Targeting TRIP13 in favorable histology Wilms tumor with nuclear export inhibitors synergizes with doxorubicin.

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies remains challenging in part due to the lack of faithful preclinical in vitro models. Here we establish twelve patient-derived WT cell lines and demonstrate that these models faithfully recapitulate WT biology using genomic and transcriptomic techniques. We then perform loss-of-function screens to identify the nuclear export gene, XPO1, as a vulnerability. We find that the FDA approved XPO1 inhibitor, KPT-330, suppresses TRIP13 expression, which is required for survival. We further identify synergy between KPT-330 and doxorubicin, a chemotherapy used in high-risk FHWT. Taken together, we identify XPO1 inhibition with KPT-330 as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.

The yes-associated protein (YAP) is associated with resistance to anti-GD2 immunotherapy in neuroblastoma through downregulation of ST8SIA1.

Pediatric patients with high-risk neuroblastoma often relapse with chemotherapy-resistant, incurable disease. Relapsed neuroblastomas harbor chemo-resistant mesenchymal tumor cells and increased expression/activity of the transcriptional co-regulator, the Yes-Associated Protein (YAP). Patients with relapsed neuroblastoma are often treated with immunotherapy such as the anti-GD2 antibody, dinutuximab, in combination with chemotherapy. We have previously shown that YAP mediates both chemotherapy and MEK inhibitor resistance in relapsed RAS mutated neuroblastoma and so posited that YAP might also be involved in anti-GD2 antibody resistance. We now show that YAP genetic inhibition significantly enhances sensitivity of mesenchymal neuroblastomas to dinutuximab and gamma delta (γδ) T cells both in vitro and in vivo. Mechanistically, YAP inhibition induces increased GD2 cell surface expression through upregulation of ST8SIA1, the gene encoding GD3 synthase and the rate-limiting enzyme in GD2 biosynthesis. The mechanism of ST8SIA1 suppression by YAP is independent of PRRX1 expression, a mesenchymal master transcription factor, suggesting YAP may be the downstream effector of mesenchymal GD2 resistance. These results therefore identify YAP as a therapeutic target to augment GD2 immunotherapy responses in patients with neuroblastoma.

Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma.

GD2-targeting immunotherapies have improved survival in children with neuroblastoma, yet on-target, off-tumor toxicities can occur and a subset of patients cease to respond. The majority of neuroblastoma patients who receive immunotherapy have been previously treated with cytotoxic chemotherapy, making it paramount to identify neuroblastoma-specific antigens that remain stable throughout standard treatment. Cell surface glycoproteomics performed on human-derived neuroblastoma tumors in mice following chemotherapy treatment identified protein tyrosine kinase 7 (PTK7) to be abundantly expressed. Furthermore, PTK7 shows minimal expression on pediatric-specific normal tissues. We developed an anti-PTK7 chimeric antigen receptor (CAR) and find PTK7 CAR T cells specifically target and kill PTK7-expressing neuroblastoma in vitro. In vivo, human/murine binding PTK7 CAR T cells regress aggressive neuroblastoma metastatic mouse models and prolong survival with no toxicity. Together, these data demonstrate preclinical efficacy and tolerability for targeting PTK7 and support ongoing investigations to optimize PTK7-targeting CAR T cells for neuroblastoma.

A phase 1 study of simvastatin in combination with topotecan and cyclophosphamide in pediatric patients with relapsed and/or refractory solid and CNS tumors.

BACKGROUND: 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) can inhibit tumor proliferation, angiogenesis, and restore apoptosis in preclinical pediatric solid tumor models. We conducted a phase 1 trial to determine the maximum tolerated dose (MTD) of simvastatin with topotecan and cyclophosphamide in children with relapsed/refractory solid and central nervous system (CNS) tumors. METHODS: Simvastatin was administered orally twice daily on days 1-21, with topotecan and cyclophosphamide intravenously on days 1-5 of a 21-day cycle. Four simvastatin dose levels (DLs) were planned, 140 (DL1), 180 (DL2), 225 (DL3), 290 (DL4) mg/m2 /dose, with a de-escalation DL of 100 mg/m2 /dose (DL0) if needed. Pharmacokinetic and pharmacodynamic analyses were performed during cycle 1. RESULTS: The median age of 14 eligible patients was 11.5 years (range: 1-23). The most common diagnoses were neuroblastoma (N = 4) and Ewing sarcoma (N = 3). Eleven dose-limiting toxicity (DLT)-evaluable patients received a median of four cycles (range: 1-6). There were three cycle 1 DLTs: one each grade 3 diarrhea and grade 4 creatine phosphokinase (CPK) elevations at DL1, and one grade 4 CPK elevation at DL0. All patients experienced at least one grade 3/4 hematologic toxicity. Best overall response was partial response in one patient with Ewing sarcoma (DL0) and stable disease for four or more cycles in four patients. Simvastatin exposure increased with higher doses and may have correlated with toxicity. Plasma interleukin 6 (IL-6) concentrations (N = 6) showed sustained IL-6 reductions with decrease to normal values by day 21 in all patients, indicating potential on-target effects. CONCLUSIONS: The MTD of simvastatin with topotecan and cyclophosphamide was determined to be 100 mg/m2 /dose.

