Genetic characterization of acute myeloid leukemia.
Leukemias account for approximately one third of all pediatric malignancies and cause the greatest number of cancer-related deaths in children. The most commonly diagnosed pediatric leukemia is acute lymphoblastic leukemia (ALL), whereas acute myeloid leukemia (AML) represents a less prevalent form in children, but the most common type in adolescents. AML is in general much less curable than ALL, and even if the outcome of children with AML has significantly improved over the past 30 years, reaching up to 60% of survival at 8 years in most of recent concluded clinical trials, these improvements have not been achieved by the introduction of new drugs. To date, prospective clinical trials indicated by cooperative groups of the international pediatric leukemia network evolved in a common strategy that consists of four to five courses of intensive myelosuppressive chemotherapy in standard risk AML patients, and considers the use of hematopoietic stem cell transplantation (HSCT) for the high risk form. Nevertheless, up to 30% of children with AML still experience relapse, and more than 50% of them die from their disease, or experience severe chemotherapy late effects and complications related to HSCT. Cytogenetic and molecular genetic permitted the identification of a great number of molecular markers in the last two decades, that have been used to guide diagnosis and clinical management of pediatric patients. We characterized new unknown mutations in leukemia at diagnosis, we established their prognostic and biological role for several of them and we created assays for the monitoring of minimal molecular disease, increasing the ability to follow patients during follow up. The new markers have been included in the new AIEOP-AML 2013 trial and will contribute to improve outcome of patients. We identified new structural alterations, such as inter- and intra-chromosomal rearrangements, which remains the most frequent mechanism of mutagenesis occurring in pediatric AML, but for a large proportion of patients, the 50%, a genetic biomarker is not present at diagnosis. Even if several efforts have been spent to identify structural alterations, intra-chromosomal rearrangements, and genetic mutations, that still constitute the most frequent mechanism of pathogenesis recognized in pediatric AML, most of them do not reconstitute an acute leukemia in vivo, suggesting that unknown mutations concur to leukemia onset. The fact is that AML is caused by a clonal transformation of myeloid cell precursors, which acquire abnormalities in their self-renewal, proliferative, and differentiation potential. These abnormalities are the result of somatic genetic alterations that occur and cooperate to establish the leukemic clone. Therefore increasing the knowledge of AML clonal heterogeneity is still necessary to explain the evolutionary process that leads to leukemogenesis. Novel data from the genomic era recently show that only a limited number of gene mutations are recurrent in pediatric AML, but the spectrum of patient specific mutations is wider and the molecular tumor diversity among children is much greater than expected.
Characterization of the cAMP response element binding protein (CREB) in acute myeloid leukemia.
cAMP response element binding protein (CREB) is one of the most important transcription factors that plays a crucial role in normal and neoplastic hematopoiesis, governing many critical cellular processes, including proliferation and differentiation of myeloid progenitor cells. We previously documented that most of the pediatric AML exhibited high levels of CREB, and the dissection of the leukemogenic mechanism revealed its that CREB is involved in leukemogenesis with important clinical relevance. We previously demonstrated that enforced expression of CREB in healthy bone marrow cells promoted aberrant growth and survival ability principally through the upregulation of specific downstream target genes. On the contrary, CREB levels downregulation forced in AML cell lines, showed a suppressed cell proliferation and clonogenic tumor ability. We generated a zebrafish model overexpressing CREB in the myeloid lineage, which showed an aberrant regulation of primitive hematopoiesis, and in most of adult CREB-zebrafish had the block of myeloid differentiation, triggering to a monocytic leukemia akin the human counterpart. We provided a new attractive in vivo model for further high-throughput drug screening, which is the main challenge to improve AML therapeutic strategies.
Anti-apoptotic factors concurring to acute myeloid leukemia progression.
