Because transgenic mice expressing an altered stoichiometry of neurofilament proteins develop a motor neuron degeneration associated with neurofilamentous aggregate formation similar to that found in amyotrophic lateral sclerosis (ALS), we studied the expression of intermediate filament proteins in sporadic ALS. Archival cervical spinal cord paraffin-embedded sections from 11 disease and 11 control cases were studied by either in situ hybridization using ³⁵S-labeled riboprobes or immunohistochemically using specific antibodies for the individual neurofilament subunit proteins, α-internexin, nestin, peripherin, vimentin, β-actin, or Tα1-tubulin. Median NFL, α-internexin, and peripherin steady-state mRNA levels were significantly reduced in the lateral motor neuron cell column (p < 0.05) of ALS cases, while neither NFM nor NFH mRNA levels were altered. ALS cases demonstrated an elevation of β-actin mRNA levels (p < 0.01) with no increase in Tα1-tubulin mRNA levels. No motor neuronal expression of nestin or vimentin was observed. Ubiquitin-immunoreactive perikaryal aggregates were immunoreactive for NFH or β-actin, but not for peripherin, α-internexin, vimentin, or nestin. In contrast, neuroaxonal spheroids were strongly immunoreactive for NFH and peripherin, but not for β-actin, α-internexin, vimentin, or nestin. These findings suggest that the stoichiometry of cytoskeletal protein expression in ALS spinal motor neurons is significantly altered in a pattern conducive to the formation of neurofilamentous aggregates.

Aberrant Notch activity is oncogenic in several malignancies, but it is unclear how expression or function of downstream elements in the Notch pathway affects tumor growth. Transcriptional regulation by Notch is dependent on interaction with the DNA-binding transcriptional repressor, RBPJ, and consequent derepression or activation of associated gene promoters. We show here that RBPJ is frequently depleted in human tumors. Depletion of RBPJ in human cancer cell lines xenografted into immunodeficient mice resulted in activation of canonical Notch target genes, and accelerated tumor growth secondary to reduced cell death. Global analysis of activated regions of the genome, as defined by differential acetylation of histone H4 (H4ac), revealed that the cell death pathway was significantly dysregulated in RBPJ-depleted tumors. Analysis of transcription factor binding data identified several transcriptional activators that bind promoters with differential H4ac in RBPJ-depleted cells. Functional studies demonstrated that NF-κB and MYC were essential for survival of RBPJ-depleted cells. Thus, loss of RBPJ derepresses target gene promoters, allowing Notch-independent activation by alternate transcription factors that promote tumorigenesis.

Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates in patients with metastatic disease. There is a pressing need to develop effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use multiomics integration of whole-exome sequencing, RNA sequencing, and mass spectrometry–based proteomics on 14 LGSOC cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. Comparison of LGSOC cell line data with LGSOC tumor data enabled predictive biomarker identification of MEK inhibitor (MEKi) efficacy, with KRAS mutations found exclusively in MEKi-sensitive cell lines and NRAS mutations found mostly in MEKi-resistant cell lines. Distinct patterns of Catalogue of Somatic Mutations in Cancer mutational signatures were identified in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes were more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. These LGSOC cell lines were representative models of the molecular aberrations found in LGSOC tumors. For prediction of in vitro MEKi efficacy, proteomic data provided better discrimination than gene expression data. Condensin, minichromosome maintenance, and replication factor C protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. This study suggests that CDKN2A/B or MTAP deficiency may be exploited using synthetically lethal treatment strategies, highlighting the importance of using proteomic data as a tool for molecular drug prediction. Multiomics approaches are crucial to improving our understanding of the molecular underpinnings of LGSOC and applying this information to develop new therapies. Significance: These findings highlight the utility of global multiomics to characterize LGSOC cell lines as research models, to determine biomarkers of MEKi resistance, and to identify potential novel therapeutic targets.

CD44 is a cell adhesion molecule implicated in leukocyte adhesion and migration, co-stimulation of T cells, and tumor metastasis. CD45 is a leukocyte-specific protein tyrosine phosphatase that dephosphorylates the Src family kinases, Lck and Fyn, in T cells. Positive regulation of Lck by CD45 is required for its effective participation in T cell receptor signaling events. Here, immobilized CD44 antibody induced a distinctive cell spreading in CD45(-), but not CD45(+), T cells, and this correlated with the induction of tyrosine-phosphorylated proteins. Two focal adhesion family kinases, Pyk2 and, to a lesser extent, FAK were inducibly phosphorylated, as was a potential substrate, Cas. CD44-mediated cell spreading and induced tyrosine phosphorylation were prevented by the Src family kinase inhibitor, PP2. Furthermore, 2-fold more Lck associated with CD44 in the low density sucrose fraction from CD45(-) T cells compared with CD45(+) T cells, suggesting that CD45 may regulate the association of Lck with CD44 in this fraction. Therefore, in CD45(-) T cells, CD44 signaling is mediated by Src family kinases, and this leads to Pyk2 phosphorylation, cytoskeletal changes, and cell spreading. This implicates CD45 in the negative regulation of Src family kinase-mediated CD44 signaling leading to T cell spreading.

Treatment-induced tumor dormancy is a state in cancer progression where residual disease is present but remains asymptomatic. Dormant cancer cells are treatment-resistant and responsible for cancer recurrence and metastasis. Prostate cancer treated with androgen-deprivation therapy (ADT) often enters a dormant state. ADT-induced prostate cancer dormancy remains poorly understood due to the challenge in acquiring clinical dormant prostate cancer cells and the lack of representative models. In this study, we aimed to develop clinically relevant models for studying ADT-induced prostate cancer dormancy. Dormant prostate cancer models were established by castrating mice bearing patient-derived xenografts (PDX) of hormonal naïve or sensitive prostate cancer. Dormancy status and tumor relapse were monitored and evaluated. Paired pre- and postcastration (dormant) PDX tissues were subjected to morphologic and transcriptome profiling analyses. As a result, we established eleven ADT-induced dormant prostate cancer models that closely mimicked the clinical courses of ADT-treated prostate cancer. We identified two ADT-induced dormancy subtypes that differed in morphology, gene expression, and relapse rates. We discovered transcriptomic differences in precastration PDXs that predisposed the dormancy response to ADT. We further developed a dormancy subtype-based, predisposed gene signature that was significantly associated with ADT response in hormonal naïve prostate cancer and clinical outcome in castration-resistant prostate cancer treated with ADT or androgen-receptor pathway inhibitors. Implications: We have established highly clinically relevant PDXs of ADT-induced dormant prostate cancer and identified two dormancy subtypes, leading to the development of a novel predicative gene signature that allows robust risk stratification of patients with prostate cancer to ADT or androgen-receptor pathway inhibitors.