Targeting CaMKK2 Inhibits Actin Cytoskeletal Assembly to Suppress Cancer Metastasis.

Triple-negative breast cancers (TNBC) tend to become invasive and metastatic at early stages in their development. Despite some treatment successes in early-stage localized TNBC, the rate of distant recurrence remains high, and long-term survival outcomes remain poor. In a search for new therapeutic targets for this disease, we observed that elevated expression of the serine/threonine kinase calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) is highly correlated with tumor invasiveness. In validation studies, genetic disruption of CaMKK2 expression or inhibition of its activity with small molecule inhibitors disrupted spontaneous metastatic outgrowth from primary tumors in murine xenograft models of TNBC. High-grade serous ovarian cancer (HGSOC), a high-risk, poor prognosis ovarian cancer subtype, shares many features with TNBC, and CaMKK2 inhibition effectively blocked metastatic progression in a validated xenograft model of this disease. Mechanistically, CaMKK2 increased the expression of the phosphodiesterase PDE1A, which hydrolyzed cyclic guanosine monophosphate (cGMP) to decrease the cGMP-dependent activity of protein kinase G1 (PKG1). Inhibition of PKG1 resulted in decreased phosphorylation of vasodilator-stimulated phosphoprotein (VASP), which in its hypophosphorylated state binds to and regulates F-actin assembly to facilitate cell movement. Together, these findings establish a targetable CaMKK2-PDE1A-PKG1-VASP signaling pathway that controls cancer cell motility and metastasis by impacting the actin cytoskeleton. Furthermore, it identifies CaMKK2 as a potential therapeutic target that can be exploited to restrict tumor invasiveness in patients diagnosed with early-stage TNBC or localized HGSOC. SIGNIFICANCE: CaMKK2 regulates actin cytoskeletal dynamics to promote tumor invasiveness and can be inhibited to suppress metastasis of breast and ovarian cancer, indicating CaMKK2 inhibition as a therapeutic strategy to arrest disease progression.

A widespread length-dependent splicing dysregulation in cancer.

Dysregulation of alternative splicing is a key molecular hallmark of cancer. However, the common features and underlying mechanisms remain unclear. Here, we report an intriguing length-dependent splicing regulation in cancers. By systematically analyzing the transcriptome of thousands of cancer patients, we found that short exons are more likely to be mis-spliced and preferentially excluded in cancers. Compared to other exons, cancer-associated short exons (CASEs) are more conserved and likely to encode in-frame low-complexity peptides, with functional enrichment in GTPase regulators and cell adhesion. We developed a CASE-based panel as reliable cancer stratification markers and strong predictors for survival, which is clinically useful because the detection of short exon splicing is practical. Mechanistically, mis-splicing of CASEs is regulated by elevated transcription and alteration of certain RNA binding proteins in cancers. Our findings uncover a common feature of cancer-specific splicing dysregulation with important clinical implications in cancer diagnosis and therapies.

Adaptive stress response genes associated with breast cancer subtypes and survival outcomes reveal race-related differences.

Aggressive breast cancer variants, like triple negative and inflammatory breast cancer, contribute to disparities in survival and clinical outcomes among African American (AA) patients compared to White (W) patients. We previously identified the dominant role of anti-apoptotic protein XIAP in regulating tumor cell adaptive stress response (ASR) that promotes a hyperproliferative, drug resistant phenotype. Using The Cancer Genome Atlas (TCGA), we identified 46-88 ASR genes that are differentially expressed (2-fold-change and adjusted p-value < 0.05) depending on PAM50 breast cancer subtype. On average, 20% of all 226 ASR genes exhibited race-related differential expression. These genes were functionally relevant in cell cycle, DNA damage response, signal transduction, and regulation of cell death-related processes. Moreover, 23% of the differentially expressed ASR genes were associated with AA and/or W breast cancer patient survival. These identified genes represent potential therapeutic targets to improve breast cancer outcomes and mitigate associated health disparities.

