Kras is required for pancreatic tumor maintenance through regulation of hexosamine biosynthesis and the non-oxidative pentose phosphate pathway. Mus musculus

The maintenance of advanced malignancies relies on continued activity of driver oncogenes, although their rate-limiting role is highly context-dependent with respect to tumor types and associated genetic alterations. Oncogenic Kras mutation is the signature event in human pancreatic ductal adenocarcinoma (PDAC), serving a critical role in tumor initiation. Here, an inducible KrasG12D-driven p53 mutant PDAC mouse model establishes that advanced PDAC remains strictly dependent on continued KrasG12D expression and that KrasG12D serves a vital role in the control of tumor metabolism, through stimulation of glucose uptake and channeling of glucose intermediates through the hexosamine biosynthesis pathway (HBP) and the pentose phosphate pathway (PPP). Notably, these studies reveal that oncogenic Kras regulates ribose biogenesis. Unlike canonical models of PPP-mediated ribose biogenesis, we demonstrate that oncogenic Kras drives intermediates from enhanced glycolytic flux into the non-oxidative arm of the PPP, thereby decoupling ribose biogenesis from NADPNADPH-mediated redox control. Together, this work provides in vivo mechanistic insights into how oncogenic Kras promotes metabolic reprogramming in native tumors and illuminates potential metabolic targets that can be exploited for therapeutic benefit in Kras-driven PDAC. Overall design: Primary pancreatic tumor lines were established from p48Cre tetO_LKrasG12D ROSA_rtTAL+ p53L+ mice. Five independent tumor lines (iKras1-5) were used for pancreatic injection into nude mice to generate orthotopic tumors. The mice were kept on doxycycline for 2 weeks until obvious tumor formation. Half of the animals were pulled off doxycycline for 24 hours. Tumors with over 75% cellularity were collected for total RNA prepartion. For in vitro expression profiles, the same five tumor lines were cultured in the presence or absence of doxycycline for 24 hours and total cellular RNA was prepared. For control samples, two independent tumor lines from LSL-KrasG12D p53L+ tumors were cultured in the presence or absence of doxycycline for 24 hours and total cellular RNA was prepared.

高级恶性肿瘤的维持依赖于驱动癌基因的持续活性，但其限速作用在很大程度上取决于肿瘤类型及相关遗传改变的背景环境。致癌性Kras突变是人类胰腺导管腺癌（pancreatic ductal adenocarcinoma, PDAC）的标志性事件，在肿瘤起始过程中发挥关键作用。本研究构建了可诱导的KrasG12D驱动型p53突变胰腺导管腺癌小鼠模型，证实晚期PDAC仍严格依赖KrasG12D的持续表达，且KrasG12D通过刺激葡萄糖摄取、将葡萄糖中间产物分流至己糖胺生物合成途径（hexosamine biosynthesis pathway, HBP）和磷酸戊糖途径（pentose phosphate pathway, PPP），在肿瘤代谢调控中发挥至关重要的作用。值得注意的是，本研究揭示致癌性Kras可调控核糖生物合成。与磷酸戊糖途径介导核糖生物合成的经典模型不同，本研究证实致癌性Kras可将糖酵解通量增强所产生的中间产物导入磷酸戊糖途径的非氧化臂，从而实现核糖生物合成与NADPH介导的氧化还原调控的解耦联。综上，本研究为致癌性Kras在原生肿瘤中促进代谢重编程的机制提供了体内实验见解，并阐明了可用于靶向治疗Kras驱动型胰腺导管腺癌的潜在代谢靶点。

整体实验设计：从p48Cre tetO_LKrasG12D ROSA_rtTAL+ p53L+小鼠中构建原代胰腺肿瘤细胞系。选取5株独立的肿瘤细胞系（iKras1-5）进行裸鼠胰腺原位注射以构建原位移植瘤模型。小鼠予以多西环素（doxycycline）喂养2周直至形成明显肿瘤，随后将半数小鼠撤除多西环素处理24小时。收集细胞占比超过75%的肿瘤组织用于总RNA制备。针对体外表达谱分析，将上述5株肿瘤细胞系在含或不含多西环素的培养基中培养24小时后提取总细胞RNA；作为对照样本，另外选取2株来自LSL-KrasG12D p53L+肿瘤的独立细胞系，在含或不含多西环素的培养基中培养24小时后提取总细胞RNA。