Ribosomal protein control of hematopoietic stem cell transformation through direct, non-canonical regulation of metabolism.

We report here that expression of the ribosomal protein, RPL22, is frequently reduced in human myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML); reduced RPL22 expression is associated with worse outcomes. Mice null for Rpl22 display characteristics of an MDS-like syndrome and develop leukemia at an accelerated rate. Rpl22-deficient mice also display enhanced hematopoietic stem cell (HSC) self-renewal and obstructed differentiation potential, which arises not from reduced protein synthesis but from increased expression of the Rpl22 target, ALOX12, an upstream regulator of fatty acid oxidation (FAO). The increased FAO mediated by Rpl22-deficiency also persists in leukemia cells and promotes their survival. Altogether, these findings reveal that Rpl22 insufficiency enhances the leukemia potential of HSC via non-canonical de-repression of its target, ALOX12, which enhances FAO, a process that may serve as a therapeutic vulnerability of Rpl22 low MDS and AML leukemia cells. Highlights: RPL22 insufficiency is observed in MDS/AML and is associated with reduced survivalRpl22-deficiency produces an MDS-like syndrome and facilitates leukemogenesisRpl22-deficiency does not impair global protein synthesis by HSCRpl22 controls leukemia cell survival by non-canonical regulation of lipid oxidation eTOC: Rpl22 controls the function and transformation potential of hematopoietic stem cells through effects on ALOX12 expression, a regulator of fatty acid oxidation.

The ERK2-DBP domain opposes pathogenesis of a mouse JAK2V617F-driven myeloproliferative neoplasm.

Although Ras/mitogen-activated protein kinase (MAPK) signaling is activated in most human cancers, attempts to target this pathway using kinase-active site inhibitors have not typically led to durable clinical benefit. To address this shortcoming, we sought to test the feasibility of an alternative targeting strategy, focused on the ERK2 substrate binding domains, D and DEF binding pocket (DBP). Disabling the ERK2-DBP domain in mice caused baseline erythrocytosis. Consequently, we investigated the role of the ERK2-D and -DBP domains in disease, using a JAK2-dependent model of polycythemia vera (PV). Of note, inactivation of the ERK2-DBP domain promoted the progression of disease from PV to myelofibrosis, suggesting that the ERK2-DBP domain normally opposes progression. ERK2-DBP inactivation also prevented oncogenic JAK2 kinase (JAK2V617F) from promoting oncogene-induced senescence in vitro. The ERK2-DBP mutation attenuated JAK2-mediated oncogene-induced senescence by preventing the physical interaction of ERK2 with the transcription factor Egr1. Because inactivation of the ERK2-DBP created a functional ERK2 kinase limited to binding substrates through its D domain, these data suggested that the D domain substrates were responsible for promoting oncogene-induced progenitor growth and tumor progression and that pharmacologic targeting of the ERK2-D domain may attenuate cancer cell growth. Indeed, pharmacologic agents targeting the ERK2-D domain were effective in attenuating the growth of JAK2-dependent myeloproliferative neoplasm cell lines. Taken together, these data indicate that the ERK-D and -DBP domains can play distinct roles in the progression of neoplasms and that the D domain has the potential to be a potent therapeutic target in Ras/MAPK-dependent cancers.

Loss of Ribosomal Protein Paralog Rpl22-like1 Blocks Lymphoid Development without Affecting Protein Synthesis.

Ribosomal proteins are thought to primarily facilitate biogenesis of the ribosome and its ability to synthesize protein. However, in this study, we show that Rpl22-like1 (Rpl22l1) regulates hematopoiesis without affecting ribosome biogenesis or bulk protein synthesis. Conditional loss of murine Rpl22l1 using stage or lineage-restricted Cre drivers impairs development of several hematopoietic lineages. Specifically, Tie2-Cre-mediated ablation of Rpl22l1 in hemogenic endothelium impairs the emergence of embryonic hematopoietic stem cells. Ablation of Rpl22l1 in late fetal liver progenitors impairs the development of B lineage progenitors at the pre-B stage and development of T cells at the CD44-CD25+ double-negative stage. In vivo labeling with O-propargyl-puromycin revealed that protein synthesis at the stages of arrest was not altered, indicating that the ribosome biogenesis and function were not generally compromised. The developmental arrest was associated with p53 activation, suggesting that the arrest may be p53-dependent. Indeed, development of both B and T lymphocytes was rescued by p53 deficiency. p53 induction was not accompanied by DNA damage as indicated by phospho-γH2AX induction or endoplasmic reticulum stress, as measured by phosphorylation of EIF2α, thereby excluding the known likely p53 inducers as causal. Finally, the developmental arrest of T cells was not rescued by elimination of the Rpl22l1 paralog, Rpl22, as we had previously found for the emergence of hematopoietic stem cells. This indicates that Rpl22 and Rpl22l1 play distinct and essential roles in supporting B and T cell development.

