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CCTG IND.236: A Phase 1b trial of combined CFI-402257 and weekly paclitaxel in patients with HER2-negative (HER2-) advanced breast cancer (aBC) Philippe L. Bedard, Mihaela Mates, John Hilton, Nathalie Levasseur, Arif Awan, Amirrtha Srikanthan, David Cescon, Karen Gelmon, Andrew Robinson, Nancy Drummond-Ivars, Irene Li, Laleh Rastgou, Jackie Edwards, Linda Hagerman, Siwei Zhang, Mark Bray, Lesley Seymour, Moira Rushton-Marovac, Pierre-Olivier Gaudreau Background: CFI-402257 is a selective oral inhibitor of TTK protein kinase, a critical regulator of the mitotic spindle assembly checkpoint overexpressed in breast cancer (BC). CFI-402257 monotherapy has anti-proliferative and cytotoxic activity and enhances antitumor activity of paclitaxel in BC xenograft models. Material and methods: Primary objectives were to establish safety and Recommended Phase 2 dose (RP2D) of CFI-402257 combined with weekly paclitaxel (Phase 1b) and Overall Response Rate (ORR) as per RECIST 1.1 (Phase 2). Patients with HER2- aBC with adequate organ function, PS 0-1, previously treated with >1 non-taxane chemotherapy, were eligible. A 3+3 design was used for Phase 1b, with dose limiting toxicities (DLTs) assessed during cycle 1 (28 days). Starting dose CFI-402257 was 84mg (DL1 = 84mg, DL2 = 112mg, DL3 = 168mg, DL4 = 210mg and DL5 = 252mg) on a 2-day on, 5-day off schedule with paclitaxel 80mg/m2 day 1, 8, 15. Safety assessments were performed weekly (CTCAE v5.0) and response every 2 cycles. A Simon 2-stage design was used for Phase 2 (stage 2 required ≥4 responses in 17 evaluable patients from stage 1). Results: 37 patients received a total of 260 cycles including all 5 dose levels. Median age was 59; 92% ER+/HER2-; 49% PS1; 22% 3 prior chemotherapy lines; 41% 4 sites of metastatic disease, and 81% had received prior CDK4/6 inhibitors. Grade 3 hematological adverse events (AEs, all dose levels) were neutropenia (70%), lymphopenia (41%) and anemia (14%). Six DLTs occurred: 5 dose-related grade 4 neutropenia and 1 febrile neutropenia. Three DLTs occurred at DL3, two at DL4, and one at DL5. Three serious AEs (two at DL3, and one at DL4) at least possibly related to treatment were seen: 2 febrile neutropenia and 1 skin infection (all grade 3). Frequent AEs (˃5%; all dose levels) considered at least possibly related to treatment were: diarrhea (38%), nausea (30%), fatigue (27%), vomiting (16%), anorexia (14%), maculo-papular rash (14%), oral mucositis (11%), alopecia (11%) and pruritus (8%). DL3 (168mg) was selected as RP2D. ORR was 3/36=8% and 1/17=5.9% in all vs Phase 2 evaluable patients, respectively. Clinical Benefit Rate (CBR; defined as complete response, partial response or stable disease ˃16 weeks in duration) was 18/33=54.6% and 10/17=58.8% in all vs Phase 2 evaluable patients, respectively. During Phase 2, the 17 evaluable patients from stage 1 did not meet pre-specified threshold for anti-tumor activity to proceed to stage 2. Conclusions: CFI-402257 and paclitaxel was well tolerated, with neutropenia as the main toxicity. DL3 (168mg) was selected as RP2D. Phase 2 ORR and CBR was 5.9% and 58.8%, respectively; during Phase 2, the 17 evaluable patients from stage 1 did not meet the pre-specified threshold for anti-tumor activity to proceed to stage 2 and the trial was closed to accrual on April 7, 2022. Final analysis and correlative analyses are ongoing. Acknowledgements: Coordinated by the CCTG. Funding supported by SU2C Canada - Canadian Cancer Society Breast Cancer Dream Team Research Funding (SU2C-AACR-DT-18-15) and OICR. CFI-402257 provided by Treadwell Therapeutics. Citation Format: Philippe Bedard, Mihaela Mates, John Hilton, Nathalie Levasseur, Arif Awan, Amirrtha Srikanthan, David W. Cescon, Karen Gelmon, Andrew Robinson, Nancy Drummond-Ivars, Irene Li, Laleh Rastgou, Jackie Edwards, Linda Hagerman, Siwei Zhang, Mark Bray, Lesley Seymour, Moira Rushton, Pierre-Olivier Gaudreau. CCTG IND.236: A Phase 1b trial of combined CFI-402257 and weekly paclitaxel in patients with HER2-negative (HER2-) advanced breast cancer (aBC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-10.