Dissecting the cellular components of ex vivo γδ T cell expansions to optimize selection of potent cell therapy donors for neuroblastoma immunotherapy trials.

γδ T lymphocytes represent an emerging class of cellular immunotherapy with preclinical promise to treat cancer, notably neuroblastoma. The innate-like immune cell subset demonstrates inherent cytoxicity toward tumor cells independent of MHC recognition, enabling allogeneic administration of healthy donor-derived γδ T cell therapies. A current limitation is the substantial interindividual γδ T cell expansion variation among leukocyte collections. Overcoming this limitation will enable realization of the full potential of allogeneic γδ T-based cellular therapy. Here, we characterize γδ T cell expansions from healthy adult donors and observe that highly potent natural killer (NK) lymphocytes expand with γδ T cells under zoledronate and IL-2 stimulation. The presence of NK cells correlates with both the expansion potential of γδ T cells and the overall potency of the γδ T cell therapy. However, the potency of the cell therapy in combination with an antibody-based immunotherapeutic, dinutuximab, appears to be independent of γδ T/NK cell content both in vitro and in vivo, which minimizes the implication of interindividual expansion differences toward efficacy. Collectively, these studies highlight the utility of maintaining the NK cell population within expanded γδ T cell therapies and suggest a synergistic action of combined innate cell immunotherapy toward neuroblastoma.

High-Throughput Screening Identifies Idasanutlin as a Resensitizing Drug for Venetoclax-Resistant Neuroblastoma Cells.

Neuroblastoma tumors frequently overexpress the anti-apoptotic protein B-cell lymphoma/leukemia 2 (BCL-2). We previously showed that treating BCL-2-dependent neuroblastoma cells with the BCL-2 inhibitor venetoclax results in apoptosis, but unfortunately partial therapy resistance is observed. The current study describes the identification of drugs capable of resensitizing venetoclax-resistant neuroblastoma cells to venetoclax. To examine these effects, venetoclax resistance was induced in BCL-2-dependent neuroblastoma cell lines KCNR and SJNB12 by continuous exposure to high venetoclax concentrations. Non-resistant and venetoclax-resistant neuroblastoma cell lines were exposed to a 209-compound library in the absence and presence of venetoclax to identify compounds that were more effective in the venetoclax-resistant cell lines under venetoclax pressure. Top hits were further validated in combination with venetoclax using BCL-2-dependent neuroblastoma model systems. Overall, high-throughput drug screening identified the MDM2 inhibitor idasanutlin as a promising resensitizing agent for venetoclax-resistant neuroblastoma cell lines. Idasanutlin treatment induced BAX-mediated apoptosis in venetoclax-resistant neuroblastoma cells in the presence of venetoclax, whereas it caused p21-mediated growth arrest in control cells. In vivo combination treatment showed tumor regression and superior efficacy over single-agent therapies in a BCL-2-dependent neuroblastoma cell line xenograft and a patient-derived xenograft. However, xenografts less dependent on BCL-2 were not sensitive to venetoclax-idasanutlin combination therapy. This study demonstrates that idasanutlin can overcome resistance to the BCL-2 inhibitor venetoclax in preclinical neuroblastoma model systems, which supports clinical development of a treatment strategy combining the two therapies.

YAP-Mediated Repression of HRK Regulates Tumor Growth, Therapy Response, and Survival Under Tumor Environmental Stress in Neuroblastoma.

Following chemotherapy and relapse, high-risk neuroblastoma tumors harbor more genomic alterations than at diagnosis, including increased transcriptional activity of the Yes-associated protein (YAP), a key downstream component of the Hippo signaling network. Although YAP has been implicated in many cancer types, its functional role in the aggressive pediatric cancer neuroblastoma is not well-characterized. In this study, we performed genetic manipulation of YAP in human-derived neuroblastoma cell lines to investigate YAP function in key aspects of the malignant phenotype, including mesenchymal properties, tumor growth, chemotherapy response, and MEK inhibitor response. Standard cytotoxic therapy induced YAP expression and transcriptional activity in patient-derived xenografts treated in vivo, which may contribute to neuroblastoma recurrence. Moreover, YAP promoted a mesenchymal phenotype in high-risk neuroblastoma that modulated tumor growth and therapy resistance in vivo. Finally, the BH3-only protein, Harakiri (HRK), was identified as a novel target inhibited by YAP, which, when suppressed, prevented apoptosis in response to nutrient deprivation in vitro and promoted tumor aggression, chemotherapy resistance, and MEK inhibitor resistance in vivo. Collectively, these findings suggest that YAP inhibition may improve chemotherapy response in patients with neuroblastoma via its regulation of HRK, thus providing a critical strategic complement to MEK inhibitor therapy. SIGNIFICANCE: This study identifies HRK as a novel tumor suppressor in neuroblastoma and suggests dual MEK and YAP inhibition as a potential therapeutic strategy in RAS-hyperactivated neuroblastomas.

