Noonan Syndrome Peripheral Blood Cells Show Enhanced Spontaneous Colonies Growth In Vitro and Hyperactive JAK and RAS Intracellular Signalling Pathways
Authors: Gaipa Giuseppe , Bugarin Cristina , Cianci Paola , Selicorni Angelo , Biondi Andrea 
Published: October 2019

Abstract
Noonan Syndrome (NS) is an autosomal dominant congenital disorder characterized by several defects at births. In addition, children with NS show splenomegaly and monocytosis, and are predisposed to develop myelo proliferative disorders (MPD) which may resolve spontaneously or progress in an aggressive clinical course similar to Juvenile Myelomonocytic Leukemia (JMML). Somatic PTPN11 mutations, which encodes the protein tyrosine phosphatase Shp2, affect around 35% of patients with JMML, while germline PTPN11 mutations cause around 50% of NS cases (Cratz et al. Blood, 2005). Moreover, mutations in other genes affecting the RAS/RAF/ERK pathway, such as SOS1 and KRAS, have been identified in PTPN11-negative NS patients (Tartaglia et al. Nature Genetics, 2007). Yet, both JMML and NS/MPD patients show hypersensitivity to GM-CSF and spontaneous colonies growth (Lavin et al. Pediatric Blood Cancer, 2008). These evidences prompted the hypothesis that children with NS could maintain an abnormal status of hematopoiesis affecting in particular the myelomonocytic lineage.
We studied 23 children with NS and 16 age-matched children with other genetic defect, affecting neither JAK-STAT nor RAS/RAF/ERK pathways. Peripheral blood (PB) cells were cultured in vitro in order to evaluate the spontaneous and factor-stimulated growth of colony-forming units. In addition, PB monocytes were evaluated for IL-6 (50 ng/mL)-induced response of pSTAT3, and GM-CSF-induced response of pSTAT5 and pERK (10 and 0.1 ng/mL respectively), measured by phosphoflow technique, as previously described (Gaipa et al, Leukemia, 2008).
We observed a significantly enhanced in vitro spontaneous growth of erythroid colony-forming units (BFU-E) in NS (n=15) as compared to control subjects (n=10) (mean ± SD: 79.67% ± 30.15% vs 22.60% ± 23.00%, respectively; p=0.0004), and an augmented growth of granulocyte-macrophage units (CFU-GM), although not significantly (19.13% ± 17.20% vs 10.90% ± 17.72%; p>0.05). This enhanced capacity of NS cells to promote colony formation was confirmed also in the presence of growth factors in the medium.
Phosphoflow assay showed an enhanced amount of stimulated pSTAT3 and pSTAT5 positive cells in NS patients as compared to control subjects (pSTAT3: 41.01% ± 32.97% (n=18) vs 22.99% ± 22.38% (n=13); p=ns, and pSTAT5: 98.20% ± 2.55% (n=5) vs 63.40% ± 40.17% (n=5), p<0.05), Interestingly, preliminary experiments showed that NS patients also have higher rate of pERK response upon stimulation with GM-CSF.
In summary, we demonstrated that children with NS, but without MPD, do have significantly enhanced capacity of both BFU-E and CFU-GM units formation in vitro. GM-CSF hypersensitivity has been demonstrated in NS/MPD (Lavin et al. Pediatric Blood Cancer, 2008), thus supporting the observed enhanced growth of CFU-GM units in NS patients with persistent monocytosis. Intriguingly, abnormal growth of BFU-E was not previously reported in these patients. The biological significance of these enhanced growth capacities, particularly in absence of recognized MPD status, should be further investigated. Signalling in transduction pathways affected by Shp2 protein seems to be latently activated in NS, although these observations need to be fully validated with further experiments. Collectively, our findings open new opportunities to investigate the almost unknown hematological alterations existing in NS patients. In addition, they may lead to an enrichment of the laboratory tools potentially able to recognize signs of progression toward malignant MPD (including JMML) in children with NS.

Disclosures:
No relevant conflicts of interest to declare.