Inhibition of BRCT(BRCA1)-phosphoprotein interaction enhances the cytotoxic effect of olaparib in breast cancer cells: a proof of concept study for synthetic lethal therapeutic option.

Synthetic lethal therapeutic strategy using poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitor olaparib in carriers of BRCA1 or BRCA2 mutation has shown promise in clinical settings. Since <5 % of patients are BRCA1 or BRCA2 mutation carriers, small molecules that functionally mimic BRCA1 or BRCA2 mutations will extend the synthetic lethal therapeutic option for non-mutation carriers. Here we provide proof of principle for this strategy using a BRCA1 inhibitor peptide 2 that targets the BRCT(BRCA1)-phosphoprotein interaction and mimics the M177R/K BRCA1 mutation. Reciprocal immunoprecipitation and immunoblotting of BRCA1 and Abraxas was used to demonstrate inhibitor 2 targets BRCT(BRCA1)-Abraxas interface. Immunostaining of γH2AX, cell cycle analysis and homologous recombination (HR) assays were conducted to confirm that inhibitor 2 functionally mimics a chemosensitizing BRCA1 mutation. The concept of synthetic lethal therapeutic strategy with the BRCA1 inhibitor 2 and the PARP inhibitor Olaparib was explored in HeLa, MDA-MB-231, and HCC1937 cell lines. The results show that inhibition of BRCA1 by 2 sensitizes HeLa and MDA-MB-231 cells but not HCC1937 to Olaparib mediated growth inhibition and apoptosis. These results provide the basis for developing high affinity BRCT(BRCA1) inhibitors as adjuvants to treat sporadic breast and ovarian cancers.

Nano pom-poms prepared exosomes enable highly specific cancer biomarker detection.

Extracellular vesicles (EVs), particularly nano-sized small EV exosomes, are emerging biomarker sources. However, due to heterogeneous populations secreted from diverse cell types, mapping exosome multi-omic molecular information specifically to their pathogenesis origin for cancer biomarker identification is still extraordinarily challenging. Herein, we introduced a novel 3D-structured nanographene immunomagnetic particles (NanoPoms) with unique flower pom-poms morphology and photo-click chemistry for specific marker-defined capture and release of intact exosome. This specific exosome isolation approach leads to the expanded identification of targetable cancer biomarkers with enhanced specificity and sensitivity, as demonstrated by multi-omic exosome analysis of bladder cancer patient tissue fluids using the next generation sequencing of somatic DNA mutations, miRNAs, and the global proteome (Data are available via ProteomeXchange with identifier PXD034454). The NanoPoms prepared exosomes also exhibit distinctive in vivo biodistribution patterns, highlighting the highly viable and integral quality. The developed method is simple and straightforward, which is applicable to nearly all types of biological fluids and amenable for enrichment, scale up, and high-throughput exosome isolation.

Evaluation of the Durability of the Immune Humoral Response to COVID-19 Vaccines in Patients With Cancer Undergoing Treatment or Who Received a Stem Cell Transplant.

