Grainyhead-like 2 interacts with noggin to regulate tissue fusion in mouse.

Defective tissue fusion during mammalian embryogenesis results in congenital anomalies, such as exencephaly, spina bifida and cleft lip and/or palate. The highly conserved transcription factor grainyhead-like 2 (Grhl2) is a crucial regulator of tissue fusion, with mouse models lacking GRHL2 function presenting with a fully penetrant open cranial neural tube, facial and abdominal clefting (abdominoschisis), and an open posterior neuropore. Here, we show that GRHL2 interacts with the soluble morphogen protein and bone morphogenetic protein (BMP) inhibitor noggin (NOG) to impact tissue fusion during development. The maxillary prominence epithelium in embryos lacking Grhl2 shows substantial morphological abnormalities and significant upregulation of NOG expression, together with aberrantly distributed pSMAD5-positive cells within the neural crest cell-derived maxillary prominence mesenchyme, indicative of disrupted BMP signalling. Reducing this elevated NOG expression (by generating Grhl2-/-;Nog+/- embryos) results in delayed embryonic lethality, partial tissue fusion rescue, and restoration of tissue form within the craniofacial epithelia. These data suggest that aberrant epithelial maintenance, partially regulated by noggin-mediated regulation of BMP-SMAD pathways, may underpin tissue fusion defects in Grhl2-/- mice.

STAT5 activation promotes progression and chemotherapy resistance in early T-cell precursor acute lymphoblastic leukemia.

Interleukin-7 (IL-7) supports the growth and chemoresistance of T-cell acute lymphoblastic leukemia (T-ALL), particularly the early T-cell precursor subtype (ETP-ALL), which frequently has activating mutations of IL-7 signaling. Signal transducer and activator of transcription (STAT5) is an attractive therapeutic target because it is almost universally activated in ETP-ALL, even in the absence of mutations of upstream activators such as the IL-7 receptor (IL-7R), Janus kinase, and Fms-like tyrosine kinase 3 (FLT3). To examine the role of activated STAT5 in ETP-ALL, we have used a Lmo2-transgenic (Lmo2Tg) mouse model in which we can monitor chemoresistant preleukemia stem cells (pre-LSCs) and leukemia stem cells (LSCs) that drive T-ALL development and relapse following chemotherapy. Using IL-7R-deficient Lmo2Tg mice, we show that IL-7 signaling was not required for the formation of pre-LSCs but essential for their expansion and clonal evolution into LSCs to generate T-ALL. Activated STAT5B was sufficient for the development of T-ALL in IL-7R-deficient Lmo2Tg mice, indicating that inhibition of STAT5 is required to block the supportive signals provided by IL-7. To further understand the role of activated STAT5 in LSCs of ETP-ALL, we developed a new transgenic mouse that enables T-cell specific and doxycycline-inducible expression of the constitutively activated STAT5B1∗6 mutant. Expression of STAT5B1∗6 in T cells had no effect alone but promoted expansion and chemoresistance of LSCs in Lmo2Tg mice. Pharmacologic inhibition of STAT5 with pimozide-induced differentiation and loss of LSCs, while enhancing response to chemotherapy. Furthermore, pimozide significantly reduced leukemia burden in vivo and overcame chemoresistance of patient-derived ETP-ALL xenografts. Overall, our results demonstrate that STAT5 is an attractive therapeutic target for eradicating LSCs in ETP-ALL.

YBX1 integration of oncogenic PI3K/mTOR signalling regulates the fitness of malignant epithelial cells.

In heterogeneous head and neck cancer (HNC), subtype-specific treatment regimens are currently missing. An integrated analysis of patient HNC subtypes using single-cell sequencing and proteome profiles reveals an epithelial-mesenchymal transition (EMT) signature within the epithelial cancer-cell population. The EMT signature coincides with PI3K/mTOR inactivation in the mesenchymal subtype. Conversely, the signature is suppressed in epithelial cells of the basal subtype which exhibits hyperactive PI3K/mTOR signalling. We further identify YBX1 phosphorylation, downstream of the PI3K/mTOR pathway, restraining basal-like cancer cell proliferation. In contrast, YBX1 acts as a safeguard against the proliferation-to-invasion switch in mesenchymal-like epithelial cancer cells, and its loss accentuates partial-EMT and in vivo invasion. Interestingly, phospho-YBX1 that is mutually exclusive to partial-EMT, emerges as a prognostic marker for overall patient outcomes. These findings create a unique opportunity to sensitise mesenchymal cancer cells to PI3K/mTOR inhibitors by shifting them towards a basal-like subtype as a promising therapeutic approach against HNC.

