RHOA-regulated IGFBP2 promotes invasion and drives progression of BCR-ABL1 chronic myeloid leukemia.

The Philadelphia 9;22 chromosome translocation has two common isoforms that are preferentially associated with distinct subtypes of leukemia. The p210 variant is the hallmark of chronic myeloid leukemia (CML) whereas p190 is frequently associated with B-cell acute lymphoblastic leukemia. The only sequence difference between the two isoforms is the guanidine exchange factor domain. This guanidine exchange factor is reported to activate RHO family GTPases in response to diverse extracellular stimuli. It is not clear whether and, if so, how RHOA contributes to progression of p210 CML. Here we show that knockout of RHOA in the K562 and KU812, p210-expressing cell lines leads to suppression of leukemogenesis in animal models in vivo. RNA-sequencing analysis of the mock control and null cells demonstrated a distinct change in the gene expression profile as a result of RHOA deletion, with significant downregulation of genes involved in cell activation and cell adhesion. Cellular analysis revealed that RHOA knockout leads to impaired cell adhesion and migration and, most importantly, the homing ability of leukemia cells to the bone marrow, which may be responsible for the attenuated leukemia progression. We also identified IGFBP2 as an important downstream target of RHOA. Further mechanistic investigation showed that RHOA activation leads to relocation of the serum response factor (SRF) into the nucleus, where it directly activates IGFBP2. Knockout of IGFBP2 in CML cells suppressed cell adhesion/invasion, as well as leukemogenesis in vivo. This elevated IGFBP2 expression was confirmed in primary CML samples. Thus, we demonstrate one mechanism whereby the RHOA-SRF-IGFBP2 signaling axis contributes to the development of leukemia in cells expressing the p210 BCR-ABL1 fusion kinase.

Variant profiles of genes mapping to chromosome 16q loss in Wilms tumors reveals link to cilia-related genes and pathways.

BACKGROUND: Wilms tumor is the most common pediatric renal tumor and the fourth most common malignancy in children. Chromosome 16q deletion(del) or loss of heterozygosity (LOH) has been correlated with recurrence and overall poor prognosis, such that patients with 16qLOH and 1p allelic loss are treated with more aggressive chemotherapeutic regimens. METHODS: In the present study, we have compared the variant profiles of Wilms tumors with and without 16q del/LOH using both data available from the TARGET database (42 samples) and tumors procured from our legacy collection (8 samples). Exome-Seq data was analyzed for tumor specific variants mapping to 16q. Whole exome analysis was also performed. An unbiased approach for somatic variant analysis was used to detect tumor-specific, somatic variants. RESULTS: Of the 72 genes mapping to 16q, 42% were cilia-related genes and 28% of these were found to carry somatic variants specific to those tumors with 16qdel/LOH. Whole exome analyses further revealed that 30% of cilia-related genes across the genome carried alterations in tumors both with and without 16qdel/LOH. Additional pathway analyses revealed that many cilia-related pathway members also carried deleterious variant in these tumors including Sonic Hedgehog (SHh), Wnt, and Notch signaling pathways. CONCLUSIONS: The data suggest that cilia-related genes and pathways are compromised in Wilms tumors. The genes on chromosome 16q that carry deleterious variants in cilia-related genes may account for the more aggressive nature of tumors with 16q del/LOH.

Distinct signaling programs associated with progression of FGFR1 driven leukemia in a mouse model of stem cell leukemia lymphoma syndrome.

Constitutive activation of FGFR1 as a result of chromosome translocations is responsible for the development of a hematopoietic stem cell disorder that progresses to AML. We have developed a syngeneic mouse model of BCR-FGFR1 driven AML and used RNASeq to define gene expression signatures associated with disease progression. The development of the leukemic stem cells (LSC) is associated with a profound downregulation of specific transcription factors that normally maintain stem cell quiescence as well as cell adhesion and motility gene sets related to confinement to the stem cell niche. A prominent feature of the LSCs is the upregulation of genes involved in T-cell function, activation, migration and development. Despite this apparent T-cell priming in the LSCs, however, the majority of these genes are subsequently inactivated in the leukemic blast cells that derive from them. These studies provide insights into the molecular etiology of development and progression of FGFR1 driven AML.

Genomic analysis of racial differences in triple negative breast cancer.