Dysregulation of the ESRP2-NF2-YAP/TAZ axis promotes hepatobiliary carcinogenesis in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD), the hepatic correlate of the metabolic syndrome, is a major risk factor for hepatobiliary cancer (HBC). Although chronic inflammation is thought to be the root cause of all these diseases, the mechanism whereby it promotes HBC in NAFLD remains poorly understood. Herein, we aim to evaluate the hypothesis that inflammation-related dysregulation of the ESRP2-NF2-YAP/TAZ axis promotes HB carcinogenesis. METHODS: We use murine NAFLD models, liver biopsies from patients with NAFLD, human liver cancer registry data, and studies in liver cancer cell lines. RESULTS: Our results confirm the hypothesis that inflammation-related dysregulation of the ESRP2-NF2-YAP/TAZ axis promotes HB carcinogenesis, supporting a model whereby chronic inflammation suppresses hepatocyte expression of ESRP2, an RNA splicing factor that directly targets and activates NF2, a tumor suppressor that is necessary to constrain YAP/TAZ activation. The resultant loss of NF2 function permits sustained YAP/TAZ activity that drives hepatocyte proliferation and de-differentiation. CONCLUSION: Herein, we report on a novel mechanism by which chronic inflammation leads to sustained activation of YAP/TAZ activity; this imposes a selection pressure that favors liver cells with mutations enabling survival during chronic oncogenic stress. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) increases the risk of hepatobiliary carcinogenesis. However, the underlying mechanism remains unknown. Our study demonstrates that chronic inflammation suppresses hepatocyte expression of ESRP2, an adult RNA splicing factor that activates NF2. Thus, inactive (fetal) NF2 loses the ability to activate Hippo kinases, leading to the increased activity of downstream YAP/TAZ and promoting hepatobiliary carcinogenesis in chronically injured livers.

Differential alternative RNA splicing and transcription events between tumors from African American and White patients in The Cancer Genome Atlas.

Individuals of African ancestry suffer disproportionally from higher incidence, aggressiveness, and mortality for particular cancers. This disparity likely results from an interplay among differences in multiple determinants of health, including differences in tumor biology. We used The Cancer Genome Atlas (TCGA) SpliceSeq and TCGA aggregate expression datasets and identified differential alternative RNA splicing and transcription events (ARS/T) in cancers between self-identified African American (AA) and White (W) patients. We found that retained intron events were enriched among race-related ARS/T. In addition, on average, 12% of the most highly ranked race-related ARS/T overlapped between any two analyzed cancers. Moreover, the genes undergoing race-related ARS/T functioned in cancer-promoting pathways, and a number of race-related ARS/T were associated with patient survival. We built a web-application, CanSplice, to mine genomic datasets by self-identified race. The race-related targets have the potential to aid in the development of new biomarkers and therapeutics to mitigate cancer disparity.

Biological Aspects of Cancer Health Disparities.

Racial and ethnic disparities span the continuum of cancer care and are driven by a complex interplay among social, psychosocial, lifestyle, environmental, health system, and biological determinants of health. Research is needed to identify these determinants of cancer health disparities and to develop interventions to achieve cancer health equity. Herein, we focus on the overall burden of ancestry-related molecular alterations, the functional significance of the alterations in hallmarks of cancer, and the implications of the alterations for precision oncology and immuno-oncology. In conclusion, we reflect on the importance of estimating ancestry, improving diverse racial and ethnic participation in cancer clinical trials, and examining the intersection among determinants of cancer health disparities.

RNA splicing and aggregate gene expression differences in lung squamous cell carcinoma between patients of West African and European ancestry.