In equilibrium, the collective behaviour of particles interacting via steep, short-ranged potentials is well captured by the virial expansion of the free energy at low density. Here, we extend this approach beyond equilibrium to the case of active matter with self-propelled particles. Given that active systems do not admit any free-energy description in general, our aim is to build the dynamics of the coarse-grained density from first principles without any equilibrium assumption. Starting from microscopic equations of motion, we obtain the hierarchy of density correlations, which we close with an ansatz for the two-point density valid in the dilute regime at small activity. This closure yields the nonlinear dynamics of the one-point density, with hydrodynamic coefficients depending explicitly on microscopic interactions, by analogy with the equilibrium virial expansion. This dynamics admits a spinodal instability for purely repulsive interactions, a signature of motility-induced phase separation. Therefore, although our approach should be restricted to dilute, weakly-active systems a priori, it actually captures the features of a broader class of active matter., 2 figures

Hypoplastic Left Heart Syndrome (HLHS) is a rare and complex congenital heart defect characterized by hypoplasia of the left ventricle, proximal aorta, as well as stenosis or atresia of the mitral and/or aortic valves. There is strong evidence for a genetic contribution to HLHS, including our lab’s previous discovery of 19 rare, predicted-damaging MYH6 variants that were significantly enriched in HLHS patients. The MYH6 gene encodes for the α-myosin heavy chain (α-MHC), a key contractile protein in cardiac sarcomere. Variants affecting cardiac MHC isoforms are thought to impact the strength, speed, and patterns of myocardial contractions. Reduced myocardial contractility due to MYH6 variants may impair cardiac blood flow and cause underdevelopment of the left heart. Mavacamten is a novel pharmacological agent that has shown promise in phase III clinical trials for treatment of hypertrophic obstructive cardiomyopathy. Mavacamten selectively acts on cardiac MHC to reduce cardiac muscle contractility. Given this specificity, Mavacamten may offer a promising way to treat diastolic dysfunction in (i.e. hypercontractility) in patients with HLHS, including those with MYH6 variants. We tested the in vitro efficacy of Mavacamten in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) exhibiting hypercontractility and investigated video-based analysis as a high-throughput method for evaluating iPSC-CM contractility, which would allow for repeated assessment of cells at different timepoints following drug treatment. Many packages for video-based contractility assessment lack precision and can be subject to disturbances such as cellular fragments, movement of cell media, and small differences in media color. They are also complex and require advanced programming knowledge. MYOCYTER is an open-source macro for ImageJ that improves upon previous plugins available by allowing for users to optimize parameters for contractility evaluation, while minimizing background noise. The aim of our study was to determine if MYOCYTER can be reliably used to detect small changes in iPSC-CM contractility following treatment with Mavacamten, and if the workflow is user-friendly enough such that reproducible analysis can be done by a high school science team.Our analysis revealed that MYOCYTER can detect baseline hypercontractility of iPSC-CMs carrying MYH6 variants compared to wild-type iPSC-CMs (p = 0.033). These differences in amplitude were eliminated following five, ten, and fifteen minutes of treatment with 500nM Mavacamten in a single experiment. Our results suggest that this method can be used to further evaluate the effect of myosin modulators, such as Mavacamten, in patient-specific iPSC-CMs. Children's Research Institute This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

