The DNA Damage Response (DDR) landscape of endometrial cancer defines discrete disease subtypes and reveals therapeutic opportunities.

Genome maintenance is an enabling characteristic that allows neoplastic cells to tolerate the inherent stresses of tumorigenesis and evade therapy-induced genotoxicity. Neoplastic cells also deploy many mis-expressed germ cell proteins termed Cancer Testes Antigens (CTAs) to promote genome maintenance and survival. Here, we present the first comprehensive characterization of the DNA Damage Response (DDR) and CTA transcriptional landscapes of endometrial cancer in relation to conventional histological and molecular subtypes. We show endometrial serous carcinoma (ESC), an aggressive endometrial cancer subtype, is defined by gene expression signatures comprising members of the Replication Fork Protection Complex (RFPC) and Fanconi Anemia (FA) pathway and CTAs with mitotic functions. DDR and CTA-based profiling also defines a subset of highly aggressive endometrioid endometrial carcinomas (EEC) with poor clinical outcomes that share similar profiles to ESC yet have distinct characteristics based on conventional histological and genomic features. Using an unbiased CRISPR-based genetic screen and a candidate gene approach, we confirm that DDR and CTA genes that constitute the ESC and related EEC gene signatures are required for proliferation and therapy-resistance of cultured endometrial cancer cells. Our study validates the use of DDR and CTA-based tumor classifiers and reveals new vulnerabilities of aggressive endometrial cancer where none currently exist.

REV7-p53 interaction inhibits ATM-mediated DNA damage signaling.

REV7 is an abundant, multifunctional protein that is a known factor in cell cycle regulation and in several key DNA repair pathways including Trans-Lesion Synthesis (TLS), the Fanconi Anemia (FA) pathway, and DNA Double-Strand Break (DSB) repair pathway choice. Thus far, no direct role has been studied for REV7 in the DNA damage response (DDR) signaling pathway. Here we describe a novel function for REV7 in DSB-induced p53 signaling. We show that REV7 binds directly to p53 to block ATM-dependent p53 Ser15 phosphorylation. We also report that REV7 is involved in the destabilization of p53. These findings affirm REV7's participation in fundamental cell cycle and DNA repair pathways. Furthermore, they highlight REV7 as a critical factor for the integration of multiple processes that determine viability and genome stability.

Trans-lesion synthesis and mismatch repair pathway crosstalk defines chemoresistance and hypermutation mechanisms in glioblastoma.

Almost all Glioblastoma (GBM) are either intrinsically resistant to the chemotherapeutical drug temozolomide (TMZ) or acquire therapy-induced mutations that cause chemoresistance and recurrence. The genome maintenance mechanisms responsible for GBM chemoresistance and hypermutation are unknown. We show that the E3 ubiquitin ligase RAD18 (a proximal regulator of TLS) is activated in a Mismatch repair (MMR)-dependent manner in TMZ-treated GBM cells, promoting post-replicative gap-filling and survival. An unbiased CRISPR screen provides an aerial map of RAD18-interacting DNA damage response (DDR) pathways deployed by GBM to tolerate TMZ genotoxicity. Analysis of mutation signatures from TMZ-treated GBM reveals a role for RAD18 in error-free bypass of O6mG (the most toxic TMZ-induced lesion), and error-prone bypass of other TMZ-induced lesions. Our analyses of recurrent GBM patient samples establishes a correlation between low RAD18 expression and hypermutation. Taken together we define molecular underpinnings for the hallmark tumorigenic phenotypes of TMZ-treated GBM.

The DNA Damage Response (DDR) landscape of endometrial cancer defines discrete disease subtypes and reveals therapeutic opportunities.

Genome maintenance is an enabling characteristic that allows neoplastic cells to tolerate the inherent stresses of tumorigenesis and evade therapy-induced genotoxicity. Neoplastic cells also deploy mis-expressed germ cell proteins termed Cancer Testes Antigens (CTAs) to promote genome maintenance and survival. Here, we present the first comprehensive characterization of the DNA Damage Response (DDR) and CTA transcriptional landscapes of endometrial cancer in relation to conventional histological and molecular subtypes. We show endometrial serous carcinoma (ESC), an aggressive endometrial cancer subtype, is defined by gene expression signatures comprising members of the Replication Fork Protection Complex (RFPC) and Fanconi Anemia (FA) pathway and CTAs with mitotic functions. DDR and CTA- based profiling also defines a subset of highly aggressive endometrioid endometrial carcinomas (EEC) with poor clinical outcomes that share similar profiles to ESC yet have distinct characteristics based on conventional histological and genomic features. Using an unbiased CRISPR-based genetic screen and a candidate gene approach, we confirm that DDR and CTA genes that constitute the ESC and related EEC gene signatures are required for proliferation and therapy-resistance of cultured endometrial cancer cells. Our study validates the use of DDR and CTA-based tumor classifiers and reveals new vulnerabilities of aggressive endometrial cancer where none currently exist.

