Taraswi Banerjee

Targeting an Achilles’ heel of cancer with a WRN helicase inhibitor

Our recently published work suggests that DNA helicases such as the Werner syndrome helicase (WRN) represent a novel class of proteins to target for anticancer therapy. Specifically, pharmacological inhibition of WRN helicase activity in human cells defective in the Fanconi anemia (FA) pathway of interstrand cross-link (ICL) repair are sensitized to the DNA cross-linking agent and chemotherapy drug mitomycin C (MMC) by the WRN helicase inhibitor NSC 617145.1 The mechanistic basis for the synergistic interaction between NSC 617145 and MMC is discussed in this paper and extrapolated to potential implications for genetic or chemically induced synthetic lethality provoked by cellular exposure to the WRN helicase inhibitor under the context of relevant DNA repair deficiencies associated with cancers or induced by small-molecule inhibitors. Experimental data are presented showing that small-molecule inhibition of WRN helicase elevates sensitivity to MMC-induced stress in human cells that are deficient in both FANCD2 and DNA protein kinase catalytic subunit (DNA-PKcs). These findings suggest a model in which drug-mediated inhibition of WRN helicase activity exacerbates the deleterious effects of MMC-induced DNA damage when both the FA and NHEJ pathways are defective. We conclude with a perspective for the FA pathway and synthetic lethality and implications for DNA repair helicase inhibitors that can be developed for anticancer strategies.

Werner Syndrome Helicase Has a Critical Role in DNA Damage Responses in the Absence of a Functional Fanconi Anemia Pathway

Werner syndrome is genetically linked to mutations in WRN that encodes a DNA helicase-nuclease believed to operate at stalled replication forks. Using a newly identified small-molecule inhibitor of WRN helicase (NSC 617145), we investigated the role of WRN in the interstrand cross-link (ICL) response in cells derived from patients with Fanconi anemia, a hereditary disorder characterized by bone marrow failure and cancer. In FA-D2(-/-) cells, NSC 617145 acted synergistically with very low concentrations of mitomycin C to inhibit proliferation in a WRN-dependent manner and induce double-strand breaks (DSB) and chromosomal abnormalities. Under these conditions, ataxia-telangiectasia mutated activation and accumulation of DNA-dependent protein kinase, catalytic subunit pS2056 foci suggested an increased number of DSBs processed by nonhomologous end-joining (NHEJ). Rad51 foci were also elevated in FA-D2(-/-) cells exposed to NSC 617145 and mitomycin C, suggesting that WRN helicase inhibition interferes with later steps of homologous recombination at ICL-induced DSBs. Thus, when the Fanconi anemia pathway is defective, WRN helicase inhibition perturbs the normal ICL response, leading to NHEJ activation. Potential implication for treatment of Fanconi anemia-deficient tumors by their sensitization to DNA cross-linking agents is discussed.

Molecular functions and cellular roles of the ChlR1 (DDX11) helicase defective in the rare cohesinopathy Warsaw breakage syndrome

In 2010, a new recessive cohesinopathy disorder, designated Warsaw breakage syndrome (WABS), was described. The individual with WABS displayed microcephaly, pre- and postnatal growth retardation, and abnormal skin pigmentation. Cytogenetic analysis revealed mitomycin C (MMC)-induced chromosomal breakage; however, an additional sister chromatid cohesion defect was also observed. WABS is genetically linked to bi-allelic mutations in the ChlR1/DDX11 gene which encodes a protein of the conserved family of Iron–Sulfur (Fe–S) cluster DNA helicases. Mutations in the budding yeast ortholog of ChlR1, known as Chl1, were known to cause sister chromatid cohesion defects, indicating a conserved function of the gene. In 2012, three affected siblings were identified with similar symptoms to the original WABS case, and found to have a homozygous mutation in the conserved Fe–S domain of ChlR1, confirming the genetic linkage. Significantly, the clinically relevant mutations perturbed ChlR1 DNA unwinding activity. In addition to its genetic importance in human disease, ChlR1 is implicated in papillomavirus genome maintenance and cancer. Although its precise functions in genome homeostasis are still not well understood, ongoing molecular studies of ChlR1 suggest the helicase plays a critically important role in cellular replication and/or DNA repair.

