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Results for “Cancer Biology”

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bioRxiv2019

The chromatin structuring protein HMGA2 influences human subtelomere stability and cancer chemosensitivity

The transient build-up of DNA supercoiling during the translocation of replication forks threatens genome stability and is controlled by DNA topoisomerases (TOPs). This crucial process has been exploited with TOP poisons for cancer chemotherapy. However, pinpointing cellular determinants of the best clinical response to TOP poisons still remains enigmatic. Here, we present an integrated approach and demonstrate that endogenous and exogenous expression of the oncofetal high-mobility group AT-hook 2 (HMGA2) protein exhibited broad protection against the formation of hydroxyurea-induced DNA breaks in various cancer cells, thus corroborating our previously proposed model in which HMGA2 functions as a replication fork chaperone that forms a protective DNA scaffold at or close to stalled replication forks. We now further demonstrate that high levels of HMGA2 also protected cancer cells against DNA breaks triggered by the clinically important TOP1 poison irinotecan. This protection is most likely due to the recently identified DNA supercoil constraining function of HMGA2 in combination with exclusion of TOP1 from binding to supercoiled substrate DNA. In contrast, low to moderate HMGA2 protein levels surprisingly potentiated the formation of irinotecan-induced genotoxic covalent TOP1-DNA cleavage complexes. Our data from cell-based and several in vitro assays indicate that, mechanistically, this potentiating role involves enhanced drug-target interactions mediated by HMGA2 in ternary complexes with supercoiled DNA. Subtelomeric regions were found to be extraordinarily vulnerable to these genotoxic challenges induced by TOP1 poisoning, pointing at strong DNA topological barriers located at human telomeres. These findings were corroborated by an increased irinotecan sensitivity of patient-derived xenografts of colorectal cancers exhibiting low to moderate HMGA2 levels. Collectively, we uncovered a therapeutically important control mechanism of transient changes in chromosomal DNA topology that ultimately leads to enhanced human subtelomere stability. Author SummaryDNA replication fork stability in rapidly dividing cancer cells is of utmost importance for the maintenance of genome stability and cancer cell viability. Cancer cells efficiently prevent fork collapse into lethal double strand breaks as a first line of defense during replication stress, but the corresponding protective mechanisms often remain elusive. Uncontrolled high levels of DNA supercoiling that are generally regulated by topoisomerases can cause replication stress and are major threats to fork stability. Using a multidisciplinary approach, we identified a possible regulatory mechanism of replication stress, which appears to involve mitigating the consequences of DNA topological changes by the oncofetal replication fork chaperone HMGA2. Our work provides mechanistic insights into the control of DNA damage triggered by clinically important anti-cancer drugs, which is mediated by the replication fork chaperone HMGA2. We thereby also identify HMGA2 expression as a predictive therapeutic marker, which could allow clinicians to take informed decisions to prevent tumor recurrence and improve survival.

cancer biology↗

bioRxiv2019

Conditional activation of immune-related signatures and prognostic significance: a pan-cancer analysis

