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Effects of trametinib and palbociclib alone and in combination on growth and expression of various signaling proteins

Supplementary Data from Intronic Cis-Element DR8 in hTERT Is Bound by Splicing Factor SF3B4 and Regulates hTERT Splicing in Non–Small Cell Lung Cancer

The ineffectiveness of chemotherapy in patients with pancreatic cancer highlights a critical unmet need in pancreatic cancer therapy. Two commonly mutated genes in pancreatic cancer, KRAS and CDKN2A, have an incidence exceeding 90%, supporting investigation of dual targeting of MEK and CDK4/6 as a potential therapeutic strategy for this patient population. An in vitro proliferation synergy screen was conducted to evaluate response of a panel of high passage and patient-derived pancreatic cancer models to the combination of trametinib and palbociclib to inhibit MEK and CDK4/6, respectively. Two adenosquamous carcinoma models, L3.6pl and UM59, stood out for their high synergy response. In vivo studies confirmed that this combination treatment approach was highly effective in subcutaneously implanted L3.6pl and UM59 tumor-bearing animals. Both models were refractory to single-agent treatment. Reverse-phase protein array analysis of L3.6pl tumors excised from treated animals revealed strong downregulation of COX-2 expression in response to combination treatment. Expression of COX-2 under a CMV-driven promoter and shRNA knockdown of COX-2 both led to resistance to combination treatment. Our findings suggest that COX-2 may be involved in the improved therapeutic outcome seen in some pancreatic tumors that fail to respond to MEK or CDK4/6 inhibitors alone but respond favorably to their combination.

Supplementary Figure from Intronic Cis-Element DR8 in hTERT Is Bound by Splicing Factor SF3B4 and Regulates hTERT Splicing in Non–Small Cell Lung Cancer

Splicing of the hTERT gene to produce the full-length (FL) transcript is necessary for telomerase enzyme activity and telomere-dependent cellular immortality in the majority of human tumors, including non–small cell lung cancer (NSCLC) cells. The molecular machinery to splice hTERT to the FL isoform remains mostly unknown. Previously, we reported that an intron 8 cis-element termed “direct repeat 8” (DR8) promotes FL hTERT splicing, telomerase, and telomere length maintenance when bound by NOVA1 and PTBP1 in NSCLC cells. However, some NSCLC cells and patient tumor samples lack NOVA1 expression. This leaves a gap in knowledge about the splicing factors and cis-elements that promote telomerase in the NOVA1-negative context. We report that DR8 regulates FL hTERT splicing in the NOVA1-negative and -positive lung cancer contexts. We identified splicing factor 3b subunit 4 (SF3B4) as an RNA trans-factor whose expression is increased in lung adenocarcinoma (LUAD) tumors compared with adjacent normal tissue and predicts poor LUAD patient survival. In contrast to normal lung epithelial cells, which continued to grow with partial reductions of SF3B4 protein, SF3B4 knockdown reduced hTERT splicing, telomerase activity, telomere length, and cell growth in lung cancer cells. SF3B4 was also demonstrated to bind the DR8 region of hTERT pre-mRNA in both NOVA1-negative and -positive NSCLC cells. These findings provide evidence that DR8 is a critical binding hub for trans-factors to regulate FL hTERT splicing in NSCLC cells. These studies help define mechanisms of gene regulation important to the generation of telomerase activity during carcinogenesis. Implications: Manipulation of a core spliceosome protein reduces telomerase/hTERT splicing in lung cancer cells and results in slowed cancer cell growth and cell death, revealing a potential therapeutic strategy.

