Search

Oncogene-induced replication stress characterizes many aggressive cancers. Several treatments are being developed that target replication stress, however, identification of tumors with high levels of replication stress remains challenging. We describe a gene expression signature of oncogene-induced replication stress. A panel of triple-negative breast cancer (TNBC) and non-transformed cell lines were engineered to overexpress CDC25A, CCNE1 or MYC, which resulted in slower replication kinetics. RNA sequencing analysis revealed a set of 52 commonly upregulated genes. In parallel, mRNA expression analysis of patient-derived tumor samples (TCGA, n = 10,592) also revealed differential gene expression in tumors with amplification of oncogenes that trigger replication stress (CDC25A, CCNE1, MYC, CCND1, MYB, MOS, KRAS, ERBB2, and E2F1). Upon integration, we identified a six-gene signature of oncogene-induced replication stress (NAT10, DDX27, ZNF48, C8ORF33, MOCS3, and MPP6). Immunohistochemical analysis of NAT10 in breast cancer samples (n = 330) showed strong correlation with expression of phospho-RPA (R = 0.451, p = 1.82 x 10(-20)) and gamma H2AX (R = 0.304, p = 2.95 x 10(-9)). Finally, we applied our oncogene-induced replication stress signature to patient samples from TCGA (n = 8,862) and GEO (n = 13,912) to define the levels of replication stress across 27 tumor subtypes, identifying diffuse large B cell lymphoma, ovarian cancer, TNBC and colorectal carcinoma as cancer subtypes with high levels of oncogene-induced replication stress.

Oncogene-induced replication stress, for instance as a result of Cyclin E1 overexpression, causes genomic instability and has been linked to tumorigenesis. To survive high levels of replication stress, tumors depend on pathways to deal with these DNA lesions, which represent a therapeutically actionable vulnerability. We aimed to uncover the consequences of Cyclin E1 or Cdc25A overexpression on replication kinetics, mitotic progression, and the sensitivity to inhibitors of the WEE1 and ATR replication checkpoint kinases. We modeled oncogene-induced replication stress using inducible expression of Cyclin E1 or Cdc25A in non-transformed RPE-1 cells, either in a TP53 wild-type or TP53-mutant background. DNA fiber analysis showed Cyclin E1 or Cdc25A overexpression to slow replication speed. The resulting replication-derived DNA lesions were transmitted into mitosis causing chromosome segregation defects. Single cell sequencing revealed that replication stress and mitotic defects upon Cyclin E1 or Cdc25A overexpression resulted in genomic instability. ATR or WEE1 inhibition exacerbated the mitotic aberrancies induced by Cyclin E1 or Cdc25A overexpression, and caused cytotoxicity. Both these phenotypes were exacerbated upon p53 inactivation. Conversely, downregulation of Cyclin E1 rescued both replication kinetics, as well as sensitivity to ATR and WEE1 inhibitors. Taken together, Cyclin E1 or Cdc25A-induced replication stress leads to mitotic segregation defects and genomic instability. These mitotic defects are exacerbated by inhibition of ATR or WEE1 and therefore point to mitotic catastrophe as an underlying mechanism. Importantly, our data suggest that Cyclin E1 overexpression can be used to select patients for treatment with replication checkpoint inhibitors.

BackgroundOncogene-induced replication stress characterizes many aggressive cancers, including triple-negative breast cancer (TNBC). Several drugs are being developed that target replication stress, although it is unclear how tumors with high levels of replication stress can be identified. We aimed to develop a gene expression signature of oncogene-induced replication stress.MethodsTNBC and non-transformed RPE1-TP53wt and RPE1-TP53mut cell lines were engineered to overexpress the oncogenes CDC25A, CCNE1 or MYC. DNA fiber analysis was used to measure replication kinetics. Analysis of RNA sequencing data of cell lines and patient-derived tumor samples (TCGA n=10,592) was used to identify differential gene expression. Immunohistochemical validation was conducted on breast cancer samples (n=330).ResultsRNA sequencing revealed 52 commonly upregulated genes after induction of CDC25A, CCNE1 or MYC in our cell line panel. Integration with gene expression data of TGCA samples with amplification of replication stress-inducing oncogenes (CDC25A, CCNE1, MYC, CCND1, MYB, MOS, KRAS, ERBB2, and E2F1), yielded a six-gene signature of oncogene-induced replication stress (NAT10, DDX27, ZNF48, C8ORF33, MOCS3, and MPP6). Expression of NAT10 in breast cancer samples was correlated with phospho-RPA (R=0.451, p=1.82×10−20) and γH2AX (R=0.304, p=2.95×10−9). Applying the oncogene-induced replication stress signature to patient samples (TCGA n=8,862 and GEO n=13,912) defined the replication stress landscape across 27 tumor subtypes, and identified diffuse large B cell lymphoma, ovarian cancer, TNBC and colorectal carcinoma as cancer subtypes with high levels of oncogene-induced replication stress.ConclusionWe developed a gene expression signature of oncogene-induced replication stress, which may facilitate patient selection for agents that target replication stress.

