Maria Trissal

Gene expression profiling-based identification of cell-surface targets for developing multimeric ligands in pancreatic cancer

Multimeric ligands are ligands that contain multiple binding domains that simultaneously target multiple cell-surface proteins. Due to cooperative binding, multimeric ligands can have high avidity for cells (tumor) expressing all targeting proteins and only show minimal binding to cells (normal tissues) expressing none or only some of the targets. Identifying combinations of targets that concurrently express in tumor cells but not in normal cells is a challenging task. Here, we describe a novel approach for identifying such combinations using genome-wide gene expression profiling followed by immunohistochemistry. We first generated a database of mRNA gene expression profiles for 28 pancreatic cancer specimens and 103 normal tissue samples representing 28 unique tissue/cell types using DNA microarrays. The expression data for genes that encode proteins with cell-surface epitopes were then extracted from the database and analyzed using a novel multivariate rule-based computational approach to identify gene combinations that are expressed at an efficient binding level in tumors but not in normal tissues. These combinations were further ranked according to the proportion of tumor samples that expressed the sets at efficient levels. Protein expression of the genes contained in the top ranked combinations was confirmed using immunohistochemistry on a pancreatic tumor tissue and normal tissue microarrays. Coexpression of targets was further validated by their combined expression in pancreatic cancer cell lines using immunocytochemistry. These validated gene combinations thus encompass a list of cell-surface targets that can be used to develop multimeric ligands for the imaging and treatment of pancreatic cancer. [Mol Cancer Ther 2008;7(9):3071–80]

Identification of novel pancreatic adenocarcinoma cell-surface targets by gene expression profiling and tissue microarray

Pancreatic cancer has a high mortality rate, which is generally related to the initial diagnosis coming at late stage disease combined with a lack of effective treatment options. Novel agents that selectively detect pancreatic cancer have potential for use in the molecular imaging of cancer, allowing for non-invasive determination of tumor therapeutic response and molecular characterization of the disease. Such agents may also be used for the targeted delivery of therapy to tumor cells while decreasing systemic effects. Using complementary assays of mRNA expression profiling to determine elevated expression in pancreatic cancer tissues relative to normal pancreas tissues, and validation of protein expression by immunohistochemistry on tissue microarray, we have identified cell-surface targets with potential for imaging and therapeutic agent development. Expression profiles of 2177 cell-surface genes for 28 pancreatic tumor specimens and 4 normal pancreas tissue samples were evaluated. Expression in normal tissues was evaluated using array data from 103 samples representing 28 organ sites as well as mining published data. One-hundred seventy unique targets were highly expressed in 2 or more of the pancreatic tumor specimens and were not expressed in the normal pancreas samples. Two targets (TLR2 and ABCC3) were further validated for protein expression by tissue microarray (TMA) based immunohistochemistry. These validated targets have potential for the development of diagnostic imaging and therapeutic agents for pancreatic cancer.

Solid-phase synthetic strategy and bioevaluation of a labeled δ-opioid receptor ligand Dmt-Tic-Lys for In Vivo imaging

A general solid-phase synthetic strategy is developed to prepare fluorescent and/or lanthanide-labeled derivatives of the delta-opioid receptor (deltaOR) ligand H-Dmt-Tic-Lys(R)-OH. The high delta-OR affinity (K(i) = 3 nM) and desirable in vivo characteristics of the Cy5 derivative 1 suggest its usefulness for structure-function studies and receptor localization and as a high-contrast noninvasive molecular marker for live imaging ex vivo or in vivo.

Acquired copy number alterations of miRNA genes in acute myeloid leukemia are uncommon

Altered microRNA (miRNA) expression is frequently observed in acute myelogenous leukemia (AML) and has been implicated in leukemic transformation. Whether somatic copy number alterations (CNAs) are a frequent cause of altered miRNA gene expression is largely unknown. Herein, we used comparative genomic hybridization with a custom high-resolution miRNA-centric array and/or whole-genome sequence data to identify somatic CNAs involving miRNA genes in 113 cases of AML, including 50 cases of de novo AML, 18 cases of relapsed AML, 15 cases of secondary AML following myelodysplastic syndrome, and 30 cases of therapy-related AML. We identified a total of 48 somatic miRNA gene-containing CNAs that were not identified by routine cytogenetics in 20 patients (18%). All these CNAs also included one or more protein coding genes. We identified a single case with a hemizygous deletion of MIR223, resulting in the complete loss of miR-223 expression. Three other cases of AML were identified with very low to absent miR-223 expression, an miRNA gene known to play a key role in myelopoiesis. However, in these cases, no somatic genetic alteration of MIR223 was identified, suggesting epigenetic silencing. These data show that somatic CNAs specifically targeting miRNA genes are uncommon in AML.

