Mad2 Induced Aneuploidy Contributes to Eml4-Alk Driven Lung Cancer by Generating an Immunosuppressive Environment

Aneuploidy, an imbalance number of chromosomes, is frequently observed in lung cancer and inversely correlates with patient survival. Paradoxically, an aneuploid karyotype has detrimental consequences on cellular fitness, and it has been proposed that aneuploid cells, at least in vitro, generate signals for their own elimination by NK cells. However, how aneuploidy affects tumor progression as well as the interplay between aneuploid tumor cells and the tumor microenvironment is still unclear. We generated a new mouse model in which overexpression of Mad2 was almost entirely restricted to normal epithelial cells of the lung, and combined it with an oncogenic Eml4-Alk chromosome inversion. This combination resulted in a higher tumor burden and an increased number of tumor nodules compared to control Eml4-Alk mice alone. The FISH analysis detected significant differences in the aneuploidy levels in the non-tumor regions of Eml4-Alk+Mad2 compared to Eml4-Alk alone, although both tumor groups presented similar levels of aneuploidy. We further show that aneuploid cells in the non-tumor areas adjacent to lung tumors recruit immune cells, such as tumor-associated macrophages. In fact, these areas presented an increase in alveolar macrophages, neutrophils, decreased cytotoxic CD8+ T cells, and IFN-γ, suggesting that aneuploid cells in the surrounding tumor areas create an immunosuppressive signature that might contribute to lung tumor initiation and progression.

Reconstructing single-cell karyotype alterations in colorectal cancer identifies punctuated and gradual diversification patterns

Central to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq—a protocol that integrates live-cell imaging of tumor organoid outgrowth and whole-genome sequencing of each imaged cell to reconstruct evolving tumor cell karyotypes across consecutive cell generations. Using patient-derived colorectal cancer organoids and fresh tumor biopsies, we demonstrate that karyotype alterations of varying complexity are prevalent and can arise within a few cell generations. Sub-chromosomal acentric fragments were prone to replication and collective missegregation across consecutive cell divisions. In contrast, gross genome-wide karyotype alterations were generated in a single erroneous cell division, providing support that aneuploid tumor genomes can evolve via punctuated evolution. Mapping the temporal dynamics and patterns of karyotype diversification in cancer enables reconstructions of evolutionary paths to malignant fitness.

Molecular architecture of the developing mouse brain

The mammalian brain develops through a complex interplay of spatial cues generated by diffusible morphogens, cell-cell interactions, and intrinsic genetic programs that result in the generation of likely more than a thousand distinct cell types. Therefore, a complete understanding of mammalian brain development requires systematic mapping of cell states covering the entire relevant spatiotemporal range. Here we report a comprehensive single-cell transcriptome atlas of mouse brain development spanning from gastrulation to birth. We identified almost a thousand distinct cellular states, including the initial emergence of the neuroepithelium, a rich set of region-specific secondary organizers and a complete developmental program for the functional elements of the brain and its enclosing membranes. We used the atlas to directly test the hypothesis that human glioblastoma reflects a return to a developmental cell state. In agreement, most aneuploid tumor cells matched embryonic rather than adult types, while karyotypically normal cells predominantly matched adult immune cell types.

Concordance of PD-L1 expression detection in non-small cell lung cancer (NSCLC) tissue biopsies between OncoTect iO single cell PD-L1 quantification assay and immunohistochemistry (IHC).

e23172 Background: PD-1/PD-L1 therapy has been shown to be effective in patients with NSCLC, specifically those with 50% PD-L1 expression or greater by IHC. Response rates for those with lower PD-L1 expression do not consistently correlate with PD-L1 amount. The OncoTect iO Lung PD-L1 Assay was designed to provide non-subjective quantification of PD-L1 expression on tumor cells and immune cells from a multitude of tumor types. The objective of this study was to compare OncoTect iO and the current method of PD-L1 testing by immunohistochemistry (IHC) in NSCLC biopsies. Methods: Eleven NSCLC tissues were obtained from two IRB approved sites. Fresh tissues were processed into single cell suspensions using the IVD IncellPREP Kit (IncellDx, Inc). Normal lung tissue adjacent to tumor sites was obtained for 9 of the samples. Cell suspensions were tested with the OncoTect iO Lung Assay which contains antibodies directed against PD-L1 (28-8), CD45, CD3, and CD8. Suspensions are fixed and permeabilized, labeled with the antibodies, and then stained with DAPI to identify intact, single cells, and to analyze cell cycle including aneuploidy.Matched FFPE sections were taken from the tissue biopsies and were tested at BioReference Laboratories with the Dako PD-L1 IHC 28-8 PharmDx Kit. Results: A cut-off of 4% was used for the OncoTect iO Assay and a positive result for the Dako assay is ≥ 5% of the tumor cells (equivalent to 5 cells out of 100). PD-L1 expression ranged from 0% to 75% in the tumor samples which is consistent with reported ranges. Seven samples were positive by OncoTect iO Lung and 6 were positive by IHC. Twelve samples were negative by OncoTect iO Lung and 13 were negative by IHC demonstrating a concordance of 95%. Of note, one tumor that was negative by OncoTect iO and IHC demonstrated 5% of aneuploidy tumor cells that were positive and most likely very rare to be detected in the assays. Conclusions: In this study, the concordance between Oncotect iO and IHC was 95%, however, the Oncotect iO Single Cell PD-L1 Quantification Assay was fast ( < 3 hours), non-subjective, and provided addition expression information for aneuploid tumor cells.

Heat Shock Factor 1 Promotes NF-Kb and B-Cell Signaling in a Preclinical Model of Chronic Lymphocytic Leukemia

Recent studies have shown that overexpression of Heat Shock Factor (HSF) 1 in aneuploid tumor cells can overcome deficiencies in heat shock protein (HSP) 90-mediated protein folding and restore protein homeostasis. In this study we determined the mechanisms by which HSF1 promotes HSP90 function and CLL pathogenesis using CLL as model system. We report that HSF1 is overexpressed in CLL and treatment with triptolide (a small molecule inhibitor of HSF1) induces apoptosis in cultured and primary CLL B-cells. Mechanistically, we demonstrate that knockdown of HSF1 or its inhibition with triptolide results in the reduced association of HSP90 with its kinase co-chaperone cell division cycle 37 (CDC37), leading to the partial depletion of HSP90 client kinases, Bruton's Tyrosine Kinase (BTK), c-RAF and cyclin-dependent kinase 4 (CDK4). RNA-sequencing of control and HSF1-knockdown CLL cells revealed that HSF1 regulates the transcription of upstream modulators of the NF-kB pathway including MYD88 and TLR1. Consequently, treatment with triptolide or knockdown of HSF1 inhibits NF-kB signaling in CLL cells. In an in vivo model of CLL, tail vein injection of luciferase-expressing Mec-1 cells into Rag2-/-IL2Rγc-/- mice followed by daily intraperitoneal injection of minnelide (a pro-drug of triptolide) for 28 days reduced in vivo disease burden and conferred significant survival advantage (p<0.0003) to treated mice compared to vehicle controls. Minnelide treatment also attenuated NF-kB and BTK signaling in vivo. In conclusion, our study provides a strong rationale to target HSF1 in CLL and test the activity of minnelide against human CLL. Disclosures Saluja: Minneamrita Therapeutics: Equity Ownership.