Noreen Fulton

A novel nuclear protein, 5qNCA (LOC51780) is a candidate for the myeloid leukemia tumor suppressor gene on chromosome 5 band q31

Interstitial deletion or loss of chromosome 5, del(5q) or −5, is a frequent finding in myeloid leukemias and myelodysplasias, suggesting the presence of a tumor suppressor gene within the deleted region. In our search for this gene, we identified a candidate, 5qNCA (LOC51780), which lies within a consistently-deleted segment of 5q31. 5qNCA expresses a 7.2-kb transcript with a 5286-bp open reading frame which is present at high levels in heart, skeletal muscle, kidney, placenta, and liver as well as CD34+ cells and AML cell lines. 5qNCA encodes a 191-kD nuclear protein which contains a highly-conserved C-terminus containing a zinc finger with the unique spacing Cys-X2-Cys-X7-His-X2-Cys-X2-Cys-X4-Cys-X2-Cys and a jmjC domain, which is often found in proteins that regulate chromatin remodeling. Expression of 5qNCA in a del(5q) cell line results in suppression of clonogenic growth. Preliminary sequence results in AML and MDS samples and cell lines has revealed a possible mutation in the KG-1 cell line resulting in a THR to ALA substitution that has not been found in over 100 normal alleles to date. We propose 5qNCA is a good candidate for the del(5q) tumor suppressor gene based on its predicted function and growth suppressive activities, and suggest that further mutational and functional study of this interesting gene is warranted.

Defined Populations of Bone Marrow Derived Mesenchymal Stem and Endothelial Progenitor Cells for Bladder Regeneration

PURPOSE Autologous sources of bone marrow mesenchymal stem cells and endothelial progenitor cells are attractive alternatives to cells currently used for bladder tissue regeneration. To evaluate the potential use of these cells we determined whether mesenchymal stem cells have contractile protein profiles and physiological functions similar to those of normal bladder smooth muscle cells, and determined the angiogenic potential of endothelial progenitor cells. MATERIALS AND METHODS Mesenchymal stem cells and smooth muscle cells (Lonza, Gaithersburg, Maryland) underwent proliferation and Western blot analyses. Immunofluorescence imaging was performed using antibodies against smooth muscle cell epitopes. Contractility was assessed by intracellular Ca(2+) release assays and confocal microscopy after carbachol stimulation. Endothelial progenitor cells were evaluated using a chicken chorioallantoic membrane model to determine neo-angiogenic potential. RESULTS Western blot and immunofluorescence data showed that mesenchymal stem cells endogenously expressed known smooth muscle cell contractile proteins at levels similar to those of smooth muscle cells. Ca(2+) release assays revealed that smooth muscle cells and mesenchymal stem cells responded to carbachol treatment with a mean +/- SD of 8.6 +/- 2.5 and 5.8 +/- 0.8 RFU, respectively, which was statistically indistinguishable. Proliferation trends of mesenchymal stem cells and control smooth muscle cells were also similar. Chorioallantoic membrane assay showed the growth of vasculature derived from endothelial progenitor cells. CONCLUSIONS Data demonstrate that mesenchymal stem cells and smooth muscle cells express the same contractile proteins and can function similarly in vitro. Endothelial progenitor cells also have the ability to form vasculature in an in vivo chorioallantoic membrane model. These findings provide evidence that mesenchymal stem cells and endothelial progenitor cells have characteristics that may be applicable for bladder tissue regeneration.

History of the Recurrent Laryngeal Nerve: From Galen to Lahey

During the second century A.D., Galen described a nerve that came from the brain on each side of the neck, went down toward the heart, and then reversed course and ascended to the larynx and caused the vocal cords to open. He called these “reversivi” (or recurrent nerves) and stated that he was the first to discover “these wonderful things.” Demonstrating before the elders of Rome, he showed that cutting the recurrent laryngeal nerve in the neck caused a live pig to stop squealing—an extraordinary feat. Because of Galen’s fame and influence, this nerve retained great importance in dissections by later anatomists and surgeons before and throughout the Renaissance. This paper documents many of these anatomical findings and highlights the importance of a careful, delicate, recurrent laryngeal nerve dissection during thyroidectomy, as popularized by Dr. Frank Lahey in 1938.