MicroRNA-223 Regulates Granulopoiesis but Is Not Required for HSC Maintenance in Mice

MIR233 is genetically or epigenetically silenced in a subset of acute myeloid leukemia (AML). MIR223 is normally expressed throughout myeloid differentiation and highly expressed in hematopoietic stem cells (HSCs). However, the contribution of MIR223 loss to leukemic transformation and HSC function is largely unknown. Herein, we characterize HSC function and myeloid differentiation in Mir223 deficient mice. We show that Mir223 loss results in a modest expansion of myeloid progenitors, but is not sufficient to induce a myeloproliferative disorder. Loss of Mir223 had no discernible effect on HSC quiescence, long-term repopulating activity, or self-renewal capacity. These results suggest that MIR223 loss is likely not an initiating event in AML but may cooperate with other AML associated oncogenes to induce leukemogenesis.

MIR142 Loss-of-Function Mutations Derepress ASH1L to Increase HOXA Gene Expression and Promote Leukemogenesis

Point mutations in the seed sequence of miR-142-3p are present in a subset of acute myelogenous leukemia (AML) and in several subtypes of B-cell lymphoma. Here, we show that mutations associated with AML result both in loss of miR-142-3p function and in decreased miR-142-5p expression. Mir142 loss altered the hematopoietic differentiation of multipotent hematopoietic progenitors, enhancing their myeloid potential while suppressing their lymphoid potential. During hematopoietic maturation, loss of Mir142 increased ASH1L protein expression and consequently resulted in the aberrant maintenance of Hoxa gene expression in myeloid-committed hematopoietic progenitors. Mir142 loss also enhanced the disease-initiating activity of IDH2-mutant hematopoietic cells in mice. Together these data suggest a novel model in which miR-142, through repression of ASH1L activity, plays a key role in suppressing HOXA9/A10 expression during normal myeloid differentiation. AML-associated loss-of-function mutations of MIR142 disrupt this negative signaling pathway, resulting in sustained HOXA9/A10 expression in myeloid progenitors/myeloblasts and ultimately contributing to leukemic transformation.Significance: These findings provide mechanistic insights into the role of miRNAs in leukemogenesis and hematopoietic stem cell function. Cancer Res; 78(13); 3510-21. ©2018 AACR.

Expression profiling of snoRNAs in normal hematopoiesis and AML

Small nucleolar RNAs (snoRNAs) are noncoding RNAs that contribute to ribosome biogenesis and RNA splicing by modifying ribosomal RNA and spliceosome RNAs, respectively. We optimized a next-generation sequencing approach and a custom analysis pipeline to identify and quantify expression of snoRNAs in acute myeloid leukemia (AML) and normal hematopoietic cell populations. We show that snoRNAs are expressed in a lineage- and development-specific fashion during hematopoiesis. The most striking examples involve snoRNAs located in 2 imprinted loci, which are highly expressed in hematopoietic progenitors and downregulated during myeloid differentiation. Although most snoRNAs are expressed at similar levels in AML cells compared with CD34+, a subset of snoRNAs showed consistent differential expression, with the great majority of these being decreased in the AML samples. Analysis of host gene expression, splicing patterns, and whole-genome sequence data for mutational events did not identify transcriptional patterns or genetic alterations that account for these expression differences. These data provide a comprehensive analysis of the snoRNA transcriptome in normal and leukemic cells and should be helpful in the design of studies to define the contribution of snoRNAs to normal and malignant hematopoiesis.

Comprehensive discovery of noncoding RNAs in acute myeloid leukemia cell transcriptomes

To detect diverse and novel RNA species comprehensively, we compared deep small RNA and RNA sequencing (RNA-seq) methods applied to a primary acute myeloid leukemia (AML) sample. We were able to discover previously unannotated small RNAs using deep sequencing of a library method using broader insert size selection. We analyzed the long noncoding RNA (lncRNA) landscape in AML by comparing deep sequencing from multiple RNA-seq library construction methods for the sample that we studied and then integrating RNA-seq data from 179 AML cases. This identified lncRNAs that are completely novel, differentially expressed, and associated with specific AML subtypes. Our study revealed the complexity of the noncoding RNA transcriptome through a combined strategy of strand-specific small RNA and total RNA-seq. This dataset will serve as an invaluable resource for future RNA-based analyses.

Abstract 3042: MIR142 loss-of-function mutations promote leukemogenesis through derepression of ASH1L resulting in increased HOX gene expression

