FAK is required for c-Met/ß-catenin-driven hepatocarcinogenesis.

UNLABELLED: Hepatocellular carcinoma (HCC) is the third most common cause of cancer death worldwide and most patients with HCC have limited treatment options. Focal adhesion kinase (FAK) is overexpressed in many HCC specimens, offering a potential target for HCC treatment. However, the role of FAK in hepatocarcinogenesis remains elusive. Establishing whether FAK expression plays a role in HCC development is necessary to determine whether it is a viable therapeutic target. In this study, we generated mice with hepatocyte-specific deletion of Fak and investigated the role of Fak in an oncogenic (c-MET/ß-catenin, MET/CAT)-driven HCC model. We found that deletion of Fak in hepatocytes did not affect morphology, proliferation, or apoptosis. However, Fak deficiency significantly repressed MET/CAT-induced tumor development and prolonged survival of animals with MET/CAT-induced HCC. In mouse livers and HCC cell lines, Fak was activated by MET, which induced the activation of Akt/Erk and up-regulated cyclin D1 and tumor cell proliferation. CAT enhanced MET-stimulated FAK activation and synergistically induced the activation of the AKT/ERK-cyclin D1 signaling pathway in a FAK kinase-dependent manner. In addition, FAK was required for CAT-induced cyclin D1 expression in a kinase-independent fashion. CONCLUSION: Fak is required for c-Met/ß-catenin-driven hepatocarcinogenesis. Inhibition of FAK provides a potential strategy to treat HCC.

Functional specificity of CpG DNA-binding CXXC domains in mixed lineage leukemia.

The MLL CXXC domain binds nonmethylated CpG-containing DNA and is essential for the oncogenic properties of MLL fusion proteins. To determine potential functional promiscuity of similar DNA binding domains, we replaced the MLL CXXC domain in the context of the leukemogenic MLL-AF9 fusion with CXXC domains from DNMT1, CGBP (CFP1), and MBD1, or with a methyl-CpG-binding domain (MBD) from MBD1. MLL(DNMT1 CXXC)-AF9 shows robust in vitro colony forming activity and in vivo leukemogenesis, similar to MLL-AF9. However, colony forming ability and leukemogenicity are abrogated in MLL-AF9 containing either the CGBP or MBD1 CXXC domains or the MBD1 MBD domain. Direct comparison of in vitro DNA binding affinity of the isolated CXXC or MBD domains demonstrated that MLL, DNMT1, and CGBP CXXC domains could each bind to unmethylated DNA but with differing affinity. In contrast, the isolated MBD1 CXXC and MBD1 MBD domains were unable to bind to the same DNA. However, all substituted domains still allowed targeting of the MLL fusions to the functionally important Hoxa9 locus in primary bone marrow progenitor cells. In addition to DNA binding activity, it was critical that specific CpG residues in the Hoxa9 locus were protected from methylation for leukemia development. This ultimately prevented histone 3 lysine 9 trimethylation (H3K9me3) of the locus and enabled Hoxa9 expression. These were properties shared by MLL and DNMT1 CXXC domains but not by CGBP CXXC or the other swapped fusions tested. We demonstrate that similar CXXC domains can be mechanistically distinguished by specificity of CpG nucleotides preferentially protected from DNA methylation.

Overexpression of Foxn1 attenuates age-associated thymic involution and prevents the expansion of peripheral CD4 memory T cells.

The forkhead box n1 (Foxn1) transcription factor is essential for thymic organogenesis during embryonic development; however, a functional role of Foxn1 in the postnatal thymus is less well understood. We developed Foxn1 transgenic mice (Foxn1Tg), in which overexpression of Foxn1 is driven by the human keratin-14 promoter. Expression of the Foxn1 transgene increased the endogenous Foxn1 levels. In aged mice, overexpression of Foxn1 in the thymus attenuated the decline in thymocyte numbers, prevented the decline in frequency of early thymic progenitors, and generated a higher number of signal joint TCR excised circle. Histologic studies revealed that structural alterations associated with thymic involution were diminished in aged Foxn1 Tg. Total numbers of EpCAM+ MHC II+ and MHC II(hi) thymic epithelial cells were higher in young and old Foxn1Tg and more EpCAM+ MHC II(hi) TEC expressed Ki-67 in aged Foxn1Tg compared with WT. Furthermore, Foxn1Tg displayed a significant reduction in the expansion of splenic CD4+ memory compartments and attenuated the decline in CD4+ and CD8+ naive compartments. Our data indicate that manipulation of Foxn1 expression in the thymus ameliorates thymopoiesis in aged mice and offer a strategy to combat the age-associated decline in naive T-cell production and CD4 naive/memory ratios in the elderly.

