**1. Introduction**

Infantile myofibromatosis (IM; [MIM#228550]) is a disorder of mesenchymal proliferation characterized by the development of nonmetastatic tumors [1] that present as firm, flesh-colored to purple nodules usually located in the skin, subcutaneous tissues, bone, muscle or visceral organs [2,3]. This disease was described under different names, the name "infantile myofibromatosis" was first used in 1981 [4]. Although rare, with an incidence of 1 in 400,000 children, IM represents the most common proliferative fibrous tumor of infancy [5,6]. Myofibromas are usually present at birth or develop shortly thereafter, and almost 90% of the tumors are diagnosed before the age of two years,

with a median age of three months [6–8]. A male predominance has been reported, and the ratio of male to female patients varies from 1.5:1 to 1.8:1 [5].

IM clinically presents in three main forms: (1) Solitary, (2) multicentric without visceral involvement, and (3) multicentric with visceral involvement [6]. The prognosis is excellent in solitary or multicentric nonvisceral forms with a possibility of spontaneous regression of the lesions but is poor when detected in the viscera [9]. Surgical excision of a single lesion is the standard of care [8]. Multiple lesions or surgically unresectable lesions are treated using various therapeutics, such as anti-inflammatory drugs, interferon-alpha, vinblastine, vincristine, dactinomycin, cyclophosphamide and methotrexate [6,8].

The molecular pathogenesis of IM is not completely understood. Familial forms exhibiting autosomal dominant and recessive transmission have been reported over the past two decades [10]. In 2013, several point mutations in the platelet-derived growth factor receptor beta (PDGFR-beta) gene (*PDGFRB*) were identified to be associated with familial IM. A study of nine unrelated families diagnosed with IM revealed two disease-causing mutations in *PDGFRB*: c.1978C>A (p.P660T) and c.1681C>T (p.R561C) [1]. Interestingly, one family did not have either of these *PDGFRB* mutations, but all affected individuals had a c.4556T>C (p.L1519P) mutation in *NOTCH3*. The germline mutation c.1681C>T (p.R561C) in *PDGFRB* was also detected in 11 individuals with familial IM [7]. In addition, one individual harbored a c.1998C>A (p.N666K) somatic mutation. Very recently, a novel *PDGFRB* mutation (c.1679C>T; p.P560L) was identified in a 3-generation family with multicentric IM [11].

Platelet-derived growth factors (PDGFs) and PDGF receptors (PDGFRs) have important functions in the regulation of cell growth and survival [12]. The PDGF family consists of four structurally related single polypeptide units that constitute five functional homo- or heterodimers: PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, and PDGF-DD [13]. PDGFs act via two receptor tyrosine kinases (RTKs), PDGFR-alpha and PDGFR-beta [14]. Both receptors can activate many major signal transduction pathways, including the Ras/MAPK, PI3K/Akt and phospholipase C-gamma pathways [15].

Moreover, other genes were associated with IM etiology, which demonstrates the possible genetic heterogeneity of this disease. As mentioned above, a connection between a c.4556T>C (p.L1519P) mutation in *NOTCH3* and IM was described in one study [1]. Human cells express four different Notch receptors, Notch 1–4, each encoded by a different gene [16]. The expression of *PDGFRB* can be regulated by Notch activity, as PDGFR-beta expression can be robustly upregulated by Notch 1 and Notch 3 signaling [17]. Another example is a c.511G>C (p.V171L) mutation in the potential tumor suppressor *NDRG4* that was associated with IM in one case [18]. In the same year, it was demonstrated that the c.1276G>A (p.V426M) mutation in *PTPRG* (protein tyrosine phosphatase, receptor type G) was able to substantially influence the penetrance of a c.1681C>T (p.R561C) mutation in *PDGFRB* [19]. *PTPRG* encodes an enzyme that could dephosphorylate PDGFR-beta and thus reduce PDGFR-beta activity [19,20].

A recent work revealed that two IM-associated mutations in *PDGFRB*, c.1681C>T (p.R561C) and c.1998C>A (p.N666K), constitutively activate PDGFR-beta and can induce cancer development in vivo [21]. The same study showed that cells harboring p.R561C and p.N666K mutations are sensitive to specific tyrosine kinase inhibitors, which were able to decrease PDGFR-beta phosphorylation and downstream signaling. These results suggested that blocking PDGFR-beta activity would offer a therapeutic option for IM treatment. Indeed, in a recently published study, targeted treatment with sunitinib and low-dose vinblastine led to a robust response in a child with refractory multiple IM and a c.1681C>T (p.R561C) mutation in *PDGFRB* [8].

In this work, we demonstrate for the first time the efficacy of sunitinib, erlotinib, U0126 and FR180204 on the cell line harboring a c.1681C>T (p.R561C) *PDGFRB* mutation found in patients with IM. Sunitinib is known as an inhibitor of several kinases, including PDGFR-beta [22], erlotinib is an inhibitor of epidermal growth factor receptor (EGFR) [23], U0126 inhibits MEK1/2 phosphorylation [24], and FR180204 inhibits ERK1/2 phosphorylation. These inhibitors were chosen on the basis of our previous findings [8] as well as on the results of subsequent phosphoprotein profiling of the NSTS-47 cell line.

#### **2. Results**

*2.1. Germline Mutations in PDGFRB Were Identified in Both Children, and the Same Mutation in PDGFRB Was Confirmed in NSTS-47 Cells*

Genetic analyses revealed that both siblings harbor a heterozygous germline c.1681C>T (p.R561C) mutation in the *PDGFRB* gene (Table 1). It was also confirmed that NSTS-47 cell line harbors the same heterozygous germline mutation c.1681C>T (p.R561C) in *PDGFRB*.


**Table 1.** Germline mutations identified in patients.
