AKR1C enzymes sustain therapy resistance in paediatric T-ALL

BACKGROUND: Despite chemotherapy intensification, a subgroup of high-risk paediatric T-cell acute lymphoblastic leukemia (TALL) patients still experience treatment failure. In this context, we hypothesised that therapy resistance in T-ALL might involve aldo-keto reductase 1C (AKR1C) enzymes as previously reported for solid tumors.METHODS: Expression of NRF2-AKR1C signaling components has been analysed in paediatric T-ALL samples endowed with different treatment outcomes as well as in patient-derived xenografts of T-ALL. The effects of AKR1C enzyme modulation has been investigated in T-ALL cell lines and primary cultures by combining AKR1C inhibition, overexpression, and gene silencing approaches.RESULTS: We show that T-ALL cells overexpress AKR1C1-3 enzymes in therapy-resistant patients. We report that AKR1C1-3 enzymes play a role in the response to vincristine (VCR) treatment, also ex vivo in patient-derived xenografts. Moreover, we demonstrate that the modulation of AKR1C1-3 levels is sufficient to sensitise T-ALL cells to VCR. Finally, we show that T-ALL chemotherapeutics induce overactivation of AKR1C enzymes independent of therapy resistance, thus establishing a potential resistance loop during T-ALL combination treatment.CONCLUSIONS: Here, we demonstrate that expression and activity of AKR1C enzymes correlate with response to chemotherapeutics in T-ALL, posing AKR1C1-3 as potential targets for combination treatments during T-ALL therapy

