSCs correlates with the reduced quiescent stem cell pool thus suggesting that Prep1 regulates the self-renewal ability by controlling the quiescence/proliferation balance. Finally, we show that in FL HSCs Prep1 absence induces the interferon signaling pathway leading to premature cycling and exhaustion of fetal HSCs.</p></div

Prep1 influences HSC quiescent pool rather than HSCs apoptosis.

<p>(A) Representative FACS contour plots to identify apoptotic <i>Prep1^+/+</i> and <i>Prep1^i/i</i> HSCs are shown on the left. The represented plots refer to L⁻S⁺K⁺CD150⁺ gate and numbers in the FACS plots indicate the percentage of cells in parental gates. On the right, the graph represents the mean of apoptotic HSCs (Annexin⁺ DAPI⁻) (n = 3; p = not significant). (B) Representative FACS contour plots to identify the cell cycle distribution of <i>Prep1^+/+</i> and <i>Prep1^i/i</i> HSCs are shown on the left. The represented plots refer to L⁻S⁺K⁺CD150⁺ gate and numbers in the FACS plots indicate the percentage of cells in parental gates. On the right, the graph represents the mean of G0 (Ki67⁻Hoechst^l°^w), G1 (Ki67⁺Hoechst^l°^w) and S/G2/M (Ki67⁺Hoechst^hi) HSCs (n = 3; p = not significant) (n = 3; G0 p = 0.02; G1 and S/G2/M p = not significant).</p

Prep1 controls the number and the functionality of HSCs in E14.5 FLs.

<p>(A) 50, 100 or 200 HSCs sorted from <i>Prep1^+/+</i> and <i>Prep1^i/i</i> FLs were transplanted into lethally irradiated mice in competition with 2×10⁵ CD45.1 BM cells. Mice showing more than 2% CD45.2⁺ cells in the PB were considered as positively repopulated. HSCs are identified as CD45.2⁺ L⁻S⁺K⁺CD150⁺CD48⁻CD41⁻cells. The graph represents the percentage of positively repopulated mice 16 weeks after transplantation (n = 4 for each genotype) (B) The graph indicates the mean chimerism shown by transplanted mice at each cell dosage in the PB 16 weeks after transplantation. (C) 2000 LSK cells purified form <i>Prep1^+/+</i> or <i>Prep1^i/i</i> FLs were transplanted in competition with 1×10⁶ BM cells into lethally irradiated CD45.1 recipients. (D) PB analyses to detect donor-derived (CD45.2⁺) cells performed at 7, 12,16 and 20 weeks after transplantation. In the graph, black bars represent the mean of CD45.2⁺ cells in the PB of <i>Prep1^+/+</i> or <i>Prep1^i/i</i> reconstituted mice (n = 4 for each genotype; p<0.001). (E–F) RUs (on the left) were calculated on FACS data (on the right) obtained from BMs of repopulated mice 20 weeks after transplantation. (E) Mean of RUs in Prep1^+/+ or Prep1^i/i-transplanted mice; p = 0.05. (F) Mean of HSCs RUs in <i>Prep1^+/+</i> or <i>Prep1^i/i</i>-transplanted mice; p = 0.01. HSCs are identified as CD45.2⁺ L⁻S⁺K⁺CD150⁺CD48⁻CD41⁻cells.</p

Prep1 does not affect Smads expression but modulates their phosphorylation in the EML1 progenitor cell line.

<p>(A) Prep1 knock-down (KD) was assessed by Immuno-blotting analysis. Actin was used as loading control. (B–C) Prep1 KD or control EML1 cells were incubated 4 h with (+) or without (−) TGFβ (10 ng/ml). (B) Histograms show fold induction of the indicated transcripts in Prep1 KD or control EML1 cells as measured by qRT-PCR. The data represent 2 independent experiments. (C) Smads and their phosphorylated forms (as indicated) were detected by Western blot analyses. Vinculin was used as loading control. The data were reproduced in 2 independent experiments.</p

Prep1^i/i HSCs undergo exhaustion faster than wt in serial transplantation assay.