MIR142 mutations have been identified in acute myeloid leukemia (AML) and non-Hodgkins lymphoma. In AML, all MIR142 mutations localize to the miR-142-3p seed sequence. We show that mutated MIR142 is unable to suppress several well-known targets of miR-142-3p. Interestingly, the mutations of miR-142-3p result in their preferential loading into the RNA-induced silencing complex, leading to the degradation of miR-142-5p. Accordingly, miR-142-5p expression is decreased in MIR142 mutated AML. Hence, MIR142 mutations in AML disrupt both miR-142-3p/5p functions. Thus, we modeled the effect of MIR142 mutations on hematopoiesis using Mir142-/- mice. We show that loss of miR142 results in significant increases in myeloid hematopoietic stem/progenitor cells (HSPCs), including granulocyte-macrophage progenitors, myeloid-biased multipotent progenitors (CD150- CD48+ Flk2- Kit+ Sca+ lineage-) and CD229- myeloid-biased HSCs (CD150+ CD48- Kit+ Sca+ lineage-). In contrast, there are significant decreases of megakaryocyte-erythroid progenitors and erythroid precursors. Although the number of HSCs is normal in Mir142-/- mice, HSC transplantation suggest that they are myeloid-biased. In AML, MIR142 mutations are commonly found in conjunction with mutations of IDH1/2. To assess the importance of this association, we transduced wildtype or Mir142-/- HSPCs with retrovirus expressing IDH2 R172K and then transplanted into lethally irradiated recipients. Expression of IDH2 R172K alone was sufficient to induce a lethal myeloproliferative neoplasm (MPN). In contrast, Mir142-/- alone did not result in MPN. However, loss of Mir142 cooperates with IDH2 R172K to produce a more severe MPN, with increased CD34+ blasts and more severe anemia. Moreover, secondary transplantation shows that Mir142-/- x IDH2 R172K cells but not IDH2 R172K cells efficiently engraft and induce MPN, suggesting that loss of miR142 increases leukemia-initiating activity. We identify the histone methyltransferase ASH1L as a target gene of miR142 that contributes to altered hematopoiesis in Mir142-/- mice. The 3’-untranslated region of ASH1L has four miR-142-3p binding sites, and luciferase reporter assay shows that miR142 suppresses its translation by 80%. Consistent with this observation, Ashl1 protein expression is 3-fold higher in Mir142-/- bone marrow. ASH1L is a key positive regulator of HOX gene expression. Accordingly, we observed markedly (5-10 fold) increased HoxA9/A10 expression in myeloid progenitors in Mir142-/- mice. Likewise, HoxA9/A10 expression is increased in CD34+ blasts from Mir142-/- x IDH2 R172K transplanted mice. Of note, increased HoxA9 expression has been shown to cooperate with mutant IDH1 to induce AML in mice. Together, these findings support a model in which loss-of-function mutations of MIR142 contribute to hematopoietic malignancies by derepressing ASH1L and inducing HOXA9/10 gene expression. Citation Format: Juo-Chin Yao, Terrence N. Wong, Maria Trissal, Rahul Ramaswamy, Yaping Sun, Pavan R. Reddy, Daniel C. Link. MIR142 loss-of-function mutations promote leukemogenesis through derepression of ASH1L resulting in increased HOX gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3042. doi:10.1158/1538-7445.AM2017-3042

Abstract B12: Complete characterization of the "microRNAome" of a human acute myeloid leukemia

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression and have been implicated in the pathogenesis of human cancer. Most current studies utilize array-based or quantitative reverse-transcription-polymerase chain reaction (RT-PCR) approaches to measure miRNA expression. However, these approaches do not interrogate all known (or predicted) miRNAs and are unable to detect mutations in miRNAs. Herein, we use next-generation sequencing approaches to comprehensively assess miRNA expression, to identify genetic variants of all miRNA genes and miRNA binding sites in a patient with AML. Methods: This patient (AML1) was a female in her 50s. Routine cytogenetics revealed a normal 46 XX karyotype, and highresolution comparative genomic hybridization studies revealed no somatic copy number alterations at a resolution of ~5kb. We previously reported the sequence of genic regions in the cancer genome of this patient (Nature 456:66, 2008). Massively parallel sequencing of small RNAs isolated from the myeloblasts of AML1 was performed using the ABI SOLiD sequencing platform. Pooled RNA isolated from CD34+ bone marrow cells of 4 healthy volunteers (CD34) was used as control. To detect genetic variants of miRNA genes, we used 454-based sequencing of all 695 miRNA genes in the Sanger miR database (version 12.0). Finally, we analyzed the previously generated whole genome sequence for AML1 for genetic variants in the 39-untranslated regions (39-UTR) of all coding genes. Results: 28×10 and 20×10 small RNA sequence reads were obtained from AML1 and CD34 respectively. 8 novel miRNAs were identified from sequences that mapped to unannotated regions of human genome. Expression of 498 known miRNAs was detected with miR-233 being the most highly expressed miRNA in both AML1 and CD34; remarkably, it represented 47.3% of all miRNA reads in AML1. MiRNA gene sequencing of AML1 leukemic blast identified several single nucleotide variants. The whole genome sequence of AML1 skin DNA was used to differentiate germline polymorphism (SNPs) from somatic mutations. 13 novel SNPs and no somatic mutation were detected. Analysis of the 39UTR of all coding genes in leukemic blasts and skin of AML1 revealed a single somatic mutation in the 39-UTR of TNFAIP2. This mutation results in suppression of TNFAIP2 protein expression in a miRNA dependent fashion possibly by creating a new miRNA binding site. However, no recurrent mutations in the 39-UTR of TNFAIP2 were detected in an additional 180 patients with AML. Conclusions: These data demonstrate the feasibility of ‘next generation’ sequencing technologies to identify novel miRNAs, accurately measure mature miRNA expression, and identify both somatic and germline genetic variants of miRNA genes and miRNA binding sites in primary cancer. Using this platform, studies are underway to comprehensively characterize miRNAs in additional human AML samples. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B12.