Selective targeting of IRAK1 attenuates low molecular weight hyaluronic acid-induced stemness and non-canonical STAT3 activation in epithelial ovarian cancer.

Advanced epithelial ovarian cancer (EOC) survival rates are dishearteningly low, with ~25% surviving beyond 5 years. Evidence suggests that cancer stem cells contribute to acquired chemoresistance and tumor recurrence. Here, we show that IRAK1 is upregulated in EOC tissues, and enhanced expression correlates with poorer overall survival. Moreover, low molecular weight hyaluronic acid, which is abundant in malignant ascites from patients with advanced EOC, induced IRAK1 phosphorylation leading to STAT3 activation and enhanced spheroid formation. Knockdown of IRAK1 impaired tumor growth in peritoneal disease models, and impaired HA-induced spheroid growth and STAT3 phosphorylation. Finally, we determined that TCS2210, a known inducer of neuronal differentiation in mesenchymal stem cells, is a selective inhibitor of IRAK1. TCS2210 significantly inhibited EOC growth in vitro and in vivo both as monotherapy, and in combination with cisplatin. Collectively, these data demonstrate IRAK1 as a druggable target for EOC.

DCLK1-Mediated Regulation of Invadopodia Dynamics and Matrix Metalloproteinase Trafficking Drives Invasive Progression in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a major health concern due to its high mortality from poor treatment responses and locoregional tumor invasion into life sustaining structures in the head and neck. A deeper comprehension of HNSCC invasion mechanisms holds the potential to inform targeted therapies that may enhance patient survival. We previously reported that doublecortin like kinase 1 (DCLK1) regulates invasion of HNSCC cells. Here, we tested the hypothesis that DCLK1 regulates proteins within invadopodia to facilitate HNSCC invasion. Invadopodia are specialized subcellular protrusions secreting matrix metalloproteinases that degrade the extracellular matrix (ECM). Through a comprehensive proteome analysis comparing DCLK1 control and shDCLK1 conditions, our findings reveal that DCLK1 plays a pivotal role in regulating proteins that orchestrate cytoskeletal and ECM remodeling, contributing to cell invasion. Further, we demonstrate in TCGA datasets that DCLK1 levels correlate with increasing histological grade and lymph node metastasis. We identified higher expression of DCLK1 in the leading edge of HNSCC tissue. Knockdown of DCLK1 in HNSCC reduced the number of invadopodia, cell adhesion and colony formation. Using super resolution microscopy, we demonstrate localization of DCLK1 in invadopodia and colocalization with mature invadopodia markers TKS4, TKS5, cortactin and MT1-MMP. We carried out phosphoproteomics and validated using immunofluorescence and proximity ligation assays, the interaction between DCLK1 and motor protein KIF16B. Pharmacological inhibition or knockdown of DCLK1 reduced interaction with KIF16B, secretion of MMPs, and cell invasion. This research unveils a novel function of DCLK1 within invadopodia to regulate the trafficking of matrix degrading cargo. The work highlights the impact of targeting DCLK1 to inhibit locoregional invasion, a life-threatening attribute of HNSCC.

Role of STAT3 in pancreatic cancer.

Pancreatic cancer remains a serious and deadly disease, impacting people globally. There remain prominent gaps in the current understanding of the disease, specifically regarding the role of the signal transducer and activator of transcription (STAT) family of proteins in pancreatic tumors. STAT proteins, particularly STAT3, play important roles in pancreatic cancer, especially pancreatic ductal adenocarcinoma (PDAC), which is the most prevalent histotype. The role of STAT3 across a continuum of molecular processes, such as PDAC tumorigenesis and progression, immune escape, drug resistance and stemness, and modulation of the tumor microenvironment (TME), are only a tip of the iceberg. In some ways, the role of STAT3 in PDAC may hold greater importance than that of oncogenic Kirsten rat sarcoma virus (KRAS). This makes STAT3 a highly attractive target for developing targeted therapies for the treatment of pancreatic cancer. In this review, the current knowledge of STAT3 in pancreatic cancer has been summarized, particularly relating to STAT3 activation in cancer cells, cells of the TME, and the state of targeting STAT3 in pre-clinical and clinical trials of PDAC.

