H. Daniel Lacorazza

Sulforaphane Induces Cell Cycle Arrest and Apoptosis in Acute Lymphoblastic Leukemia Cells

Acute lymphoblastic leukemia (ALL) is the most common hematological cancer in children. Although risk-adaptive therapy, CNS-directed chemotherapy, and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs are needed as frontline treatments in high-risk disease and as salvage agents in relapsed ALL. In this study, we report that purified sulforaphane, a natural isothiocyanate found in cruciferous vegetables, has anti-leukemic properties in a broad range of ALL cell lines and primary lymphoblasts from pediatric T-ALL and pre-B ALL patients. The treatment of ALL leukemic cells with sulforaphane resulted in dose-dependent apoptosis and G2/M cell cycle arrest, which was associated with the activation of caspases (3, 8, and 9), inactivation of PARP, p53-independent upregulation of p21CIP1/WAF1, and inhibition of the Cdc2/Cyclin B1 complex. Interestingly, sulforaphane also inhibited the AKT and mTOR survival pathways in most of the tested cell lines by lowering the levels of both total and phosphorylated proteins. Finally, the administration of sulforaphane to the ALL xenograft models resulted in a reduction of tumor burden, particularly following oral administration, suggesting a potential role as an adjunctive agent to improve the therapeutic response in high-risk ALL patients with activated AKT signaling.

The Cytosolic Protein G0S2 Maintains Quiescence in Hematopoietic Stem Cells

Bone marrow hematopoietic stem cells (HSCs) balance proliferation and differentiation by integrating complex transcriptional and post-translational mechanisms regulated by cell intrinsic and extrinsic factors. We found that transcripts of G0/G1 switch gene 2 (G0S2) are enriched in lineage− Sca-1+ c-kit+ (LSK) CD150+ CD48− CD41− cells, a population highly enriched for quiescent HSCs, whereas G0S2 expression is suppressed in dividing LSK CD150+ CD48− cells. Gain-of-function analyses using retroviral expression vectors in bone marrow cells showed that G0S2 localizes to the mitochondria, endoplasmic reticulum, and early endosomes in hematopoietic cells. Co-transplantation of bone marrow cells transduced with the control or G0S2 retrovirus led to increased chimerism of G0S2-overexpressing cells in femurs, although their contribution to the blood was reduced. This finding was correlated with increased quiescence in G0S2-overexpressing HSCs (LSK CD150+ CD48−) and progenitor cells (LS−K). Conversely, silencing of endogenous G0S2 expression in bone marrow cells increased blood chimerism upon transplantation and promoted HSC cell division, supporting an inhibitory role for G0S2 in HSC proliferation. A proteomic study revealed that the hydrophobic domain of G0S2 interacts with a domain of nucleolin that is rich in arginine-glycine-glycine repeats, which results in the retention of nucleolin in the cytosol. We showed that this cytosolic retention of nucleolin occurs in resting, but not proliferating, wild-type LSK CD150+ CD48− cells. Collectively, we propose a novel model of HSC quiescence in which elevated G0S2 expression can sequester nucleolin in the cytosol, precluding its pro-proliferation functions in the nucleolus.

Cutting Edge: Expression of the Transcription Factor E74-Like Factor 4 Is Regulated by the Mammalian Target of Rapamycin Pathway in CD8+ T Cells

T cell receptor activation inhibits expression of the E74-like factor (ELF) 4 and Krüppel-like factor 4 genes to release naive CD8+ T cells from their quiescent state. In this study, we show that ELF4 controls the ERK-mediated proliferative response by maintaining normal levels of dual-specificity phosphatases 1 and 5 in CD8+ T cells. In activated CD8+ T cells, the mammalian target of rapamycin pathway inhibits ELF4 and Krüppel-like factor 4 expression downstream of ERK and PI3K signaling. Our findings demonstrate that rapamycin could be used to modulate expression of this transcriptional network involved in cell-cycle regulation.

