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.

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.

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.

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.

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.

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.

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.

Importance of a specific amino acid pairing for murine MLL leukemias driven by MLLT1/3 or AFF1/4.

Acute leukemias caused by translocations of the MLL gene at chromosome 11 band q23 (11q23) are characterized by a unique gene expression profile. More recently, data from several laboratories indicate that the most commonly encountered MLL fusion proteins, MLLT1, MLLT3, and AFF1 are found within a molecular complex that facilitates the elongation phase of mRNA transcription. Mutational analyses suggest that interaction between the MLLT1/3 proteins and AFF family proteins are required for experimental transformation of hematopoietic progenitor cells (HPCs). Here, we define a specific pairing of two amino acids that creates a salt bridge between MLLT1/3 and AFF proteins that is critically important for MLL-mediated transformation of HPCs. Our findings, coupled with the newly defined structure of MLLT3 in complex with AFF1, should facilitate the development of small molecules that block this amino acid interaction and interfere with the activity of the most common MLL oncoproteins.

Therapeutic antagonists of microRNAs deplete leukemia-initiating cell activity.

Acute myelogenous leukemia (AML) subtypes that result from oncogenic activation of homeobox (HOX) transcription factors are associated with poor prognosis. The HOXA9 transcription activator and growth factor independent 1 (GFI1) transcriptional repressor compete for occupancy at DNA-binding sites for the regulation of common target genes. We exploited this HOXA9 versus GFI1 antagonism to identify the genes encoding microRNA-21 and microRNA-196b as transcriptional targets of HOX-based leukemia oncoproteins. Therapeutic inhibition of microRNA-21 and microRNA-196b inhibited in vitro leukemic colony forming activity and depleted in vivo leukemia-initiating cell activity of HOX-based leukemias, which led to leukemia-free survival in a murine AML model and delayed disease onset in xenograft models. These data establish microRNA as functional effectors of endogenous HOXA9 and HOX-based leukemia oncoproteins, provide a concise in vivo platform to test RNA therapeutics, and suggest therapeutic value for microRNA antagonists in AML.

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.

p27kip1 maintains a subset of leukemia stem cells in the quiescent state in murine MLL-leukemia.

MLL (mixed-lineage leukemia)-fusion genes induce the development of leukemia through deregulation of normal MLL target genes, such as HOXA9 and MEIS1. Both HOXA9 and MEIS1 are required for MLL-fusion gene-induced leukemogenesis. Co-expression of HOXA9 and MEIS1 induces acute myeloid leukemia (AML) similar to that seen in mice in which MLL-fusion genes are over-expressed. p27(kip1) (p27 hereafter), a negative regulator of the cell cycle, has also been defined as an MLL target, the expression of which is up-regulated in MLL leukemic cells (LCs). To investigate whether p27 plays a role in the pathogenesis of MLL-leukemia, we examined the effects of p27 deletion (p27(-/-)) on MLL-AF9 (MA9)-induced murine AML development. HOXA9/MEIS1 (H/M)-induced, p27 wild-type (p27(+/+)) and p27(-/-) AML were studied in parallel as controls. We found that LCs from both MA9-AML and H/M-AML can be separated into three fractions, a CD117(-)CD11b(hi) differentiated fraction as well as CD117(+)CD11b(hi) and CD117(+)CD11b(lo), two less differentiated fractions. The CD117(+)CD11b(lo) fraction, comprising only 1-3% of total LCs, expresses higher levels of early hematopoietic progenitor markers but lower levels of mature myeloid cell markers compared to other populations of LCs. p27 is expressed and is required for maintaining the quiescent and drug-resistant states of the CD117(+)CD11b(lo) fraction of MA9-LCs but not of H/M-LCs. p27 deletion significantly compromises the leukemogenic capacity of CD117(+)CD11b(lo) MA9-LCs by reducing the frequency of leukemic stem cells (LSCs) but does not do so in H/M-LCs. In addition, we found that p27 is highly expressed and required for cell cycle arrest in the CD117(-)CD11b(hi) fraction in both types of LCs. Furthermore, we found that c-Myc expression is required for maintaining LCs in an undifferentiated state independently of proliferation. We concluded that p27 represses the proliferation of LCs, which is specifically required for maintaining the quiescent and drug-resistant states of a small subset of MA9-LSCs in collaboration with the differentiation blockage function of c-Myc.

Breaking the LSD1/KDM1A addiction: therapeutic targeting of the epigenetic modifier in AML.

KDM1A/LSD1, a histone H3K4/K9 demethylase and epigenetic regulator with roles in both gene activation and repression, has increased expression in multiple cancer types. Harris et al., in this issue of Cancer Cell, and Schenk et al. show that KDM1A may be a viable therapeutic target in treating AML.

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

HOXA9 and MEIS1 have essential oncogenic roles in mixed lineage leukaemia (MLL)-rearranged leukaemia. Here we show that they are direct targets of miRNA-196b, a microRNA (miRNA) located adjacent to and co-expressed with HOXA9, in MLL-rearranged leukaemic cells. Forced expression of miR-196b significantly delays MLL-fusion-mediated leukemogenesis in primary bone marrow transplantation through suppressing Hoxa9/Meis1 expression. However, ectopic expression of miR-196b results in more aggressive leukaemic phenotypes and causes much faster leukemogenesis in secondary transplantation than MLL fusion alone, likely through the further repression of Fas expression, a proapoptotic gene downregulated in MLL-rearranged leukaemia. Overexpression of FAS significantly inhibits leukemogenesis and reverses miR-196b-mediated phenotypes. Targeting Hoxa9/Meis1 and Fas by miR-196b is probably also important for normal haematopoiesis. Thus, our results uncover a previously unappreciated miRNA-regulation mechanism by which a single miRNA may target both oncogenes and tumour suppressors, simultaneously, or, sequentially, in tumourigenesis and normal development per cell differentiation, indicating that miRNA regulation is much more complex than previously thought.

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.