G-quadruplex DNA is a barrier to replication, but how the replisome couples G4 bypass with fork progression is not well-defined. Here, we establish that REV1 is central to coordination of G4 resolution, replication fidelity, and tolerance of G4 stabilization. REV1 loss switched fork elongation to a PrimPol-driven mechanism and resulted in defective ssDNA gap suppression in cells treated with pyridostatin (PDS). Mutagenic G4 replication on the leading strand was more impacted by REV1 loss than lagging strand bypass, but only lagging strand mutagenesis was sensitive to PDS. REV1 deficiency increased nuclear G4 signal, amplified ATM/ATR signaling, and sensitized cells to G4-stabilizing agents. We discovered that the REV1 C-terminal domain interacts with the G4 helicase DHX36 to restrain PrimPol activity. The REV1-DHX36 interaction is direct and requires a newly defined REV1-interacting region at the C-terminus of DHX36. Prolonged G4 stabilization uncoupled REV1 and DHX36, with the G4 helicase accumulating at a site distal from REV1 and the DNA synthesis machinery. Our findings establish a two-tiered mechanism for REV1 action that coordinates helicase-dependent G4 unwinding with suppression of ssDNA gaps in response to G4 stabilization.

Monensin ( MON ) is a polyether ionophore antibiotic of natural origin and is an FDA-approved drug for veterinary use. Recent studies have highlighted its potential anti-cancer activity in various in vitro and in vivo models. In this study, we evaluated the anti-breast cancer activity of MON and 37 synthetic analog compounds using cell monolayer and organoid models. Through a mini-ring cell viability assay, several compounds were identified that were more potent and selective against breast cancer cells compared to non-cancerous cells, surpassing the activity of parent MON . MON and these compounds induced significant DNA fragmentation, reduced cell migration, and downregulated SOX2 expression. Furthermore, MON and the most potent analog, compound 12 , reduced the percentage of CD44 + /CD24 -/low stem-like cells and diminished cell self-renewal properties. Proteomics analyses revealed that several pathways, including extracellular matrix organization, were significantly dysregulated by MON and compound 12 in breast cancer cells. Among these, TIMP2, a protein associated with the suppression of tumor growth and metastasis, was identified as one of the most prominently upregulated proteins by MON and compound 12 in MDA-MB-231 cells. This finding was also validated in other breast cancer and melanoma cell lines. To simulate breast cancer metastasis to the brain, a human Hybrid Organoid System: Tumor in Brain Organoid (HOSTBO) model was developed. MON and compound 12 significantly reduced Ki-67 expression within the HOSTBOs, and compound 12 significantly downregulated SOX2 expression. Collectively, MON and compound 12 significantly reduced the proliferation of breast cancer stem-like cells in the organoid models, inhibited their migration, and dysregulated markers associated with stemness, demonstrating their potential as anti-metastatic agents and warranting further clinical development. Abstract Figure:

