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Patients with acute lymphoblastic leukemia have experienced significantly improved outcomes due to the advent of chimeric antigen receptor (CAR) T cells and bispecific T-cell engagers, although a proportion of patients still relapse despite these advances. T-cell exhaustion has been recently suggested to be an important driver of relapse in these patients. Indeed, phenotypic exhaustion of CD4+ T cells is predictive of relapse and poor overall survival in B-cell acute lymphoblastic leukemia (B-ALL). Thus, therapies that counter T-cell exhaustion, such as immune checkpoint blockade, may improve leukemia immunosurveillance and prevent relapse. Here, we used a murine model of Ph+ B-ALL as well as human bone marrow biopsy samples to assess the fundamental nature of CD4+ T-cell exhaustion and the preclinical therapeutic potential for combining anti–PD-L1 based checkpoint blockade with tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein. Single-cell RNA-sequence analysis revealed that B-ALL induces a unique subset of CD4+ T cells with both cytotoxic and helper functions. Combination treatment with the tyrosine kinase inhibitor nilotinib and anti–PD-L1 dramatically improves long-term survival of leukemic mice. Depletion of CD4+ T cells prior to therapy completely abrogates the survival benefit, implicating CD4+ T cells as key drivers of the protective anti-leukemia immune response. Indeed, treatment with anti–PD-L1 leads to clonal expansion of leukemia-specific CD4+ T cells with the aforementioned helper/cytotoxic phenotype as well as reduced expression of exhaustion markers. These findings support efforts to use PD1/PD-L1 checkpoint blockade in clinical trials and highlight the importance of CD4+ T-cell dysfunction in limiting the endogenous anti-leukemia response.

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy associated with poor outcomes despite intensive therapies. The genomic landscape of pediatric AML has been well characterized and has led to translational benefits, such as improved relapse predication and effective targeted therapies for a subset of individuals (e.g., FLT3-ITD AML). Despite these advances, the majority of patients continue to receive therapeutic interventions that have not changed in three decades. This underscores the need to advance our understanding of AML biology beyond genomic alterations (e.g., somatic mutations). Here, we present the largest (N = 858 patients) integrative analysis of pediatric AML paired transcriptomes and methylomes to date, performed at diagnosis for patients enrolled on Children's Oncology Group clinical trials AAML0531 (NCT00372593) or AAML1031 (NCT01371981). We also analyzed N = 400 and 253 corresponding transcriptomes and methylomes at relapse, respectively. Additionally, we performed comprehensive mutation and fusion calling from NGS data and integrated clinical covariates and outcomes. This robust dataset allowed us to uncover how pediatric AML epigenetics are perturbed from normal hematopoiesis, characterize the evolution of transcriptional and methylation changes from leukemia diagnosis to relapse, and identify more accurate relapse prediction models for pediatric AML.

Outcomes for patients with acute lymphoblastic leukemia (ALL) continue to improve in the era of immune- therapies. Despite this, measurable residual disease (MRD) frequently persists after frontline treatment, necessitating allogeneic hematopoietic stem cell transplantation or prolonged chemotherapy lasting multiple years. Relapse remains common and is associated with long-term survival rates of <25%. This emphasizes the need for research into fundamental mechanisms that enable the persistence of leukemic blasts.

Introduction:

Introduction:

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by erythroid hypoplasia, usually without perturbation of other hematopoietic lineages. Approximately 65% of DBA patients with autosomal dominant inheritance have heterozygous mutations or deletions in ribosomal protein (RP) genes while <1% of patients with X-linked inheritance have been identified with mutations in the transcription factor GATA1. Erythroid cells from patients with DBA have not been well characterized, and the mechanisms underlying the erythroid specific effects of either RP or GATA1associated DBA remain unclear. We have developed an ex vivo culture system to expand peripheral blood CD34+progenitor cells from patients with DBA and differentiate them into erythroid cells. Cells from patients with RP or GATA1mutations showed decreased proliferation and delayed erythroid differentiation in comparison with controls. RNA transcript analyses of erythroid cells from controls and patients with RP or GATA1mutations showed distinctive differences, with upregulation of heme biosynthesis genes prominently in RP-mediated DBA and failure to upregulate components of the translational apparatus in GATA1-mediated DBA. Our data show that dysregulation of translation is a common feature of DBA caused by both RP and GATA1mutations. This trial was registered at www.clinicaltrials.govas #NCT00106015.

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by erythroid hypoplasia, usually without perturbation of other hematopoietic lineages. Approximately 65% of DBA patients with autosomal dominant inheritance have heterozygous mutations or deletions in ribosomal protein (RP) genes while <1% of patients with X-linked inheritance have been identified with mutations in the transcription factor GATA1. Erythroid cells from patients with DBA have not been well characterized, and the mechanisms underlying the erythroid specific effects of either RP or GATA1 associated DBA remain unclear. We have developed an ex vivo culture system to expand peripheral blood CD34+ progenitor cells from patients with DBA and differentiate them into erythroid cells. Cells from patients with RP or GATA1 mutations showed decreased proliferation and delayed erythroid differentiation in comparison with controls. RNA transcript analyses of erythroid cells from controls and patients with RP or GATA1 mutations showed distinctive differences, with upregulation of heme biosynthesis genes prominently in RP-mediated DBA and failure to upregulate components of the translational apparatus in GATA1-mediated DBA. Our data show that dysregulation of translation is a common feature of DBA caused by both RP and GATA1 mutations. This trial was registered at www.clinicaltrials.gov as #NCT00106015.