BackgroundIt is becoming clear that tumor immune T cell infiltration and its functional orientation have substantial effect on cancer progression, influencing both response to therapy and prognosis. In this pan-cancer study, the previously described Immunologic Constant of Rejection (ICR) signature is used to define opposing immune phenotypes (i.e., immuneactive and immune-silent) across 31 different histologies. We systematically analyze the interconnections between the genetic programming of neoplasms and their immune orientation across different histologies, and the prognostic impact of such interplay. Moreover, we investigated the predictive value of ICR classification across various public datasets of immune checkpoint inhibition therapy. MethodsRNA-seq data of samples from a total of 9,282 patient tumor samples representing 31 cancer types were obtained from The Cancer Genome Atlas (TCGA). We classified each cancer type based on the expression of the ICR gene signature. Oncogenic pathway gene set enrichment and mutational status were analyzed in relation to ICR phenotypes. To explore whether tumorintrinsic attributes associate with the prognostic value of ICR across cancers, we compared mutational load, oncogenic alterations and expression of oncogenic pathways between cancer types using an integrative bioinformatic pipeline. ResultsOur analyses identified a distinct prognostic connotation of ICR depending on cancer histology. We identified several oncogenic pathways whose enrichment inversely correlated with ICR in multiple tumor types. We found several cancer specific pathways that were differentially enriched between tumors in which ICR had a prognostic impact versus the ones in which ICR did not bear any prognostic connotation such as proliferation and TGF-beta signaling. Importantly, this conditional impact of ICR was also validated in the context of immune checkpoint inhibition treatment. ConclusionsWe identified tumor-intrinsic attributes that correlate with immune phenotypes and potentially influence their development. In addition, a relationship was observed between the enrichment of oncogenic pathways and the prognostic significance of the ICR and its predictive value for patients treated with anti-CTLA4 immune checkpoint inhibition. Such information can be used to prioritize potential candidates for therapies aimed at converting immune-silent into immuneactive tumors and to refine stratification algorithms.

cancer biology↗

bioRxiv2019

Biomarker-based outcome prediction in prostate adenocarcinoma depends on the TMPRSS2-ERG status

BackgroundProstate adenocarcinoma (PCa) with/without the TMPRSS2-ERG (T2E)-fusion represent distinct molecular subtypes. ObjectiveTo investigate gene-signatures associated with metastasis in T2E-positive and -negative PCa, and to identify and validate subtype-specific prognostic biomarkers. Design, setting and participantsGene expression and clinicopathological data of two discovery PCa cohorts (total n=783) were separately analyzed regarding the T2E-status. Selected subtype-specific biomarkers were validated in two additional cohorts (total n=405). Outcome measurements and statistical analysisFrom both discovery cohorts, we generated two gene lists ranked by their differential intratumoral expression in patients with/without metastases stratified by T2E-status, which were subjected to gene set enrichment and leading-edge analyses. The resulting top 20 gene-signatures of both gene lists associated with metastasis were analyzed for overlaps between T2E-positive and -negative cases. Genes shared by several functional gene-signatures were tested for their association with event-free survival using the Kaplan-Meier method in a validation cohort. Immunohistochemistry was performed in another validation cohort. Results and limitationsMetastatic T2E-positive and -negative PCa are characterized by different gene-signatures. Five genes (ASPN, BGN, COL1A1, RRM2 and TYMS) were identified whose high expression was significantly associated with worse outcome exclusively in T2E-negative PCa. This was validated in an independent cohort for all genes and additionally for RRM2 by immunohistochemistry in a separate validation cohort. No prognostic biomarkers were identified exclusively for T2E-positive tumors. ConclusionsOur study demonstrates that the prognostic value of biomarkers critically depends on the molecular subtype, i.e. the T2E-status, which should be considered when screening for and applying novel prognostic biomarkers for outcome prediction in PCa. Patient summaryOutcome prediction for PCa is complex. The results of this study highlight that the validity of prognostic biomarkers depends on the molecular subtype, specifically the presence/absence of T2E. The reported new subtype-specific biomarkers exemplify that biomarker-based outcome prediction in PCa should consider the T2E-status.

cancer biology↗

bioRxiv2019

CXCR3-expressing metastasis-initiating cells induce and exploit a fibroblast niche in the lungs to fuel metastatic colonization

