MEK Inhibition Synergizes with TYK2 Inhibitors in NF1-Associated Malignant Peripheral Nerve Sheath Tumors

Abstract

Purpose: Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas with limited treatment options and poor survival rates. About half of MPNST cases are associated with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome. Overexpression of TYK2 occurs in the majority of MPNST, implicating TYK2 as a therapeutic target.

Experimental design: The effects of pharmacologic TYK2 inhibition on MPNST cell proliferation and survival were examined using IncuCyte live cell assays in vitro, and downstream actions were analyzed using RNA-sequencing (RNA-seq), qPCR arrays, and validation of protein changes with the WES automated Western system. Inhibition of TYK2 alone and in combination with MEK inhibition was evaluated in vivo using both murine and human MPNST cell lines, as well as MPNST PDX.

Results: Pharmacologic inhibition of TYK2 dose-dependently decreased proliferation and induced apoptosis over time. RNA-seq pathway analysis on TYK2 inhibitor-treated MPNST demonstrated decreased expression of cell cycle, mitotic, and glycolysis pathways. TYK2 inhibition resulted in upregulation of the MEK/ERK pathway gene expression, by both RNA-seq and qPCR array, as well as increased pERK1/2 levels by the WES Western system. The compensatory response was tested with dual treatment with TYK2 and MEK inhibitors, which synergistically decreased proliferation and increased apoptosis in vitro. Finally, combination therapy was shown to inhibit growth of MPNST in multiple in vivo models.

Conclusions: These data provide the preclinical rationale for the development of a phase I clinical trial of deucravacitinib and mirdametinib in NF1-assosciated MPNST.

Figure 1.

TYK2 inhibitors reduce proliferation in MPNST cells. A, TYK2 protein levels were visualized by IHC in MPNST and plexiform neurofibroma, with positive staining scored on a 0 to 3 scale. B, Relative TYK2 protein levels in MPNST cell lines by WES Western blot analysis. Protein bands analyzed by densitometry, with TYK2 normalized to β-actin. C, JW23.3 and JH-2–002 cells were treated with TYK2 inhibitors (WU-12, WU-76) or inactive control (WU-18) for 3 days, and cell confluence was determined by IncuCyte assay. D, Representative images of IncuCyte assay at 72 hours, with YOYO-1 green fluorescence as indicator of apoptosis.

Figure 2.

TYK2 inhibition leads to compensatory stimulation of the MAPK pathway in MPNST cells. A, JH-2–002 cells were treated with 40 μmol/L WU-12 or control for 48 hours, and gene expression was analyzed by qPCR array for JAK/STAT pathway–related genes. Log₂ fold changes of significantly changed genes are graphed. B, JW23.3 cells were treated with control or 40 μmol/L WU-12 or WU-76 for 48 hours. Global gene expression was determined by RNA-seq pathway analysis. Changes in gene expression for WU-12 versus control were examined by RNA-seq for genes downstream of STAT3 in a heatmap (C) and boxplot (D), as well as MEK/MAPK pathway genes in a volcano plot (E), enrichment plot (F), and boxplot (G). P < 0.05 vs. vehicle control.

Figure 3.

TYK2 inhibitory drugs decrease activation of STAT3 while increasing activation of ERK1/2 in MPNST cells. A, JW23.3 cells and (B) JH-2–002 cells were incubated with a TYK2 inhibitor (40 μmol/L WU-12) or vehicle control for the indicated times. Phosphorylated and total protein levels for STAT3 and ERK1/2 were analyzed by the WES Western system. Bands were analyzed by densitometry in the WES software, with phosphorylated protein normalized to the matching total protein and expressed as percent of control at the same time point. *, P < 0.05 vs. vehicle control at the same time point.

Figure 4.

The specific TYK2 inhibitor deucravacitinib (BMS-986165) decreases MPNST cell proliferation at lower doses. The MPNST cell lines JW23.3 (A and B), JH-2–002 (C and D), and MPNST-724 (E and F) were treated with the indicated doses of the specific TYK2 inhibitor deucravacitinib or the pan-JAK inhibitor baricitinib for 3 days in IncuCyte live cell proliferation assays.

Figure 5.

Inhibitors of TYK2 (deucravacitinib) and MEK (mirdametinib) act synergistically to reduce proliferation and increase apoptosis in JW23.3 MPNST cells. Cell confluence and apoptosis were analyzed by the IncuCyte assay after 48-hour incubation with drugs. Synergy was analyzed using Synergy Finder software by the HSA method for (A) inhibition of cell proliferation or (B) apoptosis, and mean synergy score is reported (P < 0.05 vs. drug alone). Intensity of red color indicates synergy score for each dose combination, whereas green indicates antagonism. C, Representative images of JW23.3 cells treated for 48 hours. YOYO-1 green fluorescence indicates apoptotic cells.

Figure 6.

The combination of drugs inhibiting TYK2 and MEK block MPNST tumor growth in mice. A, Schematic diagram of treatment paradigm. Mice with JW23.3 MPNST xenograft tumors (n = 6 per group; B), WU-386 MPNST PDX tumors (n = 3 per group; C), or JH-2–002 MPNST xenograft tumors (n = 5 per group; D) were treated daily with 1.5 mg/kg mirdametinib (Mirda), 30 mg/kg deucravacitinib (Deucra, BMS-986165), the combination of drugs, or vehicle control for 3 weeks or until tumors reached the maximum allowed volume. *, P < 0.05 vs. vehicle control; ^a, P < 0.05 for drug combination vs. drugs alone. E and F, Diagram of TYK2/STAT3 and MEK/ERK pathways after treatment with Deucra and/or Mirda in MPNST cells. (Illustrations were created with BioRender.com.)