KLF4 Research Articles

Construction of thalassemic mouse induced pluripotent stem cells for disease modeling

Induced pluripotent stem cells (iPSCs) have a great potential for disease modeling and therapeutic applications. One of the most common genetic diseases found in South East Asia is thalassemia. β-Thalassemia is an inherited single gene disorder caused by reduction or absence of β-globin chain production. The purpose of this study is to generate mouse iPSCs with β-thalassemia background. Mouse embryonic fibroblast (MEF) cells were obtained from transgenic β-thalassemic mouse, transduced with lentiviral vector carrying 4 transcription factors, Oct4, Klf4, Sox2 and c-Myc (OKSM), and cultured on a feeder layer. The embryonic stem (ES)-like colonies were manually picked up and expanded. The generated mouse iPSCs were similar to mouse embryonic stem cells (mESCs) in morphology, alkaline phosphatase activity and pattern of pluripotent gene expression and able to form teratoma containing tissues derived from all three germ layers. Therefore, we reported here an establishment of β-thalassemic mouse iPSCs which might be used for further disease modeling and therapeutic applications.

In vitro effects of zinc and folic acid on the expressions of Neurogenin 3, Kruppel-like factor 4, c-Myc, Nanog, Nestin and POU class 5 homeobox 1 genes

Objective: Genes are regulated at multiple levels by using nutritional factors during neurogenesis and gliogenesis in brain development. Folic acid (FA)and zinc regulate the expressions of some genes which participate in brain development as nutritional factors. So, we aimed to find the effect of FA and zinc on the expression levels of neurogenin 3 (NGN3), Kruppel-like factor 4 (Klf4), c-Myc, nanog, POU class 5 homeobox 1-Pou5F1 (Oct4), and Nestin in mouse fetal brain tissue in neural plate phase at gestational day 7 (GD7)and in fetal brain tissue at GD20. Methods: All the tissues were cultured primarily. FA and zinc solutions were added. Real time-polymerase chain reaction was performed in RNA obtained from in each sample. Results: FA has no effect on GD7 mouse neural plate tissue and GD20 mouse fetal brain tissue. Zinc has no effect on GD7 mouse neural plate tissue. Zinc increased the expressions of NGN3, Klf4, nanog, Nestin, and Oct4 genes on GD20 mouse fetal brain tissue. Conclusion: This in vitro study represents that zinc is important in the expressions of NGN3, Klf4, Nanog, Nestin, and Oct4 genes in the late phase of pregnancy. The stimulator effect of Zinc on the expression levels of these genes may show us the possible role of zinc in fetal brain development in the late phase of pregnancy.

Increases in iPS Transcription Factor (Oct4, Sox2, c-Myc, and Klf4) Gene Expression after Modified Electroconvulsive Therapy.

ObjectiveElectroconvulsive therapy (ECT) is a reasonable option for intractable depression or schizophrenia, but a mechanism of action has not been established. One credible hypothesis is related to neural plasticity. Three genes (Oct4, Sox2, c-Myc) involved in the induction of induced pluripotent stem (iPS) cells are Wnt-target genes, which constitute a key gene group involved in neural plasticity through the TCF family. Klf4 is the other gene among Yamanaka's four transcription factors, and increases in its expression are induced by stimulation of the canonical Wnt pathway.MethodsWe compared the peripheral blood gene expression of the four iPS genes (Oct4, Sox2, c-Myc, and Klf4) before and after modified ECT (specifically ECT with general anesthesia) of patients with intractable depression (n=6) or schizophrenia (n=6). Using Thymatron ten times the total bilateral electrical stimulation was evoked.ResultsBoth assessments of the symptoms demonstrated significant improvement after mECT stimulation. Expression of all four genes was confirmed to increase after initial stimulation. The gene expression levels after treatment were significantly different from the initial gene expression in all twelve cases at the following treatment stages: at the 3rd mECT for Oct4; at the 6th and 10th mECT for Sox2; and at the 3rd, 6th and 10th mECT for c-Myc.ConclusionThese significant differences were not present after correction for multiple testing; however, our data have the potential to explain the molecular mechanisms of mECT from a unique perspective. Further studie should be conducted to clarify the pathophysiological involvement of iPS-inducing genes in ECT.

Structure and functional evaluation of porcine NANOG that is a single-exon gene and has two pseudogenes

Nanog plays an important role in maintaining the pluripotency of murine and human embryonic stem cells. However, the molecular features and transcriptional regulation of the NANOG gene have not been well investigated in pig. Here, we report, for the first time, that porcine NANOG is encoded by a single exon gene (SEG) mapped on chromosome 1 and has two daughter genes, one pseudogene NANOGP1 on chromosome 5 and one tandem duplicate on chromosome 1. The duplicated pseudogene NANOGP2 has high sequence similarity to NANOG, but does not encode a functional protein due to deletions and in-frame stop codons. The NANOGP1 contains four exons and three introns, but is short of the homeodomain sequence. Transcriptome analysis confirmed that NANOG mRNA in porcine iPS cells is transcribed from the SEG NANOG, but not from NANOGP1, because the NANOGP1 promoter is highly methylated, as confirmed by global DNA methylation analysis. The NANOG protein encoded by NANOG retains N, H, and C1/W/C2 domains. The H domain is required for nuclear translocation, while the C1/W/C2 domain ensures the NANOG regulatory function. Overexpression of NANOG in porcine embryonic fibroblasts promoted upregulation of its target genes SOX2, KLF4, and c-MYC. In conclusion, the functional porcine NANOG that is different in chromosomal structure from mouse and human genes is a single exon gene and encodes the functional NANOG protein that can be specifically regulated by OCT4/SOX2, and can promote the activation of target pluripotent factors in vivo.