Supplementary Table S5 from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

Supplementary Figure S3 from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

Supplementary Data from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

Supplementary Table S2 S4 S6 S7 from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

Purpose:Lineage plasticity in prostate cancer—most commonly exemplified by loss of androgen receptor (AR) signaling and a switch from a luminal to alternate differentiation program—is now recognized as a treatment resistance mechanism. Lineage plasticity is a spectrum, but neuroendocrine prostate cancer (NEPC) is the most virulent example. Currently, there are limited treatments for NEPC. Moreover, the incidence of treatment-emergent NEPC (t-NEPC) is increasing in the era of novel AR inhibitors. In contradistinction to de novo NEPC, t-NEPC tumors often express the AR, but AR's functional role in t-NEPC is unknown. Furthermore, targetable factors that promote t-NEPC lineage plasticity are also unclear.Experimental Design:Using an integrative systems biology approach, we investigated enzalutamide-resistant t-NEPC cell lines and their parental, enzalutamide-sensitive adenocarcinoma cell lines. The AR is still expressed in these t-NEPC cells, enabling us to determine the role of the AR and other key factors in regulating t-NEPC lineage plasticity.Results:AR inhibition accentuates lineage plasticity in t-NEPC cells—an effect not observed in parental, enzalutamide-sensitive adenocarcinoma cells. Induction of an AR-repressed, lineage plasticity program is dependent on activation of the transcription factor E2F1 in concert with the BET bromodomain chromatin reader BRD4. BET inhibition (BETi) blocks this E2F1/BRD4-regulated program and decreases growth of t-NEPC tumor models and a subset of t-NEPC patient tumors with high activity of this program in a BETi clinical trial.Conclusions:E2F1 and BRD4 are critical for activating an AR-repressed, t-NEPC lineage plasticity program. BETi is a promising approach to block this program.

Supplemental Figures 1-15 and Supplemental Tables 1-32

Supplementary Table S8-S12 from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

GST omega 1 (GSTO1) is an atypical GST isoform that is overexpressed in several cancers and has been implicated in drug resistance. Currently, no small-molecule drug targeting GSTO1 is under clinical development. Here we have validated GSTO1 as an impactful target in oncology. Transcriptional profiling coupled with proteomics uncovered novel pharmacodynamic markers and cellular pathways regulated by GSTO1. CRISPR/Cas9 GSTO1 knockout (KO) cell lines failed to form tumors or displayed growth delay in vivo; they also formed smaller 3D spheroids in vitro. Multiomics analysis in GSTO1 KO cells found a strong positive correlation with cell adhesion molecules and IFN response pathways and a strong negative correlation with Myc transcriptional signature. In addition, several clinically used drugs showed significant synthetic lethality with loss or inhibition of GSTO1. Transcription and protein expression of tissue factor (gene name, F3) were downregulated in response to GSTO1 KO. F3 is associated with poor patient survival and promotion of tumor progression in multiple cancers and is a known risk factor for metastasis. Transcription of F3 was regulated by IL1β, whose secretion decreased upon inhibition of GSTO1, suggesting that IL1β links GSTO1 expression and F3 transcription. In summary, our results implicate GSTO1 as a potential therapeutic target in cancer and offer new mechanistic insights into its significant role in cancer progression.Significance:These findings validate GSTO1 as a therapeutic target in cancer and implicate GSTO1 in the modulation of tumor growth, immune responses, and expression of F3.

PDF file - 2.3MB, (S1) Scheme for profiling leukemia patient samples with a panel of kinase inhibitors; (S2) Rank ordering of 151 sample IC50 values for each small-molecule kinase inhibitor; (S3) One-way clustering of leukemia patient sample response to small-molecule kinase inhibitors; (S4) Two-way clustering of leukemia patient sample response to small-molecule kinase inhibitors