Metastatic colonization relies on interactions between disseminated cancer cells and the microenvironment in secondary organs. Here, we show that disseminated breast cancer cells evoke major phenotypic changes in lung fibroblasts to form a metastatic niche that supports malignant growth. Colonization of the lungs by cancer cells confers an inflammatory phenotype in associated fibroblasts, where IL-1 and IL-1{beta}, secreted by breast cancer cells, induce CXCL9 and CXCL10 production in metastasis-associated fibroblasts via NF-{kappa}B signaling. These paracrine interactions fuel the growth of lung metastases. Notably, we find that the chemokine receptor CXCR3, that binds CXCL9/10, is specifically expressed in a small subset of breast cancer cells with stem/progenitor cell properties and high tumor-initiating ability when co-transplanted with fibroblasts. CXCR3-expressing cancer cells show high JNK signaling that drives IL-1/{beta} expression. Thus, CXCR3 marks a population of breast cancer cells that induces CXCL9/10 production in fibroblast, but can also respond to and benefit from these chemokines. Importantly, disruption of this intercellular JNK-IL-1-CXCL9/10-CXCR3 axis significantly reduces metastatic colonization in xenograft and syngeneic mouse models. These data mechanistically demonstrate an essential role for this molecular crosstalk between breast cancer cells and their fibroblast niche in the progression of metastasis.

cancer biology↗

bioRxiv2019

Deep proteomic analysis of Dnmt1 mutant/hypomorphic colorectal cancer cells reveals dys-regulation of Epithelial-Mesenchymal Transition and subcellular re-localization of Beta-Catenin

BackgroundDNA methyltransferase I is the primary eukaryotic DNA methyltransferase engaged in maintenance of CpG DNA methylation patterns across the genome. Alteration of CpG methylation patterns and levels is a frequent and significant occurrence across many cancers, and targeted inhibition of Dnmt1 has become an approach of choice for select malignancies. There has been significant interest both in the methyltransferase activity as well as methylation-independent functions of Dnmt1. A previously generated hypomorphic allele of Dnmt1 in HCT116 colorectal cancer cells has become an important tool for understanding Dnmt1 function and how CpG methylation patterns are modulated across the genome. Colorectal cancer cells with the Dnmt1 hypomorphic allele carry a homozygous deletion of exons 3 to 5 of Dnmt1, resulting in greatly reduced Dnmt1 protein expression whilst still exhibiting a limited functional activity and methyltransferase ability. Although this cell model of reduced Dnmt1 levels and function have been used to study the downstream effects on the epigenome and transcriptome, the broader effects of the Dnmt1 hypomorph on the proteome and wider cell signalling are largely unknown. ResultsIn this study, we used quantitative proteomic analysis of nuclear-enriched samples of HCT116 Dnmt1 hypomorph cells to identify signalling pathways and processes dysregulated in the hypomorph cells as compared to wild-type HCT116 cells. Unexpectedly, we observed a clear signature of increased expression of Epithelial-to-Mesenchymal (EMT) in Dnmt1 hypomorph cells. We also observed reduced expression and sub-cellular re-localization of Beta-Catenin in Dnmt1 hypomorph cells. Expression of wild-type Dnmt1 in hypomorph cells or knock-down of wild-type Dnmt1 did not recapitulate or rescue the observed protein profiles in Dnmt1 hypomorph cells suggesting that hypomorphic Dnmt1 causes changes not solely attributable ConclusionsIn summary we present the first comprehensive proteomic analysis of the widely studied Dnmt1 hypomorph colorectal cancer cells and identify redistribution of Dnmt1 and its interaction partner Beta-Catenin as well as the dysregulation of EMT related processes and signalling pathways related to the development of a cancer stem cell phenotype.

cancer biology↗

bioRxiv2019

Investigation of the effect of estradiol and subculturing on the receptor expression within hormone-dependent breast cancer cells