KLF4 Regulates Self-Renewal of Leukemic Stem Cells in Chronic Myeloid Leukemia By Repressing Gbl Expression and Altering mTORC2 Activity

Abstract Tyrosine kinase inhibitors (TKIs) are the standard treatment for eradicating BCR-ABL-positive progenitor cells in chronic myeloid leukemia (CML); however, disease often relapses upon drug discontinuation because TKIs do not effectively eliminate leukemic stem cells (LSC). The development of novel strategies aimed at eradicating LSC without harming normal hematopoietic stem cells (HSC) is essential for the cure of CML patients. The generation of LSC-directed therapy relies on the identification of novel molecular pathways that selectively regulate LSC function independent of BCR-ABL. The Krüppel-like factor 4(KLF4) is a transcription factor that can either activate or repress gene transcription acting as an oncogene or a tumor suppressor depending on the cellular context. Analysis of a published dataset from chronic phase CML patients revealed elevated levels of KLF4 in LSC compared to progenitor cells indicating that KLF4 is likely implicated in LSC regulation. To study the role of KLF4 in LSC function, we used a CML mouse model combining somatic deletion of the Klf4 gene and retroviral transduction and transplantation of HSC. In contrast to mice receiving BCR-ABL-transduced Klf4fl/fl HSC that developed and succumbed to CML, mice transplanted with BCR-ABL-transduced Klf4Δ/Δ (Klf4fl/fl Vav-iCre+) HSC showed a progressive loss of leukemia despite an initial expansion of myeloid leukemic cells, which led to increased overall survival. This inability to sustain CML in the absence of KLF4 was caused by attrition of LSC in bone marrow and the spleen. Furthermore, deletion of KLF4 impaired the ability of LSC to recapitulate leukemia in secondary recipients suggesting a loss of self-renewal capacity. In contrast to LSC, KLF4 deletion led to increased self-renewal of normal HSC assessed by serial competitive transplantation. To identify KLF4 target genes involved in LSC self-renewal, we performed a global gene expression analysis using Klf4Δ/Δ LSC purified by cell sorting from leukemic mice. Analysis of gene expression in Klf4Δ/Δ LSC revealed significant upregulation of GβL, a component of mTOR complexes. Finally, we identified that KLF4 binds to GβL promoter by Chip-Seq analysis and that silencing resulted in inhibition of mTORC2 but not mTORC1 activity in 32D-BCR-ABL-positive CML cells. Our findings suggest that KLF4 transcriptionally represses GβL expression in LSC and that mTORC2 inhibition has the potential to completely eradicate LSC and induce treatment-free remission. Disclosures No relevant conflicts of interest to declare.

Erythroid-Specific Expression of LIN28A Is Sufficient for Robust Gamma-Globin Gene and Protein Expression in Adult Erythroblasts

LIN28 proteins bind to RNA and regulate developmental timing events in multicellular organisms, in part, by reducing cellular levels of the let-7 family of microRNAs. High-level LIN28 expression in stem cells promotes their self-renewal. Over-expression of the LIN28 proteins causes suppression of let-7 in hematopoietic stem and early progenitor cell populations (CD34+) from adult donors and manifests a more fetal-like phenotype in the erythroid lineage. Here we explore LIN28expression that is restricted to erythroid cells, rather than stem or multi-potential progenitor cells.For this purpose, lentiviral transduction vectors were produced with LIN28A expression driven by erythroid-specific gene promoter regions of the human KLF1 or SPTA1 genes, as well as an internal ribosomal entry site for puromycin selection (vectors: KLF1-LIN28A-OE and SPTA1-LIN28A-OE). Viral supernatants from these constructs were compared with empty-vector controls in matched transductions of CD34+ cells from three adult human volunteers. The cells were transduced and cultured using a three-phase, serum-free model for ex vivo erythropoiesis. Erythroblast proliferation and differentiation were comparable between control and LIN28-transduced cells assessed by cell counting and flow cytometry with staining for CD71, glycophorin A and thiazole orange. To validate restricted expression of LIN28 in the erythroid lineage, colony formation assays were performed in semisolid methylcellulose containing 1.0 ug/ml puromycin. BFU-E, CFU-GM, CFU-G, CFU-M and GEMM colonies were enumerated 14 days after plating. Puromycin addition to KLF1-LIN28A-OE and SPTA1-LIN28A-OE transductions resulted in selection of the erythroid colonies (BFU-E as a percentage of total colonies: Control: 44.6 ± 6.1%; KLF1-LIN28A-OE: 98.4 ± 0.7%, p=0.003; SPTA1-LIN28A-OE: 95.2 ± 1.1%, p=0.005).LIN28A over-expression was confirmed by RT-QPCR (KLF1-LIN28A-OE: 2.1E+05 ± 7.0E+04 copies/ng; SPTA1-LIN28A-OE: 2.2E+05 ± 8.3E+04 copies/ng; Controls: below detection limits) and Western analyses after transduction. Suppression of all let-7 miRNA family members to less than 30% control levels were detected for both vectors resulting in a reduction in total let-7 miRNA (RT-QPCR: Control: 2.0E+07 ± 9.7E+05 copies/ng; KLF1-LIN28A-OE: 5.6E+06 ± 5.6E+05 copies/ng, p=0.003; SPTA1-LIN28A-OE: 4.6E+06 ± 6.2E+05 copies/ng, p=0.003). BCL11A expression levels were also measured by RT-QPCR and Western analyses. While BCL11A showed no significant change at the mRNA level (Control: 1.2E+03 ± 4.5E+02 copies/ng; KLF1-LIN28A-OE: 2.9E+02 ± 7.4E+01 copies/ng, p=0.07; SPTA1-LIN28A-OE: 4.2E+02 ± 3.3E+02 copies/ng, p=0.07), protein analyses of nuclear BCL11A showed moderately reduced levels after KLF1-LIN28A-OE and SPTA1-LIN28A-OE transductions.Globin mRNA and protein levels were investigated and compared with controls. Gamma-globin mRNA was significantly increased in LIN28A-OE samples (Control: 3.6E+06 ± 8.2E+05 copies/ng; KLF1-LIN28A-OE: 1.9E+07 ± 1.7E+06 copies/ng, p=0.007; SPTA1-LIN28A-OE: 1.7E+07 ± 8.9E+05 copies/ng, p=0.003). Fetal hemoglobin (HbF) production was measured at the end of the culture period using High Performance Liquid Chromatography, and was increased in the KLF1-LIN28A-OE and SPTA1-LIN28A-OE samples compared to the control (Control: 7.0 ± 1.4%; KLF1-LIN28A-OE: 31.9 ± 2.7%, p=0.004; SPTA1-LIN28A-OE: 43.0 ± 6.2%, p=0.004). Flow cytometry analyses demonstrated a pan-cellular HbF distribution.In contrast to promoting self-renewal in stem cells, these data suggest that adult erythroblast-restricted LIN28 functions to partially reverse the fetal-to-adult developmental transition in hemoglobin expression. DisclosuresNo relevant conflicts of interest to declare.