"High Risk" Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is a neoplasia characterized by an abnormal, clonal and self-maintaining proliferation of lymphoid cells. In this three year of phd I tried to add some information to understand the complex molecular mechanisms underlying this disease. I performed my studies in the laboratory of "Oncoematologia Pediatrica, Dipartimento di Pediatria dell'Università  degli studi di Padova" and for three months in the laboratory of Prof. A. A. Ferrando, Columbia University, Irving Cancer Center, New York. The recombination, insertion and deletion of immunoglobulin (Ig) and T cell receptors (TCR) gene segments results in an individual gene sequence unique for each lymphocytes, named N-region. This genes junctional-region can be considered a fingerprint-marker specific of each lymphocytes and, consequentially, of each lymphoid neoplasia. It can be used to study the biological characteristics of leukemia or to analyze the minimal residual disease (MRD). The initial period of my Phd has been dedicated to the study of the t(4;11) positive ALL, in 32 children above the age of 1 year through the survival, immunophenotype and Ig/TCR pattern analysis. Immunophenotype data in t(4;11)-positive ALL from children 1 year or older demonstrate a more mature pattern of IGH rearrangements as compared to infant cases as like as the more mature age goes with a more mature lymphocyte (Chapter 1.1). Fundamental for the Ig/TCR analysis is to have enough exordia DNA to execute the panel of screening PCR. The screening of diagnosis DNA is hampered by lack of fresh material and consequent limited amount of genomic DNA available. We evaluate the possibility to perform the Ig/TCR screening starting from unstained glass slide smear of cells from bone marrow aspirate or from extracted small amount of DNA after a whole genome amplification with ?29 polymerase and random primers. We execute 476 PCR before and after the whole genome amplification. Results of PCR analysis, confirmed sequencing the PCR products, have a concordance of 98%. No false-positive results were obtained by PCR analysis after whole genome amplification (Chapter 1.2). The main topic of my Phd studies have been the prognostic value of MRD analysis in relapsed pediatric ALL patients. The treatment of ALL in children has improved over the last decades, nevertheless, disease recurrence remains the leading cause of treatment failure in the 20% of patients. In the AIEOP LAL REC2003 protocol, relapsed patient are stratified in class of risk: low-medium (S1-S2) and high risk (S3-S4) based on immunophenotype, time and site of relapse. We evaluate the prognostic value of MRD during the therapeutic treatment in high risk relapsed patients. We studied 60 relapsed patients classified in S3-S4, treated with AIEOP REC2003 protocol. The Ig/TCR clonal profile have been studied with screening PCR and homo-heteroduplex analysis in each patient; the MRD have been exanimate with RQ-PCR in different time-point during therapy: (TP1) after induction, (TP2) after re-induction, (TP3) and after consolidation after relapse. At three year, the EFS is 73, 45 and 19% respectively for patients with TP1 negative, positive not quantifiable (MRD < 10-4) or positive (MRD ? 10-4), (p < 0.05). The statistical significance of MRD predictive prognostic value have been confirmed by multivariate analysis. In high-risk relapsed patients we demonstrated that MRD evaluation was able to identify patients who cold benefit from the chemotherapy treatment and subset of patients who seem not to benefit from further consolidations, including allogenic HSCT (Chapter 2.1). Medullary (BM) relapse is currently defined still according to morphological criteria, when a blast count ? 25% is detected in a bone marrow sample after the achievement of complete remission. We evaluate the clinical significance of MRD monitoring in ALL pediatric patients as an indicator of impending hematological disease recurrence and determinate the critical levels of MRD which can predict relapse. The cumulative incidence of relapse in case of detection of positive and quantifiable MRD finding resulted 85.7%. The value is statistically significant in prediction when compared with the presence of a MRD negative or positive below the quantitative range. The prior identification of MRD as an signal of subsequent morphological hematological relapse could help to decide for a preventive chemotherapeutic treatment (Chapter 2.1). To understand the way to develop new therapeutic treatment for patient with high-risk