Impact of Decalcification, Cold Ischemia, and Deglycosylation on Performance of Programmed Cell Death Ligand-1 Antibodies With Different Binding Epitopes: Comparison of 7 Clones.

Programmed cell death ligand-1 (PD-L1) expression levels in patients' tumors have demonstrated clinical utility across many cancer types and are used to determine treatment eligibility. Several independently developed PD-L1 immunohistochemical (IHC) predictive assays are commercially available and have demonstrated different levels of staining between assays, generating interest in understanding the similarities and differences between assays. Previously, we identified epitopes in the internal and external domains of PD-L1, bound by antibodies in routine clinical use (SP263, SP142, 22C3, and 28-8). Variance in performance of assays utilizing these antibodies, observed following exposure to preanalytical factors such as decalcification, cold ischemia, and duration of fixation, encouraged additional investigation of antibody-binding sites, to understand whether binding site structures/conformations contribute to differential PD-L1 IHC assay staining. We proceeded to further investigate the epitopes on PD-L1 bound by these antibodies, alongside the major clones utilized in laboratory-developed tests (E1L3N, QR1, and 73-10). Characterization of QR1 and 73-10 clones demonstrated that both bind the PD-L1 C-terminal internal domain, similar to SP263/SP142. Our results also demonstrate that under suboptimal decalcification or fixation conditions, the performance of internal domain antibodies is less detrimentally affected than that of external domain antibodies 22C3/28-8. Furthermore, we show that the binding sites of external domain antibodies are susceptible to deglycosylation and conformational structural changes, which directly result in IHC staining reduction or loss. The binding sites of internal domain antibodies were unaffected by deglycosylation or conformational structural change. This study demonstrates that the location and conformation of binding sites, recognized by antibodies employed in PD-L1 diagnostic assays, differ significantly and exhibit differing degrees of robustness. These findings should reinforce the need for vigilance when performing clinical testing with different PD-L1 IHC assays, particularly in the control of cold ischemia and the selection of fixation and decalcification conditions.

Mapping the binding sites of antibodies utilized in programmed cell death ligand-1 predictive immunohistochemical assays for use with immuno-oncology therapies.

Programmed cell death ligand-1 (PD-L1) expression levels in patient tumor samples have proven clinical utility across various cancer types. Several independently developed PD-L1 immunohistochemical (IHC) predictive assays are commercially available. Published studies using the VENTANA PD-L1 (SP263) Assay, VENTANA PD-L1 (SP142) Assay, Dako PD-L1 IHC 22C3 pharmDx assay, Dako PD-L1 IHC 28-8 pharmDx assay, and laboratory-developed tests utilizing the E1L3N antibody (Cell Signaling Technology), have demonstrated differing levels of PD-L1 staining between assays, resulting in conjecture as to whether antibody-binding epitopes could be responsible for discordance between assays. Therefore, to understand the performance of different PD-L1 predictive immunohistochemistry assays, we aimed to distinguish the epitopes within the PD-L1 protein responsible for antibody binding. The sites at which antibody clones SP263, SP142, 22C3, 28-8, and E1L3N bind to recombinant PD-L1 were assessed using several methods, including conformational peptide array, surface plasmon resonance, and/or hydrogen/deuterium exchange mass spectrometry. Putative binding sites were confirmed by site-directed mutagenesis of PD-L1, followed by western blotting and immunohistochemical analysis of cell lines expressing mutant constructs. Our results demonstrate that clones SP263 and SP142 bind to an identical epitope in the cytoplasmic domain at the extreme C-terminus of PD-L1, distinct from 22C3 and 28-8. Using mutated PD-L1 constructs, an additional clone, E1L3N, was also found to bind to the cytoplasmic domain of PD-L1. The E1L3N binding epitope overlaps considerably with the SP263/SP142 binding site but is not identical. Clones 22C3 and 28-8 have binding profiles in the extracellular domain of PD-L1, which differ from one another. Despite identifying epitope binding variance among antibodies, evidence indicates that only the SP142 assay generates significantly discordant immunohistochemical staining, which can be resolved by altering the assay protocol. Therefore, inter-assay discordances are more likely attributable to tumor heterogeneity, assay, or platform variables rather than antibody epitope.

