Giorgia Simonetti

Targeting the p53-MDM2 interaction by the small-molecule MDM2 antagonist Nutlin-3a: a new challenged target therapy in adult Philadelphia positive acute lymphoblastic leukemia patients

MDM2 is an important negative regulator of p53 tumor suppressor. In this study, we sought to investigate the preclinical activity of the MDM2 antagonist, Nutlin-3a, in Philadelphia positive (Ph+) and negative (Ph−) leukemic cell line models, and primary B-acute lymphoblastic leukemia (ALL) patient samples. We demonstrated that Nutlin-3a treatment reduced viability and induced p53-mediated apoptosis in ALL cells with wild-type p53 protein, in a time and dose-dependent manner, resulting in the increased expression of pro-apoptotic proteins and key regulators of cell cycle arrest. The dose-dependent reduction in cell viability was confirmed in primary blast cells from B-ALL patients, including Ph+ ALL resistant patients carrying the T315I BCR-ABL1 mutation. Our findings provide a strong rational for further clinical investigation of Nutlin-3a in Ph+ and Ph− ALL.

Revealing very small FLT3 ITD mutated clones by ultra-deep sequencing analysis has important clinical implications in AML patients

FLT3 internal tandem duplication (ITD), one of the most frequent mutations in Acute Myeloid Leukemia (AML), is reported to be an unstable marker, as it can evolve from FLT3 ITD- to ITD+ during the disease course. A single-gene sensitive mutational screening approach may be helpful for better clarifying the exact timing of mutation occurrence, especially when FLT3 ITD appears to occur late, at disease progression. We developed an amplicon-based ultra-deep-sequencing (UDS) approach for FLT3 mutational screening. We exploited this highly sensitive technology for the retrospective screening of diagnosis, relapse and follow-up samples of 5 out of 256 cytogenetically normal (CN-) AML who were FLT3 wild-type at presentation, but tested ITD+ at relapse or disease progression. Our study revealed that all patients carried a small ITD+ clone at diagnosis, which was undetectable by routine analysis (0,2–2% abundance). The dynamics of ITD+ clones from diagnosis to disease progression, assessed by UDS, reflected clonal evolution under treatment pressure. UDS appears as a valuable tool for FLT3 mutational screening and for the assessment of minimal residual disease (MRD) during follow-up, by detecting small ITD+ clones that may survive chemotherapy, evolve over time and definitely worsen the prognosis of CN-AML patients.

5′UTR point substitutions and N-terminal truncating mutations of ANKRD26 in acute myeloid leukemia

Thrombocytopenia 2 (THC2) is an inherited disorder caused by monoallelic single nucleotide substitutions in the 5’UTR of the ANKRD26 gene. Patients have thrombocytopenia and increased risk of myeloid malignancies, in particular, acute myeloid leukemia (AML). Given the association of variants in the ANKRD26 5’UTR with myeloid neoplasms, we investigated whether, and to what extent, mutations in this region contribute to apparently sporadic AML. To this end, we studied 250 consecutive, non-familial, adult AML patients and screened the first exon of ANKRD26 including the 5’UTR. We found variants in four patients. One patient had the c.−125T>G substitution in the 5’UTR, while three patients carried two different variants in the 5’ end of the ANKRD26 coding region (c.3G>A or c.105C>G). Review of medical history showed that the patient carrying the c.−125T>G was actually affected by typical but unrecognized THC2, highlighting that some apparently sporadic AML cases represent the evolution of a well-characterized familial predisposition disorder. As regards the c.3G>A and the c.105C>G, we found that both variants result in the synthesis of N-terminal truncated ANKRD26 isoforms, which are stable and functional in cells, in particular, have a strong ability to activate the MAPK/ERK signaling pathway. Moreover, investigation of one patient with the c.3G>A showed that mutation was associated with strong ANKRD26 overexpression in vivo, which is the proposed mechanism for predisposition to AML in THC2 patients. These data provide evidence that N-terminal ANKRD26 truncating mutations play a potential pathogenetic role in AML. Recognition of AML patients with germline ANKRD26 pathogenetic variants is mandatory for selection of donors for bone marrow transplantation.