Identification of a Novel GRHL3/HOPX/Wnt/ß-Catenin Proto-oncogenic Axis in Squamous Cell Carcinoma of the Esophagus.

BACKGROUND & AIMS: Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with a poor long-term prognosis. The molecular mechanisms underlying the initiation and progression of this tumor are largely unknown. The transcription factor GRHL3 functions as a potent tumor suppressor in SCC of skin, head, and neck. This study aims to determine whether GRHL3 also plays a role in the homeostasis of the esophageal epithelium and in the development of ESCC. METHODS: The effects of Grhl3 deletion on squamous epithelial homeostasis in embryos and adult mice were examined using immunohistochemistry, transmission electron microscopy, and real-time polymerase chain reaction. The conditionally deleted mice were subsequently used to determine susceptibility to ESCC. Whole-transcriptome sequencing (RNA-seq) was performed on ESCC in wild-type and Grhl3 deleted animals. To decipher the signaling pathways, real-time polymerase chain reaction, immunohistochemistry, analysis of chromatin immunoprecipitation sequencing, chromatin immunoprecipitation-polymerase chain reaction, and RNA seq datasets were used. Primary human samples were used to validate the findings in the mouse model. RESULTS: Loss of Grhl3 perturbs the proliferation-differentiation balance in the esophageal epithelium, thereby increasing the susceptibility to esophageal carcinogenesis in adult mice. Grhl3 imparts its tumor suppressor function by regulating the expression of HOPX. We have identified the Wnt/ß-catenin pathway as the downstream effectors of GRHL3 and HOPX through our integrated approach using patient-derived ESCC samples and mouse models. CONCLUSIONS: GRHL3 conveys its tumor suppressor function in ESCC through regulating its target gene HOPX, which limits Wnt/ß-catenin signaling. Targeted therapies to inhibit this pathway could be a potential treatment strategy for ESCC patients with reduced GRHL3 expression.

Inhibition of retinoic acid signaling impairs cranial and spinal neural tube closure in mice lacking the Grainyhead-like 3 transcription factor.

Neural tube closure is a dynamic morphogenic event in early embryonic development. Perturbations of this process through either environmental or genetic factors induce the severe congenital malformations known collectively as neural tube defects (NTDs). Deficiencies in maternal folate intake have long been associated with NTDs, as have mutations in critical neurulation genes that include the Grainyhead-like 3 (Grhl3) gene. Mice lacking this gene exhibit fully penetrant thoraco-lumbo-sacral spina bifida and a low incidence of exencephaly. Previous studies have shown that exposure of pregnant mice carrying hypomorphic Grhl3 alleles to exogenous retinoic acid (RA) increases the incidence and severity of NTDs in their offspring. Here, we demonstrate that inhibition of RA signaling using a high affinity pan-RA receptor antagonist administered to pregnant mice at E7.5 induces fully penetrant exencephaly and more severe spina bifida in Grhl3-null mice. Later administration, although prior to neural tube closure has no effect. Similarly, blockade of RA in the context of reduced expression of Grhl2, a related gene known to induce NTDs, has no effect. Taken together, these findings provide new insights into the complexities of the interplay between RA signaling and Grhl3-induced neurulation.

Grainyhead-like transcription factors: guardians of the skin barrier.