Triple negative breast cancer (TNBC) is more prevalent in African Americans (AAs), has a more aggressive clinical course including a higher mortality rate and an increased occurrence of metastases. This study was designed to determine if racial differences at the molecular level might explain the more aggressive phenotype in AAs. Mutation profiling, was performed on 51 AA and 77 CA tumor/ normal pairs. Transcript expression analysis was performed on 35AA and 37CA. Genes with high frequency mutation rates such as MUC4 and TP53 were common to both racial populations, however genes that were less frequently mutated differed between the races suggesting that those cause the more aggressive nature of TNBC in AA women. JAK-Stat and HER2 signaling were unique to the AA and PTEN and mTOR were unique to the CA profiles. Many pathways identified by the mutational profiles were predicted to be down-regulated by the transcript expression profiles.

miR-339 Promotes Development of Stem Cell Leukemia/Lymphoma Syndrome via Downregulation of the BCL2L11 and BAX Proapoptotic Genes.

The development of myeloid and lymphoid neoplasms related to overexpression of FGFR1 kinases as a result of chromosome translocations depends on the promotion of a stem cell phenotype, suppression of terminal differentiation, and resistance to apoptosis. These phenotypes are related to the stem cell leukemia/lymphoma syndrome (SCLL), which arises through the effects of the activated FGFR1 kinase on gene transcription, which includes miRNA dysregulation. In a screen for miRNAs that are directly regulated by FGFR1, and which stimulate cell proliferation and survival, we identified miR-339-5p, which is highly upregulated in cells carrying various different chimeric kinases. Overexpression of miR-339-5p in SCLL cell types enhances cell survival and inhibition of its function leads to reduced cell viability. miR-339-5p overexpression protects cells from the consequences of FGFR1 inactivation, promoting cell-cycle progression and reduced apoptosis. Transient luciferase reporter assays and qRT-PCR detection of endogenous miR-339-5p expression in stably transduced cell lines demonstrated that BCR-FGFR1 can directly regulate miR-339-5p expression. This correlation between miR-339-5p and FGFR1 expression is also seen in primary human B-cell precursor acute lymphoblastic leukemia. In a screen to identify targets of miR-339-5p, we identified and verified the BCL2L11 and BAX genes, which can promote apoptosis. In vivo, SCLL cells forced to overexpress miR-339-5p show a more rapid onset of disease and poorer survival compared with parental cells expressing endogenous levels of miR-339-5p. Analysis of human primary B-cell precursor ALL shows a significant higher expression of miR339-5p compared with the two cohorts of CLL patient samples, suggesting direct roles in disease progression and supporting the evidence generated in mouse models of SCLL.Significance: Proapoptiotic genes that are direct targets of miR-339-5p significantly influence promotion and aggressive development of leukemia/lymphomas associated with FGFR1 overexpression. Cancer Res; 78(13); 3522-31. ©2018 AACR.

The miR-17/92 cluster is involved in the molecular etiology of the SCLL syndrome driven by the BCR-FGFR1 chimeric kinase.

MicroRNAs (miRNAs) have pathogenic roles in the development of a variety of leukemias. Here we identify miRNAs that have important roles in the development of B lymphomas resulting from the expression of the chimeric BCR-FGFR1 kinase. The miR-17/92 cluster was particularly implicated and forced expression resulted in increased cell proliferation, while inhibiting its function using miRNA sponges reduced cell growth and induced apoptosis. Cells treated with the potent BGJ389 FGFR1 inhibitor led to miR-17/92 downregulation, suggesting regulation by FGFR1. Transient luciferase reporter assays and qRT-PCR detection of endogenous miR-17/92 expression in stable transduced cell lines demonstrated that BCR-FGFR1 can regulate miR-17/92 expression. This positive association of miR-17/92 with BCR-FGFR1 was also confirmed in primary mouse SCLL tissues and primary human CLL samples. miR-17/92 promotes cell proliferation and survival by targeting CDKN1A and PTEN in B-lymphoma cell lines and primary tumors. An inverse correlation in expression levels was seen between miR-17/92 and both CDKN1A and PTEN in two cohorts of CLL patients. Finally, in vivo engraftment studies demonstrated that manipulation of miR-17/92 was sufficient to affect BCR-FGFR1-driven leukemogenesis. Overall, our results define miR-17/92 as a downstream effector of FGFR1 in BCR-FGFR1-driven B-cell lymphoblastic leukemia.

Genome-wide analysis of HOXC9-induced neuronal differentiation of neuroblastoma cells.