Novel Function of the Fanconi Anemia Group J or RECQ1 Helicase to Disrupt Protein-DNA Complexes in a Replication Protein A-stimulated Manner

Background: DNA unwinding by helicases is blocked by proteins bound to duplex DNA. Results: The single-stranded DNA-binding protein RPA stimulates FANCJ or RECQ1 helicase to disrupt protein-DNA complexes. Conclusion: Helicases partner with RPA to dislodge proteins bound to duplex DNA. Significance: Regulation of helicase-catalyzed protein displacement is highly relevant in cellular nucleic acid metabolic processes that require remodeling of chromatinized genomic DNA. Understanding how cellular machinery deals with chromosomal genome complexity is an important question because protein bound to DNA may affect various cellular processes of nucleic acid metabolism. DNA helicases are at the forefront of such processes, yet there is only limited knowledge how they remodel protein-DNA complexes and how these mechanisms are regulated. We have determined that representative human RecQ and Fe-S cluster DNA helicases are potently blocked by a protein-DNA interaction. The Fanconi anemia group J (FANCJ) helicase partners with the single-stranded DNA-binding protein replication protein A (RPA) to displace BamHI-E111A bound to duplex DNA in a specific manner. Protein displacement was dependent on the ATPase-driven function of the helicase and unique properties of RPA. Further biochemical studies demonstrated that the shelterin proteins TRF1 and TRF2, which preferentially bind the telomeric repeat found at chromosome ends, effectively block FANCJ from unwinding the forked duplex telomeric substrate. RPA, but not the Escherichia coli single-stranded DNA-binding protein or shelterin factor Pot1, stimulated FANCJ ejection of TRF1 from the telomeric DNA substrate. FANCJ was also able to displace TRF2 from the telomeric substrate in an RPA-dependent manner. The stimulation of helicase-catalyzed protein displacement is also observed with the DNA helicase RECQ1, suggesting a conserved functional interaction of RPA-interacting helicases. These findings suggest that partnerships between RPA and interacting human DNA helicases may greatly enhance their ability to dislodge proteins bound to duplex DNA, an activity that is likely to be highly relevant to their biological roles in DNA metabolism.

Impact of age-associated cyclopurine lesions on DNA repair helicases.

8,5′ cyclopurine deoxynucleosides (cPu) are locally distorting DNA base lesions corrected by nucleotide excision repair (NER) and proposed to play a role in neurodegeneration prevalent in genetically defined Xeroderma pigmentosum (XP) patients. In the current study, purified recombinant helicases from different classifications based on sequence homology were examined for their ability to unwind partial duplex DNA substrates harboring a single site-specific cPu adduct. Superfamily (SF) 2 RecQ helicases (RECQ1, BLM, WRN, RecQ) were inhibited by cPu in the helicase translocating strand, whereas helicases from SF1 (UvrD) and SF4 (DnaB) tolerated cPu in either strand. SF2 Fe-S helicases (FANCJ, DDX11 (ChlR1), DinG, XPD) displayed marked differences in their ability to unwind the cPu DNA substrates. Archaeal Thermoplasma acidophilum XPD (taXPD), homologue to the human XPD helicase involved in NER DNA damage verification, was impeded by cPu in the non-translocating strand, while FANCJ was uniquely inhibited by the cPu in the translocating strand. Sequestration experiments demonstrated that FANCJ became trapped by the translocating strand cPu whereas RECQ1 was not, suggesting the two SF2 helicases interact with the cPu lesion by distinct mechanisms despite strand-specific inhibition for both. Using a protein trap to simulate single-turnover conditions, the rate of FANCJ or RECQ1 helicase activity was reduced 10-fold and 4.5-fold, respectively, by cPu in the translocating strand. In contrast, single-turnover rates of DNA unwinding by DDX11 and UvrD helicases were only modestly affected by the cPu lesion in the translocating strand. The marked difference in effect of the translocating strand cPu on rate of DNA unwinding between DDX11 and FANCJ helicase suggests the two Fe-S cluster helicases unwind damaged DNA by distinct mechanisms. The apparent complexity of helicase encounters with an unusual form of oxidative damage is likely to have important consequences in the cellular response to DNA damage and DNA repair.

Getting Ready for the Dance: FANCJ Irons Out DNA Wrinkles

Mounting evidence indicates that alternate DNA structures, which deviate from normal double helical DNA, form in vivo and influence cellular processes such as replication and transcription. However, our understanding of how the cellular machinery deals with unusual DNA structures such as G-quadruplexes (G4), triplexes, or hairpins is only beginning to emerge. New advances in the field implicate a direct role of the Fanconi Anemia Group J (FANCJ) helicase, which is linked to a hereditary chromosomal instability disorder and important for cancer suppression, in replication past unusual DNA obstacles. This work sets the stage for significant progress in dissecting the molecular mechanisms whereby replication perturbation by abnormal DNA structures leads to genomic instability. In this review, we focus on FANCJ and its role to enable efficient DNA replication when the fork encounters vastly abundant naturally occurring DNA obstacles, which may have implications for targeting rapidly dividing cancer cells.