KLF1 Null Neonates Display Hydrops Fetalis and a Deranged Erythroid Transcriptome

We describe a case of severe neonatal anemia with kernicterus due to compound heterozygosity for null mutations in KLF1, each inherited from asymptomatic parents. One of the mutations is novel. This is the first described case of a KLF1 null human. The phenotype of severe DAT-negative non-spherocytic hemolytic anaemia (NSHA), jaundice, hepato-splenomegaly, and marked erythroblastosis is more severe than that present in CDA type IV due to dominant mutations in the second zinc-finger of KLF1. There was a very high level of HbF expression into childhood (>70%), consistent with a key role for KLF1 in human hemoglobin switching. We performed RNA-seq on circulating erythroblasts and found human KLF1 acts like mouse Klf1 to coordinate expression of >500 genes required to build a red cell including those encoding globins, cytoskeletal components, AHSP, heme synthesis enzymes, cell cycle regulators, and blood group antigens. We interrogated recently published KLF1 ChIP-seq to determine directly regulated KLF1 genes in man. We identify novel KLF1 target genes including KIF23 and KIF11 which are required for proper cytokinesis. KIF23 mutations result in CDA type III so our finding provides a direct link btween CDA IV adn CDA III. We also identify new roles for KLF1 in autophagy, global transcriptional control and RNA splicing. We suggest loss of KLF1 should be considered in otherwise unexplained cases of severe neonatal NSHA or hydrops fetalis. DisclosuresNo relevant conflicts of interest to declare.

Krüppel-like Factor 4 (KLF4) Suppresses T-Cell Acute Lymphoblastic Leukemia By Inhibiting Expression of MAP2K7 and Expansion of Leukemia Initiating Cells

