Mechanistic and pharmacodynamic effects of nintedanib

Metastasis thick tissue epithelial z-stack

Ligand-Receptor-Target (NicheNET) gene expression analysis

Unaffected thick tissue epithelial z-stack

Tumor cell-intrinsic effects of nintedanib

Niche-wide changes in target signaling pathway activity with immediate and delayed nintedanib

Chronic wound epithelial cells (KRT8+) accumulate in human patient osteosarcoma lung metastasis

Osteosarcoma is an aggressive and deadly bone tumor, primarily afflicting children, adolescents, and young adults. Poor outcomes for patients with osteosarcoma are intricately linked with the development of lung metastasis, which is responsible for nearly all deaths caused by osteosarcoma. Identification of the underlying cellular and molecular mechanisms that govern the metastatic colonization of circulating tumor cells to the lung is needed to develop biologically defined, metastasis-targeting therapies. In this study, using a combination of an in vitro organotypic metastasis model, single-cell RNA sequencing, human xenograft models, and murine immunocompetent osteosarcoma models, we found that metastasis is initiated by a subpopulation of hypoproliferative cells with the unique capacity to sustain the production of metastasis-promoting cytokines, such as IL6 and CXCL8, in response to lung epithelial cell-derived IL1α. Critically, genomic and pharmacologic disruption of IL1 signaling in osteosarcoma cells significantly reduced metastatic progression. Collectively, this study shows that tumor-stromal interactions are important for metastasis and suggests that metastatic competency is driven in part by the ability of tumor cells to respond to cues from the metastatic niche. Disruption of tumor-stromal signaling is a promising therapeutic approach to interrupt metastasis progression. SIGNIFICANCE:Elucidation of key traits of metastasis-competent cancer cells identified cross-talk between osteosarcoma and lung epithelial cells that mediates metastasis and revealed IL1 inhibition as a promising therapeutic strategy for blocking metastasis.

Immediate vs delayed treatment, tumor burden-based randomization

Purpose: Lung metastasis is responsible for nearly all deaths caused by osteosarcoma, the most common pediatric bone tumor. How malignant bone cells coerce the lung microenvironment to support metastatic growth is unclear. The purpose of this study is to identify metastasis-specific therapeutic vulnerabilities by delineating the cellular and molecular mechanisms underlying osteosarcoma lung metastatic niche formation.Experimental Design: Using single-cell RNA sequencing, we characterized genome- and tissue-wide molecular changes induced within lung tissues by disseminated osteosarcoma cells in both immunocompetent murine models of metastasis and patient samples. We confirmed transcriptomic findings at the protein level and determined spatial relationships with multiparameter immunofluorescence and spatial transcriptomics. Based on these findings, we evaluated the ability of nintedanib, a kinase inhibitor used to treat patients with pulmonary fibrosis, to impair metastasis progression in both immunocompetent murine osteosarcoma and immunodeficient human xenograft models. Single-nucleus and spatial transcriptomics were used to perform molecular pharmacodynamic studies that define the effects of nintedanib on tumor and nontumor cells within the metastatic microenvironment.Results: Osteosarcoma cells induced acute alveolar epithelial injury upon lung dissemination. Single-cell RNA sequencing demonstrated that the surrounding lung stroma adopts a chronic, nonresolving wound-healing phenotype similar to that seen in other models of lung injury. Accordingly, the metastasis-associated lung demonstrated marked fibrosis, likely because of the accumulation of pathogenic, profibrotic, partially differentiated epithelial intermediates and macrophages. Our data demonstrated that nintedanib prevented metastatic progression in multiple murine and human xenograft models by inhibiting osteosarcoma-induced fibrosis.Conclusions: Fibrosis represents a targetable vulnerability to block the progression of osteosarcoma lung metastasis. Our data support a model wherein interactions between osteosarcoma cells and epithelial cells create a prometastatic niche by inducing tumor deposition of extracellular matrix proteins such as fibronectin that is disrupted by the antifibrotic tyrosine kinase inhibitor (TKI) nintedanib. Our data shed light on the non-cell-autonomous effects of TKIs on metastasis and provide a roadmap for using single-cell and spatial transcriptomics to define the mechanism of action of TKI on metastases in animal models.

K7M2 shows evidence of acute alveolar injury during osteosarcoma metastasis

Many datasets are being produced by consortia that seek to characterize healthy and disease tissues at single-cell resolution. While biospecimen and experimental information is often captured, detailed metadata standards related to data matrices and analysis workflows are currently lacking. To address this, we develop the matrix and analysis metadata standards (MAMS) to serve as a resource for data centers, repositories, and tool developers. We define metadata fields for matrices and parameters commonly utilized in analytical workflows and developed the rmams package to extract MAMS from single-cell objects. Overall, MAMS promotes the harmonization, integration, and reproducibility of single-cell data across platforms.