Comprehensive mutational analysis of primary and relapse acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by differentiation block at the promyelocyte stage. Besides the presence of chromosomal rearrangement t(15;17), leading to the formation of PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha) fusion, other genetic alterations have also been implicated in APL. Here, we performed comprehensive mutational analysis of primary and relapse APL to identify somatic alterations, which cooperate with PML-RARA in the pathogenesis of APL. We explored the mutational landscape using whole-exome (n=12) and subsequent targeted sequencing of 398 genes in 153 primary and 69 relapse APL. Both primary and relapse APL harbored an average of eight non-silent somatic mutations per exome. We observed recurrent alterations of FLT3, WT1, NRAS and KRAS in the newly diagnosed APL, whereas mutations in other genes commonly mutated in myeloid leukemia were rarely detected. The molecular signature of APL relapse was characterized by emergence of frequent mutations in PML and RARA genes. Our sequencing data also demonstrates incidence of loss-of-function mutations in previously unidentified genes, ARID1B and ARID1A, both of which encode for key components of the SWI/SNF complex. We show that knockdown of ARID1B in APL cell line, NB4, results in large-scale activation of gene expression and reduced in vitro differentiation potential.

Targeting Suppressor of Variegation 3-9 Homologue 2 (SUV39H2) in Acute Lymphoblastic Leukemia (ALL).

Although recent progress in understanding the biology and optimizing the treatment of acute lymphoblastic leukemia (ALL) has improved cure rates of childhood ALL to nearly 90%, the cure rate in adult ALL remains less than 50%. The poor prognosis in adult ALL has in part been attributed to larger proportion of high-risk leukemia showing drug resistance. Thus, identifying novel therapeutic targets in ALL is needed for further improvements in treatment outcomes of adult ALL. Genetic aberration of chromatin-modifying molecules has been recently reported in subtypes of ALL, and targeting components of chromatin complexes has shown promising efficacy in preclinical studies. Suppressor of variegation 3-9 homologue 2 (SUV39H2), also known as KMT1B, is a SET-domain–containing histone methyltransferase that is upregulated in solid cancers, but its expression is hardly detectable in normal tissues. Here, we show that SUV39H2 is highly expressed in ALL cells but not in blood cells from healthy donors and also that SUV39H2 mRNA is expressed at significantly higher levels in bone marrow or blood cells from patients with ALL obtained at diagnosis compared with those obtained at remission (P = .007). In four ALL cell lines (Jurkat and CEM derived from T-ALL and RS4;11 and REH derived from B-ALL), SUV39H2 knockdown resulted in a significant decrease in cell viability (~ 77%, P < .001), likely through induction of apoptosis. On the other hand, SUV39H2 overexpression made cells more resistant to chemotherapy. We conclude that SUV39H2 is a promising therapeutic target and further investigation of this therapeutic approach in ALL is warranted.

Corrigendum: Comprehensive mutational analysis of primary and relapse acute promyelocytic leukemia (Leukemia (2016) 30 (1672-1681) DOI: 10.1038/leu.2016.69)

V Madan, P Shyamsunder, L Han, A Mayakonda, Y Nagata, J Sundaresan, D Kanojia, K Yoshida, S Ganesan, N Hattori, N Fulton, KT Tan, T Alpermann, MC Kuo, S Rostami, J Matthews, M Sanada, L-Z Liu, Y Shiraishi, S Miyano, E Chendamarai, HA Hou, G Malnassy, T Ma, M Garg, LW Ding, QY Sun, W Chien, T Ikezoe, M Lill, A Biondi, RA Larson, BL Powell, M Lübbert, WJ Chng, HF Tien, M Heuser, A Ganser, M Koren-Michowitz, SM Kornblau, HM Kantarjian, D Nowak, WK Hofmann, H Yang, W Stock, A Ghavamzadeh, K Alimoghaddam, T Haferlach, S Ogawa, LY Shih, V Mathews and HP Koeffler

T-LAK cell-originated protein kinase presents a novel therapeutic target in FLT3 -ITD mutated acute myeloid leukemia

Gain-of-function mutations of FLT3 (FLT3-ITD), comprises up to 30% of normal karyotype acute myeloid leukemia (AML) and is associated with an adverse prognosis. Current FLT3 kinase inhibitors have been tested extensively, but have not yet resulted in a survival benefit and novel therapies are awaited. Here we show that T-LAK cell-originated protein kinase (TOPK), a mitotic kinase highly expressed in and correlated with more aggressive phenotype in several types of cancer, is expressed in AML but not in normal CD34+ cells and that TOPK knockdown decreased cell viability and induced apoptosis. Treatment of AML cells with TOPK inhibitor (OTS514) resulted in a dose-dependent decrease in cell viability with lower IC50 in FLT3-mutated cells, including blasts obtained from patients relapsed after FLT3-inhibitor treatment. Using a MV4-11-engrafted mouse model, we found that mice treated with 7.5 mg/kg IV daily for 3 weeks survived significantly longer than vehicle treated mice (median survival 46 vs 29 days, P < 0.001). Importantly, we identified TOPK as a FLT3-ITD and CEBPA regulated kinase, and that modulating TOPK expression or activity resulted in significant decrease of FLT3 expression and CEBPA phosphorylation. Thus, targeting TOPK in FLT3-ITD AML represents a novel therapeutic approach for this adverse risk subset of AML.

