Dimitrios Papaioannou

Expression and prognostic impact of lncRNAs in acute myeloid leukemia

Significance Long noncoding RNAs (lncRNAs) are involved in numerous biological roles including epigenetic regulation, apoptosis, and cell cycle. Whereas lncRNAs contribute to epigenetic gene regulation, metastasis, and prognosis in solid tumors, their role in acute myeloid leukemia (AML) has not been hitherto reported. Here, we show that lncRNA expression profiles are associated with recurrent mutations, clinical features, and outcome in AML. A fraction of these lncRNAs may have a functional role in leukemogenesis. Furthermore, lncRNAs could be used as biomarkers for outcome in AML. The identification of patients likely to achieve complete remission with standard therapy alone, based on lncRNA expression, is a significant advance potentially sparing such patients from other toxicities and focusing investigational approaches on postremission studies. Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides, located within the intergenic stretches or overlapping antisense transcripts of protein coding genes. LncRNAs are involved in numerous biological roles including imprinting, epigenetic regulation, apoptosis, and cell cycle. To determine whether lncRNAs are associated with clinical features and recurrent mutations in older patients (aged ≥60 y) with cytogenetically normal (CN) acute myeloid leukemia (AML), we evaluated lncRNA expression in 148 untreated older CN-AML cases using a custom microarray platform. An independent set of 71 untreated older patients with CN-AML was used to validate the outcome scores using RNA sequencing. Distinctive lncRNA profiles were found associated with selected mutations, such as internal tandem duplications in the FLT3 gene (FLT3-ITD) and mutations in the NPM1, CEBPA, IDH2, ASXL1, and RUNX1 genes. Using the lncRNAs most associated with event-free survival in a training cohort of 148 older patients with CN-AML, we derived a lncRNA score composed of 48 lncRNAs. Patients with an unfavorable compared with favorable lncRNA score had a lower complete response (CR) rate [P < 0.001, odds ratio = 0.14, 54% vs. 89%], shorter disease-free survival (DFS) [P < 0.001, hazard ratio (HR) = 2.88] and overall survival (OS) (P < 0.001, HR = 2.95). The validation set analyses confirmed these results (CR, P = 0.03; DFS, P = 0.009; OS, P = 0.009). Multivariable analyses for CR, DFS, and OS identified the lncRNA score as an independent marker for outcome. In conclusion, lncRNA expression in AML is closely associated with recurrent mutations. A small subset of lncRNAs is correlated strongly with treatment response and survival.

HDAC Inhibition Induces MicroRNA-182, which Targets RAD51 and Impairs HR Repair to Sensitize Cells to Sapacitabine in Acute Myelogenous Leukemia.

Purpose: The double-strand breaks elicited by sapacitabine, a clinically active nucleoside analogue prodrug, are repaired by RAD51 and the homologous recombination repair (HR) pathway, which could potentially limit its toxicity. We investigated the mechanism by which histone deacetylase (HDAC) inhibitors targeted RAD51 and HR to sensitize acute myelogenous leukemia (AML) cells to sapacitabine. Experimental Design: Chromatin immunoprecipitation identified the role of HDACs in silencing miR-182 in AML. Immunoblotting, gene expression, overexpression, or inhibition of miR-182 and luciferase assays established that miR-182 directly targeted RAD51. HR reporter assays, apoptotic assays, and colony-forming assays established that the miR-182, as well as the HDAC inhibition–mediated decreases in RAD51 inhibited HR repair and sensitized cells to sapacitabine. Results: The gene repressors, HDAC1 and HDAC2, became recruited to the promoter of miR-182 to silence its expression in AML. HDAC inhibition induced miR-182 in AML cell lines and primary AML blasts. miR-182 targeted RAD51 protein both in luciferase assays and in AML cells. Overexpression of miR-182, as well as HDAC inhibition–mediated induction of miR-182 were linked to time- and dose-dependent decreases in the levels of RAD51, an inhibition of HR, increased levels of residual damage, and decreased survival after exposure to double-strand damage-inducing agents. Conclusions: Our findings define the mechanism by which HDAC inhibition induces miR-182 to target RAD51 and highlights a novel pharmacologic strategy that compromises the ability of AML cells to conduct HR, thereby sensitizing AML cells to DNA-damaging agents that activate HR as a repair and potential resistance mechanism. Clin Cancer Res; 22(14); 3537–49. ©2016 AACR.