Classical 5q− syndrome is an acquired macrocytic anemia of the elderly. Similar to Diamond Blackfan anemia (DBA), an inherited red cell aplasia, the bone marrow is characterized by a paucity of erythroid precursors. RPS14 deletions in combination with other deletions in the region have been implicated as causative of the 5q− syndrome phenotype. We asked whether smaller, less easily detectable deletions could account for a syndrome with a modified phenotype. We employed single-nucleotide polymorphism array genotyping to identify small deletions in patients diagnosed with DBA and other anemias lacking molecular diagnoses. Diminutive mosaic deletions involving RPS14 were identified in a 5-year-old patient with nonclassical DBA and in a 17-year-old patient with myelodysplastic syndrome. Patients with nonclassical DBA and other hypoproliferative anemias may have somatically acquired 5q deletions with RPS14 haploinsufficiency not identified by fluorescence in situ hybridization or cytogenetic testing, thus refining the spectrum of disorders with 5q− deletions.

Classical 5q− syndrome is an acquired macrocytic anemia of the elderly. Similar to Diamond Blackfan anemia (DBA), an inherited red cell aplasia, the bone marrow is characterized by a paucity of erythroid precursors. RPS14deletions in combination with other deletions in the region have been implicated as causative of the 5q− syndrome phenotype. We asked whether smaller, less easily detectable deletions could account for a syndrome with a modified phenotype. We employed single-nucleotide polymorphism array genotyping to identify small deletions in patients diagnosed with DBA and other anemias lacking molecular diagnoses. Diminutive mosaic deletions involving RPS14were identified in a 5-year-old patient with nonclassical DBA and in a 17-year-old patient with myelodysplastic syndrome. Patients with nonclassical DBA and other hypoproliferative anemias may have somatically acquired 5q deletions with RPS14 haploinsufficiency not identified by fluorescence in situ hybridization or cytogenetic testing, thus refining the spectrum of disorders with 5q− deletions.

Diamond-Blackfan anemia (DBA) is a congenital BM failure syndrome characterized by hypoproliferative anemia, associated physical abnormalities, and a predisposition to cancer. Perturbations of the ribosome appear to be critically important in DBA; alterations in 9 different ribosomal protein genes have been identified in multiple unrelated families, along with rarer abnormalities of additional ribosomal proteins. However, at present, only 50% to 60% of patients have an identifiable genetic lesion by ribosomal protein gene sequencing. Using genome-wide single-nucleotide polymorphism array to evaluate for regions of recurrent copy variation, we identified deletions at known DBA-related ribosomal protein gene loci in 17% (9 of 51) of patients without an identifiable mutation, including RPS19, RPS17, RPS26, and RPL35A. No recurrent regions of copy variation at novel loci were identified. Because RPS17 is a duplicated gene with 4 copies in a diploid genome, we demonstrate haploinsufficient RPS17 expression and a small subunit ribosomal RNA processing abnormality in patients harboring RPS17 deletions. Finally, we report the novel identification of variable mosaic loss involving known DBA gene regions in 3 patients from 2 kindreds. These data suggest that ribosomal protein gene deletion is more common than previously suspected and should be considered a component of the initial genetic evaluation in cases of suspected DBA.

CD8+ cytotoxic T lymphocytes play a major role in defense against intracellular pathogens, and their functions are specified by antigen recognition and innate cytokines. IL-12 and IFN-α/β are potent “signal 3” cytokines that are involved in both effector and memory cell development. Although the majority of effector cells are eliminated as inflammation resolves, some survive within the pool of memory cells and retain immediate effector function. In this study, we demonstrate that IL-12 instructs a unique program of effector cell differentiation that is distinct from IFN-α/β. Moreover, effector memory (TEM) cells within peripheral blood display many common attributes of cells differentiated in vitro in response to IL-12, including proinflammatory cytokine secretion and lytic activity. A pattern of IL-12–induced genes was identified that demarcate TEM from central memory cells, and the ontologies of these genes correlated precisely with their effector functions. Further, we uncovered a unique program of gene expression that was acutely regulated by IL-12 and reflected in stable gene expression patterns within TEM, but not T central memory cells in vivo. Thus, this study directly links a selective set of IL-12–induced genes to the programming of effector functions within the stable population of human CD8+ TEM cells in vivo.

CD8+cytotoxic T lymphocytes play a major role in defense against intracellular pathogens, and their functions are specified by antigen recognition and innate cytokines. IL-12 and IFN-α/β are potent “signal 3” cytokines that are involved in both effector and memory cell development. Although the majority of effector cells are eliminated as inflammation resolves, some survive within the pool of memory cells and retain immediate effector function. In this study, we demonstrate that IL-12 instructs a unique program of effector cell differentiation that is distinct from IFN-α/β. Moreover, effector memory (TEM) cells within peripheral blood display many common attributes of cells differentiated in vitro in response to IL-12, including proinflammatory cytokine secretion and lytic activity. A pattern of IL-12–induced genes was identified that demarcate TEMfrom central memory cells, and the ontologies of these genes correlated precisely with their effector functions. Further, we uncovered a unique program of gene expression that was acutely regulated by IL-12 and reflected in stable gene expression patterns within TEM, but not T central memory cells in vivo. Thus, this study directly links a selective set of IL-12–induced genes to the programming of effector functions within the stable population of human CD8+TEMcells in vivo.

