CDK4/6 inhibition enhances SHP2 inhibitor efficacy and is dependent upon RB function in malignant peripheral nerve sheath tumors

Abstract

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft tissue sarcomas with limited treatment options, and new effective therapeutic strategies are desperately needed. We observe antiproliferative potency of genetic depletion of PTPN11 or pharmacological inhibition using the SHP2 inhibitor (SHP2i) TNO155. Our studies into the signaling response to SHP2i reveal that resistance to TNO155 is partially mediated by reduced RB function, and we therefore test the addition of a CDK4/6 inhibitor (CDK4/6i) to enhance RB activity and improve TNO155 efficacy. In combination, TNO155 attenuates the adaptive response to CDK4/6i, potentiates its antiproliferative effects, and converges on enhancement of RB activity, with greater suppression of cell cycle and inhibitor-of-apoptosis proteins, leading to deeper and more durable antitumor activity in in vitro and in vivo patient-derived models of MPNST, relative to either single agent. Overall, our study provides timely evidence to support the clinical advancement of this combination strategy in patients with MPNST and other tumors driven by loss of NF1.

Fig. 1.. PTPN11 genetic depletion reduces MPNST cell growth and alters MEK/ERK activity.

(A) ST8814 and JH-2-002 transduced with doxycycline (Dox)–inducible constructs expressing shPTPN11 #818 or #5003 were treated with vehicle or Dox (300 ng/ml) for 2 weeks. Cells were fixed with 10% neutral-buffered formalin (NBF) and then stained with crystal violet. (B) Cells as in (A) were treated with vehicle or Dox (300 ng/ml) for up to 7 days. The phase confluence was monitored by IncuCyte real-time imaging system and normalized to corresponding 0-hour scan. (C) Cells as in (A) were treated with vehicle or Dox (300 ng/ml) for 72 hours, and the indicated proteins were assessed using immunoblot. (D) JH-2-002 transduced with Dox-inducible shPTPN11 #818 was treated with vehicle or Dox (300 ng/ml) for 72 hours, and, then, three replicates were collected for RNA-seq, and the fourth replicate was collected for immunoblotting validation. RNA-seq result revealed a significant decrease in PTPN11 RNA expression [n = 3, P adj = 0, and log₂ fold change (LFC) = −4.69] when comparing shPTPN11 v. ctrl. A total of 51 genes representing MEK/ERK transcriptional output (35) were assessed for their expression after PTPN11 knockdown. A volcano plot demonstrating LFC of shPTPN11 v. ctrl as a function of −log₁₀ (P adj) is shown. Black dots, not significant (ns; P adj > 0.05); red dots, LFC > 0; and blue dots, LFC < 0 (P adj < 0.05). Thirty-seven of the 51 genes (73%) in the set were significantly transcriptionally down-regulated following PTPN11 genetic depletion.

Fig. 2.. SHP2i TNO155 alters growth and gene expression in NF1-MPNST cells.

(A) Onco-print of key driver genes (NF1, CDKN2A, SUZ12, and EED) in MPNST and putative others is shown. (B) Ten NF1-MPNST cell lines were treated with increasing doses of the SHP2i TNO155 for 5 days. Cell viability was evaluated using the cell counting kit-8 (CCK-8) assay measuring metabolic activity. (C) Three NF1-MPNST cell lines were treated with dimethyl sulfoxide (DMSO) or TNO155 (0.3, 1, and 3 μM) for about 2 weeks. Cells were fixed with 10% NBF and then stained with crystal violet. (D and E) ST8814 and JH-2-002 were treated with increasing doses (30 to 3000 nM) of TNO155 for 24 hours (D) or 0.3 μM TNO155 over a time course (E). (F) Eight NF1-MPNST cell lines were treated with DMSO or 0.3 μM TNO155 for 48 hours. Representative signaling intermediates in ERK and cell cycle pathways were detected using immunoblot. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (G) ST8814 and NF90.8 transduced with Dox-inducible NF1-GRD were pretreated with vehicle or Dox (300 ng/ml), followed by treatment with DMSO or TNO155 (0.3, 1, and 3 μM) for 5 days. Cell viability was assessed using CCK-8. (H) ST8814 and NF90.8 transduced with (marked with +) Dox-inducible green fluorescent protein (GFP) or V5-tagged NF1-GRD were treated with Dox (300 ng/ml) for 24 hours. RAS activity and signaling intermediates were detected using immunoblot following the active RAS pull-down assay. PD, pull down; WCL, whole-cell lysate. (I) Venn diagram showing the number of significant overlapping genes (P adj < 0.05 and |fold change| > 1.5) between shPTPN11, TNO155, and trametinib at 6- and 24-hour time points. (J) Heatmap demonstrating significant changes (P adj < 0.05 marked with “+”) in transcriptional output of the 51 ERK signature genes, derived from RNA-seq analysis of JH-2-002 after DMSO, 0.3 μM TNO155, or 20 nM trametinib treatment (6 and 24 hours).