Childhood acute myeloid leukemia (AML) is a heterogeneous disease with a high incidence of recurrent genetic aberrations. Today, many of these aberrations have been reported to sustain an uncontrolled proliferation of malignant hematopoietic progenitors along myeloid lineage. In addition to mutations, damaged apoptotic processes have been documented frequently within leukemic cells. Evasion of cell death is a common cause of malignant transformation. In particular, deregulated expression of the B-cell lymphoma 2 (BCL-2) family of proteins is a frequent event in tumors, where an improper tuning of the apoptotic signals occurs owing to misbalanced expression of antiapoptotic (BCL-2, BCL-XL, MCL-1), versus pro-apoptotic (BAX, BAK, BAD) members. Nowadays, it is widely accepted that high expression of antiapoptotic proteins can determine resistance to chemotherapy, with clear influences on complete remission and overall survival of patients, suggesting them to be a novel candidates for targeting. Tumor cells can also avoid apoptosis normally activated after prolonged stress conditions, such as endoplasmic reticulum (ER) stress, which is triggered by increased protein overtake. We previously studied BAG-1, a pro-survival protein being frequently used by the tumor cells to sustain their uncontrolled expansion, and found that it worked in a synergic action with BCL-2, one of the most relevant anti-apoptotic molecules in cancer. BAG-1 impeded the induction of cell death during the use of anti-cancer drugs, decreasing in that way the efficiency of administered therapy. In pediatric AML, we have determined the importance of upregulated BAG-1 protein expression for patients’ overall survival. In addition, using the in vitro approaches, we have established a link between increased BAG-1 level in leukemic cells and their poor response to several cytotoxic drugs such as doxorubicin, VP16 and ABT-737. We demonstrated that after abrogation of BAG-1 expression in these cells, the cytotoxicity of anti-cancer drugs was significantly improved. These findings gave further proof that upregulated levels of anti-apoptotic factors sustained leukemia cells survival and implied that a combined therapeutic strategy with new compounds and conventional chemotherapics might help in the eradication of tumor cells. Thus, we moved towards apoptosis phenomenon to be dissected in AML. We uncovered that together with anti-apoptotic factors expression, leukemia modulated several pathways to progress, among them we focused on cell adhesion ability and mitochondria. We had preliminary data suggesting that RHOB protein overexpression over-activates its downstream pathway converging to COFILIN phosphorylation, this causing the arrest of the dynamic actin depolymerization, leading to cytoskeleton aberrancies through stress fiber formation and a lower cell death after chemotherapy treatment. We confirmed that RHOB overexpression favored the persistence of COFILIN in its phosphorylated form, which is physiologically maintained into the cytoplasm, thus preventing its translocation to mitochondria, where it is known to trigger apoptosis. These findings indicate that the active network controlled by RHOB increases blast adhesion and promotes resistance to drugs in vitro, enhancing the possibility that this mechanism play a critical role over AML cell survival and resistance to chemotherapy. A large body of evidence has highlighted the existence of a close interconnection between the cancer cells fate and the two apparently unrelated cellular processes of mitochondrial dynamics and autophagy.
Role of epigenetic in pediatric acute myeloid leukemia.
Among the events that concur to leukemogenesis, epigenetic modifications have been found to cooperate with somatic rearrangements and mutations during development of several cancers. Nevertheless, mutations of epigenetic genes have rarely been found in pediatric AML. In light of all these considerations, we considered to study the t(8;21)-rearranged AML cases addressing the hypothesis whether the methylation profile may contribute to further stratify patients carrying the same genetic lesion. The translocation t(8;21)(q22;q22)RUNX1-RUNX1T1 is a recurrent somatic lesion detected at diagnosis in approximately 12-15% of children with acute myeloid leukemia (AML). Children with this isolated translocation are usually considered at standard risk, but our last multicenter trial revealed a higher than expected cumulative incidence of relapse for these patients. Genetic and epigenetic heterogeneity is emerging as a fundamental property of AML in the dynamic evolution of the clonal architecture. In view of this observation, we hypothesized that within t(8;21) patients there may coexist a complex mosaic of cells containing combinations of the same genetic t(8,21) lesion together with different epigenetic variants, and that epigenetic complexity may play a crucial role in predisposing patients to relapse. We had performed genetic and epigenetic sequencing analysis and then integrated results to identify new molecular markers distinctive of t(8,21)-rearranged patients with a different outcome, looking for new predictive markers of high risk features, such as an higher risk to relapse. By high-throughput profiling of DNA methylation and gene expression analysis we revealed novel signatures for the t(8,21) rearranged patients, and we open for further re-evaluation of patients’ genetic cohorts to distinguish those with different characteristics, such as a higher risk of relapse, or to be refractory to therapy in order to pre-emptively modulate clinical strategies.
New therapeutic opportunities for pediatric patients affected by acute myeloid leukemia.
