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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.

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.