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Synthetic lethality is a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone, which can be co-opted for cancer therapeutics. Pairs of paralog genes are among the most straightforward potential synthetic–lethal interactions by virtue of their redundant functions. Here, we demonstrate a paralog-based synthetic lethality by targeting vaccinia-related kinase 1 (VRK1) in glioblastoma (GBM) deficient of VRK2, which is silenced by promoter methylation in approximately two thirds of GBM. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells resulted in decreased activity of the downstream substrate barrier to autointegration factor (BAF), a regulator of post-mitotic nuclear envelope formation. Reduced BAF activity following VRK1 knockdown caused nuclear lobulation, blebbing, and micronucleation, which subsequently resulted in G2–M arrest and DNA damage. The VRK1–VRK2 synthetic–lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic expression of VRK2. In VRK2-methylated GBM cell line–derived xenograft and patient-derived xenograft models, knockdown of VRK1 led to robust tumor growth inhibition. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM. Significance: A paralog synthetic–lethal interaction between VRK1 and VRK2 sensitizes VRK2-methylated glioblastoma to perturbation of VRK1 kinase activity, supporting VRK1 as a drug discovery target in this disease.

Supplementary Data from VRK1 Is a Synthetic–Lethal Target in VRK2-Deficient Glioblastoma

Supplementary Figure from VRK1 Is a Synthetic–Lethal Target in VRK2-Deficient Glioblastoma

Synthetic lethality is a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone, which can be co-opted for cancer therapeutics. Pairs of paralog genes are among the most straightforward potential synthetic–lethal interactions by virtue of their redundant functions. Here, we demonstrate a paralog-based synthetic lethality by targeting vaccinia-related kinase 1 (VRK1) in glioblastoma (GBM) deficient of VRK2, which is silenced by promoter methylation in approximately two thirds of GBM. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells resulted in decreased activity of the downstream substrate barrier to autointegration factor (BAF), a regulator of post-mitotic nuclear envelope formation. Reduced BAF activity following VRK1 knockdown caused nuclear lobulation, blebbing, and micronucleation, which subsequently resulted in G2–M arrest and DNA damage. The VRK1–VRK2 synthetic–lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic expression of VRK2. In VRK2-methylated GBM cell line–derived xenograft and patient-derived xenograft models, knockdown of VRK1 led to robust tumor growth inhibition. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM.Significance:A paralog synthetic–lethal interaction between VRK1 and VRK2 sensitizes VRK2-methylated glioblastoma to perturbation of VRK1 kinase activity, supporting VRK1 as a drug discovery target in this disease.

Synthetic lethality — a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone — can be co-opted for cancer therapeutics. A pair of paralog genes is among the most straightforward synthetic lethal interaction by virtue of their redundant functions. Here we demonstrate a paralog-based synthetic lethality by targeting Vaccinia-Related Kinase 1 (VRK1) in Vaccinia-Related Kinase 2 (VRK2)-methylated glioblastoma (GBM). VRK2 is silenced by promoter methylation in approximately two-thirds of GBM, an aggressive cancer with few available targeted therapies. Genetic knockdown of VRK1 in VRK2-methylated GBM cell lines and patient-derived models was lethal and resulted in decreased activity of the downstream substrate Barrier to Autointegration Factor (BAF), a regulator of post-mitotic nuclear envelope formation. VRK1 knockdown, and thus reduced BAF activity, caused nuclear lobulation, blebbing and micronucleation, which subsequently resulted in G2/M arrest and DNA damage. The VRK1-VRK2 synthetic lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic VRK2 expression. Knockdown of VRK1 led to robust tumor growth inhibition in VRK2-methylated GBM xenografts. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM.

Equal opportunity tissue regeneration Tissue regeneration is thought to be driven primarily by rare stem cells with distinctive properties. Single-cell RNA sequencing allows rigorous testing of this hypothesis. Karthaus et al. examined the regeneration of normal prostate tissue in mice after androgen ablation, a common treatment for prostate cancer (see the Perspective by Kelly). Unexpectedly, they found that in addition to rare stem cells, a large population of differentiated cells was a major contributor to prostate regeneration, a result that they confirmed in a study of human prostate tissue. Investigation of the molecular mechanism by which the differentiated cells acquired regenerative potential yielded insights that could potentially lead to improved therapies for prostate cancer. Science , this issue p. 497 ; see also p. 467