Thiamine mimetics sulbutiamine and benfotiamine as a nutraceutical approach to anticancer therapy.

Malignant cells frequently demonstrate an oncogenic-driven reliance on glycolytic metabolism to support their highly proliferative nature. Overexpression of pyruvate dehydrogenase kinase (PDK) may promote this unique metabolic signature of tumor cells by inhibiting mitochondrial function. PDKs function to phosphorylate and inhibit pyruvate dehydrogenase (PDH) activity. Silencing of PDK expression has previously been shown to restore mitochondrial function and reduce tumor cell proliferation. High dose Vitamin B1, or thiamine, possesses antitumor properties related to its capacity to reduce PDH phosphorylation and promote its enzymatic activity, presumably through PDK inhibition. Though a promising nutraceutical approach for cancer therapy, thiamine's low bioavailability may limit clinical effectiveness. Here, we have demonstrated exploiting the commercially available lipophilic thiamine analogs sulbutiamine and benfotiamine increases thiamine's anti-cancer effect in vitro. Determined by crystal violet proliferation assays, both sulbutiamine and benfotiamine reduced thiamine's millimolar IC50 value to micromolar equivalents. HPLC analysis revealed that sulbutiamine and benfotiamine significantly increased intracellular thiamine and TPP concentrations in vitro, corresponding with reduced levels of PDH phosphorylation. Through an ex vitro kinase screen, thiamine's activated cofactor form thiamine pyrophosphate (TPP) was found to inhibit the function of multiple PDK isoforms. Attempts to maximize intracellular TPP by exploiting thiamine homeostasis gene expression resulted in enhanced apoptosis in tumor cells. Based on our in vitro evaluations, we conclude that TPP serves as the active species mediating thiamine's inhibitory effect on tumor cell proliferation. Pharmacologic administration of benfotiamine, but not sulbutiamine, reduced tumor growth in a subcutaneous xenograft mouse model. It remains unclear if benfotiamine's effects in vivo are associated with PDK inhibition or through an alternative mechanism of action. Future work will aim to define the action of lipophilic thiamine mimetics in vivo in order to translate their clinical usefulness as anticancer strategies.

Development of an IPRP-LC-MS/MS method to determine the fate of intracellular thiamine in cancer cells.

Understanding the mechanisms underlying cancer cell survival is critical toward advancing drug discovery efforts in this field. Supplemental vitamins have been proposed to play a role in cancer cell metabolism because the increased supply of nutrients is thought to provide cofactors supporting the higher metabolic rate of cancer cells. Particularly, the role of thiamine (vitamin B1) in many biochemical pathways that supports cancer cell metabolism has been investigated. Consequently, the analysis of thiamine and its derivatives in a manner that reflects its dynamic response to genetic modification and pathophysiological stimuli is essential. In this work, we developed a mass spectrometry based-analytical method to track metabolites derived from stable isotope tracers for a better understanding of the metabolic fate of thiamine in cancer cells. This method used ion-pair reversed phase liquid chromatography to simultaneously quantify underivatized thiamine, thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP) in cells. Hexylamine was used as an ion-pairing agent. The method was successfully validated for accuracy, precision and selectivity in accordance with U.S. FDA guidance. Furthermore, the method was then applied for the determination of thiamine and its derivatives with stable isotope labeling to explore the metabolic fate of intracellular thiamine in cancer cells. The finding shows that thiamine is rapidly converted to TPP however, the TPP does not return to thiamine. It appears that TPP may be utilized for other purposes rather than simply being an enzyme cofactor, suggesting unexplored roles for thiamine in cancer.

The adaptive regulation of thiamine pyrophosphokinase-1 facilitates malignant growth during supplemental thiamine conditions.

Supplemental levels of vitamin B1 (thiamine) have been implicated in tumor progression. Tumor cells adaptively up-regulate thiamine transport during hypoxic stress. Upon uptake, thiamine pyrophosphokinase-1 (TPK1) facilitates the rapid phosphorylation of thiamine into thiamine pyrophosphate (TPP). However, the regulation of TPK1 during hypoxic stress is undefined. Understanding how thiamine homeostasis changes during hypoxia will provide critical insight into the malignant advantage supplemental thiamine may provide cancer cells. Using Western blot analysis and RT-PCR, we have demonstrated the post-transcriptional up-regulation of TPK1 in cancer cells following hypoxic exposure. TPK1 expression was also adaptively up-regulated following alterations of redox status by chemotherapeutic and antioxidant treatments. Although TPK1 was functionally up-regulated by hypoxia, HPLC analysis revealed a reduction in intracellular TPP levels. This loss was reversed by treatment with cell-permeable antioxidants and corresponded with reduced ROS production and enhanced cellular proliferation during supplemental thiamine conditions. siRNA-mediated knockdown of TPK1 directly enhanced basal ROS levels and reduced tumor cell proliferation. These findings suggest that the adaptive regulation of TPK1 may be an essential component in the cellular response to oxidative stress, and that during supplemental thiamine conditions its expression may be exploited by tumor cells for a redox advantage contributing to tumor progression.