It is now known that a very crucial role in breast cancer development, prognosis and occurrence is played by the estrogen receptor (ER). The steroid hormone estradiol (E2) acts via two nuclear receptors, estrogen receptor- (ER) and estrogen receptor-{beta} (ER{beta}). E2 was shown previously to increase breast cancer cell proliferation in a dose-dependent manner and also induce apoptosis in long term estrogen deprived breast cancer cells. Studies have also shown that the degree of subculturing affects cell line property including gene expression. The aim of this study was to investigate the effect of E2 concentration on cell proliferation, morphology and ER expression and to investigate the effect of subculturing on the expression of ER. Our results have shown that an increase in E2 concentration was found to increase MCF-7 cell proliferation, but extreme concentrations caused significantly low cell proliferation and induced apoptosis. Moreover, ER expression was significantly upregulated with an increase in E2 concentration, whereas ER{beta}2 expression was found to be unchanged at low E2 concentration and significantly upregulated at higher E2 concentration. ER expression at passage 3 ([E2]=1nM) was significantly downregulated compared to the cells at passage 0, in addition to the significant downregulation of the same at E2 concentrations of 1nM and 10{micro}M compared to the untreated control sample. Overall, our data suggests that high concentration of E2 can reduce proliferation and induce apoptosis in the breast cancer cells. Increased E2 exposure and subculturing also appear to change the ER expression significantly in the breast cancer cell line.

cancer biology↗

bioRxiv2019

The Genetic Basis of Transcriptional and Spatial Heterogeneity of Squamous Features in Pancreatic Ductal Adenocarcinoma

Recent studies indicate that pancreatic cancer expression profiles are variable and largely reflect a classical or basal-type phenotype. We performed genetic sequencing, RNA-seq, and histologic review of multiregion sampled pancreatic cancers and found that squamous and squamoid features, indicators of poor prognosis, correlate with a "basal-like" expressional type. Cancers with squamous features were more likely to have truncal mutations in chromatin modifier genes and intercellular heterogeneity for MYC amplification that was associated with entosis. In most patients the basal phenotype coexisted with a glandular component, and phylogenetic studies indicated that it arose from a subclonal population in the tumor. These data provide a unifying paradigm for understanding the interrelationship of basal-type features, squamous histology, and somatic mutations in chromatin modifier genes in the context of the clonal evolution of pancreatic cancer.

cancer biology↗

bioRxiv2019

Thermal cycling-hyperthermia in combination with polyphenols, epigallocatechin gallate and chlorogenic acid, exerts synergistic anticancer effect against human pancreatic cancer PANC-1 cells

Hyperthermia (HT) has shown feasibility and potency as an anticancer therapy. Administration of HT in the chemotherapy has previously enhanced the cytotoxicity of drugs against pancreatic cancer. However, the drugs used when conducting these studies are substantially conventional chemotherapeutic agents that may cause unwanted side effects. Additionally, the thermal dosage in the treatment of cancer cells could also probably harm the healthy cells. The purpose of this work was to investigate the potential of the two natural polyphenolic compounds, epigallocatechin gallate (EGCG) and chlorogenic acid (CGA), as heat synergizers in the thermal treatment of the PANC-1 cells. Furthermore, we have introduced a novel strategy entitled the thermal cycling-hyperthermia (TC-HT) that is capable of providing a maximum synergy and minimal side effect with the anticancer compounds. Our results demonstrate that the combination of the TC-HT and the CGA or EGCG markedly exerts the anticancer effect against the PANC-1 cells, while none of the single treatment induced such changes. The synergistic activity was attributed to the cell cycle arrest at the G2/M phase and the induction of the ROS-dependent mitochondria-mediated apoptosis. These findings not only represent the first thermal synergistic study of natural compounds in the treatment of pancreatic cancer, but also highlight the potential application of the TC-HT as an alternative strategy in anticancer treatment.

cancer biology↗

bioRxiv2019

MiR-590-5p sensitises pancreatic ductal adenocarcinoma cells by blocking autophagy via targeting ATG3