Trans-Lesion Synthesis and Mismatch Repair Pathway Crosstalk Defines Chemoresistance and Hypermutation Mechanisms in Glioblastoma.

Almost all Glioblastoma (GBM) are either intrinsically resistant to the chemotherapeutical drug temozolomide (TMZ) or acquire therapy-induced mutations that cause chemoresistance and recurrence. The genome maintenance mechanisms responsible for GBM chemoresistance and hypermutation are unknown. We show that the E3 ubiquitin ligase RAD18 (a proximal regulator of TLS) is activated in a Mismatch repair (MMR)-dependent manner in TMZ-treated GBM cells, promoting post-replicative gap-filling and survival. An unbiased CRISPR screen provides a new aerial map of RAD18-interacting DNA damage response (DDR) pathways deployed by GBM to tolerate TMZ genotoxicity. Analysis of mutation signatures from TMZ-treated GBM reveals a role for RAD18 in error-free bypass of O6mG (the most toxic TMZ-induced lesion), and error-prone bypass of other TMZ-induced lesions. Our analyses of recurrent GBM patient samples establishes a correlation between low RAD18 expression and hypermutation. Taken together we define novel molecular underpinnings for the hallmark tumorigenic phenotypes of TMZ-treated GBM.

Trans-Lesion Synthesis and Mismatch Repair Pathway Crosstalk Defines Chemoresistance and Hypermutation Mechanisms in Glioblastoma.

Almost all Glioblastoma (GBM) are either intrinsically resistant to the chemotherapeutical drug temozolomide (TMZ) or acquire therapy-induced mutations that cause chemoresistance and recurrence. The genome maintenance mechanisms responsible for GBM chemoresistance and hypermutation are unknown. We show that the E3 ubiquitin ligase RAD18 (a proximal regulator of TLS) is activated in a Mismatch repair (MMR)-dependent manner in TMZ-treated GBM cells, promoting post-replicative gap-filling and survival. An unbiased CRISPR screen provides a new aerial map of RAD18-interacting DNA damage response (DDR) pathways deployed by GBM to tolerate TMZ genotoxicity. Analysis of mutation signatures from TMZ-treated GBM reveals a role for RAD18 in error-free bypass of O6mG (the most toxic TMZ-induced lesion), and error-prone bypass of other TMZ-induced lesions. Our analyses of recurrent GBM patient samples establishes a correlation between low RAD18 expression and hypermutation. Taken together we define novel molecular underpinnings for the hallmark tumorigenic phenotypes of TMZ-treated GBM.

The TRIM69-MST2 signaling axis regulates centrosome dynamics and chromosome segregation.

Stringent control of centrosome duplication and separation is important for preventing chromosome instability. Structural and numerical alterations in centrosomes are hallmarks of neoplastic cells and contribute to tumorigenesis. We show that a Centrosome Amplification 20 (CA20) gene signature is associated with high expression of the Tripartite Motif (TRIM) family member E3 ubiquitin ligase, TRIM69. TRIM69-ablation in cancer cells leads to centrosome scattering and chromosome segregation defects. We identify Serine/threonine-protein kinase 3 (MST2) as a new direct binding partner of TRIM69. TRIM69 redistributes MST2 to the perinuclear cytoskeleton, promotes its association with Polo-like kinase 1 (PLK1) and stimulates MST2 phosphorylation at S15 (a known PLK1 phosphorylation site that is critical for centrosome disjunction). TRIM69 also promotes microtubule bundling and centrosome segregation that requires PRC1 and DYNEIN. Taken together, we identify TRIM69 as a new proximal regulator of distinct signaling pathways that regulate centrosome dynamics and promote bipolar mitosis.

Identification of Covalent Cyclic Peptide Inhibitors in mRNA Display.