The E protein-TCF1 axis controls γδ T cell development and effector fate.

TCF1 plays a critical role in T lineage commitment and the development of αß lineage T cells, but its role in γδ T cell development remains poorly understood. Here, we reveal a regulatory axis where T cell receptor (TCR) signaling controls TCF1 expression through an E-protein-bound regulatory element in the Tcf7 locus, and this axis regulates both γδ T lineage commitment and effector fate. Indeed, the level of TCF1 expression plays an important role in setting the threshold for γδ T lineage commitment and modulates the ability of TCR signaling to influence effector fate adoption by γδ T lineage progenitors. This finding provides mechanistic insight into how TCR-mediated repression of E proteins promotes the development of γδ T cells and their adoption of the interleukin (IL)-17-producing effector fate. IL-17-producing γδ T cells have been implicated in cancer progression and in the pathogenesis of psoriasis and multiple sclerosis.

c-Myb Coordinates Survival and the Expression of Genes That Are Critical for the Pre-BCR Checkpoint.

The c-Myb transcription factor is required for adult hematopoiesis, yet little is known about c-Myb function during lineage-specific differentiation due to the embryonic lethality of Myb-null mutations. We previously used tissue-specific inactivation of the murine Myb locus to demonstrate that c-Myb is required for differentiation to the pro-B cell stage, survival during the pro-B cell stage, and the pro-B to pre-B cell transition during B lymphopoiesis. However, few downstream mediators of c-Myb-regulated function have been identified. We demonstrate that c-Myb regulates the intrinsic survival of CD19+ pro-B cells in the absence of IL-7 by repressing expression of the proapoptotic proteins Bmf and Bim and that levels of Bmf and Bim mRNA are further repressed by IL-7 signaling in pro-B cells. c-Myb regulates two crucial components of the IL-7 signaling pathway: the IL-7Rα-chain and the negative regulator SOCS3 in CD19+ pro-B cells. Bypassing IL-7R signaling through constitutive activation of Stat5b largely rescues survival of c-Myb-deficient pro-B cells, whereas constitutively active Akt is much less effective. However, rescue of pro-B cell survival is not sufficient to rescue proliferation of pro-B cells or the pro-B to small pre-B cell transition, and we further demonstrate that c-Myb-deficient large pre-B cells are hypoproliferative. Analysis of genes crucial for the pre-BCR checkpoint demonstrates that, in addition to IL-7Rα, the genes encoding λ5, cyclin D3, and CXCR4 are downregulated in the absence of c-Myb, and λ5 is a direct c-Myb target. Thus, c-Myb coordinates survival with the expression of genes that are required during the pre-BCR checkpoint.

Role of a selecting ligand in shaping the murine γδ-TCR repertoire.

Unlike αß-T lineage cells, where the role of ligand in intrathymic selection is well established, the role of ligand in the development of γδ-T cells remains controversial. Here we provide evidence for the role of a bona fide selecting ligand in shaping the γδ-T cell-receptor (TCR) repertoire. Reactivity of the γδ-TCR with the major histocompatibility complex (MHC) Class Ib ligands, H2-T10/22, is critically dependent upon the EGYEL motif in the complementarity determining region 3 (CDR3) of TCRδ. In the absence of H2-T10/22 ligand, the commitment of H2-T10/22 reactive γδ-T cells to the γδ fate is diminished, and the specification of those γδ committed cells to the IFN-γ or interleukin-17 effector fate is altered. Furthermore, those cells that do adopt the γδ fate and mature exhibit a profound alteration in the γδTCR repertoire, including depletion of the EGYEL motif and reductions in both CDR3δ length and charge. Taken together, these data suggest that ligand plays an important role in shaping the TCR repertoire of γδ-T cells.

Non-coding Transcription Instructs Chromatin Folding and Compartmentalization to Dictate Enhancer-Promoter Communication and T Cell Fate.

