Krüppel-like Factor 4 Supports the Expansion of Leukemia Stem Cells in MLL-AF9-driven Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow with 5-year overall survival of less than 10% in patients over the age of 65. Limited progress has been made in the patient outcome because of the inability to selectively eradicate the leukemic stem cells (LSC) driving the refractory and relapsed disease. Herein, we investigated the role of the reprogramming factor KLF4 in AML because of its critical role in the self-renewal and stemness of embryonic and cancer stem cells. Using a conditional Cre-lox Klf4 deletion system and the MLL-AF9 retroviral mouse model, we demonstrated that loss-of-KLF4 does not significantly affect the induction of leukemia but markedly decreased the frequency of LSCs evaluated in limiting-dose transplantation studies. Loss of KLF4 in leukemic granulocyte-macrophage progenitors (L-GMP), a population enriched for AML LSCs, showed lessened clonogenicity and percentage in the G2/M phase of the cell cycle. RNAseq analysis of purified L-GMPs revealed decreased expression of stemness genes and MLL-target genes and upregulation of the RNA sensing helicase DDX58. However, silencing of DDX58 in KLF4 knockout leukemia indicated that DDX58 is not mediating this phenotype. CRISPR/Cas9 deletion of KLF4 in MOLM13 cell line and AML patient-derived xenograft cells showed impaired expansion in vitro and in vivo associated with a defective G2/M checkpoint. Collectively, our data suggest a mechanism in which KLF4 promotes leukemia progression by establishing a gene expression profile in AML LSCs supporting cell division and stemness.

A KLF4-DYRK2-mediated pathway regulating self-renewal in CML stem cells.

Leukemia stem cells are a rare population with a primitive progenitor phenotype that can initiate, sustain, and recapitulate leukemia through a poorly understood mechanism of self-renewal. Here, we report that Krüppel-like factor 4 (KLF4) promotes disease progression in a murine model of chronic myeloid leukemia (CML)-like myeloproliferative neoplasia by repressing an inhibitory mechanism of preservation in leukemia stem/progenitor cells with leukemia-initiating capacity. Deletion of the Klf4 gene severely abrogated the maintenance of BCR-ABL1(p210)-induced CML by impairing survival and self-renewal in BCR-ABL1+ CD150+ lineage-negative Sca-1+ c-Kit+ leukemic cells. Mechanistically, KLF4 repressed the Dyrk2 gene in leukemic stem/progenitor cells; thus, loss of KLF4 resulted in elevated levels of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2), which were associated with inhibition of survival and self-renewal via depletion of c-Myc protein and p53 activation. In addition to transcriptional regulation, stabilization of DYRK2 protein by inhibiting ubiquitin E3 ligase SIAH2 with vitamin K3 promoted apoptosis and abrogated self-renewal in murine and human CML stem/progenitor cells. Altogether, our results suggest that DYRK2 is a molecular checkpoint controlling p53- and c-Myc-mediated regulation of survival and self-renewal in CML cells with leukemic-initiating capacity that can be targeted with small molecules.

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-ß (or IL-6, IL-23, and IL-1ß) 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-ß 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.

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.

G0S2 modulates homeostatic proliferation of naïve CD8⁺ T cells and inhibits oxidative phosphorylation in mitochondria.

Since its discovery, diverse functions have been attributed to the G0/G1 switch gene 2 (G0S2), from lipid metabolism to control of cell proliferation. Our group showed for the first time that G0S2 promotes quiescence in hematopoietic stem cells by interacting with and retaining nucleolin around the nucleus. Herein, we report the role of G0S2 in the differentiation and function of CD8(+) T cells examined in mice with an embryonic deletion of the G0s2 gene. G0S2 expression in naïve CD8(+) T cells decreased immediately after T-cell receptor activation downstream of the mitogen-activated protein kinase, calcium/calmodulin, phosphatidylinositol 3'-kinase and mammalian target of rapamycin pathways. Surprisingly, G0S2-null naïve CD8(+) T cells displayed increased basal and spare respiratory capacity that was not associated with increased mitochondrial biogenesis but with increased phosphorylation of AMP-activated protein kinase α. Naïve CD8(+) T cells showed increased proliferation in response to in vitro activation and in vivo lymphopenia; however, naïve CD8(+) T cells expressing the OT-1 transgene exhibited normal differentiation of naïve cells to effector and memory CD8(+) T cells upon infection with Listeria monocytogenes in a wild-type or a G0s2-null environment, with increased circulating levels of free fatty acids. Collectively, our results suggest that G0S2 inhibits energy production by oxidative phosphorylation to fine-tune proliferation in homeostatic conditions.

Antileukemic properties of the kinase inhibitor OTSSP167 in T-cell acute lymphoblastic leukemia.

