Upregulated heme biosynthesis, an exploitable vulnerability in MYCN-driven leukemogenesis.

The increased heme biosynthesis long observed in leukemia was previously of unknown significance. Heme, synthesized from porphyrin precursors, plays a central role in oxygen metabolism and mitochondrial function, yet little is known about its role in leukemogenesis. Here, we show increased expression of heme biosynthetic genes, including UROD, only in pediatric AML samples that have high MYCN expression. High expression of both UROD and MYCN predicts poor overall survival and unfavorable outcomes in adult AML. Murine leukemic progenitors derived from hematopoietic progenitor cells (HPCs) overexpressing a MYCN cDNA (MYCN-HPCs) require heme/porphyrin biosynthesis, accompanied by increased oxygen consumption, to fully engage in self-renewal and oncogenic transformation. Blocking heme biosynthesis reduced mitochondrial oxygen consumption and markedly suppressed self-renewal. Leukemic progenitors rely on balanced production of heme and heme intermediates, the porphyrins. Porphyrin homeostasis is required because absence of the porphyrin exporter, ABCG2, increased death of leukemic progenitors in vitro and prolonged the survival of mice transplanted with Abcg2-KO MYCN-HPCs. Pediatric AML patients with elevated MYCN mRNA display strong activation of TP53 target genes. Abcg2-KO MYCN-HPCs were rescued from porphyrin toxicity by p53 loss. This vulnerability was exploited to show that treatment with a porphyrin precursor, coupled with the absence of ABCG2, blocked MYCN-driven leukemogenesis in vivo, thereby demonstrating that porphyrin homeostasis is a pathway crucial to MYCN leukemogenesis.

Leukemia and ABC transporters.

Acute myeloid leukemia (AML) is a heterogeneous disease caused by aberrant proliferation and/or differentiation of myeloid progenitors. However, only ~65% of AML patients respond to induction chemotherapy and the overall survival rate for AML remains low (~24% for 5-year survival). The conventional view suggests that ATP-binding cassette (ABC) transporters contribute to treatment failure due to their drug-effluxing capabilities. This might be overly simplistic. Some ABC transporters export endogenous substrates that have defined roles in normal hematopoietic progenitors. It is conceivable that these substances also provide an advantage to leukemic progenitors. This review will highlight how certain endogenous substrates impact normal hematopoietic cells and suggest that ABC transporters facilitate export of these substances to affect both normal hematopoietic and leukemic progenitors. For example, the ability to export certain endogenous ligands may facilitate leukemogenesis by modifying leukemic progenitor cell proliferation or survival. If so, the addition of ABC transporter inhibitors to traditional chemotherapy might improve therapeutic efficacy by not just increasing intracellular drug accumulation but also blocking the beneficial effects ABC transporter ligands have on cell survival.

Mina: a Th2 response regulator meets TGFß.

The JmjC protein Mina is an important immune response regulator. Classical forward genetics first discovered its immune role in 2009 in connection with the development of T helper 2 (Th2) cells. This prompted investigation into Mina's role in the two best-studied contexts where Th2 responses are essential: atopic asthma and helminth expulsion. In work focused on a mouse model of atopic asthma, Mina deficiency was found to ameliorate airway hyper-resistance and pulmonary inflammation. And, in a case-control study genetic variation at the human MINA locus was found to be associated with the development of childhood atopic asthma. Although the underlying cellular and molecular mechanism of Mina's involvement in pulmonary inflammation remains unknown, our recent work on parasitic helminth expulsion suggests the possibility that, rather than T cells, epithelial cells responding to TGFß may play the dominant role. Here we review the growing body of literature on the emerging Mina pathway in T cells and epithelial cells and attempt to set these into a broader context.

Transcriptional activation of Mina by Sp1/3 factors.