Correction: Mapping the binding sites of antibodies utilized in programmed cell death ligand-1 predictive immunohistochemical assays for use with immuno-oncology therapies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Immunophenotypic analysis of cell cycle status in acute myeloid leukaemia: relationship to cytogenetics, genotype and clinical outcome.

Cell cycle status may play an important role in directing patient therapy. We therefore determined the cell cycle status of leukaemic cells by immunophenotypic analysis of bone marrow trephine biopsies from 181 patients with acute myeloid leukaemia (AML) and correlated the results with biological features and clinical outcome. There was considerable heterogeneity between patients. The presenting white cell count significantly correlated with the proportion of non-quiescent cells (P < 0·0001), of cycling cells beyond G1 (P < 0·0001) and the speed of cycling (P < 0·0001). Profiles in acute promyelocytic leukaemia (APL) differed from non-APL and were consistent with more differentiated cells with reduced proliferative potential, but no significant differences were observed between non-APL cytogenetic risk groups. NPM1 mutations but not FLT3 internal tandem duplication (FLT3ITD ) were significantly associated with a higher proportion of cells beyond G1 (P = 0·002) and faster speed of cycling (P = 0·003). Resistance to standard cytosine arabinoside and daunorubicin induction chemotherapy was significantly related to a slower speed of cycling (P = 0·0002), as was a higher relapse rate (P = 0·05), but not with the proportion of non-quiescent cells or actively cycling cells. These results show a link between the cycling speed of AML cells and the response to chemotherapy, and help to identify a group with a very poor prognosis.

Molecular architecture of the DNA replication origin activation checkpoint.

Perturbation of DNA replication initiation arrests human cells in G1, pointing towards an origin activation checkpoint. We used RNAi against Cdc7 kinase to inhibit replication initiation and dissect this checkpoint in fibroblasts. We show that the checkpoint response is dependent on three axes coordinated through the transcription factor FoxO3a. In arrested cells, FoxO3a activates the ARF-∣Hdm2-∣p53 → p21 pathway and mediates p15(INK4B) upregulation; p53 in turn activates expression of the Wnt/ß-catenin signalling antagonist Dkk3, leading to Myc and cyclin D1 downregulation. The resulting loss of CDK activity inactivates the Rb-E2F pathway and overrides the G1-S transcriptional programme. Fibroblasts concomitantly depleted of Cdc7/FoxO3a, Cdc7/p15, Cdc7/p53 or Cdc7/Dkk3 can bypass the arrest and proceed into an abortive S phase followed by apoptosis. The lack of redundancy between the checkpoint axes and reliance on several tumour suppressor proteins commonly inactivated in human tumours provides a mechanistic basis for the cancer-cell-specific killing observed with emerging Cdc7 inhibitors.

Cell cycle markers in clinical oncology.

Analysis of complex and redundant pathways that control proliferation, differentiation, apoptosis and DNA damage response by global genome wide analysis is an intensive area of investigation aimed at identifying unique molecular signatures of prognostic significance in cancer. An alternative approach is to focus on the cell cycle machinery, which acts as an integration point for information transduced through upstream signalling pathways. Analysis of the DNA replication licensing pathway and the mitotic regulatory machinery in tumour biopsy material is now leading to the identification of novel biomarkers that are being exploited in cancer detection and prognostic assessment.

Two ubiquitin ligases, APC/C-Cdh1 and SKP1-CUL1-F (SCF)-beta-TrCP, sequentially regulate glycolysis during the cell cycle.