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy 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 or novel multi-drug combinations are needed as frontline treatments for high-risk patients and as salvage agents for relapsed disease. T-cell ALL (T-ALL) is a subset of ALL that exhibits activating mutations of NOTCH1 in more than 50% of the patients. However, the use of gamma-secretase inhibitors to reduce NOTCH1 activity has not been successful in patients due to limited response and toxicity. Therefore, identification of genetic factors that cooperate with T-ALL leukemogenesis is needed for the development of alternative therapies.KLF4 is a transcription factor that functions as a tumor suppressor or an oncogene depending on cellular context. Our data showed significant reduction of KLF4 transcripts in lymphoblasts from T-ALL patients compared to blood and bone marrow cells from healthy individuals. In consistent with reduced KLF4 levels, these patients exhibit hyper-methylation of CpG islands located between nt -811 and +1190 relative to KLF4 transcription start site. From these findings we hypothesized that KLF4 has tumor suppressor function in T-ALL leukemogenesis. To test our hypothesis, we transduced 5-FU treated bone marrow (BM) cells from control (Klf4fl/fl), Klf4 null (Klf4fl/fl; Vav-iCre) and Klf4 heterozygous (Klf4fl/+; Vav-iCre) mice with retrovirus carrying a NOTCH1 activating mutant (L1601P-ΔP) and then transplanted these BM cells into irradiated recipient mice. In contrast to controls, mice transplanted with transduced Klf4-null BM cells developed T-ALL with significantly higher penetrance (Klf4 null 76.5% v.s. control 21.3%) and shorter latency (Klf4 null 93 days v.s. control 130 days). Interestingly, Klf4 heterozygous group shows similar survival kinetics as Klf4 null group, suggesting that Klf4 haploinsufficiency is enough to accelerate onset of leukemia. To investigate the effect of Klf4 deletion in established leukemia cells, we transplanted NOTCH1 L1601P-ΔP transduced BM cells from Klf4fl/fl; CreER+ mice to induce leukemia. Post-transplantation deletion of the Klf4 gene by tamoxifen administration was able to accelerate T-ALL development compared to mice injected with vehicle. On the cellular level, loss of KLF4 led to increased proliferation of leukemia cells as assessed by in vivo BrdU incorporation, which correlated with decreased levels of p21 protein. Limited dilution transplantation of primary leukemia cells into secondary recipients showed a 9-fold increase of leukemia initiating cells (LIC) frequency in Klf4null leukemia cells compared to controls, suggesting that KLF4 controls expansion of LIC in T-ALL.To elucidate molecular mechanism underlying KLF4 regulation in T-ALL cells, we performed microarray and ChIP-Seq in control and Klf4 null CD4+CD8+ leukemia cells. Combined analyses revealed 202 genes as KLF4 direct targets, of which 11 genes are also deregulated in human T-ALL cells by comparing with published microarray datasets. One of the top upregulated genes is Map2k7, which encodes a kinase upstream of the JNK pathway. Immunoblots in leukemia cells confirmed increased expression of MAP2K7 protein and enhanced phosphorylation of its downstream targets JNK and ATF2. To further investigate the role of JNK pathway in T-ALL, we tested JNK inhibitor SP600125 in human T-ALL cell lines (KOPTK1, DND41, CCRF-CEM, MOLT3). Interestingly, SP600125 showed dose-dependent cytotoxicity in all human T-ALL cell lines tested regardless of their NOTCH1 status.Overall our results showed for the first time that KLF4 functions as a tumor suppressor in T-ALL by regulating proliferation of leukemia cells and frequency of LIC. Additional study elucidated that KLF4 suppresses the JNK pathway via direct transcriptional regulation of MAP2K7. Moreover, the vulnerability of human T-ALL cell lines to JNK inhibition provides a novel target for future therapy in T-ALL patients. DisclosuresNo relevant conflicts of interest to declare.

CDX2 regulates Human Leukemic Cell Clonogenicity and in Vivo Repopulation Capacity

Objectives: The caudal-type homeobox (CDX) gene family has been mainly studied during early development for its role in axial elongation and antero-posterior patterning. More recently, CDX genes were shown to regulate embryonic hematopoiesis via downstream HOX genes and interactions with the WNT signaling pathway. The role of CDX genes in adult hematopoiesis is poorly understood and almost no data exists on human cells. Healthy bone marrow (BM) derived hematopoietic cells express low levels of CDX1 and CDX4 but lack CDX2 expression. However, CDX2 expression is found in >80% of human acute myeloid (AML) and lymphoid leukemia (ALL) and its induction in murine BM cells results in myeloid leukemia. Here, we explore the role of CDX2 in human healthy hematopoietic and leukemic cells.Methods: CDX2 expression was modulated via lentiviral treatment in human BM CD34+ and SKM-1, EOL-1 and NALM16 human leukemic cell lines. CDX2 knockdown experiments were performed using two different shRNAs against CDX2 to control for potential off-target effects. To generate cultures displaying a very strong knockdown, individual clones were generated from single transduced cells and analyzed also separately. Efficient modulation of gene and protein CDX2 expression was analyzed by qRT-PCR and respectively immunoblot analyses. CDX2-modified (overexpressing or knockdown) or control cells were subjected to growth, colony forming (CFU), cell cycle, flow cytometry and qRT-PCR assays and analyzed in vivo upon xenotransplantation in NOD/SCID/IL2Rγnull (NSG) mice. Human recombinant DKK-1 protein was supplemented in CFU assays to CDX2 knockdown and respectively control cells.Results: shRNA-mediated CDX2 knockdown performed on SKM-1 as well as EOL-1 cells strongly reduced clonogenic capacity in CFU assays while only slightly reducing growth. Consistently, proliferation, apoptosis sensitivity and cell cycle were not influenced by CDX2 downregulation in any of the three analyzed leukemic cell lines. Importantly, CDX2 knockdown SKM-1 as well as NALM16 cells transplanted into immunopermissive NSG mice showed profoundly suppressed in vivo leukemogenic properties compared to control cells. However, overexpression of the human CDX2 gene using an SFFV-promotor driven lentiviral vector in human healthy CD34+ bone marrow-derived and also in leukemic cells resulted in a G0/G1 cell cycle arrest, reducing in vitro growth and CFU formation. Consistent with these results and in contrast to previous results reported in mice, CDX2 overexpression did not confer in vitro serial replating capacity or in vivo leukemogenic properties to healthy human CD34+ cells.To further investigate the molecular mechanisms underlying these CDX2-mediated effects in human leukemia, we analyzed the expression of HOX and Wnt-pathway associated genes via qRT-PCR. Modulation of HOX gene expression was indeed observed upon CDX2 knockdown or overexpression. Previously reported repressive effects of CDX2 on Klf4 gene expression were confirmed. Surprisingly however, we found that CDX2 expression positively regulates the expression of the WNT-inhibitory molecule DKK-1 in both leukemic and healthy CD34+ stem/progenitor cells. Supplementation of DKK-1 was able to rescue the clonogenic capacity of CDX2 knockdown leukemic cells in CFU-assays while opposite effects were noted in control cells.Conclusion: Our data suggest that CDX2 regulates in vivo leukemogenesis by inducing clonogenic properties in human leukemic cells. Its downstream molecular pathways in human leukemic cells may include, next to Klf4, HOX gene family members and the Wnt-inhibitor DKK-1. Leukemic cells might use DKK-1 expression to fine-tune their Wnt-signaling activity to an optimal dosage required for leukemia initiation and growth. Work underway in our laboratory is further investigating this hypothesis. DisclosuresNo relevant conflicts of interest to declare.