Replication fork stalling in response to replication stress triggers the activation of replication stressresponse (RSR) pathways to facilitate repair and restart replication processes, including fork reversal andtranslesion DNA synthesis (TLS). During fork reversal, the replication fork undergoes a structural rearrangementto form a four-stranded structure, known as a “chicken foot, ” which helps regulate fork speed by recruitment ofdifferent fork reversal factors, including HLTF, and SMARCAL1. Optimal replication conditions ensure a relativelyconstant and rapid fork progression rate, which is essential for timely genome duplication. TLS requiresspecialized DNA polymerases that assist fork progression by promoting direct bypass of replication blocks. Inglioblastoma, the most aggressive and resistant form of brain cancer, the TLS polymerase kappa (Pol κ) isoverexpressed. This overexpression causes resistance to standard therapeutics and is associated with poorprognosis. A major goal of our study is to understand how Pol κ protects glioma cells from endogenous oncogenicstressors and the DNA-damaging effects of chemotherapeutics. Results from Eoff lab indicate that Pol κ slowsreplication fork speed in glioma cells (T98G, U118-MG) without significantly affecting non-glioma cells (HAP-1, U2OS, RPE). We hypothesized that Pol κ slows replication fork speed by promoting fork reversal in glioma cells.To test this hypothesis, we used a proximity ligation assay (PLA) to verify the colocalization of fork reversalfactors SMARACL1, and HLTF to sites of DNA synthesis in glioma cells. PLA measures the proximity betweenproteins and newly synthesized DNA labeled with nucleotide analog 5-ethynyl-2′-deoxyuridine (EdU), indicatingtheir potential interaction or colocalization, as evidenced by the appearance of fluorescent foci. EdU-SMARCAL1, or EdU-HLTF PLA foci were increased in T98G WT without a significant increase in POLK KO T98G cellsfollowing treatment with 50 nM CPT with no considerable increase in POLK KO T98G cells. These resultssuggest that Pol κ has a role in promoting the colocalization of fork reversal proteins to sites of DNA synthesisin T98G (glioma) cells. To support this conclusion and to study how this affects fork speed, we used DNA fiberspreading (DFS) to determine fork speed with/without depletion of the fork reversal factor SMARCAL1 and HLTF.Depletion of SMARCAL1 and HLTF produced longer IdU tracts in T98G WT cells, indicating faster fork speed.The fork acceleration phenotype in POLK-KO T98G cells remained unaffected by the knock-down ofSMARCAL1, suggesting that these proteins work in the same pathway to regulate fork dynamics in glioblastomacells. These results are the first to establish a link between Pol κ and fork reversal, helping to explain how glioma-specific mechanisms tolerate high rates of DNA damage in the tumor microenvironment. Reham S. Sewilam, Megan R. Reed, Robert L. Eoff. DNA polymerase kappa slows replication fork speed by promoting fork reversal in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1488.

Monensin (MON) is a polyether ionophore antibiotic of natural origin and is an FDA-approved drug for veterinary use. Recent studies have highlighted its potential anticancer activity in various in vitro and in vivo models. In this study, we evaluated the anti-breast cancer activity of MON and 37 synthetic analog compounds using cell monolayer and organoid models. Through a mini-ring cell viability assay, several compounds were identified that were more potent and selective against breast cancer cells compared to non-cancerous cells, surpassing the activity of parent MON. MON and these compounds induced significant DNA fragmentation, reduced cell migration, and downregulated SOX2 expression. Furthermore, MON and the most potent analog, compound 12, reduced the percentage of CD44+/CD24-/low stem-like cells and diminished colony formation properties. Proteomics analyses revealed that several pathways, including extracellular matrix organization, were significantly dysregulated by MON and compound 12 in breast cancer cells. Among these, TIMP2, a protein associated with the suppression of tumor growth and metastasis, was identified as one of the most prominently upregulated proteins by MON and compound 12 in MDA-MB-231 cells. This finding was also validated in other breast cancer and melanoma cell lines. To simulate breast cancer metastasis to the brain, a human hybrid organoid system: tumor in brain organoid (HOSTBO) model was developed. MON and compound 12 significantly reduced Ki-67 expression within the HOSTBOs, and compound 12 significantly downregulated SOX2 expression. Collectively, MON and compound 12 significantly reduced the proliferation of breast cancer stem-like cells in the organoid models, inhibited their migration, and dysregulated markers associated with stemness, demonstrating their potential as anti-metastatic agents and warranting further clinical development.