S1A. Plasmids containing single guide RNAs targeted against the IRE1 locus. B. XBP1 splicing assay to assay IRE1 endoribonuclease activity in Hep3B and MDA MB 231 cells. S2. CRISPR clones obtained for MDA-MB-231 cells. S3. CRISPR clones obtained for Hep3B cells. S4. CRISPR clones obtained for HepG2 cells. S5. Representative brightfield images of Boyden's chamber migration assay. S6. Representative brightfield images of Boyden's chamber migration assay. S7. Representative brightfield images of Boyden's chamber migration assay. S8. Heatmap of standardized DSP, ZEB1 or ZEB2 expression in the TCGA PANCAN dataset. S9. Scatter plots showing the correlation between DSP methylation and the expression of DSP, ZEB1 or ZEB2 in the TCGA. S10A. Western blots showing expression levels of DSP, ZEB1, and GAPDH in MDA-MB-231 cells treated with scramble, no treatment, or siZEB1 at 100 nM (ORIGENE, cat # SR321982) for 48 hrs. The plots represent the quantification of the western blots. B-C. Barplots displaying relative gene expression levels measured using qRT-PCR of (B) ZEB1 and DSP (normalized to scramble) in MDA-MB-231 cells treated with siZEB1 at 100 nM 24 hrs, and (C) ZEB2 and DSP (normalized to scramble) in MDA-MB-231 cells treated with siZEB2 at 80 nM for 24 hrs. S11. A. Western blots showing expression levels of DSP, ZEB1, and GAPDH in Hep3B cells treated with scramble, no treatment, or siZEB1 at 100 nM (ORIGENE, cat # SR321982) for 48 hrs. The plots represent the quantification of the western blots. B-C. Barplots displaying relative gene expression levels measured using qRT-PCR of (B) ZEB1 and DSP (normalized to scramble) in Hep3B cells treated with siZEB1 at 100 nM 24 hrs, and (C) ZEB2 and DSP (normalized to scramble) in Hep3B cells treated with siZEB2 at 80 nM for 24 hrs. S12. Positive ChIP control for ZEB1 binding in Hep3B, MDA-MB-231 and HepG2 cells in the CDH1 promoter. S13. Relative ZEB1 protein expression in PA or BSA treated (48 hr) WT- or IRE1-KO MDA-MB-231 cells. S14. Relative ZEB1 protein expression in PA or BSA treated (72 hrs) WT- or IRE1-KO Hep3B cells.

COVID-19 remains a global pandemic of an unprecedented magnitude with millions of people now developing “COVID lung fibrosis.” Single-cell transcriptomics of lungs of patients with long COVID revealed a unique immune signature demonstrating the upregulation of key proinflammatory and innate immune effector genes CD47, IL-6, and JUN. We modeled the transition to lung fibrosis after COVID and profiled the immune response with single-cell mass cytometry in JUN mice. These studies revealed that COVID mediated chronic immune activation reminiscent to long COVID in humans. It was characterized by increased CD47, IL-6, and phospho-JUN (pJUN) expression which correlated with disease severity and pathogenic fibroblast populations. When we subsequently treated a humanized COVID lung fibrosis model by combined blockade of inflammation and fibrosis, we not only ameliorated fibrosis but also restored innate immune equilibrium indicating possible implications for clinical management of COVID lung fibrosis in patients.

The ability of the maternally transmitted endosymbiotic bacterium Wolbachia to induce cytoplasmic incompatibility (CI) and virus blocking makes it a promising weapon for combatting mosquito-borne diseases through either suppression or replacement of wild-type populations. Recent field trials show that both approaches significantly reduce the incidence of dengue fever in humans. However, new questions emerge about how Wolbachia-mosquito associations will co-evolve over time and whether Wolbachia-mediated virus blocking will be affected by the genetic diversity of mosquitoes and arboviruses in the real world. Here, we have compared the Wolbachia density and CI expression of two wAlbB-infected Aedes aegypti lines transinfected 15 years apart. We have also assessed wAlbB-mediated virus blocking against dengue (DENV), Zika (ZIKV), and Chikungunya (CHIKV) viruses and examined whether host genetic backgrounds modulate viral blocking effects by comparing ZIKV infection in mosquitoes with a Mexican genetic background to those with a Singaporean background. Our results show that over 15 years, wAlbB maintained the capacity to form a stable association with Ae. aegypti in terms of both density and CI expression. There were variations in wAlbB-induced virus blocking against CHIKV, DENV, and ZIKV, and higher inhibitory effects on ZIKV in mosquitoes on the Singaporean genetic background than on the Mexican background. These results provide important information concerning the robustness and long-term stability of Wolbachia as a biocontrol agent for arbovirus disease control.

