Background:Metabolic dysfunction-associated steatotic liver disease (MASLD) is a metabolic syndrome characterized by increased fat storage in hepatocytes. In the hepatocytes, autophagy protects against cytotoxic stress and harmful cellular conditions. In the hepatic stellate cells (HSCs), autophagy exerts pro-fibrotic properties and promotes the release of pro-inflammatory metabolites. We investigated the modulation of autophagy as a therapeutic approach for MASLD. Methods:Murine liver tissue and human hepatic cells were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot. Real time fluorescence was performed to monitor the autophagy maturation process. Accumulation of fat was detected by Oil Red O staining. Collagen fibers were detected by picrosirius staining under polarized light. Results:The loss of leptin in obese mice affected by metabolic dysfunction-associated steatohepatitis (MASH) promoted the over-expression of Becn1, Map1lc3b, Sqstm1, Uvrag and Prkaa1_2 and the accumulation of their proteins. The oleic acid caused an accumulation of fat, followed by the reduction of the autophagy proteins and the increase of the P-AMPK-α in LEP-/- HepG2 cells and the maturation of autophagosome vesicles in LEP-/- Hep3B cells. Oleic acid increased the accumulation of fat in human HSCs and the COL1A1 transcript level, but not the collagen I fibers. BECN1 and MAP1LC3B were up-regulated. Instead, all the autophagy proteins were downregulated, but not P-AMPK-α. Instead, the treatment with caffeine prompted neither the transactivation nor the autophagy. Conclusions:Leptin loss contributes to the autophagy process in obese mice. The administration of oleic acid in LEP-/- cells prompted autophagy not only in hepatocyte-like cells but also in human HSCs.

Breast cancer (BC) is the most prevalent malignancy in women, with hormone receptor-positive, HER2-negative (HR+/HER2−) tumors representing ~70% of cases. While CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy have transformed treatment for metastatic HR+/HER2− BC, acquired resistance remains a major obstacle. Using HR+/HER2− BC models with acquired resistance to the CDK4/6 inhibitors Palbociclib or Ribociclib, we uncovered a metabolic vulnerability in highly resistant clones, mediated by mTORC1 hyperactivation and autophagy suppression. Gene expression profiling revealed enrichment of glycolysis and mTORC1 pathways in CDK4/6i-resistant cells, which manifested as heightened sensitivity to the metabolic inhibitors Metformin and Dichloroacetate (DCA). Mechanistically, mTORC1 overactivation impaired autophagy via ULK1-Ser757 phosphorylation, as confirmed by LC3 flux assays, leaving resistant cells unable to adapt to energy stress. Treatment with metabolic drugs triggered AMPK activation, ACC inhibition, and PARP cleavage, culminating in apoptosis. Clinically, immunohistochemical analysis of a BC cohort revealed a significant correlation between mTORC1 activity (p4E-BP1T37/46) and autophagy suppression (p62 accumulation), supporting the translational relevance of this axis. Our findings propose mTORC1-mediated autophagy defects as a biomarker for metabolic vulnerability in CDK4/6i-resistant BC, offering a rationale for targeting these tumors with metabolic therapies to overcome resistance.

Activating PIK3CA mutations, present in up to 40% of hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (Her2−) breast cancer (BC) patients, can be effectively targeted with the alpha isoform-specific PI3K inhibitor Alpelisib. This treatment significantly improves outcomes for HR+, Her2−, and PIK3CA-mutated metastatic BC patients. However, acquired resistance, often due to aberrant activation of the mTOR complex 1 (mTORC1) pathway, remains a significant clinical challenge. Our study, using in vitro and orthotopic xenograft mouse models, demonstrates that constitutively active mTORC1 signaling renders PI3K inhibitor-resistant BC exquisitely sensitive to various drugs targeting cancer metabolism. Mechanistically, mTORC1 suppresses the induction of autophagy during metabolic perturbation, leading to energy stress, a critical depletion of aspartate, and ultimately cell death. Supporting this mechanism, BC cells with CRISPR/Cas9-engineered knockouts of canonical autophagy genes showed similar vulnerability to metabolically active drugs. In BC patients, high mTORC1 activity, indicated by 4E-BP1T37/46 phosphorylation, correlated with p62 accumulation, a sign of impaired autophagy. Together, these markers predicted poor overall survival in multiple BC subgroups. Our findings reveal that aberrant mTORC1 signaling, a common cause of PI3K inhibitor resistance in BC, creates a druggable metabolic vulnerability by suppressing autophagy. Additionally, the combination of 4E-BP1T37/46 phosphorylation and p62 accumulation serves as a biomarker for poor overall survival, suggesting their potential utility in identifying BC patients who may benefit from metabolic therapies.