characteristics, we embrace the hypothesis that a rare cancer stem cell population would be the origin of many malignant neoplasy, responsible of the growth, diffusion and resistance to treatment of tumor, providing a key target for novel curative therapies. In ALL patients this sub-population has not been clearly identify and characterized, anyway recent studies move the attention to the subpopulations characterized by surface antigens CD34/CD38/CD19. We analyzed the Ig/TCR clonal profile of CD34+CD38-CD19+ and CD34+CD38-CD19- subpopulations after sorting, comparing with the Ig/TCR pattern of patients' unsorted blasts. In 9/10 patients the subpopulations of progenitors, isolated and analyzed, share with total blast cells at least one specific clonal rearrangement (finger-print marker). This is a direct demonstration, through a genetic marker and not though a functional assay, of the origin fo blast cell from the subpopulation CD34+CD38-, in the 90% of cases from the sub-fraction CD34+CD38-CD19- (Chapter 3.1). We also studied the correlation between the frequency of CD34+CD38- at the diagnosis of ALL and the level of MRD after treatment. We analyzed 133 ALL patients finding and statistical significant association between the percentage of CD34+CD38- <1% and a low level of MRD at day 33 of chemotherapy (Chapter 3.1). During the last period of the phd I approached the study of WT1, a transcription factor important for normal cellular development and cell survival. The molecular mechanisms involved in disease progression and recurrence of T-ALL are poorly understood. Starting from the observation that in the 10% of T-ALL patients WT1 is mutated, the attention move toward it with genetic and proteomic studies to comprehend its involvement in the development of leukemia. My contribute concerned the study of WT1 gene promoter methylation status (Chapter 4.1).La leucemia linfoblastica acuta (LLA) è una neoplasia caratterizzata da una proliferazione abnorme, clonale ed auto-mantenuta di cellule linfoidi. In questi tre anni di dottorato ho cercato di aggiungere qualche nozione per la comprensione dei complessi meccanismi molecolari che sottendono a questa malattia. L'attività  di ricerca si è svolta presso il Laboratorio di Oncoematologia Pediatrica, Dipartimento di Pediatria dell'Università  degli Studi di Padova e per tre mesi presso il Laboratorio del Prof. A. A. Ferrando, Columbia University Irving Cancer Research Center, New York. Ogni linfocita è contraddistinto a livello dei siti di ricombinazione, inserzione e delezione dei segmenti genici delle regioni variabili delle immunoglobuline (Ig) e del recettore delle cellule T (TCR), da una regione giunzionale detta N-region. Le regioni giunzionali possono essere considerate un fingerprint-marker specifico di ogni linfocita e di ogni neoplasia linfoide ed essere utilizzate per studiare le caratteristiche biologiche della leucemia o per l'analisi della malattia residua minima (MRD). I primi mesi di dottorato sono stati dedicati allo studio della LLA positiva per t(4;11) in 32 pazienti pediatrici di età  superiore a 1 anno attraverso l'analisi dell'immunofenotipo, riarrangiamenti Ig/TCR e prognosi. Dall'analisi è stato identificato un pattern di riarrangiamenti diverso tra i pazienti pediatrici e gli infant dovuto alla diversa maturità  della cellula nelle due classi di pazienti come se la maggiore età  dei pazienti andasse a pari passo con lo stadio maturativo del blasto leucemico (Capitolo 1.1). Fondamentale per l'analisi dei riarrangiamenti Ig/TCR è la disponibilità di materiale per l'estrazione del DNA e l'esecuzione del pannello di PCR di screening. Se si ha una quantità limitata di DNA non è quindi sempre possibile ricavare il pattern di clonalità . Abbiamo eseguito uno studio per valutare la possibilità di eseguire le PCR di screening della regione Ig/TCR su DNA amplificato con una tecnica di "whole genome amplification" basata sull'utilizzo della DNA polimerasi ?29 e di random primer. Il DNA utilizzato era estratto da cellule in sospensione di asiprato midollare, anche in quantità limitata, o da cellule su vetrino non colorato. Abbiamo eseguito 476 PCR prima e dopo "whole genome amplification". Il confronto dei risultati, dopo sequenziamento dei prodotti di PCR, ha mostrato una concordanza dei risultati del 98%. L'amplificazione dell'intero genoma non ha inficiato i risultati di PCR nella regione Ig/TCR (Capitolo 1.2). Il principale campo di interesse in questi anni di dottorato è stata lo studio del valore prognostico della MRM nei pazienti pediatrici LLA