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International audience; The persistence of cancer cells resistant to therapy remains a major clinical challenge. In triple-negative breast cancer, resistance to chemotherapy results in the highest recurrence risk among breast cancer subtypes. The drug-tolerant state seems largely defined by nongenetic features, but the underlying mechanisms are poorly understood. Here, by monitoring epigenomes, transcriptomes and lineages with single-cell resolution, we show that the repressive histone mark H3K27me3 (trimethylation of histone H3 at lysine 27) regulates cell fate at the onset of chemotherapy. We report that a persister expression program is primed with both H3K4me3 (trimethylation of histone H3 at lysine 4) and H3K27me3 in unchallenged cells, with H3K27me3 being the lock to its transcriptional activation. We further demonstrate that depleting H3K27me3 enhances the potential of cancer cells to tolerate chemotherapy. Conversely, preventing H3K27me3 demethylation simultaneously to chemotherapy inhibits the transition to a drug-tolerant state, and delays tumor recurrence in vivo. Our results highlight how chromatin landscapes shape the potential of cancer cells to respond to initial therapy.

International audience; The segmented RNA genome of influenza A viruses (IAVs) enables viral evolution through genetic reassortment after multiple IAVs coinfect the same cell, leading to viruses harboring combinations of eight genomic segments from distinct parental viruses. Existing data indicate that reassortant genotypes are not equiprobable; however, the low throughput of available virology techniques does not allow quantitative analysis. Here, we have developed a high-throughput single-cell droplet microfluidic system allowing encapsulation of IAV-infected cells, each cell being infected by a single progeny virion resulting from a coinfection process. Customized barcoded primers for targeted viral RNA sequencing enabled the analysis of 18,422 viral genotypes resulting from coinfection with two circulating human H1N1pdm09 and H3N2 IAVs. Results were highly reproducible, confirmed that genetic reassortment is far from random, and allowed accurate quantification of reassortants including rare events. In total, 159 out of the 254 possible reassortant genotypes were observed but with widely varied prevalence (from 0.038 to 8.45%). In cells where eight segments were detected, all 112 possible pairwise combinations of segments were observed. The inclusion of data from single cells where less than eight segments were detected allowed analysis of pairwise cosegregation between segments with very high confidence. Direct coupling analysis accurately predicted the fraction of pairwise segments and full genotypes. Overall, our results indicate that a large proportion of reassortant genotypes can emerge upon coinfection and be detected over a wide range of frequencies, highlighting the power of our tool for systematic and exhaustive monitoring of the reassortment potential of IAVs.

SummaryTriple-negative breast cancer is associated with the worst prognosis and the highest risk of recurrence among all breast cancer subtypes1. Residual disease, formed by cancer cells persistent to chemotherapy, remains one of the major clinical challenges towards full cure2,3. There is now consensus that non-genetic processes contribute to chemoresistance in various tumor types, notably through the initial emergence of a reversible chemotolerant state4–6. Understanding non-genetic tumor evolution stands now as a prerequisite for the design of relevant combinatorial approaches to delay recurrence. Here we show that the repressive histone mark H3K27me3 is a determinant of cell fate under chemotherapy exposure, monitoring epigenomes, transcriptomes and lineage with single-cell resolution. We identify a reservoir of persister basal cells with EMT markers and activated TGF-β pathway leading to multiple chemoresistance phenotypes. We demonstrate that, in unchallenged cells, H3K27 methylation is a lock to the expression program of persister cells. Promoters are primed with both H3K4me3 and H3K27me3, and removing H3K27me3 is sufficient for their transcriptional activation. Leveraging lineage barcoding, we show that depleting H3K27me3 alters tumor cell fate under chemotherapy insult – a wider variety of tumor cells tolerate chemotherapy. Our results highlight how chromatin landscapes shape the potential of unchallenged cancer cells to respond to therapeutic stress.