Tumor evolution from a single cell into a malignant, heterogeneous tissue remains poorly understood. Here, we profile single-cell transcriptomes of genetically engineered mouse lung tumors at seven stages, from pre-neoplastic hyperplasia to adenocarcinoma. The diversity of transcriptional states increases over time and is reproducible across tumors and mice. Cancer cells progressively adopt alternate lineage identities, computationally predicted to be mediated through a common transitional, high-plasticity cell state (HPCS). Accordingly, HPCS cells prospectively isolated from mouse tumors and human patient-derived xenografts display high capacity for differentiation and proliferation. The HPCS program is associated with poor survival across human cancers and demonstrates chemoresistance in mice. Our study reveals a central principle underpinning intra-tumoral heterogeneity and motivates therapeutic targeting of the HPCS. Cellular states capable of promoting tumor progression and resisting therapies exist in heterogeneous tumors. Marjanovic et al. discover that a high-plasticity cell state common to mouse and human lung tumors drives cellular heterogeneity, is highly tumorigenic and drug resistant, and associates with poor patient prognosis.

Background Tumors comprise a complex microenvironment of interacting malignant and stromal cell types. Much of our understanding of the tumor microenvironment comes from in vitro studies isolating the interactions between malignant cells and a single stromal cell type, often along a single pathway. Result To develop a deeper understanding of the interactions between cells within human lung tumors, we perform RNA-seq profiling of flow-sorted malignant cells, endothelial cells, immune cells, fibroblasts, and bulk cells from freshly resected human primary non-small-cell lung tumors. We map the cell-specific differential expression of prognostically associated secreted factors and cell surface genes, and computationally reconstruct cross-talk between these cell types to generate a novel resource called the Lung Tumor Microenvironment Interactome (LTMI). Using this resource, we identify and validate a prognostically unfavorable influence of Gremlin-1 production by fibroblasts on proliferation of malignant lung adenocarcinoma cells. We also find a prognostically favorable association between infiltration of mast cells and less aggressive tumor cell behavior. Conclusion These results illustrate the utility of the LTMI as a resource for generating hypotheses concerning tumor-microenvironment interactions that may have prognostic and therapeutic relevance.

Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks.

For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and is the most important prognostic factor. Yet, LN metastases can harbor distinct tumor subclones from those within distant metastases, calling into question their role in distant metastasis. Here, we develop a syngeneic mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that a tumor-intrinsic interferon response program confers enhanced LN metastatic potential by upregulating MHC-I and PD-L1, enabling evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce regulatory T cells (Tregs), and generate tumor-specific immunosuppression that subsequently facilitates colonization of distant tissues. These effects extend to human cancers as well as additional cancers in mice, implicating a conserved mechanism by which malignancies spread to distant sites through systemic suppression of anti-tumor immunity.

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Supplementary figures.

Tumors comprise a complex microenvironment of interacting malignant and stromal cell types. Much of our understanding of the tumor microenvironment comes from in vitro studies isolating the interactions between malignant cells and a single stromal cell type, often along a single pathway. To develop a deeper understanding of the interactions between cells within human lung tumors we performed RNA-seq profiling of flow-sorted malignant cells, endothelial cells, immune cells, fibroblasts, and bulk cells from freshly resected human primary non-small-cell lung tumors. We mapped the cell-specific differential expression of prognostically-associated secreted factors and cell surface genes, and computationally reconstructed cross-talk between these cell types to generate a novel resource we call the Lung Tumor Microenvironment Interactome (LTMI). Using this resource, we identified and validated a prognostically unfavorable influence of Gremlin-1 production by fibroblasts on proliferation of malignant lung adenocarcinoma cells. We also found a prognostically favorable association between infiltration of mast cells and less aggressive tumor cell behavior. These results illustrate the utility of the LTMI as a resource for generating hypotheses concerning tumor-microenvironment interactions that may have prognostic and therapeutic relevance.SummaryRNA-seq profiling of sorted populations from primary lung cancer samples identifies prognostically relevant cross-talk between cell types in the tumor microenvironment.