The mutational landscape of TP53, a tumor suppressor mutated in about half of all cancers, includes over 2,000 known missense mutations. To fully leverage TP53 mutation status for personalized medicine, a thorough understanding of the functional diversity of these mutations is essential. We conducted a deep mutational scan using saturation genome editing with CRISPR-mediated homology-directed repair to engineer 9,225 TP53 variants in cancer cells. This high-resolution approach, covering 94.5% of all cancer-associated TP53 missense mutations, precisely mapped the impact of individual mutations on tumor cell fitness, surpassing previous deep mutational scan studies in distinguishing benign from pathogenic variants. Our results revealed even subtle loss-of-function phenotypes and identified promising mutants for pharmacological reactivation. Moreover, we uncovered the roles of splicing alterations and nonsense-mediated messenger RNA decay in mutation-driven TP53 dysfunction. These findings underscore the power of saturation genome editing in advancing clinical TP53 variant interpretation for genetic counseling and personalized cancer therapy.

Four molecules of the tumor suppressor p53 assemble to cooperatively bind proapoptotic target genes. The structural basis for cooperativity consists of interactions between adjacent DNA binding domains. Mutations at the interaction interface that compromise cooperativity were identified in cancer patients, suggesting a requirement of cooperativity for tumor suppression. We report on an analysis of cooperativity mutant p53E177R mice. Apoptotic functions of p53 triggered by DNA damage and oncogenes were abolished in these mice, whereas functions in cell-cycle control, senescence, metabolism, and antioxidant defense were retained and were sufficient to suppress development of spontaneous T cell lymphoma. Cooperativity mutant mice are nevertheless highly cancer prone and susceptible to different oncogene-induced tumors. Our data underscore the relevance of DNA binding cooperativity for p53-dependent apoptosis and tumor suppression and highlight cooperativity mutations as a class of p53 mutations that result in a selective loss of apoptotic functions due to an altered quaternary structure of the p53 tetramer.

The tumor suppressor gene TP53 is the most frequently mutated gene in various cancers. Unlike other tumor suppressors, TP53 is mostly hit by missense mutations, of which more than 2,000 have been described in cancer patients. To take advantage of TP53 mutation status for personalized therapy, a deeper knowledge of the functional ramifications of specific mutations is required as evidence of the functional heterogeneity of mutant p53 proteins mounts. Here, we report on a CRISPR-based saturation mutagenesis screen of 9,225 variants expressed from the endogenous TP53 gene locus of a cancer cell. By tracking changes in the abundance of individual variants in response to specific p53-pathway stimulation, we were able to construct high-resolution functional activity maps of the TP53 mutome, covering ∼94.5% of all cancer-associated missense mutations. The results demonstrate the impact of individual mutations on tumor cell fitness with unprecedented precision and coverage, even revealing underlying mechanisms such as apoptosis. The high discriminatory power also resolves subtle loss-of-function phenotypes and highlights a subset of mutants as particularly promising targets for pharmacological reactivation. Moreover, the data offer intriguing insight into the role of aberrant splicing and nonsense-mediated mRNA decay in clearing truncated proteins due to not only nonsense, frameshift, and splice-site mutations but also missense and synonymous mutations. Surprisingly, no missense mutation provided an immediate proliferative advantage over a null mutation. Nonetheless, cells with a missense, but not null mutations, acquired pro-metastatic properties after prolonged growth in mice, emphasizing the significance of mutant p53-directed clonal evolution in the progression of tumors towards metastasis.

