Sylvia Ispasanie’s research while affiliated with Charité Universitätsmedizin Berlin and other places

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Publications (6)


SFPQ Depletion Is Synthetically Lethal with BRAFV600E in Colorectal Cancer Cells
  • Article
  • Full-text available

September 2020

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79 Reads

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28 Citations

Cell Reports

Kathleen Klotz-Noack

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Christine Sers

Oncoproteins such as the BRAFV600E kinase endow cancer cells with malignant properties, but they also create unique vulnerabilities. Targeting of BRAFV600E-driven cytoplasmic signaling networks has proved ineffective, as patients regularly relapse with reactivation of the targeted pathways. We identify the nuclear protein SFPQ to be synthetically lethal with BRAFV600E in a loss-of-function shRNA screen. SFPQ depletion decreases proliferation and specifically induces S-phase arrest and apoptosis in BRAFV600E-driven colorectal and melanoma cells. Mechanistically, SFPQ loss in BRAF-mutant cancer cells triggers the Chk1-dependent replication checkpoint, results in decreased numbers and reduced activities of replication factories, and increases collision between replication and transcription. We find that BRAFV600E-mutant cancer cells and organoids are sensitive to combinations of Chk1 inhibitors and chemically induced replication stress, pointing toward future therapeutic approaches exploiting nuclear vulnerabilities induced by BRAFV600E.

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Fig. 1 (See legend on next page.)
Fig. 2 Consistent growth reduction of MCM7-suppressed colorectal cancer cell lines. a Clonogenic assay quantification of SW480 (KRAS G12V ), HCT-8 (KRAS G13A ), HT-29 (BRAF V600E ), WiDr (BRAF V600E ). All cell lines showed decreased growth after doxycycline induction of MCM7.sh3 compared to the control shRNA (scrbl). Mean percentage of the relative area covered by cells ± SEM is shown (n = 3 per group). Student's t-test (two-sided); *p < 0.05; ***p < 0.001. b Clonogenic assay quantification of DLD1 KRAS(wt/−) and its parental cell line DLD1 KRAS(wt/G13D) . Both cell lines are sensitive to MCM7 suppression, but DLD1 cells without mutated KRAS become more resistant to low levels of MCM7. Mean percentage of the relative area covered by cells ± SEM is shown (n = 3 per group). Student's t-test (two-sided); NS = not significant; *p < 0.05; ****p < 0.0001. c Quantification of soft agar assays confirmed decreased anchorage-independent colony formation in KRAS mutated DLD1 and SW480 cells. Mean fold change of colonies ± SEM is shown (n = 6 per group). Student's t-test (two-sided); **p < 0.01; ****p < 0.0001.
Fig. 3 (See legend on next page.)
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Reduced replication origin licensing selectively kills KRAS-mutant colorectal cancer cells via mitotic catastrophe

July 2020

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83 Reads

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6 Citations

Cell Death and Disease

To unravel vulnerabilities of KRAS-mutant CRC cells, a shRNA-based screen specifically inhibiting MAPK pathway components and targets was performed in CaCo2 cells harboring conditional oncogenic KRASG12V. The custom-designed shRNA library comprised 121 selected genes, which were previously identified to be strongly regulated in response to MEK inhibition. The screen showed that CaCo2 cells expressing KRASG12V were sensitive to the suppression of the DNA replication licensing factor minichromosome maintenance complex component 7 (MCM7), whereas KRASwt CaCo2 cells were largely resistant to MCM7 suppression. Similar results were obtained in an isogenic DLD-1 cell culture model. Knockdown of MCM7 in a KRAS-mutant background led to replication stress as indicated by increased nuclear RPA focalization. Further investigation showed a significant increase in mitotic cells after simultaneous MCM7 knockdown and KRASG12V expression. The increased percentage of mitotic cells coincided with strongly increased DNA damage in mitosis. Taken together, the accumulation of DNA damage in mitotic cells is due to replication stress that remained unresolved, which results in mitotic catastrophe and cell death. In summary, the data show a vulnerability of KRAS-mutant cells towards suppression of MCM7 and suggest that inhibiting DNA replication licensing might be a viable strategy to target KRAS-mutant cancers.