ricaduti. La quasi totalità dei pazienti pediatrici con leucemia linfoblastica acuta raggiunge la remissione completa continua ma esiste ancora un 20% di questi che va incontro ad una recidiva di malattia. Il protocollo AIEOP LAL REC 2003 stratifica i pazienti ricaduti in classi di rischio: basso-medio (S1-S2) e alto (S3-S4) a seconda dell'immunofenotipo, del tempo e della sede di ricaduta. Abbiamo valutato il valore prognostico della malattia residua minima durante la terapia nella classe di pazienti ad alto rischio. Sono stati studiati 60 pazienti ricaduti classificati come S3-S4, arruolati nel protocollo AIEOP LAL REC 2003. Per ogni paziente E' stato analizzato il profilo di clonalità  con PCR di screening e l'analisi heteroduplex dei riarrangiamenti Ig/TCR; è stata analizzata la malattia residua minima (MRM) attraverso RQ-PCR in diverse fasi della terapia: dopo l'induzione (TP1), dopo la re-induzione (TP2) e dopo il consolidamento (TP3) post-ricaduta. L'EFS a tre anni è del 73, 45 e 19% rispettivamente per i pazienti con MRD al TP1 negativa, positiva non quantificabile (MRD < 10-4) o positivo (MRD ? 10-4), (P < 0.05). Il valore prognostico predittivo statisticamente significativo dell'MRD è stato confermato dall'analisi multivariata. Abbiamo dimostrato che la quantificazione delle malattia residua minima permette di differenziare i pazienti precocemente e in modo efficace comprendendo quali pazienti rispondono alla terapia convenzionale e possano ricevere il trapianto di cellule staminali allogeniche e quali invece necessitino di terapie innovative (Capitolo 2.1). La ricaduta midollare è attualmente definita secondo criteri morfologici quando la conta dei blasti è ?25% dopo che il paziente ha raggiunto la remissione completa. Abbiamo valutato il potere della presenza di MRM come indicatore di successiva ricorrenza ematologica di ALL determinando se vi siano livelli critici di MRM predittivi di ricaduta. Abbiamo trovato che rilevare un prelievo positivo quantificabile durante il follow-up del paziente è associato a una cumulative relapse incidence dell'85.7%. Questo dato ha valore statisticamente significativo se confrontato con il valore predittivo di un prelievo negativo o positivo non quantificabile per MRM. Identificare anticipatamente la ricaduta potrebbe aiutare nel progettare un trattamento terapeutico preventivo di ricaduta morfologica (Capitolo 2.1). Nella prospettiva di comprendere su quali vie si potrebbero spostare le future strategie terapeutiche per pazienti che presentino caratteristiche di alto rischio, abbiamo abbracciato l'ipotesi attuale che l'origine di molte neoplasie maligne risieda in una ristretta popolazione di cellule staminali tumorali responsabile della crescita, diffusione tumorale e resistenza alla terapia. Nella LLA questa popolazione non è stata ancora chiaramente identificata e caratterizzata anche se studi recenti spingono l'attenzione sulle subpopolazioni caratterizzate dagli antigeni di superficie CD34/CD38/CD19. Abbiamo analizzato in 10 pazienti il profilo di clonalità  Ig/TCR delle popolazioni CD34+CD38-CD19+ e CD34+CD38-CD19- dopo sorting, confrontandolo con quello identificato nella popolazione totale dei blasti leucemici del paziente. In 9/10 pazienti le subpopolazioni di progenitori, isolate e analizzate, condividevano almeno un riarrangiamento genico clonale (fingerprint- marker) con i blasti leucemici. Questa è una dimostrazione diretta, attraverso un marcatore genetico, e non funzionale, dell'origine del blasti leucemici dalla popolazione CD34+CD38-, nel 90% dei casi nella sottopolazione CD34+CD38-CD19- (Capitolo 3.1). Abbiamo inoltre studiato se la frequenza del compartimento CD34+CD38- all'esordio LLA correlasse con il livello di MRM dopo chemioterapia. Abbiamo analizzato 133 pazienti LLA rilevando che una percentuale <1% di CD34+CD38- correla in modo statisticamente significativo con un basso livello di MRM rilevato dopo 33 giorni di terapia (Capitolo 3.1). Durante gli ultimi mesi di dottorato mi sono avvicinata allo studio di WT1, un fattore di regolazione dello sviluppo. I meccanismi molecolari implicati nello sviluppo e nella ricaduta della LLA ad immunofenotipo T sono scarsamente compresi. Partendo dall'osservazione che nel 10% dei pazienti con LLA-T il gene WT1 è mutato, abbiamo approcciato lo studio di questo fattore dal punto di vista genico e proteico, per comprendere il suo coinvolgimento nello sviluppo della leucemia. La parte del lavoro a cui ho contribuito ha riguardato l'analisi dello stato di metilazione del promotore del gene WT1 (Capitolo 4.1)