OBJECTIVES: Despite disparities in lung cancer incidence and mortality, the molecular landscape of lung cancer in patients of African ancestry remains underexplored, and race-related differences in RNA splicing remain unexplored. MATERIALS AND METHODS: We identified differentially spliced genes (DSGs) and differentially expressed genes (DEGs) in biobanked lung squamous cell carcinoma (LUSC) between patients of West African and European ancestry, using ancestral genotyping and Affymetrix Clariom D array. DSGs and DEGs were validated independently using the National Cancer Institute Genomic Data Commons. Associated biological processes, overlapping canonical pathways, enriched gene sets, and cancer relevance were identified using Gene Ontology Consortium, Ingenuity Pathway Analysis, Gene Set Enrichment Analysis, and CancerMine, respectively. Association with LUSC survival was conducted using The Cancer Genome Atlas. RESULTS: 4,829 DSGs and 267 DEGs were identified, including novel targets in NSCLC as well as genes identified previously to have relevance to NSCLC. RNA splicing events within 3 DSGs as well as 1 DEG were validated in the independent cohort. 853 DSGs and 29 DEGs have been implicated as potential drivers, oncogenes and/or tumor suppressor genes. Biological processes enriched among DSGs and DEGs included metabolic process, biological regulation, and multicellular organismal process and, among DSGs, ion transport. Overlapping canonical pathways among DSGs included neuronal signaling pathways and, among DEGs, cell metabolism involving biosynthesis. Gene sets enriched among DSGs included KRAS Signaling, UV Response, E2 F Targets, Glycolysis, and Coagulation. 355 RNA splicing events within DSGs and 18 DEGs show potential association with LUSC patient survival. CONCLUSION: These DSGs and DEGs, which show potential biological and clinical relevance, could have the ability to drive novel biomarker and therapeutic development to mitigate LUSC disparities.

Alternative RNA Splicing as a Potential Major Source of Untapped Molecular Targets in Precision Oncology and Cancer Disparities.

Studies of alternative RNA splicing (ARS) have the potential to provide an abundance of novel targets for development of new biomarkers and therapeutics in oncology, which will be necessary to improve outcomes for patients with cancer and mitigate cancer disparities. ARS, a key step in gene expression enabling individual genes to encode multiple proteins, is emerging as a major driver of abnormal phenotypic heterogeneity. Recent studies have begun to identify RNA splicing-related genetic and genomic variation in tumors, oncogenes dysregulated by ARS, RNA splice variants driving race-related cancer aggressiveness and drug response, spliceosome-dependent transformation, and RNA splicing-related immunogenic epitopes in cancer. In addition, recent studies have begun to identify and test, preclinically and clinically, approaches to modulate and exploit ARS for therapeutic application, including splice-switching oligonucleotides, small molecules targeting RNA splicing or RNA splice variants, and combination regimens with immunotherapies. Although ARS data hold such promise for precision oncology, inclusion of studies of ARS in translational and clinical cancer research remains limited. Technologic developments in sequencing and bioinformatics are being routinely incorporated into clinical oncology that permit investigation of clinically relevant ARS events, yet ARS remains largely overlooked either because of a lack of awareness within the clinical oncology community or perceived barriers to the technical complexity of analyzing ARS. This perspective aims to increase such awareness, propose immediate opportunities to improve identification and analysis of ARS, and call for bioinformaticians and cancer researchers to work together to address the urgent need to incorporate ARS into cancer biology and precision oncology.

SLFN11 Blocks Stressed Replication Forks Independently of ATR.

SLFN11 sensitizes cancer cells to a broad range of DNA-targeted therapies. Here we show that, in response to replication stress induced by camptothecin, SLFN11 tightly binds chromatin at stressed replication foci via RPA1 together with the replication helicase subunit MCM3. Unlike ATR, SLFN11 neither interferes with the loading of CDC45 and PCNA nor inhibits the initiation of DNA replication but selectively blocks fork progression while inducing chromatin opening across replication initiation sites. The ATPase domain of SLFN11 is required for chromatin opening, replication block, and cell death but not for the tight binding of SLFN11 to chromatin. Replication stress by the CHK1 inhibitor Prexasertib also recruits SLFN11 to nascent replicating DNA together with CDC45 and PCNA. We conclude that SLFN11 is recruited to stressed replication forks carrying extended RPA filaments where it blocks replication by changing chromatin structure across replication sites.

TDP1 is Critical for the Repair of DNA Breaks Induced by Sapacitabine, a Nucleoside also Targeting ATM- and BRCA-Deficient Tumors.

