Identification of CD44 As a RAS-MEK-Regulated Invasion Receptor That Is Overexpressed in Neoplastic Mast Cells and Triggers Disease Expansion in Advanced Systemic Mastocytosis
Authors: Mueller Niklas , Wicklein Daniel , Eisenwort Gregor , Schwaab Juliana , Jawhar Mohamad , Boehm Alexandra , Herrmann Harald , Stefanzl Gabriele , Hoermann Gregor , Sperr Wolfgang R. , Arock Michel , Reiter Andreas , Schumacher Udo , Valent Peter 
Published: October 2019

Abstract
CD44, also known as Hermes antigen, is a multifunctional invasion receptor that mediates homing and expansion of normal and neoplastic stem- and progenitor cells in various organs including the bone marrow (BM). Mast cells (MC) and their progenitors also express CD44. However, little is known about the impact and regulation of CD44 in neoplastic cells in systemic mastocytosis (SM).
We examined the expression, regulation, and functional role of CD44 in neoplastic cells in SM. As assessed by multi-color flow cytometry, CD34+/CD38- stem cells (SC), CD34+/CD38+ progenitor cells (PC), and KIT+/CD34- MC invariably expressed CD44 in all SM variants, including patients with indolent SM (ISM, 11/11), SM with associated hematologic neoplasm (SM-AHN, 7/7), aggressive SM (ASM, 3/3), and MC leukemia (MCL, 8/8). Expression of CD44 on SC, PC, and MC increased significantly with the aggressiveness of SM. Moreover, soluble CD44 levels measured in the sera of patients with SM by ELISA were found to correlate with the WHO type of SM. In particular, significantly higher levels of soluble CD44 were measured in advanced SM compared to ISM, cutaneous mastocytosis (CM), or healthy controls (p<0.05). In addition, all human MC lines examined, including HMC-1.1, HMC-1.2, ROSAKIT WT, ROSAKIT D816V, MCPV-1.1, MCPV-1.2, MCPV-1.3, and MCPV-1.4 expressed cytoplasmic and cell surface CD44. To study the mechanisms underlying CD44 expression on MC, we applied various targeted drugs. Incubation with the demethylating agents decitabine (0.1-5 µM) or 5-azacytidine (0.1-5 µM) for 96 hours resulted in a dose-dependent upregulation of CD44 surface expression compared to baseline levels (100%) in all MC lines examined (decitabine, 5 µM: 210±50% in HMC-1.1, 294±76% in HMC-1.2, 236±56% in ROSAKIT WT, 210±54% in ROSAKIT D816V, 242±47% in MCPV-1.1, 179±23% in MCPV-1.2, 207±17% in MCPV-1.3, and 152±5% in MCPV-1.4 cells, p<0.05; 5-azacytidine, 5 µM: 355±104% in HMC-1.1, 429±105% in HMC-1.2, 412±135% in ROSAKIT WT, 292±136% in ROSAKIT D816V, 365±55% in MCPV-1.1, 345±106% in MCPV-1.2, 325±87% in MCPV-1.3, and 278±38% in MCPV-1.4 cells, p<0.05). In contrast, incubation with the MEK-inhibitor refametinib (RDEA119) (0.1-5 µM) or the STAT5 blocker pimozide (2.5-10 µM) for 48 hours resulted in a dose-dependent downregulation of CD44 surface expression compared to baseline levels (100%) in all MC lines examined (refametinib, 2.5 µM: 71±9% in HMC-1.1, 82±3% in HMC-1.2, 33±13% in ROSAKIT WT, 31±3% in ROSAKIT D816V, 62±6% in MCPV-1.1, 82±13% in MCPV-1.2, 84±5% in MCPV-1.3, and 87±1% in MCPV-1.4 cells, p<0.05; pimozide, 7.5 µM: 59±7% HMC-1.1, 68±3% in HMC-1.2, 62±16% in ROSAKIT WT, and 80±3% in ROSAKIT D816V, 62±5% in MCPV-1.1, 72±4% in MCPV-1.2, 73±4% in MCPV-1.3, and 63±9% in MCPV-1.4 cells, p<0.05). These data suggest that the RAS/MEK pathway and the STAT5 pathway are involved in expression of CD44 in neoplastic MC. In order to confirm this hypothesis we transduced HMC-1.2 cells and ROSAKIT WT cells with KRASWT or oncogenic KRASG12V. In all transduced cell lines, KRAS overexpression resulted in upregulation of CD44 surface expression compared to empty vector (100%) (HMC-1.2: 143±17% with KRASWT, 249±53% with KRASG12V, p<0.05; ROSAKIT WT: 170±13% with KRASWT, 403±64% with KRASG12V, p<0.05). As expected, refametinib was found to suppress RAS-induced CD44 expression in these cells. To study the functional role of CD44 in neoplastic MC, we employed a mouse xenotransplantation model using severe combined immunodeficient (SCID) mice, HMC-1.2 cells, and shRNA against CD44. In this model, the shRNA-induced knock-down of CD44 in HMC-1.2 cells resulted in reduced MC expansion, reduced tumor formation, delayed ulceration, and prolonged survival compared to cells transduced with control shRNA (median survival in the CD44 shRNA group: 110 days vs 97 days in the control group, p<0.05). The formation of lung metastasis, quantified by human Alu-sequence-specific qPCR, was found to be particularly decreased (15-fold) in the CD44 knock-down group compared to control mice.
In conclusion, CD44 is expressed in neoplastic MC as well as in neoplastic stem- and progenitor cells in patients with advanced SM. Our data also suggest that CD44 is an important mediator of evolution and of malignant spread of neoplastic MC into various organs in SM. Future studies will show whether CD44 can serve as a therapeutic target in patients with advanced SM.

Disclosures
Hoermann: Gilead: Research Funding; Novartis: Honoraria; Amgen: Honoraria; Ariad: Honoraria. Sperr:Novartis: Honoraria; Amgen: Honoraria, Research Funding. Arock:Agensys, Inc: Research Funding. Valent:Ariad: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria; Deciphera Pharmaceuticals: Research Funding; Celgene: Honoraria, Research Funding.