Don Eslin

A Phase I Trial of DFMO Targeting Polyamine Addiction in Patients with Relapsed/Refractory Neuroblastoma

Background Neuroblastoma (NB) is the most common cancer in infancy and most frequent cause of death from extracranial solid tumors in children. Ornithine decarboxylase (ODC) expression is an independent indicator of poor prognosis in NB patients. This study investigated safety, response, pharmacokinetics, genetic and metabolic factors associated with ODC in a clinical trial of the ODC inhibitor difluoromethylornithine (DFMO) ± etoposide for patients with relapsed or refractory NB. Methods and Findings Twenty-one patients participated in a phase I study of daily oral DFMO alone for three weeks, followed by additional three-week cycles of DFMO plus daily oral etoposide. No dose limiting toxicities (DLTs) were identified in patients taking doses of DFMO between 500-1500 mg/m2 orally twice a day. DFMO pharmacokinetics, single nucleotide polymorphisms (SNPs) in the ODC gene and urinary levels of substrates for the tissue polyamine exporter were measured. Urinary polyamine levels varied among patients at baseline. Patients with the minor T-allele at rs2302616 of the ODC gene had higher baseline levels (p=0.02) of, and larger decreases in, total urinary polyamines during the first cycle of DFMO therapy (p=0.003) and had median progression free survival (PFS) that was over three times longer, compared to patients with the major G allele at this locus although this last result was not statistically significant (p=0.07). Six of 18 evaluable patients were progression free during the trial period with three patients continuing progression free at 663, 1559 and 1573 days after initiating treatment. Median progression-free survival was less among patients having increased urinary polyamines, especially diacetylspermine, although this result was not statistically significant (p=0.056). Conclusions DFMO doses of 500-1500mg/m2/day are safe and well tolerated in children with relapsed NB. Children with the minor T allele at rs2302616 of the ODC gene with relapsed or refractory NB had higher levels of urinary polyamine markers and responded better to therapy containing DFMO, compared to those with the major G allele at this locus. These findings suggest that this patient subset may display dependence on polyamines and be uniquely susceptible to therapies targeting this pathway. Trial Registration Clinicaltrials.gov NCT#01059071

Feasibility of implementing molecular-guided therapy for the treatment of patients with relapsed or refractory neuroblastoma

The primary objective of the study was to evaluate the feasibility and safety of a process which would utilize genome‐wide expression data from tumor biopsies to support individualized treatment decisions. Current treatment options for recurrent neuroblastoma are limited and ineffective, with a survival rate of <10%. Molecular profiling may provide data which will enable the practitioner to select the most appropriate therapeutic option for individual patients, thus improving outcomes. Sixteen patients with neuroblastoma were enrolled of which fourteen were eligible for this study. Feasibility was defined as completion of tumor biopsy, pathological evaluation, RNA quality control, gene expression profiling, bioinformatics analysis, generation of a drug prediction report, molecular tumor board yielding a treatment plan, independent medical monitor review, and treatment initiation within a 21 day period. All eligible biopsies passed histopathology and RNA quality control. Expression profiling by microarray and RNA sequencing were mutually validated. The average time from biopsy to report generation was 5.9 days and from biopsy to initiation of treatment was 12.4 days. No serious adverse events were observed and all adverse events were expected. Clinical benefit was seen in 64% of patients as stabilization of disease for at least one cycle of therapy or partial response. The overall response rate was 7% and the progression free survival was 59 days. This study demonstrates the feasibility and safety of performing real‐time genomic profiling to guide treatment decision making for pediatric neuroblastoma patients.

A Phase 1 Trial of TPI 287 as a Single Agent and in Combination With Temozolomide in Patients with Refractory or Recurrent Neuroblastoma or Medulloblastoma

The primary aim of this Phase I study was to determine the maximum tolerated dose (MTD) of TPI 287 and the safety and tolerability of TPI 287 alone and in combination with temozolomide (TMZ) in pediatric patients with refractory or recurrent neuroblastoma or medulloblastoma. The secondary aims were to evaluate the pharmacokinetics of TPI 287 and the treatment responses.

Combination of 13 cis-retinoic acid and tolfenamic acid induces apoptosis and effectively inhibits high-risk neuroblastoma cell proliferation.

