Is there a cloud in the silver lining for imatinib?

Imatinib mesylate (Gleevec® or Glivec®), a small molecule tyrosine kinase inhibitor for the treatment of chronic myeloid leukaemia, has been said to herald the dawn of a new er-a of rationally designed, molecularly targeted oncotherapy. Lurking on the same new horizon, however, is the age-old spectre of drug resistance. This review sets the intoxicating clinical perspective against the more sobering laboratory evidence of such divergent mechanisms of imatinib resistance as gene amplification and stem cell quiescence. Polychemotherapy has already been considered to combat resistance, but a more innovative, as yet unformulated, approach may be advocated

il CAI modula l'espressione di Bcr-Abl tramite l'aumento dei Ros in cellule di leucemia mieloide cronica Imatinib-resistenti

La leucemia mieloide cronica (LMC) \ue8 una neoplasia causata da una traslocazione reciproca non bilanciata tra il braccio lungo del cromosoma 9 e quello del cromosoma 22. Tale traslocazione determina la formazione dell\u2019oncogene di fusione bcr-abl codificante per un\u2019oncoproteina con attivit\ue0 tirosin-chinasica costitutiva. Le conoscenze dei meccanismi molecolari alla base della neoplasia, acquisite negli ultimi anni, hanno permesso di sviluppare terapie volte all\u2019inibizione dell\u2019attivit\ue0 chinasica della chimera BCR-ABL. Tra queste, l\u2019imatinib mesilato (IM), inibitore selettivo della protein chinasi, ha rivoluzionato le terapie per la LMC. Sebbene numerosi pazienti in fase cronica, trattati con IM, abbiano raggiunto la remissione completa della neoplasia, in diversi casi si \ue8 avuto ricaduta e/o progressione in fase accelerata o blastica. Da qui la necessit\ue0 di individuare nuovi farmaci che agiscano su bersagli differenti. Il carbossiamidotriazolo (CAI) \ue8 un nuovo farmaco citostatico anticancro in fase II dei clinical trials al NCI. Tale farmaco \ue8 un inibitore dei canali del Ca2+ non voltaggio dipendenti dunque agisce inibendo la proliferazione, la motilit\ue0 e la sopravvivenza di numerose linee tumorali. Il nostro gruppo di ricerca ha dimostrato che dosi sub-tossiche di CAI inibiscono la proliferazione di cellule LMC IM-resistenti (LAMA84R, K562R e KCL22R) ed inducono apoptosi. Tali effetti sono associati alla capacit\ue0 di questo farmaco di determinare un drastico decremento dei livelli generali di fosforilazione in tirosina, inibendo vie di trasduzione del segnale BCR/ABL-dipendenti ed indipendenti, ma anche un drastico decremento dei livelli totali dell\u2019 oncoproteina (Alessandro et al., 2008). Al fine di comprendere in modo pi\uf9 approfondito il meccanismo molecolare d\u2019azione del CAI, abbiamo utilizzato, come modello sperimentale, linee cellulari mieloidi murine (32D) trasfettate con plasmidi codificanti per BCR/ABL-p210 (Full lenght), BCR/ABL-T315I e BCR/ABL-E255K. T315I ed E255K sono mutazioni in Bcr-Abl tra le pi\uf9 rappresentate nella popolazione di pazienti affetti da LMC IM-resistenti. I nostri risultati hanno mostrato che il CAI riduce sia la proliferazione delle tre linee cellulari, in modo dose e tempo dipendente, sia la fosforilazione di Bcr-Abl, ma soprattutto i livelli totali di proteina; il CAI agisce anche sul mutante T315I, su cui persino i farmaci di nuova generazione non hanno mostrato alcuna efficacia. Poich\ue9 il CAI non sembra agire sui livelli di proteina totale tramite una prematura degradazione ubiquitina-proteasoma dipendente n\ue9 sembra regolare in modo significativo il ciclo cellulare, abbiamo ipotizzato che possa agire inducendo un aumento delle specie reattive dell\u2019ossigeno (ROS) all\u2019interno della cellula. A tale scopo abbiamo sottoposto le linee LAMA84R, 32D p210 e 32D T315I a trattamenti con dosi e tempi crescenti di CAI per valutare in tali cellule i livelli di ROS. I risultati ottenuti consentono di concludere che il CAI induce sulle cellule Bcr-Abl+ ed IM-resistenti un aumento della produzione di ROS, in grado di indurre uno stress cellulare. Poich\ue9 il CAI \ue8 gi\ue0 in fase II di sperimentazione clinica, per il trattamento di altre neoplasie, questi risultati forniscono indicazioni rilevanti per un uso razionale di questo farmaco nel trattamento di pazienti affetti da LMC che non rispondono alla monoterapia con IM