Overexpression of a constitutively active form of Stat5b (Stat5b-CA) increases regulatory T cells (Tregs). We show that Stat5b-CA transgenic (TG) CD4+ T cells had a markedly reduced graft-versus-host disease (GVHD) capacity versus wild-type (WT) T cells. Stat5b-CA TG versus WT CD4+ T cells had a higher proportion of Tregs, which were superior in suppressing alloresponses mediated by CD4+CD25− effector T cells (Teffs). By day 5 after transplantation, Stat5b-CA TG Tregs had expanded approximately 3-fold more than WT Tregs. Purified Stat5b-CA TG Tregs added to WT CD4+CD25− Teffs were superior on a per-cell basis for inhibiting GVHD versus WT Tregs. Surprisingly, rigorously Treg-depleted Stat5b-CA TG versus WT CD4+CD25− Teffs caused less GVHD lethality associated with diminished Teff proinflammatory and increased Th2 anti-inflammatory cytokine responses. Reduced GVHD by Stat5b-CA TG versus WT Teffs could not be explained by conversion into Tregs in day 10 posttransplantation spleen or small intestine. In addition, Stat5b-CA TG Teffs retained a graft-versus-leukemia response. These results indicate a major role for Stat5 in Treg expansion and potency along with a lesser but significant role in Teff activation and suggest a strategy of pharmacologic Stat5b up-regulation as a means of decreasing GVHD while retaining a graft-versus-leukemia effect.

Overexpression of a constitutively active form of Stat5b (Stat5b-CA) increases regulatory T cells (Tregs). We show that Stat5b-CA transgenic (TG) CD4+T cells had a markedly reduced graft-versus-host disease (GVHD) capacity versus wild-type (WT) T cells. Stat5b-CA TG versus WT CD4+T cells had a higher proportion of Tregs, which were superior in suppressing alloresponses mediated by CD4+CD25−effector T cells (Teffs). By day 5 after transplantation, Stat5b-CA TG Tregs had expanded approximately 3-fold more than WT Tregs. Purified Stat5b-CA TG Tregs added to WT CD4+CD25−Teffs were superior on a per-cell basis for inhibiting GVHD versus WT Tregs. Surprisingly, rigorously Treg-depleted Stat5b-CA TG versus WT CD4+CD25−Teffs caused less GVHD lethality associated with diminished Teff proinflammatory and increased Th2 anti-inflammatory cytokine responses. Reduced GVHD by Stat5b-CA TG versus WT Teffs could not be explained by conversion into Tregs in day 10 posttransplantation spleen or small intestine. In addition, Stat5b-CA TG Teffs retained a graft-versus-leukemia response. These results indicate a major role for Stat5 in Treg expansion and potency along with a lesser but significant role in Teff activation and suggest a strategy of pharmacologic Stat5b up-regulation as a means of decreasing GVHD while retaining a graft-versus-leukemia effect.

Chronic lymphocytic leukemia (CLL) is the most prevalent human leukemia and is characterized by the progressive accumulation of long-lived malignant B cells. Here we show that human B-CLL cells selectively express high levels of Toll-like receptor 9 (TLR9) mRNA and proteins. Treating B-CLL cells with TLR9 agonists, type B CpG oligodeoxynucleotides (CpG-B ODNs), induces significant morphologic and phenotypic activation, altered cytokine production, reversal of signal transducer, and activator of transcription 1 (STAT1) phosphorylation state, followed by profound apoptosis of B-CLL cells that is CpG-B ODN treatment time- and dose-dependent. TLR9-CpG ODN ligation-induced apoptosis of B-CLL cells is confirmed by viable cell counts, annexin V/propidium iodide and tetramethyl-rhodamine ethylester staining, Western blots of the activation, and cleaved caspases and poly (ADP-ribose) polymerase. Triggering TLR9 by CpG-B ODN leads to nuclear factor-κB-dependent production of autocrine interleukin-10, which activates JAK/STAT pathway-dependent tyrosine phosphorylation of STAT1 proteins and thereby provokes an apoptosis pathway in B-CLL cells. Treating B-CLL cells in vitro or in vivo with CpG-B ODN reduces the number of leukemia cells that engraft in NOD-scid mice. These findings provide new understanding of CpG ODN-mediated antitumor effects and support for the development of TLR9-targeted therapy for human CLL.

