Shwachman-Diamond Syndrome (SDS) is a rare autosomal recessive disease, characterized by exocrine pancreatic disorder, hematological aberrancies, bone marrow failure and cognitive impairment. In 90% of patients the SBDS gene is found mutated. Similar to other marrow failure syndromes, SDS patients have an increased risk for developing myelodysplastic syndrome and AML. To date, the mechanisms underlying the bone marrow failure in SDS patients are not fully understood. Microenvironment constituents and in particular mesenchymal stromal cells (MSCs) are considered the pivotal organizers for the generation, maintenance and plasticity of the hematopoietic stem cell niche. Recent studies show that specific changes in MSCs may be sufficient to initiate a complex phenotype of disordered homeostasis with similarities to myelodysplasia. We have demonstrated that MSCs obtained from SDS patients were comparable in vitro to HD but gene expression analysis of 16 SDS-MSCs showed that these cells had a specific gene expression signature compared to HD. These results suggest that it is possible that MSCs could be involved in the pathogenesis of the SDS marrow disorders. We increased our patients cohort and investigated whether SDS-MSCs were able to sustain malignant evolution using an innovative scaffold-free in vivo system based on the ex vivo generation of semi-cartilaginous pellets (SCPs) from human MSCs. We obtained SCPs stimulating MSCs for 21 days with a specific differentiating medium and a complete and correct formation of cartilaginous tissues both in HD and SDS samples. These SCPs were transplanted heterotopically into subcutaneous tissue of immunocompromised mice. After 60 days, we sacrificed mice and collected ossicles. We found that in 90% of cases, HD were able to recreate the hematopoietic microenvironment, with the establishment of a complete marrow niche, while none of the transplanted SDS-SCPs was able to recreate the hematopoietic microenvironment, revealing a defect in these differentiating process. The second part of our study was focused on testing a specific drug able to act on nonsense stop codon mutation, one of the most diffuse alterations in SDS patients, linked to risk of developing myelodysplastic syndrome. We successfully obtained restoration of SBDS protein in different cell lineages deriving from patients (Lymphoblastoids, MSCs, mononuclear cells from bone marrow). Protein restoration was also accompanied in some cases with an improvement of functionality. In particular, mononuclear cells from bone marrow treated with drug showed an increase in their ability to form colonies when cultured in a specific assay. This represents a powerful result, due to the potential clinical consequences related to possible therapeutic strategy. Indeed, SDS patients in future could take advantage of this drug to ameliorate their hematological defects and abolish other symptoms.

La Sindrome di Shwachman (SDS) è una rara malattia genetica, autosomica recessiva, caratterizzata da insufficienza pancreatica, disfunzioni ematologiche, displasie scheletriche e disordini cognitivi. Nel 90% dei pazienti vengono riscontrate mutazioni a carico del gene SBDS. Similarmente ad altre sindromi midollari, i pazienti affetti da SDS hanno un aumentato rischio di insorgenza di mielodisplasie e leucemia, ma i meccanismi responsabili di questa predisposizione non sono ancora stati indagati in modo approfondito. Le cellule mesenchimali stromali (MSCs) vengono considerate fattori con un ruolo fondamentale nel mantenere e sostenere la plasticità e la sopravvivenza delle cellule staminali all’interno della nicchia midollare. Studi recenti hanno dimostrato inoltre come mutazioni specifiche a livello delle MSCs possono essere fattori sufficienti per disregolare i sottili equilibri omeostatici all’interno della nicchia e dare inizio ad un processo di trasformazione neoplastica. Il nostro gruppo ha dimostrato che MSCs derivate da pazienti affetti da SDS erano comparabili a quelli di donatori sani per quanto riguarda le loro caratteristiche in vitro (marcatori di superficie, capacità di differenziare in diversi lineages, abilità nel sostenere la vitalità di cellule CD34). La gene expression analysis condotta su 16 SDS-MSCs in realtà mostra come queste cellule avessero un pattern di espressione genica differente da quello delle mesenchimali di donatori sani, suggerendo come le mesenchimali SDS potessero avere un ruolo nei disordini ematologici riscontrati nella malattia. In questo studio abbiamo aumentato la corte di pazienti e, avvalendoci di un modello in vivo, abbiamo studiato il possibile coinvolgimento delle MSCs nei disordini ematopoietici. Il nostro modello prevedeva l’impianto sottocutaneo in topi immunocompromessi di pellet cartilaginei derivanti da MSCs da donatori sani e pazienti stimolate per 21 giorni con un particolare medium di differenziamento. Dopo 60 giorni, gli animali sono stati sacrificati e gli ossicoli recuperati per l’analisi istologica. Dai nostri dati emerge come, al termine del periodo sperimentale, solo i pellet derivati da MSCs di donatore sano siano stati in grado di formare una nicchia midollare completa, con presenza di trabecole ossee, adipociti e cellule ematopoietiche murine. Di contro, nessuno dei pellet derivati da paziente è stato ritrovato vascolarizzato o colonizzato da cellule ematopoietiche. L’analisi a time point precoci ci ha permesso di individuare dei difetti nel processo differenziativo dei pellet derivati da pazienti, che non mostravano riassorbimento cartilagineo, né deposizione di matrice ossea o processi di vascolarizzazione. Questo dato ci suggerisce come nel nostro modello le mesenchimali da paziente mostrino difetti nel loro processo differenziativo e di conseguenza possano essere coinvolte anche nei disordini ematologici a carico del midollo. Nella seconda parte del nostro studio abbiamo testato un farmaco su cellule ematologiche e non ematologiche di paziente. Questo farmaco agisce sulle nonsense stop codon mutation, una delle mutazioni più diffuse nei pazienti SDS a carico del gene SBDS, consentendo il read-through della mutazione non senso e quindi la produzione di una proteina completa. I nostri risultati hanno mostrato l’azione positiva di questo farmaco in diverse linee cellulari (linfoblastoidi, mesenchimali e mononucleate da midollo), restorando la produzione della proteina. Inoltre, il trattamento con questo farmaco ha anche prodotto miglioramenti a livello funzionale nelle cellule mononucleate. In particolare queste cellule, in seguito al trattamento, hanno mostrato un significativo aumento nella capacità di dare colonie CFU-GM. Questo risultato ha forti conseguenze a livello clinico poiché, non avendo mostrato effetti tossici, questo farmaco potrebbe essere proposto per la cura dei disordini ematologici in questi pazienti.