Acryl-3,5-bis(2,4-difluorobenzylidene)-4-piperidone targeting cellular JUN proto-oncogene, AP-1 transcription factor subunit inhibits head and neck squamous cell carcinoma progression.

Aim: Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide with a survival rate below fifty percent. Addressing meager therapeutic options, a series of small molecule inhibitors were screened for antitumor efficacy. The most potent analog, acryl-3,5-bis(2,4-difluorobenzylidene)-4-piperidone (DiFiD; A-DiFiD), demonstrated strong cellular JUN proto-oncogene, activator protein 1 (AP-1) transcription factor subunit (JUN, c-Jun) antagonism. c-Jun, an oncogenic transcription factor, promotes cancer progression, invasion, and adhesion; high (JUN) mRNA expression correlates with poorer HNSCC survival. Methods: Four new small molecules were generated for cytotoxicity screening in HNSCC cell lines. A-DiFiD-treated HNSCC cells were assessed for cytotoxicity, colony formation, invasion, migration, and adhesion. Dot blot array was used to identify targets. Phospho-c-Jun (p-c-Jun) expression was analyzed using immunoblotting. The Cancer Genome Atlas (TCGA) head and neck cancer datasets were utilized to determine overall patient survival. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) datasets interfaced with University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) were analyzed to determine protein levels of c-Jun in HNSCC patients and correlate levels with patient. Results: Of the small molecules tested, A-DiFiD was the most potent in HNSCC lines, while demonstrating low half-maximal drug inhibitory concentration (IC50) in non-malignant Het-1A cells. Additionally, A-DiFiD abrogated cell invasion, migration, and colony formation. Phospho-kinase in vitro array demonstrated A-DiFiD reduced p-c-Jun. Likewise, a time dependent reduction in p-c-Jun was observed starting at 3 min post A-DiFiD treatment. TCGA Firehose Legacy vs. recurrent and metastatic head and neck cancer reveal a nearly 3% DNA amplification in recurrent/metastatic tumor compared to below 1% in primary tumors that had no lymph node metastasis. CPTAC analysis show higher tumor c-Jun levels compared to normal. Patients with high JUN expression had significantly reduced 3-year survival. Conclusions: A-DiFiD targets c-Jun, a clinical HNSCC driver, with potent anti-tumor effects.

The Role of STATs in Ovarian Cancer: Exploring Their Potential for Therapy.

Ovarian cancer (OvCa) is a deadly gynecologic malignancy that presents many clinical challenges due to late-stage diagnoses and the development of acquired resistance to standard-of-care treatment protocols. There is an increasing body of evidence suggesting that STATs may play a critical role in OvCa progression, resistance, and disease recurrence, and thus we sought to compile a comprehensive review to summarize the current state of knowledge on the topic. We have examined peer reviewed literature to delineate the role of STATs in both cancer cells and cells within the tumor microenvironment. In addition to summarizing the current knowledge of STAT biology in OvCa, we have also examined the capacity of small molecule inhibitor development to target specific STATs and progress toward clinical applications. From our research, the best studied and targeted factors are STAT3 and STAT5, which has resulted in the development of several inhibitors that are under current evaluation in clinical trials. There remain gaps in understanding the role of STAT1, STAT2, STAT4, and STAT6, due to limited reports in the current literature; as such, further studies to establish their implications in OvCa are necessitated. Moreover, due to the deficiency in our understanding of these STATs, selective inhibitors also remain elusive, and therefore present opportunities for discovery.

