Oncogene activation and loss of tumor suppressor function changes the metabolic activity of cancer cells to drive unrestricted proliferation. Moreover, cancer cells adapt their metabolism to sustain growth and survival when access to oxygen and nutrients is restricted, such as in poorly vascularized tumor areas. We show here that p53-deficient colon cancer cells exposed to tumor-like metabolic stress in spheroid culture activated the mevalonate pathway to promote the synthesis of ubiquinone. This was essential to maintain mitochondrial electron transport for respiration and pyrimidine synthesis in metabolically compromised environments. Induction of mevalonate pathway enzyme expression in the absence of p53 was mediated by accumulation and stabilization of mature SREBP2. Mevalonate pathway inhibition by statins blocked pyrimidine nucleotide biosynthesis and induced oxidative stress and apoptosis in p53-deficient cancer cells in spheroid culture. Moreover, ubiquinone produced by the mevalonate pathway was essential for the growth of p53-deficient tumor organoids. In contrast, inhibition of intestinal hyperproliferation by statins in an Apc/KrasG12D-mutant mouse model was independent of de novo pyrimidine synthesis. Our results highlight the importance of the mevalonate pathway for maintaining mitochondrial electron transfer and biosynthetic activity in cancer cells exposed to metabolic stress. They also demonstrate that the metabolic output of this pathway depends on both genetic and environmental context. Significance: These findings suggest that p53-deficient cancer cells activate the mevalonate pathway via SREBP2 and promote the synthesis of ubiquinone that plays an essential role in reducing oxidative stress and supports the synthesis of pyrimidine nucleotide.

Highlights d FADS2 expression is prognostic in some cancers d FADS2 is a target of SREBP-1 and SREBP-2 d SREBP activity and mTOR signaling regulate FADS2mediated sapienate metabolism d Torin1 treatment reduces FADS2 expression and sapienate metabolism in xenografts

Lung cancer is the most common cancer worldwide and the leading cause of cancer-related deaths in both men and women. Despite the development of novel therapeutic interventions, the 5-year survival rate for non-small cell lung cancer (NSCLC) patients remains low, demonstrating the necessity for novel treatments. One strategy to improve translational research is the development of surrogate models reflecting somatic mutations identified in lung cancer patients as these impact treatment responses. With the advent of CRISPR-mediated genome editing, gene deletion as well as site-directed integration of point mutations enabled us to model human malignancies in more detail than ever before. Here, we report that by using CRISPR/Cas9-mediated targeting of Trp53 and KRas, we recapitulated the classic murine NSCLC model Trp53fl/fl:lsl-KRasG12D/wt. Developing tumors were indistinguishable from Trp53fl/fl:lsl-KRasG12D/wt-derived tumors with regard to morphology, marker expression, and transcriptional profiles. We demonstrate the applicability of CRISPR for tumor modeling in vivo and ameliorating the need to use conventional genetically engineered mouse models. Furthermore, tumor onset was not only achieved in constitutive Cas9 expression but also in wild-type animals via infection of lung epithelial cells with two discrete AAVs encoding different parts of the CRISPR machinery. While conventional mouse models require extensive husbandry to integrate new genetic features allowing for gene targeting, basic molecular methods suffice to inflict the desired genetic alterations in vivo. Utilizing the CRISPR toolbox, in vivo cancer research and modeling is rapidly evolving and enables researchers to swiftly develop new, clinically relevant surrogate models for translational research.