Chemotherapeutic regimens used for the treatment of Neuroblastoma (NB) cause long‐term side effects in pediatric patients. NB arises in immature sympathetic nerve cells and primarily affects infants and children. A high rate of relapse in high‐risk neuroblastoma (HRNB) necessitates the development of alternative strategies for effective treatment. This study investigated the efficacy of a small molecule, tolfenamic acid (TA), for enhancing the anti‐proliferative effect of 13 cis‐retinoic acid (RA) in HRNB cell lines. LA1‐55n and SH‐SY5Y cells were treated with TA (30 μM) or RA (20 μM) or both (optimized doses, derived from dose curves) for 48 h and tested the effect on cell viability, apoptosis and selected molecular markers (Sp1, survivin, AKT and ERK1/2). Cell viability and caspase activity were measured using the CellTiter‐Glo and Caspase‐Glo kits. The apoptotic cell population was determined by flow cytometry with Annexin‐V staining. The expression of Sp1, survivin, AKT, ERK1/2 and c‐PARP was evaluated by Western blots. The combination therapy of TA and RA resulted in significant inhibition of cell viability (p < 0.0001) when compared to individual agents. The anti‐proliferative effect is accompanied by a decrease in Sp1 and survivin expression and an increase in apoptotic markers, Annexin‐V positive cells, caspase 3/7 activity and c‐PARP levels. Notably, TA + RA combination also caused down regulation of AKT and ERK1/2 suggesting a distinct impact on survival and proliferation pathways via signaling cascades. This study demonstrates that the TA mediated inhibition of Sp1 in combination with RA provides a novel therapeutic strategy for the effective treatment of HRNB in children.

Maintenance DFMO Increases Survival in High Risk Neuroblastoma

High risk neuroblastoma (HRNB) accounts for 15% of all pediatric cancer deaths. Despite aggressive therapy approximately half of patients will relapse, typically with only transient responses to second-line therapy. This study evaluated the ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) as maintenance therapy to prevent relapse following completion of standard therapy (Stratum 1) or after salvage therapy for relapsed/refractory disease (Stratum 2). This Phase II single agent, single arm multicenter study enrolled from June 2012 to February 2016. Subjects received 2 years of oral DFMO (750u2009±u2009250u2009mg/m2 twice daily). Event free survival (EFS) and overall survival (OS) were determined on an intention-to-treat (ITT) basis. 101 subjects enrolled on Stratum 1 and 100 were eligible for ITT analysis; two-year EFS was 84% (±4%) and OS 97% (±2%). 39 subjects enrolled on Stratum 2, with a two-year EFS of 54% (±8%) and OS 84% (±6%). DFMO was well tolerated. The median survival time is not yet defined for either stratum. DFMO maintenance therapy for HRNB in remission is safe and associated with high EFS and OS. Targeting ODC represents a novel therapeutic mechanism that may provide a new strategy for preventing relapse in children with HRNB.

Abstract 2467: Association of specificity protein 1 and survivin expression in medulloblastoma: Identifying effective therapeutic targets

Medulloblastoma (MB) is an aggressive malignant brain tumor diagnosed mostly in children. MB is typically treated using a multimodal approach consisting of surgery, craniospinal irradiation, and chemotherapy. These treatments cause delayed consequences in pediatric patients. In order to treat this malignancy effectively, it is important to identify critical markers associated with the disease and finding specific agents to target such markers. The transcription factor, Specificity protein 1 (Sp1) and an inhibitor of apoptosis protein, survivin are over expressed in many cancers and associated with poor prognosis. Sp1 mediates the expression of several oncogenes including survivin. Even though some evidence exists for the expression of survivin, the information on Sp1 is still limited in MB. The primary objective of this study was to determine the association of Sp1 and survivin in MB and developing the strategies to target these candidates using less toxic compounds. A human MB tumor tissue array consisting of 20 tumor and 3 normal controls was stained for Sp1 and survivin using specific antibodies. Tumor specimens showed distinct expression for both Sp1 and survivin in majority of tumor tissues. Using small interference RNA (siRNA) technology, the expression of Sp1 and survivin was inhibited in MB cell line, DAOY, and the cell viability was determined at 24 and 48 h using CellTiter-Glo kit. We observed that the inhibition of Sp1 and survivin by siRNA correlated with a decrease in DAOY cell viability. Previously, our laboratory showed that a small molecule, Tolfenamic Acid (TA) inhibits cell proliferation and tumor growth in mice via targeting Sp1 and anti-apoptotic protein, survivin. Taken together these results highlight the significance of targeting Sp1 and survivin for inhibiting MB cell proliferation and tumor growth in mouse model. Survivin has also been demonstrated to play a role in chemoresistance. We therefore, tested the combination treatment(s) involving TA and standard chemotherapeutic agents, Vincristine (Vinc) and Cisplatin (Cis) using MB cell lines, DAOY and D283. Cells were treated with TA, Vinc, Cis and combination of TA+Vinc and TA+Cis and the effect on cell viability and apoptosis was determined. Each drug alone caused a time and dose dependent decrease in cell viability that was enhanced in the presence of TA. The combination treatment also resulted in a 2-3 fold increase in apoptosis as determined by annexin V and propidium iodide staining. It is plausible that targeting Sp1 and/or survivin induces the susceptibility of MB cells to chemotherapeutic drugs. These preliminary results demonstrate the efficacy of combining inhibitors of Sp1 and survivin with chemotherapeutic agents to enhance their therapeutic response while limiting the toxicity and side-effects. Citation Format: Umesh T. Sankpal, Don Eslin, W. Paul Bowman, Jeffrey C. Murray, Irene Sanchez, Michelle Jones, Sagar Shelake, Yazmin Hernandez, Anmol Wadwani, Hassaan Patel, Ashni Dudhia, Riyaz M. Basha. Association of specificity protein 1 and survivin expression in medulloblastoma: Identifying effective therapeutic targets. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2467.