Anticancer Activity of Novel Difluorinated Curcumin Analog and Its Inclusion Complex with 2-Hydroxypropyl-ß-Cyclodextrin against Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the primary reason for cancer-related deaths in the US. Genetic mutations, drug resistance, the involvement of multiple signaling pathways, cancer stem cells (CSCs), and desmoplastic stroma, which hinders drug penetrance, contribute to poor chemotherapeutic efficacy. Hence, there is a need to identify novel drugs with improved delivery to improve treatment outcomes. Curcumin is one such compound that can inhibit multiple signaling pathways and CSCs. However, curcumin's clinical applicability for treating PDAC is limited because of its poor solubility in water and metabolic instability. Hence, we developed a difluorinated curcumin (CDF) analog that accumulates selectively in the pancreas and inhibits PDAC growth in vitro and in vivo. In the present work, we developed its 2-hydroxy-propyl-ß-cyclodextrin (HCD) inclusion complex to increase its water solubility and hydrolytic stability. The CDFHCD inclusion complex was characterized by spectroscopic, thermal, and microscopic techniques. The inclusion complex exhibited increased aqueous solubility, hydrolytic stability, and antiproliferative activity compared to parent CDF. Moreover, CDF and CDFHCD inhibited colony and spheroid formation, and induced cell cycle and apoptosis in PDAC cell lines. Hence, CDFHCD self-assembly is an efficient approach to increase water solubility and anticancer therapeutic efficacy, which now warrants advancement towards a clinical proof of concept in PDAC patients.

Doublecortin-like kinase 1 is a therapeutic target in squamous cell carcinoma.

Doublecortin like kinase 1 (DCLK1) plays a crucial role in several cancers including colon and pancreatic adenocarcinomas. However, its role in squamous cell carcinoma (SCC) remains unknown. To this end, we examined DCLK1 expression in head and neck SCC (HNSCC) and anal SCC (ASCC). We found that DCLK1 is elevated in patient SCC tissue, which correlated with cancer progression and poorer overall survival. Furthermore, DCLK1 expression is significantly elevated in human papilloma virus negative HNSCC, which are typically aggressive with poor responses to therapy. To understand the role of DCLK1 in tumorigenesis, we used specific shRNA to suppress DCLK1 expression. This significantly reduced tumor growth, spheroid formation, and migration of HNSCC cancer cells. To further the translational relevance of our studies, we sought to identify a selective DCLK1 inhibitor. Current attempts to target DCLK1 using pharmacologic approaches have relied on nonspecific suppression of DCLK1 kinase activity. Here, we demonstrate that DiFiD (3,5-bis [2,4-difluorobenzylidene]-4-piperidone) binds to DCLK1 with high selectivity. Moreover, DiFiD mediated suppression of DCLK1 led to G2/M arrest and apoptosis and significantly suppressed tumor growth of HNSCC xenografts and ASCC patient derived xenografts, supporting that DCLK1 is critical for SCC growth.

Mutant p53 Depletion by Novel Inhibitors for HSP40/J-Domain Proteins Derived from the Natural Compound Plumbagin.

Accumulation of missense mutant p53 (mutp53) in cancers promotes malignant progression. DNAJA1, a member of HSP40 (also known as J-domain proteins: JDPs), is shown to prevent misfolded or conformational mutp53 from proteasomal degradation. Given frequent addiction of cancers to oncogenic mutp53, depleting mutp53 by DNAJA1 inhibition is a promising approach for cancer therapy. However, there is no clinically available inhibitor for DNAJA1. Our in silico molecular docking study with a natural compound-derived small molecule library identified a plumbagin derivative, PLIHZ (plumbagin-isoniazid analog), as a potential compound binding to the J domain of DNAJA1. PLIHZ efficiently reduced the levels of DNAJA1 and several conformational mutp53 with minimal impact on DNA contact mutp53 and wild-type p53 (wtp53). An analog, called PLTFBH, which showed a similar activity to PLIHZ in reducing DNAJA1 and mutp53 levels, inhibited migration of cancer cells specifically carrying conformational mutp53, but not DNA contact mutp53, p53 null, and wtp53, which was attenuated by depletion of DNAJA1 or mutp53. Moreover, PLTFBH reduced levels of multiple other HSP40/JDPs with tyrosine 7 (Y7) and/or tyrosine 8 (Y8) but failed to deplete DNAJA1 mutants with alanine substitution of these amino acids. Our study suggests PLTFBH as a potential inhibitor for multiple HSP40/JDPs.