Squamous cell carcinomas (SCC) frequently have an exceptionally high mutational burden. As consequence, they rapidly develop resistance to platinum-based chemotherapy and overall survival is limited. Novel therapeutic strategies are therefore urgently required. SCC express ∆Np63, which regulates the Fanconi Anemia (FA) DNA-damage response in cancer cells, thereby contributing to chemotherapy-resistance. Here we report that the deubiquitylase USP28 is recruited to sites of DNA damage in cisplatin-treated cells. ATR phosphorylates USP28 and increases its enzymatic activity. This phosphorylation event is required to positively regulate the DNA damage repair in SCC by stabilizing ∆Np63. Knock-down or inhibition of USP28 by a specific inhibitor weakens the ability of SCC to cope with DNA damage during platin-based chemotherapy. Hence, our study presents a novel mechanism by which ∆Np63 expressing SCC can be targeted to overcome chemotherapy resistance. Limited treatment options and low response rates to chemotherapy are particularly common in patients with squamous cancer. The SCC specific transcription factor ∆Np63 enhances the expression of Fanconi Anemia genes, thereby contributing to recombinational DNA repair and Cisplatin resistance. Targeting the USP28-∆Np63 axis in SCC tones down this DNA damage response pathways, thereby sensitizing SCC cells to cisplatin treatment.

Squamous cell carcinomas (SCC) frequently have a limited response to or develop resistance to platinum-based chemotherapy, and have an exceptionally high tumor mutational burden. As a consequence, overall survival is limited and novel therapeutic strategies are urgently required, especially in light of a rising incidences. SCC tumors express ΔNp63, a potent regulator of the Fanconi Anemia (FA) DNA-damage response pathway during chemotherapy, thereby directly contributing to chemotherapy-resistance. Here we report that the deubiquitylase USP28 affects the FA DNA repair pathway during cisplatin treatment in SCC, thereby influencing therapy outcome. In an ATR-dependent fashion, USP28 is phosphorylated and activated to positively regulate the DNA damage response. Inhibition of USP28 reduces recombinational repair via an ΔNp63-Fanconi Anemia pathway axis, and weakens the ability of tumor cells to accurately repair DNA. Our study presents a novel mechanism by which tumor cells, and in particular ΔNp63 expressing SCC, can be targeted to overcome chemotherapy resistance.SignificanceLimited treatment options and low response rates to chemotherapy are particularly common in patients with squamous cancer. The SCC specific transcription factor ΔNp63 enhances the expression of Fanconi Anemia genes, thereby contributing to recombinational DNA repair and Cisplatin resistance. Targeting the USP28-ΔNp63 axis in SCC tones down this DNA damage response pathways, thereby sensitizing SCC cells to cisplatin treatment.

<p>(1) Figure S1 shows Metabolic flux analysis of p53+/+ and p53-/- cells in monolayer and spheroid culture. (2) Figure S2 shows Mechanism of induction of SREBP2 target genes in p53-deficient spheroid cultures. (3) Figure S3 shows Induction of cell cycle arrest and apoptosis by simvastatin in spheroid cultures. (4) Figure S4 shows Blockade of cholesterol and ubiquinone synthesis by statins. (5) Figure S5 shows Effect of simvastatin on TCA cycle flux and failure of ubiquinone to restore viability in monolayer cells. (6) Figure S6 shows Inhibition of apoptosis in p53-deficient spheroid cultures by uridine. (7) Figure S7 shows Genotyping of cells used for organoid cultures and lack of effect of leflunomide on intestinal hyperproliferation.</p>

<p>This file includes extended experimental procedures including 1. Analysis of RNA-sequencing and Cancer Genome Atlas data 2. Generation of shRNA Cell Lines, 3. Automated cell counting, 4. Liquid chromatography and mass spectrometry, 5. BrdU staining of murine tissue; supplementary references and supplementary figure legends</p>

<p>(1) Figure S1 shows Metabolic flux analysis of p53+/+ and p53-/- cells in monolayer and spheroid culture. (2) Figure S2 shows Mechanism of induction of SREBP2 target genes in p53-deficient spheroid cultures. (3) Figure S3 shows Induction of cell cycle arrest and apoptosis by simvastatin in spheroid cultures. (4) Figure S4 shows Blockade of cholesterol and ubiquinone synthesis by statins. (5) Figure S5 shows Effect of simvastatin on TCA cycle flux and failure of ubiquinone to restore viability in monolayer cells. (6) Figure S6 shows Inhibition of apoptosis in p53-deficient spheroid cultures by uridine. (7) Figure S7 shows Genotyping of cells used for organoid cultures and lack of effect of leflunomide on intestinal hyperproliferation.</p>