Over the past decades, significant improvements in survival for children with acute myeloid leukemia (AML) have been reached, with current overall survival ranging to 60 to 70%. However, despite the use of intensive treatments, a third of children still die from AML, and late effects adversely damage their adult life. Novel therapies are therefore desperately needed. Till now, the intensification of standard cytotoxic chemotherapy, a more precise genetic risk classification, improvements in supportive care, and the HSCT have been used to overcome relapse and death, but with the exception of the retinoic acid–sensitive acute promyelocytic leukemia subtype, molecularly targeted therapeutic approaches for pediatric acute leukemia are few and far from being translated into clinical advances. Thus, pediatric leukemia is still a prototype example of the challenges of modern cancer drug discovery. The common and persistent failure to translate promising preclinical drug candidates into clinical success is principally due to the limited effectiveness of disease models currently used in the preclinical setting. The failure of these models might be found in the inability to recapitulate key pathophysiological features of the human disease, including complex inter-and intratumour genetic heterogeneity, host-stroma–tumour cell interactions, and the cancer stem cell niche, having profound effects on the therapeutic response in vivo. Thus, more predictive models would certainly lay the groundwork for successful clinical translation and improved patient care, since the survival rates for more aggressive subtypes of childhood AML remain unacceptably low (below 20%). The goal of our research is the development of three dimensional humanized bone marrow niche in vitro model to be implanted in vivo, in NSG mice, to improve patient’s leukemia engraftment and produce Patient derived Xenografts (PdX). The PdXs will be used to advance the development of new therapeutic options for kids with AML because they will be used for testing new putative active compounds, pre-selected in vitro. Commercial libraries of FDA-approved molecules will be tested in cell lines or primary AML cultures from patients to define novel potentially active drugs. Their efficacy will be then evaluated in vivo on PdXs. This repurposing approach represents an attractive method for drug discovery, as it can enable rapid clinical translation (AIRC-IG project ID: 20562).
- Claudia Tregnago
- Valeria Bisio
- Maddalena Benetton
- Giulia Borella
- Katia Polato
- Ambra Dal Ros
Selected PublicationsZampini M, Tregnago C, Bisio V, Simula L, Borella G, Manara E, Zanon C, Zonta F, Serafin V, Accordi B, Campello S, Buldini B, Pession A, Locatelli F, Basso G, Pigazzi M. . 2018. Epigenetic heterogeneity affects the risk of relapse in children with t(8;21)RUNX1-RUNX1T1-rearranged AML. Leukemia, doi:10.1038/s41375-017-0003-y.
de Rooij JD, Branstetter C, Ma J, Li Y, Walsh MP, Cheng J, Obulkasim A, Dang J, Easton J, Verboon LJ, Mulder HL, Zimmermann M, Koss C, Gupta P, Edmonson M, Rusch M, Lim JY, Reinhardt K, Pigazzi M, Song G, Yeoh AE, Shih LY, Liang DC, Halene S, Krause DS, Zhang J, Downing JR, Locatelli F, Reinhardt D, van den Heuvel-Eibrink MM, Zwaan CM, Fornerod M, Gruber TA. 2017 Mar. Pediatric non-Down syndrome acute megakaryoblastic leukemia is characterized by distinct genomic subsets with varying outcomes. Nat Genet, 49(3):451-456
Manara E, Basso G, Zampini M, Buldini B, Tregnago C, Rondelli R, Masetti R, Bisio V, Frison M, Polato K, Cazzaniga G, Menna G, Fagioli F, Merli P, Biondi A, Pession A, Locatelli F, Pigazzi M. 2017 Jan. Characterization of children with FLT3-ITD acute myeloid leukemia: a report from the AIEOP AML-2002 study group. Leukemia. , 31(1):18-25.
Tregnago C, Manara E, Zampini M, Bisio V, Borga C, Bresolin S, Aveic S, Germano G, Basso G, Pigazzi M.. 2016 Sep. CREB engages C/EBPδ to initiate leukemogenesis. Leukemia, 30(9):1887-96
Manara E, Baron E, Tregnago C, Aveic S, Bisio V, Bresolin S, Masetti R, Locatelli F, Basso G, Pigazzi M. . 2014 Jul. MLL-AF6 fusion oncogene sequesters AF6 into the nucleus to trigger RAS activation in myeloid leukemia. Blood, 10;124(2):263-72
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