The Multifaceted Roles of Lamins in Lung Cancer and DNA Damage Response.

Emerging evidence suggests that lamin functions are not limited to maintaining the structural integrity of the nucleus in eukaryotic cells but that these functions affect many facets of cancer biology. An increasing number of reports suggest that adaptive changes in the lamin subtype composition within the nuclear lamina could affect essential features of cancer development and aggressiveness. These include regulation of cellular stiffness and mobility as well as epithelial-to-mesenchymal transition (EMT), all of which directly impact the metastatic properties of cancer cells. Additionally, insights from studies on the physiological functions of lamins suggest that cancer cells could hijack the ability of lamins to modify chromatin accessibility, cell cycle regulation, and DNA damage response. Here, we present a comprehensive overview of the role of lamins in lung cancer and DNA damage response, which is commonly evoked by lung cancer therapies. Collectively, this information should help better understand the sometimes-conflicting reports on lamin functions in lung cancer as well as in other cancer types.

Nanopore Sequencing Techniques: A Comparison of the MinKNOW and the Alignator Sequencers.

Whole genome and transcriptome analyses represent powerful tools. Despite improvements in sequencing methodology, whole transcriptome analyses are still tedious, especially for methodologies producing long reads. Here, we compare the sequence data analysis software MinKNOW and our tool Alignator. Furthermore, we provide a walk-through from RNA isolation and preparation for MinION sequencing as well as insides in the processing of sequencing data using both tools.

Syntaxin 18 regulates the DNA damage response and epithelial-to-mesenchymal transition to promote radiation resistance of lung cancer.

Radiotherapy is an important modality in lung cancer treatment. Despite advances in treatment planning and dose delivery, patient benefit is still limited by in-field relapse and metastatic recurrence. Simultaneous application of cisplatinum-based chemotherapy leads to moderately improved outcomes, thus providing proof-of-concept for radiosensitization strategies in lung cancer. In an unbiased functional genetic screen for radiosensitization targets in lung cancer, we identified syntaxin 18, a protein involved in retrograde vesicular transport between the Golgi apparatus and endoplasmic reticulum, as mediator of radioresistance. Downregulation of endogenous syntaxin 18 specifically reduced clonogenic survival of radioresistant and radiosensitive lung cancer cells following X-radiation. Gene expression programs regulating DNA repair, mitotic checkpoints and mitosis were altered in isogenic cells with reduced syntaxin 18 expression. Functionally, this translated into impaired DNA damage-induced cell cycle checkpoints leading to cell death by mitotic catastrophe. Interestingly, downregulation of syntaxin 18 in lung cancer cells also impaired expression of markers of epithelial-mesenchymal-transition, and reduced migration and invasion capacity. These findings suggest that syntaxin 18 is a key player regulating genes responsible for controlling the growth of the primary tumor as well as metastases upon radiotherapy of lung cancer. They provide a promising lead for biologically rational radiosensitization strategies impacting on radiation-induced cell death as well as metastasis.

Topical Capsaicin in Poly(lactic-co-glycolic)acid (PLGA) Nanoparticles Decreases Acute Itch and Heat Pain.

BACKGROUND: Capsaicin, the hot pepper agent, produces burning followed by desensitization. To treat localized itch or pain with minimal burning, low capsaicin concentrations can be repeatedly applied. We hypothesized that alternatively controlled release of capsaicin from poly(lactic-co-glycolic acid) (PLGA) nanoparticles desensitizes superficially terminating nociceptors, reducing burning. METHODS: Capsaicin-loaded PLGA nanoparticles were prepared (single-emulsion solvent evaporation) and characterized (size, morphology, capsaicin loading, encapsulation efficiency, in vitro release profile). Capsaicin-PLGA nanoparticles were applied to murine skin and evaluated in healthy human participants (n = 21) for 4 days under blinded conditions, and itch and nociceptive sensations evoked by mechanical, heat stimuli and pruritogens cowhage, ß-alanine, BAM8-22 and histamine were evaluated. RESULTS: Nanoparticles (loading: 58 µg capsaicin/mg) released in vitro 23% capsaicin within the first hour and had complete release at 72 h. In mice, 24 h post-application Capsaicin-PLGA nanoparticles penetrated the dermis and led to decreased nociceptive behavioral responses to heat and mechanical stimulation (desensitization). Application in humans produced a weak to moderate burning, dissipating after 3 h. A loss of heat pain up to 2 weeks was observed. After capsaicin nanoparticles, itch and nociceptive sensations were reduced in response to pruritogens cowhage, ß-alanine or BAM8-22, but were normal to histamine. CONCLUSIONS: Capsaicin nanoparticles could be useful in reducing pain and itch associated with pruritic diseases that are histamine-independent.