Epigenetic regulation of Amphiregulin and Epiregulin in colorectal cancer

November 2018

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79 Reads

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23 Citations

Expression of the epidermal growth factor ligands amphiregulin (AREG) and epiregulin (EREG) is positively correlated with a response to EGFR‐targeted therapies in colorectal cancer. Gene‐body methylation sites, which show a strong inverse correlation with AREG and EREG gene expression, were identified in cell lines using targeted 454 FLX‐bisulfite sequencing and SIRPH analyses for AREG/EREG promoters and intragenic CpGs. Upon treatment of colorectal cancer cells with 5‐aza‐2′‐desoxycytidine, methylation decreases at specific intragenic CpGs accompanied by upregulation of AREG and EREG gene expression. The same AREG gene‐body methylation was also found in human colorectal cancer samples and is independent of KRAS and NRAS mutations. Methylation is specifically decreased in the tumor epithelial compartment as compared to stromal tissue and normal epithelium. Investigation of a promoter/enhancer function of the AREG exon 2 region revealed a potential promoter function in reverse orientation. Retrospective comparison of the predictive power of AREG gene‐body methylation versus AREG gene expression using samples from colorectal cancer patients treated with anti‐EGFR inhibitors with complete clinical follow‐up revealed that AREG expression is superior to AREG gene methylation. AREG and EREG genes undergo a complex regulation involving both intragenic methylation and promoter‐dependent control.


PO-503 Hdac inhibitor resistance in colorectal cancer: ras and amp; myc – the partners in crime

ESMO Open

Introduction Oncogenic KRAS is widely acknowledged as a critical determinant of the therapeutic response of colorectal cancer (CRC) – a fact that to date is pivotal for defining an appropriate treatment strategy. Whether this also extends beyond RTK- and MAPK- targeted molecular cancer therapies, to a novel and promising class of anti-cancer drugs, namely HDAC inhibitors (HDACi), is an aspect that until now has remained largely undefined. Our aim is, therefore, to establish whether RAS is an effective predictor of response to HDACi in CRC. Ultimately, we intend to shed light on the cause underlying the limited clinical benefits of HDACi as a treatment option for solid tumours, including CRC and thereby identify opportunities to improve the prospects of HDACi treatment. Material and methods We investigated the presence of an oncogenic-RAS dependency against a wide range of different HDAC inhibitors using model systems with intrinsic and conditionally active RAS oncogenes. Results and discussions We uncovered an oncogenic RAS-dependent ‘safeguard’ mechanism imposed in order to evade the cytotoxic effect of HDACi and thereby apoptosis. Cells harbouring oncogenic RAS were observed to undergo a reversible senescence-like growth arrest in G2 phase, allowing for re-entry into cell cycle following a withdrawal of the inhibitor. This mechanism is implemented as a consequence of the inhibition of the RAS deacetylase, namely HDAC2, which in turn result in the generation of (hyper)acetylated RAS with increased binding affinity to BRAF and CRAF. This translates to a further amplification in MAPK-signalling and thus an increase in the priming of c-MYC for ubiquitin-mediated proteasomal degradation, thereby enabling the cells to exit the cell cycle and enter the defined protective state of G2 arrest. The prospect of HDACi treatment was effectively improved using current MAPK-targeted therapy and senolytic drugs by effectively preventing the observed pro-oncogenic effect of the HDACi treatment alone. Conclusion Our study reveals an oncogenic RAS-dependent resistance mechanism, enabling cells harbouring oncogenic RAS, to establish a favourable cellular state of prolonged pharmacological hideout - a phenomenon that is replicated in patient-derived 3D cell culture models of CRC. This highlights the potential clinical relevance of our findings and thus the importance of a rational mechanism-based combinatorial therapeutic design in order to realise the true therapeutic potential of HDACi.