There has been selective pressure to maintain a skin barrier since terrestrial animals evolved 360 million years ago. These animals acquired an unique integumentary system with a keratinized, stratified, squamous epithelium surface barrier. The barrier protects against dehydration and entry of microbes and toxins. The skin barrier centres on the stratum corneum layer of the epidermis and consists of cornified envelopes cemented by the intercorneocyte lipid matrix. Multiple components of the barrier undergo cross-linking by transglutaminase (TGM) enzymes, while keratins provide additional mechanical strength. Cellular tight junctions also are crucial for barrier integrity. The grainyhead-like (GRHL) transcription factors regulate the formation and maintenance of the integument in diverse species. GRHL3 is essential for formation of the skin barrier during embryonic development, whereas GRHL1 maintains the skin barrier postnatally. This is achieved by transactivation of Tgm1 and Tgm5, respectively. In addition to its barrier function, GRHL3 plays key roles in wound repair and as an epidermal tumour suppressor. In its former role, GRHL3 activates the planar cell polarity signalling pathway to mediate wound healing by providing directional migration cues. In squamous epithelium, GRHL3 regulates the balance between proliferation and differentiation, and its loss induces squamous cell carcinoma (SCC). In the skin, this is mediated through increased expression of MIR21, which reduces the expression levels of GRHL3 and its direct target, PTEN, leading to activation of the PI3K-AKT signalling pathway. These data position the GRHL family as master regulators of epidermal homeostasis across a vast gulf of evolutionary history.

Il y a eu une pression de sélection pour maintenir la barrière cutanée depuis l'évolution des animaux terrestres pendant 360 millions d'années. Ces animaux ont acquis un système tégumentaire unique avec un épithélium squameux, stratifié, kératinisé comme barrière de surface. La barrière protège contre la déshydratation et l'entée de microbes et de toxines. La barrière cutanée est centrée sur la couche du stratum corneum de l'épiderme et consiste en des enveloppes cimentées par une matrice lipidique intercornéocytaire. Les composants multiples de la barrière subissent des remaniements par les enzymes transglutaminases (TGM) tandis que la kératine fournit un soutien mécanique supplémentaire. Les jonctions serrées cellulaires jouent aussi un rôle crucial pour l'intégrité de barrière. Les facteurs de transcriptions GRHL (grainyhead-like) régulent la formation et le maintien du tégument dans différentes espèces. GRHL3 est essentielle pour la formation de la barrière cutanée au cours du développement embryonnaire tandis que GRHL1 maintient la barrière cutanée après la naissance. Ceci est permis respectivement par transactivation de Tgm1 et Tgm5. En plus de cette fonction barrière, GRHL3 joue un rôle clé dans la cicatrisation et en tant que suppresseur de tumeur épidermique. Dans ses rôles principaux, GRHL3 active la voie de signal de polarité cellulaire plane pour soutenir la cicatrisation en fournissant des repaires directionnels de migration. Dans les épithéliums squameux, GRHL3 régule la balance entre prolifération et différentiation, et sa perte induit le carcinome épidermoïde (SCC). Dans la peau ceci est médié par une augmentation de l'expression de MIR21, qui réduit le niveau d'expression de GRHL3 et sa cible directe, PTEN, menant à l'activation de la voie de signal PI3K-AKT. Ces données positionnent la famille GRHL comme régulatrice majeure de l'homéostasie épidermique à travers le vaste gouffre de l'histoire de l'évolution.

Ha habido una presión selectiva para mantener una barrera cutánea desde que los animales terrestres evolucionaron hace 360 ​​millones de años. Estos animales adquirieron un sistema tegumentario único con una barrera superficial de epitelio escamoso estratificado queratinizado. La barrera protege contra la deshidratación y la entrada de microbios y toxinas. La barrera cutánea se centra en la capa de estrato córneo de la epidermis y consta de membranas cornificadas cementadas por una matriz lipídica intercorneocitaria. Múltiples componentes de la barrera se unen por la actividad de enzimas transglutaminasas (TGM), mientras que las queratinas proporcionan resistencia mecánica adicional. Las uniones celulares estrechas también son cruciales para la integridad de la barrera. Los factores de transcripción similares a grainyhead (cabeza granulada) (GRHL) regulan la formación y mantenimiento del tegumento en diversas especies. GRHL3 es esencial para la formación de la barrera cutánea durante el desarrollo embrionario, mientras que GRHL1 mantiene la barrera cutánea postnatal. Esto se logra mediante la transactivación de Tgm1 y Tgm5, respectivamente. Además de su función de barrera, GRHL3 juega un papel clave en la reparación de heridas y como supresor de tumores epidérmicos. En su función de cicatrización, GRHL3 activa la vía de señalización de la polaridad celular plana para mediar en la cicatrización de heridas proporcionando señales de migración direccional. En el epitelio escamoso, GRHL3 regula el equilibrio entre la proliferación y la diferenciación, y su pérdida induce el carcinoma de células escamosas (SCC). En la piel, esto está mediado por una mayor expresión de MIR21, que reduce los niveles de expresión de GRHL3 y su sustrato directo, PTEN, lo que lleva a la activación de la vía de señal intracelular PI3K-AKT. Estos datos colocan la familia de factores de transcripción GRHL como reguladores críticos de la homeostasis epidérmica a través de una extensa historia evolutiva.

