Despite major advances in our understanding of the biology of childhood acute myeloid leukemia (AML) and the development of new cytotoxic drugs, the prognosis of long-term survival is still only 60-65 %. In the present research, we studied the pharmacokinetics of drugs used in the induction therapy of childhood AML and performed in vitro drug sensitivity testing of leukemic cells from children with AML. The aims of the studies were to correlate the results of the analysis to biological and clinical parameters and to identify subgroups of AML with specific drug sensitivity profiles in order to better understand why treatment fails in some patients and how therapy may be improved. Blood samples were analysed to study the pharmacokinetics of doxorubicin (n=41), etoposide (n=45) and 6-thioguanine (n=50). Doxorubicin plasma concentration and total body clearance were correlated to the effect of induction therapy, and doxorubicin plasma concentration was an independent factor for complete remission, both in univariate and multivariate analysis including sex, age, and white blood cell count at diagnosis. For etoposide and 6-thioguanine no correlation was found between pharmacokinetics and clinical effect. Children with Down syndrome (DS) tended to reach higher blood concentrations of etoposide and thioguanine nucleotides, indicating that dose reduction may be reasonable to reach the same drug exposure as in children without DS. Leukemic cells from 201 children with newly diagnosed AML, 15 of whom had DS, were successfully analysed for in vitro drug sensitivity by the fluorometric microculture cytotoxicity assay (FMCA). We found that samples from children with DS were highly sensitive to most drugs used in AML treatment. In non-DS children, the t(9;11) samples were significantly more sensitive to cytarabine (p=0.03) and doxorubicin (p=0.035) than other samples. The findings might explain the very favorable outcome reported in children with DS and t(9;11)-positive AML. A specific drug resistance profile was found for several other genetic subgroups as well. A detailed study of MLL-rearranged leukemia showed that cellular drug sensitivity is correlated both to partner genes and cell lineage, findings that support the strategy of contemporary protocols to include high-dose cytarabine in the treatment of patients with MLL-rearrangement, both in AML and acute lymphoblastic leukemia (ALL). Our results indicate that drug resistance and pharmacokinetic studies may yield important information regarding drug response in different sub-groups of childhood AML, helping us to optimize future chemotherapy in childhood AML.