Synthetic adiponectin-receptor agonist, AdipoRon, induces glycolytic dependence in pancreatic cancer cells.

Obesity creates a localized inflammatory reaction in the adipose, altering secretion of adipocyte-derived factors that contribute to pathologies including cancer. We have previously shown that adiponectin inhibits pancreatic cancer by antagonizing leptin-induced STAT3 activation. Yet, the effects of adiponectin on pancreatic cancer cell metabolism have not been addressed. In these studies, we have uncovered a novel metabolic function for the synthetic adiponectin-receptor agonist, AdipoRon. Treatment of PDAC cells with AdipoRon led to mitochondrial uncoupling and loss of ATP production. Concomitantly, AdipoRon-treated cells increased glucose uptake and utilization. This metabolic switch further correlated with AMPK mediated inhibition of the prolipogenic factor acetyl coenzyme A carboxylase 1 (ACC1), which is known to initiate fatty acid catabolism. Yet, measurements of fatty acid oxidation failed to detect any alteration in response to AdipoRon treatment, suggesting a deficiency for compensation. Additional disruption of glycolytic dependence, using either a glycolysis inhibitor or low-glucose conditions, demonstrated an impairment of growth and survival of all pancreatic cancer cell lines tested. Collectively, these studies provide evidence that pancreatic cancer cells utilize metabolic plasticity to upregulate glycolysis in order to adapt to suppression of oxidative phosphorylation in the presence of AdipoRon.

Immunoglobin A Deficiency and Squamous Cell Carcinoma With a Rare Presentation as Anal Cancer.

Selective immunoglobin A deficiency (IgAD) is the most common immunodeficiency disorder in the western world. Cancer is the most common cause of death in these individuals. Various cases have been reported of squamous cell carcinoma (SCC) in IgAD at sites like skin, oral cavity, and lung. Here we present a rare case of SCC occurring as anal cancer. No other reports to our knowledge describe this rare presentation. A 54-year-old Caucasian woman with asymptomatic partial IgAD presented with a palpable anal mass. Further evaluation showed stage IIIa SCC anal cancer (T1N1M0). Additional workup showed positive human papilloma virus (HPV) serology and positive HPV immunohistochemistry studies. The patient achieved complete response with chemoradiation with her most recent imaging and anorectal exam showing no evidence of cancer recurrence at 3 years follow-up. This case highlights the association between IgAD and malignancy. Although IgAD is the most common primary antibody deficiency, this patient's case presents a rare instance of anal SCC in an IgA-deficient individual. Studies show an association between HPV infection and SCC, but few include IgA-deficient individuals. Patients with IgAD and other immunodeficiencies are at higher risk for HPV infection and therefore may be at a higher risk of SCC. With widespread use of the HPV vaccine, the medical community should be aware of its importance in cancer prevention for these patients. Further studies are needed to evaluate relationships between IgAD, HPV infections, SCC cancer, and the role that the HPV vaccine has in cancer prophylaxis.

Prolactin receptor signaling: A novel target for cancer treatment - Exploring anti-PRLR signaling strategies.