The human Smoothened inhibitor PF-04449913 induces exit from quiescence and loss of multipotent Drosophila hematopoietic progenitor cells.

The efficient treatment of hematological malignancies as Acute Myeloid Leukemia, myelofibrosis and Chronic Myeloid Leukemia, requires the elimination of cancer-initiating cells and the prevention of disease relapse through targeting pathways that stimulate generation and maintenance of these cells. In mammals, inhibition of Smoothened, the key mediator of the Hedgehog signaling pathway, reduces Chronic Myeloid Leukemia progression and propagation. These findings make Smo a candidate target to inhibit maintenance of leukemia-initiating cells. In Drosophila melanogaster the same pathway maintains the hematopoietic precursor cells of the lymph gland, the hematopoietic organ that develops in the larva. Using Drosophila as an in vivo model, we investigated the mode of action of PF-04449913, a small-molecule inhibitor of the human Smo protein. Drosophila larvae fed with PF-04449913 showed traits of altered hematopoietic homeostasis. These include the development of melanotic nodules, increase of circulating hemocytes, the size increase of the lymph gland and accelerated differentiation of blood cells likely due to the exit of multi-potent precursors from quiescence. Importantly, the Smo inhibition can lead to the complete loss of hematopoietic precursors. We conclude that PF-04449913 inhibits Drosophila Smo blocking the Hh signaling pathway and causing the loss of hematopoietic precursor cells. Interestingly, this is the effect expected in patients treated with PF-04449913: number decrease of cancer initiating cells in the bone marrow to reduce the risk of leukemia relapse. Altogether our results indicate that Drosophila comprises a model system for the in vivo study of molecules that target evolutionary conserved pathways implicated in human hematological malignancies.

Chromothripsis in acute myeloid leukemia: biological features and impact on survival

Chromothripsis is a one-step genome-shattering catastrophe resulting from disruption of one or few chromosomes in multiple fragments and consequent random rejoining and repair. This study define incidence of chromothripsis in 395 newly-diagnosed adult acute myeloid leukemia (AML) patients from three institutions, its impact on survival and its genomic background. SNP 6.0 or CytoscanHD Array (Affymetrix®) were performed on all samples. We detected chromothripsis with a custom algorithm in 26/395 patients. Patients harboring chromothripsis had higher age (p=.002), ELN high risk (HR) (p<.001), lower white blood cell (WBC) count (p=.040), TP53 loss and/or mutations (p<.001) while FLT3 (p=.025) and NPM1 (p=.032) mutations were mutually exclusive with chromothripsis. Chromothripsis-positive patients showed a worse overall survival (OS) (p<.001) compared with HR patients (p=.011) and a poor prognosis in a COX-HR optimal regression model. Chromothripsis presented the hallmarks of chromosome instability [i.e. TP53 alteration, 5q deletion, higher mean of copy number alteration (CNA), complex karyotype, alterations in DNA repair and cell cycle] and focal deletions on chromosomes 4, 7, 12, 16, 17. CBA. FISH showed that chromothripsis is associated with marker, derivative and ring chromosomes. In conclusion, chromothripsis frequently occurs in AML (6.6%) and influences patient prognosis and disease biology.

Epigenetically induced ectopic expression of uncx impairs the proliferation and differentiation of myeloid cells