XLS file - 48K, Sources of Small-Molecule Kinase Inhibitors

XLS file - 552K, 151 Patient Drug Curve Raw Data Points

XLS file - 114K, Kinase Inhibitor Gene Targets

XLS file - 132K, 151 Patient IC50s

XLS file - 73K, Cumulative Gene Target Scores

PDF file - 378K

XLS file - 79K, Patient Clinical Information

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Treatment of locally advanced rectal cancer includes chemotherapy, radiation, and surgery but patient responses to neoadjuvant treatment are variable. We have shown that rectal tumors are comprised of multiple genetically distinct sub-clones. Unique sub-clones within tumors may harbor mutations which contribute to inter-patient variation in response to neoadjuvant chemoradiotherapy (nCRT). Analysis of the influence of nCRT on the extent and nature of intra-tumoral genetic heterogeneity in rectal cancer may provide insights into mechanisms of resistance. Locally advanced rectal cancer patients underwent pre-treatment biopsies. At the time of surgery, tissue from the treated tumor was obtained and analyzed. Pre- and post-treatment specimens were subjected to whole exome and confirmatory deep sequencing for somatic mutations. Copy number variation was assessed using OncoScan SNP arrays. Genomic data were analyzed using PyClone to identify sub-clonal tumor population following nCRT. Alterations that persisted or were enriched in the post-treatment tumor specimen following nCRT were defined for each patient. Thirty-two samples were obtained from ten patients. PyClone identified 2 to 10 genetic sub-clones per tumor. Substantial changes in the proportions of individual sub-clones in pre- versus post-treatment tumor material were found in all patients. Resistant sub-clones recurrently contained mutations in TP53, APC, ABCA13, MUC16, and THSD4. Recurrent copy number variation was observed across multiple chromosome regions after nCRT. Pathway analysis including variant alleles and copy number changes associated with resistant sub-clones revealed significantly altered pathways, especially those linked to the APC and TP53 genes, which were the two most frequently mutated genes. Intra-tumoral heterogeneity is evident in pre-treatment rectal cancer. Following treatment, sub-clonal populations are selectively modified and enrichment of a subset of pre-treatment sub-clones is seen. Further studies are needed to define recurrent alterations at diagnosis that may contribute to resistance to nCRT.

Aberrant overexpression of endoplasmic reticulum (ER)-resident oxidoreductase protein disulfide isomerase (PDI) plays an important role in cancer progression. In this study, we demonstrate that PDI promotes glioblastoma (GBM) cell growth and describe a class of allosteric PDI inhibitors that are selective for PDI over other PDI family members. Methods: We performed a phenotypic screening triage campaign of over 20,000 diverse compounds to identify PDI inhibitors cytotoxic to cancer cells. From this screen, BAP2 emerged as a lead compound, and we assessed BAP2-PDI interactions with gel filtration, thiol-competition assays, and site-directed mutagenesis studies. To assess selectivity, we compared BAP2 activity across several PDI family members in the PDI reductase assay. Finally, we performed in vivo studies with a mouse xenograft model of GBM combining BAP2 and the standard of care (temozolomide and radiation), and identified affected gene pathways with nascent RNA sequencing (Bru-seq). Results: BAP2 and related analogs are novel PDI inhibitors that selectively inhibit PDIA1 and PDIp. Though BAP2 contains a weak Michael acceptor, interaction with PDI relies on Histidine 256 in the b' domain of PDI, suggesting allosteric binding. Furthermore, both in vitro and in vivo, BAP2 reduces cell and tumor growth. BAP2 alters the transcription of genes involved in the unfolded protein response, ER stress, apoptosis and DNA repair response. Conclusion: These results indicate that BAP2 has anti-tumor activity and the suppressive effect on DNA repair gene expression warrants combination with DNA damaging agents to treat GBM.

Basal subtype cancers are deadly malignancies but the molecular events driving tumor lethality are not completely understood. Ataxia-telangiectasia group D complementing gene (ATDC, also known as TRIM29), is highly expressed and drives tumor formation and invasion in human bladder cancers but the factor(s) regulating its expression in bladder cancer are unknown. Molecular subtyping of bladder cancer has identified an aggressive basal subtype, which shares molecular features of basal/squamous tumors arising in other organs and is defined by activation of a TP63-driven gene program. Here, we demonstrate that ATDC is linked with expression of TP63 and highly expressed in basal bladder cancers. We find that TP63 binds to transcriptional regulatory regions of ATDC and KRT14 directly, increasing their expression, and that ATDC and KRT14 execute a TP63-driven invasive program. In vivo, ATDC is required for TP63-induced bladder tumor invasion and metastasis. These results link TP63 and the basal gene expression program to ATDC and to aggressive tumor behavior. Defining ATDC as a molecular determinant of aggressive, basal cancers may lead to improved biomarkers and therapeutic approaches.

