SMO inhibitor specifically targets the Hedgehog Pathway and reverts the drug-resistance of Leukemic Stem Cells

Abnormal Hedgehog signaling is associated with human malignancies. Smo, a key player of that signaling, is the most suitable target to inhibit this pathway. To this aim several molecules, antagonists of Smo, have been synthesized, and some of them have started the phase I in clinical trials. Our hospital participated to one of these studies which investigated the oral administration of a new selective inhibitor of Smo (SMOi). To evaluate ex vivo SMOi efficacy and to identify new potential clinical biomarkers of responsiveness, we separated bone marrow CD34+ cells from 5 acute myeloid leukemia (AML), 1 myelofibrosis (MF), 2 blastic phases chronic myeloid leukemia (CML) patients treated with SMOi by immunomagnetic separation, and we analysed their gene expression profile using Affimetrix HG-U133 Plus 2.0 platform. This analysis, showed differential expression after 28 days start of therapy (p-value ≤ 0.05) of 1,197 genes in CML patients and 589 genes in AML patients. This differential expression is related to Hedgehog pathway with a p-value = 0.003 in CML patients and with a p-value = 0.0002 in AML patients, suggesting that SMOi targets specifically this pathway. Among the genes differentially expressed we observed strong up-regulation of Gas1 and Kif27 genes, which may work as biomarkers of responsiveness of SMOi treatment in CML CD34+ cells whereas Hedgehog target genes (such as Smo, Gli1, Gli2, Gli3), Bcl2 and Abca2 were down-regulated, in both AML and CML CD34+ cells. It has been reported that Bcl-2 expression could be correlated with cancer therapy resistance and that Hedgehog signaling modulate ATP-binding (ABC) cassette transporters, whose expression has been correlated with chemoresistance. Moreover we confirmed that in vitro SMOi treatment targets Hedgehog pathway, down-regulate ABC transporters, Abcg2 and Abcb1 genes, and in combination with tyrosine kinase inhibitors (TKIs) could revert the chemoresistance mechanism in K562 TKIs-resistant cell line.

Leucemia Acuta Mieloide e signaling purinergico: possibili sviluppi terapeutici

I nucleotidi trifosfato sono, dal punto di vista evoluzionistico, tra le molecole più antiche e conservate tra le specie. Oltre al ruolo che ricoprono nella sintesi degli acidi nucleici e nel metabolismo energetico della cellula, negli ultimi anni è emerso sempre di più il loro coinvolgimento nella regolazione di numerose funzioni cellulari. Questi importanti mediatori cellulari sono presenti nel microambiente e cambiamenti nella loro concentrazione extracellulare possono modulare la funzionalità cellulare. I nucleotidi trifosfato ATP e UTP, presenti nel microambiente midollare, sono dei potenti stimolatori dei progenitori emopoietici. Essi stimolano la proliferazione e l’attecchimento delle cellule staminali emopoietiche, così come la loro capacità migratoria, attraverso l’attivazione di specifici recettori di membrana, i recettori purinergici (P2R). In questo studio abbiamo dimostrato che ATP e UTP esercitano un effetto opposto sul compartimento staminale leucemico di leucemia acuta mieloide (LAM). Abbiamo dimostrato che le cellule leucemiche esprimono i recettori P2 funzionalmente attivi. Studi di microarray hanno evidenziato che, a differenza di ciò che avviene nelle CD34+, la stimolazione di cellule leucemiche con ATP induce la down-regolazione dei geni coinvolti nella proliferazione e nella migrazione, mentre up-regola geni inibitori del ciclo cellulare. Abbiamo poi confermato a livello funzionale, mediante test in vitro, gli effetti osservati a livello molecolare. Studi di inibizione farmacologica, ci hanno permesso di capire che l’attività inibitoria dell’ATP sulla proliferazione si esplica attraverso l’attivazione del recettore P2X7, mentre i sottotipi recettoriali P2 prevalentemente coinvolti nella regolazione della migrazione sono i recettori P2Y2 e P2Y4. Esperimenti di xenotrapianto, hanno evidenziato che l’esposizione ad ATP e UTP sia dei blasti leucemici sia delle cellule staminali leucemiche CD38-CD34+ diminuisce la loro capacità di homing e di engraftment in vivo. Inoltre, il trattamento farmacologico con ATP, di topi ai quali è stata indotta una leucemia umana, ha diminuito lo sviluppo della leucemia in vivo.

Azacitidine and Lenalidomide Combination Therapy in Myelodysplastic Syndromes: Topography and Translational Relevance of Nuclear Phospholipase C β - dependent Signalling

Nuclear inositide signalling pathways, and particularly those regulated by PI-PLCβ1, are associated with cell proliferation and differentiation. Myelodysplastic syndromes (MDS) are a heterogeneous spectrum of chronic myeloid hemopathies with associated symptomatic cytopenias and substantial potential for evolution to acute myeloid leukemia (AML). MDS patients are currently treated with two main approaches, epigenetic (Azacitidine) and immunomodulatory (Lenalidomide: above all in cell clones bearing a deletion of the long arm of the chromosome 5 [del(5q)]). As Azacitidine and Lenalidomide alone can show adverse effects or patients can be refractory, an experimental current approach is the combination of the two drugs. Clinically, this combination therapy is promising, while its molecular effect has to be clarified. Stemming from these data, in this study the effect of an Azacitidine-Lenalidomide combination therapy was studied, in both MDS patients and hematopoietic cell lines. The specific aims of this study were to evaluate the effect of Azacitidine and Lenalidomide MDS therapy on: cell cycle regulation, hematopoietic differentiation, gene mutation and miR expression. Lenalidomide alone, via PI-PLCβ1/PKC pathway, was able to induce a selective G0/G1 arrest of the cell cycle in del(5q) cells, slowing down their rate proliferation and favouring erythropoiesis activation. In addition, although the mutation profile at baseline was not entirely capable of predicting the clinical effect of Azacitidine and Lenalidomide therapy, the presence of specific point mutations affecting three inositide genes (PI3KCD, AKT3, PLCG2) was correlated to and anticipated a negative clinical outcome. Moreover, the differential miR expression was detectable even from the 4th cycle of therapy in responder patients, as compared to non-responders. In MDS, this is the first evidence that the molecular mutation profiling of inositide genes or a specific mini-cluster of differentially expressed miRs, targeting inositide signaling molecules, can be associated with the clinical response, thus possibly predicting the effect of the therapy.