Abstract 5170: HDAC inhibitors and the mechanism of resistance in colorectal cancer: RAS and MYC - the partners in crime

July 2017

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35 Reads

Cancer Research

Although histone deacetylase inhibitors (HDACi) are considered a promising novel therapeutic approach in the light of their potent tumour-selective effects, the use of these inhibitors for treatment of colorectal cancer (CRC) have thus far demonstrated limited success as a monotherapy. What this eventually boils down to is our incomplete understanding of the molecular mechanisms, the impact of oncogenes, and thus the key pathways through which HDACi affect tumour cell growth. To shed further light on this, the involvement of oncogenic RAS - a key driver of CRC, in determining the responsiveness to HDACi has been explored. By using cell line model systems harbouring conditional oncogenic NRAS, KRAS and HRAS, we uncovered an oncogenic RAS-dependent “safeguard” mechanism imposed in order to evade the cytotoxic effect of HDACi and therefore apoptosis. Characteristically, cells harbouring oncogenic RAS were observed to undergo a reversible senescence-like growth arrest in G2, allowing for re-entry into cell cycle following the withdrawal of HDACi. This mechanism is implemented as a consequence of the direct targeting of RAS by HDAC inhibition, which resulted in a further amplified GTP-binding activity and subsequent signalling through the MAPK pathway. The observed outcome was an increase in the priming of MYC for ubiquitin-mediated proteasomal degradation, thereby enabling the cells to exit the cell cycle and enter the protective state of G2 arrest. This process was functionally reversed with a conditional non-degradable MYC (T58A/S62A), which in turn rendered the cells more susceptible to undergo apoptosis. Conclusively, in the context of a constitutively activating RAS mutation, the prospect of HDACi treatment was effectively improved using current MAPK-targeted therapy by preventing the observed pro-oncogenic effect of the HDACi treatment alone. Note: This abstract was not presented at the meeting. Citation Format: Sylvia S. Ispasanie, Lena Boehme, Martin Eilers, Tilman Brummer, Kathleen Klotz-Noack, Natalia Kuhn, Bastian Gastl, Christine Sers. HDAC inhibitors and the mechanism of resistance in colorectal cancer: RAS and MYC - the partners in crime [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5170. doi:10.1158/1538-7445.AM2017-5170


Citations (3)


... This regulatory mechanism is mediated by direct binding of SCIRT to SFPQ, which is involved in the DNA damage repair pathway. These findings provide novel insights into the oncogenic role of SCIRT and suggest critical for stem cell development, DNA damage, cell proliferation, apoptosis, and migration [32][33][34]. ...

Reference:

MA -mediated lncRNA SCIRT stability promotes NSCLC progression through binding to SFPQ and activating the PI3K/Akt pathway
SFPQ Depletion Is Synthetically Lethal with BRAFV600E in Colorectal Cancer Cells

Cell Reports

... Although these oncogenic events produce DNA damage that facilitates tumorigenesis and tumor heterogeneity, they also create a reduction in MCM/CMG functional fidelity in tumor cells that is likely exploitable with CMG inhibitors (1). Consistent with these concepts, oncogenic K-Ras also causes fork stalling and RS (18,19), and cells expressing mutant K-Ras are selectively sensitive to the reduction of MCM licensing (20), suggesting that future MCM/ CMG inhibitors will synergize with K-Ras mutations to suppress tumor growth. In addition, inhibition of MCM/CMG reserves can selectively sensitize tumor cells (including those with mutant K-Ras) to fork-stalling chemotherapy drugs (11,12), suggesting that CMG inhibitors will have the potential to overcome chemo-resistance in the management of cancer. ...

Reduced replication origin licensing selectively kills KRAS-mutant colorectal cancer cells via mitotic catastrophe

Cell Death and Disease

... Ligand-mediated EGFR activation and MAPK pathway signaling induce auto-and cross-expression of EGFR ligands that promote sustained signaling [29]. As such, EREG and AREG can regulate the expression of one another and are epigenetically co-regulated by DNA methylation [31][32][33][34][35]. EREG and AREG are also released and/or transcriptionally upregulated in response to G-protein coupled receptor (GPCR) agonists, such as lysophosphatidic acid, thrombin, and estradiol [36][37][38]. ...

Epigenetic regulation of Amphiregulin and Epiregulin in colorectal cancer