Tumor MYCN amplification is seen in high-risk neuroblastoma, yet direct targeting of this oncogenic transcription factor has been challenging. Here, we take advantage of the dependence of MYCN-amplified neuroblastoma cells on increased protein synthesis to inhibit the activity of eukaryotic translation initiation factor 4A1 (eIF4A1) using an amidino-rocaglate, CMLD012824. Consistent with the role of this RNA helicase in resolving structural barriers in 5' untranslated regions (UTRs), CMLD012824 increased eIF4A1 affinity for polypurine-rich 5' UTRs, including that of the MYCN and associated transcripts with critical roles in cell proliferation. CMLD012824-mediated clamping of eIF4A1 spanned the full lengths of mRNAs, while translational inhibition was mediated through 5' UTR binding in a cap-dependent and -independent manner. Finally, CMLD012824 led to growth inhibition in MYCN-amplified neuroblastoma models without generalized toxicity. Our studies highlight the key role of eIF4A1 in MYCN-amplified neuroblastoma and demonstrate the therapeutic potential of disrupting its function.

Cancer disparities arise from structural racism in the United States, which affects overall standard of living due to unequal access to crucial resources. Barriers in access to clinical care among under-served populations in combination with a vast underrepresentation at a scientific research level further perpetuate inequalities in cancer treatment. Specifically, Black women in the US are approximately twice as likely as Caucasian women to die of Triple-Negative Breast Cancer (TNBC), a particularly aggressive breast cancer subset, lacking expression of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor-2. Our previous work and that of others has shown racial differences in TNBC tumor biology, with preference of basal-like subtypes in African American (AA) women. Herein, we present the Women’s Health Initiative for Triple-Negative Breast Cancer Equity (WHITNEY) study, a first of its kind, coordinated effort between Massachusetts General Hospital, and Boston Medical Center, the largest safety-net hospital in New England. The WHITNEY study seeks to understand factors contributing to biological differences and outcome of TNBC diagnoses in AA women. Response to neoadjuvant chemotherapy (NACT) response is a strong predictor of TNBC outcomes and failure to achieve a pathological complete response (pCR) in the breast following NACT is associated with a high probability of metastatic relapse. As Black women with TNBC have lower pCR rates, WHITNEY has collected pre-treatment biopsies from AA and other TNBC patients undergoing NACT to understand actionable insights into the biological differences in TNBC. Our prior published work has suggested that intratumoral heterogeneity is greater in TNBCs among AA patients than others, and that epigenetic silencing of key tumor suppressors is selectively increased in these TNBCs. Core needle baseline biopsies were obtained from consented patients diagnosed with TNBC at either MGH or BMC. We utilized single-cell RNA sequencing, spatial transcriptomics, genomic and epigenetic profiling to characterize minor cell subpopulations that drive poor outcomes among African American TNBC patients. Here, we outline our preliminary tissue collection, processing and analysis methods used in the WHITNEY study to better understand the biology of TNBC in African American women. Citation Format: Malalage N. Peiris, Emma Kelly, Christina Ennis, Kiana Mahdaviani, Aylin Dedeoglu, Adrian Ilinski, Esther Rheinbay, Ruben Dries, Naomi Ko, Leif Ellisen. Unraveling the biology of TNBC in African American women through the WHITNEY study [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C051.

