Differential effects of selective inhibitors targeting the PI3K/AKT/mTOR pathway in acute lymphoblastic leukemia

Purpose: Aberrant PI3K/AKT/mTOR signaling has been linked to oncogenesis and therapy resistance in various malignancies including leukemias. In Philadelphia chromosome (Ph) positive leukemias, activation of PI3K by dysregulated BCR-ABL tyrosine kinase (TK) contributes to the pathogenesis and development of resistance to ABL-TK inhibitors (TKI). The PI3K pathway thus is an attractive therapeutic target in BCR-ABL positive leukemias, but its role in BCR-ABL negative ALL is conjectural. Moreover, the functional contribution of individual components of the PI3K pathway in ALL has not been established. Experimental design: We compared the activity of the ATP-competitive pan-PI3K inhibitor NVP-BKM120, the allosteric mTORC1 inhibitor RAD001, the ATP-competitive dual PI3K/mTORC1/C2 inhibitors NVP-BEZ235 and NVP-BGT226 and the combined mTORC1 and mTORC2 inhibitors Torin 1, PP242 and KU-0063794 using long-term cultures of ALL cells (ALL-LTC) from patients with B-precursor ALL that expressed the BCR-ABL or TEL-ABL oncoproteins or were BCR-ABL negative. Results: Dual PI3K/mTOR inhibitors profoundly inhibited growth and survival of ALL cells irrespective of their genetic subtype and their responsiveness to ABL-TKI. Combined suppression of PI3K, mTORC1 and mTORC2 displayed greater antileukemic activity than selective inhibitors of PI3K, mTORC1 or mTORC1 and mTORC2. Conclusions: Inhibition of the PI3K/mTOR pathway is a promising therapeutic approach in patients with ALL. Greater antileukemic activity of dual PI3K/mTORC1/C2 inhibitors appears to be due to the redundant function of PI3K and mTOR. Clinical trials examining dual PI3K/mTORC1/C2 inhibitors in patients with B-precursor ALL are warranted, and should not be restricted to particular genetic subtypes