NTRK1/TrkA Activation Overrides the G2/M-Checkpoint upon Irradiation.

High expression of the receptor tyrosine kinase TrkA/NTRK1 is associated with a favorable outcome in several solid tumors of childhood including neuroblastoma. During development, TrkA/NTRK1 governs migration and differentiation of neuronal precursor cells, while it is associated with mitotic dysfunction and altered DNA damage response, among others, in neuroblastoma. Here, we used human neuroblastoma cell lines with inducible TrkA/NTRK1 expression to mechanistically explore the role of TrkA/NTRK1 signaling in checkpoint activation after DNA damage induced by ionizing radiation (IR). TrkA/NTRK1 activated cells showed increased short-term cell viability upon IR compared to vector control cells. This was accompanied by a deficient G2/M-checkpoint at both low (1 Gy) and high doses (4 Gy) of IR. In a tightly controlled setting, we confirmed that this effect was strictly dependent on activation of TrkA/NTRK1 by its ligand, nerve growth factor (NGF). TrkA/NTRK1-expressing cells displayed impaired ATM and CHK1 phosphorylation, resulting in stabilization of CDC25B. In line with these findings, ATM or ATR inhibition recapitulated the effects of TrkA/NTRK1 activation on the IR-induced G2/M-checkpoint. In conclusion, we here provide first evidence for a previously unrecognized function of NTRK signaling in checkpoint regulation and the response to IR.

Statins affect cancer cell plasticity with distinct consequences for tumor progression and metastasis.

Statins are among the most commonly prescribed drugs, and around every fourth person above the age of 40 is on statin medication. Therefore, it is of utmost clinical importance to understand the effect of statins on cancer cell plasticity and its consequences to not only patients with cancer but also patients who are on statins. Here, we find that statins induce a partial epithelial-to-mesenchymal transition (EMT) phenotype in cancer cells of solid tumors. Using a comprehensive STRING network analysis of transcriptome, proteome, and phosphoproteome data combined with multiple mechanistic in vitro and functional in vivo analyses, we demonstrate that statins reduce cellular plasticity by enforcing a mesenchymal-like cell state that increases metastatic seeding ability on one side but reduces the formation of (secondary) tumors on the other due to heterogeneous treatment responses. Taken together, we provide a thorough mechanistic overview of the consequences of statin use for each step of cancer development, progression, and metastasis.

NTRK1/TrkA Signaling in Neuroblastoma Cells Induces Nuclear Reorganization and Intra-Nuclear Aggregation of Lamin A/C.

(1) Background: Neuroblastomas (NBs) are the most common extracranial solid tumors of children. The amplification of the Myc-N proto-oncogene (MYCN) is a major driver of NB aggressiveness, while high expression of the neurotrophin receptor NTRK1/TrkA is associated with mild disease courses. The molecular effects of NTRK1 signaling in MYCN-amplified NB, however, are still poorly understood and require elucidation. (2) Methods: Inducible NTRK1 expression was realized in four NB cell lines with (IMR5, NGP) or without MYCN amplification (SKNAS, SH-SY5Y). Proteome and phosphoproteome dynamics upon NTRK1 activation by its ligand, NGF, were analyzed in a time-dependent manner in IMR5 cells. Target validation by immunofluorescence staining and automated image processing was performed using the three other NB cell lines. (3) Results: In total, 230 proteins and 134 single phosphorylated class I phosphosites were found to be significantly regulated upon NTRK1 activation. Among known NTRK1 targets, Stathmin and the neurosecretory protein VGF were recovered. Additionally, we observed the upregulation and phosphorylation of Lamin A/C (LMNA) that accumulated inside nuclear foci. (4) Conclusions: We provide a comprehensive picture of NTRK1-induced proteome and phosphoproteome dynamics. The phosphorylation of LMNA within nucleic aggregates was identified as a prominent feature of NTRK1 signaling independent of the MYCN status of NB cells.