Fig. 3.. Loss of function in RB1 confers resistance to TNO155.

(A) Volcano plot from RNA-seq analysis revealing cell cycle dysregulation comparing MPNST v. pNF. (B) Selected cell cycle regulators were evaluated in the nine NF1-associated and one sporadic (STS26T) MPNST cell lines. (C) ST8814 and JH-2-002 transduced with Dox-inducible constructs expressing shPTPN11 #5003 or #818 were treated with vehicle or Dox (300 ng/ml) for 72 hours, and RB phosphorylation at serine-780 or serine-807/811 was assessed. (D) ST8814 parental (Par), or stable lines transduced with pLXSN-neomycin-E7 WT or E7 d21-24 mutant; and JH-2-002 parental, or stable lines transduced with pMSCV-puromycin-E7 WT or E7 d21-24 mutant, were treated with increasing dose of TNO155 for 5 days. Cell viability was evaluated by using the CCK-8 assay. (E) Cells as in (D) were treated with DMSO or 0.3 μM TNO155 for 48 hours.

Fig. 4.. Responses of NF1-MPNST cells to the CDK4/6i ribociclib.

(A) Ten NF1-associated MPNST cell lines were treated with increasing doses of the CDK4/6i ribociclib for 5 days. Cell viability was evaluated by using the CCK-8 assay. (B) Two NF1-MPNST cell lines were treated with DMSO or 1 μM ribociclib over a time course. Signal intermediates in ERK and cell cycle pathways were assessed. (C) JH-2-002 cells were treated with DMSO or 1 μM ribociclib for 24 hours. Seventy-one phosphorylated human RTKs were evaluated using human RTK phosphorylation array C1. Signal intensity from technical duplicates was quantified using densitometry analysis and normalized to ribociclib v. DMSO, and, notably, altered RTKs are shown. (D) Cells as in Fig. 3E were treated with increasing doses of ribociclib for 5 days. Cell viability was evaluated by using the CCK-8 assay.

Fig. 5.. Combined inhibition of SHP2 and CDK4/6 effectively suppresses MPNST cell growth.

(A) ST8814 and NF90.8 parental (Par) and trametinib-resistant (Res) lines and eight NF1-MPNST cell lines were treated with DMSO, TNO155 (0.3, 1, and 3 μM), ribociclib (1 and 3 μM), and their combination for 7 to 10 days. Direct cell counting using trypan blue exclusion assay was performed by TC20 automated cell counter. (B) Cells as in (A) were treated with drugs for 2 to 3 weeks, and colony formation was evaluated using crystal violet assay. (C) Area under the curve (AUC) is calculated on the basis of the IncuCyte cell confluence monitoring of the seven cell lines as shown, treated with DMSO, TNO155 (0.3 μM), ribociclib (1 μM), and their combination for 6 days. (D) Two NF1-MPNST cell lines were treated with DMSO, TNO155 (0.3 μM), ribociclib (1 μM), and their combination for 6 days. Cell confluence was monitored using IncuCyte imaging systems. (E) Four NF1-MPNST cell lines were treated with DMSO, 0.3 μM TNO155, and/or 1 μM ribociclib for 72 and 96 hours. ERK signaling and cell cycle regulators were evaluated using immunoblot. (F) Three NF1-MPNST cell lines transduced with shPTPN11 #818 were pretreated with vehicle or Dox (300 ng/ml) for 72 hours, followed by treatment with DMSO or 1 μM ribociclib for additional 72 hours. Cell lysates were assessed for expression of the indicated proteins.