Introduction: Bladder cancer (BC) is the most frequent urinary system cancer in the US. Neoadjuvant chemotherapy before cystectomy for muscle-invasive BC is standard management, though the absolute survival benefit is small, with many patients progressing during chemotherapy. Identifying therapies with a high probability of specific activity against each patient’s tumor remains a critical need. Methods: Following informed consent, from patients undergoing a transurethral resection of bladder tumor or cystectomy, 1+ gram of tumor was procured and divided between DNA/RNA sequencing, organoid drug-screening, and single-cell sequencing of cells surviving chemotherapy. Tissue was dissociated, filtered, and resuspended in organoid media for minimal passaging and drug screening. Drugs were tested at the maximum plasma concentration (Cmax) in human trials, so to provide physiologic relevance. When material was sufficient, cells were additionally tested using dose response. Cmax screening results were normalized to control such that a value of “100” indicated no difference in organoid viability compared to control, and a value of 0 indicated complete response. The number of drugs screened was dependent upon tissue available, with up to 34 drugs screened at Cmax and 9 drugs screened in dose response format. Results: Thus far, >100 patient-derived organoids have undergone collection and development with ~ 2/3 samples collected resulting in organoid development and drug screening. RNA sequencing and GSEA enriched pathways across organoids was compared to other published datasets, noting high levels of correlation between our organoid models and in particular, the Cancer Cell Line Encyclopedia urethral lines. Moreover, analysis of subtype and mutation/copy number data further indicate that our organoid dataset is representative of the full spectrum of disease. RNA sequencing and subtyping of cultured organoids compared to patient tissue indicate that organoid models are representative of patient tissue and do not undergo subtype shifts in our short-term 3D cultures. Our drug screening results highlight the large spectrum of response to chemotherapies that in fact, clinically benefit only a small proportion of patients in the neoadjuvant setting. Crucially, we identify promising targeted therapies to consider for patients who progress following resection and adjuvant chemotherapy. Conclusions: Rapid organoid development, characterization, and drug screening allows for the prediction of therapeutic response in ~10 days following sample collection. Use of this technique on tissue provided during disease work-up may further guide selection of effective therapeutic agents in patients with bladder cancer. This would overall minimize the morbidity of standard of care therapies and could be used to identify alternative therapeutics for patients who progress on standard therapies. Citation Format: Nathan M. Merrill, Liwei Bao, Xu Cheng, Nathalie Vandecan, Zhaoping Qin, Albert Liu, Athena Apfel, Laura Goo, Lila Tudrick, Armand Bankhead III, Phil Palmbos, Samuel Kaffenberger, Khaled Hafez, Jeffrey Montgomery, Todd Morgan, Ajjai Alva, Aaron Udager, Matthew Soellner, Sofia Merajver. Functional assays of drug sensitivity in real-time from patient material for precision oncology in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3072.

Purpose: Lineage plasticity in prostate cancer—most commonly exemplified by loss of androgen receptor (AR) signaling and a switch from a luminal to alternate differentiation program—is now recognized as a treatment resistance mechanism. Lineage plasticity is a spectrum, but neuroendocrine prostate cancer (NEPC) is the most virulent example. Currently, there are limited treatments for NEPC. Moreover, the incidence of treatment-emergent NEPC (t-NEPC) is increasing in the era of novel AR inhibitors. In contradistinction to de novo NEPC, t-NEPC tumors often express the AR, but AR's functional role in t-NEPC is unknown. Furthermore, targetable factors that promote t-NEPC lineage plasticity are also unclear. Experimental Design: Using an integrative systems biology approach, we investigated enzalutamide-resistant t-NEPC cell lines and their parental, enzalutamide-sensitive adenocarcinoma cell lines. The AR is still expressed in these t-NEPC cells, enabling us to determine the role of the AR and other key factors in regulating t-NEPC lineage plasticity. Results: AR inhibition accentuates lineage plasticity in t-NEPC cells—an effect not observed in parental, enzalutamide-sensitive adenocarcinoma cells. Induction of an AR-repressed, lineage plasticity program is dependent on activation of the transcription factor E2F1 in concert with the BET bromodomain chromatin reader BRD4. BET inhibition (BETi) blocks this E2F1/BRD4-regulated program and decreases growth of t-NEPC tumor models and a subset of t-NEPC patient tumors with high activity of this program in a BETi clinical trial. Conclusions: E2F1 and BRD4 are critical for activating an AR-repressed, t-NEPC lineage plasticity program. BETi is a promising approach to block this program.