Mina is an epigenetic gene regulatory protein known to function in multiple physiological and pathological contexts, including pulmonary inflammation, cell proliferation, cancer and immunity. We showed previously that the level of Mina gene expression is subject to natural genetic variation linked to 21 SNPs occurring in the Mina 5' region. In order to explore the mechanisms regulating Mina gene expression, we set out to molecularly characterize the Mina promoter in the region encompassing these SNPs. We used three kinds of assays--reporter, gel shift and chromatin immunoprecipitation--to analyze a 2 kb genomic fragment spanning the upstream and intron 1 regions flanking exon 1. Here we discovered a pair of Mina promoters (P1 and P2) and a P1-specific enhancer element (E1). Pharmacologic inhibition and siRNA knockdown experiments suggested that Sp1/3 transcription factors trigger Mina expression through additive activity targeted to a cluster of four Sp1/3 binding sites forming the P1 promoter. These results set the stage for comprehensive analysis of Mina gene regulation from the context of tissue specificity, the impact of inherited genetic variation and the nature of upstream signaling pathways.

Small interfering RNA-mediated silencing induces target-dependent assembly of GW/P bodies.

Gene silencing using small interfering RNA (siRNA) is a valuable laboratory tool and a promising approach to therapeutics for a variety of human diseases. Recently, RNA interference (RNAi) has been linked to cytoplasmic GW bodies (GWB). However, the correlation between RNAi and the formation of GWB, also known as mammalian processing bodies, remains unclear. In this report, we show that transfection of functional siRNA induced larger and greater numbers of GWB. This siRNA-induced increase of GWB depended on the endogenous expression of the target mRNA. Knockdown of GW182 or Ago2 demonstrated that the siRNA-induced increase of GWB required these two proteins and correlated with RNAi. Furthermore, knockdown of rck/p54 or LSm1 did not prevent the reassembly of GWB that were induced by and correlated with siRNA-mediated RNA silencing. We propose that RNAi is a key regulatory mechanism for the assembly of GWB, and in some cases, GWB may serve as markers for RNAi in mammalian cells.

The role of GW/P-bodies in RNA processing and silencing.

GW bodies, also known as mammalian P-bodies, are cytoplasmic foci involved in the post-transcriptional regulation of eukaryotic gene expression. Recently, GW bodies have been linked to RNA interference and demonstrated to be important for short-interfering-RNA- and microRNA-mediated mRNA decay and translational repression. Evidence indicates that both passenger and guide strands of short-interfering RNA duplexes can localize to GW bodies, thereby indicating that RNA-induced silencing complexes may be activated within these cytoplasmic centers. Formation of GW bodies appears to depend on both specific protein factors and RNA, in particular, microRNA. Work over the past few years has significantly increased our understanding of the biology of GW bodies, revealing that they are specialized cell components that spatially regulate mRNA turnover in various biological processes. The formation of GW bodies appears to depend on both specific protein factors and RNA, in particular, microRNA. Here, we propose a working model for GW body assembly in terms of its relationship to RNA interference. In this process, one or more heteromeric protein complexes accumulate in successive steps into larger ribonucleoprotein structures.

GW bodies, microRNAs and the cell cycle.

GW bodies (GWBs) are cytoplasmic foci initially identified through the use of an autoimmune serum targeting the marker protein, GW182. GWBs were first considered as both storage centers for a specific subset of mRNAs and degradation sites for mRNAs. Interestingly, they are known to vary in size and number throughout the cell cycle and are largest in size and most abundant in number during the late S and G2 phases. Recent studies have linked RNA interference to GWBs, in that disruption or disassembly of GWBs was demonstrated to impair siRNA and miRNA silencing activity. As miRNAs are implicated in the regulation of cell cycle progression and cell proliferation, it is very likely that GWBs, the critical intracellular structures for miRNA function, may very well be also linked to this cellular process.

Disruption of GW bodies impairs mammalian RNA interference.

The GW182 RNA-binding protein was initially shown to associate with a specific subset of mRNAs and to reside within discrete cytoplasmic foci named GW bodies (GWBs). GWBs are enriched in proteins that are involved in mRNA degradation. Recent reports have shown that exogenously introduced human Argonaute-2 (Ago2) is also enriched in GWBs, indicating that RNA interference function may be somehow linked to these structures. In this report, we demonstrate that endogenous Ago2 and transfected small interfering RNAs (siRNAs) are also present within these same cytoplasmic bodies and that the GW182 protein interacts with Ago2. Disruption of these cytoplasmic foci in HeLa cells interferes with the silencing capability of a siRNA that is specific to lamin-A/C. Our data support a model in which GW182 and/or the microenvironment of the cytoplasmic GWBs contribute to the RNA-induced silencing complex and to RNA silencing.