Chronic lymphocytic leukemia (CLL) is the most prevalent human leukemia and is characterized by the progressive accumulation of long-lived malignant B cells. Here we show that human B-CLL cells selectively express high levels of Toll-like receptor 9 (TLR9) mRNA and proteins. Treating B-CLL cells with TLR9 agonists, type B CpG oligodeoxynucleotides (CpG-B ODNs), induces significant morphologic and phenotypic activation, altered cytokine production, reversal of signal transducer, and activator of transcription 1 (STAT1) phosphorylation state, followed by profound apoptosis of B-CLL cells that is CpG-B ODN treatment time- and dose-dependent. TLR9-CpG ODN ligation-induced apoptosis of B-CLL cells is confirmed by viable cell counts, annexin V/propidium iodide and tetramethyl-rhodamine ethylester staining, Western blots of the activation, and cleaved caspases and poly (ADP-ribose) polymerase. Triggering TLR9 by CpG-B ODN leads to nuclear factor-κB-dependent production of autocrine interleukin-10, which activates JAK/STAT pathway-dependent tyrosine phosphorylation of STAT1 proteins and thereby provokes an apoptosis pathway in B-CLL cells. Treating B-CLL cells in vitro or in vivo with CpG-B ODN reduces the number of leukemia cells that engraft in NOD-scidmice. These findings provide new understanding of CpG ODN-mediated antitumor effects and support for the development of TLR9-targeted therapy for human CLL.

Multiple innate signals regulate the genesis of effector and memory CD8+T cells. In this study, we demonstrate that the innate cytokines interleukin (IL)–12 and interferon (IFN)–α/β regulate distinct aspects of effector and memory human CD8+T-cell differentiation. IL-12 exclusively promoted the development of IFN-γ– and tumor necrosis factor (TNF)–α–secreting T effector memory (TEM) cells, whereas IFN-α drove the development of T central memory (TCM) cells. The development of TEMand TCMwas linked to cell division. In rapidly dividing cells, IL-12 programmed TEMthrough induction of the IL-12 receptor β2. In contrast, IFN-α regulated TCMdevelopment by slowing the progression of cell division in a subpopulation of cells that selectively expressed elevated IFN-α/β receptor-2. The strength of signal delivered through T-cell receptor (TCR) engagement regulated the responsiveness of cells to IL-12 and IFN-α. In the presence of both IL-12 and IFN-α, these cytokine signals were amplified as the strength of the TCR signal was increased, promoting the simultaneous development of both TCMand TEM. Together, our results support a novel model in which IL-12 and IFN-α act in a nonredundant manner to regulate the colinear generation of both effector and memory cells.

Multiple innate signals regulate the genesis of effector and memory CD8+ T cells. In this study, we demonstrate that the innate cytokines interleukin (IL)–12 and interferon (IFN)–α/β regulate distinct aspects of effector and memory human CD8+ T-cell differentiation. IL-12 exclusively promoted the development of IFN-γ– and tumor necrosis factor (TNF)–α–secreting T effector memory (TEM) cells, whereas IFN-α drove the development of T central memory (TCM) cells. The development of TEM and TCM was linked to cell division. In rapidly dividing cells, IL-12 programmed TEM through induction of the IL-12 receptor β2. In contrast, IFN-α regulated TCM development by slowing the progression of cell division in a subpopulation of cells that selectively expressed elevated IFN-α/β receptor-2. The strength of signal delivered through T-cell receptor (TCR) engagement regulated the responsiveness of cells to IL-12 and IFN-α. In the presence of both IL-12 and IFN-α, these cytokine signals were amplified as the strength of the TCR signal was increased, promoting the simultaneous development of both TCM and TEM. Together, our results support a novel model in which IL-12 and IFN-α act in a nonredundant manner to regulate the colinear generation of both effector and memory cells.

Pre–B-cell leukemia spontaneously develops in BLNK-deficient mice, and pre–B-cell acute lymphoblastic leukemia cells in children often lack BLNK protein expression, demonstrating that BLNK functions as a tumor suppressor. However, the mechanism by which BLNK suppresses pre–B-cell leukemia, as well as the identification of other genetic alterations that collaborate with BLNK deficiency to cause leukemogenesis, are still unknown. Here, we demonstrate that the JAK3/STAT5 signaling pathway is constitutively activated in pre-B leukemia cells derived from BLNK−/−mice, mostly due to autocrine production of IL-7. Inhibition of IL-7R signaling or JAK3/STAT5 activity resulted in the induction of p27kip1expression and cell-cycle arrest, accompanied by apoptosis in the leukemia cells. Transgene-derived constitutively active STAT5 (STAT5b-CA) strongly synergized with the loss of BLNK to initiate leukemia in vivo. In the leukemia cells, exogenously expressed BLNK inhibited autocrine JAK3/STAT5 signaling, resulting in p27kip1induction, cell-cycle arrest, and apoptosis. BLNK-inhibition of JAK3 was dependent on the binding of BLNK to JAK3. These data indicate that BLNK normally regulates IL-7–dependent proliferation and survival of pre–B cells through direct inhibition of JAK3. Thus, somatic loss of BLNK and concomitant mutations leading to constitutive activation of Jak/STAT5 pathway result in the generation of pre–B-cell leukemia.