[Image: see text] Metabolic reprogramming is a key hallmark of cancer and shifts cellular metabolism to meet the demands of biomass production necessary for abnormal cell reproduction. One-carbon metabolism (1CM) contributes to many biosynthetic pathways that fuel growth and is comprised of a complex network of enzymes. Methotrexate and 5-fluorouracil were pioneering drugs in this field and are still widely used today as anticancer agents as well as for other diseases such as arthritis. Besides dihydrofolate reductase and thymidylate synthase, two other enzymes of the folate cycle arm of 1CM have not been targeted clinically: serine hydroxymethyltransferase (SHMT) and methylenetetrahydrofolate dehydrogenase (MTHFD). An increasing body of literature suggests that the mitochondrial isoforms of these enzymes (SHMT2 and MTHFD2) are clinically relevant in the context of cancer. In this review, we focused on the 1CM pathway as a target for cancer therapy and, in particular, SHMT2 and MTHFD2. The function, regulation, and clinical relevance of SHMT2 and MTHFD2 are all discussed. We expand on previous clinical studies and evaluate the prognostic significance of these critical enzymes by performing a pan-cancer analysis of patient data from the The Cancer Genome Atlas and a transcriptional coexpression network enrichment analysis. We also provide an overview of preclinical and clinical inhibitors targeting the folate pathway, the methionine cycle, and folate-dependent purine biosynthesis enzymes.

Certain subtypes of cancer cells require oxidative phosphorylation (OXPHOS) to survive. Increased OXPHOS dependency is frequently a hallmark of cancer stem cells and cells resistant to chemotherapy and targeted therapies. Suppressing the OXPHOS function might also influence the tumor microenvironment by alleviating hypoxia and improving the antitumor immune response. Thus, targeting OXPHOS is a promising strategy to treat various cancers. A growing arsenal of therapeutic agents is under development to inhibit this biological process. This Perspective provides an overview of the structure and function of OXPHOS complexes, their biological functions in cancer, relevant research tools and models, as well as the limitations of OXPHOS as drug targets. We also focus on the current development status of OXPHOS inhibitors and potential therapeutic strategies to strengthen their clinical applications.

GST omega 1 (GSTO1) is an atypical GST isoform that is overexpressed in several cancers and has been implicated in drug resistance. Currently, no small-molecule drug targeting GSTO1 is under clinical development. Here we have validated GSTO1 as an impactful target in oncology. Transcriptional profiling coupled with proteomics uncovered novel pharmacodynamic markers and cellular pathways regulated by GSTO1. CRISPR/Cas9 GSTO1 knockout (KO) cell lines failed to form tumors or displayed growth delay in vivo; they also formed smaller 3D spheroids in vitro. Multiomics analysis in GSTO1 KO cells found a strong positive correlation with cell adhesion molecules and IFN response pathways and a strong negative correlation with Myc transcriptional signature. In addition, several clinically used drugs showed significant synthetic lethality with loss or inhibition of GSTO1. Transcription and protein expression of tissue factor (gene name, F3) were downregulated in response to GSTO1 KO. F3 is associated with poor patient survival and promotion of tumor progression in multiple cancers and is a known risk factor for metastasis. Transcription of F3 was regulated by IL1β, whose secretion decreased upon inhibition of GSTO1, suggesting that IL1β links GSTO1 expression and F3 transcription. In summary, our results implicate GSTO1 as a potential therapeutic target in cancer and offer new mechanistic insights into its significant role in cancer progression. Significance: These findings validate GSTO1 as a therapeutic target in cancer and implicate GSTO1 in the modulation of tumor growth, immune responses, and expression of F3.