Importance: The durability of the antibody response to COVID-19 vaccines in patients with cancer undergoing treatment or who received a stem cell transplant is unknown and may be associated with infection outcomes. Objective: To evaluate anti-SARS-CoV-2 spike protein receptor binding domain (anti-RBD) and neutralizing antibody (nAb) responses to COVID-19 vaccines longitudinally over 6 months in patients with cancer undergoing treatment or who received a stem cell transplant (SCT). Design, Setting, and Participants: In this prospective, observational, longitudinal cross-sectional study of 453 patients with cancer undergoing treatment or who received an SCT at the University of Kansas Cancer Center in Kansas City, blood samples were obtained before 433 patients received a messenger RNA (mRNA) vaccine (BNT162b2 or mRNA-1273), after the first dose of the mRNA vaccine, and 1 month, 3 months, and 6 months after the second dose. Blood samples were also obtained 2, 4, and 7 months after 17 patients received the JNJ-78436735 vaccine. For patients receiving a third dose of an mRNA vaccine, blood samples were obtained 30 days after the third dose. Interventions: Blood samples and BNT162b2, mRNA-1273, or JNJ-78436735 vaccines. Main Outcomes and Measures: Geometric mean titers (GMTs) of the anti-RBD; the ratio of GMTs for analysis of demographic, disease, and treatment variables; the percentage of neutralization of anti-RBD antibodies; and the correlation between anti-RBD and nAb responses to the COVID-19 vaccines. Results: This study enrolled 453 patients (mean [SD] age, 60.4 [13,1] years; 253 [56%] were female). Of 450 patients, 273 (61%) received the BNT162b2 vaccine (Pfizer), 160 (36%) received the mRNA-1273 vaccine (Moderna), and 17 (4%) received the JNJ-7846735 vaccine (Johnson & Johnson). The GMTs of the anti-RBD for all patients were 1.70 (95% CI, 1.04-2.85) before vaccination, 18.65 (95% CI, 10.19-34.11) after the first dose, 470.38 (95% CI, 322.07-686.99) at 1 month after the second dose, 425.80 (95% CI, 322.24-562.64) at 3 months after the second dose, 447.23 (95% CI, 258.53-773.66) at 6 months after the second dose, and 9224.85 (95% CI, 2423.92-35107.55) after the third dose. The rate of threshold neutralization (≥30%) was observed in 203 of 252 patients (80%) 1 month after the second dose and in 135 of 166 patients (81%) 3 months after the second dose. Anti-RBD and nAb were highly correlated (Spearman correlation coefficient, 0.93 [0.92-0.94]; P < .001). Three months after the second dose, anti-RBD titers were lower in male vs female patients (ratio of GMTs, 0.52 [95% CI, 0.34-0.81]), patients older than 65 years vs patients 50 years or younger (ratio of GMTs, 0.38 [95% CI, 0.25-0.57]), and patients with hematologic malignant tumors vs solid tumors (ratio of GMTs, 0.40 [95% CI, 0.20-0.81]). Conclusions and Relevance: In this cross-sectional study, after 2 doses of an mRNA vaccine, anti-RBD titers peaked at 1 month and remained stable over the next 6 months. Patients older than 65 years of age, male patients, and patients with a hematologic malignant tumor had low antibody titers. Compared with the primary vaccine course, a 20-fold increase in titers from a third dose suggests a brisk B-cell anamnestic response in patients with cancer.

Microfluidic affinity selection of active SARS-CoV-2 virus particles.

We report a microfluidic assay to select active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles (VPs), which were defined as intact particles with an accessible angiotensin-converting enzyme 2 receptor binding domain (RBD) on the spike (S) protein, from clinical samples. Affinity selection of SARS-CoV-2 particles was carried out using injection molded microfluidic chips, which allow for high-scale production to accommodate large-scale screening. The microfluidic contained a surface-bound aptamer directed against the virus's S protein RBD to affinity select SARS-CoV-2 VPs. Following selection (~94% recovery), the VPs were released from the chip's surface using a blue light light-emitting diode (89% efficiency). Selected SARS-CoV-2 VP enumeration was carried out using reverse transcription quantitative polymerase chain reaction. The VP selection assay successfully identified healthy donors (clinical specificity = 100%) and 19 of 20 patients with coronavirus disease 2019 (COVID-19) (95% sensitivity). In 15 patients with COVID-19, the presence of active SARS-CoV-2 VPs was found. The chip can be reprogrammed for any VP or exosomes by simply changing the affinity agent.

WJMSC-derived small extracellular vesicle enhance T cell suppression through PD-L1.

Both mesenchymal stem cells (MSCs) and their corresponding small extracellular vesicles (sEVs, commonly referred to as exosomes) share similar immunomodulatory properties that are potentially beneficial for the treatment of acute graft versus host disease (aGvHD). We report that clinical grade Wharton's Jelly-derived MSCs (WJMSCs) secrete sEVs enriched in programmed death-ligand 1 (PD-L1), an essential ligand for an inhibitory immune checkpoint. A rapid increase in circulating sEV-associated PD-L1 was observed in patients with aGvHD and was directly associated with the infusion time of clinical grade WJMSCs. In addition, in vitro inhibitory antibody mediated blocking of sEV-associated PD-L1 restored T cell activation (TCA), suggesting a functional inhibitory role of sEVs-PD-L1. PD-L1-deficient sEVs isolated from WJMSCs following CRISPR-Cas9 gene editing fail to inhibit TCA. Furthermore, we found that PD-L1 is essential for WJMSC-derived sEVs to modulate T cell receptors (TCRs). Our study reveals an important mechanism by which therapeutic WJMSCs modulate TCR-mediated TCA through sEVs or sEV-carried immune checkpoints. In addition, our clinical data suggest that sEV-associated PD-L1 may be not only useful in predicting the outcomes from WJMSC clinical administration, but also in developing cell-independent therapy for aGvHD patients.