Untersuchungen zu Veränderungen des Zytostatika-Resistenz-Phänotyps bei Philadelphia-Chromosom positiven Leukämien unter Imatinib-Therapie

Bei vielen malignen Erkrankungen sind deregulierte Signalübertragungswege bedeutsam für die Pathogenese. Modellcharakter hierfür haben die Philadelphia (Ph) Chromosom positiven Leukämien, zu denen 90 % der chronisch myeloischen Leukämien und etwa 15 - 30 % akuten lymphatischen Leukämien des Erwachsenen gehören. Das Philadelphia Chromosom, das durch die reziproke Translokation von Chromosom 9 und 22 entsteht, codiert für ein tumorspezifisches Fusionsprotein, die BCR-ABL Tyrosinkinase. Diese ist konstitutiv überaktiviert und führt zu autonomen Wachstum und maligner Entartung der Philadelphia positiven Zellen. Ein neuer therapeutischer Ansatz, die Hemmung des pathogenetisch bedeutsamen Signalweges, ist mit dem BCR-ABL spezifischen Tyrosinkinase Inhibitor Imatinib möglich. Der starke antileukämische Effekt von Imatinib gegenüber BCR-ABL positiven Leukämien konnte in klinischen Studien mit Ansprechraten von etwa 60 % bei der Ph+ALL gezeigt werden. Dieser Erfolg wird jedoch von der Resistenzentwicklung gegenüber Imatinib getrübt. So entwickelt die Mehrzahl der Patienten mit Ph+ALL nach einer medianen Behandlungszeit von 2 Monaten ein Rezidiv. Ein häufiger Resistenzmechanismus bei Polychemotherapien ist die erhöhte Expression der pleiotrope Zytostatikaresistenz vermittelnden Proteine P-gp und MRP-1. Diese Arbeit sollte untersuchen, ob die ABC-Transportproteine P-gp und MRP-1 bei der Imatinibresistenz beteiligt sind. MRP-1 und das durch das MDR-1 Gen codierte P-gp sind membranständige Transportproteine, die Tumorzellen durch Auswärtstransport von vielen unterschiedlichen, chemisch nicht verwandten Zytostatika, Resistenzen gegenüber diesen Substanzen verleihen. Um die Rolle von P-gp und MRP-1 bei der Entwicklung der Imatinibresistenz zu bestimmen, wurde untersucht, ob Imatinib ein Substrat von P-gp oder MRP-1 ist, und ob es während der Behandlung mit Imatinib zu einem Anstieg der funktionellen Aktivität dieser Proteine kommt. Mittels transfizierter Zelllinien ließ sich zeigen, dass die P-gp Substrate Calcein AM und Tritium-markiertes Vinblastin in Anwesenheit von Imatinib schlechter transportiert werden. Bei Annahme eines kompetetiven Mechanismus lag die Vermutung nahe, dass Imatinib nicht nur ein Inhibitor des P-gp bedingten Transports von Calcein AM und Vinblastin ist, sondern selbst ein Substrat. Durch Verwendung einer P-gp transfizierten Zelllinie in einem Transwellassay, in dem diese einen vektoriellen Substrattransport bewirkt, konnte direkt gezeigt werden, dass Imatinib durch P-gp nicht aber durch MRP-1 und MRP-2 transportiert wird. Somit ist P-gp potentiell in der Lage, Resistenzen gegen Imatinib auszulösen. Um die funktionelle Aktivität von P-gp in den mononukleären Zellen der mit Imatinib behandelten Patienten bestimmen zu können, wurde eine durchflusszytometrische Methode unter Verwendung von Calcein AM etabliert. Die untersuchten Patientenzellen zeigten jedoch bei niedrigem Ausgangsniveau keinen Anstieg der funktionellen P-gp Aktivität nach Resistenzentwicklung, so dass weder MRP1 noch P-gp Anteil an der Resistenzentwicklung bei Ph+ALL gegenüber Imatinib zu haben scheinen. Die Möglichkeit, die konstitutiv aktive Tyrosinkinase von BCR-ABL mit Imatinib zu inhibieren, ermöglicht es, pathogenetische Charakteristika der BCR-ABL positiven Leukämien zu untersuchen. Die Ph+ALL ist charakterisiert durch ihre schlechte Prognose mit einem Langzeitüberleben von weniger als 10 % bei konventionellen chemotherapeutischen Regimes. Ob die Zytostatikaresistenz vermittelnden Proteine Pgp und MRP-1 hierfür ursächlich sind, sollte in einem weiteren Teil der Arbeit untersucht werden. Durch Hemmung der BCR-ABL spezifischen Tyrosinkinase von Philadelphia Chromosom positiven Zelllinien mit Imatinib kam es bei einem Teil dieser Zelllinien zur Abnahme des MRP-1 Proteinniveaus und der MRP-1 mRNA Expression. Dies begründete die Hypotheose, dass die aktivierte BCR-ABL Tyrosinkinase zu einer Hochregulation der MRP-1 Transkription