Abstract A02: Chemo-sensitization of medulloblastoma cells using small molecule tolfenamic acid

Introduction: Medulloblastomas (MB) are the most common malignant brain tumors in children and constitute 20% of all pediatric brain tumors. Significant developments in the areas of neurosurgical techniques, dosing and delivery of radiation and optimized chemotherapy have limited response on improving the five year survival rates. Because of the aggressive multimodality therapy, survivors frequently suffer numerous long-term side effects. There is an urgent need for novel strategies for enhancing and optimizing the therapeutic efficacy of current MB treatments. Studies have shown that the Specificity Protein (Sp) transcription factors regulate critical genes that are involved in regulation of proliferation, survival, angiogenesis and resistance to chemo-/radiation therapy. Rationale: Research from our laboratory and others showed the anti-cancer activity of Tolfenamic acid (TA), a small molecule and non-steroidal anti-inflammatory drug in several adult malignancies. Recently, we demonstrated for the first time that TA inhibits MB cell proliferation and tumor growth in mice xenografts. Mechanistic studies revealed that TA acts via downregulation of Sp proteins and its downstream targets like survivin and VEGF. In this study, we tested the efficacy of TA for enhancing the response of chemotherapeutic agents used as standard therapy for MB. We hypothesize that TA sensitizes MB cells to chemotherapeutic drugs via down-regulation of Sp1 and survivin. Methods: Human MB cell lines, DAOY and D283 were used to test the anti-proliferative response of TA, Irinotecan, Topotecan (TOPO), Temozolomide (TMZ) and Doxorubicin (DOXO). Subsequently, the combination of TA with TOPO was tested using the optimized doses of TA (10 µg/ml) or TOPO (20 nM for DAOY; 25 nM for D283). Cell viability was measured at 24, 48, and 72h post-treatment using CellTiter-Glo kit. To correlate the anti-proliferative response with the induction of apoptotic pathways we analyzed apoptosis by flow cytometric analysis of Annexin V stained cells, PARP cleavage, and the activation of caspase3/7. The expression of Sp1, survivin, and cleaved-PARP was determined by Western blot. qPCR analysis using TaqMan assays was used to detect microRNAs (miR-20a and miR-27a), which act as Sp repressor proteins. Results: TA and chemotherapeutic agents caused a dose and time-dependent inhibition of cell viability. The combination of TA and TOPO caused significantly higher inhibition when compared to either TA or TOPO alone confirming the efficacy of this combination therapy in pre-clinical models for MB. The effect on cell apoptosis was evaluated at 48 h post-treatment. Annexin V staining for apoptotic cells showed a significant increase in the apoptotic fraction (annexin V positive) following combination therapy when compared to individual treatment of TA or TOPO. These results were further supported by significantly upregulated caspase 3/7 activity when compared to individual treatment. DAOY and D283 were treated with vehicle (DMSO) or 10 or 20 µg/ml TA for 48 h. Whole cell lysates were prepared and analyzed by Western blot analysis. TA inhibited both Sp1 and survivin in MB cell lines. Consistent with Annexin V staining and caspase 3/7 results, TA augmented PARP cleavage confirming the activation of apoptotic pathways. TA treatment also downregulated mirR-20a and miR-27a. Conclusion: Overall, these results demonstrate that the combination of TA with standard chemotherapeutic agents effectively inhibits MB cell growth. This chemo-sensitization can be correlated with the downregulation of survivin by TA, in part due to its effects on Sp1 transcription factor either directly or through miRNAs. These preliminary results strongly demonstrate the potential of targeting Sp1 and survivin using TA to enhance the therapeutic response in pre-clinical model of MB. Financial Support: HyundaiHopeOnWheels; UFHCC, Orlando Health, Orlando, FL and Department of Pediatrics, UNT Health Science Center, Fort Worth, TX. Citation Format: Don Eslin, Umesh T. Sankpal, Jeffrey C. Murray, W. Paul Bowman, Robert Sutphin, Riyaz Basha. Chemo-sensitization of medulloblastoma cells using small molecule tolfenamic acid. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A02.