During cell proliferation, the abundance of the glycolysis-promoting enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3), is controlled by the ubiquitin ligase APC/C-Cdh1 via a KEN box. We now demonstrate in synchronized HeLa cells that PFKFB3, which appears in mid-to-late G1, is essential for cell division because its silencing prevents progression into S phase. In cells arrested by glucose deprivation, progression into S phase after replacement of glucose occurs only when PFKFB3 is present or is substituted by the downstream glycolytic enzyme 6-phosphofructo-1-kinase. PFKFB3 ceases to be detectable during late G1/S despite the absence of Cdh1; this disappearance is prevented by proteasomal inhibition. PFKFB3 contains a DSG box and is therefore a potential substrate for SCF-ß-TrCP, a ubiquitin ligase active during S phase. In synchronized HeLa cells transfected with PFKFB3 mutated in the KEN box, the DSG box, or both, we established the breakdown routes of the enzyme at different stages of the cell cycle and the point at which glycolysis is enhanced. Thus, the presence of PFKFB3 is tightly controlled to ensure the up-regulation of glycolysis at a specific point in G1. We suggest that this up-regulation of glycolysis and its associated events represent the nutrient-sensitive restriction point in mammalian cells.

Subcellular proteomics reveals a role for nucleo-cytoplasmic trafficking at the DNA replication origin activation checkpoint.

Depletion of DNA replication initiation factors such as CDC7 kinase triggers the origin activation checkpoint in healthy cells and leads to a protective cell cycle arrest at the G1 phase of the mitotic cell division cycle. This protective mechanism is thought to be defective in cancer cells. To investigate how this checkpoint is activated and maintained in healthy cells, we conducted a quantitative SILAC analysis of the nuclear- and cytoplasmic-enriched compartments of CDC7-depleted fibroblasts and compared them to a total cell lysate preparation. Substantial changes in total abundance and/or subcellular location were detected for 124 proteins, including many essential proteins associated with DNA replication/cell cycle. Similar changes in protein abundance and subcellular distribution were observed for various metabolic processes, including oxidative stress, iron metabolism, protein translation and the tricarboxylic acid cycle. This is accompanied by reduced abundance of two karyopherin proteins, suggestive of reduced nuclear import. We propose that altered nucleo-cytoplasmic trafficking plays a key role in the regulation of cell cycle arrest. The results increase understanding of the mechanisms underlying maintenance of the DNA replication origin activation checkpoint and are consistent with our proposal that cell cycle arrest is an actively maintained process that appears to be distributed over various subcellular locations.

The cell cycle and cancer.

Deregulation of the cell cycle underlies the aberrant cell proliferation that characterizes cancer and loss of cell cycle checkpoint control promotes genetic instability. During the past two decades, cancer genetics has shown that hyperactivating mutations in growth signalling networks, coupled to loss of function of tumour suppressor proteins, drives oncogenic proliferation. Gene expression profiling of these complex and redundant mitogenic pathways to identify prognostic and predictive signatures and their therapeutic targeting has, however, proved challenging. The cell cycle machinery, which acts as an integration point for information transduced through upstream signalling networks, represents an alternative target for diagnostic and therapeutic interventions. Analysis of the DNA replication initiation machinery and mitotic engine proteins in human tissues is now leading to the identification of novel biomarkers for cancer detection and prognostication, and is providing target validation for cell cycle-directed therapies.

Targeting DNA replication before it starts: Cdc7 as a therapeutic target in p53-mutant breast cancers.

Treatment options for triple-receptor negative (ER-/PR-/Her2-) and Her2-overexpressing (ER-/PR-/Her2+) breast cancers with acquired or de novo resistance are limited, and metastatic disease remains incurable. Targeting of growth signaling networks is often constrained by pathway redundancy or growth-independent cancer cell cycles. The cell-cycle protein Cdc7 regulates S phase by promoting DNA replication. This essential kinase acts as a convergence point for upstream growth signaling pathways and is therefore an attractive therapeutic target. We show that increased Cdc7 expression during mammary tumorigenesis is linked to Her2-overexpressing and triple-negative subtypes, accelerated cell cycle progression (P < 0.001), arrested tumor differentiation (P < 0.001), genomic instability (P = 0.019), increasing NPI score (P < 0.001), and reduced disease-free survival (HR = 1.98 [95% CI: 1.27-3.10]; P = 0.003), thus implicating its deregulation in the development of aggressive disease. Targeting Cdc7 with RNAi, we demonstrate that p53-mutant Her2-overexpressing and triple-negative breast cancer cell lines undergo an abortive S phase and apoptotic cell death due to loss of a p53-dependent Cdc7-inhibition checkpoint. In contrast, untransformed breast epithelial cells arrest in G1, remain viable, and are able to resume cell proliferation on recovery of Cdc7 kinase activity. Thus, Cdc7 appears to represent a potent and highly specific anticancer target in Her2-overexpressing and triple-negative breast cancers. Emerging Cdc7 kinase inhibitors may therefore significantly broaden the therapeutic armamentarium for treatment of the aggressive p53-mutant breast cancer subtypes identified in this study.