Acute myeloid leukemia or myelodysplastic syndrome with chromosome 17 abnormalities and long-term outcomes with or without hematopoietic stem cell transplantation.

INTRODUCTION: Chromosome 17 abnormalities, especially disorders of the 17p region and including TP53 gene mutations, result in very low rates of cure for patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) treated with conventional chemotherapy or allogeneic hematopoietic cell transplant (allo-HCT). Our retrospective study analyzed outcomes in patients with chromosome 17 (ch17) abnormalities who received conventional chemotherapy followed by allo-HCT versus those who did not receive a transplant. We analyzed whether poor outcomes extend to patients with all types of ch17 abnormalities and the impact of concomitant TP53 gene mutations assessed by next-generation sequencing (NGS) on prognosis. METHODS: We retrospectively analyzed diagnostic and outcome data on 98 patients treated at our institution from 2012 to 2018 with AML or MDS who possessed ch17 abnormalities by cytogenetic analysis. The presence of TP53 mutations was analyzed by NGS. Primary endpoint of our study was overall survival (OS). RESULTS: 61 patients with AML and 37 with MDS were included. Complete remission (CR) with first line treatment was similar between induction chemotherapy or hypomethylating agents (HMA), 22.9 % versus 21.6 % (p = 0.33). Median OS for all patients (with or without transplant) was 10 months. Patients with abnormal ch17 in conjunction with any TP53 mutation(s) exhibited worse OS compared to patients without a TP53 mutation (10 versus 23 months, p = 0.02). 30 patients (19 AML, 11 MDS) underwent HCT, with a median OS of 11 months. For AML patients who underwent allo-HCT, 18 were in CR (13 with cytogenetic remission) and 1 had persistent disease at transplant. In the MDS cohort, 3 patients were in CR (2 with cytogenetic remission) and 8 had stable disease. Post allo-HCT survival of AML and MDS cohorts did not differ (p = 0.6), although cytogenetic CR at time of HCT trended towards improved OS (17 versus 8 months; p = 0.6). CONCLUSIONS: AML/MDS patients with ch17 abnormalities have poor outcomes with or without HCT. Our results show that patients with ch17 abnormalities and TP53 mutations have a significantly poorer survival compared to patients who have ch17 abnormalities but no TP53 mutations. Drugs targeting abnormalities of the p53 pathway, improvement in depth of response prior to HCT, and novel maintenance strategies are needed for improved outcomes in these patients.

Adherent cell depletion promotes the expansion of renal cell carcinoma infiltrating T cells with optimal characteristics for adoptive transfer.

BACKGROUND: Tumor-infiltrating lymphocyte (TIL) therapy is a personalized cancer treatment which involves generating ex vivo cultures of tumor-reactive T cells from surgically resected tumors and administering the expanded TILs as a therapeutic infusion. Phase 1 of many TIL production protocols use aldesleukin (IL-2) alone to establish TIL cultures (termed "PreREP" (Pre-Rapid Expansion Protocol)); however, this fails to consistently produce TIL cultures from renal cell carcinoma (RCC) in a timely manner. Adding mitogenic stimulation via anti-CD3/anti-CD28 beads along with IL-2 to the fresh tumor digest (FTD) during TIL generation (termed "FTD+ beads") increases successful TIL culture rates; however, T cells produced by this method may be suboptimal for adoptive transfer. We hypothesize that adherent cell depletion (ACD) before TIL expansion will produce a superior TIL product by removing the immunosuppressive signals originating from adherent tumor and stromal cells. Here we investigate if "panning," a technique for ACD prior to TIL expansion, will impact the phenotype, functionality and/or clonality of ex vivo expanded RCC TILs. METHODS: Tumor specimens from 55 patients who underwent radical or partial nephrectomy at the University of Kansas Medical Center (KUMC) were used to develop the panning method and an additional 19 specimens were used to validate the protocol. Next-generation sequencing, immunohistochemistry/immunocytochemistry and flow cytometry were used during method development. The phenotype, functionality and clonality of autologous TILs generated in parallel by panning, PreREP, and FTD+ beads were assessed by flow cytometry, in vitro co-culture assays, and TCRB CDR3 sequencing. RESULTS: TIL cultures were successfully generated using the panning protocol from 15/16 clear cell, 0/1 chromophobe, and 0/2 papillary RCC samples. Significantly fewer regulatory (CD4+/CD25+/FOXP3+) (p=0.049, p=0.005), tissue-resident memory (CD8+/CD103+) (p=0.027, p=0.009), PD-1+/TIM-3+ double-positive (p=0.009, p=0.011) and TIGIT+ T cells (p=0.049, p=0.026) are generated by panning relative to PreREP and FTD+ beads respectively. Critically, a subset of TILs generated by panning were able to degranulate and/or produce interferon gamma in response to autologous tumor cells and the average tumor-reactive TIL yield was greatest when using the panning protocol. CONCLUSIONS: Removing immunosuppressive adherent cells within an RCC digest prior to TIL expansion allow for the rapid production of tumor-reactive T cells with optimal characteristics for adoptive transfer.

