Effects of different MEK1/2 inhibitors on proliferation of NF1 knockdown Schwann cells

doi:10.19438/j.cjoms.2020.04.003

Abstract

Abstract: PURPOSE: To establish a stable RSC96 cell line with knockdown of neurofibromin 1(NF1) gene by lentiviral vectors, and assess the influence of different MEK1/2 inhibitors on proliferation of NF1 knockdown RSC96 Schwann cell. METHODS: Lentivirus was used to construct a Schwann cell model with decreased expression of NF1 gene. qRT-PCR and Western blot (WB) were used to verify the expression level of NF1 gene. Five MEK1/2 inhibitors were included in this experiment: Selumetinib, Binimetinib, Cobimetinib, Trametinib and PD0325901. CCK-8 assay was used to detect the inhibition of different MEK1/2 inhibitors under time gradient and concentration gradient. IC₅₀ values in the blank control group and NF1^kd group of 5 inhibitors were calculated by Graphpad Prism software package. SPSS 22.0 software package was used for statistical analysis. RESULTS: All MEK1/2 inhibitors could inhibit the proliferation of NF1^kd Schwann cells (P<0.05). At 72 h, PD0325901, Cobimetinib and Trametinib had stronger inhibitory effects than Selumetinib and Binimetinib (P<0.05). In IC₅₀ experiment, the drug concentrations of Cobimetinib in NF1^kd group and blank control group were (2.35±0.98) mol/L and (14.22±1.67) mol/L, respectively(P<0.05); the drug concentrations of Binimetinib in NF1^kd group and blank control group were (18.96±2.37) mol/L and (114.1±5.98) mol/L, respectively; and the differences between the groups were significant (P<0.05). There was no significant difference in IC₅₀ values within Selumetinib, Trametinib and PD0325901 in NF1^kd group and control group (P>0.05). CONCLUSIONS: Stable Schwann cell line with knockdown of NF1 gene was successfully constructed. All five MEK inhibitors can inhibit the proliferation of NF1^kd RSC96 cell line; PD0325901, Trametinib and Cobimetinib processed longer lasting inhibiting ability. Cobimetinib and Binimetinib can both kill NF1^kd Schwann cells more specifically than the blank control group at the same drug concentration. Cobimetinib has both long-lasting efficacy and killing specificity, which may have more clinical prospects.

Key words: NF1, Lentivirus, Schwann cells, MEK1/2 inhibitors, Neurofibromatosis type 1

CLC Number:

LIU Jia-liang, DU Zhong, ZHENG Jia-wei, YOU Yuan-he, WANG Yan-an. Effects of different MEK1/2 inhibitors on proliferation of NF1 knockdown Schwann cells[J]. China Journal of Oral and Maxillofacial Surgery, 2020, 18(4): 302-307.