Aberrant overexpression and function of the miR-17-92 cluster in MLL-rearranged acute leukemia.

MicroRNA (miRNA)-17-92 cluster (miR-17-92), containing seven individual miRNAs, is frequently amplified and overexpressed in lymphomas and various solid tumors. We have found that it is also frequently amplified and the miRNAs are aberrantly overexpressed in mixed lineage leukemia (MLL)-rearranged acute leukemias. Furthermore, we show that MLL fusions exhibit a much stronger direct binding to the locus of this miRNA cluster than does wild-type MLL; these changes are associated with elevated levels of histone H3 acetylation and H3K4 trimethylation and an up-regulation of these miRNAs. We further observe that forced expression of this miRNA cluster increases proliferation and inhibits apoptosis of human cells. More importantly, we show that this miRNA cluster can significantly increase colony-forming capacity of normal mouse bone marrow progenitor cells alone and, particularly, in cooperation with MLL fusions. Finally, through combinatorial analysis of miRNA and mRNA arrays of mouse bone marrow progenitor cells transfected with this miRNA cluster and/or MLL fusion gene, we identified 363 potential miR-17-92 target genes that exhibited a significant inverse correlation of expression with the miRNAs. Remarkably, these potential target genes are significantly enriched (P < 0.01; >2-fold) in cell differentiation, hematopoiesis, cell cycle, and apoptosis. Taken together, our studies suggest that overexpression of miR-17-92 cluster in MLL-rearranged leukemias is likely attributed to both DNA copy number amplification and direct up-regulation by MLL fusions, and that the miRNAs in this cluster may play an essential role in the development of MLL-associated leukemias through inhibiting cell differentiation and apoptosis, while promoting cell proliferation, by regulating relevant target genes.

Regulation of mir-196b by MLL and its overexpression by MLL fusions contributes to immortalization.

Chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene produce chimeric proteins that cause abnormal expression of a subset of HOX genes and leukemia development. Here, we show that MLL normally regulates expression of mir-196b, a hematopoietic microRNA located within the HoxA cluster, in a pattern similar to that of the surrounding 5' Hox genes, Hoxa9 and Hoxa10, during embryonic stem (ES) cell differentiation. Within the hematopoietic lineage, mir-196b is most abundant in short-term hematopoietic stem cells and is down-regulated in more differentiated hematopoietic cells. Leukemogenic MLL fusion proteins cause overexpression of mir-196b, while treatment of MLL-AF9 transformed bone marrow cells with mir-196-specific antagomir abrogates their replating potential in methylcellulose. This demonstrates that mir-196b function is necessary for MLL fusion-mediated immortalization. Furthermore, overexpression of mir-196b was found specifically in patients with MLL associated leukemias as determined from analysis of 55 primary leukemia samples. Overexpression of mir-196b in bone marrow progenitor cells leads to increased proliferative capacity and survival, as well as a partial block in differentiation. Our results suggest a mechanism whereby increased expression of mir-196b by MLL fusion proteins significantly contributes to leukemia development.

MLL protects CpG clusters from methylation within the Hoxa9 gene, maintaining transcript expression.