It is now established that Bcl11b specifies T cell fate. Here, we show that in developing T cells, the Bcl11b enhancer repositioned from the lamina to the nuclear interior. Our search for factors that relocalized the Bcl11b enhancer identified a non-coding RNA named ThymoD (thymocyte differentiation factor). ThymoD-deficient mice displayed a block at the onset of T cell development and developed lymphoid malignancies. We found that ThymoD transcription promoted demethylation at CTCF bound sites and activated cohesin-dependent looping to reposition the Bcl11b enhancer from the lamina to the nuclear interior and to juxtapose the Bcl11b enhancer and promoter into a single-loop domain. These large-scale changes in nuclear architecture were associated with the deposition of activating epigenetic marks across the loop domain, plausibly facilitating phase separation. These data indicate how, during developmental progression and tumor suppression, non-coding transcription orchestrates chromatin folding and compartmentalization to direct with high precision enhancer-promoter communication.

Multisystem Anomalies in Severe Combined Immunodeficiency with Mutant BCL11B.

BACKGROUND: Severe combined immunodeficiency (SCID) is characterized by arrested T-lymphocyte production and by B-lymphocyte dysfunction, which result in life-threatening infections. Early diagnosis of SCID through population-based screening of newborns can aid clinical management and help improve outcomes; it also permits the identification of previously unknown factors that are essential for lymphocyte development in humans. METHODS: SCID was detected in a newborn before the onset of infections by means of screening of T-cell-receptor excision circles, a biomarker for thymic output. On confirmation of the condition, the affected infant was treated with allogeneic hematopoietic stem-cell transplantation. Exome sequencing in the patient and parents was followed by functional analysis of a prioritized candidate gene with the use of human hematopoietic stem cells and zebrafish embryos. RESULTS: The infant had "leaky" SCID (i.e., a form of SCID in which a minimal degree of immune function is preserved), as well as craniofacial and dermal abnormalities and the absence of a corpus callosum; his immune deficit was fully corrected by hematopoietic stem-cell transplantation. Exome sequencing revealed a heterozygous de novo missense mutation, p.N441K, in BCL11B. The resulting BCL11B protein had dominant negative activity, which abrogated the ability of wild-type BCL11B to bind DNA, thereby arresting development of the T-cell lineage and disrupting hematopoietic stem-cell migration; this revealed a previously unknown function of BCL11B. The patient's abnormalities, when recapitulated in bcl11ba-deficient zebrafish, were reversed by ectopic expression of functionally intact human BCL11B but not mutant human BCL11B. CONCLUSIONS: Newborn screening facilitated the identification and treatment of a previously unknown cause of human SCID. Coupling exome sequencing with an evaluation of candidate genes in human hematopoietic stem cells and in zebrafish revealed that a constitutional BCL11B mutation caused human multisystem anomalies with SCID and also revealed a prethymic role for BCL11B in hematopoietic progenitors. (Funded by the National Institutes of Health and others.).

Ribosomal Protein Rpl22 Controls the Dissemination of T-cell Lymphoma.

Mutations in ribosomal proteins cause bone marrow failure syndromes associated with increased cancer risk, but the basis by which they do so remains unclear. We reported previously that the ribosomal protein Rpl22 is a tumor suppressor in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), and that loss of just one Rpl22 allele accelerates T-cell lymphomagenesis by activating NF-κB and inducing the stem cell factor Lin28B. Here, we show that, paradoxically, loss of both alleles of Rpl22 restricts lymphoma progression through a distinct effect on migration of malignant cells out of the thymus. Lymphoma-prone AKT2-transgenic or PTEN-deficient mice on an Rpl22(-/-) background developed significantly larger and markedly more vascularized thymic tumors than those observed in Rpl22(+/+) control mice. But, unlike Rpl22(+/+) or Rpl22(+/-) tumors, Rpl22(-/-) lymphomas did not disseminate to the periphery and were retained in the thymus. We traced the defect in the Rpl22(-/-) lymphoma migratory capacity to downregulation of the KLF2 transcription factor and its targets, including the key migratory factor sphingosine 1-phosphate receptor 1 (S1PR1). Indeed, reexpression of S1PR1 in Rpl22-deficient tumor cells restores their migratory capacity in vitro The regulation of KLF2 and S1PR1 by Rpl22 appears to be proximal as Rpl22 reexpression in Rpl22-deficient lymphoma cells restores expression of KLF2 and S1P1R, while Rpl22 knockdown in Rpl22-sufficient lymphomas attenuates their expression. Collectively, these data reveal that, while loss of one copy of Rpl22 promotes lymphomagenesis and disseminated disease, loss of both copies impairs responsiveness to migratory cues and restricts malignant cells to the thymus. Cancer Res; 76(11); 3387-96. ©2016 AACR.