A close association between active fibroblastic foci (desmoplasia) and distant metastasis in the lung adenocarcinoma (LUAD) has been established decades ago and yet, the pro- and anti-tumor properties of cancer-associated fibroblasts (CAFs) are still being resolved in the hope of preventing LUAD progression. To better understand the role of stromal cells in progression, there is a growing interest in 3D patient-derived models for their translational and throughput advantages. In parallel, the emergence of multiplexed spatial biology platforms is advancing translational oncology forward by providing a more global understanding of the tumor microenvironment (TME). Despite the enthusiasm for these new technologies, there are no study showing the applicability and significance of using multiplexed spatial biology to characterized 3D patient-derived models. Here, we modeled spatial heterogeneity by assembling EGFR mutated patient-derived organoids (PDOs) with fibroblasts harvested from the tumor edge (tumor-adjacent fibroblasts; TAFs) or tumor core (tumor core fibroblasts; TCFs) referred here as tumor-stroma assembloids. Using these assembloids, we generated an extensive dataset with CODEX imaging technology and used a quantitative approach to dissect tumor-stroma crosstalk. Our results show that TAFs expressing the myofibroblast marker aSMA were strictly found at the periphery of the hydrogel dome containing the assembloid, while aSMA- fibroblasts were found strictly in the center. In contrast, we did not observe a similar spatial organization in TCF assembloids. Next, we used CELESTA, an unsupervised machine learning algorithm that uses prior knowledge of the cell expression and spatial information to dissect cell heterogeneity, then performed co-localization analyses. We also leveraged the EGFR mutation status of the PDOs and observed the protective effect of the fibroblasts from erlotinib. Both assembloid conditions showed drastically different tumor-stroma spatial changes including gains and losses of colocalizations even though erlotinib did not significantly change the subpopulation fractions, cell heterogeneity, nor cell density. The top tumor-stroma colocalization pairs that persisted after erlotinib involved either myofibroblasts and PanCK-Vim+MUC1+ cancer cells or were found in the center of the assembloids, shielded by a stromal barrier of myofibroblasts. Taken together, our results show that the assembloids undergo drastic spatial reorganization following erlotinib treatment even though subpopulation fractions and cell heterogeneity remain unchanged. This study provided new insights on tumor-stroma crosstalk in the LUAD and motivates the use 3D patient-derived models coupled with multiplex imaging to answer complex spatial biology questions. Citation Format: Gina Bouchard, Irene Li, Weiruo Zhang, Sylvia Plevritis. Spatial biology of tumor-stroma assembloids reveals that nothing is lost, nothing is created, everything is reorganized. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4712.

Elevated uptake of saturated fatty acid palmitate is associated with metastatic progression of cancer cells; however, the precise signaling mechanism behind the phenomenon is unclear. The loss of cell adhesion proteins, such as desmoplakin (DSP), is a key driving event in the transformation of cancer cells to more aggressive phenotypes. Here, we investigated the mechanism by which palmitate induces the loss of DSP in liver and breast cancer cells. We propose that palmitate activates the IRE1–XBP1 branch of the endoplasmic reticulum (ER) stress pathway to upregulate the ZEB transcription factor, leading to transcriptional repression of DSP. Using liver and breast cancer cells treated with palmitate, we found loss of DSP leads to increased cell migration independent of E-cadherin. We report that the ZEB family of transcription factors function as direct transcriptional repressors of DSP. CRISPR-mediated knockdown of IRE1 confirmed that the transcription of ZEB, loss of DSP, and enhanced migration in the presence of palmitate is dependent on the IRE1–XBP1 pathway. In addition, by analyzing the somatic expression and copy number variation profiles of over 11,000 tumor samples, we corroborate our hypothesis and establish the clinical relevance of DSP loss via ZEB in human cancers. Implications: Provides mechanistic link on palmitate-induced activation of IRE1α to cancer cell migration.