Radio-resistance is a growing concern in treating patients with pancreatic cancer (PC). Here we investigated the role of miR-590-5p in the radio-resistance of PC cells. We developed radioresistant PC cell lines and followed by microarray analysis and levels of miRs compared to parental cell lines. PC cells were transfected using either miR mimics or inhibitors followed by clonogenic survival assays. We also studied the effect of miR-590-5p on autophagy using electron microscopy and immunoblot analysis. In addition, the luciferase assay was used to identify potential targets. The radio-resistant PC cells exhibited decreased expression of miR-590-5p, with elevated autophagy against the parental cells. The over-expression of miR-590-5p inhibited radiation-mediated autophagy, while inhibitors induced autophagy in PC cells. The up-regulation of miR-590-5p enhanced the radio-sensitivity of PC cells. We confirmed ATG-3 as a target of miR-590-5p, whose levels were unregulated in radio-resistant cells. We also found that levels of ATG-3 were associated with autophagy. Expression of miR-590-5p inhibited radiation-mediated autophagy and enhanced the radio-sensitivity of PC cells.

cancer biology↗

bioRxiv2019

Exosome-mediated MIR211 modulates tumor microenvironment via the DUSP6-ERK5 axis and contributes to BRAFV600E inhibitor resistance in melanoma.

The microRNA MIR211 is an important regulator of melanoma tumor cell behavior. Previous studies suggested that in certain tumors, MIR211 acted as a tumor suppressor while in others it behaved as an oncogenic regulator. When MIR211 is expressed in BRAFV600E-mutant A375 melanoma cells in mouse xenografts, it promotes aggressive tumor growth accompanied by increased cellular proliferation and angiogenesis. We demonstrate that MIR211 is transferred to adjacent cells in the tumor micro-environment via exosomes. Cross-species genome-wide transcriptomic analysis showed that human tumor-derived MIR211 interacts with the mouse transcriptome in the tumor microenvironment, and activates ERK5 signaling in human tumor cells via the modulation of a feedback loop. Human miR211 directly inhibits human DUSP6 protein phosphatase at the post-transcriptional level. We provide support for the hypothesis that DUSP6 inhibition conferred resistance of the human tumor cells to the BRAF inhibitor vemurafenib and to the MEK inhibitor cobimetinib, with associated increases in ERK5 phosphorylation. These findings are consistent with a model in which MIR211 regulates melanoma tumor proliferation and BRAF inhibitor resistance by inducing ERK5 signaling within the complex tumor microenvironment. We propose that the MIR211-ERK5 axis represents an important and sensitive regulatory arm in melanoma with potential theranostic applications.

cancer biology↗

bioRxiv2019

Protoporphyrin IX is a dual inhibitor of p53/MDM2 and p53/MDM4 interactions and induces apoptosis in B-cell chronic lymphocytic leukaemia cells

p53 is a tumor suppressor, which belongs to the p53 family of proteins. The family consists of p53, p63 and p73 proteins, which share similar structure and function. Activation of wild-type p53 or TAp73 in tumors leads to tumor regression, and small molecules restoring the p53 pathway are in clinical development. Protoporphyrin IX (PpIX), a metabolite of aminolevulinic acid, is a clinically approved drug applied in photodynamic diagnosis and therapy. PpIX induces p53- and TAp73-dependent apoptosis and inhibits TAp73/MDM2 and TAp73/MDM4 interactions. Here we demonstrate that PpIX is a dual inhibitor of p53/MDM2 and p53/MDM4 interactions and activates apoptosis in B-cell chronic lymphocytic leukaemia cells without illumination and without affecting normal cells. PpIX stabilizes p53 and TAp73 proteins, induces p53-downstream apoptotic targets and provokes cancer cell death at doses non-toxic to normal cells. Our findings open up new opportunities for repurposing PpIX for treating lymphoblastic leukaemias with wtTP53.

cancer biology↗

bioRxiv2019

Variable Retention of Differentiation-specific DNA Replication Timing in Human Pediatric Leukemia