Abstract 3267: Novel combination treatment with 13-cis-retinoic acid and tolfenamic acid induces anti-proliferative response in high-risk neuroblastoma cells

High-risk neuroblastoma (HRNB) is an aggressive extra-cranial solid tumor of childhood. 13-cis-retinoic acid (RA) is commonly used in adjuvant therapy and helpful in reducing the reoccurrence of this malignancy. Due to the very poor prognosis, there is an urgent need to establish more effective strategies for treating NB. In this study we evaluated the effect of the small molecule Tolfenamic Acid (TA) for enhancing the anti-proliferative effect of RA in NB cells, LA1-55n and SH-SY5Y. TA is known to induce degradation of Specificity protein1 (Sp1) transcription factor which regulates several genes associated with cell proliferation and cell cycle. After testing the dose/time-dependent response of individual agents, the optimized doses were used for the combination experiments. LA1-55n and SH-SY5Y cells were treated with TA (30 uM) or RA (20 uM) or both for 48 hour and tested to assess the effect on cell growth, apoptosis and selected molecular markers including Sp1, survivin, AKT and ERK1/2. Cell viability and caspase activity were measured with Cell TiterGlo and Caspase Glo kits. The apoptotic cell population was determined by flow cytometry using Annexin-V staining. The expression of Sp1, survivin, AKT, ERK1/2 and c-PARP were evaluated by western blot analysis. Combination treatment with TA and RA caused significant inhibition of cell viability (p Citation Format: Sagar Shelake, Don Eslin, Robert Sutphin, Anmol Wadhwani, Laura E. Kenyon, W. Paul Bowman, Riyaz Basha. Novel combination treatment with 13-cis-retinoic acid and tolfenamic acid induces anti-proliferative response in high-risk neuroblastoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3267. doi:10.1158/1538-7445.AM2015-3267

Abstract B11: Nifurtimox and radiation combination increases reactive oxygen species and induces apoptosis in medulloblastoma cells

