Inducing and exploiting vulnerabilities for the treatment of liver cancer.

Liver cancer remains difficult to treat, owing to a paucity of drugs that target critical dependencies1,2; broad-spectrum kinase inhibitors such as sorafenib provide only a modest benefit to patients with hepatocellular carcinoma3. The induction of senescence may represent a strategy for the treatment of cancer, especially when combined with a second drug that selectively eliminates senescent cancer cells (senolysis)4,5. Here, using a kinome-focused genetic screen, we show that pharmacological inhibition of the DNA-replication kinase CDC7 induces senescence selectively in liver cancer cells with mutations in TP53. A follow-up chemical screen identified the antidepressant sertraline as an agent that kills hepatocellular carcinoma cells that have been rendered senescent by inhibition of CDC7. Sertraline suppressed mTOR signalling, and selective drugs that target this pathway were highly effective in causing the apoptotic cell death of hepatocellular carcinoma cells treated with a CDC7 inhibitor. The feedback reactivation of mTOR signalling after its inhibition6 is blocked in cells that have been treated with a CDC7 inhibitor, which leads to the sustained inhibition of mTOR and cell death. Using multiple in vivo mouse models of liver cancer, we show that treatment with combined inhibition of of CDC7 and mTOR results in a marked reduction of tumour growth. Our data indicate that exploiting an induced vulnerability could be an effective treatment for liver cancer.

SCAI promotes error-free repair of DNA interstrand crosslinks via the Fanconi anemia pathway.

DNA interstrand crosslinks (ICLs) are cytotoxic lesions that threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI-FANCD2 (ID2) marking the activation step that triggers incisions on DNA to unhook the ICL. Restoration of intact DNA requires the coordinated actions of polymerase ζ (Polζ)-mediated translesion synthesis (TLS) and homologous recombination (HR). While the proteins mediating FA pathway activation have been well characterized, the effectors regulating repair pathway choice to promote error-free ICL resolution remain poorly defined. Here, we uncover an indispensable role of SCAI in ensuring error-free ICL repair upon activation of the FA pathway. We show that SCAI forms a complex with Polζ and localizes to ICLs during DNA replication. SCAI-deficient cells are exquisitely sensitive to ICL-inducing drugs and display major hallmarks of FA gene inactivation. In the absence of SCAI, HR-mediated ICL repair is defective, and breaks are instead re-ligated by polymerase θ-dependent microhomology-mediated end-joining, generating deletions spanning the ICL site and radial chromosomes. Our work establishes SCAI as an integral FA pathway component, acting at the interface between TLS and HR to promote error-free ICL repair.

What are the DNA lesions underlying formaldehyde toxicity?

Formaldehyde is a highly reactive organic compound. Humans can be exposed to exogenous sources of formaldehyde, but formaldehyde is also produced endogenously as a byproduct of cellular metabolism. Because formaldehyde can react with DNA, it is considered a major endogenous source of DNA damage. However, the nature of the lesions underlying formaldehyde toxicity in cells remains vastly unknown. Here, we review the current knowledge of the different types of nucleic acid lesions that are induced by formaldehyde and describe the repair pathways known to counteract formaldehyde toxicity. Taking this knowledge together, we discuss and speculate on the predominant lesions generated by formaldehyde, which underly its natural toxicity.

VCF1 is a p97/VCP cofactor promoting recognition of ubiquitylated p97-UFD1-NPL4 substrates.

The hexameric AAA+ ATPase p97/VCP functions as an essential mediator of ubiquitin-dependent cellular processes, extracting ubiquitylated proteins from macromolecular complexes or membranes by catalyzing their unfolding. p97 is directed to ubiquitylated client proteins via multiple cofactors, most of which interact with the p97 N-domain. Here, we discover that FAM104A, a protein of unknown function also named VCF1 (VCP/p97 nuclear Cofactor Family member 1), acts as a p97 cofactor in human cells. Detailed structure-function studies reveal that VCF1 directly binds p97 via a conserved α-helical motif that recognizes the p97 N-domain with unusually high affinity, exceeding that of other cofactors. We show that VCF1 engages in joint p97 complex formation with the heterodimeric primary p97 cofactor UFD1-NPL4 and promotes p97-UFD1-NPL4-dependent proteasomal degradation of ubiquitylated substrates in cells. Mechanistically, VCF1 indirectly stimulates UFD1-NPL4 interactions with ubiquitin conjugates via its binding to p97 but has no intrinsic affinity for ubiquitin. Collectively, our findings establish VCF1 as an unconventional p97 cofactor that promotes p97-dependent protein turnover by facilitating p97-UFD1-NPL4 recruitment to ubiquitylated targets.