Rare cell types in the prostate are reported to have stem cell properties based on organ regeneration potential following castration. Here, we use single cell RNA-seq (scRNA-Seq) to characterize these populations from the murine and human prostate in hormonally intact and androgen deprived conditions. Prostate cells from hormonally intact mice partitioned into one large subset of basal epithelial cells, another large subset of luminal epithelial cells, which we designate luminal 1 and two rare luminal populations: luminal 2 and luminal 3. Luminal cells that persist following castration display enhanced organoid regeneration potential, particularly within 1-2 days of androgen addback, and contribute equipotently to prostatic regeneration as revealed by lineage tracing. This regeneration is mediated, in part, through the orchestrated expression of Nrg2, Igf1, Fgf10 and Rspo3 by distinct populations of androgen-responsive mesenchymal and smooth muscle cells. Thus, luminal cells that persist post-castration undergo a cell state change that primes a proliferative response to microenvironment signals, analogous to other models of tissue injury such as liver damage. Citation Format: Wouter Karthaus, Matan Hofree, Danielle Choi, Eliot L. Linton, Mesruh Turkekul, Alborz Bejnood, Brett Carver, Anuhandra Gopalan, Vincent Laudone, Moshe Biton, Ojasvi Chaudhary, Ignas Masilionis, Linas Mazutis, Dana Pe'er, Aviv Regev, Charles Sawyers. Acquired stemness by luminal cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5722.

The majority of cancer-associated deaths result from distant organ metastasis rather than the primary tumor, yet the mechanisms that enable this process remain poorly understood. For most solid tumors, colonization of regional or distant lymph nodes (LNs) typically precedes the formation of distant organ metastases, yet it remains unclear whether LN metastasis plays a functional role in disease progression. LNs are education hubs of the adaptive immune system wherein antigens derived from pathogens or malignancies are presented to lymphocytes to elicit an adaptive immune response. Nonetheless, LN metastasis, which is typically attributed to passive drainage of tumor cells through lymphatics, frequently does not lead to the generation of an antitumor immune response but instead correlates with further disease progression. Here, using a novel mouse model, we find that LN metastasis represents a critical step in tumor progression through the capacity of such metastases to induce tumor-specific immunosuppression in a manner that promotes further dissemination of tumors to distant organs. We use an in vivo passaging approach of a nonmetastatic syngeneic melanoma to generate 300 unique cell lines of known phylogenetic relationships that exhibit varying degrees of LN metastatic capacity. Transcriptional profiling of the lines reveals a conserved enrichment for immune-related programs and constitutive activation of an interferon signaling axis, and using ATAC-seq, we find that these programs are rendered stable through alterations in chromatin accessibility. We show that the presence of these LN metastases enables distant organ seeding of metastases in a manner that the parental tumor cannot, and this differential seeding is eliminated in mice lacking an adaptive immune response. Using mass cytometry to perform organism-wide immune profiling, we identify multiple cellular mediators of tolerance. In particular, we find that LN metastases have the capacity to both resist NK cell cytotoxicity and induce regulatory T cells (Tregs) in vitro. Furthermore, depletion of NK cells in vivo enables nonmetastatic tumors to disseminate to LNs, and ablation of Tregs using FoxP3-DTR mice eliminates the occurrence of lymphatic metastases. Through the use of whole-exome sequencing, we show that neither the metastatic proclivity nor immunosuppression evolves through the acquisition of driver mutations, loss of neoantigens, loss of MHC class I presentation, or decreases in melanoma antigen expression. Rather, using CRISPR/Cas9, we find that key interferon-induced molecules are required for LN metastatic seeding, and using additional mouse models of pancreatic ductal adenocarcinoma and head and neck squamous cell carcinoma (HNSCC), along with RNA-seq analysis of malignant populations sorted from HNSCC patients, we find that these effects are conserved across multiple malignancies and in humans. Together, these findings demonstrate a critical role for LN metastasis in promoting tumor immune tolerance. This abstract is also being presented as Poster B25. Citation Format: Nathan E. Reticker-Flynn, Pamela A. Basto, Weiruo Zhang, Alborz Bejnood, Justin A. Kenkel, Maria M. Martins, Serena Chang, Andrew J. Gentles, John B. Sunwoo, Sylvia K. Plevritis, Edgar G. Engleman. Lymph node colonization promotes distant tumor metastasis through the induction of tumor-specific immunosuppression [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR04.