340 BIOLOGICAL CHARACTERISTICS AND FUNCTIONAL CAPABILITY OF FELINE ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS

Chronic kidney disease is a major cause of mortality in cats (Boyd et al. 2008J. Vet. Intern. Med. 22, 1111–1117). Similarly, the black-footed cat (Felis nigripes; BFC) frequently suffers from kidney failure caused by amyloidosis (Terio et al. 2008 Vet. Pathol. 45, 393–400). Adipose tissue-derived mesenchymal stem cells (AMSC) are a valuable cell source in regenerative medicine for treating certain diseases, including those suffered by endangered species. In the domestic cat (DSH), AMSC have been isolated from subcutaneous (SQ; Quimby et al. 2011 J. Feline Med. Surg. 13, 418–426) and epididymal adipose tissue (Zhang et al. 2014 Stem Cell Rev. Rep. 10, 600–611). Whether AMSC isolated from visceral fat of the abdominal cavity (AB) have similar developmental potential has not been studied. In this study, we (1) compared the biological characteristics of DSH-AMSC isolated from AB and SQ adipose tissue, and (2) evaluated the functional capability of DSH and BFC-AMSC to differentiate into other cell types. The AB and SQ adipose tissues were harvested via laparoscopy or from an incision in the ventral abdomen, respectively. Tissues were digested with collagenase II (1 mg mL–1) at 37°C for 20 to 40 min with shaking at 150 rpm for 20 to 40 min. Cells from the stromal vascular fraction were cultured in DMEM-F12 medium with 12% fetal bovine serum under 5% CO2 in air at 38°C. Results showed that AB biopsies were smaller (1.2 ± 0.2 g) than that of SQ biopsies (3.6 ± 0.7 g). The mean number of nucleated cells per gram from AB biopsies (0.6 to 22 × 106) was similar to that of SQ biopsies (0.4 to 24 ×106). The cell-doubling numbers (days) per passage (P1 to P5) in both cell types remained constant (0.9 to 2.6), but SQ-AMSC at P5 required more cell doublings (4.5 ± 2.1) to reach 50% confluence. The AB-AMSC showed more colony-forming units (CFU; 7.0%) after 8 to 10 days of seeding at 8000 per cm2 than did SQ-AMSC (1.5% CFU). The SQ-AMSC did not form colonies at cell densities below 4000 per cm2. However, AB-AMSC colony formation only substantially decreased when the cell densities were below 1000 per cm2 (0.1%). Flow cytometry analysis revealed higher percentages of CD90+ (92%), CD105+ (80%), and CD146+ (17%) cells in AB-AMSC than in SQ-AMSC (77, 57, and 9%, respectively). Both AB and SQ-AMSC showed negative expressions of CD14–, CD45–, CD73–, and HLA-DR–. Gene expression analysis revealed that pluripotent genes Nanog, KLF4, Oct-4, and proto-oncogene C-Myc were expressed by both cell types, while Sox2 was not expressed in either type of AMSC. Under appropriate stimuli, DSH- and BFC-AMSC demonstrated differentiation potential towards adipogenic, osteogenic, chondrogenic, and neurogenic lineages. The AMSC from both species were less responsive towards osteogenesis than adipogenesis, and BFC cells had more capability to differentiate towards chondrocytes. These results suggest that the defined AMSC population (regardless of site of collection) could potentially be employed as a therapeutic agent for diseased or injured felids, both domestic and endangered.

Abstract 17827: Klf5 Mediates Glucose-driven Changes ofCcardiac Pparα in Diabetes

Krüppel-like factors (KLF) affect metabolism. Lipopolysaccharide-induced sepsis reduced cardiac PPARα and increased KLF5 (8-fold) more than any cardiac KLF isoform detected by whole genome microarray analysis. In silico analysis of ppara gene promoter predicted two KLF5 binding sites that overlap with c-Jun (AP-1) binding sites: -792/-772 bp and -719/-698 bp. Infection of a mouse cardiomyocyte cell line (HL-1) with adenovirus expressing constitutively active c-Jun reduced, while Ad-KLF5 increased PPARα mRNA in a dose-dependent manner. Chromatin immunoprecipitation (ChIP) showed that c-Jun binds both -792/-772 bp and -719/-698 on ppara promoter while KLF5 binds on -792/-772 bp. ChIP on LPS-treated HL-1 cells showed that c-Jun binding on -792/-772 bp prohibits KLF5 binding. We generated a cardiomyocyte-specific KLF5 knockout mouse (αMHC-KLF5-/-), which had 50% normal cardiac function. Cardiomyocyte-specific KLF5 ablation reduced PPARα (50%) and several fatty acid metabolism-associated genes such as CD36 (40%), LpL (20%), PGC1α (45%), AOX (28%) and Cpt1 (45%). As PPARα regulates cardiac fatty acid metabolism, we tested whether cardiac KLF5 is modulated in diabetes, when cardiac PPARα and lipid changes occur. I.p. injection of streptozotocin (STZ) in C57BL/6 mice increased plasma glucose (2.9-fold) and reduced cardiac KLF5 and PPARα gene expression; similar to STZ-treated rats but unlike what had been found in a different mouse strain (C57BL/6 x DBA2) treated with STZ. Treatment of HL-1 cells with increased glucose-containing medium (1 mg/ml) reduced KLF5 (80%) and PPARα (65%), as well as fatty acid metabolism markers, such as AOX (85%), Cpt1β (70%), LCAD (80%) and VLCAD (85%). On the other hand GLUT1 and GLUT4 were increased (30% and 20%) and PDK4 was reduced (65%) indicating increased glucose utilization. A model of non-insulin dependent hyperglycemia (ob/ob mice) had reduced cardiac KLF5 (60%) and PPARα (65%). Correction of hyperglycemia in STZ-treated C57BL/6 mice by pharmacological (dapagliflozin) or antisense oligonucleotide inhibition of kidney’s sodium glucose transporter 2 (SGLT2), restored KLF5 and PPARα gene expression. Thus, KLF5 is a transcriptional regulator of cardiac PPARα that is driven by changes in plasma glucose levels

