The androgen receptor (AR) is a pivotal regulator of growth and survival of prostate cancer (PCa) and the majority of lethal castration-resistant prostate cancers (CRPC) remain reliant on AR signaling. PCa exhibits variability in progression and responses to treatment suggesting genetic heterogeneity. Two independent studies identified PCa predisposing single nucleotide polymorphisms (SNPs) within the FAM111A protease gene, but the mechanistic basis of this association remained elusive. Our in vitro and in vivo studies uncovered that AR represses FAM111A in castration sensitive and resistant cells via an AR binding site within the FAM111A gene. FAM111A levels are significantly lower in matched castration-resistant than in castration-sensitive cells and xenografts, and lower in metastatic lesions than in primary tumors. We discovered that FAM111A is AR-repressed in castration sensitive PCa xenograft and multiple PCa cells. Additionally, FAM111A subcellular localization changes dramatically with acquisition of castration resistance, where in castration sensitive cells FAM111A is predominantly in the nucleoli, but with castration resistance it becomes more dispersed in the nucleus and in the cytoplasm. FAM111A depletion in castration sensitive and resistant cells enhances the efficacy of PARP1 inhibitors olaparib and niraparib, consistent with its role in DNA repair. Moreover, FAM111A depletion reduces AR target gene prostate specific antigen (PSA) and transmembrane serine protease 2 (TMPRSS2) transcription, indicating that FAM111A modulates AR-dependent gene expression forming a FAM111A-AR co-regulatory loop in PCa. Our studies argue that AR-dependent FAM111A regulation modulates PCa gene expression, acquisition of castration resistance, and sensitivity to agents that target DNA damage repair.

Canine oral squamous cell carcinoma (COSCC) is the second most common oral tumor in dogs and the most relevant for comparative human trials as a spontaneous large animal model of disease. Historical genomic work has focused primarily on bulk sequencing. The present study describes the complete transcriptomic landscape of COSCC with spatial distinction between the surface tumor, deep invasive tumor, peritumoral dysplastic epithelium, and tumor microenvironment compared to matched normal oral samples. Each region demonstrated distinct molecular signatures. Genes related to epithelial growth factor (EGFR) and epithelial-mesenchymal transformation (EMT) were upregulated in both peritumoral dysplasia and surface cancer. Additionally, the KRAS gene set, KRT17, and SSP1 were enriched in cancer. We identified five genes that represent dysplastic lesion with high potential for malignant transformation (FZD4, GAS1, HACD2, NOG, and SLC39A6). Also, three genes, SFRP4, FZD1, and IL34 represented a specific signature of the invasive portion of the COSCC that should be explored for prognostic value as a biomarker of malignancy. Lastly, we verified the immunomodulatory tumor microenvironment detecting an increase in macrophages and an abundance of IL-10 secretion. The other predominant leukocytes were T-cells, with CD4+ T-cells being the most prevalent. CD4+ T cells expressed transcripts for both stimulatory (Inducible T-cell Co-Stimulator (ICOS) and inhibitory molecules (CTLA4). The observed high CTLA4 suggests that this inhibitory signal may be preventing a robust antitumor immune response. Taken together, this study identified multiple targets to be explored for biomarkers of malignancy, prediction of tumor behavior, and potential targets for development of novel therapies.

High-grade soft tissue sarcomas (STS) are a heterogeneous and aggressive set of cancers. Failure to respond anthracycline chemotherapy, standard first-line treatment, is associated with poor outcomes. We investigated the contribution of STS cancer stem cells (STS-CSCs) to doxorubicin resistance. We identified a positive correlation between CSC abundance and doxorubicin IC50. Utilizing patient-derived samples from five sarcoma subtypes we investigated if a common genetic signature across STS-CSCs could be targeted. We identified Enhancer of Zeste homolog 2 (EZH2), a member of the polycomb repressive complex 2 (PRC2) responsible for H3K27 methylation as being enriched in CSCs. EZH2 activity and a shared epigenetic profile was observed across subtypes and targeting of EZH2 ablated the STS-CSC population. Treatment of doxorubicin-resistant cell lines with tazemetostat resulted in a decrease in the STS-CSC population. These data confirm the presence of shared genetic programs across distinct subtypes of CSC-STS that can be therapeutically targeted.

