Combined CDK4/6 and ERK1/2 inhibition enhances anti-tumor activity in NF1-associated plexiform neurofibroma [trigeminal nerve]

Purpose: Plexiform neurofibromas (PNF) are peripheral nerve sheath tumors that cause significant morbidity in persons with neurofibromatosis type 1 (NF1), yet treatment options remain limited. To identify novel therapeutic targets for PNF, we applied an integrated multi-omics approach to quantitatively profile kinome activation in a mouse model that has predicted therapeutic responses in clinical trials for NF1-associated PNF with high fidelity. Experimental Design: Utilizing RNA sequencing combined with chemical proteomic profiling of the functionally enriched kinome using multiplexed inhibitor beads coupled with mass spectrometry, we identified molecular signatures predictive of response to CDK4/6 and RAS/MAPK pathway inhibition in PNF. Informed by these results, we evaluated the effectivity of the CDK4/6 inhibitor, abemaciclib, and the ERK1/2 inhibitor (LY3214996) alone and in combination to reduce PNF tumor burden in Nf1flox/flox;PostnCre mice. Results: Converging signatures of CDK4/6 and RAS/MAPK pathway activation were identified within the transcriptome and kinome that were conserved in both murine and human PNF. We observed robust synergism of the CDK4/6 inhibitor, abemaciclib, in combination with the ERK1/2 inhibitor (LY3214996) in primary Nf1-/- Schwann cell cultures. Consistent with these findings, the combination of abemaciclib (CDK4/6i) and LY3214996 (ERK1/2i) synergized to suppress molecular signatures of MAPK activation and exhibited enhanced anti-tumor activity in Nf1flox/flox;PostnCre mice in vivo. Conclusion: These findings provide rationale for the clinical translation of CDK4/6 inhibitors alone and in combination with therapies targeting the RAS/MAPK pathway for the treatment of PNF and other peripheral nerve sheath tumors in persons with NF1. Overall design: RNAseq of trigeminal nerve from Nf1 flox/flox PostnCre - (WT) and PostnCre+ (Nf1) mice 12 experimental samples

研究目的：丛状神经纤维瘤（Plexiform neurofibromas, PNF）是一类外周神经鞘肿瘤，可导致Ⅰ型神经纤维瘤病（neurofibromatosis type 1, NF1）患者出现显著的致病负担，然而当前其治疗手段仍十分有限。为识别丛状神经纤维瘤的新型治疗靶点，我们采用整合多组学方法，对一个能高度精准预测NF1相关丛状神经纤维瘤临床试验治疗反应的小鼠模型中的激酶组（kinome）激活情况进行定量分析。

实验设计：本研究结合RNA测序（RNA sequencing）与基于多重抑制剂磁珠（multiplexed inhibitor beads）耦联质谱（mass spectrometry）技术的功能富集激酶组化学蛋白质组分析，鉴定出可预测丛状神经纤维瘤对CDK4/6及RAS/MAPK通路抑制剂治疗反应的分子特征。基于上述结果，我们评估了CDK4/6抑制剂阿贝西利（abemaciclib）与ERK1/2抑制剂LY3214996单药及联合使用时，在Nf1flox/flox;PostnCre小鼠中降低丛状神经纤维瘤肿瘤负荷的效果。

结果：我们在转录组与激酶组中均鉴定出CDK4/6与RAS/MAPK通路激活的趋同特征，该特征在小鼠与人类丛状神经纤维瘤中均保守存在。我们在原代Nf1-/-雪旺细胞（Schwann cell）培养体系中观察到，CDK4/6抑制剂阿贝西利与ERK1/2抑制剂LY3214996联合使用具有显著的协同效应。与上述发现一致，阿贝西利（CDK4/6i）与LY3214996（ERK1/2i）联合使用可协同抑制MAPK激活的分子特征，并在体内Nf1flox/flox;PostnCre小鼠中展现出增强的抗肿瘤活性。

结论：本研究结果为单独使用CDK4/6抑制剂，以及联合靶向RAS/MAPK通路的疗法治疗NF1患者的丛状神经纤维瘤及其他外周神经鞘肿瘤提供了临床转化依据。

整体实验设计：对Nf1 flox/flox PostnCre-（野生型，WT）与PostnCre+（Nf1模型）小鼠的三叉神经进行RNA测序，共包含12个实验样本。