Homeobox (HOX) genes play a definitive role in determination of cell fate during embryogenesis and hematopoiesis. MLL-related leukemia is coincident with increased expression of a subset of HOX genes, including HOXA9. MLL functions to maintain, rather than initiate, expression of its target genes. However, the mechanism of MLL maintenance of target gene expression is not understood. Here, we demonstrate that Mll binds to specific clusters of CpG residues within the Hoxa9 locus and regulates expression of multiple transcripts. The presence of Mll at these clusters provides protection from DNA methylation. shRNA knock-down of Mll reverses the methylation protection status at the previously protected CpG clusters; methylation at these CpG residues is similar to that observed in Mll null cells. Furthermore, reconstituting MLL expression in Mll null cells can reverse DNA methylation of the same CpG residues, demonstrating a dominant effect of MLL in protecting this specific region from DNA methylation. Intriguingly, an oncogenic MLL-AF4 fusion can also reverse DNA methylation, but only for a subset of these CpGs. This method of transcriptional regulation suggests a mechanism that explains the role of Mll in transcriptional maintenance, but it may extend to other CpG DNA binding proteins. Protection from methylation may be an important mechanism of epigenetic inheritance by regulating the function of both de novo and maintenance DNA methyltransferases.

BCOR Binding to MLL-AF9 Is Essential for Leukemia via Altered EYA1, SIX, and MYC Activity.

MLL is a target of chromosomal translocations in acute leukemias with poor prognosis. The common MLL fusion partner AF9 (MLLT3) can directly bind to AF4, DOT1L, BCOR, and CBX8. To delineate the relevance of BCOR and CBX8 binding to MLL-AF9 for leukemogenesis, here we determine protein structures of AF9 complexes with CBX8 and BCOR, and show that binding of all four partners to AF9 is mutually exclusive. Using the structural analyses, we identify point mutations that selectively disrupt AF9 interactions with BCOR and CBX8. In bone marrow stem/progenitor cells expressing point mutant CBX8 or point mutant MLL-AF9, we show that disruption of direct CBX8/MLL-AF9 binding does not impact in vitro cell proliferation, whereas loss of direct BCOR/MLL-AF9 binding causes partial differentiation and increased proliferation. Strikingly, loss of MLL-AF9/BCOR binding abrogated its leukemogenic potential in a mouse model. The MLL-AF9 mutant deficient for BCOR binding reduces the expression of the EYA1 phosphatase and the protein level of c-Myc. Reduction in BCOR binding to MLL-AF9 alters a MYC-driven gene expression program, as well as altering expression of SIX-regulated genes, likely contributing to the observed reduction in the leukemia-initiating cell population.

NKG2D signaling certifies effector CD8 T cells for memory formation.

BACKGROUND: The development of memory responses is an evolutionary function of the adaptive immune system. We propose that for the immune system to populate the memory compartment with the best-suited CD8 T cells it utilizes a process of certification or molecular accreditation mediated through Natural Killer Group 2D (NKG2D). This process of certification assures that the memory compartment is filled with CD8 T cells that have demonstrated their ability to kill their cognate targets through a two-step process that utilizes T cell receptor (TCR) and NKG2D signaling. METHODS: One week after immunization with peptide-pulsed dendritic cells, NKG2D signaling was transiently blocked in vivo with a single injection of neutralizing antibodies. Under such conditions, we determined the importance of NKG2D signaling during the effector phase for memory formation without compromising NKG2D signaling at the memory phase. Both open (polyclonal) and closed (monoclonal) CD8 T cell repertoires were studied. RESULTS: We show that signaling through NKG2D mediated this certification. Temporary blockade of NKG2D signaling during the effector phase resulted in the formation of highly defective memory CD8 T cells characterized by altered expression of the ribosomal protein S6 and epigenetic modifiers, suggesting modifications in the T cell translational machinery and epigenetic programming. Finally, these uncertified memory cells were not protective against a B16 tumor challenge. CONCLUSION: Signaling through NKG2D during the effector phase (certification) favors the development of functional memory CD8 T cells, a previously undescribed role for NKG2D. Temporary blockade of NKG2D signaling during the effector phase results in the formation of highly defective memory CD8 T cells potentially by affecting the expression of the ribosomal protein S6 and epigenetic modifiers, suggesting alterations in T cell translational machinery and epigenetic programming.

Bromodomain and histone acetyltransferase domain specificities control mixed lineage leukemia phenotype.