Rpl22 Loss Impairs the Development of B Lymphocytes by Activating a p53-Dependent Checkpoint.

Although ribosomal proteins facilitate the ribosome's core function of translation, emerging evidence suggests that some ribosomal proteins are also capable of performing tissue-restricted functions either from within specialized ribosomes or from outside of the ribosome. In particular, we have previously demonstrated that germline ablation of the gene encoding ribosomal protein Rpl22 causes a selective and p53-dependent arrest of ab T cell progenitors at the b-selection checkpoint. We have now identified a crucial role for Rpl22 during early B cell development. Germline ablation of Rpl22 results in a reduction in the absolute number of B-lineage progenitors in the bone marrow beginning at the pro­B cell stage. Although Rpl22-deficient pro­B cells are hyporesponsive to IL-7, a key cytokine required for early B cell development, the arrest of B cell development does not result from disrupted IL-7 signaling. Instead, p53 induction appears to be responsible for the developmental defects, as Rpl22 deficiency causes increased expression of p53 and activation of downstream p53 target genes, and p53 deficiency rescues the defect in B cell development in Rpl22-deficient mice. Interestingly, the requirement for Rpl22 in the B cell lineage appears to be developmentally restricted, because Rpl22-deficient splenic B cells proliferate normally in response to Ag receptor and Toll receptor stimuli and undergo normal class-switch recombination. These results indicate that Rpl22 performs a critical, developmentally restricted role in supporting early B cell development by preventing p53 induction.

Regulatory Roles of Rpl22 in Hematopoiesis: An Old Dog with New Tricks.

Ribosomal proteins have long been known to serve critical roles in facilitating the biogenesis of the ribosome and its ability to synthesize proteins. However, evidence is emerging that suggests ribosomal proteins are also capable of performing tissue-restricted, regulatory functions that impact normal development and pathological conditions, including cancer. The challenge in studying such regulatory functions is that elimination of many ribosomal proteins also disrupts ribosome biogenesis and/or function. Thus, it is difficult to determine whether developmental abnormalities resulting from ablation of a ribosomal protein result from loss of core ribosome functions or from loss of the regulatory function of the ribosomal protein. Rpl22, a ribosomal protein component of the large 60S subunit, provides insight into this conundrum; Rpl22 is dispensable for both ribosome biogenesis and protein synthesis yet its ablation causes tissue-restricted disruptions in development. Here we review evidence supporting the regulatory functions of Rpl22 and other ribosomal proteins.

Noncanonical mode of ERK action controls alternative αß and γδ T cell lineage fates.

Gradations in extracellular regulated kinase (ERK) signaling have been implicated in essentially every developmental checkpoint or differentiation process encountered by lymphocytes. Yet, despite intensive effort, the molecular basis by which differences in ERK activation specify alternative cell fates remains poorly understood. We report here that differential ERK signaling controls lymphoid-fate specification through an alternative mode of action. While ERK phosphorylates most substrates, such as RSK, by targeting them through its D-domain, this well-studied mode of ERK action was dispensable for development of γδ T cells. Instead, development of γδ T cells was dependent upon an alternative mode of action mediated by the DEF-binding pocket (DBP) of ERK. This domain enabled ERK to bind a distinct and select set of proteins required for specification of the γδ fate. These data provide the first in vivo demonstration for the role of DBP-mediated interactions in orchestrating alternate ERK-dependent developmental outcomes.

Origins of γδ T cell effector subsets: a riddle wrapped in an enigma.

αß and γδ T cells are thought to arise from a common precursor in the thymus but play distinct roles in pathogen resistance. Although conventional αß T cells exit the thymus in a naive state and acquire effector function in the periphery, the effector fate of many γδ T cells is specified in the thymus and exhibits limited plasticity thereafter. This review describes the current models that have been proposed to explain the acquisition of effector fate by γδ T cells, as well as the apparent linkage to Vγ gene usage. The two predominant models are the predetermination model, which suggests that effector fate is determined prior to TCR expression, perhaps in association with the developmental timing of Vγ rearrangement, and the TCR-dependence model, which proposes that the nature of the TCR signal, particularly its intensity or duration, plays an important role in influencing effector fate.

