A gemcitabine sensitivity screen identifies a role for NEK9 in the replication stress response.

The Replication Stress Response (RSR) is a signaling network that recognizes challenges to DNA replication and coordinates diverse DNA repair and cell-cycle checkpoint pathways. Gemcitabine is a nucleoside analogue that causes cytotoxicity by inducing DNA replication blocks. Using a synthetic lethal screen of a RNAi library of nuclear enzymes to identify genes that when silenced cause gemcitabine sensitization or resistance in human triple-negative breast cancer cells, we identified NIMA (never in mitosis gene A)-related kinase 9 (NEK9) as a key component of the RSR. NEK9 depletion in cells leads to replication stress hypersensitivity, spontaneous accumulation of DNA damage and RPA70 foci, and an impairment in recovery from replication arrest. NEK9 protein levels also increase in response to replication stress. NEK9 complexes with CHK1, and moreover, NEK9 depletion impairs CHK1 autophosphorylation and kinase activity in response to replication stress. Thus, NEK9 is a critical component of the RSR that promotes CHK1 activity, maintaining genome integrity following challenges to DNA replication.

Regulation of antigen-specific T cell infiltration and spatial architecture in multiple myeloma and premalignancy.

Entry of antigen-specific T cells into human tumors is critical for immunotherapy, but the underlying mechanisms are poorly understood. Here, we combined high-dimensional spatial analyses with in vitro and in vivo modeling to study the mechanisms underlying immune infiltration in human multiple myeloma (MM) and its precursor monoclonal gammopathy of undetermined significance (MGUS). Clustered tumor growth was a feature of MM but not MGUS biopsies, and this growth pattern was reproduced in humanized mouse models. MM biopsies exhibited intralesional as well as spatial heterogeneity, with coexistence of T cell-rich and T cell-sparse regions and the presence of areas of T cell exclusion. In vitro studies demonstrated that T cell entry into MM clusters was regulated by agonistic signals and CD2-CD58 interactions. Upon adoptive transfer, antigen-specific T cells localized to the tumor site but required in situ DC-mediated antigen presentation for tumor entry. C-type lectin domain family 9 member A-positive (CLEC9A+) DCs appeared to mark portals of entry for gradients of T cell infiltration in MM biopsies, and their proximity to T cell factor 1-positive (TCF1+) T cells correlated with disease state and risk status. These data illustrate a role for tumor-associated DCs and in situ activation in promoting the infiltration of antigen-specific T cells in MM and provide insights into spatial alterations in tumor/immune cells with malignant evolution.

Ten-eleven translocation protein 1 modulates medulloblastoma progression.

BACKGROUND: Medulloblastoma (MB) is the most common malignant pediatric brain tumor that originates in the cerebellum and brainstem. Frequent somatic mutations and deregulated expression of epigenetic regulators in MB highlight the substantial role of epigenetic alterations. 5-hydroxymethylcytosine (5hmC) is a highly abundant cytosine modification in the developing cerebellum and is regulated by ten-eleven translocation (TET) enzymes. RESULTS: We investigate the alterations of 5hmC and TET enzymes in MB and their significance to cerebellar cancer formation. We show total abundance of 5hmC is reduced in MB, but identify significant enrichment of MB-specific 5hmC marks at regulatory regions of genes implicated in stem-like properties and Nanog-binding motifs. While TET1 and TET2 levels are high in MBs, only knockout of Tet1 in the smoothened (SmoA1) mouse model attenuates uncontrolled proliferation, leading to a favorable prognosis. The pharmacological Tet1 inhibition reduces cell viability and platelet-derived growth factor signaling pathway-associated genes. CONCLUSIONS: These results together suggest a potential key role of 5hmC and indicate an oncogenic nature for TET1 in MB tumorigenesis, suggesting it as a potential therapeutic target for MBs.

Subtype and grade-dependent spatial heterogeneity of T-cell infiltration in pediatric glioma.

Brain tumors are the leading cause of cancer-related mortality in children and have distinct genomic and molecular features compared with adult glioma. However, the properties of immune cells in these tumors has been vastly understudied compared with their adult counterparts. We combined multiplex immunofluorescence immunohistochemistry coupled with machine learning and single-cell mass cytometry to evaluate T-cells infiltrating pediatric glial tumors. We show that low-grade tumors are characterized by greater T-cell density compared with high-grade glioma (HGG). However, even among low-grade tumors, T-cell infiltration can be highly variable and subtype-dependent, with greater T-cell density in pleomorphic xanthoastrocytoma and ganglioglioma. CD3+ T-cell infiltration correlates inversely with the expression of SOX2, an embryonal stem cell marker commonly expressed by glial tumors. T-cells within both HGG and low-grade glioma (LGG) exhibit phenotypic heterogeneity and tissue-resident memory T-cells consist of distinct subsets of CD103+ and TCF1+ cells that exhibit distinct spatial localization patterns. TCF1+ T-cells are located closer to the vessels while CD103+ resident T-cells reside within the tumor further away from the vasculature. Recurrent tumors are characterized by a decline in CD103+ tumor-infiltrating T-cells. BRAFV600E mutation is immunogenic in children with LGG and may serve as a target for immune therapy. These data provide several novel insights into the subtype-dependent and grade-dependent changes in immune architecture in pediatric gliomas and suggest that harnessing tumor-resident T-cells may be essential to improve immune control in glioma.