Developmental transcription programs are epigenetically regulated by multi-protein complexes, including the menin- and MLL-containing trithorax (TrxG) complexes, which promote gene transcription by depositing the H3K4me3 activating mark at target gene promoters. We recently reported that in Ewing sarcoma, MLL1 (lysine methyltransferase 2A, KMT2A) and menin are overexpressed and function as oncogenes. Small molecule inhibition of the menin-MLL interaction leads to loss of menin and MLL1 protein expression, and to inhibition of growth and tumorigenicity. Here, we have investigated the mechanistic basis of menin-MLL-mediated oncogenic activity in Ewing sarcoma. Bromouridine sequencing (Bru-seq) was performed to identify changes in nascent gene transcription in Ewing sarcoma cells, following exposure to the menin-MLL interaction inhibitor MI-503. Menin-MLL inhibition resulted in early and widespread reprogramming of metabolic processes. In particular, the serine biosynthetic pathway (SSP) was the pathway most significantly affected by MI-503 treatment. Baseline expression of SSP genes and proteins (PHGDH, PSAT1, and PSPH), and metabolic flux through the SSP were confirmed to be high in Ewing sarcoma. In addition, inhibition of PHGDH resulted in reduced cell proliferation, viability, and tumor growth in vivo, revealing a key dependency of Ewing sarcoma on the SSP. Loss of function studies validated a mechanistic link between menin and the SSP. Specifically, inhibition of menin resulted in diminished expression of SSP genes, reduced H3K4me3 enrichment at the PHGDH promoter, and complete abrogation of de novo serine and glycine biosynthesis, as demonstrated by metabolic tracing studies with 13 C-labeled glucose. These data demonstrate that the SSP is highly active in Ewing sarcoma and that its oncogenic activation is maintained, at least in part, by menin-dependent epigenetic mechanisms involving trithorax complexes. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Glioblastoma multiforme (GBM), the most common and aggressive primary brain tumor, has a high mortality rate despite extensive efforts to develop new treatments. GBM exhibits both intra- and intertumor heterogeneity, lending to resistance and eventual tumor recurrence. Large-scale genomic and proteomic analysis of GBM tumors has uncovered potential drug targets. Effective and “druggable” targets must be validated to embark on a robust medicinal chemistry campaign culminating in the discovery of clinical candidates. Here, we review recent developments in GBM drug discovery and delivery. To identify GBM drug targets, we performed extensive bioinformatics analysis using data from The Cancer Genome Atlas project. We discovered 20 genes, BOC, CLEC4GP1, ELOVL6, EREG, ESR2, FDCSP, FURIN, FUT8-AS1, GZMB, IRX3, LITAF, NDEL1, NKX3-1, PODNL1, PTPRN, QSOX1, SEMA4F, TH, VEGFC, and C20orf166AS1 that are overexpressed in a subpopulation of GBM patients and correlate with poor survival outcomes. Importantly, nine of these genes exhibit higher expression in GBM versus low-grade glioma and may be involved in disease progression. In this review, we discuss these proteins in the context of GBM disease progression. We also conducted computational multi-parameter optimization to assess the blood-brain barrier (BBB) permeability of small molecules in clinical trials for GBM treatment. Drug delivery in the context of GBM is particularly challenging because the BBB hinders small molecule transport. Therefore, we discuss novel drug delivery methods, including nanoparticles and prodrugs. Given the aggressive nature of GBM and the complexity of targeting the central nervous system, effective treatment options are a major unmet medical need. Identification and validation of biomarkers and drug targets associated with GBM disease progression present an exciting opportunity to improve treatment of this devastating disease.