A critical unanswered question about mixed lineage leukemia (MLL) is how specific MLL fusion partners control leukemia phenotype. The MLL-cyclic AMP-responsive element binding protein-binding protein (CBP) fusion requires both the CBP bromodomain and histone acetyltransferase (HAT) domain for transformation and causes acute myelogenous leukemia (AML), often preceded by a myelodysplastic phase. We did domain-swapping experiments to define whether unique specificities of these CBP domains drive this specific MLL phenotype. Within MLL-CBP, we replaced the CBP bromodomain or HAT domain with P300/CBP-associated factor (P/CAF) or TAF(II)250 bromodomains or the P/CAF or GCN5 HAT domains. HAT, but not bromodomain, substitutions conferred enhanced proliferative capacity in vitro but lacked expression of myeloid cell surface markers normally seen with MLL-CBP. Mice reconstituted with domain-swapped hematopoietic progenitors developed different disease from those with MLL-CBP. This included development of lymphoid disease and lower frequency of the myelodysplastic phase in those mice developing AML. We conclude that both the CBP bromodomain and HAT domain play different but critical roles in determining the phenotype of MLL-CBP leukemia. Our results support an important role for MLL partner genes in determining the leukemia phenotype besides their necessity in leukemogenesis. Here, we find that subtleties in MLL fusion protein domain specificity direct cells toward a specific disease phenotype.

Publisher Correction: miR-196b directly targets both HOXA9/MEIS1 oncogenes and FAS tumour suppressor in MLL-rearranged leukaemia.

This corrects the article DOI: 10.1038/ncomms1681.

Mammalian septins nomenclature.

There are 10 known mammalian septin genes, some of which produce multiple splice variants. The current nomenclature for the genes and gene products is very confusing, with several different names having been given to the same gene product and distinct names given to splice variants of the same gene. Moreover, some names are based on those of yeast or Drosophila septins that are not the closest homologues. Therefore, we suggest that the mammalian septin field adopt a common nomenclature system, based on that adopted by the Mouse Genomic Nomenclature Committee and accepted by the Human Genome Organization Gene Nomenclature Committee. The human and mouse septin genes will be named SEPT1-SEPT10 and Sept1-Sept10, respectively. Splice variants will be designated by an underscore followed by a lowercase "v" and a number, e.g., SEPT4_v1.

Adolescent binge-pattern alcohol exposure alters genome-wide DNA methylation patterns in the hypothalamus of alcohol-naïve male offspring.

Teenage binge drinking is a major health concern in the United States, with 21% of teenagers reporting binge-pattern drinking behavior in the previous 30 days. Recently, our lab showed that alcohol-naïve offspring of rats exposed to alcohol during adolescence exhibited altered gene expression profiles in the hypothalamus, a brain region involved in stress regulation. We employed Enhanced Reduced Representation Bisulfite Sequencing as an unbiased approach to test the hypothesis that parental exposure to binge-pattern alcohol during adolescence alters DNA methylation profiles in their alcohol-naïve offspring. Wistar rats were administered a repeated binge-ethanol exposure paradigm during early (postnatal day (PND) 37-44) and late (PND 67-74) adolescent development. Animals were mated 24 h after the last ethanol dose and subsequent offspring were produced. Analysis of male PND7 offspring revealed that offspring of alcohol-exposed parents exhibited differential DNA methylation patterns in the hypothalamus. The differentially methylated cytosines (DMCs) were distinct between offspring depending on which parent was exposed to ethanol. Moreover, novel DMCs were observed when both parents were exposed to ethanol and many DMCs from single parent ethanol exposure were not recapitulated with dual parent exposure. We also measured mRNA expression of several differentially methylated genes and some, but not all, showed correlative changes in expression. Importantly, methylation was not a direct predictor of expression levels, underscoring the complexity of transcriptional regulation. Overall, we demonstrate that adolescent binge ethanol exposure causes altered genome-wide DNA methylation patterns in the hypothalamus of alcohol-naïve offspring.

One Step Forward in the Challenging Arena of MLL-AF4 Leukemia.

MLL-AF4 leukemia is the predominant infant acute leukemia and has a poor prognosis. No current experimental models accurately reflect the human disease. Lin et al., in this issue of Cancer Cell, describe their model that recapitulates multiple key aspects of this aggressive disease, facilitating future mechanistic and preclinical studies.

The miR-17∼92 cluster contributes to MLL leukemia through the repression of MEIS1 competitor PKNOX1.