Peptides have historically been underutilized for covalent inhibitor discovery, despite their unique abilities to interact with protein surfaces and interfaces. This is in part due to a lack of methods for screening and identifying covalent peptide ligands. Here, we report a method to identify covalent cyclic peptide inhibitors in mRNA display. We combine co- and post-translational library diversification strategies to create cyclic libraries with reactive dehydroalanines (Dhas), which we employ in selections against two model targets. The most potent hits exhibit low nanomolar inhibitory activities and disrupt known protein-protein interactions with their selected targets. Overall, we establish Dhas as electrophiles for covalent inhibition and showcase how separate library diversification methods can work synergistically to dispose mRNA display to novel applications like covalent inhibitor discovery.

A living ex vivo platform for functional, personalized brain cancer diagnosis.

Functional precision medicine platforms are emerging as promising strategies to improve pre-clinical drug testing and guide clinical decisions. We have developed an organotypic brain slice culture (OBSC)-based platform and multi-parametric algorithm that enable rapid engraftment, treatment, and analysis of uncultured patient brain tumor tissue and patient-derived cell lines. The platform has supported engraftment of every patient tumor tested to this point: high- and low-grade adult and pediatric tumor tissue rapidly establishes on OBSCs among endogenous astrocytes and microglia while maintaining the tumor's original DNA profile. Our algorithm calculates dose-response relationships of both tumor kill and OBSC toxicity, generating summarized drug sensitivity scores on the basis of therapeutic window and allowing us to normalize response profiles across a panel of U.S. Food and Drug Administration (FDA)-approved and exploratory agents. Summarized patient tumor scores after OBSC treatment show positive associations to clinical outcomes, suggesting that the OBSC platform can provide rapid, accurate, functional testing to ultimately guide patient care.

VCP/p97, a pleiotropic protein regulator of the DNA damage response and proteostasis, is a potential therapeutic target in KRAS-mutant pancreatic cancer.

We and others have recently shown that proteins involved in the DNA damage response (DDR) are critical for KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cell growth in vitro. However, the CRISPR-Cas9 library that enabled us to identify these key proteins had limited representation of DDR-related genes. To further investigate the DDR in this context, we performed a comprehensive, DDR-focused CRISPR-Cas9 loss-of-function screen. This screen identified valosin-containing protein (VCP) as an essential gene in KRAS-mutant PDAC cell lines. We observed that genetic and pharmacologic inhibition of VCP limited cell growth and induced apoptotic death. Addressing the basis for VCP-dependent growth, we first evaluated the contribution of VCP to the DDR and found that loss of VCP resulted in accumulation of DNA double-strand breaks. We next addressed its role in proteostasis and found that loss of VCP caused accumulation of polyubiquitinated proteins. We also found that loss of VCP increased autophagy. Therefore, we reasoned that inhibiting both VCP and autophagy could be an effective combination. Accordingly, we found that VCP inhibition synergized with the autophagy inhibitor chloroquine. We conclude that concurrent targeting of autophagy can enhance the efficacy of VCP inhibitors in KRAS-mutant PDAC.

Roles of trans-lesion synthesis (TLS) DNA polymerases in tumorigenesis and cancer therapy.

DNA damage tolerance and mutagenesis are hallmarks and enabling characteristics of neoplastic cells that drive tumorigenesis and allow cancer cells to resist therapy. The 'Y-family' trans-lesion synthesis (TLS) DNA polymerases enable cells to replicate damaged genomes, thereby conferring DNA damage tolerance. Moreover, Y-family DNA polymerases are inherently error-prone and cause mutations. Therefore, TLS DNA polymerases are potential mediators of important tumorigenic phenotypes. The skin cancer-propensity syndrome xeroderma pigmentosum-variant (XPV) results from defects in the Y-family DNA Polymerase Pol eta (Polη) and compensatory deployment of alternative inappropriate DNA polymerases. However, the extent to which dysregulated TLS contributes to the underlying etiology of other human cancers is unclear. Here we consider the broad impact of TLS polymerases on tumorigenesis and cancer therapy. We survey the ways in which TLS DNA polymerases are pathologically altered in cancer. We summarize evidence that TLS polymerases shape cancer genomes, and review studies implicating dysregulated TLS as a driver of carcinogenesis. Because many cancer treatment regimens comprise DNA-damaging agents, pharmacological inhibition of TLS is an attractive strategy for sensitizing tumors to genotoxic therapies. Therefore, we discuss the pharmacological tractability of the TLS pathway and summarize recent progress on development of TLS inhibitors for therapeutic purposes.