Supplementary Table 1. HDAC inhibitors tested with healthy donor PBMCs and their biochemical potency (nM) across HDACs 1, 2, 3, and 6. Supplementary Table 2. Information for consented Non-small cell lung cancer (NSCLC) patients that underwent surgical resectioning as part of their treatment plan and whose tumor specimen and blood samples obtained after surgery were analyzed. Supplementary Figure 1. Reduced CD4+FOXP3+ Treg cells in healthy donor and NSCLC patient PBMC in the presence of ricolinostat. Supplementary Figure 2. Up-regulation of CD69 on T cells in healthy donor PBMC cultures in the presence of ricolinostat. Supplementary Figure 3. Viability of immune cells within dissociated tumor specimens cultured in the presence of ricolinostat or entinostat. Supplementary Figure 4. Effector function of T cells within 2-D cultures of disaggregated tumor specimens from NSCLC patients. Supplementary Figure 5. Increased expression of MHC class II and CD86 on monocytes in healthy donor PBMC in the presence of ricolinostat. Supplementary Figure 6. Phenotype of T cells infiltrating lung tumors of genetically engineered mice treated with ricolinostat. Supplementary Figure 7. Gene expression profile of Tumor-infiltrating T cells. Supplementary Figure 8. Ricolinostat promotes up-regulation of MHC class II and CD86 on tumor-associated macrophages. Supplementary figure 9. Immunohistochemical and flow cytometric analyses of acetylated α-tubulin in lung tumors of KP mice. Supplementary Figure 10. Kinetics of tumor growth in KP mice treated with ricolinostat. Supplementary Figure 11. Phenotype of tumor-infiltrating T cell subsets in lung tumors of KP mice treated with JQ1. Supplementary Figure 12. Immunohistochemical and flow cytometric analyses of phospho-STAT5 levels in lung tumors of KP mice. Supplementary Figure 13. Suppressive function of Tregs isolated from the spleen of lung tumor-bearing KP mice treated with JQ1. Supplementary Figure 14. Phenotype of Tregs in the spleen or lung tumors of genetically engineered mouse models (GEMM) of NSCLC. Supplementary Figure 15. Phenotype of Tregs within cultures of dissociated tumor specimen from NSCLC patients. Supplementary Figure 17. Histology and immunohistochemical staining of lung tumor sections from treated KP mice. Supplementary Figure 18. Kinetics of tumor growth in TL or wild-type mice. Supplementary Figure 19. Quantification of T cell subsets and TAMs in tumors of KP mice. Supplementary Figure 20. Phenotype of CD8+T cells infiltrating lung tumors of treated KP mice. Supplementary Figure 21. Gene expression profile of tumor-infiltrating macrophages (TAMs) in treated KP mice. Supplementary Figure 22. Gene expression profile of tumor-infiltrating macrophages T cells in treated KP mice. Supplementary Figure 23. Proportion of CD4+Foxp3+ Tregs present within tumor-infiltrating T cells utilized in gene expression studies. Supplementary Figure 24. Gating strategy. Supplementary Figure 25. Correlation plot for the expression of indicated genes in CD45+ leukocytes in KP tumors as evaluated by single cell RNA-Sequencing.

List of epigenetic genes and related shRNAs

Figure S1. Characterization of cells and CDK4/6 inhibitors; Figure S2. CDK6 phosphorylates serine residues of the regulatory domain of NFAT4 (NFATc3); Figure S3. Analysis of lung tumor immune infiltrates after CDK4/6 inhibition from KrasG12D (Kras), KrasG12DLkb1 (KL) or KrasG12DTrp53fl/fl (KP) mice; Figure S4. T cell proliferation and cytokine/chemokine profiling of KrasG12DTrp53fl/fl GEMM mice; Figure S5. Tumor antigen experienced T cells are more sensitive to CDK4/6 inhibition; Figure S6. Short-term CDK4/6 inhibition alters the cell cycle status of tumor infiltrating T cells; Figure S7. CDK4/6 inhibition induces changes in the expression of activation and suppression marker genes in tumor-infiltrating T cells; Figure S8. Combination treatment of CDK4/6 inhibitor and anti-PD-1 antibody elicits anti-tumor immunity; Figure S9. Combination treatment of CDK4/6 inhibitor and anti-PD-1 antibody on established tumor; Figure S10. Effect of TCR stimulation and CDK4/6 inhibition on phosphorylation of NFkB; Supplementary Table S1; Supplmentary Table S2; Supplementary Table S3: Selected genes reported to be regulated by NFAT

Characteristics of SCLC cell lines

List of hits in the top 10% of screen hits from NCI-H69 and GLC-16 xenograft tumors

Abstract Immune checkpoint blockade, exemplified by antibodies targeting the PD-1 receptor, can induce durable tumor regressions in some patients. To enhance the efficacy of existing immunotherapies, we screened for small molecules capable of increasing the activity of T cells suppressed by PD-1. Here, we show that short-term exposure to small-molecule inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) significantly enhances T-cell activation, contributing to antitumor effects in vivo, due in part to the derepression of NFAT family proteins and their target genes, critical regulators of T-cell function. Although CDK4/6 inhibitors decrease T-cell proliferation, they increase tumor infiltration and activation of effector T cells. Moreover, CDK4/6 inhibition augments the response to PD-1 blockade in a novel ex vivo organotypic tumor spheroid culture system and in multiple in vivo murine syngeneic models, thereby providing a rationale for combining CDK4/6 inhibitors and immunotherapies. Significance: Our results define previously unrecognized immunomodulatory functions of CDK4/6 and suggest that combining CDK4/6 inhibitors with immune checkpoint blockade may increase treatment efficacy in patients. Furthermore, our study highlights the critical importance of identifying complementary strategies to improve the efficacy of immunotherapy for patients with cancer. Cancer Discov; 8(2); 216–33. ©2017 AACR. See related commentary by Balko and Sosman, p. 143. See related article by Jenkins et al., p. 196. This article is highlighted in the In This Issue feature, p. 127