References

[1] Gutmann DH, Ferner RE, Listernick RH, et al.Neurofibromatosis type 1[J]. Nat Rev Dis Primers, 2017, 3: 17004.
[2] 郑家伟, 孙坚, 张志愿, 等. I型神经纤维瘤病诊治进展[J].中华口腔医学研究杂志, 2007, 16(6): 561-569
[3] Pemov A, Li H, Patidar R, et al.The primacy of NF1 loss as the driver of tumorigenesis in neurofibromatosis type 1-associated plexiform neurofibromas[J]. Oncogene, 2017, 36(22): 3168-3177.
[4] Ratner N, Miller SJ.A RASopathy gene commonly mutated in cancer: the neurofibromatosis type 1 tumour suppressor[J]. Nat Rev Cancer, 2015, 15(5): 290-301.
[5] Radomska KJ, Coulpier F, Gresset A, et al.Cellular origin, tumor progression, and pathogenic mechanisms of cutaneous neurofibromas revealed by mice with knockout in boundary cap cells[J]. Cancer Discov, 2019, 9(1): 130-147.
[6] Yu Y, Choi K, Wu J, et al.NF1 patient missense variants predict a role for ATM in modifying neurofibroma initiation[J]. Acta Neuropathol, 2020, 139(1): 157-174.
[7] Jessen WJ, Miller SJ, Jousma E, et al.MEK inhibition exhibits efficacy in human and mouse neurofibromatosis tumors[J]. J Clin Invest, 2013, 123(1): 340-347.
[8] Jousma E, Rizvi TA, Wu J, et al.Preclinical assessments of the MEK inhibitor PD-0325901 in a mouse model of neurofibromatosis type 1[J]. Pediatr Blood Cancer, 2015, 62(10): 1709-1716.
[9] Dombi E, Baldwin A, Marcus LJ, et al.Activity of selumetinib in neurofibromatosis type 1-related plexiform neurofibromas[J]. N Engl J Med, 2016, 375(26): 2550-2560.
[10] Liao CP, Booker RC, Brosseau JP, et al.Contributions of inflammation and tumor microenvironment to neurofibroma tumorigenesis[J]. J Clin Invest, 2018, 128(7): 2848-2861.
[11] Johannessen CM, Reczek EE, James MF, et al.The NF1 tumor suppressor critically regulates TSC2 and mTOR[J]. Proc Natl Acad Sci USA, 2005, 102(24): 8573-8578.
[12] Chen S, Burgin S, McDaniel A, et al. Nf1-/- Schwann cell-conditioned medium modulates mast cell degranulation by c-Kit-mediated hyperactivation of phosphatidylinositol 3-kinase[J]. Am J Pathol, 2010,177(6): 3125-3132.
[13] Widemann BC, Babovic-Vuksanovic D, Dombi E, et al.Phase Ⅱ trial of pirfenidone in children and young adults with neurofibromatosis type 1 and progressive plexiform neurofibromas[J]. Pediatr Blood Cancer, 2014, 61(9): 1598-1602.
[14] Babovic-Vuksanovic D, Widemann BC, Dombi E, et al.Phase Ⅰ trial of pirfenidone in children with neurofibromatosis 1 and plexiform neurofibromas[J]. Pediatr Neurol, 2007,36(5): 293-300.
[15] Babovic-Vuksanovic D, Ballman K, Michels V, et al.Phase II trial of pirfenidone in adults with neurofibromatosis type 1[J]. Neurology, 2006,67(10): 1860-1862.
[16] Widemann BC, Dombi E, Gillespie A, et al.Phase 2 randomized, flexible-crossover, double-blinded, placebo-controlled trial of the farnesyl transferase inhibitor tipifarnib in children and young adults with neurofibromatosis type 1 and progressive plexiform neurofibromas[J]. Neuro Oncol, 2014, 16(5): 707-718.
[17] Weiss B, Widemann BC, Wolters P, et al.Sirolimus for progressive neurofibromatosis type 1-associated plexiform neurofibromas: a neurofibromatosis clinical trials consortium phase Ⅱ study[J]. Neuro Oncol, 2015, 17(4): 596-603.
[18] Jakacki RI, Dombi E, Steinberg SM, et al.Phase Ⅱ trial of pegylated interferon alfa-2b in young patients with neurofibromatosis type 1 and unresectable plexiform neurofibromas[J]. Neuro Oncol, 2017, 19(2): 289-297.
[19] Robertson KA, Nalepa G, Yang FC, et al.Imatinib mesylate for plexiform neurofibromas in patients with neurofibromatosis type 1: a phase 2 trial[J]. Lancet Oncol, 2012, 13(12): 1218-1224.
[20] Flaherty KT, Robert C, Hersey P, et al.Improved survival with MEK inhibition in BRAF-mutated melanoma[J]. N Engl J Med, 2012, 367(2): 107-114.
[21] Ascierto PA, Schadendorf D, Berking C, et al.MEK162 for patients with advanced melanoma harbouring NRAS or Val600 BRAF mutations: a non-randomised, open-label phase 2 study[J]. Lancet Oncol, 2013, 14(3): 249-256.
[22] Rosen LS, LoRusso P, Ma WW, et al. A first-in-human phase I study to evaluate the MEK1/2 inhibitor, cobimetinib, administered daily in patients with advanced solid tumors[J]. Invest New Drugs, 2016, 34(5): 604-613.