Expression and polymorphism (rs4880) of mitochondrial superoxide dismutase (SOD2) and asparaginase induced hepatotoxicity in adult patients with acute lymphoblastic leukemia

Asparaginase, which depletes asparagine and glutamine, activates amino-acid stress response. Oxidative stress mediated by excessive reactive oxygen species (ROS) causes enhanced mitochondrial permeabilization and subsequent cell apoptosis and is considered as a plausible mechanism for drug-induced hepatotoxicity, a common toxicity of asparaginase in adults with acute lymphoblastic leukemia (ALL). Studies investigating the pharmacogenetics of asparaginase in ALL are limited and focused on asparaginase-induced allergic reaction common in pediatric patients. Here, we sought to determine a potential association between the variant rs4880 in SOD2 gene, a key mitochondrial enzyme that protects cells against ROS, and hepatotoxicity during asparaginase-based therapy in 224 patients enrolled on CALGB-10102, a treatment trial for adults with ALL. We report that the CC genotype of rs4880 is associated with increased hepatotoxicity following asparaginase-based treatment. Thus, rs4880 likely contributes to asparaginase-induced hepatotoxicity, and functional studies investigating this single-nucleotide polymorphism (SNP) are needed to develop therapeutic approaches that mitigate this toxicity.

WT1 peptide vaccine in Montanide in contrast to poly ICLC, is able to induce WT1-specific immune response with TCR clonal enrichment in myeloid leukemia

BackgroundThe optimal strategy for vaccination to induce CD8+ T cell responses against WT1 is not known.MethodsA pilot randomized study in HLA-A02+ patients to receive vaccination with WT1 in Montanide or in poly ICLC, a TLR3 agonist, to explore the novel immune adjuvant was conducted. Seven patients were randomized. Four patients received WT1 in Montanide, and three with WT1 in poly ICLC. Five patients were in morphologic remission and two had residual morphologic disease at the study entry.ResultsAll patients finished the induction phase without any major toxicity except mild transient local injection reaction. One patient on the Montanide arm developed aseptic ulceration at two vaccine sites which healed without antibiotics. Three of 4 patients on the Montanide arm had a decreased expression of WT1 after WT1 vaccination, and two of them demonstrated generation of WT1-specific cytotoxic CD8+ T cell responses with biased TCR beta chain enrichment. In contrast, no obvious WT1-specific immune responses were detected in two patients on the poly ICLC arm, nor was there clonal enrichment by TCR alpha/beta sequencing; however, these patients did also have decreased WT1 expression and remained in remission several years after the initiation of treatment.ConclusionsWT1 peptide vaccine with Montanide as an adjuvant induces detectable WT1-specific CD8+ T cell responses with clonal TCR enrichment, which may be capable of controlling leukemia recurrence in the setting of minimal residual disease. Poly ICLC may induce anti-leukemic activity in the absence of detectable WT1 specific CD8+ T cell responses.Trial registration NCT01842139, 7/3/2012 retrospectively registered; https://clinicaltrials.gov/ct2/show/NCT01842139.

Ras-Onkogene und DNA-Cytometrie: prognostischer Wert beim oxyphilen Schilddrüsentumor

Die morphologisch benignen oxyphilen Schilddrusentumore (OST) werden in manchen Studien als potentiell maligne betrachtet, da sie zu einem spateren Zeitpunkt metastasieren konnen [5]. Einige Autoren konnten dies in ihren Studien nicht bestatigen [3]. Diese Falle halten allerdings eine Kontroverse uber die richtige Behandlung vor allem benigner OSTs in Gang. Daher ist es zusatzlich zur histologischen Einteilung wichtig, effektive diagnostische Methoden zu suchen. Die Ras-Protoonkogene nehmen eine zentrale Rolle in der Zellregulation ein. Sie sind u.a. an der Zell-Proliferation, -Transformation und -Differenzierung beteiligt [2]. Den Ras-Onkogenen wurden bei vielen Tumoren einschlieslich den Schilddrusentumoren [2] eine Rolle bei der Tumorinitiierung und Progression zugeschrieben. Der prognostische Wert der DNA-Cytometrie wurde beim OST bereits mehrfach untersucht. Die Ergebnisse bleiben allerdings kontrovers [1, 3]. Ziel dieser Studie war es herauszuarbeiten, ob die Ras-Mutationenanalyse und nucleare DNA-Messung hilfreich sind, die Tumoren zu erkennen, die metastasieren und sich aggressiv verhalten.