Published the 2015 Report on the scientific research activity funded by Fondazione Ginevra Caltagirone

The line of research developed in 2015 by Dr. Masetti is part of a broader planning already started in 2012, thanks to the support of Caltagirone Foundation, focused on deep sequencing of the genome of pediatric patients with acute myeloid leukemia, still a very aggressive disease and now burdened with a disease-free survival at 5 years still does not exceed 65%.

The objective, as explained above, is to threefold of:

1) identify new molecular markers for diagnosis and disease monitoring,

2) understanding the biological behavior of the disease and its evolution,

3) identify molecular targets that may be susceptible to an integrated targeted biological treatment to conventional chemotherapy.

The first years of the project focused mainly on the first point. mutations have been identified in genes that regulate cell proliferation and cell death, in genes that code for enzymes that serve to DNA to divide, and others that serve for transcribing the DNA, in subgroups of AML in particularly unfavorable prognosis.

The ultimate fusion gene identified in 2015 shall be those resulting from NUP98 and PHF23 genes and was published in the Journal of Hematology and Oncology. For the first time, in the article mentioned, it is demonstrated recurrence in approximately 3% of all AML in normal karyotype of this fusion gene.

In 2015, in addition to having produced this important result in the identification of new markers, they were also achieved significant results with respect to the understanding of the biological behavior of the disease and the testing of new pharmacological agents targeted at specific molecular targets of the disease.

The massive exome sequencing of onset conditions, remission and relapse of four pediatric patients with AML with normal karyotype has identified mutations in each present only debut, only to relapse and mutations present in both of the same patient samples . These results confirm an eminently polyclonal nature of pediatric AML, extending the validity of the clonal evolution model of the disease, already validated by Ding et al for adult AML. It thus configures the evidence of a genomic complexity considerably higher than previously thought. In particular, while one or a few mutational events are initially responsible for the leukemic transformation (primary events), following the neoplastic population differs in subclones through the acquisition of additional mutations (secondary events). The evolution of the disease is then the effect of dynamic interaction of these mutations with the microenvironment and treatment to which the patient is subjected.

Among secondary events highlighted in one of the analyzed patients, include the A72V mutation of PTPN11, which appears to relapse in 64% of the blast population, and the D835E mutation of FLT3 clone detected in a small (6%) at the onset that goes expanding the relapsed (26%). These events, known to be associated with an increase in cellular proliferative signals, although not appear decisive in the onset of the disease, certainly favor its perpetuation and survival to traditional therapies.

In the light of the availability of specific pharmacological inhibitors, stressing the need to characterize the molecular point of view not only the disease onset, as practiced routinely, but also to recurrence, to use an individualized therapy, just after bankruptcy conventional therapies.

Although the mutations of WT1, a transcription factor particularly expressed in leukemia cells and mutated in approximately 10% of AML, they are highly unstable revealed during the course of disease, both in terms of acquisition of that loss, reflecting the role not primary and still obscure these events in the pathogenesis of AML. On the contrary, the CEBPA mutation, a transcription factor mutated in 4.5% of pediatric AML, is present in homozygosity in the totality of the leukemic population is at the onset that the recurrence of one of the patients analyzed, in accordance with the primary role attributed to this event so that the LAM with biallelic mutation CEBPA is inserted as a provisional entity in the WHO classification of AML.

Other molecular events that we have identified as possibly contributing to recurrence of the disease, including a mutation of the tyrosine kinase TYK2, associated with an increased resistance to apoptosis, and a SETD2 mutation, a methyltransferase involved in mismatch repair whose inactivation is associated with a accumulation of somatic mutations, mechanism able to further increase the genomic complexity of the disease and promote its plasticity, and then the survival to therapies.

The drug used in the study is funded GANT61 an inhibitor of the GLI family proteins used in preclinical in some solid tumors such as neuroblastoma that the Hedgehog pathway is abnormally active.

During the first four months l 'research has focused primarily in preclinical research models suitable for testing the efficacy of the drug. In this case, two cell lines were chosen: the M07 and WSU-AML granted by St. Jude Children's Research Hospital, Memphis in addition to a series of negative AML cell lines for fusion gene CBFA2T3-GLIS2.

The positive and negative cell lines to the translocation in question have been treated for 72h with the 'inhibitor of the pathway Hedgeogh, the GANT61. 8 were tested concentrations of the drug from which it is derived the average dose that kills 50% of the cells (IC50). From the statistical analysis it showed that the positive cell lines translocation of GLIS2 are more sensitive to treatment with GANT61 than other negative LAM lines (Figure 1A).

This was an important result because it has already given a first idea on the mechanism of action of the drug that acts in some way on the specifically chosen target (in this case GLIS2 that is indispensable for the process of leukemogenesis) and at the same time the non-specific toxicity is low for the other cell lines in which it is not present the same translocation of GLIS2 same.

In the same way by treating with the drug the same cell lines with GLIS2 translocation of the gene was conducted a study of gene expression in order to determine the pharmacological effect on the transcriptional activity mediated by GLIS2. The two lines were treated with GANT61 for 48h, then was extracted the total RNA of the cell and the gene expression was quantified through affymetrix platform. Analysis of the data was confirmed the activation of some specific genes of Hedgeogh signaling pathway as BMP2, GATA3, CCND2 or NCAM1 cited in the two previous studies, some of which undergoes a clear decrease of the expression following treatment pharmacological. But in addition to these genes the statistical analysis of the data showed for the first time the expression of other genes with important functions within the neoplastic cell. Among these were some involved in the regulation of proliferation processes (kIF14, MELK, MCM10, NUF2), cell cycle regulation (CCNA2, CDKN3, CDC7, PRC1), repair (BRCA1, BRCA2) and especially genes involved in epigenetic processes such as methylation DNA (DNMT1, DNMT3B). All of these genes undergo a statistically significant drop following treatment with GANT61.

This is a very interesting result as it helps to better understand tumor biology of this subgroup of AML. The decrease of the expression of all these genes after treatment with GANT61 strengthens the hypothesis that the drug is specifically for the aberration in question and above could be a first-line treatment in the treatment of this subgroup of AML with poor prognosis. In addition, since the drug acts negatively on the expression of genes involved in DNA repair mechanisms, the GANT61 may potentiate the effect of conventional chemotherapeutic agents used today in the treatment protocols.