HER2 mediates clinical resistance to the KRASG12C inhibitor sotorasib, which is overcome by co-targeting SHP2.

INTRODUCTION: Mutant RAS guanosine triphosphate hydrolases (GTPases) are key oncogenic drivers in many cancers. The KRASG12C variant has recently become targetable by a new drug class specifically locking KRASG12C in its inactive guanosine diphosphate (GDP)-bound state. Clinical activity was demonstrated in patients with advanced lung cancers harbouring KRASG12C mutations but was limited by the development of resistance. METHODS: A biopsy from progressing lung cancer of a patient treated with the KRASG12C inhibitor sotorasib was obtained, and the underlying resistance factors were analysed. Mechanistic studies were performed in vitro and in vivo to uncover strategies to overcome resistance to KRASG12C inhibition. RESULTS: We demonstrated acquisition of HER2 copy number gain and KRASG12C mutation retention in the post-progression biopsy. To explore HER2 gain as the relevant resistance mechanism, we generated KRASG12C lung cancer models overexpressing HER2. MAPK pathway signalling remained active despite KRASG12C inhibitor treatment. Combined pharmacological inhibition of KRASG12C and SHP2 synergistically overcame HER2-mediated resistance in vitro and in vivo. CONCLUSIONS: These findings establish HER2 copy number gain as a clinically relevant mechanism of resistance to pharmacological KRASG12C inhibition that can be overcome by co-targeting SHP2.

Differential immunomodulatory effect of PARP inhibition in BRCA1 deficient and competent tumor cells.

Poly-ADP-ribose polymerase (PARP) inhibitors are active against cells and tumors with defects in homology-directed repair as a result of synthetic lethality. PARP inhibitors (PARPi) have been suggested to act by either catalytic inhibition or by PARP localization in chromatin. In this study, we treat BRCA1 mutant cells derived from a patient with triple negative breast cancer and control cells for three weeks with veliparib, a PARPi, to determine if treatment with this drug induces increased levels of mutations and/or an inflammatory response. We show that long-term treatment with PARPi induces an inflammatory response in HCC1937 BRCA1 mutant cells. The levels of chromatin-bound PARP1 in the BRCA1 mutant cells correlate with significant upregulation of inflammatory genes and activation of the cyclic GMP-AMP synthase (cGAS)/signaling effector stimulator of interferon genes (STING pathway). In contrast, an increased mutational load is induced in BRCA1-complemented cells treated with a PARPi. Our results suggest that long-term PARP inhibitor treatment may prime both BRCA1 mutant and wild-type tumors for positive responses to immune checkpoint blockade, but by different underlying mechanisms.

Streamlining Quantitative Analysis of Long RNA Sequencing Reads.

Transcriptome analyses allow for linking RNA expression profiles to cellular pathways and phenotypes. Despite improvements in sequencing methodology, whole transcriptome analyses are still tedious, especially for methodologies producing long reads. Currently, available data analysis software often lacks cost- and time-efficient workflows. Although kit-based workflows and benchtop platforms for RNA sequencing provide software options, e.g., cloud-based tools to analyze basecalled reads, quantitative, and easy-to-use solutions for transcriptome analysis, especially for non-human data, are missing. We therefore developed a user-friendly tool, termed Alignator, for rapid analysis of long RNA reads requiring only FASTQ files and an Ensembl cDNA database reference. After successful mapping, Alignator generates quantitative information for each transcript and provides a table in which sequenced and aligned RNA are stored for further comparative analyses.

Hypoxia Induces Resistance to EGFR Inhibitors in Lung Cancer Cells via Upregulation of FGFR1 and the MAPK Pathway.