Tem havido uma pressão seletiva para manter a barreira cutânea desde a evolução dos animais terrestres há 360 milhões de anos. Estes animais adquiriram um sistema tegumentar único com uma barreira de superfície escamosa, estratificada e queratinizada. A barreira protege contra a desidratação e entrada de micróbios e toxinas. A barreira cutânea é centrada na camada do estrato córneo da epiderme e consiste em envelopes cornificados revestidos pela matriz lipídica intercorneocítica. Vários componentes da barreira sofrem ligação cruzada por enzimas transglutaminase (TGM), enquanto as queratinas fornecem resistência mecânica adicional. As junções celulares também são cruciais para a integridade da barreira. Os fatores de transcrição do tipo grainyhead (GRHL) regulam a formação e manutenção do tegumento em diversas espécies. GRHL3 é essencial para a formação da barreira cutânea durante o desenvolvimento embrionário, enquanto GRHL1 mantém a barreira cutânea pós-natal. Isso é obtido pela transativação de Tgm1 e Tgm5, respectivamente. Além de sua função de barreira, GRHL3 desempenha papéis importantes no reparo de feridas e como supressor de tumor epidérmico. Em sua função anterior, GRHL3 ativa a via de sinalização de polaridade celular planar para mediar a cicatrização de feridas, fornecendo pistas de migração direcional. No epitélio escamoso, o GRHL3 regula o equilíbrio entre a proliferação e a diferenciação, e sua perda induz o carcinoma de células escamosas (CCE). Na pele, isso é mediado pelo aumento da expressão de MIR21, que reduz os níveis de expressão de GRHL3 e seu alvo direto, PTEN, levando à ativação da via de sinalização PI3K-AKT. Esses dados posicionam a família GRHL como reguladores mestres da homeostase epidérmica em um vasto abismo da história evolutiva.

Small molecule inhibition of Dynamin-dependent endocytosis targets multiple niche signals and impairs leukemia stem cells.

Intensive chemotherapy for acute leukemia can usually induce complete remission, but fails in many patients to eradicate the leukemia stem cells responsible for relapse. There is accumulating evidence that these relapse-inducing cells are maintained and protected by signals provided by the microenvironment. Thus, inhibition of niche signals is a proposed strategy to target leukemia stem cells but this requires knowledge of the critical signals and may be subject to compensatory mechanisms. Signals from the niche require receptor-mediated endocytosis, a generic process dependent on the Dynamin family of large GTPases. Here, we show that Dynole 34-2, a potent inhibitor of Dynamin GTPase activity, can block transduction of key signalling pathways and overcome chemoresistance of leukemia stem cells. Our results provide a significant conceptual advance in therapeutic strategies for acute leukemia that may be applicable to other malignancies in which signals from the niche are involved in disease progression and chemoresistance.

Inactivation of Zeb1 in GRHL2-deficient mouse embryos rescues mid-gestation viability and secondary palate closure.