TP53, encoding the tumor suppressor p53, is frequently mutated in various cancers, producing mutant p53 proteins (mutp53) which can exhibit neomorphic, gain-of-function properties. The latter transform p53 into an oncoprotein that promotes metastatic tumor progression via downstream effectors such as ENTPD5, an endoplasmic reticulum UDPase involved in the calnexin/calreticulin cycle of N-glycoprotein biosynthesis. Elucidating the mechanisms underlying the pro-metastatic functions of the mutp53-ENTPD5 axis is crucial for developing targeted therapies for aggressive metastatic cancer.We analyzed pancreatic, lung, and breast adenocarcinoma cells with p53 missense mutations to study the impact of mutp53 and ENTPD5 on the N-glycoproteins integrin-α5 (ITGA5) and integrin-β1 (ITGB1), which heterodimerize to form the key fibronectin receptor. We assessed the role of the mutp53-ENTPD5 axis in integrin-dependent tumor-stroma interactions and tumor cell motility using adhesion, migration, and invasion assays, identifying and validating therapeutic intervention targets. We employed an orthotopic xenograft model of pancreatic ductal adenocarcinoma to examine in vivo targeting of mutp53-ENTPD5-mediated ITGA5 regulation for cancer therapy.Mutp53 depletion diminished ITGA5 and ITGB1 expression and impaired tumor cell adhesion, migration, and invasion, rescued by ENTPD5. The mutp53-ENTPD5 axis maintained ITGA5 expression and function via the calnexin/calreticulin cycle. Targeting this axis using ITGA5-blocking antibodies, α-glucosidase inhibitors, or pharmacological degradation of mutp53 by HSP90 inhibitors, such as Ganetespib, effectively inhibited ITGA5-mediated cancer cell motility in vitro. In the orthotopic xenograft model, Ganetespib reduced ITGA5 expression and metastasis in an ENTPD5-dependent manner.The mutp53-ENTPD5 axis fosters ITGA5 and ITGB1 expression and tumor cell motility through the calnexin/calreticulin cycle, contributing to cancer metastasis. ITGA5-blocking antibodies or α-glucosidase inhibitors target this axis and represent potential therapeutic options worth exploring in preclinical models. The pharmacologic degradation of mutp53 by HSP90 inhibitors effectively blocks ENTPD5-ITGA5-mediated cancer cell motility and metastasis in vivo, warranting further clinical evaluation in p53-mutant cancers. This research underscores the significance of understanding the complex interplay between mutp53, ENTPD5, and the calnexin/calreticulin cycle in integrin-mediated metastatic tumor progression, offering valuable insights for the development of potential therapeutic strategies.

BACKGROUND:In vivo gene editing of somatic cells with CRISPR nucleases has facilitated the generation of autochthonous mouse tumors, which are initiated by genetic alterations relevant to the human disease and progress along a natural timeline as in patients. However, the long and variable, orthotopic tumor growth in inner organs requires sophisticated, time-consuming and resource-intensive imaging for longitudinal disease monitoring and impedes the use of autochthonous tumor models for preclinical studies.METHODS:To facilitate a more widespread use, we have generated a reporter mouse that expresses a Cre-inducible luciferase from Gaussia princeps (GLuc), which is secreted by cells in an energy-consuming process and can be measured quantitatively in the blood as a marker for the viable tumor load. In addition, we have developed a flexible, complementary toolkit to rapidly assemble recombinant adenoviruses (AVs) for delivering Cre recombinase together with CRISPR nucleases targeting cancer driver genes.RESULTS:We demonstrate that intratracheal infection of GLuc reporter mice with CRISPR-AVs efficiently induces lung tumors driven by mutations in the targeted cancer genes and simultaneously activates the GLuc transgene, resulting in GLuc secretion into the blood by the growing tumor. GLuc blood levels are easily and robustly quantified in small-volume blood samples with inexpensive equipment, enable tumor detection already several months before the humane study endpoint and precisely mirror the kinetics of tumor development specified by the inducing gene combination.CONCLUSIONS:Our study establishes blood-based GLuc monitoring as an inexpensive, rapid, high-throughput and animal-friendly method to longitudinally monitor autochthonous tumor growth in preclinical studies.

The processes leading from disturbed B-cell development to adult B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) remain poorly understood. Here, we describe Irf4 −/− mice as prone to developing BCP-ALL with age. Irf4 −/− preB-I cells exhibited impaired differentiation but enhanced proliferation in response to IL-7, along with reduced retention in the IL-7 providing bone marrow niche due to decreased CXCL12 responsiveness. Thus selected, preB-I cells acquired Jak3 mutations, probably following irregular AID activity, resulting in malignant transformation. We demonstrate heightened IL-7 sensitivity due to Jak3 mutants, devise a model to explain it, and describe structural and functional similarities to Jak2 mutations often occurring in human Ph-like ALL. Finally, targeting JAK signaling with Ruxolitinib in vivo prolonged survival of mice bearing established Irf4 −/− leukemia. Intriguingly, organ infiltration including leukemic meningeosis was selectively reduced without affecting blood blast counts. In this work, we present spontaneous leukemogenesis following IRF4 deficiency with potential implications for high-risk BCP-ALL in adult humans.