Human B-lineage precursor acute lymphoid leukemias (BCP-ALLs) comprise a group of genetically and clinically distinct disease entities with features of differentiation arrest at known stages of normal B-lineage differentiation. We previously showed BCP-ALL cells display unique and clonally heritable DNA-replication timing (RT) programs; i.e., programs describing the variable order of replication of megabase-scale chromosomal units of DNA in different cell types. To determine the extent to which BCP-ALL RT programs mirror or deviate from specific stages of normal human B-cell differentiation, we transplanted immunodeficient mice with quiescent normal human CD34+ cord blood cells and obtained RT signatures of the regenerating B-lineage populations. We then compared these with RT signatures for leukemic cells from a large cohort of BCP-ALL patients. The results identify BCP-ALL subtype-specific features that resemble specific stages of B-cell differentiation and features that appear associated with relapse. These results suggest the genesis of BCP-ALL involves alterations in RT that reflect clinically relevant leukemia-specific genetic and/or epigenetic changes. SUMMARYGenome-wide DNA replication timing profiles of >100 pediatric leukemic samples and normally differentiating human B-lineage cells isolated from xenografted immunodeficient mice were generated. Comparison of these identified potentially clinically relevant features that both match and deviate from the normal profiles.

cancer biology↗

bioRxiv2019

Aicardi-Goutieres Syndrome gene Rnaseh2c is a metastasis susceptibility gene in breast cancer

Breast cancer is the second leading cause of cancer-related deaths in the United States, with the majority of these deaths due to metastatic lesions rather than the primary tumor. Thus, a better understanding of the etiology of metastatic disease is crucial for improving survival. Using a haplotype mapping strategy in mouse and shRNA-mediated gene knockdown, we identified Rnaseh2c, a scaffolding protein of the heterotrimeric RNase H2 endoribonuclease complex, as a novel metastasis susceptibility factor. We found that the role of Rnaseh2c in metastatic disease is independent of RNase H2 enzymatic activity, and immunophenotyping and RNA-sequencing analysis revealed engagement of the T cell-mediated adaptive immune response. Furthermore, the cGAS-Sting pathway was not activated in the metastatic cancer cells used in this study, suggesting that the mechanism of immune response in breast cancer is different from the mechanism proposed for Aicardi-Goutieres Syndrome, a rare interferonopathy caused by RNase H2 mutation. These results suggest an important novel, non-enzymatic role for RNASEH2C during breast cancer progression and add Rnaseh2c to a panel of genes we have identified that together could determine patients with high risk for metastasis. These results also highlight a potential new target for combination with immunotherapies and may contribute to a better understanding of the etiology of Aicardi-Goutieres Syndrome autoimmunity. Author SummaryThe majority of breast cancer-associated deaths are due to metastatic disease, the process where cancerous cells leave the primary tumor in the breast and spread to a new location in the body. To better understand the etiology of this process, we investigate the effects of gene expression changes in the primary tumor. In this study, we found that changing the expression of the gene Rnaseh2c changed the number of metastases that developed in the lungs of tumor-bearing mice. By investigating the enzyme complex Rnaseh2c is part of, RNase H2, we determined that Rnaseh2cs effects may be independent of RNase H2 enzyme activity. Because Rnaseh2c is known to cause the autoimmune disease Aicardi-Goutieres Syndrome (AGS), we tested whether the immune system is involved in the metastatic effect. Indeed, we found that the cytotoxic T cell response is important for mediating the effect that Rnaseh2c has on metastasis. Together these data indicate that Rnaseh2c expression contributes to a patients susceptibility to developing breast cancer metastasis and demonstrate that the immune system is involved in this outcome. The implications of this study suggest immunotherapy could be a viable treatment for breast cancer metastasis and may help inform the understanding of AGS and RNase H2 in cancer.