Design, Optimization, and Multisite Evaluation of a Targeted Next-Generation Sequencing Assay System for Chimeric RNAs from Gene Fusions and Exon-Skipping Events in Non-Small Cell Lung Cancer.

Lung cancer accounts for approximately 14% of all newly diagnosed cancers and is the leading cause of cancer-related deaths. Chimeric RNA resulting from gene fusions (RNA fusions) and other RNA splicing errors are driver events and clinically addressable targets for non-small cell lung cancer (NSCLC). The reliable assessment of these RNA markers by next-generation sequencing requires integrated reagents, protocols, and interpretive software that can harmonize procedures and ensure consistent results across laboratories. We describe the development and verification of a system for targeted RNA sequencing for the analysis of challenging, low-input solid tumor biopsies that includes reagents for nucleic acid quantification and library preparation, run controls, and companion bioinformatics software. Assay development reconciled sequence discrepancies in public databases, created predictive formalin-fixed, paraffin-embedded RNA qualification metrics, and eliminated read misidentification attributable to index hopping events on the next-generation sequencing flow cell. The optimized and standardized system was analytically verified internally and in a multiphase study conducted at five independent laboratories. The results show accurate, reproducible, and sensitive detection of RNA fusions, alternative splicing events, and other expression markers of NSCLC. This comprehensive approach, combining sample quantification, quality control, library preparation, and interpretive bioinformatics software, may accelerate the routine implementation of targeted RNA sequencing of formalin-fixed, paraffin-embedded samples relevant to NSCLC.

Developing a genetic signature to predict drug response in ovarian cancer.

There is a lack of personalized treatment options for women with recurrent platinum-resistant ovarian cancer. Outside of bevacizumab and a group of poly ADP-ribose polymerase inhibitors, few options are available to women that relapse. We propose that efficacious drug combinations can be determined via molecular characterization of ovarian tumors along with pre-established pharmacogenomic profiles of repurposed compounds. To that end, we selectively performed multiple two-drug combination treatments in ovarian cancer cell lines that included reactive oxygen species inducers and HSP90 inhibitors. This allowed us to select cell lines that exhibit disparate phenotypes of proliferative inhibition to a specific drug combination of auranofin and AUY922. We profiled altered mechanistic responses from these agents in both reactive oxygen species and HSP90 pathways, as well as investigated PRKCI and lncRNA expression in ovarian cancer cell line models. Generation of dual multi-gene panels implicated in resistance or sensitivity to this drug combination was produced using RNA sequencing data and the validity of the resistant signature was examined using high-density RT-qPCR. Finally, data mining for the prevalence of these signatures in a large-scale clinical study alluded to the prevalence of resistant genes in ovarian tumor biology. Our results demonstrate that high-throughput viability screens paired with reliable in silico data can promote the discovery of effective, personalized therapeutic options for a currently untreatable disease.

Licofelone Enhances the Efficacy of Paclitaxel in Ovarian Cancer by Reversing Drug Resistance and Tumor Stem-like Properties.

