PI3Kγ/δ and NOTCH1 Cross-Regulate Pathways That Define the T-cell Acute Lymphoblastic Leukemia Disease Signature.

PI3K/AKT and NOTCH1 signaling pathways are frequently dysregulated in T-cell acute lymphoblastic leukemias (T-ALL). Although we have shown that the combined activities of the class I PI3K isoforms p110γ and p110δ play a major role in the development and progression of PTEN-null T-ALL, it has yet to be determined whether their contribution to leukemogenic programing is unique from that associated with NOTCH1 activation. Using an Lmo2-driven mouse model of T-ALL in which both the PI3K/AKT and NOTCH1 pathways are aberrantly upregulated, we now demonstrate that the combined activities of PI3Kγ/δ have both overlapping and distinct roles from NOTCH1 in generating T-ALL disease signature and in promoting tumor cell growth. Treatment of diseased animals with either a dual PI3Kγ/δ or a γ-secretase inhibitor reduced tumor burden, prolonged survival, and induced proapoptotic pathways. Consistent with their similar biological effects, both inhibitors downregulated genes involved in cMYC-dependent metabolism in gene set enrichment analyses. Furthermore, overexpression of cMYC in mice or T-ALL cell lines conferred resistance to both inhibitors, suggesting a point of pathway convergence. Of note, interrogation of transcriptional regulators and analysis of mitochondrial function showed that PI3Kγ/δ activity played a greater role in supporting the disease signature and critical bioenergetic pathways. Results provide insight into the interrelationship between T-ALL oncogenic networks and the therapeutic efficacy of dual PI3Kγ/δ inhibition in the context of NOTCH1 and cMYC signaling. Mol Cancer Ther; 16(10); 2069-82. ©2017 AACR.

The transcription factor GATA4 is required for follicular development and normal ovarian function.

Sex determination in mammals requires interaction between the transcription factor GATA4 and its cofactor FOG2. We have recently described the function of both proteins in testis development beyond the sex determination stage; their roles in the postnatal ovary, however, remain to be defined. Here, we use gene targeting in mice to determine the requirement of GATA4 and FOG2 in ovarian development and folliculogenesis. The results from this study identify an essential role of the GATA4 protein in the ovarian morphogenetic program. We show that in contrast to the sex determination phase, which relies on the GATA4-FOG2 complex, the subsequent regulation of ovarian differentiation is dependent upon GATA4 but not FOG2. The loss of Gata4 expression within the ovary results in impaired granulosa cell proliferation and theca cell recruitment as well as fewer primordial follicles in the ovarian cortex, causing a failure in follicular development. Preantral follicular atresia is observed within the few follicles that develop despite Gata4 deficiency. The depletion of the follicular pool in GATA4 deficient ovary results in the formation of ovarian cysts and sterility.

Targeting nonclassical oncogenes for therapy in T-ALL.

Constitutive phosphoinositide 3-kinase (PI3K)/Akt activation is common in T cell acute lymphoblastic leukemia (T-ALL). Although four distinct class I PI3K isoforms (α, ß, γ, δ) could participate in T-ALL pathogenesis, none has been implicated in this process. We report that in the absence of PTEN phosphatase tumor suppressor function, PI3Kγ or PI3Kδ alone can support leukemogenesis, whereas inactivation of both isoforms suppressed tumor formation. The reliance of PTEN null T-ALL on the combined activities of PI3Kγ/δ was further demonstrated by the ability of a dual inhibitor to reduce disease burden and prolong survival in mice as well as prevent proliferation and promote activation of proapoptotic pathways in human tumors. These results support combined inhibition of PI3Kγ/δ as therapy for T-ALL.

GATA transcription factors in the developing reproductive system.

Previous work has firmly established the role for both GATA4 and FOG2 in the initial global commitment to sexual fate, but their (joint or individual) function in subsequent steps remained unknown. Hence, gonad-specific deletions of these genes in mice were required to reveal their roles in sexual development and gene regulation. The development of tissue-specific Cre lines allowed for substantial advances in the understanding of the function of GATA proteins in sex determination, gonadal differentiation and reproductive development in mice. Here we summarize the recent work that examined the requirement of GATA4 and FOG2 proteins at several critical stages in testis and ovarian differentiation. We also discuss the molecular mechanisms involved in this regulation through the control of Dmrt1 gene expression in the testis and the canonical Wnt/ß-catenin pathway in the ovary.

Transcriptional profiling of C. elegans DAF-19 uncovers a ciliary base-associated protein and a CDK/CCRK/LF2p-related kinase required for intraflagellar transport.