Novel vertebrate- and brain-specific driver of neuronal outgrowth.

During the process of neuronal outgrowth, developing neurons produce new projections, neurites, that are essential for brain wiring. Here, we discover a relatively late-evolved protein that we denote Ac45-related protein (Ac45RP) and that, surprisingly, drives neuronal outgrowth. Ac45RP is a paralog of the Ac45 protein that is a component of the vacuolar proton ATPase (V-ATPase), the main pH regulator in eukaryotic cells. Ac45RP mRNA expression is brain specific and coincides with the peak of neurogenesis and the onset of synaptogenesis. Furthermore, Ac45RP physically interacts with the V-ATPase V0-sector and colocalizes with V0 in unconventional, but not synaptic, secretory vesicles of extending neurites. Excess Ac45RP enhances the expression of V0-subunits, causes a more elaborate Golgi, and increases the number of cytoplasmic vesicular structures, plasma membrane formation and outgrowth of actin-containing neurites devoid of synaptic markers. CRISPR-cas9n-mediated Ac45RP knockdown reduces neurite outgrowth. We conclude that the novel vertebrate- and brain-specific Ac45RP is a V0-interacting constituent of unconventional vesicular structures that drives membrane expansion during neurite outgrowth and as such may furnish a tool for future neuroregenerative treatment strategies.

Targeting CDC7 potentiates ATR-CHK1 signaling inhibition through induction of DNA replication stress in liver cancer.

BACKGROUND: Liver cancer is one of the most commonly diagnosed cancers and the fourth leading cause of cancer-related death worldwide. Broad-spectrum kinase inhibitors like sorafenib and lenvatinib provide only modest survival benefit to patients with hepatocellular carcinoma (HCC). This study aims to identify novel therapeutic strategies for HCC patients. METHODS: Integrated bioinformatics analyses and a non-biased CRISPR loss of function genetic screen were performed to identify potential therapeutic targets for HCC cells. Whole-transcriptome sequencing (RNA-Seq) and time-lapse live imaging were performed to explore the mechanisms of the synergy between CDC7 inhibition and ATR or CHK1 inhibitors in HCC cells. Multiple in vitro and in vivo assays were used to validate the synergistic effects. RESULTS: Through integrated bioinformatics analyses using the Cancer Dependency Map and the TCGA database, we identified ATR-CHK1 signaling as a therapeutic target for liver cancer. Pharmacological inhibition of ATR or CHK1 leads to robust proliferation inhibition in liver cancer cells having a high basal level of replication stress. For liver cancer cells that are resistant to ATR or CHK1 inhibition, treatment with CDC7 inhibitors induces strong DNA replication stress and consequently such drugs show striking synergy with ATR or CHK1 inhibitors. The synergy between ATR-CHK1 inhibition and CDC7 inhibition probably derives from abnormalities in mitosis inducing mitotic catastrophe. CONCLUSIONS: Our data highlights the potential of targeting ATR-CHK1 signaling, either alone or in combination with CDC7 inhibition, for the treatment of liver cancer.

The RECQL helicase prevents replication fork collapse during replication stress.

Most tumors lack the G1/S phase checkpoint and are insensitive to antigrowth signals. Loss of G1/S control can severely perturb DNA replication as revealed by slow replication fork progression and frequent replication fork stalling. Cancer cells may thus rely on specific pathways that mitigate the deleterious consequences of replication stress. To identify vulnerabilities of cells suffering from replication stress, we performed an shRNA-based genetic screen. We report that the RECQL helicase is specifically essential in replication stress conditions and protects stalled replication forks against MRE11-dependent double strand break (DSB) formation. In line with these findings, knockdown of RECQL in different cancer cells increased the level of DNA DSBs. Thus, RECQL plays a critical role in sustaining DNA synthesis under conditions of replication stress and as such may represent a target for cancer therapy.