Unusual Complication of a Pancreatic Neuroendocrine Tumor Presenting with Malignant Hypercalcemia

CONTEXT Hypersecretion of PTHrP is a relatively common cause of malignancy-related hypercalcemia but has only been described in a few cases of neuroendocrine tumors (NET). OBJECTIVE The aim of this case report is to describe the clinical syndrome, complex therapeutic interventions, and unusual complications caused by persistent PTHrP hypersecretion in a patient with a pancreatic NET. CASE ILLUSTRATION A 58-yr-old male patient presented with nonspecific abdominal pain and was found to have severe hypercalcemia secondary to a well-differentiated NET of the pancreas associated with extensive liver metastases. Elevated ionized calcium levels accompanied by low serum PTH and remarkably elevated PTHrP concentrations were consistent with PTHrP-related hypercalcemia that proved to be resistant to various chemotherapeutic regimens and supportive therapy. Partial control of the humoral syndrome was obtained only after the application of cytoreductive interventions and the introduction of various molecular targeted therapies. Due to persistent PTHrP action, bone disease emerged in the form of brown tumors. DISCUSSION The manifestation of paraneoplastic syndrome due to PTHrP hypersecretion, despite its rareness in NET, should be considered in the differential diagnosis of hypercalcemia in such tumors. Moreover, the appearance of bone lesions in this setting may be in the context of metabolic bone disease and could be misdiagnosed as bone metastases.

MicroRNA fingerprints in juvenile myelomonocytic leukemia (JMML) identified miR-150-5p as a tumor suppressor and potential target for treatment

Juvenile myelomonocytic leukemia (JMML) is an aggressive leukemia of early childhood characterized by aberrant proliferation of myelomonocytic cells and hypersensitivity to GM-CSF stimulation. Mutually exclusive mutations in the RAS/ERK pathway genes such as PTPN11, NRAS, KRAS, CBL, or NF1 are found in ~90% of the cases. These mutations give rise to disease at least in part by activating STAT5 through phosphorylation and by promoting cell growth. MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression, which are often deregulated in leukemia. However, little is known about their role in JMML. Here, we report distinctive miR expression signatures associated with the molecular subgroups of JMML. Among the downregulated miRs in JMML, miR-150-5p was found to target STAT5b, a gene which is often over-activated in JMML, and contributes to the characteristic aberrant signaling of this disorder. Moreover, loss of miR-150-5p and upregulation of STAT5b expression were also identified in a murine model of JMML. Ectopic overexpression of miR-150-5p in mononuclear cells from three JMML patients significantly decreased cell proliferation. Altogether, our data indicate that miR expression is deregulated in JMML and may play a role in the pathogenesis of this disorder by modulating key effectors of cytokine receptor pathways.

Prognostic and biologic significance of long non-coding RNA profiling in younger adults with cytogenetically normal acute myeloid leukemia

Long non-coding ribonucleic acids (RNAs) are a novel class of RNA molecules, which are increasingly recognized as important molecular players in solid and hematologic malignancies. Herein we investigated whether long non-coding RNA expression is associated with clinical and molecular features, as well as outcome of younger adults (aged <60 years) with de novo cytogenetically normal acute myeloid leukemia. Whole transcriptome profiling was performed in a training (n=263) and a validation set (n=114). Using the training set, we identified 24 long non-coding RNAs associated with event-free survival. Linear combination of the weighted expression values of these transcripts yielded a prognostic score. In the validation set, patients with high scores had shorter disease-free (P<0.001), overall (P=0.002) and event-free survival (P<0.001) than patients with low scores. In multivariable analyses, long non-coding RNA score status was an independent prognostic marker for disease-free (P=0.01) and event-free survival (P=0.002), and showed a trend for overall survival (P=0.06). Among multiple molecular alterations tested, which are prognostic in cytogenetically normal acute myeloid leukemia, only double CEBPA mutations, NPM1 mutations and FLT3-ITD associated with distinct long non-coding RNA signatures. Correlation of the long non-coding RNA scores with messenger RNA and microRNA expression identified enrichment of genes involved in lymphocyte/leukocyte activation, inflammation and apoptosis in patients with high scores. We conclude that long non-coding RNA profiling provides meaningful prognostic information in younger adults with cytogenetically normal acute myeloid leukemia. In addition, expression of prognostic long non-coding RNAs associates with oncogenic molecular pathways in this disease. clinicaltrials.gov Identifier: 00048958 (CALGB-8461), 00899223 (CALGB-9665), and 00900224 (CALGB-20202).

Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia.

Significance In this work we report on the previously uncharacterized clinical and biological role for EGFL7 in acute myeloid leukemia (AML). Patients with increased EGFL7 mRNA expression had lower complete remission rates and shorter overall and event-free survival, demonstrating the clinical relevance of EGFL7 expression in cytogenetically normal AML. Our results show that AML blasts are able to synthesize and secrete EGFL7 protein, promoting autocrine blast cell growth. Inhibition of EGFL7 results in decreased proliferation and induces apoptosis of AML cells. Taken together, our data provide the rationale for targeting EGFL7 using blocking antibodies as a therapy for patients with AML. Epithelial growth factor-like 7 (EGFL7) is a protein that is secreted by endothelial cells and plays an important role in angiogenesis. Although EGFL7 is aberrantly overexpressed in solid tumors, its role in leukemia has not been evaluated. Here, we report that levels of both EGFL7 mRNA and EGFL7 protein are increased in blasts of patients with acute myeloid leukemia (AML) compared with normal bone marrow cells. High EGFL7 mRNA expression associates with lower complete remission rates, and shorter event-free and overall survival in older (age ≥60 y) and younger (age <60 y) patients with cytogenetically normal AML. We further show that AML blasts secrete EGFL7 protein and that higher levels of EGFL7 protein are found in the sera from AML patients than in sera from healthy controls. Treatment of patient AML blasts with recombinant EGFL7 in vitro leads to increases in leukemic blast cell growth and levels of phosphorylated AKT. EGFL7 blockade with an anti-EGFL7 antibody reduced the growth potential and viability of AML cells. Our findings demonstrate that increased EGFL7 expression and secretion is an autocrine mechanism supporting growth of leukemic blasts in patients with AML.