In response to replication stress, replication fork stalls, and cells activate replication stress response (RSR) pathways to repair and restart replications. Among these RSR pathways, translesion synthesis is performed by translesion synthesis polymerases. The human Y-family translesion polymerase kappa (pol k) is overexpressed in glioblastoma multiforme (GBM), the most aggressive, invasive, and heterogenous form of brain cancer. GBM tumors typically have a high degree of resistance to standard therapeutics, which contributes to poor prognosis. In this study, we explore the mechanism by which pol k acts as a barrier to protect tumor cells from DNA damaging effects of chemotherapeutics. Results from our laboratory demonstrated a role for pol k in controlling fork speed and suppression of genomic single-stranded DNA (ssDNA) gap formation in GBM cells. By performing DNA fiber experiments (± S1 nuclease that cleaves single stranded DNA allowing determination of ssDNA gaps) in multiple cell lines, it was determined that pol k slows fork elongation rate and prevents ssDNA gaps accumulation in GBM-derived cells (e.g., T98G, U118-MG, and primary cultures from GBM specimens) with no considerable effect on several non-GBM cell lines, including HAP-1 cells, a commercially available near-haploid cell line derived from chronic myeloid leukemia patient. Results with cells co-depleted of pol k and another y family translesion polymerase Rev1 support the idea that these TLS enzymes both function to promote replication gap suppression in GBM cells, with pol k exerting a slightly more pronounced effect on fork speed and gap formation than Rev1. Conversely, loss of pol k is dispensable for replication gap suppression in HAP-1 cells unless Rev1 is also depleted. To begin to understand the source of ssDNA formation in pol k-deficient GBM cells, I used RNA interference to deplete PrimPol expression and then performed DNA fiber analysis. My results are supportive of the idea that ssDNA gap accumulation in pol k-depleted GBM cells was dependent upon repriming mediated by PrimPol, with no discernable impact of PrimPol depletion on gaps formation in either WT or POLK-KO HAP-1 cells. To explore the possibility that pol k could also be preventing ssDNA formation through its fork protection activity, I performed a modified DNA fiber assay that includes treatment with high dose hydroxyurea (HU) after labeling with CldU and IdU. Depletion of pol k led to degradation of nascent strand DNA in both GBM and non GBM cells. Therefore, it seems unlikely that this function of pol k is what aids in control of replication fork elongation and prevention of ssDNA gap formation in GBM cells. In conclusion, pol k has a heightened impact on controlling fork speed and ssDNA gap formation in GBM cells, a role that is not apparent in HAP-1 cells unless cells are also depleted of Rev1. Importantly, pol k suppresses ssDNA gaps in GBM cells by opposing repriming mediated by PrimPol, but the exact mechanistic basis of pol k-mediated fork slowing remains unknown. In contrast to the GBM-specific role in replication gap suppression, pol k was found to have a general role in replication fork protection in cell lines originating from different tumor types. These findings have important implications for understanding how tumor-specific mechanisms promote tolerance of DNA damage and survival of genotoxic therapies. This work was supported by a grant from the National Science Foundation (MCB 1903357 to R.L.E) along with a Barton Bridging Award from the UAMS College of Medicine to R.L.E.

In some organisms, the replication of G-quadruplex (G4) structures is supported by the Rev1 DNA polymerase. We previously showed that residues in the insert-2 motif of human Rev1 (hRev1) increased the affinity of the enzyme for G4 DNA and mediated suppression of mutagenic replication near G4 motifs. We have now investigated the conservation of G4-selective properties in Rev1 from other species. We compared Rev1 from Danio rerio (zRev1), Saccharomyces cerevisiae (yRev1), and Leishmania donovani (lRev1) with hRev1, including an insert-2 mutant form of hRev1 (E466A/Y470A or EY). We found that zRev1 retained all of the G4-selective prowess of the human enzyme, but there was a marked attenuation of G4 binding affinity for the EY hRev1 mutant and the two Rev1 proteins lacking insert-2 (yRev1 and lRev1). Perhaps most strikingly, we found that insert-2 was important for disruption of the G4 structure and optimal stimulation of processive DNA synthesis across the guanine-rich motif by DNA polymerase kappa (pol κ). Our findings have implications for how Rev1 might contribute to G4 replication in different species spanning the evolutionary tree – signaling the importance of selection for enzymes with robust G4-selective properties in organisms where these non-B DNA structures may fulfill taxa-specific physiological functions.