Expressions- und Funktionsanalysen von Neuroglobin und Cytoglobin

Im Rahmen der vorliegenden Dissertation wurden Untersuchungen zur Expression und Funktion der respiratorischen Proteine Neuroglobin (Ngb) und Cytoglobin (Cygb) in Vertebraten durchgeführt. Beide Globine wurden erst kürzlich entdeckt, und ihre Funktionen konnten trotz vorliegender Daten zur Struktur und biochemischen Eigenschaften dieser Proteine bisher nicht eindeutig geklärt werden. Im ersten Abschnitt der vorliegenden Arbeit wurde die zelluläre und subzelluläre Lokalisation von Neuroglobin und Cytoglobin in murinen Gewebeschnitten untersucht. Die Expression von Ngb in neuronalen und endokrinen Geweben hängt offensichtlich mit den hohen metabolischen Aktivitäten dieser Organe zusammen. Insbesondere im Gehirn konnten regionale Unterschiede in der Ngb-Expression beobachtet werden. Dabei korrelierte eine besonders starke Neuroglobin-Expression mit Gehirnbereichen, die bekanntermaßen die höchsten Grundaktivitäten aufweisen. In Anbetracht dessen liegt die Funktion des Neuroglobins möglicherweise im basalen O2-Metabolismus dieser Gewebe, wobei Ngb als O2-Lieferant und kurzfristiger O2-Speicher den vergleichsweise hohen Sauerstoffbedarf vor Ort sicherstellen könnte. Weitere Funktionen in der Entgiftung von ROS bzw. RNS oder die kürzlich publizierte mögliche Rolle des Ngb bei der Verhinderung der Mitochondrien-vermittelten Apoptose durch eine Reduktion des freigesetzten Cytochrom c wären darüber hinaus denkbar. Die Cygb-Expression im Gehirn beschränkte sich auf relativ wenige Neurone in verschiedenen Gehirnbereichen und zeigte dort vorwiegend eine Co-Lokalisation mit der neuronalen NO-Synthase. Dieser Befund legt eine Funktion des Cytoglobins im NO-Metabolismus nahe. Quantitative RT-PCR-Experimente zur mRNA-Expression von Ngb und Cygb in alternden Säugern am Bsp. der Hamsterspezies Phodopus sungorus zeigten keine signifikanten Änderungen der mRNA-Mengen beider Globine in alten im Vergleich zu jungen Tieren. Dies widerspricht publizierten Daten, in denen bei der Maus anhand von Western Blot-Analysen eine Abnahme der Neuroglobin-Menge im Alter gezeigt wurde. Möglicherweise handelt es sich hierbei um speziesspezifische Differenzen. Die im Rahmen dieser Arbeit durchgeführte vergleichende Sequenzanalyse der humanen und murinen NGB/Ngb-Genregion liefert zum einen Hinweise auf die mögliche Regulation der Ngb-Expression und zum anderen eine wichtige Grundlage für die funktionellen Analysen dieses Gens. Es konnte ein minimaler Promotorbereich definiert werden, der zusammen mit einigen konservierten regulatorischen Elementen als Basis für experimentelle Untersuchungen der Promotoraktivität in Abhängigkeit von äußeren Einflüssen dienen wird. Bioinformatische Analysen führten zur Identifizierung des sog. „neuron restrictive silencer element“ (NRSE) im Ngb-Promotor, welches vermutlich für die vorwiegend neuronale Expression des Proteins verantwortlich ist. Die kontrovers diskutierte O2-abhängige Regulation der Ngb-Expression konnte hingegen anhand der durchgeführten komparativen Sequenzanalysen nicht bestätigt werden. Es wurden keine zwischen Mensch und Maus konservierten Bindestellen für den Transkriptionsfaktor HIF-1 identifiziert, der die Expression zahlreicher hypoxieregulierter Gene, z.B. Epo und VEGF, vermittelt. Zusammen mit den in vivo-Daten spricht dies eher gegen eine Regulation der Ngb-Expression bei verminderter Verfügbarkeit von Sauerstoff. Die Komplexität der Funktionen von Ngb und Cygb im O2-Stoffwechsel der Vertebraten macht den Einsatz muriner Modellsysteme unerlässlich, die eine sukzessive Aufklärung der Funktionen beider Proteine erlauben. Die vorliegende Arbeit liefert auch dazu einen wichtigen Beitrag. Die hergestellten „gene-targeting“-Vektorkonstrukte liefern in Verbindung mit den etablierten Nachweisverfahren zur Genotypisierung von embryonalen Stammzellen die Grundlage zur erfolgreichen Generierung von Ngb-knock out sowie Ngb- und Cygb-überexprimierenden transgenen Tieren. Diese werden für die endgültige Entschlüsselung funktionell relevanter Fragestellungen von enormer Bedeutung sein.In the context of this thesis the expression and function of the respiratory proteins Neuroglobin (Ngb) and Cytoglobin (Cygb) in vertebrates were investigated. Both globins were recently discovered and their functions are, despite available data on the structure and biochemical characteristics of these proteins, not yet clear. In the first part of this work the cellular and subcellular localization of Neuroglobin and Cytoglobin in mouse tissue sections were examined. The expression of Neuroglobin in neuronal and endocrine tissues is obviously connected with the high metabolic activities of these organs. In particular regional differences to the Ngb expression in the brain could be observed. A strong Neuroglobin expression correlated thereby with brain regions with the highest basic metabolic rates. This points to towards a possible role of Neuroglobin close to the basal O2 metabolism of this tissues, whereby Ngb could guarantee the comparatively high oxygen demand as local O2 supplier or short term O2 storage. Further functions in the detoxification of reactive oxygen and nitrogen species (ROS/RNS) or the recently published putative role in the prevention of mitochondria mediated apoptosis by reduction of relased cytochrom c are also conceivable. The Cygb expression in the brain was limited to distinct neurons in different brain areas, where predominantly a colocalization with the neural NO Synthase could be shown. These findings suggest a function of Cytoglobin in the NO metabolism. Quantitative Realtime PCR analyses of Ngb and Cygb expression in senescent mammals at the example of the hamster species Phodopus sungorus did not show significant changes in the mRNA amount of both globins among young and old animals. This is contradictory to published data, where a reduction of the Neuroglobin expression was shown in aged mice. This discrepancies might be due to species specific differences. Furthermore a comparative sequence analysis of the human and mouse NGB/Ngb gene region has been carried out, which is required for the understanding of the regulatory mechanism of the Ngb gene expression and on the other hand an important prerequisite for the functional analysis of this gene. A minimal promotor region could be defined, which will together with other conserved regulatory elements provide the basis for experimental investigations for the promotor activity against external stimuli. Bioinformatical analysis led to the identification of the so called "neuron restrictive silencer element" (NRSE) in the Ngb promotor region, which is probably responsible for the predominant neuronal expression of this protein. Comparative sequence analysis did not confirm the controversially discussed O2 dependent regulation of the Ngb gene expression. Conserved binding sites for the transcription factor HIF-1, which madiate the expression of numerous hypoxia responsible genes, e.g. Epo and VEGF, could not be identified in the mouse and human gene sequences. Together with further in vivo data this speaks against a regulation of the Ngb gene expression in response to decreased availability of oxygen. The complexity of the functions of Ngb and Cygb in the O2 metabolism of the vertebrates makes the application of murine model systems essential, which enable a gradual clearing up of the functions of both proteins. This work supplies an important contribution to this. The designed gene-targeting vector constructs build together with the established procedures for genotyping of embryonic stem cells the basis for a successful generation of Ngb knock out as well as Ngb and Cygb overexpressing transgenic animals.These will be of enormous importance for the final decoding of functional relevant questions