Cilia are ubiquitous cell surface projections that mediate various sensory- and motility-based processes and are implicated in a growing number of multi-organ genetic disorders termed ciliopathies. To identify new components required for cilium biogenesis and function, we sought to further define and validate the transcriptional targets of DAF-19, the ciliogenic C. elegans RFX transcription factor. Transcriptional profiling of daf-19 mutants (which do not form cilia) and wild-type animals was performed using embryos staged to when the cell types developing cilia in the worm, the ciliated sensory neurons (CSNs), still differentiate. Comparisons between the two populations revealed 881 differentially regulated genes with greater than a 1.5-fold increase or decrease in expression. A subset of these was confirmed by quantitative RT-PCR. Transgenic worms expressing transcriptional GFP fusions revealed CSN-specific expression patterns for 11 of 14 candidate genes. We show that two uncharacterized candidate genes, termed dyf-17 and dyf-18 because their corresponding mutants display dye-filling (Dyf) defects, are important for ciliogenesis. DYF-17 localizes at the base of cilia and is specifically required for building the distal segment of sensory cilia. DYF-18 is an evolutionarily conserved CDK7/CCRK/LF2p-related serine/threonine kinase that is necessary for the proper function of intraflagellar transport, a process critical for cilium biogenesis. Together, our microarray study identifies targets of the evolutionarily conserved RFX transcription factor, DAF-19, providing a rich dataset from which to uncover-in addition to DYF-17 and DYF-18-cellular components important for cilium formation and function.

The retrograde IFT machinery of C. elegans cilia: two IFT dynein complexes?

We analyzed the relatively poorly understood IFT-dynein (class DYNC2)-driven retrograde IFT pathway in C. elegans cilia, which yielded results that are surprising in the context of current models of IFT. Assays of C. elegans dynein gene expression and intraflagellar transport (IFT) suggest that conventional IFT-dynein contains essential heavy (CHE-3), light-intermediate (XBX-1), plus three light polypeptide chains that participate in IFT, but no "essential" intermediate chain. IFT assays of XBX-1::YFP suggest that IFT-dynein is transported as cargo to the distal tip of the cilium by kinesin-2 motors, but independent of the IFT-particle/BBSome complexes. Finally, we were surprised to find that the subset of cilia present on the OLQ (outer labial quadrant) neurons assemble independently of conventional "CHE-3" IFT-dynein, implying that there is a second IFT-dynein acting in these cilia. We have found a novel gene encoding a dynein heavy chain, DHC-3, and two light chains, in OLQ neurons, which could constitute an IFT-dynein complex in OLQ neuronal cilia. Our results underscore several surprising features of retrograde IFT that require clarification.

An essential role for DYF-11/MIP-T3 in assembling functional intraflagellar transport complexes.

MIP-T3 is a human protein found previously to associate with microtubules and the kinesin-interacting neuronal protein DISC1 (Disrupted-in-Schizophrenia 1), but whose cellular function(s) remains unknown. Here we demonstrate that the C. elegans MIP-T3 ortholog DYF-11 is an intraflagellar transport (IFT) protein that plays a critical role in assembling functional kinesin motor-IFT particle complexes. We have cloned a loss of function dyf-11 mutant in which several key components of the IFT machinery, including Kinesin-II, as well as IFT subcomplex A and B proteins, fail to enter ciliary axonemes and/or mislocalize, resulting in compromised ciliary structures and sensory functions, and abnormal lipid accumulation. Analyses in different mutant backgrounds further suggest that DYF-11 functions as a novel component of IFT subcomplex B. Consistent with an evolutionarily conserved cilia-associated role, mammalian MIP-T3 localizes to basal bodies and cilia, and zebrafish mipt3 functions synergistically with the Bardet-Biedl syndrome protein Bbs4 to ensure proper gastrulation, a key cilium- and basal body-dependent developmental process. Our findings therefore implicate MIP-T3 in a previously unknown but critical role in cilium biogenesis and further highlight the emerging role of this organelle in vertebrate development.

Identification of novel regulatory factor X (RFX) target genes by comparative genomics in Drosophila species.