Abstract The outcome for children with high-risk B-cell acute lymphoblastic leukemia (B-ALL) is poor. Disease relapse is speculated to be due to leukemia cells escaping treatment. Our group discovered a unique cell subpopulation in B-ALL that has the capacity to initiate leukemia and is resistant to treatment. Transcriptome studies of this cell population highlight a unique RNA regulation process, specifically RNA splicing. In B-ALL, dysregulated splicing is reported to be associated with drug resistance and disease relapse. SF3B1 is a core component of the spliceosome and an essential protein in the RNA splicing process. SF3B1 inhibition is therapeutic in many cancers. In our current studies, we investigated the therapeutic potential of pladienolide B (Plad-B), an SF3B1 inhibitor, in B-ALL. SF3B1 protein expression was significantly higher in cell lines (Reh and JM1) and 28 primary B-ALL samples (14 each for standard-risk and high-risk), regardless of the risk group, than in normal B-cells (NBs) and hematopoietic stem cells (HSCs). Plad-B showed significant dose-dependent cytotoxicity in the cell lines with IC50 of 1.2nM and 0.6nM, respectively, and three harvested high-risk patient-derived xenograft (PDX) samples with almost the same IC50 as the cell lines. Plad-B did not show cytotoxicity in NBs and HSCs at the same tested concentrations. In vivo efficacy of Plad-B was tested using an Reh xenograft mouse model and a high-risk PDX model. Plad-B, as a single drug treatment, significantly prolonged survival in both models (p<0.01). G2/M cell cycle arrest and apoptosis induction were observed at 24 hours after Plad-B treatment in Reh and JM1. Splicing events were examined by RNA-seq in the treated cells at different time points (15 min, 30 min, and 60 min). Plad B demonstrated rapid splicing inhibition as early as 15 min post-treatment and, at 60 min, splicing was inhibited in 1,669 genes, including apoptosis-associated genes. 2,625 differential splicing events were observed in these genes with ~96% from intron retention and exon skipping. Furthermore, 202 genes showed significant changes in their expression over this time frame. Most of the genes were rapidly downregulated in the treatment group compared to the control, and some were slowly downregulated. There were also some genes which were upregulated, either rapidly or slowly, compared to the control. We demonstrated that Plad-B induced short pro-apoptotic spliced isoforms, instead of anti-apoptotic forms, in BCL2L1 and MCL-1, as early as 60 min after treatment. In conclusion, these data demonstrated the therapeutic potential of SF3B1 inhibition in high-risk B-ALL. Plad-B rapidly inhibited splicing in many genes and downregulated the anti-apoptotic forms of apoptosis-associated genes, leading to cell apoptosis. In future studies, we will identify the downstream targets of Plad-B and further investigate the mechanism of rapid apoptosis induction by SF3B1 inhibition. Citation Format: Yuki Murakami, Hiroaki Konishi, Clifford Tepper, John McPherson, Noriko Satake. Targeting the spliceosome in high-risk B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5987.