Pre–B-cell leukemia spontaneously develops in BLNK-deficient mice, and pre–B-cell acute lymphoblastic leukemia cells in children often lack BLNK protein expression, demonstrating that BLNK functions as a tumor suppressor. However, the mechanism by which BLNK suppresses pre–B-cell leukemia, as well as the identification of other genetic alterations that collaborate with BLNK deficiency to cause leukemogenesis, are still unknown. Here, we demonstrate that the JAK3/STAT5 signaling pathway is constitutively activated in pre-B leukemia cells derived from BLNK−/− mice, mostly due to autocrine production of IL-7. Inhibition of IL-7R signaling or JAK3/STAT5 activity resulted in the induction of p27kip1 expression and cell-cycle arrest, accompanied by apoptosis in the leukemia cells. Transgene-derived constitutively active STAT5 (STAT5b-CA) strongly synergized with the loss of BLNK to initiate leukemia in vivo. In the leukemia cells, exogenously expressed BLNK inhibited autocrine JAK3/STAT5 signaling, resulting in p27kip1 induction, cell-cycle arrest, and apoptosis. BLNK-inhibition of JAK3 was dependent on the binding of BLNK to JAK3. These data indicate that BLNK normally regulates IL-7–dependent proliferation and survival of pre–B cells through direct inhibition of JAK3. Thus, somatic loss of BLNK and concomitant mutations leading to constitutive activation of Jak/STAT5 pathway result in the generation of pre–B-cell leukemia.

Patients with acute lymphoblastic leukemia have experienced significantly improved outcomes due to the advent of chimeric antigen receptor T-cells and bispecific T-cell engagers, although a proportion of patients still relapse despite these advances. T-cell exhaustion has been recently suggested to be an important driver of relapse in these patients. Indeed, phenotypic exhaustion of CD4+T-cells is predictive of relapse and poor overall survival in B-ALL. Thus, therapies that counter T-cell exhaustion, such as immune checkpoint blockade, may improve leukemia immunosurveillance and prevent relapse. Here, we used a murine model of Ph+B-ALL as well as human bone marrow biopsy samples to assess the fundamental nature of CD4+T-cell exhaustion and the preclinical therapeutic potential for combining anti-PD-L1 based checkpoint blockade with tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein. scRNA-seq analysis revealed that B-ALL induces a unique subset of CD4+T-cells with both cytotoxic and helper functions. Combination treatment with the tyrosine kinase inhibitor nilotinib and anti-PD-L1 dramatically improves long-term survival of leukemic mice. Depletion of CD4+T-cells prior to therapy completely abrogates the survival benefit, implicating CD4+T-cells as key drivers of the protective anti-leukemia immune response. Indeed, treatment with anti-PD-L1 leads to clonal expansion of leukemia-specific CD4+T-cells with the afore-mentioned helper/cytotoxic phenotype, as well as reduced expression of exhaustion markers. These findings support efforts to utilize PD1/PD-L1 checkpoint blockade in clinical trials and highlight the importance of CD4+T-cell dysfunction in limiting the endogenous anti-leukemia response.

Hematopoietic stem and progenitor cells were previously found to express Toll-like receptors (TLRs), suggesting that bacterial/viral products may influence blood cell formation. We now show that common lymphoid progenitors (CLPs) from mice with active HSV-1 infection are biased to dendritic cell (DC) differentiation, and the phenomenon is largely TLR9 dependent. Similarly, CLPs from mice treated with the TLR9 ligand CpG ODN had little ability to generate CD19+ B lineage cells and had augmented competence to generate DCs. TNFa mediates the depletion of late-stage lymphoid progenitors from bone marrow in many inflammatory conditions, but redirection of lymphopoiesis occurred in TNFa-/- mice treated with CpG ODN. Increased numbers of DCs with a lymphoid past were identified in Ig gene recombination substrate reporter mice treated with CpG ODN. TLR9 is highly expressed on lymphoid progenitors, and culture studies revealed that those receptors, rather than inflammatory cytokines, accounted for the production of several types of functional DCs. Common myeloid progenitors are normally a good source of DCs, but this potential was reduced by TLR9 ligation. Thus, alternate differentiation pathways may be used to produce innate effector cells in health and disease.