(2017). SHWACHMAN-DIAMOND SYNDROME: FROM PATHOGENESIS TO DRUG TARGETING. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).

SHWACHMAN-DIAMOND SYNDROME: FROM PATHOGENESIS TO DRUG TARGETING

BARDELLI, DONATELLA
2017

Abstract

Shwachman-Diamond Syndrome (SDS) is a rare autosomal recessive disease, characterized by exocrine pancreatic disorder, hematological aberrancies, bone marrow failure and cognitive impairment. In 90% of patients the SBDS gene is found mutated. Similar to other marrow failure syndromes, SDS patients have an increased risk for developing myelodysplastic syndrome and AML. To date, the mechanisms underlying the bone marrow failure in SDS patients are not fully understood. Microenvironment constituents and in particular mesenchymal stromal cells (MSCs) are considered the pivotal organizers for the generation, maintenance and plasticity of the hematopoietic stem cell niche. Recent studies show that specific changes in MSCs may be sufficient to initiate a complex phenotype of disordered homeostasis with similarities to myelodysplasia. We have demonstrated that MSCs obtained from SDS patients were comparable in vitro to HD but gene expression analysis of 16 SDS-MSCs showed that these cells had a specific gene expression signature compared to HD. These results suggest that it is possible that MSCs could be involved in the pathogenesis of the SDS marrow disorders. We increased our patients cohort and investigated whether SDS-MSCs were able to sustain malignant evolution using an innovative scaffold-free in vivo system based on the ex vivo generation of semi-cartilaginous pellets (SCPs) from human MSCs. We obtained SCPs stimulating MSCs for 21 days with a specific differentiating medium and a complete and correct formation of cartilaginous tissues both in HD and SDS samples. These SCPs were transplanted heterotopically into subcutaneous tissue of immunocompromised mice. After 60 days, we sacrificed mice and collected ossicles. We found that in 90% of cases, HD were able to recreate the hematopoietic microenvironment, with the establishment of a complete marrow niche, while none of the transplanted SDS-SCPs was able to recreate the hematopoietic microenvironment, revealing a defect in these differentiating process. The second part of our study was focused on testing a specific drug able to act on nonsense stop codon mutation, one of the most diffuse alterations in SDS patients, linked to risk of developing myelodysplastic syndrome. We successfully obtained restoration of SBDS protein in different cell lineages deriving from patients (Lymphoblastoids, MSCs, mononuclear cells from bone marrow). Protein restoration was also accompanied in some cases with an improvement of functionality. In particular, mononuclear cells from bone marrow treated with drug showed an increase in their ability to form colonies when cultured in a specific assay. This represents a powerful result, due to the potential clinical consequences related to possible therapeutic strategy. Indeed, SDS patients in future could take advantage of this drug to ameliorate their hematological defects and abolish other symptoms.
BIONDI, ANDREA
D'AMICO, GIOVANNA
Shwachman-Diamond; Mesenchimali; PTC124; niche; ossicle
Shwachman-Diamond; Mesenchimali; PTC124; niche; ossicle
MED/04 - PATOLOGIA GENERALE
English
27-giu-2017
MEDICINA TRASLAZIONALE E MOLECOLARE - DIMET - 76R
29
2015/2016
open
(2017). SHWACHMAN-DIAMOND SYNDROME: FROM PATHOGENESIS TO DRUG TARGETING. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/170787
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