Genomic instability is a hallmark of cancer for which an important cause is the DNA damage generated by oncogene-induced replication stress (RS). Genomically instable cancers increasingly depend on checkpoint kinases, including ATR and WEE1, to activate the DNA damage response, pointing towards therapeutic strategies that target elevated levels of RS as an Achilles’ heel in cancer. However, the cellular consequences of RS remain unclear, and tools to identify tumors with high levels of RS are lacking.In this thesis we aimed to uncover the biological effects of oncogene-induced RS on tumor cells, uncover therapeutic opportunities of cancers that are characterized by high levels of RS, and develop tools to improve the identification of tumors with RS.Immunohistochemical analysis of RS levels in relation to oncogene expression in breast cancer tissues, revealed that TNBCs exhibited the highest levels of RS, which were correlated with expression of the Cyclin E1 and c-Myc oncogenes.In parallel, we studied the consequences of oncogene overexpression on replication kinetics and mitotic progression in experimental models, which show that Cyclin E1 or Cdc25A-induced RS leads to genomic instability, exacerbated by inhibition of ATR or WEE1.Finally, we developed an mRNA expression-based signature for oncogene-induced RS to categorize tumors based on their RS levels, which identified DLBCL, ovarian cancer and TNBC as cancers with elevated RS. Taken together, we have mapped the cellular consequences of oncogene-induced RS in experimental models and patient samples, and have developed a tool to facilitate patient selection for therapeutic approaches that target RS.

Replication stress entails the improper progression of DNA replication. In cancer cells, including breast cancer cells, an important cause of replication stress is oncogene activation. Importantly, tumors with high levels of replication stress may have different clinical behavior, and high levels of replication stress appear to be a vulnerability of cancer cells, which may be therapeutically targeted by novel molecularly targeted agents. Unfortunately, data on replication stress is largely based on experimental models. Further investigation of replication stress in clinical samples is required to optimally implement novel therapeutics. To uncover the relation between oncogene expression, replication stress, and clinical features of breast cancer subgroups, we immunohistochemically analyzed the expression of a panel of oncogenes (Cyclin E, c-Myc, and Cdc25A,) and markers of replication stress (phospho-Ser33-RPA32 and γ-H2AX) in breast tumor tissues prior to treatment (n = 384). Triple-negative breast cancers (TNBCs) exhibited the highest levels of phospho-Ser33-RPA32 (P P P P r = 0.37, P r = 0.63, P

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

Summary: The brain cancer medulloblastoma consists of different transcriptional subgroups. To characterize medulloblastoma at the phosphoprotein-signaling level, we performed high-throughput peptide phosphorylation profiling on a large cohort of SHH (Sonic Hedgehog), group 3, and group 4 medulloblastomas. We identified two major protein-signaling profiles. One profile was associated with rapid death post-recurrence and resembled MYC-like signaling for which MYC lesions are sufficient but not necessary. The second profile showed enrichment for DNA damage, as well as apoptotic and neuronal signaling. Integrative analysis demonstrated that heterogeneous transcriptional input converges on these protein-signaling profiles: all SHH and a subset of group 3 patients exhibited the MYC-like protein-signaling profile; the majority of the other group 3 subset and group 4 patients displayed the DNA damage/apoptotic/neuronal signaling profile. Functional analysis of enriched pathways highlighted cell-cycle progression and protein synthesis as therapeutic targets for MYC-like medulloblastoma. : Using peptide phosphorylation profiling, Zomerman et al. identify two medulloblastoma phosphoprotein-signaling profiles that have prognostic value and are potentially targetable. They find that these profiles extend across transcriptome-based subgroup borders. This suggests that diverse genetic information converges on common protein-signaling pathways and highlights protein-signaling as a unique information layer. Keywords: medulloblastoma, protein-signaling, protein synthesis, MYC, TP53, proteome, phosphoproteome