Potential systemic factors contributing to aging-associated breast cancer (BC) remain elusive. Here, we reveal that the polyploid giant cells (PGCs) that contain more than two sets of genomes prevailing in aging and cancerous tissues constitute 5-10% of healthy female bone marrow mesenchymal stromal cells (fBMSCs). The PGCs can repair DNA damage and stimulate neighboring cells for clonal expansion. However, dying PGCs in advanced-senescent fBMSCs can form "spikings" which are then separated into membraned mtDNA-containing vesicles (Senescent PGC-Spiking Bodies; SPSBs). SPSB-phagocytosed macrophages accelerate aging with diminished clearance on BC cells and protumor M2 polarization. SPSB-carried mitochondrial OXPHOS components are enriched in BC of elder patients and associated with poor prognosis. SPSB-incorporated breast epithelial cells develop aggressive characteristics and PGCs resembling the polyploid giant cancer cells (PGCCs) in clonogenic BC cells and cancer tissues. These findings highlight an aging BMSC-induced BC risk mediated by SPSB-induced macrophage dysfunction and epithelial cell precancerous transition. SIGNIFICANCE:Mechanisms underlying aging-associated cancer risk remain unelucidated. This work demonstrates that polyploid giant cells (PGCs) in bone marrow mesenchymal stromal cells (BMSCs) from healthy female bone marrow donors can boost neighboring cell proliferation for clonal expansion. However, the dying-senescent PGCs in the advanced-senescent fBMSCs can form "spikings" which are separated into mitochondrial DNA (mtDNA)-containing spiking bodies (senescent PGC-spiking bodies; SPSBs). The SPSBs promote macrophage aging and breast epithelial cell protumorigenic transition and form polyploid giant cancer cells. These results demonstrate a new form of ghost message from dying-senescent BMSCs, that may serve as a systemic factor contributing to aging-associated immunosuppression and breast cancer risk. Graphic Abstract:

N-Myc is a key driver of neuroblastoma and neuroendocrine prostate cancer (NEPC). One potential way to circumvent the challenge of undruggable N-Myc is to target the protein homeostasis (proteostasis) system that maintains N-Myc levels. Here, we identify heat shock protein 70 (HSP70) as a top partner of N-Myc, which binds a conserved "SELILKR" motif and prevents the access of E3 ubiquitin ligase, STIP1 homology and U-box containing protein 1 (STUB1), possibly through steric hindrance. When HSP70's dwell time on N-Myc is increased by treatment with the HSP70 allosteric inhibitor, STUB1 is in close proximity with N-Myc and becomes functional to promote N-Myc ubiquitination on the K416 and K419 sites and forms polyubiquitination chains linked by the K11 and K63 sites. Notably, HSP70 inhibition significantly suppressed NEPC tumor growth, increased the efficacy of aurora kinase A (AURKA) inhibitors, and limited the expression of neuroendocrine-related pathways.

Kaposi’s sarcoma-associated herpesvirus (KSHV) establishes a latent infection, and viral genes are poised to be transcribed in the latent chromatin. In the poised chromatins, KSHV latency-associated nuclear antigen (LANA) interacts with cellular chromodomain-helicase-DNA-binding protein 4 (CHD4) and inhibits viral promoter activation. CHD4 is known to regulate cell differentiation by preventing enhancers from activating promoters. Here, we identified a putative CHD4 inhibitor peptide (VGN73) from the LANA sequence corresponding to the LANA-CHD4 interaction surface. The VGN73 interacts with CHD4 at its PHD domain with a dissociation constant (KD) of 14 nM. Pre-treatment with VGN73 enhanced monocyte differentiation into macrophages and globally altered the repertoire of activated genes in U937 cells. Furthermore, the introduction of the peptide into the cancer cells induced caspase-mediated CHD4 cleavage, triggered cell death, and inhibited tumor growth in a xenograft mouse model. The VGN73 may facilitate cell differentiation therapy.