Prolactin (PRL) is a peptide hormone mainly secreted from the anterior pituitary gland. PRL is reported to play a role in pregnancy, mammary gland development, immune modulation, reproduction, and differentiation of islet cells. PRL binds to its receptor PRLR, which belongs to a superfamily of the class I cytokine receptor that has no intrinsic kinase activity. In canonical signaling, PRL binding to PRLR induces downstream signaling including JAK-STAT, AKT and MAPK pathways. This leads to increased cell proliferation, stemness, migration, apoptosis inhibition, and resistance to chemotherapy. PRL-signaling is upregulated in numerous hormone-dependent cancers including breast, prostate, ovarian, and endometrial cancer. However, more recently, the pathway has been reported to play a tumor-promoting role in other cancer types such as colon, pancreas, and hepatocellular cancers. Hence, the signaling pathway is an attractive target for drug development with blockade of the receptor being a potential therapeutic approach. Different strategies have been developed to target this receptor including modification of PRL peptides (Del1-9-G129R-hPRL, G129R-Prl), growth hormone receptor/prolactin receptor bispecific antibody antagonist, neutralizing antibody LFA102, an antibody-drug conjugate (ABBV-176) of the humanized antibody h16f (PR-1594804) and pyrrolobenzodiazepine dimer, a bispecific antibody targeting both PRLR and CD3, an in vivo half-life extended fusion protein containing PRLR antagonist PrlRA and albumin binding domain. There have also been attempts to discover and develop small molecular inhibitors targeting PRLR. Recently, using structure-based virtual screening, we identified a few antipsychotic drugs including penfluridol as a molecule that inhibits PRL-signaling to inhibit PDAC tumor progression. In this review, we will summarize the recent advances in the biology of this receptor in cancer and give an account of PRLR antagonist development for the treatment of cancer.

Enhanced IFNα Signaling Promotes Ligand-Independent Activation of ERα to Promote Aromatase Inhibitor Resistance in Breast Cancer.

Aromatase inhibitors (AIs) reduce estrogen levels up to 98% as the standard practice to treat postmenopausal women with estrogen receptor-positive (ER+) breast cancer. However, approximately 30% of ER+ breast cancers develop resistance to treatment. Enhanced interferon-alpha (IFNα) signaling is upregulated in breast cancers resistant to AIs, which drives expression of a key regulator of survival, interferon-induced transmembrane protein 1 (IFITM1). However, how upregulated IFNα signaling mediates AI resistance is unknown. In this study, we utilized MCF-7:5C cells, a breast cancer cell model of AI resistance, and demonstrate that these cells exhibit enhanced IFNα signaling and ligand-independent activation of the estrogen receptor (ERα). Experiments demonstrated that STAT1, the mediator of intracellular signaling for IFNα, can interact directly with ERα. Notably, inhibition of IFNα signaling significantly reduced ERα protein expression and ER-regulated genes. In addition, loss of ERα suppressed IFITM1 expression, which was associated with cell death. Notably, chromatin immunoprecipitation experiments validated that both ERα and STAT1 associate with ERE sequences in the IFITM1 promoter. Overall, hyperactivation of IFNα signaling enhances ligand-independent activation of ERα, which promotes ER-regulated, and interferon stimulated gene expression to promote survival in AI-resistant breast cancer cells.

Tumor-initiating stem cell shapes its microenvironment into an immunosuppressive barrier and pro-tumorigenic niche.

Tumor-initiating stem cells (TSCs) are critical for drug resistance and immune escape. However, the mutual regulations between TSC and tumor microenvironment (TME) remain unclear. Using DNA-label retaining, single-cell RNA sequencing (scRNA-seq), and other approaches, we investigated intestinal adenoma in response to chemoradiotherapy (CRT), thus identifying therapy-resistant TSCs (TrTSCs). We find bidirectional crosstalk between TSCs and TME using CellPhoneDB analysis. An intriguing finding is that TSCs shape TME into a landscape that favors TSCs for immunosuppression and propagation. Using adenoma-organoid co-cultures, niche-cell depletion, and lineaging tracing, we characterize a functional role of cyclooxygenase-2 (Cox-2)-dependent signaling, predominantly occurring between tumor-associated monocytes and macrophages (TAMMs) and TrTSCs. We show that TAMMs promote TrTSC proliferation through prostaglandin E2 (PGE2)-PTGER4(EP4) signaling, which enhances ß-catenin activity via AKT phosphorylation. Thus, our study shows that the bidirectional crosstalk between TrTSC and TME results in a pro-tumorigenic and immunosuppressive contexture.