WAPL-Dependent Repair of Damaged DNA Replication Forks Underlies Oncogene-Induced Loss of Sister Chromatid Cohesion.

Premature loss of sister chromatid cohesion at metaphase is a diagnostic marker for different cohesinopathies. Here, we report that metaphase spreads of many cancer cell lines also show premature loss of sister chromatid cohesion. Cohesion loss occurs independently of mutations in cohesion factors including SA2, a cohesin subunit frequently inactivated in cancer. In untransformed cells, induction of DNA replication stress by activation of oncogenes or inhibition of DNA replication is sufficient to trigger sister chromatid cohesion loss. Importantly, cell growth under conditions of replication stress requires the cohesin remover WAPL. WAPL promotes rapid RAD51-dependent repair and restart of broken replication forks. We propose that active removal of cohesin allows cancer cells to overcome DNA replication stress. This leads to oncogene-induced cohesion loss from newly synthesized sister chromatids that may contribute to genomic instability and likely represents a targetable cancer cell vulnerability.

Celastrol-induced degradation of FANCD2 sensitizes pediatric high-grade gliomas to the DNA-crosslinking agent carboplatin.

BACKGROUND: Pediatric high-grade gliomas (pHGG) are the leading cause of cancer-related death during childhood. Due to their diffuse growth characteristics, chemoresistance and location behind the blood-brain barrier (BBB), the prognosis of pHGG has barely improved in the past decades. As such, there is a dire need for new therapies that circumvent those difficulties. Since aberrant expression of DNA damage-response associated Fanconi anemia proteins play a central role in the onset and therapy resistance of many cancers, we here investigated if FANCD2 depletion could sensitize pHGG to additional DNA damage. METHODS: We determined the capacity of celastrol, a BBB-penetrable compound that degrades FANCD2, to sensitize glioma cells to the archetypical DNA-crosslinking agent carboplatin in vitro in seven patient-derived pHGG models. In addition, we tested this drug combination in vivo in a patient-derived orthotopic pHGG xenograft model. Underlying mechanisms to drug response were investigated using mRNA expression profiling, western blotting, immunofluorescence, FANCD2 knockdown and DNA fiber assays. FINDINGS: FANCD2 is overexpressed in HGGs and depletion of FANCD2 by celastrol synergises with carboplatin to induce cytotoxicity. Combination therapy prolongs survival of pHGG-bearing mice over monotherapy and control groups in vivo (P<0.05). In addition, our results suggest that celastrol treatment stalls ongoing replication forks, causing sensitivity to DNA-crosslinking in FANCD2-dependent glioma cells. INTERPRETATION: Our results show that depletion of FANCD2 acts as a chemo-sensitizing strategy in pHGG. Combination therapy using celastrol and carboplatin might serve as a clinically relevant strategy for the treatment of pHGG. FUNDING: This study was funded by a grant from the Children Cancer-Free Foundation (KIKA, project 210). The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Loss of p53 suppresses replication-stress-induced DNA breakage in G1/S checkpoint deficient cells.

In cancer cells, loss of G1/S control is often accompanied by p53 pathway inactivation, the latter usually rationalized as a necessity for suppressing cell cycle arrest and apoptosis. However, we found an unanticipated effect of p53 loss in mouse and human G1-checkpoint-deficient cells: reduction of DNA damage. We show that abrogation of the G1/S-checkpoint allowed cells to enter S-phase under growth-restricting conditions at the expense of severe replication stress manifesting as decelerated DNA replication, reduced origin firing and accumulation of DNA double-strand breaks. In this system, loss of p53 allowed mitogen-independent proliferation, not by suppressing apoptosis, but rather by restoring origin firing and reducing DNA breakage. Loss of G1/S control also caused DNA damage and activation of p53 in an in vivo retinoblastoma model. Moreover, in a teratoma model, loss of p53 reduced DNA breakage. Thus, loss of p53 may promote growth of incipient cancer cells by reducing replication-stress-induced DNA damage.