<p>(A) 2×10⁶ unfractionated BM cells from primary recipients were injected into secondary hosts. Primary recipients had received 1×10⁶ unfractionated CD45.2⁺ FL cells together with 1×10⁶ unfractionated CD45.1⁺ wt BM cells (chimerism shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107916#pone.0107916.s004" target="_blank">FigureS4</a>). (B) PB analyses of secondary recipients were performed 8 and 20 weeks after transplantation to detect CD45.2⁺ cells. Each recipient is depicted with a separate symbol, blue and red colors indicating <i>Prep1^+/+</i> and <i>Prep1^i/i</i> reconstituted mice, respectively. Black bars represent the mean values in <i>Prep1^+/+</i> or <i>Prep1^i/i</i> reconstituted mice (n = 8 for each genotype at both time points; p-value<0.001 at both time points). (C) 20 weeks after transplantation, PB of secondary hosts was analysed for donor-derived myeloid cells (CD45.2⁺Gr1⁺Mac1⁺; p = 0.000017), B lymphoid cells (CD45.2⁺B220⁺; p = 0.000007) and T lymphoid cells (CD45.2⁺CD3⁺; p = 0.000017). Black bars represent mean values (n = 8 for each genotype). (D) Tertiary transplantations were performed injecting 2×10⁶ BM cells from secondary recipients into tertiary hosts. (E) PB of tertiary hosts was analyzed 20 weeks after transplantation to detect CD45.2⁺ cells. Black bars represent the mean values (n = 10 for <i>Prep1^+/+</i> and n = 8 for <i>Prep1^i/i</i>; p-value = 0.007). On the right, representative FACS histograms obtained for CD45.2⁺ cells of control and mutant tertiary recipients. (F) The ratio between the number of positively repopulated mice (CD45.2⁺ cells >2%) and transplanted mice is represented for primary, secondary and tertiary transplantations for both genotypes (primary transplantation n = 10 for each genotype; secondary transplantation n = 8 for each genotype; tertiary transplantation n = 10 for <i>Prep1^+/+</i> and n = 8 for <i>Prep1^i/i</i>).</p

Prep1 affects stem and progenitors compartments in E14.5 FLs.

<p>(A–D) Representative FACS analyses of <i>Prep1^+/+</i> and <i>Prep1^i/i</i> FLs are shown on the left. Graphs describing percentage and absolute numbers are reported on the right. Numbers in the FACS plots indicate the percentage of cells in parental gates. (A) Lin⁻Sca1⁺cKit⁺ population is shown. FACS plots are referred to Lineage⁻ gate (n = 8 for each genotype; % L⁻S⁺K⁺ p = 0.01; # L⁻S⁺K⁺ p = 0.01). (B) HSC population is identified as L⁻S⁺K⁺CD150⁺ CD41⁻CD48⁻. FACS plots are referred to L⁻S⁺K⁺ gate (n = 8 for each genotype; % HSCs p = 0.0000056; # HSCs p = not significant). (C) CLPs are identified as Lin⁻Il7R⁺Sca1^intckit^int. FACS plots are referred to Lin⁻Il7R⁺ gate. (n = 4 for each genotype; % CLPs p = 0.00014; # CLPs p = 0.00023). (D) FACS plots regarding myeloid progenitors refer to Lin⁻Sca1⁻ckit^hi gate. GMPs are FcγR⁺CD34⁺ (n = 4 for each genotype; % GMPs p = 0.05; # GMPs p = 0.07); CMPs are FcγR^intCD34^+/l°^w (n = 4 for each genotype; % CMPs p = 0.005; # CMPs p = 0.007); MEPs are FcγR⁻CD34⁻ (n = 4 for each genotype; % MEPs p = not significant; # MEPs p = not significant).</p

Impaired clonogenic activity of Prep1 deficient stem cell populations.

<p>(A) LTC-IC assay was performed plating 100, 300 or 500 L⁻S⁺K⁺A⁺ cells purified from <i>Prep1^+/+</i> and <i>Prep1^i/i</i> FLs. Mean value of scored colonies after 12 days in methylcellulose at each cell dose are shown (n = 9 at each cell dose for each genotype; 500 L⁻S⁺K⁺A⁺ p = 0.003, 300 and 100 L⁻S⁺K⁺A⁺ p = not significant). (B) LTC-IC was performed with 50, 100, 200 L⁻S⁺K⁺CD150⁺ CD48⁻CD41⁻ from both genotypes and colonies scored after 12 days in methylcellulose. (n = 4 at each cell dose for each genotype; 200 cells p = 0.001, 100 cells p = 0.01, 50 cells p = not significant). (C) Representative images of LTC-IC colonies obtained from <i>Prep1^+/+</i> and <i>Prep1^i/i</i> L⁻S⁺K⁺A⁺ cells (<i>Prep1^+/+</i> colony: 4x original magnification; <i>Prep1^i/i</i> colony: 10x original magnification; scale bars = 100 µm).</p

Prep1 ChIP-seq tracing of the mouse embryo Irf1, Irf2 and Irf8 genes.

<p>ChIPseq tracing from the m9 mouse genome. In the <i>Irf1</i> gene a Prep1 peak is present about 10 Kbp upstream of the promoter: the peak is centered around the TGAGTGACAG Prep1 consensus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107916#pone.0107916-Penkov2" target="_blank">[36]</a>. In the <i>Irf2</i> gene, the peak is present 50 Kbp upstream of the promoter and is centered around a similar TGAATGACAA Prep1 consensus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107916#pone.0107916-Penkov2" target="_blank">[36]</a>. In the <i>Irf8</i> gene, the peak is right at the promoter, 100 bp distance, centered around a less frequent hexameric consensus, TGGCAG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107916#pone.0107916-Penkov2" target="_blank">[36]</a>. Chromatin immunoprecipitation was performed on E11.5 mouse embryos <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107916#pone.0107916-Penkov2" target="_blank">[36]</a> and was confirmed in ES cells.</p