2'-C-cyano-2'-deoxy-1-ß-d-arabino-pentofuranosylcytosine (CNDAC) is the active metabolite of the anticancer drug, sapacitabine. CNDAC is incorporated into the genome during DNA replication and subsequently undergoes ß-elimination that generates single-strand breaks with abnormal 3'-ends. Because tyrosyl-DNA phosphodiesterase 1 (TDP1) selectively hydrolyzes nonphosphorylated 3'-blocking ends, we tested its role in the repair of CNDAC-induced DNA damage. We show that cells lacking TDP1 (avian TDP1-/- DT40 cells and human TDP1 KO TSCER2 and HCT116 cells) exhibit marked hypersensitivity to CNDAC. We also identified BRCA1, FANCD2, and PCNA in the DNA repair pathways to CNDAC. Comparing CNDAC with the chemically related arabinosyl nucleoside analog, cytosine arabinoside (cytarabine, AraC) and the topoisomerase I inhibitor camptothecin (CPT), which both generate 3'-end blocking DNA lesions that are also repaired by TDP1, we found that inactivation of BRCA2 renders cells hypersensitive to CNDAC and CPT but not to AraC. By contrast, cells lacking PARP1 were only hypersensitive to CPT but not to CNDAC or AraC. Examination of TDP1 expression in the cancer cell line databases (CCLE, GDSC, NCI-60) and human cancers (TCGA) revealed a broad range of expression of TDP1, which was correlated with PARP1 expression, TDP1 gene copy number and promoter methylation. Thus, this study identifies the importance of TDP1 as a novel determinant of response to CNDAC across various cancer types (especially non-small cell lung cancers), and demonstrates the differential involvement of BRCA2, PARP1, and TDP1 in the cellular responses to CNDAC, AraC, and CPT. Mol Cancer Ther; 16(11); 2543-51. ©2017 AACR.

Drug-Driven Synthetic Lethality: Bypassing Tumor Cell Genetics with a Combination of AsiDNA and PARP Inhibitors.

Purpose: Cancer treatments using tumor defects in DNA repair pathways have shown promising results but are restricted to small subpopulations of patients. The most advanced drugs in this field are PARP inhibitors (PARPi), which trigger synthetic lethality in tumors with homologous recombination (HR) deficiency. Using AsiDNA, an inhibitor of HR and nonhomologous end joining, together with PARPi should allow bypassing the genetic restriction for PARPi efficacy.Experimental Design: We characterized the DNA repair inhibition activity of PARPi (olaparib) and AsiDNA by monitoring repair foci formation and DNA damage. We analyzed the cell survival to standalone and combined treatments of 21 tumor cells and three nontumor cells. In 12 breast cancer (BC) cell lines, correlation with sensitivity to each drug and transcriptome were statistically analyzed to identify resistance pathways.Results: Molecular analyses demonstrate that olaparib and AsiDNA respectively prevent recruitment of XRCC1 and RAD51/53BP1 repair enzymes to damage sites. Combination of both drugs increases the accumulation of unrepaired damage resulting in an increase of cell death in all tumor cells. In contrast, nontumor cells do not show an increase of DNA damage nor lethality. Analysis of multilevel omics data from BC cells highlighted different DNA repair and cell-cycle molecular profiles associated with resistance to AsiDNA or olaparib, rationalizing combined treatment. Treatment synergy was also confirmed with six other PARPi in development.Conclusions: Our results highlight the therapeutic interest of combining AsiDNA and PARPi to recapitulate synthetic lethality in all tumors independently of their HR status. Clin Cancer Res; 23(4); 1001-11. ©2016 AACR.

Novel TDP2-ubiquitin interactions and their importance for the repair of topoisomerase II-mediated DNA damage.

Tyrosyl DNA phosphodiesterase 2 (TDP2) is a multifunctional protein implicated in DNA repair, signal transduction and transcriptional regulation. In its DNA repair role, TDP2 safeguards genome integrity by hydrolyzing 5'-tyrosyl DNA adducts formed by abortive topoisomerase II (Top2) cleavage complexes to allow error-free repair of DNA double-strand breaks, thereby conferring cellular resistance against Top2 poisons. TDP2 consists of a C-terminal catalytic domain responsible for its phosphodiesterase activity, and a functionally uncharacterized N-terminal region. Here, we demonstrate that this N-terminal region contains a ubiquitin (Ub)-associated (UBA) domain capable of binding multiple forms of Ub with distinct modes of interactions and preference for either K48- or K63-linked polyUbs over monoUb. The structure of TDP2 UBA bound to monoUb shows a canonical mode of UBA-Ub interaction. However, the absence of the highly conserved MGF motif and the presence of a fourth α-helix make TDP2 UBA distinct from other known UBAs. Mutations in the TDP2 UBA-Ub binding interface do not affect nuclear import of TDP2, but severely compromise its ability to repair Top2-mediated DNA damage, thus establishing the importance of the TDP2 UBA-Ub interaction in DNA repair. The differential binding to multiple Ub forms could be important for responding to DNA damage signals under different contexts or to support the multi-functionality of TDP2.

