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M. Arock et al.
vated in neoplastic MC in such patients and in KIT- mutated MCL-like cell lines, and inhibition of these path- ways induces growth arrest in such cells.22,24 Other intra- cellular pathways and molecules, such as the Feline sarco- ma oncoprotein,25 or the mechanistic target of rapamycin (mTOR) complex,26 are also potential triggers of oncogen- esis. In addition, the KIT mutant activates ERK independ- ently of SRC, in contrast to KIT WT.27 Finally, LYN and BTK are found activated in neoplastic MC in a KIT-inde- pendent manner.28
Because KIT-activating mutations are found in most SM patients, several KIT-targeted tyrosine kinase inhibitors (KIT-TKI) have been developed. However, the nature of the mutation influences the sensitivity of the mutant to these TKI. For instance, the KIT D816V mutant is insensi- tive to imatinib.29 By contrast, patients presenting with KIT WT, or KIT mutant outside exon 17, may potentially respond to imatinib.30 While in ISM the KIT D816V mutant seems to be the unique molecular abnormality found, addi- tional and recurrent somatic mutations of myeloid malig- nancy-related genes have been reported in advanced SM. The genes most frequently affected are TET2, SRSF2, ASXL1, RUNX1, JAK2, N/KRAS and CBL,31-35 while EZH2, IDH2, ETV6, U2AF or SF3B1 are less often affected.36 All these mutations may be co-expressed with KIT D816V in the same cells or may be expressed in other myeloid cells but not in MC, especially in (A)SM-AHN with TET2, SRSF2 and ASXL1 mutants, in which acquisition of KIT D816V is often a late event conferring a mastocytosis phe- notype on a pre-existing clonal condition.37 These defects, and particularly the SRSF2, ASXL1 and RUNX1 (S/A/R) mutations, have a negative impact on the disease progno- sis.31-34,35 Thus, targets other than KIT and drug combina- tions might be considered in order to develop more effec- tive therapies for advanced SM. The potential of new tar- gets and of new targeted drugs or drug combinations is cur- rently investigated using the available human MCL-like MC lines, which will be described hereafter.
Major characteristics of available human mast cell leukemia-like mast cell lines
It is beyond the scope of this review to develop all the applications of the available human MC lines. We will only provide a detailed description of the cell lines that qualify as MCL-like, namely HMC-1, ROSA and MCPV- 1. Indeed, these cell models have been and are still used in vitro and/or in vivo to evaluate the potential effects of drugs and drug combinations to treat mastocytosis. Table 1 shows a summary of the major characteristics of all available human MC lines, while Tables 2 and 3 provide detailed phenotypic information.
The HMC-1 cell line and subclones (HMC-1.1 and HMC-1.2)
The origin, major characteristics and phenotype of HMC-1 cells are presented in Tables 1 through 4. HMC-1 cells are metachromatic cells containing histamine and tryptase.11 The original cell line presented with a complex karyotype (Table 1), which might have potentially played a role in the cells’ immortalization. However, despite this fact, HMC-1 cells remained sensitive to KIT inhibitors, in favor of a critical role of the KIT mutants in their mainte- nance. Indeed, in HMC-1 cells, KIT is constitutively phos- phorylated on tyrosine residues in the absence of SCF.17 Sequencing of the coding region of KIT cDNA revealed that KIT in HMC-1 cells is composed of a normal WT
allele and a mutant allele with two point mutations, KIT V560G (NM_000222.2(KIT):c.1679T>G, p.Val560Gly) and KIT D816V.38
Seven years later, two HMC-1 subclones, namely HMC-1.1 and HMC-1.2, were described.12 Both sub- clones have a heterozygous KIT V560G mutation,12 but only HMC-1.2 cells display the KIT D816V mutation.12 In both subclones, KIT was found constitutively phosphory- lated in the absence of SCF, although the presence of the KIT D816V mutation seemed to confer a slight growth advantage to HMC-1.2 cells over HMC-1.1 cells.12
HMC-1 cells have been extensively used to study KIT mutant-related and KIT mutant-independent signaling pathways and to evaluate anti-neoplastic effects of cytore- ductive/targeted drugs developed to treat advanced SM.
The ROSAKIT WT and ROSAKIT D816V subclones
The SCF-dependent ROSAKIT WT cell line was established from a CD34+ fraction of normal umbilical cord blood cells.15 CD34+ cord blood cells were cultured in the pres- ence of human SCF and, after an 8-week culture period, cells continued to proliferate, with virtually all cells being MC. The doubling time was relatively short (48-72 h) in the presence of SCF.15 ROSAKIT WT cells are round cells with a relatively high nuclear-to-cytoplasm ratio and metachromatic cytoplasmic granules.15 ROSAKIT WT cells stain strongly positive for tryptase and KIT, but express only little if any chymase.15
KIT WT
ROSA cells were found to express FcεRI, KIT
(CD117), CD33, CD4, CD9, CD203c, and CD300a, con- sistent with a MC phenotype, while they did not express CD2 or CD25 (Tables 2 and 3).15 Moreover, similar to pri- mary cord blood-derived MC, incubation of ROSA cells with interleukin-4 and IgE for 4-5 days enhanced surface expression of FcεRI. In addition, ROSA cells sensitized with interleukin-4 and IgE were fully activated by anti- IgE.15 However, over long periods of continuous culture, expression of FcεRI tends to fade on the cells, which become less sensitive to FcεRI cross-linking (unpublished observation).
ROSAKIT WT cells have a normal KIT structure, but harbor a complex karyotype, with a derivative chromosome 1 [der(1)inv(1)(p31q21)del(1)(q24q32)]. In fact, the cell line consists of two subclones: one minor subclone carrying a complete trisomy 5, and the other predominant subclone carrying a partial trisomy 5 [+del(5)(q14q34)].15 In addi- tion, molecular studies revealed that both ROSAKIT WT sub- clones have a P53 deletion and a hot spot K700E mutation in SF3B1 (unpublished observation). We assume that these alterations contributed to the immortalization of ROSAKIT WT cells and provide a premalignant (permissive) cellular background sufficient to trigger proliferation when a driv- er, such as KIT D816V, is introduced.
Indeed, when ROSAKIT WT cells were further transfected with a lentivirus encoding for GFP + KIT D816V, the resulting subclone proliferated independently of SCF. This subclone, termed ROSAKIT D816V, has the same dou- bling time as ROSAKIT WT cells cultured in SCF.15 Similar to their parental cells, ROSAKIT D816V cells have a rather mature morphology with numerous cytoplasmic granules.15
ROSAKIT D816V cells exhibit the same complex karyotype and the same SF3B1 K700E mutation as ROSAKIT WT cells.15 Moreover, the phenotype of ROSAKIT D816V cells is similar to that of the parental cell line, including expression of the FcεRI and negativity for CD2 and CD25 (detailed in
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haematologica | 2018; 103(11)