Drug development for first-line treatment of epithelial ovarian cancer (EOC) has been stagnant for almost three decades. Traditional cell culture methods for primary drug screening do not always accurately reflect clinical disease. To overcome this barrier, we grew a panel of EOC cell lines in three-dimensional (3D) cell cultures to form multicellular tumor spheroids (MCTS). We characterized these MCTS for molecular and cellular features of EOC and performed a comparative screen with cells grown using two-dimensional (2D) cell culture to identify previously unappreciated anticancer drugs. MCTS exhibited greater resistance to chemotherapeutic agents, showed signs of senescence and hypoxia, and expressed a number of stem cell-associated transcripts including ALDH1A and CD133, also known as PROM1 Using a library of clinically repurposed drugs, we identified candidates with preferential activity in MCTS over 2D cultured cells. One of the lead compounds, the dual COX/LOX inhibitor licofelone, reversed the stem-like properties of ovarian MCTS. Licofelone also synergized with paclitaxel in ovarian MCTS models and in a patient-derived tumor xenograft model. Importantly, the combination of licofelone with paclitaxel prolonged the median survival of mice (>141 days) relative to paclitaxel (115 days), licofelone (37 days), or vehicle (30 days). Increased efficacy was confirmed by Mantel-Haenszel HR compared with vehicle (HR = 0.037) and paclitaxel (HR = 0.017). These results identify for the first time an unappreciated, anti-inflammatory drug that can reverse chemotherapeutic resistance in ovarian cancer, highlighting the need to clinically evaluate licofelone in combination with first-line chemotherapy in primary and chemotherapy-refractory EOC.Significance: This study highlights the use of an in vitro spheroid 3D drug screening model to identify new therapeutic approaches to reverse chemotherapy resistance in ovarian cancer. Cancer Res; 78(15); 4370-85. ©2018 AACR.

Role of miR-139 as a surrogate marker for tumor aggression in breast cancer.

MicroRNAs are non-protein coding molecules that play a key role in oncogenesis, tumor progression, and metastasis in many types of malignancies including breast cancer. In the current study, we studied the expression of microRNA-139-5p (miR-139) in invasive ductal carcinoma (IDC) of the breast and correlated its expression with tumor grade, molecular subtype, hormonal status, human epidermal growth factor receptor 2 status, proliferation index, tumor size, lymph node status, patient's age, and overall survival in 74 IDC cases. In addition, we compared and correlated miR-139 expression in 18 paired serum and tissue samples from patients with IDC to assess its value as a serum marker. Our data showed that miR-139 was down-regulated in all tumor tissue samples compared with control. More pronounced down-regulation was seen in tumors that were higher grade, estrogen receptor negative, progesterone receptor negative, more proliferative, or larger in size (P < .05). Although not statistically significant, lower miR-139 level was frequently associated with human epidermal growth factor receptor 2 overexpression. In addition, significantly lower miR-139 tissue level was seen in patients who were deceased (P = .027), although older age (>50 years) and positive local nodal disease did not adversely affect miR-139 expression. In contrast, serum miR-139 profile of the patients appeared similar to that of normal control. In conclusion, our study demonstrated that down-regulation of miR-139 was associated with aggressive tumor behavior and disease progression in breast cancer. miR-139 may serve as a risk assessment biomarker in tailoring treatment options.

High Dose Parenteral Ascorbate Inhibited Pancreatic Cancer Growth and Metastasis: Mechanisms and a Phase I/IIa study.

Pancreatic cancer is among the most lethal cancers with poorly tolerated treatments. There is increasing interest in using high-dose intravenous ascorbate (IVC) in treating this disease partially because of its low toxicity. IVC bypasses bioavailability barriers of oral ingestion, provides pharmacological concentrations in tissues, and exhibits selective cytotoxic effects in cancer cells through peroxide formation. Here, we further revealed its anti-pancreatic cancer mechanisms and conducted a phase I/IIa study to investigate pharmacokinetic interaction between IVC and gemcitabine. Pharmacological ascorbate induced cell death in pancreatic cancer cells with diverse mutational backgrounds. Pharmacological ascorbate depleted cellular NAD+ preferentially in cancer cells versus normal cells, leading to depletion of ATP and robustly increased α-tubulin acetylation in cancer cells. While ATP depletion led to cell death, over-acetylated tubulin led to inhibition of motility and mitosis. Collagen was increased, and cancer cell epithelial-mesenchymal transition (EMT) was inhibited, accompanied with inhibition in metastasis. IVC was safe in patients and showed the possibility to prolong patient survival. There was no interference to gemcitabine pharmacokinetics by IVC administration. Taken together, these data revealed a multi-targeting mechanism of pharmacological ascorbate's anti-cancer action, with minimal toxicity, and provided guidance to design larger definitive trials testing efficacy of IVC in treating advanced pancreatic cancer.