Can insulin-like growth factor 1 (IGF-1), IGF-1 receptor connective tissue growth factor and Ki-67 labelling index have a prognostic role in pulmonary carcinoids?

Introduction Altered expression of Insulin-like Growth Factor-1 (IGF-1), its receptor (IGF-1R), Connective Tissue Growth Factor (CTGF) and Hypoxia Inducible Factor-1 (HIF-1), has been implicated in tumorigenesis. So far, these factors have not been studied systematically in Pulmonary Carcinoids (PCs). Aims To examine IGF-1, IGF-1R, CTGF and HIF-1 expression in PCs, and assess their prognostic value over established factors. Materials & Methods Retrospective study of 121 PCs (104 Typical and 17 Atypical). The expression of growth factors was studied immunohistochemically and tumors were considered positive if immunoreactivity appeared in >50% of cells. Results All studied parameters were expressed in the majority of tumors (IGF-1, IGF-1R, CTGF and HIF-1, in 78.5%, 67%, 72% and 78%, respectively). Their expression tended to be more frequent in TCs and in tumors with Ki-67≤2% (significant only for HIF-1; 82 vs. 53%; p=0.023 and 83 vs. 63%; p=0.025 respectively). CTGF was the only factor correlated with more extensive disease (larger size; presence of lymph node and distant metastases). According to logistic regression analysis, only advanced age, Ki-67≥3.4% and lymph node involvement could predict the development of distant metastases. Conclusions IGF-1, IGF-1R, CTGF and HIF-1 are avidly expressed in PCs; however, their presence did not appear to be of statistically significant value over established prognostic factors.

Ten-year outcome of patients with acute myeloid leukemia not treated with allogeneic transplantation in first complete remission

The probability that adult patients with de novo acute myeloid leukemia (AML) receiving intensive chemotherapy in the absence of allogeneic hematopoietic stem cell transplantation (Allo-HCT) in first complete remission (CR1) will be disease-free at 10 years after diagnosis, a long-term surrogate of cure, is unknown. To address this question, we examined 2551 AML patients (1607 aged <60 years, and 944 aged ≥60 years) enrolled in Cancer and Leukemia Group B treatment protocols and the cytogenetics companion protocol 8461 between 1983 and 2004. At 10 years, 267 (16.6%) of patients aged <60 years and 23 (2.4%) of those aged ≥60 years were alive and disease-free. This disease-free AML group consisted predominantly of patients with core-binding factor AML with t(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22) and those with a normal karyotype. Occurrences of AML beyond 10 years were infrequent and associated with cytogenetic findings different from those at diagnosis. These data provide evidence that the frequency of long-term cure of AML is low among younger and especially older patients in the absence of Allo-HCT in CR1. In older patients not appropriate for Allo-HCT, these data provide further justification for early use of alternative treatments outside of intensive chemotherapy.

Abstract 3485: Identification and characterization of circular RNAs in cancer

Circular RNAs (circRNAs) are an abundant class of non-coding RNAs that are formed by a backsplice event resulting in formation of covalently closed circular RNA molecules. The efficiency of backsplicing is inferior to linear splicing, however, due to their long half-lives circRNAs can accumulate to high levels in cells. Functional characterization of a few circRNAs has shown that they can act as endogenous microRNA sponges and transcriptional regulators; one example is CDR1as/ciRS-7, which has been shown to act as a sponge for miR-7. However, the functions of most circRNAs are still unknown. To study the potential role of circRNAs in cancer, we identified exonic circRNA candidates by analysing ENCODE RNAseq data from a panel of cancer cell lines. We used chimeric alignment detection implemented in the STAR aligner and subsequent filtering of output files to identify chimeric alignments consistent with circRNA backsplice sites. The circular nature of the identified putative circRNAs was validated by testing their resistance to RNase R digestion, and sequences surrounding the backsplice sites were used as target recognition sequences for designing LNA-modified gapmer antisense oligonucleotides (ASOs) to specifically knock down the circular isoforms of the RNA transcripts. Data from ongoing studies on knockdown of several abundant circRNAs in cancer cell lines, assessment of the specificity of backsplice-targeting ASOs relative to the linear mRNA counterparts, and analyses to identify biological effects of modulating circRNA levels in cultured cells will be presented. Citation Format: Charlotte A. Thrue, Andreas Petri, Marianne B. Lovendorf, Karen Dybkaer, Dimitrios Papaioannou, Ramiro Garzon, Sakari Kauppinen. Identification and characterization of circular RNAs in cancer [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 3485. doi:10.1158/1538-7445.AM2017-3485