Cleft lip and palate are common birth defects resulting from failure of the facial processes to fuse during development. The mammalian grainyhead-like (Grhl1-3) genes play key roles in a number of tissue fusion processes including neurulation, epidermal wound healing and eyelid fusion. One family member, Grhl2, is expressed in the epithelial lining of the first pharyngeal arch in mice at embryonic day (E)10.5, prompting analysis of the role of this factor in palatogenesis. Grhl2-null mice die at E11.5 with neural tube defects and a cleft face phenotype, precluding analysis of palatal fusion at a later stage of development. However, in the first pharyngeal arch of Grhl2-null embryos, dysregulation of transcription factors that drive epithelial-mesenchymal transition (EMT) occurs. The aberrant expression of these genes is associated with a shift in RNA-splicing patterns that favours the generation of mesenchymal isoforms of numerous regulators. Driving the EMT perturbation is loss of expression of the EMT-suppressing transcription factors Ovol1 and Ovol2, which are direct GRHL2 targets. The expression of the miR-200 family of microRNAs, also GRHL2 targets, is similarly reduced, resulting in a 56-fold upregulation of Zeb1 expression, a major driver of mesenchymal cellular identity. The critical role of GRHL2 in mediating cleft palate in Zeb1-/- mice is evident, with rescue of both palatal and facial fusion seen in Grhl2-/-;Zeb1-/- embryos. These findings highlight the delicate balance between GRHL2/ZEB1 and epithelial/mesenchymal cellular identity that is essential for normal closure of the palate and face. Perturbation of this pathway may underlie cleft palate in some patients.

Interrogating the Grainyhead-like 2 (Grhl2) genomic locus identifies an enhancer element that regulates palatogenesis in mouse.

The highly-conserved Grainyhead-like (Grhl) transcription factors are critical regulators of embryogenesis that regulate cellular survival, proliferation, migration and epithelial integrity, especially during the formation of the craniofacial skeleton. Family member Grhl2 is expressed throughout epithelial tissues during development, and loss of Grhl2 function leads to significant defects in neurulation, abdominal wall closure, formation of the face and fusion of the maxilla/palate. Whereas numerous downstream target genes of Grhl2 have been identified, very little is known about how this crucial developmental transcription factor itself is regulated. Here, using in silico and in utero expression analyses and functional deletion in mice, we have identified a novel 2.4 â€‹kb enhancer element (mm1286) that drives reporter gene expression in a pattern that strongly recapitulates endogenous Grhl2 in the craniofacial primordia, modulates Grhl2 expression in these tissues, and augments Grhl2-mediated closure of the secondary palate. Deletion of this genomic element, in the context of inactivation of one allele of Grhl2 (through generation of double heterozygous Grhl2+/-;mm1286+/- mice), results in a significant predisposition to palatal clefting at birth. Moreover, we found that a highly conserved 325 bp region of mm1286 is both necessary and sufficient for mediating the craniofacial-specific enhancer activity of this region, and that an extremely well-conserved 12-bp sequence within this element (CTGTCAAACAGGT) substantially determines full enhancer function. Together, these data provide valuable new insights into the upstream genomic regulatory landscape responsible for transcriptional control of Grhl2 during palatal closure.

Loss of GRHL3 leads to TARC/CCL17-mediated keratinocyte proliferation in the epidermis.

Identifying soluble factors that influence epidermal integrity is critical for the development of preventative and therapeutic strategies for disorders such as ichthyosis, psoriasis, dermatitis and epidermal cancers. The transcription factor Grainyhead-like 3 (GRHL3) is essential for maintaining barrier integrity and preventing development of cutaneous squamous cell carcinoma (SCC); however, how loss of this factor, which in the skin is expressed exclusively within suprabasal epidermal layers triggers proliferation of basal keratinocytes, had thus far remained elusive. Our present study identifies thymus and activation-regulated chemokine (TARC) as a novel soluble chemokine mediator of keratinocyte proliferation following loss of GRHL3. Knockdown of GRHL3 in human keratinocytes showed that of 42 cytokines examined, TARC was the only significantly upregulated chemokine. Mouse skin lacking Grhl3 presented an inflammatory response with hallmarks of TARC activation, including heightened induction of blood clotting, increased infiltration of mast cells and pro-inflammatory T cells, increased expression of the pro-proliferative/pro-inflammatory markers CD3 and pSTAT3, and significantly elevated basal keratinocyte proliferation. Treatment of skin cultures lacking Grhl3 with the broad spectrum anti-inflammatory 5-aminosalicylic acid (5ASA) partially restored epidermal differentiation, indicating that abnormal keratinocyte proliferation/differentiation balance is a key driver of barrier dysfunction following loss of Grhl3, and providing a promising therapeutic avenue in the treatment of GRHL3-mediated epidermal disorders.