Despite the success of immune check point inhibition, identification of other pathways capable of modulating the immune response against the tumor remains challenging. T-cell co-stimulation has been investigated with limited clinical success so far due in part to the fine tuning required for agonist antibodies against those co-stimulatory receptors and to the lack of biomarkers to facilitate the selection of patients likely to benefit from T-cell co-stimulation. TNFR2 belongs to the TNFR family of costimulatory molecules, and its expression on tumor infiltrating lymphocytes across a wide range of tumors make it an attractive target for T-cell co-stimulation. Recently, we identified HFB200301, an anti-TNFR2 antibody with Fc-independent agonist activity that does not block TNFR2 interaction with TNFα. HFB200301 activates CD4+, CD8+ T cells, and NK cells in vitro. In vivo, HFB200301 demonstrated potent single agent anti-tumor activity in syngeneic tumor models and can further increase the antitumor activity in combination with PD-1 blockade. To understand the immunological basis for the anti-tumor efficacy of HFB200301, we investigated the pharmacodynamic effects of HFB200301 in syngeneic mouse tumor models, including immuno-phenotyping and receptor occupancy of tumor infiltrating cells. In hTNFR2 knock-in mice bearing MC38 tumors, HFB200301 induces expansion of CD4+ and CD8+ T cells, and NK cells in the tumor micro-environment without affecting regulatory T cell numbers. We also demonstrate that the anti-tumor efficacy of HFB200301 is correlated with receptor occupancy and circulating soluble TNFR2 in a dose-dependent manner in this model. To discover predictive biomarkers of response to HFB200301, we used primary tumor samples and our proprietary Drug Intelligent Science (DIS™) single-cell platform to establish an immune-related signature. Single-cell RNA sequencing and clonotype barcoding of ex-vivo tumor cultures treated with HFB200301 were used to identify unique T cell profiles with a T cell centric gene panel. These unique T cell profiles may help identifying patients more likely to respond to HFB200301 treatment. In summary, HFB200301 exhibits a unique mechanism of action mainly relying on its agonistic activity on several effector cell types in tumor micro-environment that we expect will benefit a patient population selected with a unique biomarker signature. HFB200301 is currently in preclinical development and a biomarker-driven Phase 1 clinical study is projected for 2021. Citation Format: Shuo Wei, Ross Fulton, Yun-Yueh Lu, Qian Zhang, He Zhou, Andreas Raue, Mingjie Chen, Wenhua Xu, Xing Cai, Juliana Crivello, Zachary Duda, Zhiyuan Wang, Rebecca Silver, Alexandra Staskus, Charina Ortega, Sami Ellouze, Carine George, Sophie Foulon, Dean Lee, Monika Manne, Nicola Beltraminelli, Jinping Gan, Francisco Adrian, Liang Schweizer, Jennifer Watkins-Yoon. Mechanism of action and biomarker strategy for HFB200301, an anti-TNFR2 agonist antibody for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1883.

Agonist OX40 antibodies have shown promising pre-clinical activities, but their clinical activities have been limited thus far. Several reasons may account for this limited clinical activity, including sub-optimal antibody design, dose selection, and lack of a biomarker strategy for indication selection and patient enrichment. Previous clinical trials selected doses that maximized receptor occupancy, but some patients responded at lower doses, indicating nuances to choosing the correct therapeutic doses for an agonist antibody. In addition, preclinical work has demonstrated a so-called “hook” effect whereby agonist activity decreases at higher concentrations, which further emphasizes the need to develop a novel anti-OX40 therapeutic antibody that addresses the previously encountered challenges. HFB301001 is a novel human IgG1 agonist antibody that binds to a unique epitope on OX40. This allows for agonistic activity that does not compete with the endogenous OX40 ligand. Relative to other clinical stage OX40 antibodies, HFB301001 has reduced OX40 downregulation following co-stimulation of T cells, and it has demonstrated superior in vivo anti-tumor activity and pharmacodynamic immune modulation in a human OX40 knock-in mouse model. HFB301001 is well tolerated in cynomolgus monkeys. To progress into clinical studies, we have determined human dose projections for clinical evaluation of HFB301001 using PKPD modeling, serum exposure in non-human primates, antitumor efficacy in mouse models and immune cell pharmacodynamics. We also took advantage of Fc variants to delineate the relative contributions of Treg depletion versus enhancing T cell activity by agonism to efficacy of HFB301001. To further enhance probability of success (POS) in clinical studies, we are applying our single-cell Drug Intelligent Science (DIS™) platform to rationally identify cancer indications and to define novel predictive response biomarkers. We have used single-cell profiling to identify unique tumor-infiltrating T cell signatures that may help identify patients more likely to response to HFB301001 treatment, inform indication selection, and establish a patient stratification biomarker strategy. Finally, we show here our phase I trial design for HFB301001 that implements these findings. In conclusion, HFB301001 is a highly differentiated therapeutic antibody which is well positioned to enter a global, multi-center Phase I clinical trial to explore optimal biologically active dose and evaluate predictive biomarker hypotheses. Here, we present results supporting the rationale for indication selection, biomarker identification, dose selection, and phase I clinical trial design. Citation Format: Ross Fulton, Jinping Gan, Yun-Yueh Lu, Julianna Crivello, Zachery Duda, Zhiyuan Wang, Rebecca Silver, Alexandra Staskus, Charina Ortega, Sami Ellouze, Carine George, Sophie Foulon, Wenhua Xu, Xing Cai, Joyce Pi, Dean Lee, Monika Manne, Ruina Jin, Yuan Wang, Hongkai Zhang, Nicola Beltraminelli, Francisco Adrian, Robert Petit, Liang Schweizer, Andreas Raue. Clinical approach and biomarker strategy for HFB301001, a novel OX40 agonistic antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1882.