Hematopoietic stem and progenitor cells were previously found to express Toll-like receptors (TLRs), suggesting that bacterial/viral products may influence blood cell formation. We now show that common lymphoid progenitors (CLPs) from mice with active HSV-1 infection are biased to dendritic cell (DC) differentiation, and the phenomenon is largely TLR9 dependent. Similarly, CLPs from mice treated with the TLR9 ligand CpG ODN had little ability to generate CD19+B lineage cells and had augmented competence to generate DCs. TNFα mediates the depletion of late-stage lymphoid progenitors from bone marrow in many inflammatory conditions, but redirection of lymphopoiesis occurred in TNFα−/−mice treated with CpG ODN. Increased numbers of DCs with a lymphoid past were identified in Ig gene recombination substrate reporter mice treated with CpG ODN. TLR9 is highly expressed on lymphoid progenitors, and culture studies revealed that those receptors, rather than inflammatory cytokines, accounted for the production of several types of functional DCs. Common myeloid progenitors are normally a good source of DCs, but this potential was reduced by TLR9 ligation. Thus, alternate differentiation pathways may be used to produce innate effector cells in health and disease.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, congenital abnormalities, and cancer predisposition. Small ribosomal subunit genes RPS19, RPS24, and RPS17 are mutated in approximately one-third of patients. We used a candidate gene strategy combining high-resolution genomic mapping and gene expression microarray in the analysis of 2 DBA patients with chromosome 3q deletions to identify RPL35A as a potential DBA gene. Sequence analysis of a cohort of DBA probands confirmed involvement RPL35A in DBA. shRNA inhibition shows that Rpl35a is essential for maturation of 28S and 5.8S rRNAs, 60S subunit biogenesis, normal proliferation, and cell survival. Analysis of pre-rRNA processing in primary DBA lymphoblastoid cell lines demonstrated similar alterations of large ribosomal subunit rRNA in both RPL35A-mutated and some RPL35A wild-type patients, suggesting additional large ribosomal subunit gene defects are likely present in some cases of DBA. These data demonstrate that alterations of large ribosomal subunit proteins cause DBA and support the hypothesis that DBA is primarily the result of altered ribosomal function. The results also establish that haploinsufficiency of large ribosomal subunit proteins contributes to bone marrow failure and potentially cancer predisposition.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, congenital abnormalities, and cancer predisposition. Small ribosomal subunit genes RPS19, RPS24, and RPS17are mutated in approximately one-third of patients. We used a candidate gene strategy combining high-resolution genomic mapping and gene expression microarray in the analysis of 2 DBA patients with chromosome 3q deletions to identify RPL35Aas a potential DBA gene. Sequence analysis of a cohort of DBA probands confirmed involvement RPL35Ain DBA. shRNA inhibition shows that Rpl35a is essential for maturation of 28S and 5.8S rRNAs, 60S subunit biogenesis, normal proliferation, and cell survival. Analysis of pre-rRNA processing in primary DBA lymphoblastoid cell lines demonstrated similar alterations of large ribosomal subunit rRNA in both RPL35A-mutated and some RPL35Awild-type patients, suggesting additional large ribosomal subunit gene defects are likely present in some cases of DBA. These data demonstrate that alterations of large ribosomal subunit proteins cause DBA and support the hypothesis that DBA is primarily the result of altered ribosomal function. The results also establish that haploinsufficiency of large ribosomal subunit proteins contributes to bone marrow failure and potentially cancer predisposition.

Donor cell expression of C3 enhances the alloimmune response and is associated with the fate of transplantation. To clarify the mechanism for enhancement of the immune response, we have explored the role of C3a receptor (C3aR)–ligand interaction on murine bone marrow dendritic cells (DCs). We show that DCs either lacked receptor for C3a (a C3 cleavage product) or were treated with C3aR antagonist, elicited defective T-cell priming against alloantigen expressed on the DCs. This was associated with reduced surface expression of major histocompatibility complex (MHC) and costimulatory molecules on the DCs, and with defective priming in skin allograft rejection. In addition, DCs lacking factor B were unable to generate potent T-cell responses against donor antigen, whereas lack of C4 had no detectable effect, suggesting a role for the alternative pathway contributing to allostimulation. Furthermore, therapeutic complement regulator can down-regulate DC allostimulatory function. These findings suggest that the capacity of DCs for allostimulation depends on their ability to express, activate, and detect relevant complement components leading to C3aR signaling. This mechanism, in addition to underpinning the cell-autonomous action of donor C3 on allostimulation, has implications for a wider range of immune responses in self-restricted T-cell priming.

Donor cell expression of C3 enhances the alloimmune response and is associated with the fate of transplantation. To clarify the mechanism for enhancement of the immune response, we have explored the role of C3a receptor (C3aR)–ligand interaction on murine bone marrow dendritic cells (DCs). We show that DCs either lacked receptor for C3a (a C3 cleavage product) or were treated with C3aR antagonist, elicited defective T-cell priming against alloantigen expressed on the DCs. This was associated with reduced surface expression of major histocompatibility complex (MHC) and costimulatory molecules on the DCs, and with defective priming in skin allograft rejection. In addition, DCs lacking factor B were unable to generate potent T-cell responses against donor antigen, whereas lack of C4 had no detectable effect, suggesting a role for the alternative pathway contributing to allostimulation. Furthermore, therapeutic complement regulator can down-regulate DC allostimulatory function. These findings suggest that the capacity of DCs for allostimulation depends on their ability to express, activate, and detect relevant complement components leading to C3aR signaling. This mechanism, in addition to underpinning the cell-autonomous action of donor C3 on allostimulation, has implications for a wider range of immune responses in self-restricted T-cell priming.