Biochemical and cell biological assays to identify and characterize DNA helicase inhibitors.

The growing number of DNA helicases implicated in hereditary disorders and cancer indicates that this particular class of enzymes plays key roles in genomic stability and cellular homeostasis. Indeed, a large body of work has provided molecular and cellular evidence that helicases act upon a variety of nucleic acid substrates and interact with numerous proteins to enact their functions in replication, DNA repair, recombination, and transcription. Understanding how helicases operate in unique and overlapping pathways is a great challenge to researchers. In this review, we describe a series of experimental approaches and methodologies to identify and characterize DNA helicase inhibitors which collectively provide an alternative and useful strategy to explore their biological significance in cell-based systems. These procedures were used in the discovery of biologically active compounds that inhibited the DNA unwinding function catalyzed by the human WRN helicase-nuclease defective in the premature aging disorder Werner syndrome. We describe in vitro and in vivo experimental approaches to characterize helicase inhibitors with WRN as the model, anticipating that these approaches may be extrapolated to other DNA helicases, particularly those implicated in DNA repair and/or the replication stress response.

RECQL: a new breast cancer susceptibility gene

Identifying and characterizing novel genetic risk factors for BRCA1/2 negative breast cancers is highly relevant for early diagnosis and development of a management plan. Mutations in a number of DNA repair genes have been associated with genomic instability and development of breast and various other cancers. Whole exome sequencing efforts by 2 groups have led to the discovery in distinct populations of multiple breast cancer susceptibility mutations in RECQL, a gene that encodes a DNA helicase involved in homologous recombination repair and response to replication stress. RECQL pathogenic mutations were identified that truncated or disrupted the RECQL protein or introduced missense mutations in its helicase domain. RECQL mutations may serve as a useful biomarker for breast cancer. Targeting RECQL associated tumors with novel DNA repair inhibitors may provide a new strategy for anti-cancer therapy.

A new development in DNA repair modulation: discovery of a BLM helicase inhibitor.