Le gène MLL (Mixed-Lineage Leukemia), un homologue du gène trithorax de la Drosophile, localisé à la bande chromosomique 11q23, est fréquemment réarrangé dans plusieurs types de leucémies, essentiellement suite à des translocations chromosomiques. Dans les différentes translocations chromosomiques, la partie N-terminale de MLL est fusionnée avec les séquences d’un gène partenaire. Malgré le grand nombre de partenaires de fusion rapportés, peu de fusions MLL ont été bien caractérisées sur le plan moléculaire. De plus, l’impact pronostique de plusieurs fusions moins fréquentes n’est pas bien établi. L’objectif de mon projet est de caractériser plusieurs translocations MLL qui ont été détectées dans 39 spécimens leucémiques collectés par la Banque de cellules leucémiques du Québec (www.bclq.gouv.qc.ca), et d’établir une corrélation entre les résultats de la cytogénétique et différents paramètres biologiques et cliniques des leucémies respectives. L’identification des gènes partenaires de fusion (GPF) dans notre série (30 échantillons étudiés), a révélé la fusion de MLL à un gène partenaire très récurrent dans 26 leucémies: MLLT3(AF9), AFF1(AF4), MLLT4(AF6), MLLT1(ENL), ELL; à un GPF modérément commun dans 1 leucémie : MLLT6(AF17); et à un partenaire rare de MLL dans 3 leucémies : GAS7 et AF15/CASC5 (2 cas). Nous avons poursuivi notre travail avec la caractérisation des points de cassure de deux fusions, soit MLL-ELL associée à un syndrome myéloprolifératif (une association rare), et MLL-GAS7 (une fusion rare de MLL), associée à une leucémie aiguë myéloïde. L’analyse des transcrits de fusion par RT-PCR et séquençage a révélé respectivement la fusion de l’exon 9 de MLL à l’exon 2 de ELL et des exons 7 ou 8 de MLL (deux transcrits) à l’exon 2 de GAS7. Ce travail permettra d’effectuer des études fonctionnelles et des projets de recherche translationnelle en utilisant ces spécimens de leucémies avec différents réarrangements de MLL, bien caractérisés sur le plan clinique et moléculaire. / The MLL (Mixed-Lineage Leukemia) gene, a human homolog of the Drosophila trithorax gene, located at chromosomal band 11q23, is frequently rearranged in several types of leukemia, mostly by chromosomal translocations. In different chromosomal translocations, the N-terminal part of MLL is fused to sequences of the partner gene. Despite the large number of fusion partners that have been reported, several gene fusions remain poorly characterized at the molecular level. Moreover, the prognostic impact of less frequent fusions is not well established. The aim of my project is to characterize different MLL fusions detected in 39 leukemic samples, collected by the Quebec Leukemia Cell Bank (www.bclq.gouv.qc.ca) and to correlate cytogenetics with the clinical and biological features of the corresponding leukemia. Identification of fusion partner genes in our series (30 samples studied), revealed fusion of MLL to one of the most frequent partners in 26 leukemias: MLLT3(AF9), AFF1(AF4), MLLT4(AF6), MLLT1(ENL), ELL; to a moderately common MLL fusion partner in 1 leukemia: MLLT6(AF17); and to a rare partner in 3 leukemias: GAS7 and AF15/CASC5 (2 cases). We have characterized the breakpoints of two fusions, MLL-ELL in a myeloproliferative syndrome (a rare association) and MLL-GAS7 (a rare MLL fusion) associated with acute myeloid leukemia. Fusion transcripts analysis by RT-PCR and sequencing revealed respectively, a fusion of MLL exon 9 to ELL exon 2 and of MLL exon 7 or exon 8 (two transcripts) to GAS7 exon 2. This study is essential to perform functional studies and translational research projects using these well characterized leukemic specimens with different MLL rearrangements.

An 80 MHz bandpass filter with a tunable quality factor of 16∼44 using an improved transconductor circuit is presented. A noise optimized biquad structure for high-Q, high- frequency bandpass filter is proposed. The quality factor of the filter is tuned using a new quality factor locked loop algorithm. It was shown that a second-order quality factor locked loop is necessary and sufficient to tune the quality factor of a bandpass filter with zero steady state error. The accuracy, mismatch, and sensitivty analysis of the new tuning scheme was performed and analyzed. Based on the proposed noise optimized filter structure and new quality factor tuning scheme, a biquad filter was designed and fabricated in 0.25 μm BiCMOS process. The measured results show that the biquad filter achieves a SNR of 45 dB at IMD of 40 dB. The P-1dB compression point and IIP3 of the filter are -10 dBm and -2.68 dBm, respectively. The proposed biquad filter and quality factor tuning scheme consumes 58mW and 13 mW of power at 3.3 V supply.