führt, und damit für die schlechte Prognose ursächlich ist, lag nahe. Weder c-kit noch die BCR-ABL nachgeschalteten Signalproteine RAS-MAP-Kinase, c-MYC und STAT 5 waren bei der Herrunterregulation von MRP-1 unter Imatinib beteiligt. Durch den Nachweis der vorwiegend zytoplasmatischen Lokalisation von MRP-1 in den untersuchten Lymphoblasten mittels konfokaler Immunfluoreszenzmikroskopie und durch den Nachweis einer sehr niedrigen funktionellen Aktivität von MRP-1 in diesen Zellen mittels Durchflusszytometrie eine Bedeutung von MRP-1 für die schlechte Prognose nicht anzunehmen.Deregulation and activation of molecular pathways is crucially involved in the pathogenesis of malignant disease. In 90 % of CML and 15 % - 30 % of ALL the Philadelphia (Ph) chromosome is found and essential for initiating and maintaining the transformed phenotype. The Ph chromosome is characterized at the molecular level by a reciprocal translocation between chromosomes 9 and 22, that leads to formation of a BCR-ABL fusion protein with enhanced and deregulated kinase activity. Recently, imatinib a competitive selective inhibitor of the abl tyrosine kinase has been shown in clinical trials to have pronounced anti-leukemic activity against BCR-ABL expressing leukemia's. In patients with relapsed or chemotherapy-refractory Ph+ALL hematologic response rates of 60-70% have been reported. These responses are generally brief, however, with resistance developing after a median of 2 months. The present study assessed if the ABC-transport proteins P-gp or MRP-1 are involved in imatinibresistance. MRP-1 and P-gp, which is encoded by the mdr-1 gene, are the most common reason for acquisition of resistance to a broad spectrum of anticancer drugs. The over expression of these membrane proteins is considered to result in multidrug resistance by energy-dependent efflux of many anticancer drugs from cell. Therefore it was the aim of the study to assess whether imatinib is a substrate of P-gp and/or MRP- 1/2, and whether clinical resistance to imatinib is associated with high-level activity of these transporters in Ph+ALL. Most substrates of P-gp or MRP-1 are competitive inhibitors of other substrates of these ABC-transporters. Using MDR-1 transfected cell lines over expressing P-gp, imatinib showed an inhibitory effect on Calcein AM and vinblastine transport mediated by P-gp. Direct evidence that imatinib is a substrate of P-gp was obtained with a transwell assay in which MDR-1 and MRP-1 transfected cell lines cause a vectorial drug transport. In this assay P-gp but not MRP-1/2 is able to transport imatinib. Thus P-gp could potentially be involved in imatinib-resistance. To measure functional activity of P-gp and MRP-1 in ALL blasts a flow cytometric method using calcein AM was established. The functional multidrug resistance was low in mononuclear cells of Ph+ALL patients. Most patients did not show an increase of activity after relapse. Therefore these data do not support the thesis of a prominent role of P-gp in imatinib resistance of patients with Ph+ALL. Inhibiting the constitutive active BCR-ABL kinase with imatinib make it possible to uncover characteristics of Ph+ cell lines. Ph+ALL is characterized by an aggressive clinical course with long-term survival of only 0 - 10 % with current chemotherapy regimes. Whether ABC-transporter like P-gp or MRP-1 are involved is only poorly understood. Interestingly, cultivating Ph+ cell lines with imatinib containing media leads to decreased MRP-1 protein and MRP-1 mRNA level in some cell lines. This suggests that a constitutive active BCR-ABL leads to up regulation of MRP-1 and thereby to the poor prognosis. C-kit and signaling pathways downstream BCR-ABL like RAS-MAPK, c- MYC and STAT 5 were not involved in the imatinib induced downregulation of MRP-1. But the low functional activity of MRP-1 shown by flow cytometry and the cytoplasmatic localization of MRP-1 demonstrated by confocal immunfluorescence microscopy in the assessed cell lines enfeeble the thesis of an involvement of MRP-1 in resistance of Ph+ALL to chemotherapy