cancer biology↗

bioRxiv2019

A Quasi Birth-and-Death Model For Tumor Recurrence

A major cause of chemoresistance and recurrence in tumors is the presence of dormant tumor foci that survive chemotherapy and can eventually transition to active growth to regenerate the cancer. In this paper, we propose a Quasi Birth-and-Death (QBD) model for the dynamics of tumor growth and recurrence/remission of the cancer. Starting from a discrete-state master equation that describes the time-dependent transition probabilities between states with different numbers of dormant and active tumor foci, we develop a framework based on a continuum-limit approach to determine the time-dependent probability that an undetectable residual tumor will become large enough to be detectable. We derive an exact formula for the probability of recurrence at large times and show that it displays a phase transition as a function of the ratio of the death rate {micro}A of an active tumor focus to its doubling rate{lambda} . We also derive forward and backward Kolmogorov equations for the transition probability density in the continuum limit and, using a first-passage time formalism, we obtain a drift-diffusion equation for the mean recurrence time and solve it analytically to leading order for a large detectable tumor size N. We show that simulations of the discrete-state model agree with the analytical results, except for O(1/N) corrections. Finally, we describe a scheme to fit the model to recurrence-free survival (Kaplan-Meier) curves from clinical cancer data, using ovarian cancer data as an example. Our model has potential applications in predicting how changing chemotherapy schedules may affect disease recurrence rates, especially in cancer types for which no targeted therapy is available.

cancer biology↗

bioRxiv2019

Cancer gene therapy by NF-κB-activated cancer cell-specific expression of CRISPR/Cas9 targeting to telomere

NF-{kappa}B has been a luring target for cancer therapy due to its over activation in all tumors. In this study, we showed that a gene therapy named as NF-{kappa}B-activated gene expression (Nage) could be used to induce cancer cell death in vitro and in vivo by utilizing the NF-{kappa}B activity in cancer cells; however, it had no effect on normal cells. In this gene therapy, we constructed a NF-{kappa}B-specific promoter by fusing a NF-{kappa}B decoy sequence to a minimal promoter, which could be bound by the intracellular over activated NF-{kappa}B and thus activate the expression of downstream effector gene in a NF-{kappa}B-specific manner. In this study, we firstly demonstrated the cancer cell-specific activation of NF-{kappa}B. We then demonstrated the cancer cell specificity of Nage vector expression by introducing a Nage vector that could express a reporter gene ZsGreen in various cell lines. We next demonstrated that a Nage vector that could express CRISPR/Cas9 protein and a telomere-targeting sgRNA could be used to specifically induce death of cancer cells. We finally showed that the Cas9/sgRNA Nage vector packaged into the adeno-associated virus (AAV) could be used to inhibit the growth of xenografted tumors in mouse by intravenously injecting recombinant AAV.

cancer biology↗

bioRxiv2019

Specific inhibition of DPY30 activity by ASH2L-derived peptides suppresses blood cancer cell growth

DPY30 facilitates H3K4 methylation by directly binding to ASH2L in the SET1/MLL complexes and plays an important role in hematologic malignancies. However, the domain on DPY30 that regulates cancer growth is not evident, and the potential of pharmacologically targeting this chromatin modulator to inhibit cancer has not been explored. Here we have developed a peptide-based strategy to specifically target DPY30 activity. We have designed cell-penetrating peptides derived from ASH2L that can either bind to DPY30 or show defective or enhanced binding to DPY30. The DPY30-binding peptides specifically inhibit its activity in interacting with ASH2L and enhancing H3K4 methylation. Treatment with the DPY30-binding peptides significantly inhibited the growth of MLL-rearranged leukemia and other MYC-dependent hematologic cancer cells. We also revealed subsets of genes that may mediate the effect of the peptides on cancer cell growth, and showed that the DPY30-binding peptide sensitized leukemia to other types of epigenetic inhibitors. These results strongly support a critical role of the ASH2L-binding groove of DPY30 in promoting blood cancers, and demonstrate a proof-of-principle for the feasibility of pharmacologically targeting the ASH2L-binding groove of DPY30 for potential cancer inhibition.

cancer biology↗

bioRxiv2019

Multi-omic analysis supports a developmental hierarchy of molecular subtypes in high-grade serous ovarian carcinoma