Development of resistance remains the key obstacle to the clinical efficacy of EGFR tyrosine kinase inhibitors (TKI). Hypoxia is a key microenvironmental stress in solid tumors associated with acquired resistance to conventional therapy. Consistent with our previous studies, we show here that long-term, moderate hypoxia promotes resistance to the EGFR TKI osimertinib (AZD9291) in the non-small cell lung cancer (NSCLC) cell line H1975, which harbors two EGFR mutations including T790M. Hypoxia-induced resistance was associated with development of epithelial-mesenchymal transition (EMT) coordinated by increased expression of ZEB-1, an EMT activator. Hypoxia induced increased fibroblast growth factor receptor 1 (FGFR1) expression in NSCLC cell lines H1975, HCC827, and YLR086, and knockdown of FGFR1 attenuated hypoxia-induced EGFR TKI resistance in each line. Upregulated expression of FGFR1 by hypoxia was mediated through the MAPK pathway and attenuated induction of the proapoptotic factor BIM. Consistent with this, inhibition of FGFR1 function by the selective small-molecule inhibitor BGJ398 enhanced EGFR TKI sensitivity and promoted upregulation of BIM levels. Furthermore, inhibition of MEK activity by trametinib showed similar effects. In tumor xenografts in mice, treatment with either BGJ398 or trametinib enhanced response to AZD9291 and improved survival. These results suggest that hypoxia is a driving force for acquired resistance to EGFR TKIs through increased expression of FGFR1. The combination of EGFR TKI and FGFR1 or MEK inhibitors may offer an attractive therapeutic strategy for NSCLC. SIGNIFICANCE: Hypoxia-induced resistance to EGFR TKI is driven by overexpression of FGFR1 to sustain ERK signaling, where a subsequent combination of EGFR TKI with FGFR1 inhibitors or MEK inhibitors reverses this resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/21/4655/F1.large.jpg.

Oncometabolites suppress DNA repair by disrupting local chromatin signalling.

Deregulation of metabolism and disruption of genome integrity are hallmarks of cancer1. Increased levels of the metabolites 2-hydroxyglutarate, succinate and fumarate occur in human malignancies owing to somatic mutations in the isocitrate dehydrogenase-1 or -2 (IDH1 or IDH2) genes, or germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase genes (SDHA, SDHB, SDHC and SDHD), respectively2-4. Recent work has made an unexpected connection between these metabolites and DNA repair by showing that they suppress the pathway of homology-dependent repair (HDR)5,6 and confer an exquisite sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) that are being tested in clinical trials. However, the mechanism by which these oncometabolites inhibit HDR remains poorly understood. Here we determine the pathway by which these metabolites disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal that is essential for the proper execution of HDR. Consequently, recruitment of TIP60 and ATM, two key proximal HDR factors, is substantially impaired at DNA breaks, with reduced end resection and diminished recruitment of downstream repair factors. These findings provide a mechanistic basis for oncometabolite-induced HDR suppression and may guide effective strategies to exploit these defects for therapeutic gain.

Proton Irradiation Increases the Necessity for Homologous Recombination Repair Along with the Indispensability of Non-Homologous End Joining.

Technical improvements in clinical radiotherapy for maximizing cytotoxicity to the tumor while limiting negative impact on co-irradiated healthy tissues include the increasing use of particle therapy (e.g., proton therapy) worldwide. Yet potential differences in the biology of DNA damage induction and repair between irradiation with X-ray photons and protons remain elusive. We compared the differences in DNA double strand break (DSB) repair and survival of cells compromised in non-homologous end joining (NHEJ), homologous recombination repair (HRR) or both, after irradiation with an equal dose of X-ray photons, entrance plateau (EP) protons, and mid spread-out Bragg peak (SOBP) protons. We used super-resolution microscopy to investigate potential differences in spatial distribution of DNA damage foci upon irradiation. While DNA damage foci were equally distributed throughout the nucleus after X-ray photon irradiation, we observed more clustered DNA damage foci upon proton irradiation. Furthermore, deficiency in essential NHEJ proteins delayed DNA repair kinetics and sensitized cells to both, X-ray photon and proton irradiation, whereas deficiency in HRR proteins sensitized cells only to proton irradiation. We assume that NHEJ is indispensable for processing DNA DSB independent of the irradiation source, whereas the importance of HRR rises with increasing energy of applied irradiation.

Optimizing biodegradable nanoparticle size for tissue-specific delivery.