Application of high resolution melting (HRM) analysis in detection of PDGFRA gene mutations among chronic myeloid leukemia patients treated with imanitib mesylate

A molecular detection of PDGFRA variants is highly relevant for prognosis and therapy prediction in chronic myeloid leukemia (CML) patients treated with Imatinib mesylate (IM). Even though IM has shown an excellent efficacy as a frontline treatment in CML, emerging of resistance towards IM in some CML patients has become a major concern. The development of resistance can be either due to BCR-ABL dependent mechanism or BCR-ABL independent mechanism. The BCR-ABL independent mechanisms include factor in pharmacokinetics of IM such as absorption and distribution of metabolism. However, resistance to IM can also due to the involvement of others IM targeted tyrosine kinases that may play a role in the IM resistance. In response to growing demand for reliable, faster and more sensitive methods, the present study used a High resolution melting (HRM) analysis to elucidate the BCR-ABL independent mechanism involving PDGFRA tyrosine kinase, in Malaysian CML patients undergoing IM therapy. A total of 86 CML patients on IM therapy (43 IM resistances and 43 IM responses) were included in this study. Using HRM analysis, 86 CML patients were screened for PDGFRA variants of exon 10 (c.1432 T>C), exon 12 (c.1701 A>G), exon 14 (c.1977 C>A) and exon 18 (c.2525 A>T). The selected samples with showing different melting curve profile from the reference genotype was subjected to DNA sequencing analysis to validate the genotype. From the data analysed, two PDGFRA variants were detected in exons 10 and 12. The established HRM profile has demonstrated 100% sensitivity and specificity when compared to DNA sequencing. PDGFRA exon 10 (c.1432 T>C) showed a significant risk factor (OR 3.797; 95% CI: 1.502 – 9.591; p = 0.005) for the development of IM resistance. PDGFRA exon 12 (c.1701 A>G) was not a significant risk factor (OR 1.597; 95% CI: 0.681 – 3.745; p = 0.281) for IM resistance. However there was no PDGFRA heterogeneity detected for both exon 14 c.1977 C>G and exon 18 c.2525 A>T. The results suggested that PDGFRA influenced IM resistance in CML patients and further analysis are warranted to confirm the role of PDGFRA in IM resistance mechanism in CML patient

Italian Proteomics Association, 5th Annual National Conference Abstract Volume

Abstract volume of the Italian Proteomics Association 5th Annual national conferenc

Targeting Drug Resistance in Chronic Myeloid Leukemia: A Dissertation

Inhibiting BCR-ABL kinase activity with tyrosine kinase inhibitors (TKIs) has been the frontline therapy for CML. Resistance to TKIs frequently occurs, but the mechanisms remain elusive. First, to uncover survival pathways involved in TKI resistance in CML, I conducted a genome-wide RNAi screen in human CML cells to identify genes governing cellular sensitivity to the first generation TKI called IM (Gleevec). I identified genes converging on and activating the MEK/ERK pathway through transcriptional up-regulation of PRKCH. Combining IM with a MEK inhibitor synergistically kills TKI-resistant CML cells and CML stem cells. Next, I performed single cell RNA-seq to compare expression profiles of CML stem cells and hematopoietic stem cells isolated from the same patient. Among the genes that are preferentially expressed in CML stem cells is PIM2, which encodes a pro-survival serine-threonine kinase that phosphorylates and inhibits the pro-apoptotic protein BAD. Inhibiting PIM2 function sensitizes CML stem cells to IM-induced apoptosis and prevents disease relapse in a CML mouse model. Last, I devised a CRISPR-Cas9 based strategy to perform insertional mutagenesis at a defined genomic location in murine hematopoietic Ba/F3 cells. As proof of principle, we showed its capability to perform unbiased, saturated point mutagenesis in a 9 amino acid region of BCR-ABL encompassing the socalled “gatekeeper” residue, an important determinant of TKI binding. We found that the ranking order of mutations from the screen correlated well with their prevalence in IM-resistant CML patients. Overall, my findings reveal novel resistance mechanisms in CML and provide alternative therapeutic strategies