Krüppel-like factor 4 (KLF4) promotes the survival of natural killer cells and maintains the number of conventional dendritic cells in the spleen

The development and survival of NK cells rely on a complex, spatiotemporal gene expression pattern regulated by specific transcription factors in NK cells and tissue‐specific microenvironments supported by hematopoietic cells. Here, we show that somatic deletion of the KLF4 gene, using inducible and lineage‐specific cre‐transgenic mice, leads to a significant reduction of NK cells (NK1.1+ TCR‐β−) in the blood and spleen but not in the BM, liver, or LNs. Functional and immunophenotypic analyses revealed increased apoptosis of CD27+/− CD11b+ NK cells in the spleen of KLF4‐deficient mice, although remaining NK cells were able to lyse tumor target cells and produce IFN‐γ. A normal recovery of adoptively transferred KLF4‐deficient NK cells in WT hosts suggested that the survival defect was not intrinsic of NK cells. However, BM chimeras using KLF4‐deficient mice as donors indicated that reduced survival of NK cells depended on BM‐derived hematopoietic cells in the spleen. The number of CD11chi DCs, which are known to support NK cell survival, was reduced significantly in the spleen of KLF4‐deficient mice, likely a result of a lower number of precDC progenitor cells in this tissue. Taken together, our data suggest that the pluripotency‐associated gene KLF4 is required for the maintenance of DCs in the spleen and consequently, survival of differentiated NK cells in this tissue.

G0S2 inhibits the proliferation of K562 cells by interacting with nucleolin in the cytosol

G0/G1 switch gene 2 (G0S2) is a basic protein with ill-defined function that inhibits the proliferation of hematopoietic stem cells. Herein, we show that treatment of K562 cells with 5-azacytidine (5-Aza) resulted in a 24-fold increase in G0S2 expression and a reduction in cell growth. Conversely, gene demethylation in the presence of G0S2-specific shRNA restored proliferation, further supporting an inhibitory role for G0S2 in cell proliferation. Elevated levels of G0S2 inhibited the division of K562 cells by sequestering the nucleolar phosphoprotein nucleolin in the cytosol. G0S2 inhibited the proliferation of leukemia cells in vivo in xenograft models. Collectively, our data identify a new mechanism that controls proliferation in K562 cells, suggesting a possible tumor suppressor function in leukemia cells.

Differential roles of KLF4 in the development and differentiation of CD8+ T cells

The transcription factor Krüppel-like factor 4 (KLF4) can activate or repress gene expression in a cell-context dependent manner. We have previously shown that KLF4 inhibits the proliferation of naïve CD8(+) T cells in vitro downstream of the transcription factor ELF4. In this work, we describe a novel role of KLF4 in the differentiation of CD8(+) T cells upon infection. Loss of KLF4 had minimal effect on thymic T cell development and distribution of mature T cells in the spleen, blood, and lymph nodes. KLF4-deficient naïve CD8(+) T cells also displayed normal homeostatic proliferation upon adoptive transfer into lymphopenic hosts. However, activation of KLF4-deficient naïve CD8(+) T cells by in vitro TCR crosslink and co-stimulation resulted in increased proliferation. Furthermore, naïve KLF4-deficient OT-I CD8(+) T cells generated increased numbers of functional memory CD8(+) T cells compared to wild type OT-I CD8(+) T cells co-injected in the same recipient in both primary and recall responses to Listeria monocytogenes-OVA. Collectively, our data demonstrate that KLF4 regulates differentiation of functional memory CD8(+) T cells while sparing development and homeostasis of naïve CD8(+) T cells.

The Transcription Factor E74-Like Factor 4 Suppresses Differentiation of Proliferating CD4+ T Cells to the Th17 Lineage

The differentiation of CD4+ T cells into different Th lineages is driven by cytokine milieu in the priming site and the underlying transcriptional circuitry. Even though many positive regulators have been identified, it is not clear how this process is inhibited at transcriptional level. In this study, we report that the E-twenty six (ETS) transcription factor E74-like factor 4 (ELF4) suppresses the differentiation of Th17 cells both in vitro and in vivo. Culture of naive Elf4−/− CD4+ T cells in the presence of IL-6 and TGF-β (or IL-6, IL-23, and IL-1β) resulted in increased numbers of IL-17A–positive cells compared with wild-type controls. In contrast, the differentiation to Th1, Th2, or regulatory T cells was largely unaffected by loss of ELF4. The increased expression of genes involved in Th17 differentiation observed in Elf4−/− CD4+ T cells suggested that ELF4 controls their programming into the Th17 lineage rather than only IL-17A gene expression. Despite normal proliferation of naive CD4+ T cells, loss of ELF4 lowered the requirement of IL-6 and TGF-β signaling for IL-17A induction in each cell division. ELF4 did not inhibit Th17 differentiation by promoting IL-2 production as proposed for another ETS transcription factor, ETS1. Elf4−/− mice showed increased numbers of Th17 cells in the lamina propria at steady state, in lymph nodes after immunization, and, most importantly, in the CNS following experimental autoimmune encephalomyelitis induction, contributing to the increased disease severity. Collectively, our findings suggest that ELF4 restrains Th17 differentiation in dividing CD4+ T cells by regulating commitment to the Th17 differentiation program.

