Modeling human MLL-AF9 translocated acute myeloid leukemia from single donors reveals RET as a potential therapeutic target.

Acute myeloid leukemias (AMLs) result from a series of genetic events occurring in a stem or progenitor hematopoietic cell that gives rise to their clonal expansion and an impaired capacity to differentiate. To circumvent the genetic heterogeneity of AML patient cohorts, we have developed a model system, driven by the MLL-AF9 (MA9) oncogene, to generate multiple human leukemias using progenitor cells from a single healthy donor. Through stepwise RNA-sequencing data generated using this model and AML patients, we have identified consistent changes associated with MA9-driven leukemogenesis and demonstrate that no recurrent secondary mutations are required. We identify 39 biomarkers whose high expression level is specific to this genetic subtype of AML and validate that many of these have diagnostic utility. We further examined one biomarker, the receptor tyrosine kinase (RTK) RET, and show through shRNA knockdowns that its expression is essential for in vivo and in vitro growth of MA9-AML. These results highlight the value of novel human models of AML derived from single donors using specific oncogenic fusions to understand their biology and to uncover potential therapeutic targets.

Epigenetic regulation of GATA2 and its impact on normal karyotype acute myeloid leukemia.

The GATA2 gene encodes a zinc-finger transcription factor that acts as a master regulator of normal hematopoiesis. Mutations in GATA2 have been implicated in the development of myelodysplastic syndrome and acute myeloid leukemia (AML). Using RNA sequencing we now report that GATA2 is either mutated with a functional consequence, or expressed at low levels in the majority of normal karyotype AML (NK-AML). We also show that low-GATA2-expressing specimens (GATA2(low)) exhibit allele-specific expression (ASE) (skewing) in more than half of AML patients examined. We demonstrate that the hypermethylation of the silenced allele can be reversed by exposure to demethylating agents, which also restores biallelic expression of GATA2. We show that GATA2(low) AML lack the prototypical R882 mutation in DNMT3A frequently observed in NK-AML patients and that The Cancer Genome Atlas AML specimens with DNMT3A R882 mutations are characterized by CpG hypomethylation of GATA2. Finally, we validate that several known missense single-nucleotide polymorphisms in GATA2 are actually loss-of-function variants, which, when combined with ASE, represent the equivalent of homozygous GATA2 mutations. From a broader perspective, this work suggests for the first time that determinants of ASE likely have a key role in human leukemia.

The TGF-ß-Smad3 pathway inhibits CD28-dependent cell growth and proliferation of CD4 T cells.

Transforming growth factor-ß (TGF-ß) maintains self-tolerance through a constitutive inhibitory effect on T-cell reactivity. In most physiological situations, the tolerogenic effects of TGF-ß depend on the canonical signaling molecule Smad3. To characterize how TGF-ß/Smad3 signaling contributes to maintenance of T-cell tolerance, we characterized the transcriptional landscape downstream of TGF-ß/Smad3 signaling in resting or activated CD4 T cells. We report that in the presence of TGF-ß, Smad3 modulates the expression of >400 transcripts. Notably, we identified 40 transcripts whose expression showed Smad3 dependence in both resting and activated cells. This 'signature' confirmed the non-redundant role of Smad3 in TGF-ß biology and identified both known and putative immunoregulatory genes. Moreover, we provide genomic and functional evidence that the TGF-ß/Smad3 pathway regulates T-cell activation and metabolism. In particular, we show that TGF-ß/Smad3 signaling dampens the effect of CD28 stimulation on T-cell growth and proliferation. The impact of TGF-ß/Smad3 signals on T-cell activation was similar to that of the mTOR inhibitor Rapamycin. Considering the importance of co-stimulation on the outcome of T-cell activation, we propose that TGF-ß-Smad3 signaling may maintain T-cell tolerance by suppressing co-stimulation-dependent mobilization of anabolic pathways.

Ly49 and CD94/NKG2: developmentally regulated expression and evolution.

Murine natural killer (NK) cells express two families of MHC class I-specific receptors, namely the Ly49 family and CD94/NKG2 heterodimers. Stochastic co-expression of these receptors generates diverse receptor repertoires in adult NK-cell populations, whereas fetal NK cells have much more limited receptor diversity as they mostly express CD94/NKG2A but not Ly49. These receptors are also expressed on CD8-T cells and NK1.1+ T cells and regulate their functions, but their expression pattern on NK cells is significantly different from those on T cells. Thus, expression of Ly49 and CD94/NKG2 is developmentally regulated. NK cells acquire the Ly49 family of receptors in an orderly manner as they differentiate from bone marrow progenitors in vitro. Similarly, acquisition of CD94 and NKG2 by NK cells as they differentiate from embryonic stem cells is also orderly To gain insight into the mechanisms regulating Ly49 expression, potential regulatory regions of several Ly49 genes have been examined. Ly49 genes with different expression patterns have remarkably similar sequences in the putative regulatory regions. Finally, a functional Ly49 gene has been identified in baboon, and primate comparisons suggest that functional extinction of the Ly49 gene in the human lineage seems to have been a relatively recent event.

Comparative analysis of the promoter regions and transcriptional start sites of mouse Ly49 genes.

Despite numerous studies on the function of Ly49 natural killer cell receptors in the mouse, relatively little is known about how these genes are regulated at the transcriptional level. In the present study, we sequenced and compared 800 bp of the promoter region of nine Ly49 genes from C57B1/6 mice. This comparison showed that there is a high degree of sequence identity between the genes, and also revealed a region which is conserved between the mouse genes and the human Ly49L gene, indicating a potential core promoter region. This analysis also found that Ly49B and H differ from the other genes in having long interspersed repetitive sequence in their promoter region which suggests a gene conversion or rearrangement involving these two genes. In addition, we performed 5' rapid amplification of cDNA ends on four Ly49 genes to localize transcriptional start sites. These experiments showed that the transcriptional initiation sites are heterogeneous for all of the genes examined, and that a large majority of Ly49G transcripts originate from the second exon as well as its first intron. Although potential TATA boxes have been previously identified for some of the genes, we did not find evidence that a majority of transcripts initiate at the expected distance downstream of these boxes. Our data suggest that differences in the location of transcriptional start sites contribute to the observed complexity in receptor repertoire patterns.

Functional analysis of 5' and 3' regions of the closely related Ly49c and j genes.

The Ly49 multigene family consists of at least 14 closely related genes located in the natural killer (NK) gene complex on mouse Chromosome 6. Reverse transcriptase (RT)-PCR on single NK cells has shown that Ly49c is expressed on approximately 50% of NK cells, whereas the closely related Ly49j gene is expressed on 5-8% of NK cells. In this study, we examined three regions to determine whether they contain cis-acting elements involved in regulating the expression of these two closely related Ly49 genes within NK cells. Luciferase reporter assays in EL-4 cells suggested that the 5' regions of Ly49c and j contain promoter elements and repressor sequences. In addition, luciferase assays suggest that Ly49j also contains an active promoter in the first intron, although the transcripts produced from this promoter appear to be severely truncated. Finally, comparisons of the 3' noncoding regions of Ly49c and j revealed that the sequence of Ly49j diverges completely from Ly49c 130 bp downstream of the termination codon. The polyadenylation signal for Ly49j is located downstream of the Ly49c poly(A) site, which results in a much longer 3' untranslated region (UTR). When the Ly49j 3'UTR was used to provide the polyadenylation signal for the green fluorescent protein (GFP) reporter gene, GFP expression was reduced twofold. These results suggest that both internal promoters, repressors, and 3' regions play a role in regulating Ly49 gene expression.