Restricted cell cycle is essential for clonal evolution and therapeutic resistance of pre-leukemic stem cells.

Pre-leukemic stem cells (pre-LSCs) give rise to leukemic stem cells through acquisition of additional gene mutations and are an important source of relapse following chemotherapy. We postulated that cell-cycle kinetics of pre-LSCs may be an important determinant of clonal evolution and therapeutic resistance. Using a doxycycline-inducible H2B-GFP transgene in a mouse model of T-cell acute lymphoblastic leukemia to study cell cycle in vivo, we show that self-renewal, clonal evolution and therapeutic resistance are limited to a rare population of pre-LSCs with restricted cell cycle. We show that proliferative pre-LSCs are unable to return to a cell cycle-restricted state. Cell cycle-restricted pre-LSCs have activation of p53 and its downstream cell-cycle inhibitor p21. Furthermore, absence of p21 leads to proliferation of pre-LSCs, with clonal extinction through loss of asymmetric cell division and terminal differentiation. Thus, inducing proliferation of pre-LSCs represents a promising strategy to increase cure rates for acute leukemia.

Bone marrow transplantation corrects haemolytic anaemia in a novel ENU mutagenesis mouse model of TPI deficiency.

In this study, we performed a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen in mice to identify novel genes or alleles that regulate erythropoiesis. Here, we describe a recessive mouse strain, called RBC19, harbouring a point mutation within the housekeeping gene, Tpi1, which encodes the glycolysis enzyme, triosephosphate isomerase (TPI). A serine in place of a phenylalanine at amino acid 57 severely diminishes enzyme activity in red blood cells and other tissues, resulting in a macrocytic haemolytic phenotype in homozygous mice, which closely resembles human TPI deficiency. A rescue study was performed using bone marrow transplantation of wild-type donor cells, which restored all haematological parameters and increased red blood cell enzyme function to wild-type levels after 7 weeks. This is the first study performed in a mammalian model of TPI deficiency, demonstrating that the haematological phenotype can be rescued.

Stage-dependent therapeutic efficacy in PI3K/mTOR-driven squamous cell carcinoma of the skin.

Cutaneous squamous cell carcinoma (SCC) is a recurrent cancer that is prevalent in predisposed subjects such as immunosuppressed patients and patients being treated for other malignancies. Model systems to trial therapies at different stages of SCC development are lacking, therefore precluding efficient therapeutic interventions. Here, we have disrupted the expression of the tumor suppressor GRHL3 to induce loss of PTEN and activation of the PI3K/mTOR signaling pathway in mice and human skin, promoting aggressive SCC development. We then examined the potential for targeting PI3K/mTOR and an oncogenic driver miR-21, alone and in combination, for the prevention and treatment of SCC during the initiation, promotion/progression and establishment stages. Treatment with PI3K/mTOR inhibitors completely prevented tumor initiation, and these inhibitors significantly delayed the course of papilloma progression to malignancy. However, established SCC did not undergo any growth regression, indicating that this therapy is ineffective in established cancers. Mechanistically, the resistant SCCs displayed increased miR-21 expression in mice and humans where antagonists of miR-21 rescued expression levels of GRHL3/PTEN, but the combination of miR-21 antagonism with PI3K/mTOR inhibition resulted in acquired SCC resistance in part via c-MYC and OCT-4 upregulation. In conclusion, our data provide molecular evidence for the efficacy of targeting oncogenic drivers of SCC during the initiation and promotion stages and indicate that combination therapy may induce an aggressive phenotype when applied in the establishment stage.

Loss of Dynamin 2 GTPase function results in microcytic anaemia.