Protein folding in the endoplasmic reticulum is an oxidative process that relies on protein disulfide isomerase (PDI) and endoplasmic reticulum oxidase 1 (ERO1). Over 30% of proteins require the chaperone PDI to promote disulfide bond formation. PDI oxidizes cysteines in nascent polypeptides to form disulfide bonds and can also reduce and isomerize disulfide bonds. ERO1 recycles reduced PDI family member PDIA1 using a FAD cofactor to transfer electrons to oxygen. ERO1 dysfunction critically affects several diseases states. Both ERO1 and PDIA1 are overexpressed in cancers and implicated in diabetes and neurodegenerative diseases. Cancer-associated ERO1 promotes cell migration and invasion. Furthermore, the ERO1-PDIA1 interaction is critical for epithelial-to-mesenchymal transition. Co-expression analysis of ERO1A gene expression in cancer patients demonstrated that ERO1A is significantly upregulated in lung adenocarcinoma (LUAD), glioblastoma and low-grade glioma (GBMLGG), pancreatic ductal adenocarcinoma (PAAD), and kidney renal papillary cell carcinoma (KIRP) cancers. ERO1Α knockdown gene signature correlates with knockdown of cancer signaling proteins including IGF1R, supporting the search for novel, selective ERO1 inhibitors for the treatment of cancer. In this review, we explore the functions of ERO1 and PDI to support inhibition of this interaction in cancer and other diseases.

Background: Half of muscle-invasive bladder cancer patients will relapse with metastatic disease and molecular tests to predict relapse are needed. TP63 has been proposed as a prognostic biomarker in bladder cancer, but reports associating it with clinical outcomes are conflicting. Since TP63 is expressed as multiple isoforms, we hypothesized that these conflicting associations with clinical outcome may be explained by distinct opposing effects of differential TP63 isoform expression. Methods: Using RNA-Seq data from The Cancer Genome Atlas (TCGA), TP63 isoform-level expression was quantified and associated with clinical covariates (e.g. survival, stage) across 8,519 patients from 29 diseases. A comprehensive catalog of TP63 isoforms was assembled using gene annotation databases and de novo discovery in bladder cancer patients. Quantifications and un-annotated TP63 isoforms were validated using quantitative RT-PCR and a separate bladder cancer cohort. Findings: DNp63 isoform expression was associated with improved bladder cancer patient survival in patients with a luminal subtype (HR = 0.89, CI 0.80–0.99, Cox p = 0.034). Conversely, TAp63 isoform expression was associated with reduced bladder cancer patient survival in patients with a basal subtype (HR = 2.35, CI 1.64–3.37, Cox p < 0.0001). These associations were observed in multiple TCGA disease cohorts and correlated with epidermal differentiation (DNp63) and immune-related (TAp63) gene signatures. Interpretation: These results comprehensively define TP63 isoform expression in human cancer and suggest that TP63 isoforms are involved in distinct transcriptional programs with opposing effects on clinical outcome. Keywords: TP63, Isoforms, Cancer, Bladder Cancer, Biomarker

Inhibiting STAT3 signaling reduces tumor progression, metastasis and chemoresistance, however the precise molecular mechanism has not been fully delineated in ovarian cancer. Methods: In this study, we generated STAT3 knockout (KO) ovarian cancer cell lines. Effects of STAT3 KO on cell proliferation, migration and spheroid formation were assessed in vitro and effects on in vivo tumor growth were tested using several tumor xenograft models. We used multi-omic genome-wide profiling to identify multi-level (Bru-Seq, RNA-Seq, and MS Proteomic) expression signatures of STAT3 KO ovarian cancer cells. Results: We observed that deletion of STAT3 blocked cell proliferation and migration in vitro and suppressed tumor growth in mice. Deletion of STAT3 transcriptionally suppressed key genes involved in EMT, cell cycle progression, E2F signaling, and altered stemness markers. Notably, KO of STAT3 resulted in modulation of the expression of other STAT family members. Conclusion: Our study presents a rich, multi-faceted summary of the molecular mechanisms impacted by STAT3 deletion and provides new insight for STAT3's potential as a therapeutic target in ovarian cancer.