Binding of multiple myeloma (MM) cells to bone marrow stromal cells (BMSCs) triggers expression of adhesive molecules and secretion of interleukin-6 (IL-6), promoting MM cell growth, survival, drug resistance, and migration, which highlights the possibility of developing and validating novel anti-MM therapeutic strategies targeting MM cells–host BMSC interactions and their sequelae. Recently, we have found that expression of the peroxisome proliferator-activated receptor γ (PPARγ) and its ligands can potently inhibit IL-6–regulated MM cell growth. Here we demonstrate that PPARγ agonists 15-d-PGJ2 and troglitazone significantly suppress cell-cell adhesive events, including expression of adhesion molecules and IL-6 secretion from BMSCs triggered by adhesion of MM cells, as well as overcome drug resistance by a PPARγ-dependent mechanism. The synthetic and natural PPARγ agonists have diverging and overlapping mechanisms blocking transactivation of transcription factors NF-κB and 5′-CCAAT/enhancer–binding protein β (C/EBPβ). Both 15-d-PGJ2 and troglitazone blocked C/EBPβ transcriptional activity by forming PPARγ complexes with C/EBPβ. 15-d-PGJ2 and troglitazone also blocked NF-κB activation by recruiting the coactivator PGC-1 from p65/p50 complexes. In addition, 15-d-PGJ2 had a non–PPARγ-dependent effect by inactivation of phosphorylation of IKK and IκB. These studies provide the framework for PPARγ-based pharmacological strategies targeting adhesive interactions of MM cells with the bone marrow microenvironment.

Binding of multiple myeloma (MM) cells to bone marrow stromal cells (BMSCs) triggers expression of adhesive molecules and secretion of interleukin-6 (IL-6), promoting MM cell growth, survival, drug resistance, and migration, which highlights the possibility of developing and validating novel anti-MM therapeutic strategies targeting MM cells–host BMSC interactions and their sequelae. Recently, we have found that expression of the peroxisome proliferator-activated receptor γ (PPARγ) and its ligands can potently inhibit IL-6–regulated MM cell growth. Here we demonstrate that PPARγ agonists 15-d-PGJ2 and troglitazone significantly suppress cell-cell adhesive events, including expression of adhesion molecules and IL-6 secretion from BMSCs triggered by adhesion of MM cells, as well as overcome drug resistance by a PPARγ-dependent mechanism. The synthetic and natural PPARγ agonists have diverging and overlapping mechanisms blocking transactivation of transcription factors NF-κB and 5′-CCAAT/enhancer–binding protein β (C/EBPβ). Both 15-d-PGJ2 and troglitazone blocked C/EBPβ transcriptional activity by forming PPARγ complexes with C/EBPβ. 15-d-PGJ2 and troglitazone also blocked NF-κB activation by recruiting the coactivator PGC-1 from p65/p50 complexes. In addition, 15-d-PGJ2 had a non–PPARγ-dependent effect by inactivation of phosphorylation of IKK and IκB. These studies provide the framework for PPARγ-based pharmacological strategies targeting adhesive interactions of MM cells with the bone marrow microenvironment.

Reactive oxygen species are known to be involved in several cellular processes, including cell signaling. SOD2 is a key enzyme in the conversion of reactive oxygen species and has been implicated in a host of disease states, including cancer. Using an integrated, whole-cell approach encompassing epigenetics, genomics, and proteomics, we have defined the role of SOD2 in multiple myeloma. We show that the SOD2 promoter is methylated in several cell lines and there is a correlative decrease in expression. Furthermore, myeloma patient samples have decreased SOD2 expression compared with healthy donors. Overexpression of SOD2 results in decreased proliferation and altered sensitivity to 2-methoxyestradiol–induced DNA damage and apoptosis. Genomic profiling revealed regulation of 65 genes, including genes involved in tumorigenesis, and proteomic analysis identified activation of the JAK/STAT pathway. Analysis of nearly 400 activated transcription factors identified 31 transcription factors with altered DNA binding activity, including XBP1, NFAT, forkhead, and GAS binding sites. Integration of data from our gestalt molecular analysis has defined a role for SOD2 in cellular proliferation, JAK/STAT signaling, and regulation of several transcription factors.

Reactive oxygen species are known to be involved in several cellular processes, including cell signaling. SOD2 is a key enzyme in the conversion of reactive oxygen species and has been implicated in a host of disease states, including cancer. Using an integrated, whole-cell approach encompassing epigenetics, genomics, and proteomics, we have defined the role of SOD2 in multiple myeloma. We show that the SOD2promoter is methylated in several cell lines and there is a correlative decrease in expression. Furthermore, myeloma patient samples have decreased SOD2 expression compared with healthy donors. Overexpression of SOD2 results in decreased proliferation and altered sensitivity to 2-methoxyestradiol–induced DNA damage and apoptosis. Genomic profiling revealed regulation of 65 genes, including genes involved in tumorigenesis, and proteomic analysis identified activation of the JAK/STAT pathway. Analysis of nearly 400 activated transcription factors identified 31 transcription factors with altered DNA binding activity, including XBP1, NFAT, forkhead, and GAS binding sites. Integration of data from our gestalt molecular analysis has defined a role for SOD2 in cellular proliferation, JAK/STAT signaling, and regulation of several transcription factors.

Acute myeloid leukemia (AML) remains a therapeutic challenge with high mortality rates despite intensive and myeloablative therapies. Structural and sequence alterations have been linked to outcomes in pediatric AML and have been used for risk-based therapy allocation with modest success. Given the vast heterogeneity of AML, conventional cytogenetic and mutational (cytomolecular) biomarkers have not yielded a robust prognostic model: nearly one-third of pediatric patients deemed “low risk” relapse and, inversely, approximately one-third of those in “high risk” categories have favorable outcomes. AML studies in adults previously identified a leukemia stem cell score (LSC17) that was highly prognostic across five independent cohorts comprised of adult patients with diverse AML subtypes (n = 908). We reasoned that incorporating a similar scoring system in pediatric AML would lead to improved prognostic risk models.