In a dominant mouse ethylnitrosurea mutagenesis screen for genes regulating erythropoiesis, we identified a pedigree with a novel microcytic hypochromia caused by a V235G missense mutation in Dynamin 2 (Dnm2). Mutations in Dnm2, a GTPase, are highly disease-specific and have been implicated in four forms of human diseases: centronuclear myopathy, Charcot-Marie Tooth neuropathy and, more recently, T-cell leukaemia and Hereditary Spastic Paraplegia, but red cell abnormalities have not been reported to date. The V235G mutation lies within a crucial GTP nucleotide-binding pocket of Dnm2, and resulted in defective GTPase activity and incompatibility with life in the homozygous state. Dnm2 is an essential mediator of clathrin-mediated endocytosis, which is required for the uptake of transferrin (Tf) into red cells for incorporation of haem. Accordingly, we observed significantly reduced Tf uptake by Dnm2+/V235G cells, which led to impaired endosome formation. Despite these deficiencies, surprisingly all iron studies were unchanged, suggesting an unexplained alternative mechanism underlies microcytic anaemia in Dnm2+/V235G mice. This study provides the first in vivo evidence for the requirements of Dnm2 in normal erythropoiesis.

Identification of a Novel Proto-oncogenic Network in Head and Neck Squamous Cell Carcinoma.

BACKGROUND: The developmental transcription factor Grainyhead-like 3 (GRHL3) plays a critical tumor suppressor role in the mammalian epidermis through direct regulation of PTEN and the PI3K/AKT/mTOR signaling pathway. GRHL3 is highly expressed in all tissues derived from the surface ectoderm, including the oral cavity, raising a question about its potential role in suppression of head and neck squamous cell carcinoma (HNSCC). METHODS: We explored the tumor suppressor role of Grhl3 in HNSCC using a conditional knockout (Grhl3 (∆/-) /K14Cre (+) ) mouse line (n = 26) exposed to an oral chemical carcinogen. We defined the proto-oncogenic pathway activated in the HNSCC derived from these mice and assessed it in primary human HNSCC samples, normal oral epithelial cell lines carrying shRNA to GRHL3, and human HNSCC cell lines. Data were analyzed with two-sided chi square and Student's t tests. RESULTS: Deletion of Grhl3 in oral epithelium in mice did not perturb PTEN/PI3K/AKT/mTOR signaling, but instead evoked loss of GSK3B expression, resulting in stabilization and accumulation of c-MYC and aggressive HNSCC. This molecular signature was also evident in a subset of primary human HNSCC and HNSCC cell lines. Loss of Gsk3b in mice, independent of Grhl3, predisposed to chemical-induced HNSCC. Restoration of GSK3B expression blocked proliferation of normal oral epithelial cell lines carrying shRNA to GRHL3 (cell no., Day 8: Scramble ctl, 616±21.8 x 10(3) vs GRHL3-kd, 1194±44 X 10(3), P < .001; GRHL3-kd vs GRHL3-kd + GSK3B, 800±98.84 X 10(3), P = .003) and human HNSCC cells. CONCLUSIONS: We defined a novel molecular signature in mammalian HNSCC, suggesting new treatment strategies targeting the GRHL3/GSK3B/c-MYC proto-oncogenic network.

Loss of Grainy head-like 1 is associated with disruption of the epidermal barrier and squamous cell carcinoma of the skin.

The Grainyhead-like 1 (GRHL1) transcription factor regulates the expression of desmosomal cadherin desmoglein 1 (Dsg1) in suprabasal layers of the epidermis. As a consequence, the epidermis of Grhl1-null mice displays fewer desmosomes that are abnormal in structure. These mice also exhibit mild chronic skin barrier defects as evidenced by altered keratinocyte terminal differentiation, increased expression of inflammatory markers and infiltration of the skin by immune cells. Exposure of Grhl1 (-/-) mice to a standard chemical skin carcinogenesis protocol results in development of fewer papillomas than in wild type control animals, but with a rate of conversion to squamous cell carcinoma (SCC) that is strikingly higher than in normal littermates. The underlying molecular mechanism differs from mice with conditional ablation of a closely related Grhl family member, Grhl3, in the skin, which develop SCC due to the loss of expression of phosphatase and tensin homolog (PTEN) and activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) signaling pathway.

Induction of human fetal hemoglobin expression by adenosine-2',3'-dialdehyde.