Neuron-specific expression of neuroglobin in mammals

Abstract Neuroglobin, a vertebrate oxygen-binding protein, is expressed in many regions of the adult brain. We examined the cell type-specific expression of neuroglobin in neurons and astroglial cells in primary cultures of fetal hippocampal cells and sections of the adult mouse brain using neuroglobin-specific polyclonal antibodies and cell type-specific markers NeuN and GFAP to differentiate between neurons and glial cells. Neuroglobin is exclusively expressed in neurons, but not in astroglial cells. Accordingly, neuroglobin was detected in two neuroblastoma cell lines (N2a, SH-SY5Y) and the pheochromocytoma cell line PC-12, but not in glioblastoma cell lines (DKMG, GAMG) or other, non-neural cells (HeLa, Vero). Analysis of the neuroglobin genomic sequence from man and mouse identifies sequence motifs with similarity to the neuron-restrictive silencer element, possibly explaining a neuron-specific expression of neuroglobin. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Globin; Neuroglobin; Oxygen-binding; Glial cells; Gene expression Neuroglobin (Ngb) is a recently discovered vertebrate heme protein We have recently analyzed the expression pattern of Ngb mRNA and protein within different regions of the murine brain, revealing regionally different Ngb expression with high levels in some brain regions, including the hippocampus, the thalamus and the hypothalamus, the preposital hypoglossal and the lateral vestibular nucleus For primary hippocampal cell culture, eight to ten hippocampi from 17-to 18-day-old rat fetuses (SpragueDawley) were prepared according to Ref. [1]. Cells were plated in a concentration of 2 £ 10 5 /ml on glass coverslips (1.2 cm Ø) precoated with poly-L-lysine (Sigma-Aldrich, Seelze, Germany) and cultured in Dulbecco's modified Eagle's medium/Nut Mix F-12 (Invitrogen), 2 mM Lglutamin (PAA Laboratories, Cölbe, Germany) and penicillin/streptomycin (50 units/ml and 50 mg/ml; PAA Laboratories) with 10% fetal calf serum (FCS) for the first 24 h. This procedure allows a limited growth of astroglial cells. Further cultivation was done in medium plus 2% B-27 Supplement (Invitrogen) for up to 14 days in a humidified atmosphere of 37 8C and 5% CO 2 . Cell lines were cultured in Dulbecco's MEM (Invitrogen) with 2 mM L-glutamin (PAA Laboratories) and penicillin/streptomycin (50 units

Effect of ABL-directed tyrosine kinase inhibitors on BCR-ABL+ ALL LTCs.

<div><p>Ph+ ALL cells with the T315I mutation showed no growth inhibition (A) or induction of cell death (B) in response to any of the TKI. The BCR-ABL+ ALL-LTC PH was used as a positive control (A and B). </p> <p>Response to ABL-directed TKI of 6 non-mutated BCR-ABL+ LTCs (BV, CM, DW, KW, PH and VB) and the LTC KÖ with the T315I mutation (C). Cell death was examined on day 4 of exposure to increasing concentrations of imatinib, dasatinib, and nilotinib. </p> <p>(A, B, C) Cell proliferation was assessed by XTT assay and induction of cell death was measured by Annexin-V/propidium iodide staining. The data shown represent the means <u>+</u> SD of 3 experimental replicates from one representative experiment out of 3 performed. </p></div

The impact of mTORC1 inhibition in B-ALL is independent of the presence of an ABL translocation.

<div><p>BCR-ABL+ (BV, PH, KW, CM, BV und DW), TEL-ABL+ (VG) and BCR-ABL- (HP, KR, RL, CR und SK) LTCs were exposed to increasing concentrations of the mTORC1 inhibitor RAD001. (A) Proliferation and (B) cell death were measured after 4 days of drug treatment. The (A) proliferation rate and (B) rate of cell death of the ABL-translocated cells (BCR-ABL+/TEL-ABL+) and the BCR-ABL- cells did not differ in their response to treatment with RAD001 at 25nM or 5µM, respectively (corresponding approximately to the IC₅₀) values determined for growth inhibition and induction of apoptosis, respectively). </p> <p>(A, B) Cell proliferation was assessed by XTT assay, induction of apoptosis was measured by Annexin-V/propidium iodide staining. The data shown represent the means + SD of 3 experimental replicates from one representative experiment out of 3 performed. </p> <p>(C) BCR-ABL+ (PH, BV and KÖ), TEL-ABL+ (VG), BCR-ABL- (HP) and Jurkat cells were treated with increasing concentrations of RAD001 for 2h. Lysates of these cells were used for the detection of phosphorylated and total AKT, S6 and 4E-BP1 by Western blotting. Lysates of untreated Jurkat cells were used as positive controls and those of cells treated with the PI3K inhibitor Wortmannin (WM) for 2h at 1µM were used as negative controls. β-Actin was used as loading control.</p></div