SARS-CoV-2 testing and sequencing for international arrivals reveals significant cross border transmission of high risk variants into the United Kingdom.

BACKGROUND: Mandatory Day 2 and Day 8 PCR testing and variant sequencing of international arrivals has been recently introduced by the UK Government to mitigate against cross-border transmission of high-risk SARS-CoV-2 variants. METHODS: SARS-CoV-2 testing and sequencing combines TaqPath CE-IVD COVID-19 RT-PCR with Ion AmpliSeq SARS-CoV-2 Next Generation Sequencing Assay. Retrospective analysis of test trending data was performed from initiation of testing on the 11th March through to the 14th April 2021. FINDINGS: During this time interval, 203,065 SARS-CoV-2 PCR tests were performed, with 3,855 samples testing positive, giving a prevalence of 1.9%. In total 1,913 SARS-CoV-2 genomes were sequenced from positive cases with Ct values < 30 and 1,635 (85.5%) sequences passed quality metrics for lineage analysis. A high diversity of 49 different SARS-CoV-2 variants were identified, including the VOCs B.1.1.7 (Kent; 80.6%), B.1.351 (South Africa; 4.2%), B.1.617.2 (India; 1.7%), P.1 (Brazil; 0.4%) and B.1.1.7 with E484K (Bristol; 0.2%). Vaccine effectiveness was age-related and dose-dependent, ranging from 5% in > 60 with a single dose to 83% in <60 with both doses of a vaccine. Viral load was variant dependent with the B.1.617.2 showing a 21 fold increase in viral copy number compared to the other variants. INTERPRETATION: The unexpectedly high prevalence of COVID-19 infection in UK arrivals is associated with a rich diversity of SARS-CoV-2 high risk variants entering the UK including the VOC B.1.617.2. Vaccination does not preclude infection and its effectiveness is significantly age-dependent and impacted by variant type. The rapid high-throughput test and sequence workflow we have adopted is particularly suited to the monitoring of cross border transmission and enables immediate public health interventions. FUNDING: Data analysis conducted in this study was limited to secondary use of information previously collected in the course of normal care.

Clinical, Radiometabolic and Immunologic Effects of Olaparib in Locally Advanced Triple Negative Breast Cancer: The OLTRE Window of Opportunity Trial.

INTRODUCTION: Olaparib is effective in metastatic triple negative breast cancer (TNBC) carrying germline mutations in DNA damage repair (DDR) genes BRCA1/2 (gBRCA-mut). The OLTRE window-of-opportunity trial preliminarily investigated potential pathologic, radiometabolic and immune biomarkers of early-response to olaparib in gBRCA-wild-type (wt) TNBC and, as proof-of-concept in gBRCA-mut HER2-negative BC. METHODS: Patients received olaparib for 3 weeks (3w) before standard neoadjuvant chemotherapy and underwent multiple FDG18-PET/CT scan (basal, after olaparib), clinical assessments (basal, every 3w), tumor biopsies and blood samplings (baseline, after olaparib). Clinical and radiometabolic responses were evaluated according to RECIST1.1 and PERCIST criteria. RESULTS: 27 patients with gBRCA-wt TNBC and 8 with gBRCA-mut BC (6 TNBC, 2 HR+/HER2-negative) were enrolled. Three (11.1%) patients showed mutations in non-BRCA1/2 DDR genes and 4 (14.8%) in other genes. 3w olaparib induced 16/35 and 15/27 partial clinical and radiometabolic responses, including in 40.7% and 50.0% gBRCA-wt patients. gBRCA-mut tumors presented numerically higher tumor-infiltrating lymphocytes (TILs) levels and PD-L1 positive tumors. Clinical responders experienced a reduction in T-regs/T-eff ratio (p=0.05), B and NK lymphocytes (p=0.003 both), with an average increase in T-helpers rate (p<0.001) and CD4/CD8 ratio (p=0.02). Ki67% and TILs did not vary significantly (p=0.67 and p=0.77). A numerical increase in PD-L1 positive cases after olaparib was observed, though non-significant (p=0.134). No differences were observed according to gBRCA status and type of response. CONCLUSIONS: Early-stage TNBC might be a target population for olaparib, irrespective of gBRCA mutations. Future trials should combine TILs, PD-L1 and gBRCA status to better identify candidates for escalated/de-escalated treatment strategies including olaparib.