Wnt/beta-Catenin-Signalgebung hat große Bedeutung für die Initiation und Progression verschiedener Krebsarten. Unser Labor hat kürzlich ein Mausmodell für Squamöse Speicheldrüsen-Karzinome etabliert, das menschliche Hals-Nasen-Ohren-Karzinome reflektiert, durch kombinierte Mutationen von beta-Catenin und dem Bmp-Rezeptor 1a. Diese Tumore enthielten hohen Level von sich selbst-erneuernden Krebs-Stammzellen. Behandlung mit den Wnt-Inhibitoren ICG-001 blockierte die Selbsterneuerung und induzierte die Differenzierung der Krebs-Stammzellen. In den Krebs-Stammzellen der Maus wurde eine globale Aufregulierung des Histonmarkers H3K4me3 beobachtet, was durch Wnt-Inhibition gehemmt werden konnte. Um die molekularen Mechnismen aufzuklären, wurden die Histon-Methyltransferasen für H3K4me3, d.h., Mitglieder der Mll-Proteinfamilie, in sphären-kultivierten Krebs-Stammzellen durch RT-PCR analysiert: Mll1 war hoch transkribiert, zusammen mit den Hoxa9- und Meis1-Zielgenen. Interessanterweise aktivierte die Expression von Mll1 durch Wnt-Signalgebung die distale Enhancer-Region von Mll1, was durch Luciferase-Reporter-Assays gemessen wurde. Immunopräzipitation zeigte weiter, dass Mll1 im beta-Catenin-Transcriptionsfaktor-Komplex involviert ist: shRNA-Behandlung von Mll1 reduzierte die Sphären-Bildung der Speicheldrüsen-Krebs-Stammzellen der Maus. In doppelt-mutanten Mäusen hat die zusätzliche genetische Ablation von Mll1 die Tumorbildung verhindert und die Selbsterneuerung der Krebs-Stammzellen reduziert. Diese Daten zeigen dass die beta-Catenin-Mll1-Achse die Selbsterneuerung der Stammzellen antreibt und deren Differenzierung verhindert, und zwar via epigenetische Mechanismen. Deshalb wird durch das Targeting von Mll1 und dessen Interaktion mit beta-Catenin und andern Komponenten den gesunden epigenetischen Zustand in den Stammzellen wieder herstellt, was eine neue und vielversprechende Möglichkeit für die Behandlung von Patienten mit Hals-Nasen-Ohren-Tumoren darstellt. / Wnt/beta-catenin signaling has been implicated in the initiation and progression of various human cancers. Our lab has recently established a mouse model of salivary gland squamous cell carcinomas (SCCs), which resembles human head and neck cancer, by combined gain- and loss-of-function mutations of beta-catenin and the Bmp receptor 1a (double mutant tumors). These tumors contained highly self-renewing cancer stem cells (CSCs) that were Wnt-dependent. Treatment with the Wnt inhibitor ICG-001 (interferes with beta-catenin-CBP-Mll1 interaction) blocked the self-renewal and induced differentiation of CSCs. In the mouse salivary gland CSCs, a global up-regulation of the histone mark H3K4me3 was observed, which could be suppressed by Wnt inhibition. To study the potential molecular mechanisms, the H3K4me3 histone methyl-transferases, i.e., members of the Mll protein family were analyzed in freshly isolated, sphere-cultured CSCs by RT-PCR: Mll1 was highly transcribed, together with its target genes Hoxa9 and Meis1. Interestingly, the expression of Mll1 was upregulated by Wnt signaling by activating its distal enhancer regions, which was seen with Luciferase reporter assays. Immuno-precipitation further showed that Mll1 is involved in the beta-catenin/Tcf4 transcription factor complex: shRNA treatment against Mll1 reduced sphere formation of mouse salivary gland CSCs. In double mutant mice, additional genetic ablation of Mll1 (triple mutant tumors) abrogated tumor formation and affected the self-renewal ability of CSCs. Collectively, the data presented in this study show that the beta-catenin-Mll1 axis drives self‐renewal and fends off differentiation of CSCs via epigenetic mechanisms. Therefore, targeting Mll1 or its interaction with beta-catenin and other components may help to restore a healthy epigenetic state in the stem cells, which represent a novel and promising therapeutic approach for the treatment of head and neck SCCs.