Induction of differentiation is a therapeutic strategy in neuroblastoma, a common pediatric cancer of the sympathetic nervous system. The homeobox protein HOXC9 is a key regulator of neuroblastoma differentiation. To gain a molecular understanding of the function of HOXC9 in promoting differentiation of neuroblastoma cells, we conducted a genome-wide analysis of the HOXC9-induced differentiation program by microarray gene expression profiling and chromatin immunoprecipitation in combination with massively parallel sequencing (ChIP-seq). Here we describe in details the experimental system, methods, and quality control for the generation of the microarray and ChIP-seq data associated with our recent publication [1].

Indoleamine 2,3-dioxygenase inhibition alters the non-coding RNA transcriptome following renal ischemia-reperfusion injury.

BACKGROUND: Indoleamine 2,3 dioxygenase (IDO) degrades the essential amino acid tryptophan and has been shown to minimize rejection in animal models of renal transplantation. Ischemia-reperfusion injury (IRI) is unavoidable in renal transplantation and correlates with shorter graft survival times. Despite its favorable effects on rejection, there is evidence that IDO may facilitate renal IRI. Differentiating the negative impact of IDO on IRI from its pro-tolerant effects in allograft rejection is of clinical relevance. In these studies we hypothesized that constitutive IDO activity may influence renal genes associated with recovery from IRI, and that IDO inhibition may unmask these effects. METHODS: We examined the renal transcriptome in a rat model of IRI with and without IDO inhibition with 1-methyl-d-tryptophan (1-MT), and assessed for alterations in the gene expression signature. RESULTS: These studies demonstrated that during recovery from renal IRI, pre-treatment with 1-MT alleviated alterations in 105 coding sequences associated with IRI, and in turn triggered new changes in 66 non-coding transcripts, the majority of which were represented by small nucleolar RNA. CONCLUSION: These results suggest a biologic role for non-coding, IDO-dependent genes in regulating the early response to IRI.

Dysregulated signaling pathways in the development of CNTRL-FGFR1-induced myeloid and lymphoid malignancies associated with FGFR1 in human and mouse models.

Myeloid and lymphoid neoplasm associated with FGFR1 is an aggressive disease, and resistant to all the current chemotherapies. To define the molecular etiology of this disease, we have developed murine models of this disease, in syngeneic hosts as well as in nonobese diabetic/severe combined immunodeficiency/interleukin 2Rγ(null) mice engrafted with transformed human CD34+ hematopoietic stem/progenitor cells. Both murine models mimic the human disease with splenohepatomegaly, hypercellular bone marrow, and myeloproliferative neoplasms that progresses to acute myeloid leukemia. Molecular genetic analyses of these model mice, as well as primary human disease, demonstrated that CNTRL-FGFR1, through abnormal activation of several signaling pathways related to development and differentiation of both myeloid and T-lymphoid cells, contribute to overt leukemogenesis. Clonal evolution analysis indicates that myeloid related neoplasms arise from common myeloid precursor cells that retain potential for T-lymphoid differentiation. These data indicate that simultaneously targeting these pathways is essential to successfully treating this almost invariably lethal disease.

Regulation of retinoschisin secretion in Weri-Rb1 cells by the F-actin and microtubule cytoskeleton.

Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process.

A novel nonsense mutation in a Japanese family with ataxia with oculomotor apraxia type 2 (AOA2).

We report a 67-year-old Japanese woman with ataxia with oculomotor apraxia type 2 (AOA2). She was born to consanguineous parents and showed a teenage onset, a slowly progressive cerebellar ataxia and sensory-motor neuropathy and an elevated level of serum alpha-fetoprotein (AFP). All of these clinical features were consistent with typical AOA2. She lacked oculomotor apraxia, as frequently observed in previously reported AOA2 patients. She was homozygous for a novel nonsense mutation, Glu385Ter (E385X), in the senataxin gene (SETX). To our knowledge, this is the fifth Japanese family with genetically confirmed AOA2. The mutations in SETX in Japanese AOA2 families are heterogeneous, except for M274I, which has been found in two unrelated families. More extensive screening by serum AFP followed by molecular genetic analysis of SETX in patients with Friedreich's ataxia-like phenotype may show that AOA2 is more common in Japan than previously thought.

Tissue-specific differentially methylated regions of the human VASA gene are potentially associated with maturation arrest phenotype in the testis.