Background More than twenty years after its discovery, the role of the importin beta superfamily member Ran GTP-binding protein (RanBP) 17 is still ill defined. Previously, we observed notable RanBP17 RNA expression levels in head and neck squamous cell carcinoma (HNSCC) cell lines with disruptive TP53 mutations. Methods We deployed HNSCC cell lines as well as cell lines from other tumor entities such as HCT116, MDA-MB-231 and H460, which were derived from colon, breast and lung cancers respectively. RNAi was used to evaluate the effect of RanBP17 on cell proliferation. FACS analysis was used for cell sorting according to their respective cell cycle phase and for BrdU assays. Immunocytochemistry was deployed for colocalization studies of RanBP17 with Nucleolin and SC35 (nuclear speckles) domains. TCGA analysis was performed for prognostic assessment and correlation analysis of RanBP17 in HNSCC patients. Results RNAi knockdown of RanBP17 , significantly reduced cell proliferation in HNSCC cell lines. This effect was also seen in the HNSCC unrelated cell lines HCT116 and MDA-MB-231. Similarly, inhibiting cell proliferation with cisplatin reduced RanBP17 in keratinocytes but lead to induction in tumor cell lines. A similar observation was made in tumor cell lines after treatment with the EGFR kinase inhibitor AG1478. In addition to previous reports, showing colocalization of RanBP17 with SC35 domains, we observed colocalization of RanBP17 to nuclear bodies that are distinct from nucleoli and SC35 domains. Interestingly, for HPV positive but not HPV negative HNSCC, TCGA data base analysis revealed a strong positive correlation of RanBP17 RNA with patient survival and CDKN2A . Conclusions Our data point to a role of RanBP17 in proliferation of HNSCC and other epithelial cells. Furthermore, RanBP17 could potentially serve as a novel prognostic marker for HNSCC patients. However, we noted a major discrepancy between RanBP17 RNA and protein expression levels with the used antibodies. These observations could be explained by the presence of additional RanBP17 splice isoforms and more so of non-coding circular RanBP17 RNA species. These aspects need to be addressed in more detail by future studies.

How disturbances of B cell development provoke adult B acute lymphoblastic leukemia (B-ALL) remains poorly understood. Here we describe Irf4–/– mice as prone to developing B-ALL with age. Irf4–/– pro/preB cells exhibited impaired differentiative but enhanced proliferative potential in vitro and accumulated in spleens of healthy Irf4–/– mice, suggesting reduced adherence to the IL-7 providing bone marrow niche. Thus selected, pro/preB cells transformed acquiring proliferative IL-7 independency through Jak3 gain-of-function mutations. Targeting JAK signaling with Ruxolitinib in vivo prolonged survival of mice bearing established Irf4–/– leukemia. Intriguingly, organ infiltration including leukemic meningeosis was selectively reduced without affecting blood blast counts. As low IRF4 expression and JAK3 mutations also characterize a subpopulation of Ph-like B-ALL in adult humans, our results imply Irf4–/– mice as a suitable model for investigating preleukemic conditions in adults. Using this model, we identified an unexpected effect of Ruxolitinib treatment in B-ALL.