Glioblastoma (GBM) remains the most frequently diagnosed primary malignant brain cancer in adults. Despite recent progress in understanding the biology of GBM, the clinical outcome for patients remains poor, with a median survival of approximately one year after diagnosis. One factor contributing to failure in clinical trials is the fact that traditional models used in GBM drug discovery poorly recapitulate patient tumors. Previous studies have shown that monensin (MON) analogs, namely esters and amides on C-26 were potent towards various types of cancer cell lines. In the present study we have investigated the activity of these molecules in GBM organoids, as well as in a host:tumor organoid model. Using a mini-ring cell viability assay we have identified seven analogs (IC50 = 91.5 ± 54.4-291.7 ± 68.8 nM) more potent than parent MON (IC50 = 612.6 ± 184.4 nM). Five of these compounds induced substantial DNA fragmentation in GBM organoids, suggestive of apoptotic cell death. The most active analog, compound 1, significantly reduced GBM cell migration, induced PARP degradation, diminished phosphorylation of STAT3, Akt and GSK3β, increased ɣH2AX signaling and upregulated expression of the autophagy associated marker LC3-II. To investigate the activity of MON and compound 1 in a tumor microenvironment, we developed human cerebral organoids (COs) from human induced pluripotent stem cells (iPSCs). The COs showed features of early developing brain such as multiple neural rosettes with a proliferative zone of neural stem cells (Nestin+), neurons (TUJ1 +), primitive ventricular system (SOX2 +/Ki67 +), intermediate zone (TBR2 +) and cortical plate (MAP2 +). In order to generate host:tumor organoids, we co-cultured RFP-labeled U87MG cells with fully formed COs. Compound 1 and MON reduced U87MG tumor size in the COs after four days of treatment and induced a significant reduction of PARP expression. These findings highlight the therapeutic potential of MON analogs towards GBM and support the application of organoid models in anti-cancer drug discovery.

OBJECTIVES/GOALS: A functional precision medicine platform to identify therapeutic targets for a glioblastoma patient with Li Fraumeni syndrome was performed. Comparative transcriptomics identified druggable targets and patient derived organoids and a 3D-PREDICT drug screening assay was used to validate the pipeline and identify further therapeutic targets. METHODS/STUDY POPULATION: A comparative transcriptomics pipeline was used to identify druggable genes that are uniquely overexpressed in our patient of interest relative to a cancer compendium of 12,747 tumor RNA sequencing datasets including 200 GBMs. Mini-ring patient derived organoid-based drug viability assays were performed to validate the comparative transcriptomics data. Additionally, a spheroid-based drug screening assay (3D-PREDICT) was performed and used to identify further therapeutic targets. RESULTS/ANTICIPATED RESULTS: Using comparative transcriptomics STAT1 and STAT2 were found to be significantly overexpressed in our patient, indicating ruxolitinib, a Janus kinase 1 and 2 inhibitor, as a potential therapy. Druggable pathways predicted using comparative transcriptomics corresponded with ruxolitinib sensitivity in a panel of patient derived organoids screened with this compound. Cells from the LFS patient were among the most sensitive to ruxolitinib compared to patient-derived cells with lower STAT1 and STAT2 expression levels. Additionally, 3D-PREDICT screening identified the mTOR inhibitor everolimus as a potential candidate. These two targeted therapies were selected for our patient and resulted in radiographic disease stability. DISCUSSION/SIGNIFICANCE: This research illustrates the use of comparative transcriptomics to identify druggable pathways irrespective of actionable DNA mutations present. Our results are promising and serve to highlight the importance of functional precision medicine in tailoring treatment regimes to specific patients.

The lipid peroxidation product malondialdehyde and the DNA peroxidation product base-propenal react with dG to generate the exocyclic adduct, M1dG. This mutagenic lesion has been found in human genomic and mitochondrial DNA. M1dG in genomic DNA is enzymatically oxidized to 6-oxo-M1dG, a lesion of currently unknown mutagenic potential. Here, we report the synthesis of an oligonucleotide containing 6-oxo-M1dG and the results of extension experiments aimed at determining the effect of the 6-oxo-M1dG lesion on the activity of human polymerase iota (hPol ι). For this purpose, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed to obtain reliable quantitative data on the utilization of poorly incorporated nucleotides. Results demonstrate that hPol ι primarily incorporates deoxycytidine triphosphate (dCTP) and thymidine triphosphate (dTTP) across from 6-oxo-M1dG with approximately equal efficiency, whereas deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) are poor substrates. Following the incorporation of a single nucleotide opposite the lesion, 6-oxo-M1dG blocks further replication by the enzyme.