A Cancer Reprogramming Method Using MicroRNAs as a Novel Therapeutic Approach against Colon Cancer: Research for Reprogramming of Cancer Cells by MicroRNAs.

We previously generated induced pluripotent stem cells by reprograming adipose stem cells through the introduction of microRNAs targeting four transcription factors (Oct3/4, Sox2, c-Myc, and Klf4). In this study, we aimed to reprogram cancer cells using microRNAs to explore their therapeutic potential. Mature microRNAs (mir-302a-d, 369-3p and 5p, and mir-200c, as needed) were introduced into colon cancer cells (DLD-1, RKO, and HCT116) using lipofection. The transfected cells exhibited an embryonic stem cell-like morphology and expressed the undifferentiated marker genes Nanog, Oct3/4, SOX2, and Klf4, as well as tumor-related antigen-1-60. These cells expressed neurogenic or adipogenic markers, indicating that reprogramming of the cancer cells was partially successful. Moreover, we found that miRNA-expressing DLD-1 cells showed low proliferative activity in vitro and in vivo, accompanied by increased expression of the tumor suppressor genes p16 (ink4a) and p21 (waf1) . miRNA-expressing DLD-1 cells also exhibited enhanced sensitivity to 5-fluorouracil, possibly through the downregulation of multidrug-resistant protein 8. The reprogrammed cells from DLD-1, RKO, and HCT116 cells exhibited reduced c-Myc expression, in contrast to the high c-Myc expression in the induced pluripotent cancer cells that were generated using four transcription factors. Our cancer reprogramming method employing simple lipofection of mature microRNAs is safe and well suited for clinical application, because it avoids integration of exogenous genes into the host genome and allows escape from augmentation of c-Myc gene expression.

Malignant meningioma with adenocarcinoma-like metaplasia: demonstration of intestinal phenotype.

Meningiomas show a diverse histopathologic appearance, often referred to as metaplastic changes; however, adenocarcinoma-like metaplasia is an extremely rare condition. Here, we present a novel case. A dura-based bulky mass located in the right frontotemporal region was identified radiologically in an 83-year-old woman. The tumor, yellow to ash-gray in color, was subtotally removed. Histopathological examination revealed robust adenocarcinoma-like structures within a conventional meningothelial neoplasm. Meningioma elements showed a WHO grade I to III histology. Morphological and immunophenotypic transition between meningothelial and columnar epithelial cells was confirmed on detailed observation. It was of note that the adenocarcinomatous components shared an immunophenotype with intestinal epithelium, expressing CDX2, MUC2 and cytokeratin 20. The present case could be differentiated from secretory meningioma based on distinct cellular atypia, lack of intracytoplasmic lumina and pseudosammoma bodies, and the intact status of the KLF4 gene. In addition, the morphological and immunophenotypic transition excluded the possibility of metastatic carcinoma within meningioma. This is the first reported case of meningioma with adenocarcinoma-like metaplasia harboring an intestinal immunophenotype.

WWOX suppresses KLF5 expression and breast cancer cell growth.

The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor in a variety of cancers, including breast cancer. Reduced WWOX expression is associated with the basal-like subtype and a relatively poor disease-free survival rate among breast cancer patients. Though several WWOX partners have been identified, the functional mechanisms of WWOX's role in cancers have not been fully addressed to date. In the current study, we found WWOX suppresses expression of KLF5-an oncogenic transcription factor-at protein level, and suppresses cancer cell proliferation in both bladder and breast cancer cell lines. Furthermore, we demonstrated that WWOX physically interacts with KLF5 via the former's WW domains and the latter's PY motifs. Interestingly, we found the expression of WWOX negatively correlates with KLF5 expression in a panel of breast cancer cell lines. Taken together, we conjecture that WWOX may suppress cancer cell proliferation partially by reducing the expression of KLF5.