Aislamiento y caracterización de un inmunógeno asociado al linfoma L5178Y

Los antígenos tumorales representan el punto partida en el desarrollo de estrategias para la prevención, diagnóstico, monitoreo y tratamiento del cáncer y por ello, es de gran interés biomédico su aislamiento, así como las metodologías que para ello se emplean. En este trabajo usamos herramientas propias de la proteómica como la electroforesis bidimensional, análisis de imagen asistido por computadora, Western blotting y espectrometría de masas para la definición de los antígenos asociados al linfoma murino L5178Y. Se llevó a cabo el análisis comparativo de los patrones bidimensionales de expresión de proteínas de membrana de las células tumorigénicas L5178Y-R y su contraparte no tumorigénica L5178Y-S; establecimos los mapas de referencia para ambas líneas y encontramos diferencias en el patrón de expresión de proteínas, que hacen distintivo el perfil de membrana de cada una de estas líneas. Adicionalmente, aprovechamos la respuesta inmune humoral contra tumores de animales inmunizados con ambas líneas para detectar antígenos potenciales en ensayos de inmunodetección. De esta forma identificamos una forma inmunogénica de la proteína sérica alfa-2-HS-glicoproteína asociada a la línea celular L5178Y-R, pero no a la sub-línea no tumorigénica L5178Y-S. Se requieren estudios adicionales para establecer el papel que juega esta forma inmunogénica de fetuina en la tumorigénesis del linfoma L5178Y-R y si esta fetuina inmunogénica se encuentra en otras formas de cáncer. Abstract Tumor antigens hold great promise to develop strategies in cancer prevention, diagnosis, monitoring and treatment. In the present thesis work we applied proteomic tools such as two-dimensional electrophoresis, computer assisted image analysis, Western blotting, and mass spectrometry, to define the antigens associated to the murine lymphoma L5178Y. In was performed comparative analysis of two-dimensional patterns of membrane proteins for the tumorigenic cells L5178Y-R (LY-R) and the non tumorigenic L5178Y-S (LY-S). The reference proteomic maps for both cell lines showed differences in protein expression patterns that make distinctive the membrane profile for each cell line. Auto-antibodies raised against LY-R and LY-S cells were employed to detect potential antigens in the surface of these cells in immunodetection assays. Through this approach it was detected an immunogenic form of alpha-2-HSglycoprotein, also known as fetuin-A, on LY-R lymphoma cells. Fetuin-A was recognized by antibodies present in the serum of LY-R tumor-bearing and immunized mice, but not by sera of mice immunized with the non-tumorigenic variant LY-S or by healthy mouse serum. Further analysis is needed in order to establish the role that immunogenic fetuin-A plays in tumorigenesis of this lymphoma and if this immunogenic form is relevant to others types of cancer

CTO inhibits the adhesion of LAMA84R cells to exosome-treated HUVEC monolayer.

<p>(a) Phase contrast micrographs showing the adhesion of LAMA84R cells (arrows) on HUVEC monolayer treated with 50 µg/ml of exosomes (Exo) and with 50 µg/ml of exosomes after pre-treatment of 24 h with 10 µM CTO. (b) Adhesion of LAMA84R cells to endothelial cell monolayer treated for 6 h with: 50 µg/ml of exosomes (Exo) and exosomes plus increasing doses of CTO (1–10 µM); (c) Adhesion of LAMA84R cells to endothelial cell monolayer treated for 6 h with: 50 µg/ml of exosomes (Exo), 50 µg/ml of exosomes plus 10 µg/ml of a neutralizing anti-IL8 antibody (N Ab IL8), 10 ng/ml of recombinant IL8 (Rec IL8), 10 ng/ml of recombinant IL8 plus 10 µg/ml of a neutralizing anti-IL8 antibody and 10 ng/ml of recombinant IL8 with increasing doses of CTO (1–10 µM). Values are representative for three independent experiments. *p≤0.05; **p≤0.01.</p

<i>in vitro</i> inhibition of exosome-stimulated angiogenesis by CTO.

<p>(a) Phase contrast micrographs showing the effects of LAMA84R exosomes and CTO treatment on endothelial network formation (matrigel assay). Few cables are observed when HUVEC are plated in low serum medium (CN). Addition to HUVEC cells of 10 ng/ml of recombinant IL8 (Rec IL8) or 50 µg/ml of LAMA84R exosomes (Exo) induces the formation of capillary-like structures. No tube formation is observed when HUVEC are plated in the presence of 50 µg/ml of exosomes plus neutralizing anti-IL8 antibody (Exo + N Ab IL8). CTO inhibits the effects of recombinant IL8 (10 µM CTO + Rec IL8) or exosomes (Exo +10 µM CTO) on tube formation by HUVEC on matrigel. (b) Histograms showing the quantitative analysis of the cables length by Image J software.</p