Expression of tryptophan 2,3-dioxygenase (TDO) is a determinant of malignancy in gliomas through kynurenine (KYN) signaling. We report that inhibition of TDO activity attenuated recovery from replication stress and increased the genotoxic effects of bis-chloroethylnitrosourea (BCNU). Activation of the Chk1 arm of the replication stress response (RSR) was reduced when TDO activity was blocked prior to BCNU treatment, whereas phosphorylation of serine 33 (pS33) on replication protein A (RPA) was enhanced-indicative of increased fork collapse. Analysis of quantitative proteomic results revealed that TDO inhibition reduced nuclear 53BP1 and sirtuin levels. We confirmed that cells lacking TDO activity exhibited elevated gamma-H2AX signal and defective recruitment of 53BP1 to chromatin following BCNU treatment, which corresponded with delayed repair of DNA breaks. Addition of exogenous KYN increased the rate of break repair. TDO inhibition diminished SIRT7 deacetylase recruitment to chromatin, which increased histone H3K18 acetylation-a key mark involved in preventing 53BP1 recruitment to sites of DNA damage. TDO inhibition also sensitized cells to ionizing radiation (IR)-induced damage, but this effect did not involve altered 53BP1 recruitment. These experiments support a model where TDO-mediated KYN signaling helps fuel a robust response to replication stress and DNA damage.

Aberrant activation of human DNA polymerase kappa (hpol κ) via the kynurenine pathway-aryl hydrocarbon receptor (KP-AhR) pathway contributes to replication stress and genome instability in glioblastoma. Our previous studies relied solely on a small-molecule inhibitor of tryptophan 2-3-deoxygenase and an AhR antagonist. Xenobiotic response elements (XREs) are short DNA sequences present in gene promoter regions that act as binding sites for the ligand complexed AhR. To elucidate hpol κ regulation by KP-AhR, we generated POLK-ΔXRE cell lines using CRISPR/Cas9 editing. XRE ablation in glioblastoma cells led to decreased hpol κ expression. Disconnecting hpol κ transcriptional regulation from AhR signaling resulted in a diminished capacity for fork restart and a slight increase in degradation of forks stalled by hydroxyurea (HU). In spite of the reduced ability to resolve replication stress, the POLK-ΔXRE T98G cell lines had fewer micronuclei and ultra-fine bridges (UFBs), indicative of less chromosomal instability. These results further support the idea that hpol κ regulation by the AhR may influence the replication stress response and overall genomic instability in glioblastoma cells.

Rev1 is a special translesion synthesis (TLS) DNA polymerase that uses a unique protein-template mechanism to bypass DNA lesions like abasic sites and guanine adducts. Earlier work from our laboratory revealed that human Rev1 (hRev1) was capable of disrupting G-quadruplex (G4) structures and preventing their refolding, and that this was independent of its nucleotidyl transfer activity. In the present study, we investigated the G4 binding specificity of hRev1 further, and demonstrated that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Site-directed mutagenesis helped identify specific amino acids in the insert-2 region of the protein to be important for high G4 DNA binding affinity. Using a forward mutagenesis assay with plasmids containing the supF gene with an engineered G4 insert, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence lacking the G4 insert. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >800-fold over control and deletions upstream of the G4 site more than doubled. Complementation experiments further showed that these mutagenic effects could be partially rescued by wild-type and E466K hRev1, while the E466A and Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in animals but not yeast, trypanosomes, or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.