Fig. 6.. Combination of TNO155 and ribociclib additively inhibits cell cycle and induces apoptosis.

(A) Upset matrix plot derived from RNA-seq analysis, showing the overlapping numbers of significant genes (P adj < 0.05 and |fold change| > 1.5) after 24-hour treatment with 0.3 μM TNO155, 1 μM ribociclib, and their combination, and normalized to DMSO control. Rows, the sets; columns, intersections between these sets. (B and C) Heatmaps demonstrating the more potent transcriptional inhibition of mitotic prometaphase (B) and cell cycle checkpoints (C) by combined TNO155 and ribociclib relative to either single agent alone, highlighting BIRC5, PLK1, CLSPN, and CCNA2 (P adj < 0.05 marked with + and LFC). (D) Five NF1-MPNST cell lines were treated with DMSO, 0.3 μM TNO155, and/or 1 μM ribociclib for 48 hours, following overnight starvation in 0.1% FBS-containing culture medium to synchronize cells. Cells were fixed in ice cold 70% ethanol and stained with propidium iodide/ribonuclease staining solution (Cell Signaling Technology, no. 4087) and then analyzed by flow cytometry. (E) ST8814 and JH-2-079c were treated with DMSO, 0.3 μM TNO155, and/or 1 μM ribociclib for 48 hours, and, then, protein lysates were assessed using human apoptosis antibody array (R&D Systems, no. ARY009). Signal intensity from technical duplicates was quantified by densitometry analysis using ImageJ and normalized to DMSO. Data represent means ± SEM. (F) Nine NF1-MPNST cell lines were treated as in (E), and the indicated proteins involved in apoptosis and ERK signaling were detected using immunoblot.

Fig. 7.. The combination of TNO155 and ribociclib is active against MPNST tumor growth in vivo.

(A and B) Five-to-6-week-old female NRG mice bearing six individual NF1-MPNST PDXs were treated with vehicle, ribociclib (75 mg/kg, once daily), TNO155 (7.5 mg/kg, twice daily), or their combination by oral gavage for 4 weeks. Average tumor volume of four to five mice per arm was plotted over the time course of treatment days. VS, very sensitive; PS, partially sensitive. (C) Tumors of each arm from WU-386 were harvested 4 hours after last dose of 4 weeks on drugs and fixed in 10% NBF. The Ki-67 expression was assessed using immunohistochemistry (IHC). (D and E) Tumors of each arm from WU-225, WU-386 were harvested 4 hours after last dose, 4 weeks on drugs (D); or 24 hours after last dose, 3 days on drugs (E). The indicated proteins involved in ERK and cell cycle signaling were detected using immunoblot. (F) Onco-print of key driver genes in MPNST and putative others is shown. For NF1 and SUZ12, both germline (G) and somatic (S) mutations are shown. (G) Volcano plot demonstrating LFC of VS/PS as a function of −log₁₀ (P adj). Blue dots represent 227 genes that were significantly altered (P adj < 0.05 and |LFC| > 0.585) when comparing the RNA-seq data of VS (JH-2-031, WU-225, and JH-2-002) v. PS (WU-545, JH-2-079c, and WU-386). (H) The mice bearing JH-2-031 were on initial treatment as in (A) for about 4 weeks and left untreated for another 4 weeks before rechallenging with the combination for additional 2 weeks. (I) The mice bearing JH-2-002 as in (A) were continuously on treatment for 10 weeks. (J) Tumors of each arm from JH-2-002 and JH-2-031 were harvested 4 hours after last dose of 10 weeks and fixed in 10% NBF. The Ki-67 expression was assessed using IHC.