Discovering autocatalytic chemistries that can evolve is a major goal in systems chemistry and a critical step towards understanding the origin of life. Autocatalytic networks have been discovered in various chemistries, but we lack a general understanding of how network topology controls the Darwinian properties of variation, differential reproduction, and heredity, which are mediated by the chemical composition. Using barcoded sequencing and droplet microfluidics, we establish a landscape of thousands of networks of RNAs that catalyze their own formation from fragments, and derive relationships between network topology and chemical composition. We find that strong variations arise from catalytic innovations perturbing weakly connected networks, and that growth increases with global connectivity. These rules imply trade-offs between reproduction and variation, and between compositional persistence and variation along trajectories of network complexification. Overall, connectivity in reaction networks provides a lever to balance variation (to explore chemical states) with reproduction and heredity (persistence being necessary for selection to act), as required for chemical evolution., Autocatalytic networks may have started evolution during the origin of life. Here, the authors establish a landscape of thousands of RNA networks by barcoded sequencing and microfluidics, and derive relationships between topology and Darwinian properties such as variation and differential reproduction.

The long and complex Trypanosoma brucei development in the tsetse fly vector culminates when parasites gain mammalian infectivity in the salivary glands. A key step in this process is the establishment of monoallelic variant surface glycoprotein (VSG) expression and the formation of the VSG coat. The establishment of VSG monoallelic expression is complex and poorly understood, due to the multiple parasite stages present in the salivary glands. Therefore, we sought to further our understanding of this phenomenon by performing single-cell RNA-sequencing (scRNA-seq) on these trypanosome populations. We were able to capture the developmental program of trypanosomes in the salivary glands, identifying populations of epimastigote, gamete, pre-metacyclic and metacyclic cells. Our results show that parasite metabolism is dramatically remodeled during development in the salivary glands, with a shift in transcript abundance from tricarboxylic acid metabolism to glycolytic metabolism. Analysis of VSG gene expression in pre-metacyclic and metacyclic cells revealed a dynamic VSG gene activation program. Strikingly, we found that pre-metacyclic cells contain transcripts from multiple VSG genes, which resolves to singular VSG gene expression in mature metacyclic cells. Single molecule RNA fluorescence in situ hybridisation (smRNA-FISH) of VSG gene expression following in vitro metacyclogenesis confirmed this finding. Our data demonstrate that multiple VSG genes are transcribed before a single gene is chosen. We propose a transcriptional race model governs the initiation of monoallelic expression., Author summary African trypanosomes are parasitic protists which cause endemic disease in sub-Saharan Africa. To evade mammalian immune responses the parasite has developed a system of antigenic variation, where the surface of the cell is covered in a tightly packed coat of variant surface glycoproteins (VSGs). Each cell expresses only one variant surface glycoprotein at a time, and this is periodically switched to evade new antibodies. The process of singular gene expression is termed monoallelic expression and this has two components, establishment and maintenance, i.e. how a single gene is selected for expression and how its singular expression is maintained throughout successive generations. The establishment of monoallelic VSG gene expression occurs in the salivary gland of the tsetse fly vector, although this process is not well understood. We used single cell gene expression profiling applied to thousands of single cells in the salivary gland of the fly. We show that in order to select a single gene, trypanosomes initially transcribe multiple VSGs before a single gene is selected for high-level expression. We propose a model where this process is driven by a race to accumulate transcription factors at a single VSG gene.