Abstract Breast cancer remains one of the leading cancers among women and an estimated 90% of breast cancer deaths are due to metastasis. Cancer stem cells (CSCs) are a sub-population of cancer cells which are responsible for its initiation, progression and metastasis. CSCs are resistant to conventional chemo- and radio-therapies and therefore therapeutic strategies targeting CSCs hold great potential for novel advances in cancer treatment. Salinomycin (SAL) is a naturally occurring polyether ionophore antibiotic which was shown to effectively target breast CSCs. A library of 17 novel SAL analogs was synthesized and screened to identify compounds with improved selectivity against breast cancer stem cells. SAL analogs were either single modified esters or amides in the C1 position or double-modified C20-oxo derivatives. Eight single- and two double-modified analogs were more potent (IC50 range of 1.13 ± 0.19 to 3.93 ± 0.39 µM) towards the breast cancer cell line MDA-MB-231 compared to parent SAL (IC50 of 4.90 ± 1.60 µM). These analogs induced DNA fragmentation suggestive of apoptotic cell death. Compounds 2 (butyl ester analog of SAL) and 17 (double-modified C20-oxosalinomycin with benzhydroxamic acid) have been chosen for follow-up screening due to their improved activity and selectivity versus parent SAL. This included clonogenic assays to assess the ability of the compounds to affect cell renewal, and wound healing assays to assess cell migratory properties. In both assays, compound 17 showed superior properties over SAL. Furthermore, analog 17 showed improved targeting of breast CSCs in both cell monolayer and organoid culture as assessed in assays measuring the CD44+/CD24- stem cell sub-population. Analogs and parent compound were further studied examining (ADP-ribose) polymerase (PARP) cleavage and Bcl-2 levels by immunoblotting. All three compounds induced loss of 116 kDa PARP expression within 48h, with the highest effect induced by analog 17. In addition, treatment with compound 17 caused a decrease in Bcl-2 expression as early as 24 h. Select analogs were next screened against the NCI-60 Human Tumor Cell Line Panel. The described above double-modified analog was found to be more potent than SAL towards all 6 breast cancer cell lines in the panel as well as other tumor types. The present findings highlight the therapeutic potential of SAL analogs towards breast stem cells and support further research and clinical development of these compounds. Funding: The present study was funded by grants (to AM and TCC) from the Arkansas Breast Cancer Research Program. Testing was performed by the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, http://dtp.cancer.gov. Citation Format: Alicja Joanna Urbaniak, Megan R. Reed, Michał Antoszczak, Michał Sulik, Adam Huczyński, Robert L. Eoff, Melanie C. MacNicol, Timothy C. Chambers, Angus M. MacNicol. Inhibition of breast cancer stem cells in 2- and 3-dimensional culture by novel salinomycin analogs [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-46.

Glioblastoma (GBM) is highly resistant to treatment and invasion into the surrounding brain is a cancer hallmark that leads to recurrence despite surgical resection. With the emergence of precision medicine, patient-derived 3D systems are considered potentially robust GBM preclinical models. In this study, we screened a library of 22 anti-invasive compounds (i.e., NF-kB, GSK-3-B, COX-2, and tubulin inhibitors) using glioblastoma U-251 MG cell spheroids. We evaluated toxicity and invasion inhibition using a 3D Matrigel invasion assay. We next selected three compounds that inhibited invasion and screened them in patient-derived glioblastoma organoids (GBOs). We developed a platform using available macros for FIJI/ImageJ to quantify invasion from the outer margin of organoids. Our data demonstrated that a high-throughput invasion screening can be done using both an established cell line and patient-derived 3D model systems. Tubulin inhibitor compounds had the best efficacy with U-251 MG cells, however, in ex vivo patient organoids the results were highly variable. Our results indicate that the efficacy of compounds is highly related to patient intra and inter-tumor heterogeneity. These results indicate that such models can be used to evaluate personal oncology therapeutic strategies.