The majority of cancer-associated deaths are the result of distant organ metastasis, an event that is typically preceded by metastasis to regional or distant lymph nodes (LNs). LNs are education hubs of the adaptive immune system wherein antigens derived from pathogens or malignancies are presented to lymphocytes in a manner that facilitates elimination of the threat. Nonetheless, LN metastasis, which is typically attributed to passive drainage of tumor cells through lymphatics, frequently does not lead to the generation of an anti-tumor immune response, but instead correlates with poor prognosis and further disease progression. Here, we find that LN metastasis represents a critical step in tumor progression through the capacity of such metastases to induce systemic immune tolerance in a manner that promotes further dissemination of tumors to distant organs. Through serial in vivo passaging of a syngeneic melanoma in mice, we generate nearly 300 unique cell lines that exhibit an enhanced capacity to metastasize to LNs. Transcriptional profiling of these lines reveals increased expression of immune-related programs. We show that the presence of these LN metastases enables distant organ seeding of metastases in a manner that the parental tumor cannot, and this differential seeding is eliminated in mice that lack an adaptive immune response. To query the effects of the LN metastases on the systemic immune response, we perform organism-wide immune profiling by mass cytometry and identify a number of cellular mediators of tolerance. In particular, we find that LN metastases have the capacity to both resist NK cell cytotoxicity and induce regulatory T cells (Tregs) in vitro. Furthermore, depletion of NK cells in vivo enables non-metastatic tumors to disseminate to LNs, and ablation of Tregs using FoxP3-DTR mice eliminates the occurrence of lymphatic metastases. We further identify an interferon signaling axis that is constitutively activated within the LN metastases in the absence of exogenous interferon signaling. Through the use of ATAC-seq, we find that this program is conferred through epigenetic regulation of chromatin accessibility. Knockout of key interferon-induced genes using CRISPR/Cas9 in the LN-metastatic cells reveals that this program is required for enhanced LN metastatic seeding in vivo, and their overexpression increases LN metastasis of the non-metastatic cells. Using additional mouse models of pancreatic ductal adenocarcinoma and head and neck squamous cell carcinoma (HNSCC), we show that these findings are conserved across multiple malignancies. Additionally, we perform RNA-seq on sorted malignant populations from node-positive and node-negative HNSCC patients and confirm that these differences in transcriptional profiles extend to the human disease. Together, these findings demonstrate a critical role for LN metastasis in promoting tumor immune tolerance. Citation Format: Nathan E. Reticker-Flynn, Maria M. Martins, Pamela A. Basto, Weiruo Zhang, Alborz Bejnood, Andrew J. Gentles, John B. Sunwoo, Sylvia K. Plevritis, Edgar G. Engleman. Lymph node colonization promotes distant tumor metastasis through the induction of systemic immune tolerance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2703.

We present an analysis of dihadron correlations using recently developed methods for background subtraction which allow for higher precision measurements with fewer assumptions about the background. These studies indicate that low momentum jets interacting with the medium do not equilibrate with the medium but rather that interactions with the medium lead to more subtle increases in their widths and fragmentation functions, consistent with observations from studies of higher momentum fully reconstructed jets. The away-side shape is not consistent with a Mach cone. The qualitatively different conclusions reached with a more careful consideration of the background subtraction call into question the complete suppression of jets in central collisions observed in earlier studies, indicating that this is also an artifact of the background subtraction.

The TALLYHO/JngJ (TH) strain is a newly established, polygenic mouse model for type 2 diabetes (T2D) and obesity, and we have previously reported some key physiological features of this model after the overt onset of diabetes. In the present work, we conducted a comprehensive phenotypic characterization of TH in order to completely characterize this new and relevant model for human T2D and obesity. We monitored the development of obesity and diabetes starting at 4 weeks of age by measuring body weight, glucose tolerance, and plasma levels of insulin, glucose, and triglyceride. Additionally, histological alterations in the pancreas and glucose uptake and glucose transporter 4 (GLUT4) content in soleus muscle were also examined. Compared with age- and sex-matched C57BL/6J (B6) mice, both male and female TH mice were significantly heavier, hyperleptinemic, and hyperinsulinemic at 4 weeks of age, without glucose intolerance or hyperglycemia. TH mice maintained higher body weights throughout the study period of 16 weeks. The hyperinsulinemia in TH mice worsened with age, but to a lesser degree in females than in males. Both the male and the female TH mice had enlarged pancreatic islets. Male TH mice showed impaired glucose tolerance at 8 weeks that became more prominent at 16 weeks. Plasma glucose levels continuously increased with age in male TH mice resulting in frank diabetes, while female TH mice remained normoglycemic throughout the study. Impaired glucose tolerance and hyperglycemia in male TH mice were accompanied by impaired 2-deoxyglucose uptake in the soleus muscle at basal and insulin-stimulated states, but without any reduction in GLUT4 content. Interestingly, male TH mice exhibited a drastic elevation in plasma triglyceride levels in the pre-diabetic stage that was maintained throughout the study. These findings suggest that obesity and insulin resistance are an inherent part of the TH phenotype and glucose intolerance is evident preceding progression to overt diabetes in male TH mice.