Indiana University-Purdue University Indianapolis (IUPUI) / The development and function of hematopoietic stem cells (HSCs) is regulated by numerous signaling pathways including Akt-mechanistic target of rapamycin complex1 (mTORC1) pathway. Dysregulation of this pathway results in impaired HSC function and contributes to the development of hematologic malignancies. Activated mTORC1 phosphorylates and subsequently activates ribosomal protein S6 kinase 1 (S6K1). To study the role of S6K1 in hematopoiesis as well as leukemogenesis, we used a genetic model of S6K1 deficient mice (S6K1-/-). We found that loss of S6K1 expression in HSCs results in reduction of absolute HSC number in bone marrow (BM). Following chemotherapy, cycling HSCs undergo apoptosis and quiescent HSCs are required to cycle to regenerate the hematopoietic system. S6K1 regulates the quiescence of HSCs and in the absence of S6K1, mice are more susceptible to repeated myeloablative stress. We also observed that loss of expression as well as gain of expression of S6K1 affects the self-renewal ability of HSCs. Interestingly, when we overexpressed S6K1, it also resulted in reduced self-renewal of HSCs. Next, we assessed the role of S6K1 in the propagation of acute myeloid leukemia (AML). The mixed-lineage leukemia (MLL) gene is required for the maintenance of adult HSCs. Translocations in MLL are detected in approximately 5-10% of adult acute leukemia patients and in approximately 70% of acute leukemias in infants. We expressed MLL-AF9 fusion oncoprotein in WT and S6K1-/- hematopoietic stem and progenitor cells (HSC/Ps) and performed serial transplantation. Upon secondary transplantation, recipients of S6K1 deficient AML cells survived significantly longer compared to controls. In vitro, pharmacological inhibition of S6K1 activity resulted in reduced growth of primary human cells expressing MLL-AF9. Both human and murine HSC/Ps expressing MLL-AF9 showed reduced mTORC1 activity upon inhibition of S6K1 suggesting that loss of S6K1 activity results in reduced Akt-mTORC1 activation both upstream and downstream of mTORC1. Overall, our studies establish a critical role of S6K1 activity in the maintenance of HSC function and in the propagation of leukemia.

No description available.

No description available.

La leucémie myéloïde aiguë (AML) est une forme agressive du cancer du sang qui est caractérisée par un haut taux de mortalité, tant chez les patients plus jeunes que plus âgés. Le développement de nouveaux traitements est ardu par l’hétérogénéité de cette maladie. Dans cette perspective, les études CCC phénotypiques menées à l’IRIC visent à déceler de nouveaux composés ayant une synergie contre des souches AML primaires de patients en vue de personnaliser les thérapies selon leur profil cytogénétique. Il a été découvert que le MG624, un antagoniste des récepteurs nicotiniques α7-nAChR inhibant la prolifération des cellules cancéreuses dans les SCLC, démontrait une activité spécifique contre la souche AML5. Dans cette recherche, les voies de synthèse des analogues du MG624 sont explorées afin de concevoir une série de sondes permettant d’identifier les cibles potentielles via une approche chimioprotéomique par affinité dans le lysat cellulaire. Cette méthode a permis d’identifier trois cibles potentielles à l’aide d’un essai par compétition et de contrôles négatifs, soit XIAP, NQO2 et U119B, toutes impliquées dans différentes formes de cancer. Ces expériences ont mené à une meilleure compréhension des motifs procurant l’activité du composé chez les cellules leucémiques. La synthèse d’une sonde par photoaffinité a ensuite été élaborée pour éventuellement lier les protéines identifiées de manière covalente dans l’environnement cellulaire natif afin de valider ces cibles. / Acute myeloid leukemia (AML) is an aggressive form of blood cancer characterized by a high mortality rate in both younger and older patients. The development of new treatments is hampered by the heterogeneity of this disease, which has led to the phenotypic CCC studies carried out at IRIC to identify new compounds that exhibit synergies against primary AML patient strains to personalize therapies according to their cytogenetic profile. MG624, an α7-nAChR nicotinic receptor antagonist that inhibits cancer cell proliferation in SCLC, was found to have specific activity against AML5. In this research, the synthetic pathways of MG624 analogues are explored to design a series of probes to identify potential targets via an affinity-based chemoproteomic approach in cell lysate. This method identified three potential targets using a competitive assay and negative controls, namely XIAP, NQO2 and U119B, all implicated in different forms of cancer. These experiments led to a better understanding of the motifs that provide the compound's activity in leukemic cells. A photo-affinity probe synthesis was then developed to covalently bind the identified proteins in the native cellular environment to eventually validate these targets.