Abstract 3016: Characterization of cancer stem-like cells derived from mouse induced pluripotent stem cells transformed by tumor-derived exosomes/microvesicles

Abstract It is widely accepted that the microenvironment/niche exert genetic and/or epigenetic effects on stem cells. Current studies have demonstrated that an aberrant microenvironment may contribute to the genesis of cancer stem cells (CSCs). In the case of cancer, growing evidence indicate a cancerous niche can play an active role in the regulation of tumor cell maintenance and progression through exosomes/microvesicles-based intercellular communication. It has not been reported, however, whether this vesicle-mediated communication induces the malignant transformation of normal stem cells/differentiating progenitors, result in CSC generation. Recentry, we have reported that the conditioned medium derived from the mouse Lewis Lung Carcinoma (LLC) cell line can convert mouse induced pluripotent stem cells (miPSCs) into CSCs. Here, we investigated the contribution of tumor-derived exosomes/microvesicles (TEMVs) that are secreted from LLC cells to induce the transformation of miPSCs into CSCs. The differentiating miPSCs were exposed to TEMVs derived from LLC cells for 4 weeks. We used Nanog-GFP miPS, in which GFP expression is under control of Nanog promoter, indicative of undifferentiation state. During this period, we observed the reemergence of the GFP-positive colonies, whereas most of cells were differentiated and GFP-negative without TEMV-treatment. The resultant TEMVs treated cells (miPS-LLCemv) expressed Nanog and Oct3/4 proteins comparable to miPSCs assessed by Western blotting. The sphere formation of the miPS-LLCemv cells in suspension culture indicated the selfrenewal capacity of the miPS-LLCemv cells. When the miPSLLCemv cells were subcutaneously allografted into immunodeficient mice, malignant tumors with extensive angiogenesis developed. Phathological features such as vementin, Ki67, PPAR gamma expression, and accumulation of lipid droplets in tumor revealed these tumors were liposarcomas. Tumor cells established from liposarcomas showed the capacity of spheroid formation in suspension culture and the capacity of differntiation into adipocytes and endothelial cells. These results indicate that miPS-LLCemv cells and established tumor cells possess CSC properties. Interestingly, these cells expressed Sox2 and Klf4 genes significantly higher than parental miPS cells, might be related to selfrenewal and addipogenesis. Thus, we concluded that TEMVs should have the potential to induce CSC properties in normal stem cells. Citation Format: Yan Ting, Junko Masuda, Akifumi Mizutani, Ling Chen, Tsukasa Shigehiro, Shuichi Matsuda, Tomonari Kasai, Takayuki Kudoh, Hiroshi Murakami, Mary J.C. Hendrix, Luigi Strizzi, David S. Salomon, Li Fu, Masaharu Seno. Characterization of cancer stem-like cells derived from mouse induced pluripotent stem cells transformed by tumor-derived exosomes/microvesicles. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3016. doi:10.1158/1538-7445.AM2014-3016

Abstract 1064: IL-6 secreted by tumor-associated macrophages promotes DLBCL chemoresistance via expansion of the CSCs population

Abstract Cancer stem-like cells (CSCs), a highly tumorigenic subpopulation of cells found in developmentally diverse tumors, are highly resistant to chemotherapeutic drugs. Tumor-associated macrophages (TAMs) are known to promote progression of tumors and chemoresistance of DLBCL (Diffuse Large B cell Lymphoma). Here, we report that TAMs promote a CSC-like phenotype in DLBCL cells. This was characterized by increased ALDH1A1, Sox-2, Oct-4, Nanog and KLF-4 gene expression. In in vitro studies, we found that co-culture with monocyte cell lines enhanced the chemoresistance and CSC-like phenotypes of DLBCL cells. RNA-seq revealed high levels of IL-6 expression following co-culture with DLBCL, and in fact IL-6 was sufficient to promote the chemoresistance and CSC-like phenotypes of DLBCL cells. Surprisingly, patients exhibited elevated IL-6 after chemotherapy relative to patients pre-chemotherapy, suggesting that chemotherapy may elicit a host response favoring chemoresistance. High levels of serum IL-6 were associated with poor prognosis. In agreement with these results, tumor infiltration by TAMs directly correlated with disease severity. Therefore, IL-6 may be a prognostic marker for outcome in DLBCL. Citation Format: Nan Bai. IL-6 secreted by tumor-associated macrophages promotes DLBCL chemoresistance via expansion of the CSCs population. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1064. doi:10.1158/1538-7445.AM2014-1064

Induced pluripotent stem cells can be reprogrammed from human umbilical cord mesenchymal cells by six transcription factors

Objective To establish and identify the induced pluripotent stem cell(iPSC) line reprogrammed from human umbilical cord mesenchymal cells(HuMSCs). Methods HuMSCs were cultured by adhesion method, and OCT4, SOX2, KLF4, c-Myc, NANOG, LIN-28 were transfected into HuMSCs with lentiviral victor to reprogramme HuMSCs into iPSC.Morphological observation, pluripotency genes(SOX2, TDGF1, THY-1, OCT4, REX1 and TERF1) expression, alkaline phosphatase detection, karyotype analysis, embryonic stem cells(ESC) specific proteins(NANOG, OCT4, SSEA-4, TRA-1-81) immunofluorescence staining, differentiated into teratomas in vivo(inject the iPSC into SCID mice) and embryniod bodies in vitro were performed to exam the pluripotency of the iPSC. Results Four days after being infected by lentivirus, the HuMSCs became round-shape; 10 days after infection, some embryonic stem(ESC)-like colonies appeared.Fourteen days after infection, picked up the regularly shaped colonies and cultured several passages.About 1.25% HuMSCs were reprogrammed into iPSC.The iPSC presented clone-like growth like ESC.All the cells were positive to alkaline phosphatase staining and expressed the pluripotency genes.The iPSC also expressed the ESC specific proteins, and karyotype analysis showed normal chromosome caryotype(46, XY). Furthermore, the iPSC could form embryoid bodies in vitro, expressed alpha fetoprotein(AFP), smooth muscle actin(SMA) and β-tubulin.The iPSC could alsoform teratomas in vivo. Conclusion OCT4, SOX2, KLF4, c-Myc, NANOG, LIN-28 can reprogram HuMSCs into iPSC efficiently. Key words: Umbilical cord mesenchymal stem cells; Induced pluripotent stem cells; Transcription factor; Lentiviral vector

16IN - Meningioma: what Can we Learn from the Biology for Precision Medicine?

