Targeting the oncogenic signaling of the transcription factor STAT3 with potent and selective monobody inhibitors

Many targeted cancer therapies fail to improve the overall survival of patients. Limitations involve low drug selectivity and the rapid development of resistance. Moreover, the majority of oncogenic drivers presently remain considered as undruggable as these proteins often lack well-defined binding pockets able to fit small molecular antagonists. The need for novel strategies to expand the number of druggable targets thus appears crucial.

STAT3 is a transcription factor that is constitutively active in a majority of solid and hematological tumors. It is considered a central target for cancer therapies, as it induces the transcription of genes essential for tumor proliferation, including anti-apoptotic genes, cell cycle regulators and angiogenic factors. Therefore, the inhibition of STAT3 represents a promising therapeutic strategy. However, the targeting of STAT3 still remains a formidable challenge due to the sparse number of chemical probes able to bind to non-enzymes. The engineering of protein binders could overcome many of the challenges in developing STAT3 antagonists. While most common approaches to preclude STAT3 activation consist in either inhibiting its phosphorylation by upstream tyrosine kinases with clinically-approved drugs, or in preventing the SH2 domain-dependent STAT3 dimerization using pre-clinical small molecule probes, the targeting of other STAT3 domains, such as its Coiled-coil or N-terminal domains, has not been thoroughly investigated.

Our lab uses small engineered antibody mimics derived from a fibronectin type 3 scaffold (termed monobodies) capable of high affinity and selective binding. In addition, their small size (~10 kDa) and lack of disulfide bonds makes them promising candidates for intracellular antagonistic use. In this work, we selected several monobodies using phage and yeast display that bind to previously untargeted domains of STAT3 with low nanomolar affinities. The monobodies MS3-6 and MS3-N3, binding to the Coiled-coil and N-terminal domain of STAT3 respectively, showed high selectivity to STAT3 as compared to other STAT family members and other unrelated proteins. Additionally, the monobodies strongly inhibited the transcriptional activity of STAT3 in luciferase reporter assays, reduced mRNA levels of STAT3 downstream genes in human lung cancer cells, decreased STAT3 phosphorylation levels upon IL-22 stimulation and interfered with STAT3 nuclear translocation. Notably, the precise blockade of these previously untargeted key domains using monobodies provide new insights into the STAT3 signaling and expand the current strategies to preclude its activity. Altogether, we have developed the first selective monobody inhibitors against a transcription factor implicated in cancer and inflammatory diseases, and used them as biochemical tools to further characterize a poorly understood alternative IL-22R signaling axis involved in STAT3-driven human disorders such as colitis and psoriasis.

Gene expression analysis of metastases and IL-1R1 characterization in a NALP3 mouse model

Heidi Fournier

Tumor microenvironment contributes strongly to tumor evolution toward metastasis, a final stage in cancer progression. It provides essential clues to promote migration of cancer cells in metastatic sites. Moreover, inflammatory cells and immunomodulatory mediators present in the microenvironment are able to polarize the host immune response, thereby potentiating primary tumor development. The reciprocal and dynamic interactions between microenvironment and tumor cells orchestrate critical steps of evolution toward metastasis. To address the role of inflammation in tumor progression, a mouse model of downregulated inflammation was used: the NALP3 gene in these mice is knocked-out, causing a impaired processing of the two inflammatory cytokines Interleukin-1[beta] and IL-18 which are essential for the recruitment and activation of immune cells. In this case, a decrease in tumor growth and metastasis was observed in previous studies, leading us to investigate the molecular and cellular interactions of tumor and immune cells in inflammatory and non-inflammatory contexts. Gene expression analysis was performed on two steps of mouse tumor cells: the B16-F10 melanoma cells seem to be more dependent on the immune cells recruitment to metastasize in vivo, while the Lewis Lung Carcinomas cells showed a quite equivalent growth in both wild-type or NALP3 knock-out mice. Different techniques were used to do so, such as standard in vitro assays and mRNA expression analyses, and in vivo tests with extraction of tumoral mRNA by Laser Capture Microdissection. Gene expression analyses of tumor cells were performed to address the influence of the inflammatory background. Comparison of these two gene expression profiles may reveal genes related to the specific reactive inflammatory response surrounding the given metastases. Another point that was investigated was the importance of the IL-1R1, receptor of interleukin-1. Since it represents the counterpart of IL-1[beta] signaling, its expression by immune and tumor cells may correlate with degree of tumor growth and invasion. In vitro and in vivo assays allowed better understanding the importance of this receptor. While a downregulation of this receptor impairs proliferation of in vitro cell cultures, at the in vivo level, the number of metastases is decreased too. These results provide potential clues indicating the important role played by the inflammatory cells, and how they can support and promote tumor progression, in particular through the expression of NALP3. The adaptation mechanisms of tumor cells to their microenvironment were also assessed, opening different insights for targeting the tumor itself as well as the inflammatory microenvironment.