BACKGROUND: Regulatory factor X (RFX) transcription factors play a key role in ciliary assembly in nematode, Drosophila and mouse. Using the tremendous advantages of comparative genomics in closely related species, we identified novel genes regulated by dRFX in Drosophila. RESULTS: We first demonstrate that a subset of known ciliary genes in Caenorhabditis elegans and Drosophila are regulated by dRFX and have a conserved RFX binding site (X-box) in their promoters in two highly divergent Drosophila species. We then designed an X-box consensus sequence and carried out a genome wide computer screen to identify novel genes under RFX control. We found 412 genes that share a conserved X-box upstream of the ATG in both species, with 83 genes presenting a more restricted consensus. We analyzed 25 of these 83 genes, 16 of which are indeed RFX target genes. Two of them have never been described as involved in ciliogenesis. In addition, reporter construct expression analysis revealed that three of the identified genes encode proteins specifically localized in ciliated endings of Drosophila sensory neurons. CONCLUSION: Our X-box search strategy led to the identification of novel RFX target genes in Drosophila that are involved in sensory ciliogenesis. We also established a highly valuable Drosophila cilia and basal body dataset. These results demonstrate the accuracy of the X-box screen and will be useful for the identification of candidate genes for human ciliopathies, as several human homologs of RFX target genes are known to be involved in diseases, such as Bardet-Biedl syndrome.

Identification of ciliary and ciliopathy genes in Caenorhabditis elegans through comparative genomics.

BACKGROUND: The recent availability of genome sequences of multiple related Caenorhabditis species has made it possible to identify, using comparative genomics, similarly transcribed genes in Caenorhabditis elegans and its sister species. Taking this approach, we have identified numerous novel ciliary genes in C. elegans, some of which may be orthologs of unidentified human ciliopathy genes. RESULTS: By screening for genes possessing canonical X-box sequences in promoters of three Caenorhabditis species, namely C. elegans, C. briggsae and C. remanei, we identified 93 genes (including known X-box regulated genes) that encode putative components of ciliated neurons in C. elegans and are subject to the same regulatory control. For many of these genes, restricted anatomical expression in ciliated cells was confirmed, and control of transcription by the ciliogenic DAF-19 RFX transcription factor was demonstrated by comparative transcriptional profiling of different tissue types and of daf-19(+) and daf-19(-) animals. Finally, we demonstrate that the dye-filling defect of dyf-5(mn400) animals, which is indicative of compromised exposure of cilia to the environment, is caused by a nonsense mutation in the serine/threonine protein kinase gene M04C9.5. CONCLUSION: Our comparative genomics-based predictions may be useful for identifying genes involved in human ciliopathies, including Bardet-Biedl Syndrome (BBS), since the C. elegans orthologs of known human BBS genes contain X-box motifs and are required for normal dye filling in C. elegans ciliated neurons.

Caenorhabditis elegans DYF-2, an orthologue of human WDR19, is a component of the intraflagellar transport machinery in sensory cilia.

The intraflagellar transport (IFT) machinery required to build functional cilia consists of a multisubunit complex whose molecular composition, organization, and function are poorly understood. Here, we describe a novel tryptophan-aspartic acid (WD) repeat (WDR) containing IFT protein from Caenorhabditis elegans, DYF-2, that plays a critical role in maintaining the structural and functional integrity of the IFT machinery. We determined the identity of the dyf-2 gene by transgenic rescue of mutant phenotypes and by sequencing of mutant alleles. Loss of DYF-2 function selectively affects the assembly and motility of different IFT components and leads to defects in cilia structure and chemosensation in the nematode. Based on these observations, and the analysis of DYF-2 movement in a Bardet-Biedl syndrome mutant with partially disrupted IFT particles, we conclude that DYF-2 can associate with IFT particle complex B. At the same time, mutations in dyf-2 can interfere with the function of complex A components, suggesting an important role of this protein in the assembly of the IFT particle as a whole. Importantly, the mouse orthologue of DYF-2, WDR19, also localizes to cilia, pointing to an important evolutionarily conserved role for this WDR protein in cilia development and function.

Analysis of xbx genes in C. elegans.

Cilia and flagella are widespread eukaryotic subcellular components that are conserved from green algae to mammals. In different organisms they function in cell motility, movement of extracellular fluids and sensory reception. While the function and structural description of cilia and flagella are well established, there are many questions that remain unanswered. In particular, very little is known about the developmental mechanisms by which cilia are generated and shaped and how their components are assembled into functional machineries. To find genes involved in cilia development we used as a search tool a promoter motif, the X-box, which participates in the regulation of certain ciliary genes in the nematode Caenorhabditis elegans. By using a genome search approach for X-box promoter motif-containing genes (xbx genes) we identified a list of about 750 xbx genes (candidates). This list comprises some already known ciliary genes as well as new genes, many of which we hypothesize to be important for cilium structure and function. We derived a C. elegans X-box consensus sequence by in vivo expression analysis. We found that xbx gene expression patterns were dependent on particular X-box nucleotide compositions and the distance from the respective gene start. We propose a model where DAF-19, the RFX-type transcription factor binding to the X-box, is responsible for the development of a ciliary module in C. elegans, which includes genes for cilium structure, transport machinery, receptors and other factors.