Quantitative proteomics reveals a "poised quiescence" cellular state after triggering the DNA replication origin activation checkpoint.

An origin activation checkpoint has recently been discovered in the G1 phase of the mitotic cell cycle, which can be triggered by loss of DNA replication initiation factors such as the Cdc7 kinase. Insufficient levels of Cdc7 activate cell cycle arrest in normal cells, whereas cancer cells appear to lack this checkpoint response, do not arrest, and proceed with an abortive S phase, leading to cell death. The differential response between normal and tumor cells at this checkpoint has led to widespread interest in the development of pharmacological Cdc7 inhibitors as novel anticancer agents. We have used RNAi against Cdc7 in combination with SILAC-based high resolution MS proteomics to investigate the cellular mechanisms underlying the maintenance of the origin activation checkpoint in normal human diploid fibroblasts. Bioinformatics analysis identified clear changes in wide-ranging biological processes including altered cellular energetic flux, moderate stress response, reduced proliferative capacity, and a spatially distributed response across the mitochondria, lysosomes, and the cell surface. These results provide a quantitative overview of the processes involved in maintenance of the arrested state, show that this phenotype involves active rather than passive cellular adaptation, and highlight a diverse set of proteins responsible for cell cycle arrest and ultimately for promotion of cellular survival. We propose that the Cdc7-depleted proteome maintains cellular arrest by initiating a dynamic quiescence-like response and that the complexities of this phenotype will have important implications for the continued development of promising Cdc7-targeted cancer therapies.

Biomarkers in bladder cancer.

Cancer biomarkers provide an opportunity to diagnose tumours earlier and with greater accuracy. They can also identify those patients most at risk of disease recurrence and predict which tumours will respond to different therapeutic approaches. Such biomarkers will be especially useful in the diagnosis and management of bladder cancer. At present, bladder tumours are diagnosed and followed-up using a combination of cystoscopic examination, cytology and histology. These are not only expensive, but also highly subjective investigations and reveal little about the underlying molecular characteristics of the tumour. In recent years numerous diagnostic and prognostic biomarkers of bladder cancer have been identified. Two separate approaches to biomarker discovery have been employed. The first is hypothesis-driven and focuses upon proteins involved in molecular pathways known to be implicated in tumorigenesis. An alternative approach has been to study the global expression of genes (so-called 'genomics') looking for characteristic signatures associated with disease outcomes. In this review we summarize the current state of biomarker development in this field, and examine why so few have made the successful transition into the clinic. Finally, we introduce a novel approach to biomarker development utilizing components of the DNA replication licensing machinery.

Cdc7 kinase is a predictor of survival and a novel therapeutic target in epithelial ovarian carcinoma.