RNA-Based Classification of Homologous Recombination Deficiency in Racially Diverse Patients with Breast Cancer.

BACKGROUND: Aberrant expression of DNA repair pathways such as homologous recombination (HR) can lead to DNA repair imbalance, genomic instability, and altered chemotherapy response. DNA repair imbalance may predict prognosis, but variation in DNA repair in diverse cohorts of breast cancer patients is understudied. METHODS: To identify RNA-based patterns of DNA repair expression, we performed unsupervised clustering on 51 DNA repair-related genes in the Cancer Genome Atlas Breast Cancer [TCGA BRCA (n = 1,094)] and Carolina Breast Cancer Study [CBCS (n = 1,461)]. Using published DNA-based HR deficiency (HRD) scores (high-HRD ≥ 42) from TCGA, we trained an RNA-based supervised classifier. Unsupervised and supervised HRD classifiers were evaluated in association with demographics, tumor characteristics, and clinical outcomes. RESULTS: : Unsupervised clustering on DNA repair genes identified four clusters of breast tumors, with one group having high expression of HR genes. Approximately 39.7% of CBCS and 29.3% of TCGA breast tumors had this unsupervised high-HRD (U-HRD) profile. A supervised HRD classifier (S-HRD) trained on TCGA had 84% sensitivity and 73% specificity to detect HRD-high samples. Both U-HRD and S-HRD tumors in CBCS had higher frequency of TP53 mutant-like status (45% and 41% enrichment) and basal-like subtype (63% and 58% enrichment). S-HRD high was more common among black patients. Among chemotherapy-treated participants, recurrence was associated with S-HRD high (HR: 2.38, 95% confidence interval = 1.50-3.78). CONCLUSIONS: HRD is associated with poor prognosis and enriched in the tumors of black women. IMPACT: RNA-level indicators of HRD are predictive of breast cancer outcomes in diverse populations.

Discovery and Structural Basis of the Selectivity of Potent Cyclic Peptide Inhibitors of MAGE-A4.

MAGE proteins are cancer testis antigens (CTAs) that are characterized by highly conserved MAGE homology domains (MHDs) and are increasingly being found to play pivotal roles in promoting aggressive cancer types. MAGE-A4, in particular, increases DNA damage tolerance and chemoresistance in a variety of cancers by stabilizing the E3-ligase RAD18 and promoting trans-lesion synthesis (TLS). Inhibition of the MAGE-A4:RAD18 axis could sensitize cancer cells to chemotherapeutics like platinating agents. We use an mRNA display of thioether cyclized peptides to identify a series of potent and highly selective macrocyclic inhibitors of the MAGE-A4:RAD18 interaction. Co-crystal structure indicates that these inhibitors bind in a pocket that is conserved across MHDs but take advantage of A4-specific residues to achieve high isoform selectivity. Cumulatively, our data represent the first reported inhibitor of the MAGE-A4:RAD18 interaction and establish biochemical tools and structural insights for the future development of MAGE-A4-targeted cellular probes.

Four-Year Disease-Free Remission in a Patient With POLE Mutation-Associated Colorectal Cancer Treated Using Anti-PD-1 Therapy.

The stability of the human genome depends upon a delicate balance between replication by high- and low-fidelity DNA polymerases. Aberrant replication by error-prone polymerases or loss of function of high-fidelity polymerases predisposes to genetic instability and, in turn, cancer. DNA polymerase epsilon (Pol ε) is a high-fidelity, processive polymerase that is responsible for the majority of leading strand synthesis, and mutations in Pol ε have been increasingly associated with various human malignancies. The clinical significance of Pol ε mutations, including how and whether they should influence management decisions, remains poorly understood. In this report, we describe a 24-year-old man with an aggressive stage IV high-grade, poorly differentiated colon carcinoma who experienced a dramatic response to single-agent checkpoint inhibitor immunotherapy after rapidly progressing on standard chemotherapy. His response was complete and durable and has been maintained for more than 48 months. Genetic testing revealed a P286R mutation in the endonuclease domain of POLE and an elevated tumor mutational burden of 126 mutations per megabase, both of which have been previously associated with response to immunotherapy. Interestingly, tumor staining for PD-L1 was negative. This case study highlights the importance of genetic profiling of both early and late-stage cancers, the clinical significance of POLE mutations, and how the interplay between genetic instability and immune-checkpoint blockade can impact clinical decision-making.