Genomically instable cancers are characterized by progressive loss and gain of chromosomal fragments, and the acquisition of complex genomic rearrangements. Such cancers, including triple-negative breast cancers and high-grade serous ovarian cancers, typically show aggressive behavior and lack actionable driver oncogenes. Increasingly, oncogene-induced replication stress or defective replication fork maintenance is considered an important driver of genomic instability. Paradoxically, while replication stress causes chromosomal instability and thereby promotes cancer development, it intrinsically poses a threat to cellular viability. Apparently, tumor cells harboring high levels of replication stress have evolved ways to cope with replication stress. As a consequence, therapeutic targeting of such compensatory mechanisms is likely to preferentially target cancers with high levels of replication stress and may prove useful in potentiating chemotherapeutic approaches that exert their effects by interfering with DNA replication. Here, we discuss how replication stress drives chromosomal instability, and the cell cycle-regulated mechanisms that cancer cells employ to deal with replication stress. Importantly, we discuss how mechanisms involving DNA structure-specific resolvases, cell cycle checkpoint kinases and mitotic processing of replication intermediates offer possibilities in developing treatments for difficult-to-treat genomically instable cancers.

Replication stress entails the improper progression of DNA replication. In cancer cells, including breast cancer cells, an important cause of replication stress is oncogene activation. Importantly, tumors with high levels of replication stress may have different clinical behavior, and high levels of replication stress appear to be a vulnerability of cancer cells, which may be therapeutically targeted by novel molecularly targeted agents. Unfortunately, data on replication stress is largely based on experimental models. Further investigation of replication stress in clinical samples is required to optimally implement novel therapeutics. To uncover the relation between oncogene expression, replication stress and clinical features of breast cancer subtypes, we immunohistochemically analyzed the expression of a panel of oncogenes (Cdc25a, Cyclin E and c-Myc) and markers of replication stress (phospho-Ser33-RPA32 and γ-H2AX) in treatment-naive breast tumor tissues (n=384). Triple-negative breast cancers (TNBCs) exhibited the highest levels of phospho-Ser33-RPA32 (PPPPPP

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have recently entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, drug resistance is a clinical hurdle, and we poorly understand how cancer cells escape the deadly effects of PARPi without restoring the HR pathway. By combining genetic screens with multi-omics analysis of matched PARPi-sensitive and -resistant Brca2-mutated mouse mammary tumors, we identified loss of PAR glycohydrolase (PARG) as a major resistance mechanism. We also found the presence of PARG-negative clones in a subset of human serous ovarian and triple-negative breast cancers. PARG depletion restores PAR formation and partially rescues PARP1 signaling. Importantly, PARG inactivation exposes vulnerabilities that can be exploited therapeutically. Gogola et al. show loss of poly(ADP-ribose) glycohydrolase (PARG) confers resistance of BRCA2-deficient tumor cells to PARP inhibition by restoring PAR formation, controlled DNA replication fork progression, and the recruitment of downstream DNA repair factors while sensitizing them to ionizing radiation and temozolomide.

The brain cancer medulloblastoma consists of different transcriptional subgroups. To characterize medulloblastoma at the phosphoprotein-signaling level, we performed high-throughput peptide phosphorylation profiling on a large cohort of SHH (Sonic Hedgehog), group 3, and group 4 medulloblastomas. We identified two major protein-signaling profiles. One profile was associated with rapid death post-recurrence and resembled MYC-like signaling for which MYC lesions are sufficient but not necessary. The second profile showed enrichment for DNA damage, as well as apoptotic and neuronal signaling. Integrative analysis demonstrated that heterogeneous transcriptional input converges on these protein-signaling profiles: all SHH and a subset of group 3 patients exhibited the MYC-like protein-signaling profile; the majority of the other group 3 subset and group 4 patients displayed the DNA damage/apoptotic/neuronal signaling profile. Functional analysis of enriched pathways highlighted cell-cycle progression and protein synthesis as therapeutic targets for MYC-like medulloblastoma.