INTRODUCTION:Autophagic cell death in cancer cells can be mediated by inhibition of deacetylases. Although extensive studies have focused on the autophagic process in cancer, little is known about the role of autophagy in degrading cytosolic and nuclear components of pancreatic neuroendocrine neoplastic (pNEN) cells leading to cell death, thus improving the therapy of patients affected by pNEN. METHODS:2D and 3D human pNEN and pancreatic stellate cells were treated with panobinostat and bafilomycin. Autophagy markers were detected by RT-qPCR, immunofluorescence, and Western blot. Autophagosomes were detected by electron microscopy and their maturation by real-time fluorescence of LC3B stable transfected cells. ChIP was performed at the cAMP responsive element. Immunofluorescence was performed in murine pancreatic tissue. RESULTS:We observed that pan-deacetylase inhibitor panobinostat treatment causes autophagic cell death in pNEN cells. We also found that although AMPK-α phosphorylation is counterbalanced by phosphorylated AKT, it is not capable to inhibiting autophagic cell death. However, the binding activity of the cAMP responsive element is prompted by panobinostat. Although autophagy inhibition prevented autophagosome synthesis, maturation, and cell death, panobinostat treatment induced the accumulation of mature autophagosomes in the cytosol and the nucleus, leading to disruption of the organelles, cellular digestion, and decay. Observation of autophagosome membrane proteins Beclin1 and LC3B aggregation in murine pancreatic islets indicates that autophagy restoration may also lead to autophagosome aggregation in murine insulinoma cells. A basal low expression of autophagy markers was detectable in patients affected by pNEN, and, interestingly, the expression of these markers was significantly lower in metastatic pNEN. DISCUSSION/CONCLUSION:Our study highlights that the autophagy functional restoration and prolongation of this catabolic process, mediated by inhibition of deacetylase, is responsible for the reduction of pNEN cells. Prompting of autophagy cell death could be a promising strategy for the therapy of pNEN.

Cancer cells have a characteristic metabolism, mostly caused by alterations in signal transduction networks rather than mutations in metabolic enzymes. For metabolic drugs to be cancer-selective, signaling alterations need to be identified that confer a druggable vulnerability. Here, we demonstrate that many tumor cells with an acquired cancer drug resistance exhibit increased sensitivity to mechanistically distinct inhibitors of cancer metabolism. We demonstrate that this metabolic vulnerability is driven by mTORC1, which promotes resistance to chemotherapy and targeted cancer drugs, but simultaneously suppresses autophagy. We show that autophagy is essential for tumor cells to cope with therapeutic perturbation of metabolism and that mTORC1-mediated suppression of autophagy is required and sufficient for generating a metabolic vulnerability leading to energy crisis and apoptosis. Our study links mTOR-induced cancer drug resistance to autophagy defects as a cause of a metabolic liability and opens a therapeutic window for the treatment of otherwise therapy-refractory tumor patients.

Engineered p53 mutant mice are valuable tools for delineating p53 functions in tumor suppression and cancer therapy. Here, we have introduced the R178E mutation into the Trp53 gene of mice to specifically ablate the cooperative nature of p53 DNA binding. Trp53(R178E) mice show no detectable target gene regulation and, at first sight, are largely indistinguishable from Trp53(-/-) mice. Surprisingly, stabilization of p53(R178E) in Mdm2(-/-) mice nevertheless triggers extensive apoptosis, indicative of residual wild-type activities. Although this apoptotic activity suffices to trigger lethality of Trp53(R178E);Mdm2(-/-) embryos, it proves insufficient for suppression of spontaneous and oncogene-driven tumorigenesis. Trp53(R178E) mice develop tumors indistinguishably from Trp53(-/-) mice and tumors retain and even stabilize the p53(R178E) protein, further attesting to the lack of significant tumor suppressor activity. However, Trp53(R178E) tumors exhibit remarkably better chemotherapy responses than Trp53(-/-) ones, resulting in enhanced eradication of p53-mutated tumor cells. Together, this provides genetic proof-of-principle evidence that a p53 mutant can be highly tumorigenic and yet retain apoptotic activity which provides a survival benefit in the context of cancer therapy.