Nanoparticles (NPs) are promising vehicles for drug delivery because of their potential to target specific tissues [1]. Although it is known that NP size plays a critical role in determining their biological activity, there are few quantitative studies of the role of NP size in determining biodistribution after systemic administration. Here, we engineered fluorescent, biodegradable poly(lactic-co-glycolic acid) (PLGA) NPs in a range of sizes (120-440nm) utilizing a microfluidic platform and used these NPs to determine the effect of diameter on bulk tissue and cellular distribution after systemic administration. We demonstrate that small NPs (∼120nm) exhibit enhanced uptake in bulk lung and bone marrow, while larger NPs are sequestered in the liver and spleen. We also show that small NPs (∼120nm) access specific alveolar cell populations and hematopoietic stem and progenitor cells more readily than larger NPs. Our results suggest that size of PLGA NPs can be used to tune delivery to certain tissues and cell populations in vivo.

Cediranib suppresses homology-directed DNA repair through down-regulation of BRCA1/2 and RAD51.

Combining the anti-angiogenic agent cediranib with the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib improves progression-free survival compared to olaparib alone in ovarian cancer patients through an unknown mechanism. PARP inhibitors are used primarily in the treatment of patients with DNA repair-associated (BRCA1/2) mutated cancers because these mutations cause a deficit in homology-directed DNA repair (HDR) that confers sensitivity to these agents. However, the combination of cediranib and olaparib was effective in patients without BRCA1/2 mutations. We report here that cediranib confers sensitivity to olaparib by down-regulating HDR in tumor cells. This occurs partially as a result of cediranib inducing hypoxia, which suppresses expression of the HDR factors BRCA1/2 and RAD51 recombinase (RAD51). However, we also observed that cediranib has a direct effect on HDR independent of its ability to induce tumor hypoxia. This direct effect occurs through platelet-derived growth factor receptor (PDGFR) inhibition, activation of protein phosphatase 2A (PP2A), and E2F transcription factor 4 (E2F4)/RB transcriptional corepressor like 2 (RB2/p130)-mediated repression of BRCA1/2 and RAD51 gene expression. This down-regulation was seen in mouse tumor xenografts but not in mouse bone marrow, providing a therapeutic window for combining cediranib and olaparib in cancer therapy. Our work reveals a treatment strategy by which DNA repair can be manipulated in human tumors to induce synthetic lethality, broadening the potential therapeutic scope of cediranib based on its activity as a DNA repair inhibitor.

High-throughput Evaluation of Protein Migration and Localization after Laser Micro-Irradiation.

DNA- and histone-related research frequently comprises the quantitative analysis of protein modifications, such as histone phosphorylation. Analysis of accumulation and disappearance of protein foci are used to monitor DNA damage and repair kinetics. If the protein of interest doesn't accumulate in foci, laser micro-irradiation of single nuclei provides an alternative method to monitor DNA repair proteins and histone dynamics at the DNA damage site. We have developed an automated evaluation tool for standardized, high-throughput analysis of micro-irradiated cells featuring single cell background subtraction and detection across multiple fluorescence channels, allowing for robust statistics.

Mitochondrial DNA Stress Signalling Protects the Nuclear Genome.

The mammalian genome comprises nuclear DNA (nDNA) derived from both parents and mitochondrial DNA (mtDNA) that is maternally inherited and encodes essential proteins required for oxidative phosphorylation. Thousands of copies of the circular mtDNA are present in most cell types that are packaged by TFAM into higher-order structures called nucleoids1. Mitochondria are also platforms for antiviral signalling2 and, due to their bacterial origin, mtDNA and other mitochondrial components trigger innate immune responses and inflammatory pathology2,3. We showed previously that instability and cytoplasmic release of mtDNA activates the cGAS-STING-TBK1 pathway resulting in interferon stimulated gene (ISG) expression that promotes antiviral immunity4. Here, we find that persistent mtDNA stress is not associated with basally activated NF-κB signalling or interferon gene expression typical of an acute antiviral response. Instead, a specific subset of ISGs, that includes Parp9, remains activated by the unphosphorylated form of ISGF3 (U-ISGF3) that enhances nDNA damage and repair responses. In cultured primary fibroblasts and cancer cells, the chemotherapeutic drug doxorubicin causes mtDNA damage and release, which leads to cGAS-STING-dependent ISG activation. In addition, mtDNA stress in TFAM-deficient mouse melanoma cells produces tumours that are more resistant to doxorubicin in vivo. Finally, Tfam +/- mice exposed to ionizing radiation exhibit enhanced nDNA repair responses in spleen. Therefore, we propose that damage to and subsequent release of mtDNA elicits a protective signalling response that enhances nDNA repair in cells and tissues, suggesting mtDNA is a genotoxic stress sentinel.