Unravelling roles of error-prone DNA polymerases in shaping cancer genomes.

Mutagenesis is a key hallmark and enabling characteristic of cancer cells, yet the diverse underlying mutagenic mechanisms that shape cancer genomes are not understood. This review will consider the emerging challenge of determining how DNA damage response pathways-both tolerance and repair-act upon specific forms of DNA damage to generate mutations characteristic of tumors. DNA polymerases are typically the ultimate mutagenic effectors of DNA repair pathways. Therefore, understanding the contributions of DNA polymerases is critical to develop a more comprehensive picture of mutagenic mechanisms in tumors. Selection of an appropriate DNA polymerase-whether error-free or error-prone-for a particular DNA template is critical to the maintenance of genome stability. We review different modes of DNA polymerase dysregulation including mutation, polymorphism, and over-expression of the polymerases themselves or their associated activators. Based upon recent findings connecting DNA polymerases with specific mechanisms of mutagenesis, we propose that compensation for DNA repair defects by error-prone polymerases may be a general paradigm molding the mutational landscape of cancer cells. Notably, we demonstrate that correlation of error-prone polymerase expression with mutation burden in a subset of patient tumors from The Cancer Genome Atlas can identify mechanistic hypotheses for further testing. We contrast experimental approaches from broad, genome-wide strategies to approaches with a narrower focus on a few hundred base pairs of DNA. In addition, we consider recent developments in computational annotation of patient tumor data to identify patterns of mutagenesis. Finally, we discuss the innovations and future experiments that will develop a more comprehensive portrait of mutagenic mechanisms in human tumors.

Rad18 mediates specific mutational signatures and shapes the genomic landscape of carcinogen-induced tumors in vivo.

The E3 ubiquitin ligase Rad18 promotes a damage-tolerant and error-prone mode of DNA replication termed trans-lesion synthesis that is pathologically activated in cancer. However, the impact of vertebrate Rad18 on cancer genomes is not known. To determine how Rad18 affects mutagenesis in vivo, we have developed and implemented a novel computational pipeline to analyze genomes of carcinogen (7, 12-Dimethylbenz[a]anthracene, DMBA)-induced skin tumors from Rad18+/+ and Rad18- / - mice. We show that Rad18 mediates specific mutational signatures characterized by high levels of A(T)>T(A) single nucleotide variations (SNVs). In Rad18- /- tumors, an alternative mutation pattern arises, which is characterized by increased numbers of deletions >4 bp. Comparison with annotated human mutational signatures shows that COSMIC signature 22 predominates in Rad18+/+ tumors whereas Rad18- / - tumors are characterized by increased contribution of COSMIC signature 3 (a hallmark of BRCA-mutant tumors). Analysis of The Cancer Genome Atlas shows that RAD18 expression is strongly associated with high SNV burdens, suggesting RAD18 also promotes mutagenesis in human cancers. Taken together, our results show Rad18 promotes mutagenesis in vivo, modulates DNA repair pathway choice in neoplastic cells, and mediates specific mutational signatures that are present in human tumors.

An introduction for the special issue on environmental health and genome integrity.

Environmental exposures and genome maintenance mechanisms that respond to environmentally-induced genotoxicity have a profound impact on human health. Eight review articles in this Special Issue (SI) titled "Environmental Health and Genome Integrity" describe emerging new mechanisms by which distinct forms of environmentally-induced DNA damage are remediated, and explain how DNA repair pathway choices impact genome integrity and disease propensity. Here, we provide an introduction to reviews from this SI. Our expanding knowledge of how genotoxic exposures impact the genome will allow us to better predict, prevent and treat environmentally-induced human diseases such as cancer and neurodegenerative disorders.

Roles of Chk2/CHEK2 in guarding against environmentally induced DNA damage and replication-stress.