A New Pentafluorothio-Substituted Curcuminoid with Superior Antitumor Activity.

A new and readily available pentafluorothiophenyl-substituted N-methyl-piperidone curcuminoid 1a was prepared and investigated for its anti-proliferative, pro-apoptotic and cancer stem cell-differentiating activities against a panel of human tumor cell lines derived from various tumor entities. The compound 1a was highly anti-proliferative and reached IC50 values in the nanomolar concentration range. 1a was superior to the known anti-tumorally active curcuminoid EF24 (2) and its known N-ethyl-piperidone analog 1b in all tested tumor cell lines. Furthermore, 1a induced a noticeable increase of intracellular reactive oxygen species in HT-29 colon adenocarcinoma cells, which possibly leads to a distinct increase in sub-G1 cells, as assessed by cell cycle analysis. A considerable activation of the executioner-caspases 3 and 7 as well as nuclei fragmentation, cell rounding, and membrane protrusions suggest the triggering of an apoptotic mechanism. Yet another effect was the re-organization of the actin cytoskeleton shown by the formation of stress fibers and actin aggregation. 1a also caused cell death in the adherently cultured glioblastoma cell lines U251 and Mz54. We furthermore observed that 1a strongly suppressed the stem cell properties of glioma stem-like cell lines including one primary line, highlighting the potential therapeutic relevance of this new compound.

Honokiol Affects Stem Cell Viability by Suppressing Oncogenic YAP1 Function to Inhibit Colon Tumorigenesis.

Honokiol (HNK) is a biphenolic compound that has been used in traditional medicine for treating various ailments, including cancers. In this study, we determined the effect of HNK on colon cancer cells in culture and in a colitis-associated cancer model. HNK treatment inhibited proliferation and colony formation while inducing apoptosis. In addition, HNK suppressed colonosphere formation. Molecular docking suggests that HNK interacts with reserve stem cell marker protein DCLK1, with a binding energy of -7.0 Kcal/mol. In vitro kinase assays demonstrated that HNK suppressed the DCLK1 kinase activity. HNK also suppressed the expression of additional cancer stem cell marker proteins LGR5 and CD44. The Hippo signaling pathway is active in intestinal stem cells. In the canonical pathway, YAP1 is phosphorylated at Ser127 by upstream Mst1/2 and Lats1/2. This results in the sequestration of YAP1 in the cytoplasm, thereby not allowing YAP1 to translocate to the nucleus and interact with TEAD1-4 transcription factors to induce gene expression. However, HNK suppressed Ser127 phosphorylation in YAP1, but the protein remains sequestered in the cytoplasm. We further determined that this occurs by YAP1 interacting with PUMA. To determine if this also occurs in vivo, we performed studies in an AOM/DSS induced colitis-associated cancer model. HNK administered by oral gavage at a dose of 5mg/kg bw for 24 weeks demonstrated a significant reduction in the expression of YAP1 and TEAD1 and in the stem marker proteins. Together, these data suggest that HNK prevents colon tumorigenesis in part by inducing PUMA-YAP1 interaction and cytoplasmic sequestration, thereby suppressing the oncogenic YAP1 activity.

DCLK1 isoforms and aberrant Notch signaling in the regulation of human and murine colitis.