Relating Linear Energy Transfer to the Formation and Resolution of DNA Repair Foci After Irradiation with Equal Doses of X-ray Photons, Plateau, or Bragg-Peak Protons.

Proton beam therapy is increasingly applied for the treatment of human cancer, as it promises to reduce normal tissue damage. However, little is known about the relationship between linear energy transfer (LET), the type of DNA damage, and cellular repair mechanisms, particularly for cells irradiated with protons. We irradiated cultured cells delivering equal doses of X-ray photons, Bragg-peak protons, or plateau protons and used this set-up to quantitate initial DNA damage (mainly DNA double strand breaks (DSBs)), and to analyze kinetics of repair by detecting γH2A.X or 53BP1 using immunofluorescence. The results obtained validate the reliability of our set-up in delivering equal radiation doses under all conditions employed. Although the initial numbers of γH2A.X and 53BP1 foci scored were similar under the different irradiation conditions, it was notable that the maximum foci level was reached at 60 min after irradiation with Bragg-peak protons, as compared to 30 min for plateau protons and photons. Interestingly, Bragg-peak protons induced larger and irregularly shaped γH2A.X and 53BP1 foci. Additionally, the resolution of these foci was delayed. These results suggest that Bragg-peak protons induce DNA damage of increased complexity which is difficult to process by the cellular repair apparatus.

Restraining Akt1 Phosphorylation Attenuates the Repair of Radiation-Induced DNA Double-Strand Breaks and Reduces the Survival of Irradiated Cancer Cells.

The survival kinase protein kinase B (Akt) participates in the regulation of essential subcellular processes, e.g., proliferation, growth, survival, and apoptosis, and has a documented role in promoting resistance against genotoxic stress including radiotherapy, presumably by influencing the DNA damage response and DNA double-strand break (DSB) repair. However, its exact role in DSB repair requires further elucidation. We used a genetic approach to explore the consequences of impaired phosphorylation of Akt1 at one or both of its key phosphorylation sites, Threonine 308 (T308) or Serine 473 (S473), on DSB repair and radiosensitivity to killing. Therefore, we overexpressed either the respective single or the double phosphorylation-deficient mutants (Akt1-T308A, Akt1-S473A, or Akt1-T308A/S473A) in TRAMPC1 murine prostate cancer cells (TrC1) and measured the DSB repair kinetics and clonogenic cell survival upon irradiation. Only the expression of the Akt1-T308A/S473A induced a significant delay in the kinetics of DSB repair in irradiated TrC1 as determined by the γH2A.X (H2A histone family, member X) assay and the neutral comet assay, respectively. Moreover, Akt1-T308A/S473A-expressing cells were characterized by increased radiosensitivity compared to Akt1-WT (wild type)-expressing cells in long-term colony formation assays. Our data reveal that Akt1's activation state is important for the cellular radiation response, presumably by modulating the phosphorylation of effector proteins involved in the regulation of DSB repair.

Krebs-cycle-deficient hereditary cancer syndromes are defined by defects in homologous-recombination DNA repair.

The hereditary cancer syndromes hereditary leiomyomatosis and renal cell cancer (HLRCC) and succinate dehydrogenase-related hereditary paraganglioma and pheochromocytoma (SDH PGL/PCC) are linked to germline loss-of-function mutations in genes encoding the Krebs cycle enzymes fumarate hydratase and succinate dehydrogenase, thus leading to elevated levels of fumarate and succinate, respectively1-3. Here, we report that fumarate and succinate both suppress the homologous recombination (HR) DNA-repair pathway required for the resolution of DNA double-strand breaks (DSBs) and for the maintenance of genomic integrity, thus rendering tumor cells vulnerable to synthetic-lethal targeting with poly(ADP)-ribose polymerase (PARP) inhibitors. These results identify HLRCC and SDH PGL/PCC as familial DNA-repair deficiency syndromes, providing a mechanistic basis to explain their cancer predisposition and suggesting a potentially therapeutic approach for advanced HLRCC and SDH PGL/PCC, both of which are incurable when metastatic.