Bloom’s syndrome (BS) is a rare autosomal recessive genetic disorder characterized by predisposition to a wide variety of cancers observed in the normal population.1 The BLM gene defective in BS encodes a RecQ DNA helicase (BLM) that is important for genomic stability by suppressing sister chromatid exchanges (SCE) that arise during homologous recombination (HR).2 In fact, SCE frequency of patient cells is used for clinical diagnosis of BS. BLM helicase is believed to suppress SCEs by channeling DNA molecules away from pathways leading to crossover products through its DNA unwinding function and interaction with protein partners (e.g., human Topoisomerase IIIα).3 Targeting DNA helicases for therapeutic purposes has attracted interest with the discovery of other DNA repair inhibitors, highlighted by poly(ADP)ribosylase (PARP) inhibitors used in synthetic lethal approaches to attenuate carcinogenesis in HR-defective BRCA1/2-deficient tumors.4 Small molecules (< 800 Daltons) can penetrate cell membranes and represent a potentially suitable class of compounds for therapeutic use, such as anti-cancer drugs. In the January 24, 2013 issue of Chemistry and Biology, Nguyen et al. reported their discovery of a small molecule inhibitor of BLM helicase.5 From a high throughput screen of a chemical compound library and medicinal chemistry optimization, a small molecule (ML216) was identified that inhibited BLM helicase activity on a forked duplex DNA substrate in vitro (IC50 ~3 μM) by preventing BLM binding to DNA.5 Cultured human fibroblasts exposed to ML216 (50 μM) displayed reduced proliferation, a statistically significant increase in SCE frequency, and elevated sensitivity to aphidicolin, an inhibitor of replicative DNA polymerases. The specificity for ML216 targeting BLM in cell-based experiments was suggested because BLM-deficient cells were resistant to the phenotypic effects of ML216. The BLM helicase inhibitor discovery may provide a new strategy for understanding molecular functions of BLM required for its role in chromosomal stability, and also potential development of a new class of chemotherapy drugs to treat tumors which rely heavily on BLM for proliferation. From a biochemist’s perspective, it is intriguing that ML216 potently inhibited BLM unwinding of a forked DNA duplex substrate, but only modestly affected unwinding of other DNA substrates (G-quadruplex, Holliday Junction, or plasmid-based D-loop) at much higher concentrations of drug.5 The specificity of ML216 (and conceivably other helicase inhibitors) may allow an experimental approach to dissect molecular requirements of the helicase for its role(s) in genome stability. Although ML216 inhibited unwinding by the sequence-related BLM and WRN helicases similarly in vitro, the apparent dependence on BLM for ML216 to exert its biological effects in human cells suggests BLM specificity for the drug’s mechanism of action in vivo. A co-crystal structure of BLM in complex with inhibitor would be informative. Cellular cues in vivo may induce a specific conformation of WRN that makes it resistant to ML216. Direct or water-mediated contacts of the small molecule with poorly conserved amino acid residues of BLM that are distal in the primary structure but proximal in the tertiary structure may be critical for drug action. Other studies reporting pharmacological inhibition of DNA repair protein function have also shown a dependence on target protein for the small molecule’s cellular effect. An inhibitor of WRN helicase (NSC 19630) was discovered that inhibited proliferation and induced DNA damage and apoptosis in human cancer cells in a WRN-dependent manner.6 Although the mechanism of action whereby NSC 19630 interferes with critical function(s) of WRN at the cellular level is unknown, there are several avenues to investigate. The WRN-inhibitor drug complex may prevent WRN from interacting favorably with its protein partners or cause formation of a static protein-DNA complex that is deleterious to normal biological DNA transactions. Since NSC 19630 exerted only a marginal effect on DNA-dependent WRN ATPase or exonuclease activity in vitro at very high drug concentrations,6 WRN inhibitor is likely to operate by a mechanism distinct from that of the BLM inhibitor which adversely affected BLM DNA binding and DNA-dependent ATPase activity at relatively low drug concentrations.5 Our current hypothesis is that the biological effects of NSC 19630 may at least partly reflect an inactive WRN helicase-drug complex trapped on DNA repair or replication intermediates. Further studies will be necessary to determine if this is the case. However, a recent study of clinical PARP inhibitors that operate in a PARP-dependent manner hinted at a provocative scenario. Small molecule inhibition of PARP1 or PARP2 became more cytotoxic than genetic depletion of PARP by causing PARP to become trapped on DNA at damaged sites.7 This finding suggests a reasonable mechanism for a class of DNA helicase inhibitors (like NSC 19630), but more research is necessary. Understanding the mechanisms of DNA repair inhibitors has potential clinical significance. Chemo- and radio-therapy approaches to combat cancer are largely based on introducing DNA damage leading to double strand breaks (DSB). Recently, a small molecule inhibitor (SCR7) of DNA Ligase IV responsible for nonhomologous end-joining (NHEJ) was discovered and found to inhibit NHEJ in a Ligase IV-dependent manner,8 reminiscent of the helicase and PARP inhibitors discussed above. Importantly, SCR7 impeded tumor progression in mouse models.8 Hopefully, further research and clinical applications for helicase inhibitors will prove to be promising.

p53 gain-of-function mutations increase Cdc7-dependent replication initiation

Cancer‐associated p53 missense mutants confer gain of function (GOF) and promote tumorigenesis by regulating crucial signaling pathways. However, the role of GOF mutant p53 in regulating DNA replication, a commonly altered pathway in cancer, is less explored. Here, we show that enhanced Cdc7‐dependent replication initiation enables mutant p53 to confer oncogenic phenotypes. We demonstrate that mutant p53 cooperates with the oncogenic transcription factor Myb in vivo and transactivates Cdc7 in cancer cells. Moreover, mutant p53 cells exhibit enhanced levels of Dbf4, promoting the activity of Cdc7/Dbf4 complex. Chromatin enrichment of replication initiation factors and subsequent increase in origin firing confirm increased Cdc7‐dependent replication initiation in mutant p53 cells. Further, knockdown of CDC7 significantly abrogates mutant p53‐driven cancer phenotypes in vitro and in vivo. Importantly, high CDC7 expression significantly correlates with p53 mutational status and predicts poor clinical outcome in lung adenocarcinoma patients. Collectively, this study highlights a novel functional interaction between mutant p53 and the DNA replication pathway in cancer cells. We propose that increased Cdc7‐dependent replication initiation is a hallmark of p53 gain‐of‐function mutations.