Reactive Oxygen Species Signaling Promotes Hypoxia-Inducible Factor 1α Stabilization in Sonic Hedgehog-Driven Cerebellar Progenitor Cell Proliferation.

Cerebellar development is a highly regulated process involving numerous factors acting with high specificity, both temporally and by location. Part of this process involves extensive proliferation of cerebellar granule neuron precursors (CGNPs) induced by Sonic Hedgehog (SHH) signaling, but downstream effectors of mitogenic signaling are still being elucidated. Using primary CGNP cultures, a well-established model for SHH-driven proliferation, we show that SHH-treated CGNPs feature high levels of hypoxia-inducible factor 1α (HIF1α), which is known to promote glycolysis, stemness, and angiogenesis. In CGNPs cultured under normoxic conditions, HIF1α is posttranslationally stabilized in a manner dependent upon reactive oxygen species (ROS) and NADPH oxidase (NOX), both of which are also upregulated in these cells. Inhibition of NOX activity resulted in HIF1α destabilization and reduced levels of cyclin D2, a marker of CGNP proliferation. As CGNPs are the putative cells of origin for the SHH subtype of medulloblastoma and aberrant SHH signaling is implicated in other neoplasms, these studies may also have future relevance in the context of cancer. Taken together, our findings suggest that a better understanding of nonhypoxic HIF1α stabilization through NOX-induced ROS generation can provide insights into normal cell proliferation in cerebellar development and SHH-driven cell proliferation in cancers with aberrant SHH signaling.

Upregulation of the chromatin remodeler HELLS is mediated by YAP1 in Sonic Hedgehog Medulloblastoma.

Medulloblastoma is a malignant pediatric tumor that arises from neural progenitors in the cerebellum. Despite a five-year survival rate of ~70%, nearly all patients incur adverse side effects from current treatment strategies that drastically impact quality of life. Roughly one-third of medulloblastoma are driven by aberrant activation of the Sonic Hedgehog (SHH) signaling pathway. However, the scarcity of genetic mutations in medulloblastoma has led to investigation of other mechanisms contributing to cancer pathogenicity including epigenetic regulation of gene expression. Here, we show that Helicase, Lymphoid Specific (HELLS), a chromatin remodeler with epigenetic functions including DNA methylation and histone modification, is induced by Sonic Hedgehog (SHH) in SHH-dependent cerebellar progenitor cells and the developing murine cerebella. HELLS is also up-regulated in mouse and human SHH medulloblastoma. Others have shown that HELLS activity generally results in a repressive chromatin state. Our results demonstrate that increased expression of HELLS in our experimental systems is regulated by the oncogenic transcriptional regulator YAP1 downstream of Smoothened, the positive transducer of SHH signaling. Elucidation of HELLS as one of the downstream effectors of the SHH pathway may lead to novel targets for precision therapeutics with the promise of better outcomes for SHH medulloblastoma patients.

Tumour-associated macrophages exhibit anti-tumoural properties in Sonic Hedgehog medulloblastoma.

Medulloblastoma, which is the most common malignant paediatric brain tumour, has a 70% survival rate, but standard treatments often lead to devastating life-long side effects and recurrence is fatal. One of the emerging strategies in the search for treatments is to determine the roles of tumour microenvironment cells in the growth and maintenance of tumours. The most attractive target is tumour-associated macrophages (TAMs), which are abundantly present in the Sonic Hedgehog (SHH) subgroup of medulloblastoma. Here, we report an unexpected beneficial role of TAMs in SHH medulloblastoma. In human patients, decreased macrophage number is correlated with significantly poorer outcome. We confirm macrophage anti-tumoural behaviour in both ex vivo and in vivo murine models of SHH medulloblastoma. Taken together, our findings suggest that macrophages play a positive role by impairing tumour growth in medulloblastoma, in contrast to the pro-tumoural role played by TAMs in glioblastoma, another common brain tumour.

Second generation benzofuranone ring substituted noscapine analogs: synthesis and biological evaluation.

Microtubules, composed of α/ß tubulin heterodimers, represent a validated target for cancer chemotherapy. Thus, tubulin- and microtubule-binding antimitotic drugs such as taxanes and vincas are widely employed for the chemotherapeutic management of various malignancies. Although quite successful in the clinic, these drugs are associated with severe toxicity and drug resistance problems. Noscapinoids represent an emerging class of microtubule-modulating anticancer agents based upon the parent molecule noscapine, a naturally occurring non-toxic cough-suppressant opium alkaloid. Here we report in silico molecular modeling, chemical synthesis and biological evaluation of novel analogs derived by modification at position-7 of the benzofuranone ring system of noscapine. The synthesized analogs were evaluated for their tubulin polymerization activity and their biological activity was examined by their antiproliferative potential using representative cancer cell lines from varying tissue-origin [A549 (lung), CEM (lymphoma), MIA PaCa-2 (pancreatic), MCF-7 (breast) and PC-3 (prostate)]. Cell-cycle studies were performed to explore their ability to halt the cell-cycle and induce subsequent apoptosis. The varying biological activity of these analogs that differ in the nature and bulk of substituent at position-7 was rationalized utilizing predictive in silico molecular modeling.