Bladder cancer will cause an estimated 17,000 deaths this year in the United States. Transformation-related protein 63 (TP63) is a p53 family transcription factor that has been shown to be important for development and is commonly expressed in bladder cancer. However, the relationship of TP63 to bladder cancer patient outcome remains unclear. Some studies have associated TP63 expression with a positive patient outcome, while others have shown TP63 expression is associated with reduced survival. One potential explanation for this discrepancy is that TP63 is expressed as one of at least 15 unique isoforms, of which there are two main types (TAp63 and DNp63) exhibiting unique functional domains. In this work we define the landscape of TP63 isoform expression and quantify its association with bladder cancer patient outcome using RNA sequencing data from the TCGA bladder cancer cohort and other human bladder cancer samples. To examine the TP63 isoform landscape in bladder cancer, we utilized annotations from Refgene and Ensembl databases and used Cufflinks to identify two potentially novel TP63 isoforms not represented in current TP63 gene annotations. To quantitate isoform expression, we used multiple RNA-Seq expression quantification algorithms (Cufflinks, Kallisto, and Salmon) to infer isoform expression estimates of 15 TP63 isoforms for 408 bladder cancer patients from TCGA. We experimentally validated our findings using a collection of 14 bladder cancer cell lines and show that results translated to an additional collection of 19 primary bladder cancer tumor samples. We find that DNp63 is significantly higher in patients with luminal papillary and basal squamous subtypes, while TAp63 was selectively expressed in patients with basal squamous bladder cancer. Analysis of TP63 isoform expression with clinical data demonstrated that DNp63 trends with improved patient survival (HR=0.94, CI=0.88,1.006, P=0.082), whereas TAp63 is associated with worse patient survival (HR = 1.8, CI=1.38,2.4, P=0.00022) in human bladder cancer. Taken together, this work represents a comprehensive assessment of TP63 isoform expression in the TCGA bladder cancer cohort using contemporary, extended gene isoform annotations and quantifications—underscoring the need for profiling additional bladder cancer cohorts with RNA-Seq for further insight to isoform-specific expression. It also implicates TP63 isoform expression as a potential biomarker of clinical patient outcomes. Citation Format: Armand Bankhead III, Thomas McMaster, Phillip L. Palmbos. TP63 isoform expression landscape associations with bladder cancer patient outcomes [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr B02.

Medical castration that interferes with androgen receptor (AR) function is the principal treatment for advanced prostate cancer. However, clinical progression is universal, and tumors with AR-independent resistance mechanisms appear to be increasing in frequency. Consequently, there is an urgent need to develop new treatments targeting molecular pathways enriched in lethal prostate cancer. Lysine-specific demethylase 1 (LSD1) is a histone demethylase and an important regulator of gene expression. Here, we show that LSD1 promotes the survival of prostate cancer cells, including those that are castration-resistant, independently of its demethylase function and of the AR. Importantly, this effect is explained in part by activation of a lethal prostate cancer gene network in collaboration with LSD1’s binding protein, ZNF217. Finally, that a small-molecule LSD1 inhibitor―SP-2509―blocks important demethylase-independent functions and suppresses castration-resistant prostate cancer cell viability demonstrates the potential of LSD1 inhibition in this disease.

The ineffectiveness of chemotherapy in patients with pancreatic cancer highlights a critical unmet need in pancreatic cancer therapy. Two commonly mutated genes in pancreatic cancer, KRAS and CDKN2A, have an incidence exceeding 90%, supporting investigation of dual targeting of MEK and CDK4/6 as a potential therapeutic strategy for this patient population. An in vitro proliferation synergy screen was conducted to evaluate response of a panel of high passage and patient-derived pancreatic cancer models to the combination of trametinib and palbociclib to inhibit MEK and CDK4/6, respectively. Two adenosquamous carcinoma models, L3.6pl and UM59, stood out for their high synergy response. In vivo studies confirmed that this combination treatment approach was highly effective in subcutaneously implanted L3.6pl and UM59 tumor-bearing animals. Both models were refractory to single-agent treatment. Reverse-phase protein array analysis of L3.6pl tumors excised from treated animals revealed strong downregulation of COX-2 expression in response to combination treatment. Expression of COX-2 under a CMV-driven promoter and shRNA knockdown of COX-2 both led to resistance to combination treatment. Our findings suggest that COX-2 may be involved in the improved therapeutic outcome seen in some pancreatic tumors that fail to respond to MEK or CDK4/6 inhibitors alone but respond favorably to their combination.