In silico and in vitro drug screening identifies new therapeutic approaches for Ewing sarcoma.

The long-term overall survival of Ewing sarcoma (EWS) patients remains poor; less than 30% of patients with metastatic or recurrent disease survive despite aggressive combinations of chemotherapy, radiation and surgery. To identify new therapeutic options, we employed a multi-pronged approach using in silico predictions of drug activity via an integrated bioinformatics approach in parallel with an in vitro screen of FDA-approved drugs. Twenty-seven drugs and forty-six drugs were identified, respectively, to have anti-proliferative effects for EWS, including several classes of drugs in both screening approaches. Among these drugs, 30 were extensively validated as mono-therapeutic agents and 9 in 14 various combinations in vitro. Two drugs, auranofin, a thioredoxin reductase inhibitor, and ganetespib, an HSP90 inhibitor, were predicted to have anti-cancer activities in silico and were confirmed active across a panel of genetically diverse EWS cells. When given in combination, the survival rate in vivo was superior compared to auranofin or ganetespib alone. Importantly, extensive formulations, dose tolerance, and pharmacokinetics studies demonstrated that auranofin requires alternative delivery routes to achieve therapeutically effective levels of the gold compound. These combined screening approaches provide a rapid means to identify new treatment options for patients with a rare and often-fatal disease.

Beyond standard therapy: drugs under investigation for the treatment of gastrointestinal stromal tumor.

INTRODUCTION: Gastrointestinal stromal tumor (GIST) is the most common nonepithelial malignancy of the GI tract. With the discovery of KIT and later platelet-derived growth factor α (PDGFRA) gain-of-function mutations as factors in the pathogenesis of the disease, GIST was the quintessential model for targeted therapy. Despite the successful clinical use of imatinib mesylate, a selective receptor tyrosine kinase (RTK) inhibitor that targets KIT, PDGFRA and BCR-ABL, we still do not have treatment for the long-term control of advanced GIST. AREAS COVERED: This review summarizes the drugs that are under investigation or have been assessed in trials for GIST treatment. The article focuses on their mechanisms of actions, the preclinical evidence of efficacy, and the clinical trials concerning safety and efficacy in humans. EXPERT OPINION: It is known that KIT and PDGFRA mutations in GIST patients influence the response to treatment. This observation should be taken into consideration when investigating new drugs. RECIST was developed to help uniformly report efficacy trials in oncology. Despite the usefulness of this system, many questions are being addressed about its validity in evaluating the true efficacy of drugs knowing that new targeted therapies do not affect the tumor size as much as they halt progression and prolong survival.

Casitas B-Lineage Lymphoma RING Domain Inhibitors Protect Mice against High-Fat Diet-Induced Obesity and Insulin Resistance.

The casitas b-lineage lymphoma (c-Cbl) is an important adaptor protein with an intrinsic E3 ubiquitin ligase activity that interacts with E2 proteins such as UbCH7. c-Cbl plays a vital role in regulating receptor tyrosine kinase signaling. c-Cbl involves in whole-body energy homeostasis, which makes it a potential target for the treatment of type 2 diabetes and obesity. In the present study, we have designed two parental peptides and 55 modified peptides based on the structure of UbCH7 loop L1 and L2. Thirteen of the modified peptides showed increased inhibitory activity in a fluorescence polarization-based assay. In the in vivo proof of study principle, mice treated with peptides 10, 34, 49 and 51 were protected against high-fat diet-induced obesity and insulin resistant. These inhibitors may potentially lead to new therapeutic alternatives for obesity and type 2 diabetes.

Drug repurposing identifies a synergistic combination therapy with imatinib mesylate for gastrointestinal stromal tumor.

Gastrointestinal stromal tumor (GIST) is a rare and therefore often neglected disease. Introduction of the kinase inhibitor imatinib mesylate radically improved the clinical response of patients with GIST; however, its effects are often short-lived, with GISTs demonstrating a median time-to-progression of approximately two years. Although many investigational drugs, approved first for other cancers, have been subsequently evaluated for the management of GIST, few have greatly affected the overall survival of patients with advanced disease. We employed a novel, focused, drug-repurposing effort for GIST, including imatinib mesylate-resistant GIST, evaluating a large library of FDA-approved drugs regardless of current indication. As a result of the drug-repurposing screen, we identified eight FDA-approved drugs, including fludarabine phosphate (F-AMP), that showed synergy with and/or overcame resistance to imatinib mesylate. F-AMP induces DNA damage, Annexin V, and caspase-3/7 activities as the cytotoxic effects on GIST cells, including imatinib mesylate-resistant GIST cells. F-AMP and imatinib mesylate combination treatment showed greater inhibition of GIST cell proliferation when compared with imatinib mesylate and F-AMP alone. Successful in vivo experiments confirmed the combination of imatinib mesylate with F-AMP enhanced the antitumor effects compared with imatinib mesylate alone. Our results identified F-AMP as a promising, repurposed drug therapy for the treatment of GISTs, with potential to be administered in combination with imatinib mesylate or for treatment of imatinib mesylate-refractory tumors.