PURPOSE: There is a lack of prognostic and predictive biomarkers in epithelial ovarian carcinoma, and the targeting of oncogenic signaling pathways has had limited impact on patient survival in this highly heterogeneous disease. The origin licensing machinery, which renders chromosomes competent for DNA replication, acts as a convergence point for upstream signaling pathways. We tested the hypothesis that Cdc7 kinase, a core component of the licensing machinery, is predictive of clinical outcome and may constitute a novel therapeutic target in epithelial ovarian carcinoma. EXPERIMENTAL DESIGN: A total of 143 cases of ovarian cancer and 5 cases of normal ovary were analyzed for Cdc7 protein expression dynamics and clinicopathologic features. To assess the therapeutic potential of Cdc7, expression was down-regulated by RNA interference in SKOV-3 and Caov-3 ovarian cancer cells. RESULTS: Increased Cdc7 protein levels were significantly associated with arrested tumor differentiation (P = 0.004), advanced clinical stage (P = 0.01), genomic instability (P < 0.001), and accelerated cell cycle progression. Multivariate analysis shows that Cdc7 predicts disease-free survival independent of patient age, tumor grade and stage (hazard ratio, 2.03; confidence interval, 1.53-2.68; P < 0.001), with the hazard ratio for relapse increasing to 10.90 (confidence interval, 4.07-29.17) for the stages 3 to 4/upper Cdc7 tertile group relative to stages 1 to 2/lower Cdc7 tertile tumors. In SKOV-3 and Caov-3 cells, Cdc7 siRNA knockdown triggered high levels of apoptosis, whereas untransformed cells arrest in G(1) phase and remain viable. CONCLUSIONS: Our findings show that Cdc7 kinase predicts survival and is a potent anticancer target in epithelial ovarian carcinoma, highlighting its potential as a predictor of susceptibility to small molecule kinase inhibitors currently in development.

Interobserver Reliability of Programmed Cell Death Ligand-1 Scoring Using the VENTANA PD-L1 (SP263) Assay in NSCLC.

INTRODUCTION: The VENTANA PD-L1 (SP263) Assay is approved for use with anti-programmed cell death-1/programmed cell death ligand-1 (PD-1/PD-L1) therapies in NSCLC and urothelial carcinoma. Here, we investigate interobserver reliability of the SP263 assay, applied to PD-L1 scoring of tumor cells (TCs) in NSCLC. METHODS: Six practicing European pulmonary pathologists independently scored the proportion of TCs expressing PD-L1 (TC score) from 200 archival, commercially sourced, formalin-fixed paraffin-embedded NSCLC resections stained using the SP263 assay. Agreement in scores was analyzed using the intraclass correlation coefficient and concordance in patient's classification using Fleiss' kappa. RESULTS: Results from 172 samples showed strong pair-wise correlations between pathologists (R2 >0.89) for TC scoring with an intraclass correlation coefficient of 0.96. Overall agreement was greater than 90% for TC of 1% and above, and greater than 94% for TCs of at least 25% and at least 50%. Fleiss' kappa showed substantial agreement for TC of 1% and above, and almost perfect agreement for TCs of at least 25% and at least 50%. CONCLUSIONS: Assessment of TC score in NSCLC was highly reproducible using the SP263 assay, building confidence in the accuracy of this assay in selection of patients for anti-PD-1/PD-L1 therapy.

Identification of an arginine-rich motif in human papillomavirus type 1 E1;E4 protein necessary for E4-mediated inhibition of cellular DNA synthesis in vitro and in cells.

Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G(2)-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1;E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.

Molecular structure of human geminin.

The origin licensing repressor geminin is a unique bifunctional protein providing a molecular link between cellular proliferation, differentiation and genomic stability. Here we report the first molecular structure of human geminin, determined by EM and image processing at a resolution of 17.5 A. The geminin molecule is a tetramer formed by two dimers with monomers interacting via coiled-coil domains. The unusual structural organization of geminin provides molecular insight into its bifunctional nature.

Modular assembly using sequential palladium coupling gives easy access to the SMoC class of cellular transporters.

The transducing ability of the third helix of transcription factor homeodomains is effectively mimicked by a biphenyl system displaying guanidine groups. The biphenyl class of small molecule carriers (SMoCs) can carry biomolecules into a wide variety of cell types. A "combinatorial" approach to the synthesis of SMoCs is described using sequential Pd(0) coupling chemistry to assemble the molecules from highly functionalized building blocks. SMoCs coupled to the DNA licensing repressor protein geminin can inhibit DNA replication in vitro. We conducted a structure-activity investigation utilizing a range of SMoC-geminin conjugates and demonstrate that both electrostatic and structural features are important for efficient uptake and functional activity. The best analogue was more efficient than either (Arg)(4) or (Arg)(8) linked to geminin. Effective inhibition of DNA synthesis was achieved in fibroblasts and osteosarcoma cell lines.