Checkpoint kinase 2 (human CHEK2; murine Chk2) is a critical mediator of the DNA damage response and has established roles in DNA double strand break (DSB)-induced apoptosis and cell cycle arrest. DSBs may be invoked directly by ionizing radiation but may also arise indirectly from environmental exposures such as solar ultraviolet (UV) radiation. The primary forms of DNA damage induced by UV are DNA photolesions (such as cyclobutane pyrimidine dimers CPD and 6-4 photoproducts) which interfere with DNA synthesis and lead to DNA replication fork stalling. Persistently stalled and unresolved DNA replication forks can "collapse" to generate DSBs that induce signaling via Chk2 and its upstream activator the ataxia telangiectasia-mutated (ATM) protein kinase. This review focuses on recently defined roles of Chk2 in protecting against DNA replication-associated genotoxicity. Several DNA damage response factors such as Rad18, Nbs1 and Chk1 suppress stalling and collapse of DNA replication forks. Defects in the primary responders to DNA replication fork stalling lead to generation of DSB and reveal "back-up" roles for Chk2 in S-phase progression and genomic stability. In humans, there are numerous variants of the CHEK2 gene, including CHEK2*1100delC. Individuals with the CHEK2*1100delC germline alteration have an increased risk of developing breast cancer and malignant melanoma. DNA replication fork-stalling at estrogen-DNA adducts and UV-induced photolesions are implicated in the etiology of breast cancer and melanoma, respectively. It is likely therefore that the Chk2/CHEK2-deficiency is associated with elevated risk for tumorigenesis caused by replication-associated genotoxicities that are exacerbated by environmental genotoxins and intrinsic DNA-damaging agents.

Irreversible JNK1-JUN inhibition by JNK-IN-8 sensitizes pancreatic cancer to 5-FU/FOLFOX chemotherapy.

Over 55,000 people in the United States are diagnosed with pancreatic ductal adenocarcinoma (PDAC) yearly, and fewer than 20% of these patients survive a year beyond diagnosis. Chemotherapies are considered or used in nearly every PDAC case, but there is limited understanding of the complex signaling responses underlying resistance to these common treatments. Here, we take an unbiased approach to study protein kinase network changes following chemotherapies in patient-derived xenograft (PDX) models of PDAC to facilitate design of rational drug combinations. Proteomics profiling following chemotherapy regimens reveals that activation of JNK-JUN signaling occurs after 5-fluorouracil plus leucovorin (5-FU + LEU) and FOLFOX (5-FU + LEU plus oxaliplatin [OX]), but not after OX alone or gemcitabine. Cell and tumor growth assays with the irreversible inhibitor JNK-IN-8 and genetic manipulations demonstrate that JNK and JUN each contribute to chemoresistance and cancer cell survival after FOLFOX. Active JNK1 and JUN are specifically implicated in these effects, and synergy with JNK-IN-8 is linked to FOLFOX-mediated JUN activation, cell cycle dysregulation, and DNA damage response. This study highlights the potential for JNK-IN-8 as a biological tool and potential combination therapy with FOLFOX in PDAC and reinforces the need to tailor treatment to functional characteristics of individual tumors.

Mass spectrometry-based selectivity profiling identifies a highly selective inhibitor of the kinase MELK that delays mitotic entry in cancer cells.

The maternal embryonic leucine zipper kinase (MELK) has been implicated in the regulation of cancer cell proliferation. RNAi-mediated MELK depletion impairs growth and causes G2/M arrest in numerous cancers, but the mechanisms underlying these effects are poorly understood. Furthermore, the MELK inhibitor OTSSP167 has recently been shown to have poor selectivity for MELK, complicating the use of this inhibitor as a tool compound to investigate MELK function. Here, using a cell-based proteomics technique called multiplexed kinase inhibitor beads/mass spectrometry (MIB/MS), we profiled the selectivity of two additional MELK inhibitors, NVS-MELK8a (8a) and HTH-01-091. Our results revealed that 8a is a highly selective MELK inhibitor, which we further used for functional studies. Resazurin and crystal violet assays indicated that 8a decreases triple-negative breast cancer cell viability, and immunoblotting revealed that impaired growth is due to perturbation of cell cycle progression rather than induction of apoptosis. Using double-thymidine synchronization and immunoblotting, we observed that MELK inhibition delays mitotic entry, which was associated with delayed activation of Aurora A, Aurora B, and cyclin-dependent kinase 1 (CDK1). Following this delay, cells entered and completed mitosis. Using live-cell microscopy of cells harboring fluorescent proliferating cell nuclear antigen, we confirmed that 8a significantly and dose-dependently lengthens G2 phase. Collectively, our results provide a rationale for using 8a as a tool compound for functional studies of MELK and indicate that MELK inhibition delays mitotic entry, likely via transient G2/M checkpoint activation.