Molecular pathogenesis and targeted therapies for NOTCH1-induced T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic tumor resulting from the malignant transformation of immature T-cell progenitors. Originally associated with a dismal prognosis, the outcome of T-ALL patients has improved remarkably over the last two decades as a result of the introduction of intensified chemotherapy protocols. However, these treatments are associated with significant acute and long-term toxicities, and the treatment of patients presenting with primary resistant disease or thoserelapsing after a transient response remains challenging. T-ALL is a genetically heterogeneous disease in which numerous chromosomal and genetic alterations cooperate to promote the aberrant proliferation and survival of leukemic lymphoblasts. However, the identification of activating mutations in the NOTCH1 gene in over 50% of T-ALL cases has come to define aberrant NOTCH signaling as a central player in this disease. Therefore, the NOTCH pathway represents an important potential therapeutic target. In this review, we will update our current understanding of the molecular basis of T-ALL, with a particular focus on the role of the NOTCH1 oncogene and the development of anti-NOTCH1 targeted therapies for the treatment of this disease

Comparative sequence analysis of incomplete DJH and TCR gene rearrangements in children with relapses of T-ALL

The detection of minimal residual disease (MRD) during the first phase of treatment can predict outcome in childhood acute lymphoblastic leukemia (ALL).1 Currently MRD detection in ALL patients provides important information in order to assign a tailored-treatment and the risk of an impending relapse. Nevertheless, the major treatment failure in ALL occurs predominantly in patients with T-cell ALL. This reflects especially a more therapy-resistance and a slower clearance of blasts of T-ALL in comparison with precursor-B-ALL.2 Therefore, the quantification of early response to therapy and the monitoring of MRD during and after treatment can greatly improve the outcome and long-term quality of life of these patients. In childhood ALL, detection of MRD with high sensitivity (ie 104, 105) can be achieved by quantitative PCR methods (RQ-PCR) of rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes.1,3 The usefulness of these specific PCR targets should give attention to the possible modification of Ig and TCR gene rearrangements that could occur during the course of disease, due to continue activity of the V(D)J recombinase enzymes in leukemic blast

Extracellular ATP signaling during differentiation of C2C12 skeletal muscle cells: role in proliferation

Evidence shows that extracellular ATP signals influence myogenesis, regeneration and physiology of skeletal muscle. Present work was aimed at characterizing the extracellular ATP signaling system of skeletal muscle C2C12 cells during differentiation. We show that mechanical and electrical stimulation produces substantial release of ATP from differentiated myotubes, but not from proliferating myoblasts. Extracellular ATP-hydrolyzing activity is low in myoblasts and high in myotubes, consistent with the increased expression of extracellular enzymes during differentiation. Stimulation of cells with extracellular nucleotides produces substantial Ca(2+) transients, whose amplitude and shape changed during differentiation. Consistently, C2C12 cells express several P2X and P2Y receptors, whose level changes along with maturation stages. Supplementation with either ATP or UTP stimulates proliferation of C2C12 myoblasts, whereas excessive doses were cytotoxic. The data indicate that skeletal muscle development is accompanied by major functional changes in extracellular ATP signaling

Either IL-7 activation of JAK-STAT or BEZ inhibition of PI3K-AKT-mTOR pathways dominates the single-cell phosphosignature of ex vivo treated pediatric T-cell acute lymphoblastic leukemia cells

: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from lymphoblasts of T-cell origin. While T-ALL accounts for only 15% of childhood and 25% of adult ALL, 30% of patients relapse with a poor outcome. Targeted therapy of resistant and high-risk pediatric T-ALLs is therefore urgently needed, together with precision medicine tools allowing the testing of efficacy in patient samples. Furthermore, leukemic cell heterogeneity requires drug response assessment at the single-cell level. Here, we used single-cell mass cytometry to study signal transduction pathways such as the JAK-STAT, PI3K-AKT-mTOR and MEK-ERK pathways in 16 diagnostic and 5 relapsed TALL primary samples and investigated the in vitro response of cells to Interleukin-7 (IL-7) and the inhibitor BEZ-235. T-ALL cells showed upregulated activity of the PI3K-AKTmTOR and MEK-ERK pathways and increased proliferation and translation markers. We found that perturbation induced by the ex vivo administration of either IL-7 or BEZ-235 reveals a high degree of exclusivity with respect to the phospho-protein responsiveness to these agents. Notably, these response signatures were maintained from diagnosis to relapse in individual patients. In conclusion, we demonstrated the power of mass cytometry single-cell profiling of signal transduction pathways in T-ALL. Taking advantage of this advanced approach, we were able to identify distinct clusters with different responsiveness to IL-7 and BEZ-235 that can persist at relapse. Collectively our observations can contribute to a better understanding of the complex signaling network governing T-ALL behavior and its correlation with influence on the response to therapy

DNA methyltransferase 3a hot-spot locus is not mutated in pediatric patients affected by acute myeloid or T-cell acute lymphoblastic leukemia: an Italian study

no abstract availabl