Alternative promoter usage generates long and short isoforms (DCLK1-L and DCLK1-S) of doublecortin-like kinase-1 (DCLK1). Tight control of Notch signaling is important to prevent and restitute inflammation in the intestine. Our aim was to investigate whether Notch1-DCLK1 axis regulates the mucosal immune responses to infection and whether this is phenocopied in human models of colitis. In the FFPE (formalin-fixed paraffin-embedded) sections prepared from the colons of ulcerative colitis (UC) and immune-mediated colitis (IRAEC) patients, expression of DCLK1 isoforms correlated positively with Notch1 and negatively with a transcriptional repressor, FoxD3 (Forkhead Box D3). DCLK1 protein staining in these sections was predominantly sub-epithelial (stromal) wherein DCLK1 co-localized with NICD, CD68, CD11c, and neutrophil elastase (NE). NE also co-stained with Citrullinated-H3 indicating the presence of neutrophil extracellular traps. In human neutrophils, elevated levels of DCLK1-S, CXCL-10, Ly6G, MPO, NE, and Notch1/2 in LPS-treated cells were inhibited when LPS was added in conjunction with Notch blocker dibenzazepine (DBZ; LPS + DBZ group). In CR-infected Rag1-/- mice, higher levels of DCLK1 in the colonic crypts were inhibited when mice received DBZ for 10 days coincident with significant dysbiosis, barrier disruption, and colitis. Concurrently, DCLK1 immunoreactivity shifted toward the stroma in CR + DBZ mice with predominance of DCLK1-S that coincided with higher Notch1 levels. Upon antibiotic treatment, partial restoration of crypt DCLK1, reduction in MPO activity, and increased survival followed. When intestinal epithelial cell-specific Dclk1-knockout (Dclk1ΔIEC) or Dclk1ΔIEC;Rag1-/- double knockout (DKO) mice were infected with CR and given a single dose of DBZ, they developed barrier defect and severe colitis with higher levels of stromal DCLK1-S, Ly6G, NE, and Notch1. We therefore propose that, by regulating the mucosal immune responses, the Notch-DCLK1 axis may be integral to the development of murine or human colitis.

Fosciclopirox suppresses growth of high-grade urothelial cancer by targeting the γ-secretase complex.

Ciclopirox (CPX) is an FDA-approved topical antifungal agent that has demonstrated preclinical anticancer activity in a number of solid and hematologic malignancies. Its clinical utility as an oral anticancer agent, however, is limited by poor oral bioavailability and gastrointestinal toxicity. Fosciclopirox, the phosphoryloxymethyl ester of CPX (Ciclopirox Prodrug, CPX-POM), selectively delivers the active metabolite, CPX, to the entire urinary tract following parenteral administration. We characterized the activity of CPX-POM and its major metabolites in in vitro and in vivo preclinical models of high-grade urothelial cancer. CPX inhibited cell proliferation, clonogenicity and spheroid formation, and increased cell cycle arrest at S and G0/G1 phases. Mechanistically, CPX suppressed activation of Notch signaling. Molecular modeling and cellular thermal shift assays demonstrated CPX binding to γ-secretase complex proteins Presenilin 1 and Nicastrin, which are essential for Notch activation. To establish in vivo preclinical proof of principle, we tested fosciclopirox in the validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) mouse bladder cancer model. Once-daily intraperitoneal administration of CPX-POM for four weeks at doses of 235 mg/kg and 470 mg/kg significantly decreased bladder weight, a surrogate for tumor volume, and resulted in a migration to lower stage tumors in CPX-POM treated animals. This was coupled with a reduction in the proliferation index. Additionally, there was a reduction in Presenilin 1 and Hes-1 expression in the bladder tissues of CPX-POM treated animals. Following the completion of the first-in-human Phase 1 trial (NCT03348514), the pharmacologic activity of fosciclopirox is currently being characterized in a Phase 1 expansion cohort study of muscle-invasive bladder cancer patients scheduled for cystectomy (NCT04608045) as well as a Phase 2 trial of newly diagnosed and recurrent urothelial cancer patients scheduled for transurethral resection of bladder tumors (NCT04525131).