Helix-loop-helix factor inhibitor of differentiation 3 regulates interleukin-5 expression and B-1a B cell proliferation.

OBJECTIVE: Natural immunity is emerging as an important mediator of protection from atherogenesis. Natural IgM antibodies that recognize oxidation-specific epitopes on low-density lipoprotein or phospholipids and the B-1a B cells that produce them attenuate atherosclerosis. We previously demonstrated that Apoe(-/-) mice globally deficient in the helix-loop-helix protein inhibitor of differentiation 3 (Id3) develop early diet-induced atherosclerosis. Furthermore, B cell-mediated attenuation of atherosclerosis in B cell-deficient mice was dependent on Id3. Here, we sought to determine whether Id3 regulates B-1a B cells and the natural antibodies that they produce and identify mechanisms mediating these effects. APPROACH AND RESULTS: Mice lacking Id3 had significantly fewer B-1a B cells in the spleen and peritoneal cavity and reduced serum levels of the natural antibody E06. B cell-specific deletion of Id3 revealed that this effect was not because of the loss of Id3 in B cells. Interleukin (IL)-33 induced abundant, Id3-dependent IL-5 production in the recently identified innate lymphoid cell, the natural helper (NH) cell, but not Th2 or mast cells. In addition, delivery of IL-5 to Id3-deficient mice restored B-1a B cell proliferation. B-1a B cells were present in aortic samples also containing NH cells. Aortic NH cells produced IL-5, a B-1a B cell mitogen in response to IL-33 stimulation. CONCLUSIONS: These studies are the first to identify NH and B-1a B cells in the aorta and provide evidence that Id3 is a key regulator of NH cell IL-5 production and B-1a B cell homeostasis.

Lymphatic endothelial cells induce tolerance via PD-L1 and lack of costimulation leading to high-level PD-1 expression on CD8 T cells.

Lymphatic endothelial cells (LECs) induce peripheral tolerance by direct presentation to CD8 T cells (T(CD8)). We demonstrate that LECs mediate deletion only via programmed cell death-1 (PD-1) ligand 1, despite expressing ligands for the CD160, B- and T-lymphocyte attenuator, and lymphocyte activation gene-3 inhibitory pathways. LECs induce activation and proliferation of T(CD8), but lack of costimulation through 4-1BB leads to rapid high-level expression of PD-1, which in turn inhibits up-regulation of the high-affinity IL-2 receptor that is necessary for T(CD8) survival. Rescue of tyrosinase-specific T(CD8) by interference with PD-1 or provision of costimulation results in autoimmune vitiligo, demonstrating that LECs are significant, albeit suboptimal, antigen-presenting cells. Because LECs express numerous peripheral tissue antigens, lack of costimulation coupled to rapid high-level up-regulation of inhibitory receptors may be generally important in systemic peripheral tolerance.

c-Myb is required for pro-B cell differentiation.

The c-Myb transcription factor is required for normal adult hematopoiesis. However, the embryonic lethality of Myb-null mutations has been an impediment to identifying roles for c-Myb during lymphocyte development. We have used tissue-specific inactivation of the Myb locus in early progenitor cells to demonstrate that c-Myb is absolutely required for the differentiation of CD19(+) B-lineage cells and B cell differentiation is profoundly blocked beyond the pre-pro-B cell stage in Myb(f/f) Mb1-cre mice. We demonstrate that c-Myb is required for the intrinsic survival of CD19(+) pro-B cells as well as the proper expression of the alpha-chain of the IL-7 receptor (CD127) and Ebf1. However, survival of c-Myb-deficient CD19(+) pro-B cells cannot be rescued by transduction with CD127-producing retrovirus, suggesting that c-Myb controls a survival pathway independent of CD127. Furthermore, c-Myb-deficient progenitor cells inefficiently generate CD19(+) B-lineage cells during stromal cell culture but this process can be partially rescued with exogenous Ebf1. Thus, c-Myb does not appear to be required for commitment to B cell differentiation but is crucial for B cell differentiation to the CD19(+) pro-B cell stage as well as survival of CD19(+) pro-B cells. Surprisingly, forced c-Myb expression in lymphoid-primed multipotent progenitors favors differentiation toward the myeloid lineage, suggesting that proper c-Myb expression is crucial for B-lineage development.