Introduction In non-small cell lung cancer (NSCLC) cisplatin is the first line chemotherapy and triggers a strong apoptotic response mainly by inducing DNA interstrand crosslinks. However, the effectiveness of cytotoxic cancer therapy is often limited by the emergence of drug resistant tumour cells. Here, we identify strategies for overcoming resistance to DNA interstrand crosslinkers such as cisplatin. Material and methods We treated the NSCLC cell line H460 continuously with DNA crosslinking agents resulting in resistant subclones that displayed cross-resistance to multiple DNA-damaging cancer drugs, which are commonly used in the clinic. Results and discussions Drug resistance was found to be mediated by mTOR-dependent upregulation of DNA repair via the Fanconi anaemia pathway. Strikingly, despite being multidrug-resistant, resistant cells were highly sensitive to treatment with the metabolic drugs 2-deoxyglucose (2-DG) and dichloroacetate (DCA), whereas parental H460 cells failed to respond. Moreover, xenograft studies in mice revealed, that DCA preferentially reduces growth of resistant tumour cells. Interestingly, mTOR inhibition by RNAi or pharmacological inhibitors not only reversed resistance to crosslinking drugs but in parallel mitigated the apoptotic response to metabolic drugs, thereby linking sensitivity to metabolic drugs with mTOR signalling in chemoresistant cells. Mechanistically, inhibition of autophagy by mTOR was found to be decisive. We observed elevated levels of the mTOR-induced inhibitory S757 phosphorylation of the central autophagy-initiating kinase Ulk1 in chemoresistant tumour cells. Matching these observations, autophagosome formation was detected only in parental, but was abrogated in resistant tumour cells upon DCA treatment. In line, knockout of ATG7 using the CRISPR/Cas9 system or direct inhibition of different steps of autophagy with pharmacological autophagy inhibitors was sufficient to render parental cells sensitive to metabolic drugs. Failure to initiate autophagy resulted in lethal energy stress indicating that autophagy is absolutely required to survive metabolic drug treatment. Conclusion Together these results demonstrate that mTOR-triggered resistance to DNA-damaging cancer drugs generates a therapeutic vulnerability to metabolic drugs due to mTOR-mediated inhibition of autophagy.

Die Inzidenz neuroendokriner Tumore des Pankreas (PNET) stieg in den letzten Jahren. Neben der chirurgischen Resektion und palliativen Therapie wurde noch keine effektive Therapie entwickelt.

NAFLD wird in eine NAFL (Fettleber) und eine NASH (Steatohepatitis) untergliedert. Definierend für die NAFLD ist die Anreicherung von Fett in den Hepatozyten, in Unabhängigkeit vom Alkoholkonsum. Beide Erkrankungen gewinnen weltweit eine immer größere sozioökonomische Bedeutung. Autophagie ist ein Selbstverdauungsmechanismus, der der Zelle hilft, metabolischen Stress zu überleben. Ihre Dysregulation verursacht verschiedene Erkrankungen.

TP63, a member of the p53 gene family gene, encodes the ΔNp63 protein and is one of the most frequently amplified genes in squamous cell carcinomas (SCC) of the head and neck (HNSCC) and lungs (LUSC). Using an epiallelic series of siRNAs with intrinsically different knockdown abilities, we show that the complete loss of ΔNp63 strongly impaired cell proliferation, whereas partial ΔNp63 depletion rendered cells hypersensitive to cisplatin accompanied by an accumulation of DNA damage. Expression profiling revealed wide-spread transcriptional regulation of DNA repair genes and in particular Fanconi anemia (FA) pathway components such as FANCD2 and RAD18 - known to be crucial for the repair of cisplatin-induced interstrand crosslinks. In SCC patients ΔNp63 levels significantly correlate with FANCD2 and RAD18 expression confirming ΔNp63 as a key activator of the FA pathway in vivo Mechanistically, ΔNp63 bound an upstream enhancer of FANCD2 inactive in primary keratinocytes but aberrantly activated by ΔNp63 in SCC. Consistently, depletion of FANCD2 sensitized to cisplatin similar to depletion of ΔNp63. Together, our results demonstrate that ΔNp63 directly activates the FA pathway in SCC and limits the efficacy of cisplatin treatment. Targeting ΔNp63 therefore would not only inhibit SCC proliferation but also sensitize tumors to chemotherapy.

Inactivation of the p53 tumor suppressor by Mdm2 is one of the most frequent events in cancer, so compounds targeting the p53-Mdm2 interaction are promising for cancer therapy. Mechanisms conferring resistance to p53-reactivating compounds are largely unknown. Here we show using CRISPR-Cas9-based target validation in lung and colorectal cancer that the activity of nutlin, which blocks the p53-binding pocket of Mdm2, strictly depends on functional p53. In contrast, sensitivity to the drug RITA, which binds the Mdm2-interacting N terminus of p53, correlates with induction of DNA damage. Cells with primary or acquired RITA resistance display cross-resistance to DNA crosslinking compounds such as cisplatin and show increased DNA cross-link repair. Inhibition of FancD2 by RNA interference or pharmacological mTOR inhibitors restores RITA sensitivity. The therapeutic response to p53-reactivating compounds is therefore limited by compound-specific resistance mechanisms that can be resolved by CRISPR-Cas9-based target validation and should be considered when allocating patients to p53-reactivating treatments.