Mixed lineage leukemias have a relatively poor prognosis and arise as a result of translocations between the MLL(KMT2A) gene and one of multiple partner genes. Downstream targets of MLL are aberrantly upregulated and include the developmentally important HOX genes and MEIS1, as well as multiple microRNAs (miRNAs), including the miR-17∼92 cluster. Here we examined the contribution of specific miRNAs to MLL leukemias through knockdown studies utilizing custom anti-microRNA oligonucleotides. Combinatorial treatment against miR-17-5p and miR-19a-3p of the miR-17∼92 cluster dramatically reduces colony forming ability of MLL-fusion containing cell lines relative to non-MLL acute myeloid leukemia (AML) controls. To determine the mechanism by which these miRNAs contribute to leukemia, we validated PKNOX1 as a target of both miR-17-5p and miR-19a-3p. MEIS1 and PKNOX1 are TALE domain proteins that participate in ternary complexes with HOX and PBX partners. Here we establish the competitive relationship between PKNOX1 and MEIS1 in PBX-containing complex formation and determine the antagonistic role of PKNOX1 to leukemia in a murine MLL-AF9 model. These data implicate the miR-17∼92 cluster as part of a regulatory mechanism necessary to maintain MEIS1/HOXA9 -mediated transformation in MLL leukemia, indicating that targeting multiple non-homologous miRNAs may be utilized as a novel therapeutic regimen.

Association between early promoter-specific DNA methylation changes and outcome in older acute myeloid leukemia patients.

Treatment options for older patients with acute myeloid leukemia (AML) range from supportive care alone to full-dose chemotherapy. Identifying factors that predict response to therapy may help increase efficacy and avoid toxicity. The phase II SWOG S0703 study investigated the use of hydroxyurea and azacitidine with gemtuzumab ozogamicin in the elderly AML population and found survival rates similar to those expected with standard AML regimens, with less toxicity. As part of this study, global DNA methylation along with promoter DNA methylation and expression analysis of six candidate genes (CDKN2A, CDKN2B, HIC1, RARB, CDH1 and APAF1) were determined before and during therapy to investigate whether very early changes are prognostic for clinical response. Global DNA methylation was not associated with a clinical response. Samples after 3 or 4 days of treatment with azacitidine showed significantly decreased CDKN2A promoter DNA methylation in patients achieving complete remission (CR) compared to those who did not. Samples from day 7 of treatment showed significantly decreased RARB, CDKN2B and CDH1 promoter DNA methylation in responders compared to nonresponders. Gene-specific DNA methylation analysis of peripheral blood samples may help early identification of those older AML patients most likely to benefit from demethylating agent therapy.

A t(11;15) fuses MLL to two different genes, AF15q14 and a novel gene MPFYVE on chromosome 15.

The mixed lineage leukemia gene (MLL, also known as HRX, ALL-1 and Htrx) located at 11q23 is involved in translocations with over 40 different chromosomal bands in a variety of leukemia subtypes. Here we report our analysis of a rare but recurring translocation, t(11;15)(q23;q14). This translocation has been described in a small subset of cases with both acute myeloblastic leukemia and ALL. Recent studies have shown that MLL is fused to AF15q14 in the t(11;15). Here we analyse a sample from another patient with this translocation and confirm the presence of an MLL-AF15q14 fusion. However, we have also identified and cloned another fusion transcript from the same patient sample. In this fusion transcript, MLL is fused to a novel gene, MLL partner containing FYVE domain (MPFYVE). Both MLL-AF15q14 and MLL-MPFYVE are in-frame fusion transcripts with the potential to code for novel fusion proteins. MPFYVE is also located on chromosome 15, approximately 170 kb telomeric to AF15q14. MPFYVE contains a highly conserved motif, the FYVE domain which, in other proteins, has been shown to bind to phosphotidyl-inositol-3 phosphate (PtdIns(3)P). The MLL-MPFYVE fusion may be functionally important in the leukemia process in at least some patients containing this translocation.

Characterization of the MLL partner gene AF15q14 involved in t(11;15)(q23;q14).