Radiation (XRT) is a part of standard therapy for treating medulloblastoma (MB) in children. Morbidities associated with irradiation often cause long-term side-effects. Here we are testing a strategy to enhance the response of radiation using pre-clinical models for MB. XRT-induced cell death is partially associated with the generation of reactive oxygen species (ROS). Since ROS production is implicated in cell apoptosis, induction of increased ROS may serve as a promising strategy for inhibiting cancer cell growth. Nifurtimox (Nfx), a nitrofuran compound used to treat the parasitic infection Chagas9 disease, is known to induce ROS in pre-clinical models for neuroblastoma and medulloblastoma and is currently in clinical trials for treating these malignancies in children. We investigated the efficacy of Nfx in augmenting the effect of XRT using MB cell lines (DAOY and D283) and a patient-derived primary culture (003BN cells). DAOY, D283, and 003BN cells were treated with vehicle (DMSO) or increasing concentrations/doses of Nfx (5-70 µg/ml) or XRT (2-10 Gy) and cell viability was measured up to 3 (MB cell lines) or 5 (primary cultures) days post-treatment. The optimized concentration/dose of Nfx and XRT were used for combination experiments. MB cells were treated with vehicle, Nfx (20 μg/ml), XRT (5 or 10 Gy) or both and the effect on cell viability, apoptosis, ROS levels and catalase activity was measured. Cell viability was measured with CellTiter Glo kit (Promega). Apoptosis was analyzed by flow cytometry (Annexin-V staining) and caspase 3/7 activity was measured using Caspase-Glo Assay (Promega). The expression of c-PARP was determined by Western blot analysis. Results revealed that Nfx and XRT combination significantly increased MB cell growth inhibition when compared to the effect of either Nfx or XRT. The anti-proliferative effect of Nfx and XRT combination was accompanied by the activation of apoptosis as determined by increased annexin V staining and caspase 3/7 activity. An increase in levels of ROS was also observed following combination treatment with Nfx and XRT. Notably, catalase activity was unaltered following the treatment with single or double agents suggesting a contribution of impairment in the redox system in causing MB cell growth inhibition. This pre-clinical study demonstrates that Nfx potentially enhances the therapeutic efficacy of radiation in MB cell lines and primary cultures. To further delineate the underlying mechanisms and to identify other potential candidates modulated by this combination therapy we are undertaking a molecular profiling approach using Affimetrix gene expression arrays. Citation Format: Don Eslin, Chris M. Lee, Giselle Sholler, Amy Smith, Robert M. Sutphin, Dennis A. Steindler, Ping Zhao, Shanshan Wang, Umesh T. Sankpal, Sarah Connelly, Riyaz Basha. Nifurtimox and radiation combination increases reactive oxygen species and induces apoptosis in medulloblastoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B11.

Abstract LB-160: A feasibility trial using molecular-guided therapy for the treatment of patients with refractory or recurrent neuroblastoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnBackground: Neuroblastoma is the most common extracranial solid tumor in children, and treatment options for recurrent neuroblastoma are limited. Using molecular profiling to target the molecular vulnerabilities of individual patient tumors with existing therapeutic agents may result in a rational, data-driven approach to treatment with potential to improve clinical outcomes. Methods: This was a multicenter study through the Neuroblastoma and Medulloblastoma Translational Research Consortium. The primary objective of this study was to evaluate the feasibility of supporting real-time treatment decisions through predictive modeling of genome-wide mRNA gene expression data from neuroblastoma tumor biopsies. Feasibility was defined as completion of tumor biopsy, histopathological evaluation, RNA extraction and quality control, gene expression profiling within a CLIA-certified laboratory, bioinformatic analysis, generation of a drug prediction report, molecular tumor board review yielding a formulated treatment plan, independent medical monitor review, treatment initiation within a 2-week period and completion of one cycle of therapy. The secondary objectives included a reproducibility study of patient biopsies, safety and response to therapy. Research validation in cell culture models of patient tumors was performed. Results: Fourteen patients with multiply relapsed neuroblastoma were enrolled between July 2011 and November 2012. All biopsies passed histopathology and RNA quality control. Generation of gene expression data and its analysis (3-8 days), reports which linked this data into medically actionable drug candidates (0-3 days), molecular tumor board (1-6 days) and independent medical monitor review (1 -4 days) were all completed in real-time. The average time was 12.4 days from biopsy to initiation of treatment. There were no unexpected serious adverse events on study and patients tolerated therapy well. Clinical benefit was seen 50% of patients. Triplicate biopsies showed reproducible sets of drugs and the comparatively large differences in drug lists between patients show that the drug lists are tailored to patients. Comparison of RNA expression profiles with RNA sequencing from each patient showed strong correlation. Conclusion: This study shows that it is feasible to create therapeutic treatment plans based on genomic profiling in real time and that patients are able to be treated safely on a tumor board derived molecular guided therapy using existing medications. Incorporation of further genomic studies to evaluate additional molecular profiling techniques in order to make more informed individual therapeutic treatment plans will be evaluated in future studies.nnCitation Format: Giselle L. Sholler, Genevieve Bergendahl, Alyssa VanderWerff, William Ferguson, William Roberts, Don Eslin, Jacqueline Kraveka, Joel Kaplan, Deanna Mitchell, Nehal Parikh, Kathleen Neville, Takamaru Ashikaga, Jeffrey Bond, Gina Hanna, Melinda Merchant, Matt Huentelman, Jason Corneveaux, Jeffrey Trent. A feasibility trial using molecular-guided therapy for the treatment of patients with refractory or recurrent neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-160. doi:10.1158/1538-7445.AM2013-LB-160