Epimorphin-induced MET sensitizes ovarian cancer cells to platinum.

Distinctive genotypic and phenotypic features of ovarian cancer via epithelial-mesenchymal transition (EMT) have been correlated with drug resistance and disease recurrence. We investigated whether therapeutic reversal of EMT could re-sensitize ovarian cancer cells (OCCs) to existing chemotherapy. We report that epimorphin, a morphogenic protein, has pivotal control over mesenchymal versus epithelial cell lineage decision of the putative OCCs. Exposure to epimorphin induced morphological changes reminiscent of mesenchymal-to-epithelial transition (MET), but in a dose dependent manner, i.e., at 10 µg/mL of epimorphin cells obtain a more mesenchymal-like morphology while at 20 µg/mL of epimorphin cells display an epithelial morphology. The latter changes were accompanied by suppression of mesenchymal markers, such as vimentin (∼8-fold↓, p<0.02), Twist1 (∼7-fold↓, p<0.03), dystroglycan (∼4-fold↓, p<0.01) and palladin (∼3-fold↓, p<0.01). Conversely, significant elevations of KLF4 (∼28-fold↑, p<0.002), ß-catenin (∼6-fold↑, p<0.004), EpCAM (∼6-fold↑, p<0.0002) and occludin (∼15-fold↑, p<0.004) mRNAs as part of the commitment to the epithelial cell lineage were detected in response to 20 µg/mL of exogenous epimorphin. Changes in occludin mRNA levels were accompanied by a parallel, albeit weaker expression at the protein level (∼5-fold↑, p<0.001). Likewise, acquisition of epithelial-like properties, including mucin1, CK19, and ß-catenin gene expression, was also obtained following epimorphin treatment. Further, MMP3 production was found to be reduced whereas laminin secretion was strongly amplified upon epimorphin-induced MET. These results suggest there is a dosage window for actions of epimorphin on cellular differentiation, wherein it can either suppress or enhance epithelial differentiation of OCCs. Importantly, induction of epithelial-like phenotypes by epimorphin led to an enhanced sensitivity to carboplatin. Overall, we demonstrate that epimorphin can revert OCCs away from their mesenchymal phenotype and toward an epithelial phenotype, thereby enhancing their sensitivity to a front-line chemotherapeutic agent.

Drug repurposing for gastrointestinal stromal tumor.

Despite significant treatment advances over the past decade, metastatic gastrointestinal stromal tumor (GIST) remains largely incurable. Rare diseases, such as GIST, individually affect small groups of patients but collectively are estimated to affect 25 to 30 million people in the United States alone. Given the costs associated with the discovery, development, and registration of new drugs, orphan diseases such as GIST are often not pursued by mainstream pharmaceutical companies. As a result, "drug repurposing" or "repositioning," has emerged as an alternative to the traditional drug development process. In this study, we screened 796 U.S. Food and Drug Administration (FDA)-approved drugs and found that two of these compounds, auranofin (Ridaura) and fludarabine phosphate, effectively and selectively inhibited the proliferation of GISTs, including imatinib-resistant cells. One of the most notable drug hits, auranofin, an oral, gold-containing agent approved by the FDA in 1985 for the treatment of rheumatoid arthritis, was found to inhibit thioredoxin reductase activity and induce reactive oxygen species (ROS) production, leading to dramatic inhibition of GIST cell growth and viability. Importantly, the anticancer activity associated with auranofin was independent of imatinib-resistant status, but was closely related to the endogenous and inducible levels of ROS. Coupled with the fact that auranofin has an established safety profile in patients, these findings suggest for the first time that auranofin may have clinical benefit for patients with GIST, particularly in those suffering from imatinib-resistant and recurrent forms of this disease.