We here describe a leukemogenic role of the homeobox gene UNCX, activated by epigenetic modifications in acute myeloid leukemia (AML). We found the ectopic activation of UNCX in a leukemia patient harboring a t(7;10)(p22;p14) translocation, in 22 of 61 of additional cases [a total of 23 positive patients out of 62 (37.1%)], and in 6 of 75 (8%) of AML cell lines. UNCX is embedded within a low-methylation region (canyon) and encodes for a transcription factor involved in somitogenesis and neurogenesis, with specific expression in the eye, brain, and kidney. UNCX expression turned out to be associated, and significantly correlated, with DNA methylation increase at its canyon borders based on data in our patients and in archived data of patients from The Cancer Genome Atlas. UNCX-positive and -negative patients displayed significant differences in their gene expression profiles. An enrichment of genes involved in cell proliferation and differentiation, such as MAP2K1 and CCNA1, was revealed. Similar results were obtained in UNCX-transduced CD34+ cells, associated with low proliferation and differentiation arrest. Accordingly, we showed that UNCX expression characterizes leukemia cells at their early stage of differentiation, mainly M2 and M3 subtypes carrying wild-type NPM1. We also observed that UNCX expression significantly associates with an increased frequency of acute promyelocytic leukemia with PML-RARA and AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 classes, according to the World Health Organization disease classification. In summary, our findings suggest a novel leukemogenic role of UNCX, associated with epigenetic modifications and with impaired cell proliferation and differentiation in AML.

Efficacy of Azacitidine in the treatment of adult patients aged 65 years or older with AML

ABSTRACT Introduction: Therapy for acute myeloid leukemia (AML) in elderly populations (>65 years) is still a challenge for scientists and hematologists worldwide, and represents an urgent medical need. Notably, the identification and the recognition of molecular and epigenetic mechanisms involved in the pathogenesis of such a heterogeneous disease, are providing new tools for a more successful and ‘targeted’ approach. Azacitidine is a hypomethylating agent (HMA) with relevant activity in patients affected by myelodysplastic syndrome (MDS) and AML with low blast cells percentage (>30%), in terms of reduction of transfusion dependence, and improvement of quality of life. Areas covered: This review summarizes the mechanism of action, safety profile and efficacy of azacitidine in the field of elderly AML populations, providing up-to-date references on this subset of high-risk patients. Expert opinion: HMAs are the first successful treatment for elderly patients with high-risk MDS and are effective for some AML subtypes. Translational studies based on gene expression profiling and molecular sequencing, would be able to identify, in the near future, patients with a favorable profile of response to these compounds suggesting new potential treatment combinations also.

Network integration of multi-tumour omics data suggests novel targeting strategies

We characterize different tumour types in search for multi-tumour drug targets, in particular aiming for drug repurposing and novel drug combinations. Starting from 11 tumour types from The Cancer Genome Atlas, we obtain three clusters based on transcriptomic correlation profiles. A network-based analysis, integrating gene expression profiles and protein interactions of cancer-related genes, allows us to define three cluster-specific signatures, with genes belonging to NF-κB signaling, chromosomal instability, ubiquitin-proteasome system, DNA metabolism, and apoptosis biological processes. These signatures have been characterized by different approaches based on mutational, pharmacological and clinical evidences, demonstrating the validity of our selection. Moreover, we define new pharmacological strategies validated by in vitro experiments that show inhibition of cell growth in two tumour cell lines, with significant synergistic effect. Our study thus provides a list of genes and pathways that could possibly be used, singularly or in combination, for the design of novel treatment strategies.Tumours of different tissues can show similarities in genomic alterations. Here, the authors combine tumour transcriptome and protein interaction data in a network-based analysis of 11 tumours types, and identify clusters of tumours with specific signatures for multi-tumour drug targeting and survival prognosis.

Aneuploidy: Cancer strength or vulnerability?

Aneuploidy is a very rare and tissue‐specific event in normal conditions, occurring in a low number of brain and liver cells. Its frequency increases in age‐related disorders and is one of the hallmarks of cancer. Aneuploidy has been associated with defects in the spindle assembly checkpoint (SAC). However, the relationship between chromosome number alterations, SAC genes and tumor susceptibility remains unclear. Here, we provide a comprehensive review of SAC gene alterations at genomic and transcriptional level across human cancers and discuss the oncogenic and tumor suppressor functions of aneuploidy. SAC genes are rarely mutated but frequently overexpressed, with a negative prognostic impact on different tumor types. Both increased and decreased SAC gene expression show oncogenic potential in mice. SAC gene upregulation may drive aneuploidization and tumorigenesis through mitotic delay, coupled with additional oncogenic functions outside mitosis. The genomic background and environmental conditions influence the fate of aneuploid cells. Aneuploidy reduces cellular fitness. It induces growth and contact inhibition, mitotic and proteotoxic stress, cell senescence and production of reactive oxygen species. However, aneuploidy confers an evolutionary flexibility by favoring genome and chromosome instability (CIN), cellular adaptation, stem cell‐like properties and immune escape. These properties represent the driving force of aneuploid cancers, especially under conditions of stress and pharmacological pressure, and are currently under investigation as potential therapeutic targets. Indeed, promising results have been obtained from synthetic lethal combinations exploiting CIN, mitotic defects, and aneuploidy‐tolerating mechanisms as cancer vulnerability.