Shifting of virus serotypes and clade replacement events are known to drive dengue epidemics. However, only a few studies have attempted to elucidate the virus attributes that contribute to such epidemics. In 2007, Singapore experienced a dengue outbreak affecting more than 8000 individuals. The outbreak ensued with the shuffling of dominant clades (from clade I to clade II) of Dengue virus 2 (DENV-2) cosmopolitan genotype, at a time when the Aedes premise index was significantly low. Therefore, we hypothesized that clade II had higher epidemic potential and fitness than clade I. To test this hypothesis, we tested the replication and apoptotic qualities of clade I and II isolates in mammalian cells and their ability to infect and disseminate in a field strain of Ae. Aegypti. Our findings indicated that clade II replicated more efficiently in mammalian cells than clade I and possessed higher transmission potential in local vectors. This could collectively improve the epidemic potential of clade II, which dominated during the outbreak in 2007. The findings exemplify complex interactions between the emergence, adaptation and transmission potential of DENV, and testify the epidemiological importance of a deeper understanding of virus and vector dynamics in endemic regions.

Cellular cross-talk in tissue microenvironments is fundamental to normal and pathological biological processes. Global assessment of cell-cell interactions (CCIs) is not yet technically feasible, but computational efforts to reconstruct these interactions have been proposed. Current computational approaches that identify CCI often make the simplifying assumption that pairwise interactions are independent of one another, which can lead to reduced accuracy. We present REMI (REgularized Microenvironment Interactome), a graph-based algorithm that predicts ligand-receptor (LR) interactions by accounting for LR dependencies on high-dimensional, small–sample size datasets. We apply REMI to reconstruct the human lung adenocarcinoma (LUAD) interactome from a bulk flow-sorted RNA sequencing dataset, then leverage single-cell transcriptomics data to increase the cell type resolution and identify LR prognostic signatures among tumor-stroma-immune subpopulations. We experimentally confirmed colocalization of CTGF:LRP6 among malignant cell subtypes as an interaction predicted to be associated with LUAD progression. Our work presents a computational approach to reconstruct interactomes and identify clinically relevant CCIs.

Advances in multiplexed in situ imaging are revealing important insights in spatial biology. However, cell type identification remains a major challenge in imaging analysis, with most existing methods involving substantial manual assessment and subjective decisions for thousands of cells. We propose a novel machine learning algorithm, CELESTA, which uses both cell’s protein expression and spatial information to identify cell type of individual cells. We demonstrate the performance of CELESTA on multiplexed immunofluorescence in situ images of colorectal cancer and head and neck cancer. Using the cell types identified by CELESTA, we identify tissue architecture associated with lymph node metastasis in HNSCC, which we validate in an independent cohort. By coupling our in situ spatial analysis with single-cell RNA-sequencing data on proximal sections of the same tissue specimens, we identify and validate cell-cell crosstalk associated with lymph node metastasis, demonstrating the power of spatial biology to reveal clinically-relevant cellular interactions.

Electronic health records (EHRs), digital collections of patient healthcare events and observations, are ubiquitous in medicine and critical to healthcare delivery, operations, and research. Despite this central role, EHRs are notoriously difficult to process automatically. Well over half of the information stored within EHRs is in the form of unstructured text (e.g. provider notes, operation reports) and remains largely untapped for secondary use. Recently, however, newer neural network and deep learning approaches to Natural Language Processing (NLP) have made considerable advances, outperforming traditional statistical and rule-based systems on a variety of tasks. In this survey paper, we summarize current neural NLP methods for EHR applications. We focus on a broad scope of tasks, namely, classification and prediction, word embeddings, extraction, generation, and other topics such as question answering, phenotyping, knowledge graphs, medical dialogue, multilinguality, interpretability, etc., Comment: 33 pages, 11 figures