2011

Quantitative analysis of gene expression and transcriptional memory in mouse embryonic stem cells

Aleksandra Mandic

Quantitative studies of gene expression in have shown widespread variability in transcript and protein product abundance at the single-cell level. Such fluctuations in gene expression have been shown to lead to heterogeneous cellular responses and phenotypes, acting in a range of systems, from immunity, cancer to development. To better understand heterogeneity in cellular populations, we developed a novel method for the precise quantification of gene expression in single living cells. Furthermore, we focused on whether and to which extent transcriptional expression profiles are transmitted during cell division. In the first section, we described a new approach for quantitative measurements of gene expression, based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse imaging. Using the currently brightest luminescent reporter, fluctuations of protein expression levels could be monitored in single living cells with high sensitivity and temporal resolution over extended time periods. Moreover, our system allowed measurement of single-cell protein degradation rates in the absence of protein synthesis inhibitors, as well as calculation of absolute synthesis rates over the cell cycle. Finally, the internal tag could be excised by inducible expression of Cre recombinase, which enabled estimation of endogenous mRNA half-lives by monitoring the decay of luminescent signal. Second, to monitor transcriptional activity and quantify memory across multiple endogenous genes in mouse embryonic stem cells, we generated cell lines in which a short-lived luciferase reporter allowed sensitive monitoring of transcriptional kinetics by luminescence imaging at high time resolution over long periods of time. By coupling live single-cell monitoring of transcriptional activity of 9 different promoters with mathematical modelling, we found that different levels of expression fluctuations shape transcriptional memory. Long fluctuations (1-10 generations) and short fluctuations (1-3 h), as well as their amplitudes, vary widely between across different genes and together shape transcriptional memory. Surprisingly, we have shown that there was a large level of gene-specific cell-to-cell synchronicity in transcriptional profiles. Overall, we uncovered that noise and timescales of slow and fast fluctuations, as well as their synchronisation over the cell cycle, defined how transcriptional dynamics were correlated within a lineage of related cells. In conclusion, this thesis described an innovative method that allows measurement of absolute protein expression levels, protein turnover, as well as mRNA half-lives for endogenously expressed genes. Our approach opens new avenues in quantitative studies of gene expression in single living cells. Furthermore, we revealed different levels of transcriptional fluctuations that shape gene specific transcriptional memory and its timescales in mouse embryonic stem cells.

EPFL2018

The role of OCT4 and SOX2 in regulating chromatin accessibility and cell fate across the cell cycle in embryonic stem cells

Torgil Elias Friman

Precise spatiotemporal regulation of gene expression is essential for development and homeostasis of complex organisms. This is achieved in large part by sequence-specific transcription factors (TF) that bind to genomic regulatory elements to activate or repress transcription. DNA in eukaryotic nuclei is wrapped around histones into nucleosomes, which are inaccessible to most proteins. However, certain TFs can access their binding sites in the presence of histones and promote nucleosome eviction to increase chromatin accessibility, allowing for binding of other protein. These TFs, called pioneer factors, include OCT4 and SOX2, which are master regulators of embryonic stem (ES) cells. Dividing cells, including stem cells, must achieve proper gene regulation despite the progression of the cell cycle, which imposes several constraints. During S phase, the replication fork passes through the genome to make a copy of each chromosome, which leads to a loss of chromatin accessibility. Furthermore, the substantial chromatin compaction that occurs during mitosis to prepare for chromosome segregation is associated with eviction of most DNA-binding proteins and reduced accessibility at distal cis-regulatory elements including enhancers. How pioneer factors operate in the context of cell cycle progression is unclear. In this thesis, I will present work studying different features of OCT4 and SOX2, including how they interplay to regulate chromatin accessibility in ES cells, the impact of small endogenous fluctuations of their protein levels on cell fate and chromatin accessibility, and how their roles in regulating cell fate and chromatin accessibility is related to different stages of the cell cycle. Using live-cell imaging of fluorescently labeled proteins, we discovered that OCT4 and SOX2 were associated to mitotic chromatin. Using cell-cycle specific degradation strategies, we could show that the presence of SOX2 and OCT4 at the mitosis-G1 (M-G1) transition contributes to maintain the pluripotency of ES cells. We further showed that ES cells with high levels of OCT4 in G1, but not in S phase, are more prone to differentiate towards neuroectoderm and mesendoderm cell fates. Cells with high levels of OCT4 displayed increased chromatin accessibility at enhancers of differentiation genes, suggesting accessibility fluctuates with OCT4 levels and can prime cells for differentiation. To explore potential cell cycle-dependent regulation of chromatin accessibility, we degraded OCT4 at the M-G1 transition, which led to a reduction in accessibility at many OCT4-bound regions. When OCT4 returned later in the cell cycle, chromatin accessibility was recovered, although at many loci this recovery was incomplete, suggesting that OCT4 is required at the M-G1 transition to maintain these regions open. We then rapidly degraded OCT4 at other phases of the cell cycle. Surprisingly, loss of chromatin accessibility was highly similar at all cell cycle phases, showing that OCT4 is constantly required to maintain regulatory elements accessible. To explore the dynamic regulation of accessibility by OCT4, we performed time-course measurements of accessibility upon its degradation. This revealed that loss of accessibility was temporally correlated to the degradation of OCT4. These results suggest that chromatin accessibility is highly dynamic and continuously dependent on the presence of OCT4 across the cell cycle in ES cells.

EPFL2019