Kathleen Klotz-Noack’s research while affiliated with Charité Universitätsmedizin Berlin and other places

In colorectal cancer, oncogenic mutations transform a hierarchically organized and homeostatic epithelium into invasive cancer tissue lacking visible organization. We sought to define transcriptional states of colorectal cancer cells and signals controlling their development by performing single-cell transcriptome analysis of tumors and matched non-cancerous tissues of twelve colorectal cancer patients. We defined patient-overarching colorectal cancer cell clusters characterized by differential activities of oncogenic signaling pathways such as mitogen-activated protein kinase and oncogenic traits such as replication stress. RNA metabolic labeling and assessment of RNA velocity in patient-derived organoids revealed developmental trajectories of colorectal cancer cells organized along a mitogen-activated protein kinase activity gradient. This was in contrast to normal colon organoid cells developing along graded Wnt activity. Experimental targeting of EGFR-BRAF-MEK in cancer organoids affected signaling and gene expression contingent on predictive KRAS/BRAF mutations and induced cell plasticity overriding default developmental trajectories. Our results highlight directional cancer cell development as a driver of non-genetic cancer cell heterogeneity and re-routing of trajectories as a response to targeted therapy.

In colorectal cancer (CRC), the prevalence of NRAS mutations (5–9%) is inferior to that of KRAS mutations (40–50%). NRAS mutations feature lately during tumour progression and drive resistance to anti-EGFR therapy in KRAS wild-type tumours. To elucidate specific functions of NRAS mutations in CRC, we expressed doxycycline-inducible G12D and Q61K mutations in the CRC cell line Caco-2. A focused phospho-proteome analysis based on the Bio-Plex platform, which interrogated the activity of MAPK, PI3K, mTOR, STAT, p38, JNK and ATF2, did not reveal significant differences between Caco-2 cells expressing NRASG12D, NRASQ61K and KRASG12V. However, phenotypic read-outs were different. The NRAS Q61K mutation promoted anchorage-independent proliferation and tumorigenicity, similar to features driven by canonical KRAS mutations. In contrast, expression of NRASG12D resulted in reduced proliferation and apoptosis. At the transcriptome level, we saw upregulation of cytokines and chemokines. IL1A, IL11, CXCL8 (IL-8) and CCL20 exhibited enhanced secretion into the culture medium. In addition, RNA sequencing results indicated activation of the IL1-, JAK/STAT-, NFκB- and TNFα signalling pathways. These results form the basis for an NRASG12D-driven inflammatory phenotype in CRC.

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.

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.

We address whether combinations with a pan-RAF inhibitor (RAFi) would be effective in KRAS mutant pancreatic ductal adenocarcinoma (PDAC). Chemical library and CRISPR genetic screens identify combinations causing apoptotic anti-tumor activity. The most potent combination, concurrent inhibition of RAF (RAFi) and ERK (ERKi), is highly synergistic at low doses in cell line, organoid, and rat models of PDAC, whereas each inhibitor alone is only cytostatic. Comprehensive mechanistic signaling studies using reverse phase protein array (RPPA) pathway mapping and RNA sequencing (RNA-seq) show that RAFi/ERKi induced insensitivity to loss of negative feedback and system failures including loss of ERK signaling, FOSL1, and MYC; shutdown of the MYC transcriptome; and induction of mesenchymal-to-epithelial transition. We conclude that low-dose vertical inhibition of the RAF-MEK-ERK cascade is an effective therapeutic strategy for KRAS mutant PDAC.

Colorectal cancer (CRC) is a substantial cause of death in the western world. With KRAS being one of the most frequently altered oncogenes in CRC, it is an obvious target for cancer therapy. But despite enormous efforts over the past three decades to target mutated RAS, not a single drug has made it to the clinic. In order to find vulnerabilities of KRAS mutant cells, we performed a shRNA-based screen. We used CaCo2 cells (wild type for KRAS/BRAF/PIK3CA) with doxycycline (DOX) inducible expression of the oncogenic KRASG12V as a model system. The custom-designed shRNA library comprised 121 selected genes encoding signaling proteins and transcription factors, which we have previously identified to be strongly up- or downregulated after treatment with MEK inhibitors. We found that CaCo2 cells expressing KRASG12V are highly sensitive to the depletion of the DNA replication licensing factor Minichromosome Maintenance Complex Component 7 (MCM7), whereas wild-type CaCo2 cells are resistant to MCM7 depletion. The screening result was verified with 3 independent shRNAs under anchorage dependent and independent conditions. Similar results were obtained with NRASG12D and in DLD1 (KRASwt/KRASG13D) and DLD1 (KRASwt/-) cells. The proposed molecular mechanism is RAS-induced genotoxic stress that cannot be resolved after MCM7 reduction. MCM7-containing complexes prime DNA for replication by binding to replication origins. However, only a few of the licensed origins are later used for DNA replication, while most origins stay dormant. The dormant origins are essential for resolving stalled replication forks after replicative stress. Unresolved stalled forks lead to DNA damage, which drives the cell into apoptosis. Further experiments will show the extent of DNA damage upon RAS induction and knock down of MCM7. Since DNA replication licensing is only required in actively dividing cells and cells with physiological RAS signaling seem to be resistant to partial depletion of MCM7, it might be an ideal target for treating RAS mutated cancers. Citation Format: Bastian Gastl, Kathleen Klotz-Noack, Bertram Klinger, Johannes Zuber, Nils Blüthgen, Reinhold Schäfer, Christine Sers. Depletion of replication factor MCM7 is synthetically lethal to oncogenic KRAS expression [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A20.

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

To prevent re-replication of DNA in a single cell cycle, the licensing of replication origins by Mcm2-7 is prevented during S and G2 phases. Animal cells achieve this by cell-cycle-regulated proteolysis of the essential licensing factor Cdt1 and inhibition of Cdt1 by geminin. Here we investigate the consequences of ablating geminin in synchronised human U2OS cells. Following geminin loss, cells complete an apparently normal S phase, but a proportion arrest at the G2-M boundary. When Cdt1 accumulates in these cells, DNA re-replicates, suggesting that the key role of geminin is to prevent re-licensing in G2. If cell cycle checkpoints are inhibited in cells lacking geminin, cells progress through mitosis and less re-replication occurs. Checkpoint kinases thereby amplify re-replication into an all-or-nothing response by delaying geminin-depleted cells in G2. Deep DNA sequencing revealed no preferential re-replication of specific genomic regions after geminin depletion. This is consistent with the observation that cells in G2 have lost their replication timing information. By contrast, when Cdt1 is overexpressed or is stabilised by the neddylation inhibitor MLN4924, re-replication can occur throughout S phase.

In this issue of Molecular Cell, Kawabata et al. (2011) show that mice hypomorphic for the replication licensing protein Mcm4 show spontaneous DNA replication defects due to a lack of dormant origins, potentially explaining why these mice are cancer prone.