Role of the reprogramming factor KLF4 in blood formation

Krüppel‐like factor 4 is a zinc finger protein with dual functions that can act as a transcriptional activator and repressor of genes involved in cell proliferation, differentiation, and apoptosis. Although most studies have focused on terminally differentiated epithelial cells, evidence suggests that Krüppel‐like factor 4 regulates the development and function of the myeloid and lymphoid blood lineages. The ability of Krüppel‐like factor 4 to dedifferentiate from somatic cells into pluripotent stem cells in cooperation with other reprogramming factors suggests its potential function in the preservation of tissue‐specific stem cells. Additionally, emerging interest in the redifferentiation of induced pluripotent stem cells into blood cells to correct hematologic deficiencies and malignancies warrants further studies on the role of Krüppel‐like factor 4 in steady‐state blood formation.

Loss of n-6 fatty acid induced pediatric obesity protects against acute murine colitis

Dietary influences may affect microbiome composition and host immune responses, thereby modulating propensity toward inflammatory bowel diseases (IBDs): Crohn disease (CD) and ulcerative colitis (UC). Dietary n‐6 fatty acids have been associated with UC in prospective studies. However, the critical developmental period when (n‐6) consumption may induce UC is not known. We examined the effects of transiently increased n‐6 consumption during pediatric development on subsequent dextran‐sulfate‐sodium (DSS)‐induced acute murine colitis. The animals transiently became obese then rapidly lost this phenotype. Interestingly, mice were protected against DSS colitis 40 days after n‐6 consumption. The transient high n‐6‐induced protection against colitis was fat type‐ and dietary reversal‐dependent and could be transferred to germ‐free mice by fecal microbiota transplantation. We also detected decreased numbers of chemokine receptor (Cxcr)5+ CD4+ T cells in the mesenteric lymph nodes (MLNs) of transiently n‐6‐fed mice. Further experiments revealed that anti‐chemokine ligand (Cxc1)13 (the ligand of Cxcr5) antibody treatment decreased DSS colitis severity, implicating the importance of the Cxcr5‐Cxcl13 pathway in mammalian colitis. Consecutively, we found elevated CXCL13 concentrations (CD: 1.8‐fold, P= 0.0077; UC: 1.9‐fold, P= 0.056) in the serum of untreated pediatric IBD patients. The human serologic observations supported the translational relevance of our findings.—Nagy‐Szakal, D., Mir, S. A. V., Harris, R. A., Dowd, S. E., Yamada, T., Lacorazza, H. D., Tatevian, N., Smith, C. W., de Zoeten, E. F., Klein, J., Kellermayer, R. Loss of n‐6 fatty acid induced pediatric obesity protects against acute murine colitis. FASEB J. 29, 3151‐3159 (2015). www.fasebj.org

Transcription factor ELF4 promotes development and function of memory CD8+ T cells in Listeria monocytogenes infection

Most differentiated CD8+ T cells die off at the end of an infection, revealing two main subsets of memory T cells — central and effector memory — which can be found in lymphoid tissues or circulating through nonlymphoid organs, respectively. The cell intrinsic regulation of the differentiation of CD8+ T cells to effector and central memory remains poorly studied. Herein, we describe a novel role of the ETS transcription factor ELF4 in the development and function of memory CD8+ T cells following infection with Listeria monocytogenes. Adoptively transferred Elf4−/− naïve CD8+ T cells produced lower numbers of effector memory CD8+ T cells despite a normal pool of central memory. This was caused by suboptimal priming and decreased survival of CD8+ T cells at the peak of response while enhanced Notch1 signaling and upregulation of eomesodermin correlated with “normal” development of Elf4−/− central memory. Finally, loss of ELF4 impaired the expansion of both central and effector memory CD8+ T cells in a recall response by also activating Notch1 signaling. Altogether, ELF4 emerges as a novel transcriptional regulator of CD8+ T‐cell differentiation in response to infection.