SUMMARY It is intuitively obvious that the ability of a cell to repair DNA damage is saturable, either by limitation of enzymatic activities, the time allotted to achieve their function, or both. However, very little is known regarding the mechanisms that establish such a threshold. Here we demonstrated that the CUL4A ubiquitin ligase restricts the cellular repair capacity by orchestrating the concerted actions of nucleotide excision repair (NER) and the DNA damage-responsive G1/S checkpoint through selective degradation of the DDB2 and XPC DNA damage sensors and the p21/CIP1/WAF1 checkpoint effector. We generated Cul4a conditional knockout mice and observed that skin-specific Cul4a ablation dramatically increased resistance to UV-induced skin carcinogenesis. Our findings reveal that wild-type cells do not operate at their full DNA repair potential, underscore the critical role of CUL4A in establishing the cellular DNA repair threshold, and highlight the potential augmentation of cellular repair proficiency by pharmacological CUL4A inhibition.

The cullin 4 subfamily of genes includes CUL4A and CUL4B, which share a mostly identical amino acid sequence aside from the elongated N-terminal region in CUL4B. Both act as scaffolding proteins for modular cullin RING ligase 4 (CRL4) complexes which promote the ubiquitination of a variety of substrates. CRL4 function is vital to cells as loss of both genes or their shared substrate adaptor protein DDB1 halts proliferation and eventually leads to cell death. Due to their high structural similarity, CUL4A and CUL4B share a substantial overlap in function. However, in some cases, differences in subcellular localization, spatiotemporal expression patterns and stress-inducibility preclude functional compensation. In this review, we highlight the most essential functions of the CUL4 genes in: DNA repair and replication, chromatin-remodeling, cell cycle regulation, embryogenesis, hematopoiesis and spermatogenesis. CUL4 genes are also clinically relevant as dysregulation can contribute to the onset of cancer and CRL4 complexes are often hijacked by certain viruses to promote viral replication and survival. Also, mutations in CUL4B have been implicated in a subset of patients suffering from syndromic X-linked intellectual disability (AKA mental retardation). Interestingly, the antitumor effects of immunomodulatory drugs are caused by their binding to the CRL4CRBN complex and re-directing the E3 ligase towards the Ikaros transcription factors IKZF1 and IKZF3. Because of their influence over key cellular functions and relevance to human disease, CRL4s are considered promising targets for therapeutic intervention.

Aims Triple-negative breast cancer comprises a clinically aggressive group of invasive carcinomas. We examined a published gene expression screen of a panel of breast cancer cell lines to identify a potential triple-negative breast cancer-specific gene signature, and attempted to verify our findings by performing immunohistochemical analysis on tissue microarrays containing a large cohort of invasive breast carcinomas. Methods The microarray dataset for a panel of human breast cancer cell lines was interrogated for triple-negative breast cancer-specific genes. Membranous immunohistochemical expression of the protein product of the AXL gene was assessed semiquantitatively in 569 invasive breast carcinomas grouped according to molecular subgroup by immunohistochemistry. Results AXL was significantly upregulated in triple-negative/basal B cell lines compared with luminal or basal A cell lines. No significant difference was observed in the level of immunohistochemical expression of Axl protein between triple-negative breast cancers and other molecular subgroups (p=0.257). Axl expression was significantly associated with lymphovascular invasion (LVI) in all subgroups combined (p=0.033), and within the luminal A (p=0.002) and triple-negative breast cancer subgroups (p=0.026). Conclusions Despite preferential upregulation of AXL in triple-negative/basal B cell lines, analysis of Axl protein expression in a large series of patients’ breast tumours revealed no association between Axl expression and triple-negative breast cancer or other subtype. The association of Axl expression with LVI supports previous work that implicates Axl as a promoter of invasiveness in breast cancer cell lines. Further studies are necessary to explore whether Axl expression of individual breast cancer tumours can be clinically useful.

Eukaryotic cells repair ultraviolet light (UV)-and chemical carcinogen-induced DNA stranddistorting damage through the nucleotide excision repair (NER) pathway. Concurrent activation of the DNA damage checkpoints is also required to arrest the cell cycle and allow time for NER action. Recent studies uncovered critical roles for ubiquitin-mediated post-translational modifications in controlling both NER and checkpoint functions. In this review, we will discuss recent progress in delineating the roles of cullin-RING E3 ubiquitin ligases in orchestrating the cellular DNA damage response through ubiquitination of NER factors, histones, and checkpoint effectors.

Cyclin-dependent kinase (CDK)4 and CDK6 are frequently overexpressed or hyperactivated in human cancers. Targeting CDK4/CDK6 in combination with cytotoxic killing therefore represents a rational approach to cancer therapy. By selective inhibition of CDK4/CDK6 with PD 0332991, which leads to early G1 arrest and synchronous S phase entry upon release of the G1 block, we have developed a novel strategy to prime acute myeloid leukemia (AML) cells for cytotoxic killing by cytarabine (Ara-C). This sensitization is achieved in part through enrichment of S-phase cells, which maximizes the AML populations for Ara-C incorporation into replicating DNA to elicit DNA damage. Moreover, PD 0332991 trigged apoptosis of AML cells through inhibition of the homeobox (HOX)A9 oncogene expression, reducing the transcription of its target PIM1. Reduced PIM1 synthesis attenuates PIM1-mediated phosphorylation of the pro-apoptotic BAD and activates BAD-dependent apoptosis. In vivo, timely inhibition of CDK4/CDK6 by PD 0332991 and release profoundly suppresses tumor growth in response to reduced doses of Ara-C in a xenograft AML model. Collectively, these data suggest selective and reversible inhibition of CDK4/CDK6 as an effective means to enhance Ara-C killing of AML cells at reduced doses, which has implications for the treatment of elderly AML patients who are unable to tolerate high dose Ara-C therapy.