Abstract 3472: Separase overexpression defines a new subset of acute myeloma leukemia patients characterized by high CD34 and MYC levels

The endopeptidase Separase, encoded by the ESPL1 gene, plays a key role in faithful segregation of sister chromatids by cleaving the cohesin complex at the metaphase to anaphase transition. Its overexpression associates with aneuploidy and bad prognosis in solid tumors. Little is known in Acute Myeloid Leukemia (AML). We profiled the genomic landscape of 405 and 78 AML cases by SNP array (SNP 6.0 and Cytoscan HD, Affymetrix) and whole exome sequencing (100 bp, paired-end, Illumina), respectively. Bone marrow blasts from 61 patients were analyzed by gene expression profiling (HTA 2.0, Affymetrix). Separase expression was determined by Immunohistochemistry (1:600 antibody dilution Abnova, clone 6H6) in 44 AML and 4 control bone marrow specimens. One patient exhibited a nonsynonimous mutation in ESPL1 (1.3%), which was predicted to alter the protein function. Moreover, ESPL1 copy number gain was observed in 5/405 cases (1.2%): 2 hyperdiploid AML, one trisomy 12 and 2 cases with a short gain at 12q. Notably, protein level detection in one of the 12q-gain cases confirmed Separase overexpression. To determine the incidence of Separase overexpression, we performed Immunohistochemistry on additional 43 AML. Separase was overexpressed in 29/44 AML (66%, Separase-high), being comparable to control marrow biopsies in the remaining 15 samples (Separase-low). Sixty-two percent of Separase-high AML were aneuploid. However, no significative association was observed, as previously reported for mutations in the cohesin genes in AML. Separase overexpression correlated with increased patients’ age (median age 64 vs. 57 years, p=.01), 17-fold upregulation of CD34 (p=.004) and a trend towards reduced overall survival (6-years follow-up). Separase overexpression was not mutually esclusive with cohesin gene mutations, it co-occurred with NPM1 and FLT3 lesions and frequent mutations in genes involved in protein post-translational modification and ubiquitination (p=.04). Separase-low cases were enriched for mutations in RAS signaling pathway (NRAS, KRAS, NF1, RIT1, GRAP2, RALGDS; p=4.5x10-5) and in cell migration-related genes (LIMS2, S1PR1, PPIA, PLXNB1, FAT1). Separase-high cases also showed a defined transcriptomic profile, characterized by reduced expression of HOXA/B family genes, the DNA damage repair gene ATM, the p53 regulator MDM2 and forced expression of the cell cycle markers CDC20, AURKB, NUSAP1 and of MYC, independently of chromosome 8 gain. Taken together, our data suggest that genomic lesions targeting ESPL1 are a rare event in AML. However, Separase overexpression is a common feature and defines a new subset of AML cases with a distinct gene expression profile, which may benefit of innovative targeted therapies including CDC20 and bromodomain inhibitors. Supported by: ELN, AIL, AIRC, progetto Regione-Universita 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Giorgia Simonetti, Antonella Padella, Simona Righi, Maria Chiara Fontana, Marco Manfrini, Cristina Papayannidis, Giovanni Marconi, Carmen Baldazzi, Marianna Garonzi, Alberto Ferrarini, Massimo Delledonne, Nicoletta Testoni, Elena Sabattini, Giovanni Martinelli. Separase overexpression defines a new subset of acute myeloma leukemia patients characterized by high CD34 and MYC levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3472. doi:10.1158/1538-7445.AM2017-3472