BACKGROUND: Pharmacologic reactivation of fetal hemoglobin expression is a promising strategy for treatment of sickle cell disease and ß-thalassemia. The objective of this study was to investigate the effect of the methyl transferase inhibitor adenosine-2',3'-dialdehyde (Adox) on induction of human fetal hemoglobin (HbF) in K562 cells and human hematopoietic progenitor cells. METHODS: Expression levels of human fetal hemoglobin were assessed by northern blot analysis and Real-time PCR. HbF and adult hemoglobin (HbA) content were analyzed using high-performance liquid chromatography (HPLC). DNA methylation levels on human gamma-globin gene promoters were determined using Bisulfite sequence analysis. Enrichment of histone marks on genes was assessed by chromosome immunoprecipitation (ChIP). RESULTS: Adox induced γ-globin gene expression in both K562 cells and in human bone marrow erythroid progenitor cells through a mechanism potentially involving inhibition of protein arginine methyltransferase 5 (PRMT5). CONCLUSIONS: The ability of methyl transferase inhibitors such as Adox to efficiently reactivate fetal hemoglobin expression suggests that these agents may provide a means of reactivating fetal globin expression as a therapeutic option for treating sickle cell disease and ß-thalassemia.

Regulation of delta-aminolevulinic acid dehydratase by krüppel-like factor 1.

Krüppel-like factor 1(KLF1) is a hematopoietic-specific zinc finger transcription factor essential for erythroid gene expression. In concert with the transacting factor GATA1, KLF1 modulates the coordinate expression of the genes encoding the multi-enzyme heme biosynthetic pathway during erythroid differentiation. To explore the mechanisms underpinning KLF1 action at the gene loci regulating the first 3 steps in this process, we have exploited the K1-ERp erythroid cell line, in which KLF1 translocates rapidly to the nucleus in response to treatment with 4-OH-Tamoxifen (4-OHT). KLF1 acts as a differentiation-independent transcriptional co-regulator of delta-aminolevulinic acid dehydratase (Alad), but not 5-aminolevulinate synthase gene (Alas2) or porphobilinogen deaminase (Pbgd). Similar to its role at the ß-globin promoter, KLF1 induces factor recruitment and chromatin changes at the Alad1b promoter in a temporally-specific manner. In contrast to these changes, we observed a distinct mechanism of histone eviction at the Alad1b promoter. Furthermore, KLF1-dependent events were not modulated by GATA1 factor promoter co-occupancy alone. These results not only enhance our understanding of erythroid-specific modulation of heme biosynthetic regulation by KLF1, but provide a model that will facilitate the elucidation of novel KLF1-dependent events at erythroid gene loci that are independent of GATA1 activity.

Inhibition of apoptosis by BCL2 prevents leukemic transformation of a murine myelodysplastic syndrome.

Programmed cell death or apoptosis is a prominent feature of low-risk myelodysplastic syndromes (MDS), although the underlying mechanism remains controversial. High-risk MDS have less apoptosis associated with increased expression of the prosurvival BCL2-related proteins. To address the mechanism and pathogenic role of apoptosis and BCL2 expression in MDS, we used a mouse model resembling human MDS, in which the fusion protein NUP98-HOXD13 (NHD13) of the chromosomal translocation t(2;11)(q31;p15) is expressed in hematopoietic cells. Hematopoietic stem and progenitor cells from 3-month-old mice had increased rates of apoptosis associated with increased cell cycling and DNA damage. Gene expression profiling of these MDS progenitors revealed a specific reduction in Bcl2. Restoration of Bcl2 expression by a BCL2 transgene blocked apoptosis of the MDS progenitors, which corrected the macrocytic anemia. Blocking apoptosis also restored cell-cycle quiescence and reduced DNA damage in the MDS progenitors. We expected that preventing apoptosis would accelerate malignant transformation to acute myeloid leukemia (AML). However, contrary to expectations, preventing apoptosis of premalignant cells abrogated transformation to AML. In contrast to the current dogma that overcoming apoptosis is an important step toward cancer, this work demonstrates that gaining a survival advantage of premalignant cells may delay or prevent leukemic progression.