Novel drugs are needed to increase treatment response in children with high-risk T-cell acute lymphoblastic leukemia (T-ALL). Following up on our previous report on the activation of the MAP2K7-JNK pathway in pediatric T-ALL, here we demonstrate that OTSSP167, recently shown to inhibit MAP2K7, has antileukemic capacity in T-ALL. OTSSP167 exhibited dose-dependent cytotoxicity against a panel of T-ALL cell lines with IC50 in the nanomolar range (10-50 nM). OTSSP167 induces apoptosis and cell cycle arrest in T-ALL cell lines, associated at least partially with the inhibition of MAP2K7 kinase activity and lower activation of its downstream substrate, JNK. Other leukemic T-cell survival pathways, such as mTOR and NOTCH1 were also inhibited. Daily intraperitoneal administration of 10 mg/kg OTSSP167 was well tolerated, with mice showing no hematological toxicity, and effective at reducing the expansion of human T-ALL cells in a cell-based xenograft model. The same dosage of OTSSP167 efficiently controlled the leukemia burden in the blood, bone marrow, and spleen of 3 patient-derived xenografts, which resulted in prolonged survival. OTSSP167 exhibited synergistic interactions when combined with dexamethasone, L-asparaginase, vincristine, and etoposide. Our findings reveal novel antileukemic properties of OTSSP167 in T-ALL and support the use of OTSSP167 as an adjuvant drug to increase treatment response and reduce relapses in pediatric T-ALL.

The transcription factor E74-like factor controls quiescence of endothelial cells and their resistance to myeloablative treatments in bone marrow.

OBJECTIVE: The regeneration of the hematopoietic system in bone marrow after chemotherapy depends on a balance between the quiescence and proliferation of lineage-specific progenitor cells. Even though the vascular network in bone is damaged by cytoablation, the transcriptional control of quiescence in endothelial cells is not well known. In this study, we investigated the role of the transcription factor E74-like factor (ELF4) in the proliferation of endothelial cells in bone marrow. METHODS AND RESULTS: Loss-of-function models were used to study the role of ELF4 in human and murine endothelial cells. ELF4 promotes cell cycle entry by activating cyclin-dependent kinase-4 in human umbilical vein endothelial cells. Elf4-null mice exhibited enhanced recovery of bone marrow CD45- CD31+ endothelial cells and sinusoidal blood vessels following administration of 5-fluorouracil. CONCLUSIONS: Loss of ELF4 leads to increased quiescence in bone marrow endothelial cells by the deregulation of cyclin-dependent kinase-4 expression and to enhanced regeneration of sinusoidal blood vessels.

Inhibition of the MAP2K7-JNK pathway with 5Z-7-oxozeaenol induces apoptosis in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric leukemia with a worse prognosis than most frequent B-cell ALL due to a high incidence of treatment failures and relapse. Our previous work showed that loss of the pioneer factor KLF4 in a NOTCH1-induced T-ALL mouse model accelerated the development of leukemia through expansion of leukemia-initiating cells and activation of the MAP2K7 pathway. Similarly, epigenetic silencing of the KLF4 gene in children with T-ALL was associated with MAP2K7 activation. Here, we showed the small molecule 5Z-7-oxozeaenol (5Z7O) induces dose-dependent cytotoxicity in a panel of T-ALL cell lines mainly through inhibition of the MAP2K7-JNK pathway, which further validates MAP2K7 as a therapeutic target. Mechanistically, 5Z7O-mediated apoptosis was caused by the downregulation of regulators of the G2/M checkpoint and the inhibition of survival pathways. The anti-leukemic capacity of 5Z7O was evaluated using leukemic cells from two mouse models of T-ALL and patient-derived xenograft cells generated using lymphoblasts from pediatric T-ALL patients. Finally, a combination of 5Z7O with dexamethasone, a drug used in frontline therapy, showed synergistic induction of cytotoxicity. In sum, we report here that MAP2K7 inhibition thwarts survival mechanisms in T-ALL cells and warrants future pre-clinical studies for high-risk and relapsed patients.

Krüppel-like factor 4 promotes survival and expansion in acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy of the bone marrow that affects mostly elderly adults. Alternative therapies are needed for AML patients because the overall prognosis with current standard of care, high dose chemotherapy and allogeneic transplantation, remains poor due to the emergence of refractory and relapsed disease. Here, we found expression of the transcription factor KLF4 in AML cell lines is not silenced through KLF4 gene methylation nor via proteasomal degradation. The deletion of KLF4 by CRISPR-CAS9 technology reduced cell growth and increased apoptosis in both NB4 and MonoMac-6 cell lines. Chemical induced differentiation of gene edited NB4 and MonoMac6 cells with ATRA and PMA respectively increased apoptosis and altered expression of differentiating markers CD11b and CD14. Transplantation of NB4 and MonoMac-6 cells lacking KLF4 into NSG mice resulted in improved overall survival compared to the transplantation of parental cell lines. Finally, loss-of-KLF4 did not alter sensitivity of leukemic cells to the chemotherapeutic drugs daunorubicin and cytarabine. These results suggest that KLF4 expression supports AML cell growth and survival, and the identification and disruption of KLF4-regulated pathways could represent an adjuvant therapeutic approach to increase response.

Predicting radiosensitivity using DNA end-binding complex analysis.

Previous reports have suggested that measuring radiosensitivity of normal and tumor cells would have significant clinical relevance for the practice of radiation oncology. We hypothesized that radiosensitivity might be predicted by analyzing DNA end-binding complexes (DNA-EBCs), which form at DNA double-strand breaks, the most important cytotoxic lesion caused by radiation. To test this hypothesis, the DNA-EBC pattern of 21 primary human fibroblast cultures and 15 tumor cell lines were studied. DNA-EBC patterns were determined using a modified electrophoretic mobility shift assay and were correlated with radiosensitivity, as measured by SF2. DNA-EBC analysis identified a rapidly migrating ATM-containing band (identified as "band-A") of which the density correlated with SF2 (0.02

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.

Kruppel-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-ß(-)) 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 CD11c(hi) 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.