Translesion DNA synthesis (TLS) polymerase kappa (hpol k) is overexpressed in glioblastoma multiforme (GBM) and further enriched in recurrent tumors. Expression of hpol k is a prognostic indicator of shorter survival and poor response to the standard-of-care chemotherapeutic, temozolomide (TMZ). However, the effect of high levels of hpol k on DNA replication programs in glioblastoma remains unclear. We have found that either pharmacological inhibition or genetic ablation of hpol k resulted in an accelerated fork rate and increased origin firing. These effects are specific to glioblastoma, as U2OS and hTERT-RPE cells did not exhibit a change in fork rate following inhibition of hpol k activity. Additionally, POLKKO GBM cells exhibited diminished Chk1 activation and increased genomic instability indicative of a defective replication stress response (RSR). Treatment with hydroxyurea (HU) further exacerbated DNA damage relative to POLKWT cells. Furthermore, we observed an increase in ssDNA gap formation, both globally and at sites of replication, in GBM cells lacking hpol k activity. From these results, we conclude that heightened hpol k activity may be an adaptive response to the chronically elevated levels of replication stress experienced by GBM cells. The fork slowing action of hpol k prevents gap synthesis, possibly preventing depletion of the RPA pool in GBM cells through Chk1-mediated resolution of replication intermediates and by limiting origin activity. Loss of hpol k activity jeopardized the genomic integrity of GBM cells by allowing unrestrained fork progress and a shift towards a gap synthesis mode of replication. These results provide new insights into tumor-specific changes in replication programs and may carry implications for how modulation of the RSR could be leveraged to sensitize GBM cells to gap-inducing therapies.

We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.

Breast cancer remains one of the leading cancers among women. Cancer stem cells (CSCs) are tumor-initiating cells which drive progression, metastasis, and reoccurrence of the disease. CSCs are resistant to conventional chemo- and radio-therapies and their ability to survive such treatment enables tumor reestablishment. Metastasis is the main cause of mortality in women with breast cancer, thus advances in treatment will depend on therapeutic strategies targeting CSCs. Salinomycin (SAL) is a naturally occurring polyether ionophore antibiotic known for its anticancer activity towards several types of tumor cells. In the present work, a library of 17 C1-single and C1/C20-double modified SAL analogs was screened to identify compounds with improved activity against breast CSCs. Six single- and two double-modified analogs were more potent (IC50 range of 1.1 +/- 0.1-1.4 +/- 0.2 mu M) toward the breast cancer cell line MDA-MB-231 compared to SAL (IC50 of 4.9 +/- 1.6 mu M). Double-modified compound 17 was found to be more efficacious than SAL against the majority of cancer cell lines in the NCI-60 Human Tumor Cell Line Panel. Compound 17 was more potent than SAL in inhibiting cell migration and cell renewal properties of MDA-MB-231 cells, as well as inducing selective loss of the CD44(+)/CD24(-/low) stem-cell-like subpopulation in both monolayer (2D) and organoid (3D) culture. The present findings highlight the therapeutic potential of SAL analogs towards breast CSCs and identify select compounds that merit further study and clinical development.

Mitochondrial dynamics regulated by mitochondrial fusion and fission maintain mitochondrial functions, whose alterations underline various human diseases. Here, we show that inositol is a critical metabolite directly restricting AMPK-dependent mitochondrial fission independently of its classical mode as a precursor for phosphoinositide generation. Inositol decline by IMPA1/2 deficiency elicits AMPK activation and mitochondrial fission without affecting ATP level, whereas inositol accumulation prevents AMPK-dependent mitochondrial fission. Metabolic stress or mitochondrial damage causes inositol decline in cells and mice to elicit AMPK-dependent mitochondrial fission. Inositol directly binds to AMPKγ and competes with AMP for AMPKγ binding, leading to restriction of AMPK activation and mitochondrial fission. Our study suggests that the AMP/inositol ratio is a critical determinant for AMPK activation and establishes a model in which AMPK activation requires inositol decline to release AMPKγ for AMP binding. Hence, AMPK is an inositol sensor, whose inactivation by inositol serves as a mechanism to restrict mitochondrial fission.

Garcinoic acid has been identified as an inhibitor of DNA polymerase β (pol β). However, no structure-activity relationship (SAR) studies of garcinoic acid as a pol β inhibitor have been conducted, in part due to the lack of an efficient synthetic method for this natural product and its analogs. We developed an efficient semi-synthetic method for garcinoic acid and its analogs by starting from natural product δ-tocotrienol. Our preliminary SAR studies provided a valuable insight into future discovery of garcinoic acid-based pol β inhibitors.