Abstract Purpose: A substantial contribution to the poor prognosis for head and neck squamous cell carcinoma (HNSCC) patients is the progression to distant metastasis. This occurs in 30% of patients over the course of their disease, and radically reduces the 5-year survival rate to under 50%. Investigations in recent years have begun to unveil the intricate interplay of the extracellular matrix (ECM) in both tumor-intrinsic signaling and immunoregulation. However, studies conducted in the context of metastatic have been limited. Here, we employ spatial transcriptomics on HNSCC patient biopsies to identify ECM components that may contribute to cancer progression and immune suppression. Experimental design: Comprehensive transcriptome profiling of HNSCC patient biopsies was conducted using the GeoMx Digital Spatial Profiler (DSP). This approach allowed us to characterize regions of the tumor microenvironment (TME) by immunofluorescent staining for subsequent bioinformatic analysis. To investigate the biological consequences of associations observed in our dataset, we performed in vivo murine studies. We have produced a series of SCC C57BL/6 murine cell lines derived from Smad4-/-KrasG12D Keratin15+ stem cells, which faithfully mimic the clinical progression of HNSCC tumors, including metastasis. Using these murine models, we applied relevant pharmacologic inhibitors and genetic knockdown of targets identified with our GeoMx DSP analysis. In vitro immune-based and functional assays were conducted to elucidate the underlying mechanisms contributing to SCC progression. Results: GeoMx DSP analysis revealed that laminins were enriched in stage IV HNSCC patient stroma, relative to lower stage patients. This coincided with TGFβ enrichment and inversely associated with signatures of anti-tumor immune infiltration. Analysis of The Cancer Genome Atlas data revealed similar inverse correlations in SCCs, distinct from breast cancer and lung adenocarcinoma. Our metastatic SCC murine lines likewise exhibited elevated levels of these laminin proteins, accompanied by high levels of laminin-binding integrins and TGFβ-Smad signaling. Independent of impacting cell proliferation, shRNA knockdown of laminin decreased tumor volume in an immune-competent setting. Targeting laminin-integrin signaling with the small molecule inhibitors Galunisertnib and Buparlisib also reduced the metastatic capacity of these cells in C57BL/6 mice. Conclusions: Spatial transcriptomic profiling of the HNSCC TME uncovers intricate associations and promising targets not readily discernible in bulk RNAseq analyses. Validation through genetic and pharmacological approaches in immune-competent models underscores their potential as strategies to reduce HNSCC burden. Citation Format: John D. Aleman, Khoa A. Nguyen, Erica Wong, Yao Ke, Hanne T. Lind, Clifford G. Tepper, Andrew C. Birkeland, Christian D. Young, Sana D. Karam, Xiao-Jing Wang. Regional transcriptomic profiling of the head and neck squamous cell carcinoma tumor microenvironment paired with murine models identify extracellular matrix components that mediate tumor progression and immune suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4206.

Supplementary Table 1, Figures 1-4, Methods from Nrdp1-Mediated Regulation of ErbB3 Expression by the Androgen Receptor in Androgen-Dependent but not Castrate-Resistant Prostate Cancer Cells

Supplementary Figures 1-20, Table 1 from A Novel Androgen Receptor Splice Variant Is Up-regulated during Prostate Cancer Progression and Promotes Androgen Depletion–Resistant Growth

S-Figure 1. KSHV reactivation by PEP005; S-Figure 2. Chromatin immunoprecipitations; S-Figure 3. Intersection analysis of genes differentially-expressed in response to each treatment in different PEL cell lines.

<p>Please note the Supplementary Methods were corrected by the authors and re-posted on March 16, 2009. The corrected file provides an updated GEO accession number. The original and revised versions are provided for reference.</p>

Tensin-1(TNS1)is a 220 kD focal adhesion protein that binds to actin filaments,integrin receptors,small GTPases,tyrosine-phosphorylated proteins,and lipids.1 These binding activities enable TNS1 to link the actin cytoskeleton to integrins and transduce outside-in and inside-out signals at focal adhesion sites,thereby regulating cell attachment,migration,prolif-eration,and mechanical sensing.

p-p70S6K, pS6 and p-ERK expression in bladder cancer cells treated with pictilisib

Cell cycle analysis, Flow cytometric analysis and p-AKT expression in TCCSUP cells treated with pictilisib

Supplementary Methods, Figure Legends 1-11 from ANCCA/ATAD2 Overexpression Identifies Breast Cancer Patients with Poor Prognosis, Acting to Drive Proliferation and Survival of Triple-Negative Cells through Control of B-Myb and EZH2

Please note the Supplementary Methods were corrected by the authors and re-posted on March 16, 2009. The corrected file provides an updated GEO accession number. The original and revised versions are provided for reference.

The mutation status and absolute IC50 of bladder cancer cells

The heat map of autophagy related genes in pictilisib (PIC), cisplatin (CIS) and combination (COM) resistant tumors of PDX BL0269 model

Legend for Supplementary Figures S1-S5.