Direct comparison of Cdkn2a and p53 loss reveals global transcriptomic differences, including tumor suppressor dependency on pentose phosphate pathway in MPNST

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are aggressive and chemo-resistant sarcomas with poor overall survival. Loss of tumor suppressors CDKN2A (~80%) or P53 (~20%) is a signature event in MPNST development. Here, we use CRISPR/Cas9 somatic tumorigenesis mouse models to mechanistically compare CDKN2A and P53 loss transcriptomic and metabolic differences, leading to therapeutic vulnerabilities in MPNSTs. Multi-omic analyses identified the pentose phosphate pathway (PPP) and regulation of NADPH metabolism as critical metabolic vulnerabilities in the CDKN2A-deleted MPNSTs. Disruption of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting step in the PPP, slowed CDKN2A-deleted MPNST growth, yet sensitized both genotypes of MPNSTs to standard-of-care chemotherapy. Moreover, the redox-regulated transcription factor NRF2 controlled G6PD activity in these tumors. Genomic analysis of patient samples showed a NRF2 gene signature that correlated with tumor transformation, further underscoring this pathway as a therapeutic target. This work identifies the G6PD/NADPH axis as a central metabolic vulnerability in MPNSTs. Overall design: To better understand the transcriptomic differences between loss of tumor suppressors CDKN2A and P53, we derived mouse Malignant Peripheral Nerve Sheath Tumors (MPNSTs) primary cell lines (Individual tumor for each cell line). Tumors were developed utilizing CRISPR/Cas9 somatic tumorigenesis models of Nf1/Cdkn2a-deleted and Nf1/p53-deleted autochthonous MPNSTs in genetically identical, wild-type mice (129 Sv/Jae). Adenovirus containing gRNAs targeting either Nf1 and Cdkn2a or Nf1 and p53 and contains full length Cas9 were injected into the sciatic nerve. Tumors develop in 3-4 months. Tumors were dissociated and plated to develop cell lines. Three cell lines of each genotype (Nf1-/-; Cdkn2a-/- and Nf1-/-; p53-/-) were submitted for RNA sequencing.

恶性周围神经鞘膜瘤（Malignant Peripheral Nerve Sheath Tumors, MPNSTs）是一类侵袭性强、化疗耐药的肉瘤，患者总体生存率较差。抑癌基因CDKN2A（约80%）或P53（约20%）的缺失是MPNST发生发展的标志性事件。本研究利用CRISPR/Cas9体细胞致瘤小鼠模型，从机制层面比较CDKN2A与P53缺失后的转录组及代谢组差异，以此探究MPNST的治疗易感靶点。多组学分析显示，磷酸戊糖途径（pentose phosphate pathway, PPP）与NADPH代谢调控是CDKN2A缺失型MPNST中关键的代谢易感靶点。作为磷酸戊糖途径的限速酶，葡萄糖-6-磷酸脱氢酶（glucose-6-phosphate dehydrogenase, G6PD）的功能抑制可减缓CDKN2A缺失型MPNST的生长，同时可使两种基因型的MPNST对标准化疗方案产生敏感性。此外，氧化还原调控转录因子核因子E2相关因子2（NRF2）可调控上述肿瘤中的G6PD活性。对患者样本的基因组分析结果表明，NRF2基因特征与肿瘤转化呈显著相关，进一步证实该通路可作为MPNST的治疗靶点。本研究明确了G6PD/NADPH轴作为MPNST核心代谢易感靶点的地位。

总体实验设计：为深入解析抑癌基因CDKN2A与P53缺失后的转录组差异，我们构建了小鼠恶性周围神经鞘膜瘤（MPNST）原代细胞系（每株细胞系对应单个体肿瘤）。本研究采用CRISPR/Cas9体细胞致瘤模型，在遗传背景一致的野生型129 Sv/Jae小鼠中，分别诱导生成Nf1/Cdkn2a缺失和Nf1/p53缺失的自发性MPNST。将靶向Nf1与Cdkn2a、或Nf1与p53且携带全长Cas9的腺病毒注射至坐骨神经，肿瘤可在3-4个月内形成。将肿瘤组织解离并培养以建立细胞系，每种基因型（Nf1-/-; Cdkn2a-/-与Nf1-/-; p53-/-）各3株细胞系提交进行RNA测序。