Despite its potential importance in obesity and related disorders, little is known about regulation of lipogenesis in human adipose tissue. To investigate this area at the molecular and mechanistic levels, we studied lipogenesis and the regulation of 1 of its core enzymes, fatty acid synthase (FAS), in human adipose tissue in response to hormonal and nutritional manipulation. As a paradigm for lipogenic genes, we cloned the upstream region of the human FAS gene, compared its sequence to that of FAS orthologs from other species, and identified important regulatory elements that lie upstream of the FAS coding region. Lipogenesis, as assessed by glucose incorporation into lipids, was increased by insulin and more so by the combination of insulin and dexamethasone (Dex, a potent glucocorticoid analogue). In parallel, FAS expression, activity, and gene transcription rate were also significantly increased by these treatments. We also showed that linoleic acid, a representative PUFA, attenuated the actions of insulin and Dex on fatty acid and lipid synthesis as well as FAS activity and expression. Using reporter assays, we determined that the regions responsible for hormonal regulation of the FAS gene lie in the proximal portion of the gene's 5'-flanking region, within which we identified an insulin response element similar to the E-box sequence we identified previously in the rat FAS gene. In summary, we demonstrated that lipogenesis occurs in human adipose tissue and can be induced by insulin, further enhanced by glucocorticoids, and suppressed by PUFA in a hormone-dependent manner.

Homologous chromosomes must pair and establish stable connections during prophase I of meiosis to segregate reliably from each other at anaphase I. In most organisms, the stable connections, called chiasmata, arise from crossovers. In Drosophila males, homologs pair and segregate without crossing over. Chiasmata are replaced by a homolog conjunction complex that includes the Stromalin in Meiosis (SNM) and Modifier of Mdg4 in Meiosis (MNM) proteins. MNM is one of 31 alternative splice products of mod(mdg4), all of which share a common 402-amino-acid N terminus and differ at their C termini. Previous data demonstrated that an MNM-specific exon is required for homolog conjunction, but did not address whether the N-terminal common region, which includes a BTB domain that can mediate coalescence of protein-DNA complexes, is also required. Here we describe a mutation in the common region of mod(mdg4), Z3-3401, that causes qualitatively similar phenotypes as the MNM-specific alleles but disrupts X–Y segregation much more drastically than autosomal segregation. The mutant MNM protein in Z3-3401 is expressed throughout prophase I in spermatocytes but the protein is confined to the cytoplasm, suggesting that the Z3-3401 mutation disrupts a signal required for nuclear localization or retention. Z3-3401 fails to complement a large battery of lethal and semilethal alleles in the common region for meiotic nondisjunction, including an allele containing an amino acid substitution at a conserved residue in the BTB/POZ domain, consistent with a general requirement for the mod(mdg4) common region in homolog segregation.

We extend our investigation of magnetic field evolution in three-dimensional flows driven by the stationary accretion shock instability (SASI) with a suite of higher-resolution idealized models of the post-bounce core-collapse supernova environment. Our magnetohydrodynamic simulations vary in initial magnetic field strength, rotation rate, and grid resolution. Vigorous SASI-driven turbulence inside the shock amplifies magnetic fields exponentially; but while the amplified fields reduce the kinetic energy of small-scale flows, they do not seem to affect the global shock dynamics. The growth rate and final magnitude of the magnetic energy are very sensitive to grid resolution, and both are underestimated by the simulations. Nevertheless our simulations suggest that neutron star magnetic fields exceeding $10^{14}$ G can result from dynamics driven by the SASI, \emph{even for non-rotating progenitors}., Comment: 28 pages, 17 figures, accepted for publication in the ApJ