Impact of ABL-kinase inhibition on PI3K/AKT/mTOR signaling in BCR-ABL and TEL-ABL positive ALL LTCs.

<p>BCR-ABL+ (BV, PH, KW, CM, BV und DW), TEL-ABL+ (VG) and BCR-ABL- (HP, KR, RL, CR und SK) LTCs were treated with 1µM Imatinib for 20h. Lysates of these cells were used for the detection of phosphorylated and total AKT, S6 and 4E-BP1 by Western blotting. Because of the constitutively activated PI3K/AKT/mTOR pathway in Jurkat cells, lysates of untreated Jurkat cells were used as positive controls and that of cells treated for 2h with 1µM Wortmannin (WM), a PI3K inhibitor, were used as negative controls. β-Actin was used as loading control.</p

The impact of additional inhibition of mTORC2 on combined PI3K and mTORC1 inhibition in B-ALL is independent of the presence of an ABL translocation.

<div><p>BCR-ABL+ (PH and BV) and BCR-ABL- (HP) cells were treated with 0.5µM or 2µM NVP-BKM120 (PI3K inhibitor), 0.5µM or 2µM RAD001 (mTORC1 inhibitor) alone or in combination. For combined PI3K/mTORC1/C2 inhibition, cells were treated with 0.5µM or 2µM of NVP-BGT226 or NVP-BEZ235. (A) Proliferation and (B) cell death were measured after 4 days of drug treatment. </p> <p>(A, B) Cell proliferation was assessed by XTT assay, induction of cell death was measured by Annexin-V/propidium iodide staining. The data shown represent the means + SD of 3 experimental replicates from one representative experiment out of 3 performed. </p> <p>(C) Lysates were prepared after 2h of drug treatment for the detection of phosphorylated and total AKT, S6 and 4E-BP1 by Western blotting. Lysates of untreated Jurkat cells were used as positive controls, those of cells treated for 2h with 1µM Wortmannin (WM) were used as negative controls. β-Actin was used as loading control. </p></div

The impact of combined PI3K, mTORC1 and mTORC2 inhibition in B-ALL is independent of the presence of an ABL translocation.

<div><p>BCR-ABL+ (BV, PH, KW, CM, BV und DW), TEL-ABL+ (VG) and BCR-ABL- (HP, KR, RL, CR und SK) LTCs were exposed to increasing concentrations of the PI3K/mTORC1/C2 inhibitors NVP-BGT226 and NVP-BEZ235. (A) Proliferation was measured after 4 days of drug treatment. Inhibition of proliferation by 10nM NVP-BGT226 (corresponding approximately to the IC₅₀) was more pronounced in the ABL-translocated cells (BCR-ABL+/TEL-ABL+) than in BCR-ABL- cells (p=0.0283 (*)). In contrast, treatment with 50nM NVP-BEZ235 (corresponding approximately to the IC₅₀) showed no difference between BCR-ABL+/TEL-ABL+ and BCR-ABL- cells. (B) Apoptosis was measured after 4 days of drug exposure. The rate of cell death induced by NVP-BEZ235 or NVP-BGT226 was not significantly different in ABL-translocated cells (BCR-ABL+/TEL-ABL+) and BCR-ABL- cells (A, B) Cell proliferation was assessed by XTT assay, induction of cell death was measured by Annexin-V/propidium iodide staining. The data shown represent the means + SD of 3 experimental replicates from one representative experiment out of 3 performed. </p> <p>(C, D) BCR-ABL+ (PH, BV and KÖ), TEL-ABL+ (VG), BCR-ABL- (HP) and Jurkat cells were treated with increasing concentrations of (C) NVP-BGT226 or (D) NVP-BEZ235 for 2h. Lysates of these cells were used for the detection of phosphorylated and total AKT, S6 and 4E-BP1 by Western blotting. Lysates of untreated Jurkat cells were used as positive controls and those of cells treated for 2h with 1µM Wortmannin (WM) served as negative controls. β-Actin was used as loading control. d = DMSO control. </p></div