No relevant conflicts of interest to declare.

Monosomy 7 (mono7) alterations in acute myeloid leukemia (AML) are associated with poor outcome and disease progression. Through advancements in multi-omic approaches, more specific treatment strategies may be available for high-risk cohorts. Here we describe pediatric cases of AML with mono7, co-occurring fusions, associated outcome, and potential therapeutic treatments.

Interleukin (IL) 4 is a potent immunomodulatory cytokine secreted by T-helper 2 (Th2) cells and Th2 mast cells that promotes the commitment of cells. However, unregulated production and release of IL-4 can exacerbate allergic reactions and increase susceptibility to infectious organisms and viruses. Here, we present evidence that AG-490, a Janus tyrosine kinase (JAK) 2-JAK3 inhibitor, effectively blocked IL-4 gene expression and secretion in the Th2 cell line D10 that was not occurring after anti-CD3 antibody stimulation, whereas AG-490 had no inhibitory effect on production of other Th2 cytokines or cytokines synthesized by the corresponding Th1 cell line clone 29. AG-490 potently inhibited IL-4–mediated proliferation of both D10 and the IL-4–dependent cell line CT.4S. Moreover, AG-490 markedly inhibited IL-4 activation of JAK3 and blocked the downstream activation of signal transducer and activator of transcription 6, as judged by tyrosine phosphorylation, DNA binding, and transcription assays. In contrast, AG-490 did not affect tumor necrosis factor ? activation of NF-?B at similar concentrations of drug. These data suggest that tyrosine kinase inhibitors that inhibit JAK3 may have previously unrecognized and selective clinical potential as immunotherapeutic drugs to treat Th2-mediated diseases driven by IL-4.

The transcription factor, signal transducer and activator of transcription (Stat) 6, regulates TH2-lymphocyte activity by controlling the expression and responsiveness to interleukin (IL)–4, which plays a key role in numerous allergic maladies. Therefore, we sought to use a phosphorothiolate cis-element decoy to target disruption of Stat6 transcriptional activity. Here we showed that the Stat6 decoy potently ablated the messenger RNA expression and production of IL-4, but not of several other cytokines. The Stat6 decoy functionally disrupted IL-4–inducible cell proliferation of murine TH2 cells and primary human CD4+ T lymphocytes. Specificity of the decoy was demonstrated by its ability to directly block Stat6 binding to a cis-element probe and transactivation, but not affect Stat6 tyrosine phosphorylation or expression of the IL-4 receptor chains. Moreover, the decoy failed to inhibit non–Stat6-dependent signaling pathways since IL-2 was competent to induce cell proliferation and activation of Stats 1, 3, and 5a/b. With the use of laser scanning confocal microscopy, fluorescently tagged Stat6 decoy was detectable in the cytoplasm and nucleus; however, greater levels of oligonucleotide were present in the latter following IL-4 treatment. Taken together, these data suggest that IL-4–driven TH2 cell activity can be preferentially restricted via targeted disruption of Stat6 by a novel and specific decoy strategy that may possess gene therapeutic potential.

The transcription factor, signal transducer and activator of transcription (Stat) 6, regulates TH2-lymphocyte activity by controlling the expression and responsiveness to interleukin (IL)–4, which plays a key role in numerous allergic maladies. Therefore, we sought to use a phosphorothiolate cis-element decoy to target disruption of Stat6 transcriptional activity. Here we showed that the Stat6 decoy potently ablated the messenger RNA expression and production of IL-4, but not of several other cytokines. The Stat6 decoy functionally disrupted IL-4–inducible cell proliferation of murine TH2 cells and primary human CD4+T lymphocytes. Specificity of the decoy was demonstrated by its ability to directly block Stat6 binding to a cis-element probe and transactivation, but not affect Stat6 tyrosine phosphorylation or expression of the IL-4 receptor chains. Moreover, the decoy failed to inhibit non–Stat6-dependent signaling pathways since IL-2 was competent to induce cell proliferation and activation of Stats 1, 3, and 5a/b. With the use of laser scanning confocal microscopy, fluorescently tagged Stat6 decoy was detectable in the cytoplasm and nucleus; however, greater levels of oligonucleotide were present in the latter following IL-4 treatment. Taken together, these data suggest that IL-4–driven TH2 cell activity can be preferentially restricted via targeted disruption of Stat6 by a novel and specific decoy strategy that may possess gene therapeutic potential.

Acute myeloid leukemia (AML) remains a therapeutic challenge with high mortality rates despite intensive and myeloablative therapies. While immunotherapies targeting CD19 have yielded remarkable outcomes in acute lymphoblastic leukemia, identifying similar antigen therapeutic targets in AML remains a challenge due to inherent heterogeneity associated with AML and overlapping immunophenotypes with normal hematopoietic stem and myeloid cell populations. Transcriptional heterogeneity within pediatric AML has primarily been linked to underlying fusion. Therefore, we integrated large transcriptomics and proteomics datasets from AML and normal tissues to identify potential targets expressed in leukemias, but not in normal bone marrow or other normal tissue types.