ABSTRACT Meningiomas differ considerably by their histological appearance, location, biological behavior, recurrence rate, and overall survival. The most frequent alteration found in sporadic meningioma corresponds to biallelic NF2 inactivation. Besides the central role of NF2, other alternative genetic alterations have been recently identified in meningioma initiation. The AKT1E17K mutation was found in 12%, predominantly in benign skull base meningiomas, occurring frequently together with mutations in the TRAF7 gene. TRAF7 mutations are exclusively of NF2 mutations and occur in about 24% of meningiomas. In addition, meningiomas with TRAF7 mutations are almost always characterized by recurrent mutation in the KLF4 gene (co-regulator of the bradykinin B2 receptor). Another non-NF2-associated mutation of meningiomas affects the SMO (Smoothened) gene which is a member of the hedgehog pathway. SMO mutations occur in 5% of grade I meningiomas and are restricted to the midline skull base. Meningioma progression and recurrence are linked to chromosome instability, CDKN2AB genes on chromosome 9p being suspected to be important. Mouse models that reproduce human meningioma tumorigenesis have been developed. Two conditional Nf2 knockout models have shown that Nf2 inactivation in leptomeningeal cells of mice is sufficient to induce meningiomas. Additional deletion of Cdkn2ab, together with Nf2 inactivation, results in increased meningioma frequency, as well as development of grade II or grade III meningiomas. All relevant growth factor receptors/kinases have been described to be expressed in meningiomas, including epidermal growth factor receptor (EGFR), platelet-derived growth factor beta receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), and insulin-like growth factor receptor (IGFR). A new mouse model surexpressing PDGF-beta has been recently developed showing an increase in frequency and malignancy in association with Nf2 and Cdkn2ab inactivation. In conclusion, increasing knowledge on the molecular mechanisms of meningioma initiation and development provides target that could be relevant to develop new therapeutic approaches, particularly in surgery and radiotherapy-refractory meningiomas. Disclosure: All authors have declared no conflicts of interest.

Trichostatin A-induced apoptosis is mediated by Kruppel-like factor 4 in ovarian and lung cancer.

The istone deacetylase (HDAC) inhibitor trichostatin A (TSA) is known to mediate the regulation of gene expression and anti proliferation activity in cancer cells. Kruppel-like factor 4 (klf4) is a zinc finger- containing transcription factor of the SP/KLF family, that is expressed in a variety of tissues and regulates cell proliferation, differentiation, tumorigenesis, and apoptosis. It may either either function as a tumor suppressor or an oncogene depending on genetic context of tumors. In this study, we tested the possibility that TSA may increase klf4 expression and cancer cell growth inhibition and apoptosis in SKOV-3 and A549 cells. The cytotoxicity of TSA was determined using the MTT assay test, while klf4 gene expression was assessed by real time PCR and to ability of TSA to induce apoptosis using a Vybrant Apoptosis Assay kit. Our results showed that TSA exerted dose and time dependent cytotoxicity effect on SKOV-3 and A549 cells. Moreover TSA up-regulated klf4 expression. Flow cytometric analysis demonstrated that apoptosis was increased after TSA treatment. Taken together, this study showed that TSA increased klf4 expression in SKOV3 and A549 cell lines, consequently, klf4 may played a tumor-suppressor role by increasing both cell growth inhibition and apoptosis. This study sheds light on the details of molecular mechanisms of HDACI-induced cell cycle arrest and apoptosis.

Krüppel-like transcription factors KLF1 and KLF2 have unique and coordinate roles in regulating embryonic erythroid precursor maturation.

The Krüppel-like transcription factors KLF1 and KLF2 are essential for embryonic erythropoiesis. They can partially compensate for each other during mouse development, and coordinately regulate numerous erythroid genes, including the β-like globins. Simultaneous ablation of KLF1 and KLF2 results in earlier embryonic lethality and severe anemia. In this study, we determine that this anemia is caused by a paucity of blood cells, and exacerbated by diminished β-like globin gene expression. The anemia phenotype is dose-dependent, and, interestingly, can be ameliorated by a single copy of the KLF2, but not the KLF1 gene. The roles of KLF1 and KLF2 in maintaining normal peripheral blood cell numbers and globin mRNA amounts are erythroid cell-specific. Mechanistic studies led to the discovery that KLF2 has an essential function in erythroid precursor maintenance. KLF1 can partially compensate for KLF2 in this role, but is uniquely crucial for erythroid precursor proliferation through its regulation of G1- to S-phase cell cycle transition. A more drastic impairment of primitive erythroid colony formation from embryonic progenitor cells occurs with simultaneous loss of KLF1 and KLF2 than with loss of a single factor. KLF1 and KLF2 coordinately regulate several proliferation-associated genes, including Foxm1. Differential expression of FoxM1, in particular, correlates with the observed KLF1 and KLF2 gene dosage effects on anemia. Furthermore, KLF1 binds to the FoxM1 gene promoter in blood cells. Thus KLF1 and KLF2 coordinately regulate embryonic erythroid precursor maturation through the regulation of multiple homeostasis-associated genes, and KLF2 has a novel and essential role in this process.