Microenvironment and prognostic factors in bone tumors: IDH mutations in chondrosarcoma

Microenvironment in bone tumors is a dynamic entity composed of cells from different origins (immune cells, stromal cells, mesenchymal stem cells, endothelial cells, pericytes) and vascular structures surrounded by a matrix of different nature (bone, cartilage, myxoid). Interactions between cancer cells and tumor microenvironment (TME) are complex and can change as tumor progress, but are also crucial in determining response to cancer therapies. Chondrosarcoma is the second most frequent bone cancer in adult age, but its treatment still represents a challenge, for the intrinsic resistance to conventional chemotherapy and radiation therapy. This resistance is mainly due to pathological features, as dense matrix, scarce mitoses and poor vascularization, sustained by biological mechanisms only partially delucidated. Somatic mutation in the Krebs cycle enzyme isocytrate dehydrogenase (IDH) have been described in gliomas, acute myeloid leukemia, cholangiocarcinoma, melanoma, colorectal, prostate cancer, thyroid carcinoma and other cancers. In mesenchymal tumors IDH mutations are present in about 50% of central chondrosarcoma. IDH mutations are an early event in chondrosarcoma-genesis, and contribute to the acquisition of malignancy through the block of cellular differentiation, hypoxia induction through HIF stabilization, DNA methylation and alteration of cellular red-ox balance. While in gliomas IDH mutations confers a good prognosis, in chondrosarcoma IDH prognostic role is controversial in different reported series. First aim of this project is to define the prevalence and the prognostic role of IDH mutation in high grade central conventional chondrosarcoma patients treated at Istituto Ortopedico Rizzoli. Second aim is the critical revision of scientific literature to understand better how a genomic event in cancer cell can trigger alteration in the TME, through immune infiltrate reshaping, angiogenesis induction, metabolic and methylation rewiring. Third aim is to screen other sarcoma histotypes for the presence of IDH mutation.

Development of machine learning methods for multi-modal biomarkers detection and integration

In medicine, innovation depends on a better knowledge of the human body mechanism, which represents a complex system of multi-scale constituents. Unraveling the complexity underneath diseases proves to be challenging. A deep understanding of the inner workings comes with dealing with many heterogeneous information. Exploring the molecular status and the organization of genes, proteins, metabolites provides insights on what is driving a disease, from aggressiveness to curability. Molecular constituents, however, are only the building blocks of the human body and cannot currently tell the whole story of diseases. This is why nowadays attention is growing towards the contemporary exploitation of multi-scale information. Holistic methods are then drawing interest to address the problem of integrating heterogeneous data. The heterogeneity may derive from the diversity across data types and from the diversity within diseases. Here, four studies conducted data integration using customly designed workflows that implement novel methods and views to tackle the heterogeneous characterization of diseases. The first study devoted to determine shared gene regulatory signatures for onco-hematology and it showed partial co-regulation across blood-related diseases. The second study focused on Acute Myeloid Leukemia and refined the unsupervised integration of genomic alterations, which turned out to better resemble clinical practice. In the third study, network integration for artherosclerosis demonstrated, as a proof of concept, the impact of network intelligibility when it comes to model heterogeneous data, which showed to accelerate the identification of new potential pharmaceutical targets. Lastly, the fourth study introduced a new method to integrate multiple data types in a unique latent heterogeneous-representation that facilitated the selection of important data types to predict the tumour stage of invasive ductal carcinoma. The results of these four studies laid the groundwork to ease the detection of new biomarkers ultimately beneficial to medical practice and to the ever-growing field of Personalized Medicine.

Pre-clinical and clinical development of leukemia stem cell inhibitors in acute leukemias

In Leukemias, recent developments have demonstrated that the Hedgehog pathway plays a key-role in the peculiar ability of self renewal of leukemia stem cells. The aim of this research activity was to investigate, through a first in man, Phase I, open label, clinical trial, the role and the impact, mainly in terms of safety profile, adverse events and pharmacokinetics, of a Sonic Hedgehog inhibitor compound on a population of heavely pretreated patients affected by AML, CML, MF, or MDS, resistant or refractory to standard chemotherapy. Thirty-five patients have been enrolled. The drug was administered orally, in 28 days cycles, without rest periods. The compound showed a good safety profile. The half life was of 17-35 hours, justifying the daily administration. Significant signs of activity, in terms of reduction of bone marrow blast cell amount were seen in most of the patients enrolled. Interestingly, correlative biological studies demonstrated that, comparing the gene expression profyiling signature of separated CD34+ cells before and after one cycle of treatment, the most variably expressed genes were involved in the Hh pathway. Moreover, we observed that many genes involved in MDR (multidrug resistance)were significantly down regulated after treatment. These data might lead to future clinical trials based on combinatory approaches, including, for instance, Hh inhibitors and conventional chemotherapy.