Translocations interrupting the mixed lineage leukemia gene (MLL) occur in 7-10% of acute lymphoblastic leukemia (ALL) and 5-6% of acute myeloid leukemia (AML) cases. One of these translocations, t(11;15)(q23;q14), occurs rarely in both ALL and AML. The gene on chromosome 15, AF15q14, was cloned recently in a patient with AML-M4. We have identified the same gene in a de novo T-ALL patient. However, both the MLL and AF15q14 breakpoints in these patients differed: in the previously reported AML-M4, both gene breaks were within exons, while in our ALL case the MLL break is intronic and the AF15q14 break is exonic. The MLL-AF15q14 fusion described previously shares no AF15q14 residues in common with the chimera reported here. The fusion proteins also differ with respect to MLL--the previously described fusion contains 55 extra amino acids as its MLL break is in exon 11, while the chimera we report breaks in intron 9. Contrary to the originally described normal AF15q14 (5925-bp cDNA encoding a 1833-aa protein), we identify a 7542-bp cDNA and a 2342-aa AF15q14 protein. AF15q14 appears identical to an mRNA previously found to be expressed in melanoma rendered nontumorigenic by microcell-mediated introduction of normal chromosome 6, suggesting the gene may function normally to suppress cell growth and/or enhance maturation.

WEE1 is a validated target of the microRNA miR-17-92 cluster in leukemia.

MicroRNAs are short single-stranded RNAs that regulate target gene expression by binding to complementary sites in the 3' untranslated region (UTR) of their mRNA targets. The polycistronic miR-17-92 cluster, which encodes miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a, was previously shown to be overexpressed in multiple types of cancer. In this study, target gene prediction algorithms were used to predict potential targets of the miR-17-92 cluster. WEE1, a kinase that inhibits cell cycle progression, was identified as a possible target of five of the six miRNAs in the cluster. Luciferase reporter assays were used to determine that miR-17, miR-20a, and miR-18a specifically target nucleotides 465-487 of the 3' UTR of WEE1, whereas miR-19a and miR-19b exert control on WEE1 by targeting nucleotides 1069-1091. A negative correlation was determined between endogenous miR-17 or miR-19a expression and endogenous WEE1 protein expression in the same panel of cell lines. We conclude that WEE1 is a valid target of the miR-17-92 cluster in leukemia.

Degree of recruitment of DOT1L to MLL-AF9 defines level of H3K79 Di- and tri-methylation on target genes and transformation potential.

The MLL gene is a common target of chromosomal translocations found in human leukemia. MLL-fusion leukemia has a consistently poor outcome. One of the most common translocation partners is AF9 (MLLT3). MLL-AF9 recruits DOT1L, a histone 3 lysine 79 methyltransferase (H3K79me1/me2/me3), leading to aberrant gene transcription. We show that DOT1L has three AF9 binding sites and present the nuclear magnetic resonance (NMR) solution structure of a DOT1L-AF9 complex. We generate structure-guided point mutations and find that they have graded effects on recruitment of DOT1L to MLL-AF9. Chromatin immunoprecipitation sequencing (ChIP-seq) analyses of H3K79me2 and H3K79me3 show that graded reduction of the DOT1L interaction with MLL-AF9 results in differential loss of H3K79me2 and me3 at MLL-AF9 target genes. Furthermore, the degree of DOT1L recruitment is linked to the level of MLL-AF9 hematopoietic transformation.

FAK mediates a compensatory survival signal parallel to PI3K-AKT in PTEN-null T-ALL cells.

Mutations and inactivation of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) are observed in 15%-25% of cases of human T cell acute lymphoblastic leukemia (T-ALL). Pten deletion induces myeloproliferative disorders (MPDs), acute myeloid leukemia (AML), and/or T-ALL in mice. Previous studies attributed Pten-loss-related hematopoietic defects and leukemogenesis to excessive activation of phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling. Although inhibition of this signal dramatically suppresses the growth of PTEN-null T-ALL cells in vitro, treatment with inhibitors of this pathway does not cause a complete remission in vivo. Here, we report that focal adhesion kinase (Fak), a protein substrate of Pten, also contributes to T-ALL development in Pten-null mice. Inactivation of the FAK signaling pathway by either genetic or pharmacologic methods significantly sensitizes both murine and human PTEN-null T-ALL cells to PI3K/AKT/mTOR inhibition when cultured in vitro on feeder layer cells or a matrix and in vivo.