Numerous CpG islands containing tissue-specific differentially methylated regions (TDMRs) are potential methylation sites in normal cells and tissues. The VASA (also known as DDX4) gene is believed to be under the control of TDMRs. A total of 131 male patients with idiopathic azoospermia or severe oligospermia were evaluated histologically, and the methylation status of CpG islands in the VASA gene was screened. Genome DNAs were obtained from testicular biopsy and modified with sodium bisulfite, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied. This system is capable of analyzing both the methylated and unmethylated CpG island in the genome. The methylation analysis is conducted by an epigram as graphic data. On histological assessment, 17 of 131 patients revealed maturation arrest (MA).In all, 6 of the 17 patients showed particularly high VASA TDMR methylation rates, whereas the remaining 11 patients and controls had low methylation rates. This study may imply that the VASA TDMR methylation is significantly higher among patients with MA, in whom the VASA gene expression was silenced. This finding represents an important contribution to the molecular basis of meiotic arrest as one possible cause of idiopathic infertility.

Quantitative analysis of human tissue-specific differences in methylation.

Tissue-specific differentially methylated regions (tDMRs) have been identified and implicated for their indispensable involvement in mammalian development and tissue differentiation. In this report, a quantitative DNA methylation analysis was performed for 13 human orthologous regions of recently confirmed mouse tDMRs by using Sequenom Mass Array, by which bisulfite-treated fragments are quantitatively detected using time of flight mass spectroscopy analysis. Eight regions were shown as tDMRs in various tissues from three independent individuals. Testis DNA samples from eight individuals were also analyzed for methylation. Interestingly, there is evidence that the DNA methylation level is divergent among individuals. DNA methylation levels of five testis-specific DMRs were significantly inversely correlated with the number of spermatocytes. However, a positive correlation was seen at tDMRs located near the TRIM38 and CASZ1 genes. Our results indicate that tDMRs are conserved between mouse and human and may have an important role in regulating tissue function, differentiation, and aging.

Development of pyrrole-imidazole polyamide for specific regulation of human aurora kinase-A and -B gene expression.

Pyrrole-imidazole polyamide (PIP) is a nuclease-resistant novel compound that inhibits gene expression through binding to the minor groove of DNA. Human aurora kinase-A (AURKA) and -B (AURKB) are important regulators in mitosis during the cell cycle. In this study, two specific PIPs (PIP-A and PIP-B) targeting AURKA and AURKB promoter regions were designed and synthesized, and their biological effects were investigated by several in vitro assays. PIP-A and PIP-B significantly inhibited the promoter activities, mRNA expression, and protein levels of AURKA and AURKB, respectively, in a concentration-dependent manner. Moreover, 1:1 combination treatment with both PIPs demonstrated prominent antiproliferative synergy (CI value [ED(50)] = 0.256) to HeLa cells as a result of inducing apoptosis-mediated severe catastrophe of cell-cycle progression. The novel synthesized PIP-A and PIP-B are potent and specific gene-silencing agents for AURKA and AURKB.

Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: clinical application for diagnosis of tuberculous meningitis.

Although the "gold standard" for diagnosis of tuberculous meningitis (TBM) is bacterial isolation of Mycobacterium tuberculosis, there are still several complex issues. Recently, we developed an internally controlled novel wide-range quantitative nested real-time PCR (WR-QNRT-PCR) assay for M. tuberculosis DNA in order to rapidly diagnose TBM. For use as an internal control calibrator to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. Due to the development of the NM-plasmid, the WR-QNRT-PCR assay demonstrated statistically significant accuracy over a wide detection range (1 to 10(5) copies). In clinical applications, the WR-QNRT-PCR assay revealed sufficiently high sensitivity (95.8%) and specificity (100%) for 24 clinically suspected TBM patients. In conditional logistic regression analysis, a copy number of M. tuberculosis DNA (per 1 ml of cerebrospinal fluid) of >8,000 was an independent risk factor for poor prognosis for TBM (i.e., death) (odds ratio, 16.142; 95% confidence interval, 1.191 to 218.79; P value, 0.0365). In addition, the copy numbers demonstrated by analysis of variance statistically significant alterations (P < 0.01) during the clinical treatment course for 10 suspected TBM patients. In simple regression analysis, the significant correlation (R(2) = 0.597; P < 0.0001) was demonstrated between copy number and clinical stage of TBM. We consider the WR-QNRT-PCR assay to be a useful and advanced assay technique for assessing the clinical treatment course of TBM.

Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: development and methodology.