RNF4-mediated polyubiquitination regulates the Fanconi anemia/BRCA pathway.

The Fanconi anemia/BRCA (FA/BRCA) pathway is a DNA repair pathway that is required for excision of DNA interstrand cross-links. The 17 known FA proteins, along with several FA-associated proteins (FAAPs), cooperate in this pathway to detect, unhook, and excise DNA cross-links and to subsequently repair the double-strand breaks generated in the process. In the current study, we identified a patient with FA with a point mutation in FANCA, which encodes a mutant FANCA protein (FANCAI939S). FANCAI939S failed to bind to the FAAP20 subunit of the FA core complex, leading to decreased stability. Loss of FAAP20 binding exposed a SUMOylation site on FANCA at amino acid residue K921, resulting in E2 SUMO-conjugating enzyme UBC9-mediated SUMOylation, RING finger protein 4-mediated (RNF4-mediated) polyubiquitination, and proteasome-mediated degradation of FANCA. Mutation of the SUMOylation site of FANCA rescued the expression of the mutant protein. Wild-type FANCA was also subject to SUMOylation, RNF4-mediated polyubiquitination, and degradation, suggesting that regulated release of FAAP20 from FANCA is a critical step in the normal FA pathway. Consistent with this model, cells lacking RNF4 exhibited interstrand cross-linker hypersensitivity, and the gene encoding RNF4 was epistatic with the other genes encoding members of the FA/BRCA pathway. Together, the results from our study underscore the importance of analyzing unique patient-derived mutations for dissecting complex DNA repair processes.

Compensatory functions and interdependency of the DNA-binding domain of BRCA2 with the BRCA1-PALB2-BRCA2 complex.

BRCA1, BRCA2, and PALB2 are key players in cellular tolerance to chemotherapeutic agents, including camptothecin, cisplatin, and PARP inhibitor. The N-terminal segment of BRCA2 interacts with PALB2, thus contributing to the formation of the BRCA1-PALB2-BRCA2 complex. To understand the role played by BRCA2 in this complex, we deleted its N-terminal segment and generated BRCA2(Δ)(N) mutant cells. Although previous studies have suggested that BRCA1-PALB2 plays a role in the recruitment of BRCA2 to DNA-damage sites, BRCA2(Δ)(N) mutant cells displayed a considerably milder phenotype than did BRCA2(-/-) null-deficient cells. We hypothesized that the DNA-binding domain (DBD) of BRCA2 might compensate for a defect in BRCA2(ΔN) that prevented stable interaction with PALB2. To test this hypothesis, we disrupted the DBD of BRCA2 in wild-type and BRCA2(Δ)(N) cells. Remarkably, although the resulting BRCA2(Δ)(DBD) cells displayed a moderate phenotype, the BRCA2(Δ)(N+ΔDBD) cells displayed a very severe phenotype, as did the BRCA2(-/-) cells, suggesting that the N-terminal segment and the DBD play a substantially overlapping role in the functionality of BRCA2. We also showed that the formation of both the BRCA1-PALB2-BRCA2 complex and the DBD is required for efficient recruitment of BRCA2 to DNA-damage sites. Our study revealed the essential role played by both the BRCA1-PALB2-BRCA2 complex and the DBD in the functionality of BRCA2, as each can compensate for the other in the recruitment of BRCA2 to DNA-damage sites. This knowledge adds to our ability to accurately predict the efficacy of antimalignant therapies for patients carrying mutations in the BRCA2 gene.