Multiple studies have identified transcriptome subtypes of high-grade serous ovarian carcinoma (HGSOC), but these have yet to impact clinical practice. Interpretation and translation of HGSOC subtypes are complicated by tumor evolution and polyclonality accompanied by accumulation of somatic aberrations, varying cell type admixtures, and different tissues of origin. The chronology of HGSOC subtype evolution was examined in the context of these factors by a novel integrative analysis of bulk absolute somatic copy number analysis and gene expression in The Cancer Genome Atlas, complemented by single-cell RNA-seq analysis of six independent tumors. The approach was validated by contrast to soft-tissue sarcoma. Genomic lesions associated with HGSOC subtypes tend to be subclonal, implying subtype divergence at later stages of tumor evolution. Subclonality of recurrent HGSOC alterations is particularly evident for proliferative tumors, characterized by extreme genomic instability, absence of immune infiltration, and greater patient age. In contrast, differentiated tumors are characterized by largely intact genome integrity, high immune infiltration, and younger patient age. We propose an alternative model to discrete subtypes of HGSOC, in which tumors develop from an early differentiated spectrum to a late proliferative spectrum, along a timeline characterized by increasing genomic instability and subclonal expansion. The proposed methods provide a new approach to investigating tumor evolution through multi-omic analysis. Statement of SignificanceThis study proposes a method to infer whether transcriptome-based groupings of tumors differentiate early in carcinogenesis and are therefore potentially appropriate targets for therapy, and demonstrates that this is not the case for high-grade serous ovarian carcinoma (HGSOC). Significant findings for HGSOC include: O_LITumor purity, ploidy, and subclonality can be reliably inferred from different genomic platforms and show marked differences between subtypes C_LIO_LIRecurrent DNA alterations are associated with subtypes and tend to occur more frequently in subclones C_LIO_LISingle-cell sequencing of 42,000 tumor cells reveals widespread heterogeneity in tumor cell type composition that drives bulk subtype calls, but demonstrates a lack of intrinsic subtypes among tumor epithelial cells C_LIO_LIFindings prompt the dismissal of discrete transcriptome subtypes for HGSOC and replacement by a more realistic model of continuous tumor development that includes mixtures of subclones, accumulation of somatic aberrations, infiltration of immune and stromal cells in proportions correlated with tissue of origin and tumor stage, and evolution between properties previously associated with discrete subtypes C_LI

cancer biology↗

bioRxiv2019

The oncoprotein DEK affects the outcome of PARP1/2 inhibition during replication stress

DNA replication stress is a major source of genomic instability and is closely linked to tumor formation and progression. Poly(ADP-ribose)polymerases1/2 (PARP1/2) enzymes are activated in response to replication stress resulting in poly(ADP-ribose) (PAR) synthesis. PARylation plays an important role in the remodelling and repair of impaired replication forks, providing a rationale for targeting highly replicative cancer cells with PARP1/2 inhibitors. The human oncoprotein DEK is a unique, non-histone chromatin architectural protein whose deregulated expression is associated with the development of a wide variety of human cancers. Recently, we showed that DEK is a high-affinity target of PARylation and that it promotes the progression of impaired replication forks. Here, we investigated a potential functional link between PAR and DEK in the context of replication stress. Under conditions of mild replication stress induced either by topoisomerase1 inhibition with camptothecin or nucleotide depletion by hydroxyurea, we found that the effect of acute PARP1/2 inhibition on replication fork progression is dependent on DEK expression. Reducing DEK protein levels also overcomes the restart impairment of stalled forks provoked by blocking PARylation. Non-covalent DEK-PAR interaction via the central PAR-binding domain of DEK is crucial for counteracting PARP1/2 inhibition as shown for the formation of RPA positive foci in hydroxyurea treated cells. Finally, we show by iPOND and super resolved microscopy that DEK is not directly associated with the replisome since it binds to DNA at the stage of chromatin formation. Our report sheds new light on the still enigmatic molecular functions of DEK and suggests that DEK expression levels may influence the sensitivity of cancer cells to PARP1/2 inhibitors.

cancer biology↗