Previously, we designed an internally controlled quantitative nested real-time (QNRT) PCR assay for Mycobacterium tuberculosis DNA in order to rapidly diagnose tuberculous meningitis. This technique combined the high sensitivity of nested PCR with the accurate quantification of real-time PCR. In this study, we attempted to improve the original QNRT-PCR assay and newly developed the wide-range QNRT-PCR (WR-QNRT-PCR) assay, which is more accurate and has a wider detection range. For use as an internal-control "calibrator" to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. It had artificial random nucleotides in five regions annealing specific primers and probes. The NM-plasmid demonstrated statistically uniform amplifications (F = 1.086, P = 0.774) against a range (1 to 10(5)) of copy numbers of mimic M. tuberculosis DNA and was regarded as appropriate for use as a new internal control in the WR-QNRT-PSR assay. In addition, by the optimization of assay conditions in WR-QNRT-PCR, two-step amplification of target DNA was completely consistent with the standard curve of this assay. Due to the development of the NM-plasmid as the new internal control, significantly improved quantitative accuracy and a wider detection range were realized with the WR-QNRT-PCR assay. In the next study, we will try to use this novel assay method with actual clinical samples and examine its clinical usefulness.

Analysis of tissue-specific differentially methylated regions (TDMs) in humans.

Alterations in DNA methylation have been implicated in mammalian development. Hence, the identification of tissue-specific differentially methylated regions (TDMs) is indispensable for understanding its role. Using restriction landmark genomic scanning of six mouse tissues, 150 putative TDMs were identified and 14 were further analyzed. The DNA sequences of the 14 mouse TDMs are analyzed in this study. Six of the human homologous regions show TDMs to both mouse and human and genes in five of these regions have conserved tissue-specific expression: preferential expression in testis. A TDM, DDX4, is further analyzed in nine testis tissues. An increase in methylation of the promoter region is significantly associated with a marked reduction of the gene expression and defects in spermatogenesis, suggesting that hypomethylation of the DDX4 promoter region regulates DDX4 gene expression in spermatogenic cells. Our results indicate that some genomic regions with tissue-specific methylation and expression are conserved between mouse and human and suggest that DNA methylation may have an important role in regulating differentiation and tissue-/cell-specific gene expression of some genes.

Disruption of the gene encoding the beta1-subunit of transducin in the Rd4/+ mouse.

PURPOSE: The Rd4/+ mouse inherits an autosomal dominant retinal degeneration that cosegregates with a large inversion spanning nearly all of mouse chromosome 4 (Chr 4). This inversion is homozygous lethal. The hypothesis for the study was that disruption of a gene at one of the two breakpoints in the Rd4 chromosome is responsible for the retinal degeneration. The purpose was to identify the disrupted gene. METHODS: Genotyping was performed by PCR and gel electrophoresis. The Rd4/+ phenotype was confirmed by ERG. Fluorescence in situ hybridization (FISH) analysis was performed with bacterial artificial chromosome (BAC) probes. Northern and quantitative PCR procedures were used to evaluate Gnb1 mRNA expression. Protein expression was measured by Western blot. RESULTS: To identify the Rd4 gene defect, the breakpoints were first localized with a testcross and the locus refined by using FISH. Genetic testcross data revealed that the inversion breakpoints are located within a few centimorgans of both the telomeric and centromeric ends of Chr 4. Initial FISH analysis showed the proximal breakpoint of the inversion to be in the centromere itself. Therefore, we focused on the distal breakpoint and found that it lies in the second intron of the gene Gnb1, coding for the transducin beta1-subunit (Tbeta1) protein that is directly involved in the response to light of rod photoreceptors. Before the beginning of retinal degeneration in Rd4/+ retina, the levels of Gnb1 mRNA and Tbeta1 protein are 50% of those in wild-type retina. CONCLUSIONS: The results suggest that disruption of the Gnb1 gene is responsible for Rd4 retinal disease.

Characterization of FAM10A4, a member of the ST13 tumor suppressor gene family that maps to the 13q14.3 region associated with B-Cell leukemia, multiple myeloma, and prostate cancer.

Using the 650-kb DNA sequence from the minimally deleted region in B-cell chronic lymphocytic leukemia (BCLL), we have identified a new gene, FAM10A4, that maps to the proximal end of the region. This gene has been shown to be part of a now six-member family of genes with high homology to the ST13 tumor suppressor gene. We have established conditions to specifically undertake mutation studies of the chromosome 13 member of this family and have identified a Ser71Leu change in BCLL samples, which is apparently a polymorphism. The characterization of this gene will permit mutation studies in other tumor cell types such as multiple myeloma and prostate cancer, which also show genetic loss in the 13q14 region.