Giselle Saulnier Sholler

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

PCI-24781 (abexinostat), a novel histone deacetylase inhibitor, induces reactive oxygen species-dependent apoptosis and is synergistic with bortezomib in neuroblastoma.

In this study, we investigated the cytotoxic effects of a broad-spectrum histone deacetylase (HDAC) inhibitor, PCI-24781, alone and in combination with the proteasome inhibitor bortezomib in neuroblastoma cell lines. The combination was shown to induce synergistic cytotoxity involving the formation of reactive oxygen species. The cleavage of caspase-3 and PARP, as determined by western blotting, indicated that cell death was primarily due to apoptosis. Xenograft mouse models indicated increased survival among animals treated with this combination. The Notch signaling pathway and MYCN gene expression were quantified by reverse transcription-polymerase chain reaction (PCR) in cells treated with PCI-24781 and bortezomib, alone and in combination. Notch pathway expression increased in response to an HDAC inhibitor. NFKB1 and MYCN were both significantly down regulated. Our results suggest that PCI-24781 and bortezomib are synergistic in neuroblastoma cell lines and may be a new therapeutic strategy for this disease.

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.

BKM120 induces apoptosis and inhibits tumor growth in medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumor in children, accounting for nearly 20 percent of all childhood brain tumors. New treatment strategies are needed to improve patient survival outcomes and to reduce adverse effects of current therapy. The phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) intracellular signaling pathway plays a key role in cellular metabolism, proliferation, survival and angiogenesis, and is often constitutively activated in human cancers, providing unique opportunities for anticancer therapeutic intervention. The aim of this study was to evaluate the pre-clinical activity of BKM120, a selective pan-class I PI3K inhibitor, on MB cell lines and primary samples. IC50 values of BKM120 in the twelve MB cell lines tested ranged from 0.279 to 4.38 μM as determined by cell viability assay. IncuCyte ZOOM Live-Cell Imaging system was used for kinetic monitoring of cytotoxicity of BKM120 and apoptosis in MB cells. BKM120 exhibited cytotoxicity in MB cells in a dose and time-dependent manner by inhibiting activation of downstream signaling molecules AKT and mTOR, and activating caspase-mediated apoptotic pathways. Furthermore, BKM120 decreased cellular glycolytic metabolic activity in MB cell lines in a dose-dependent manner demonstrated by ATP level per cell. In MB xenograft mouse study, DAOY cells were implanted in the flank of nude mice and treated with vehicle, BKM120 at 30 mg/kg and 60 mg/kg via oral gavage daily. BKM120 significantly suppressed tumor growth and prolonged mouse survival. These findings help to establish a basis for clinical trials of BKM120, which could be a novel therapy for the treatment of medulloblastoma patients.

Tolcapone induces oxidative stress leading to apoptosis and inhibition of tumor growth in Neuroblastoma

Catechol‐O‐methyltransferase (COMT) is an enzyme that inactivates dopamine and other catecholamines by O‐methylation. Tolcapone, a drug commonly used in the treatment of Parkinsons disease, is a potent inhibitor of COMT and previous studies indicate that Tolcapone increases the bioavailability of dopamine in cells. In this study, we demonstrate that Tolcapone kills neuroblastoma (NB) cells in preclinical models by inhibition of COMT. Treating four established NB cells lines (SMS‐KCNR, SH‐SY5Y, BE(2)‐C, CHLA‐90) and two primary NB cell lines with Tolcapone for 48 h decreased cell viability in a dose‐dependent manner, with IncuCyte imaging and Western blotting indicating that cell death was due to caspase‐3‐mediated apoptosis. Tolcapone also increased ROS while simultaneously decreasing ATP‐per‐cell in NB cells. Additionally, COMT was inhibited by siRNA in NB cells and showed similar increases in apoptotic markers compared to Tolcapone. In vivo xenograft models displayed inhibition of tumor growth and a significant decrease in time‐to‐event in mice treated with Tolcapone compared to untreated mice. These results indicate that Tolcapone is cytotoxic to neuroblastoma cells and invite further studies into Tolcapone as a promising novel therapy for the treatment of neuroblastoma.

Abstract 3199: BKM120 is cytotoxic in neuroblastoma targeting the PI3K pathway

Background: Neuroblastoma is the most common extracranial solid tumor found in children, accounting for approximately 15% of cancer-related deaths. Many cellular processes have been discovered to play a role in neuroblastoma9s potency, including a family of lipid kinases within the phosphoinositide 3-kinase (PI3K) signaling pathway that contribute to cell survival, proliferation, and differentiation. Targeting this pathway could unveil new treatment strategies that work to specifically treat each child9s unique disease. BKM120 is a novel cancer therapeutic that targets the PI3K/Akt/mTOR signaling pathway and has recently been shown to have great potential in the clinic by acting on Class IA PI3Ks. Though Class IA PI3Ks hold multiple types, BKM120 has been shown to act preferentially on PIK3CA mutant isoforms. In this study we show the inhibitory effect of BKM120 on neuroblastoma cell lines that over-express PI3KCA, suggesting a promising role in the clinic for children with this expression profile. It has also been suggested that inhibitors of the PI3K pathway exert their inhibitory effects on cancer cells by destabilizing MYCN, a protein found over-expressed in approximately one third of neuroblastoma patients that encourages malignant progression of the disease. Methods: Neuroblastoma (NB) cell lines BE(2)-C and SMS-KCNR cells and patient lines MGT-002-13, MGT-003-08, MGT-014-11, and MGT-015-08 were used in these studies. Cell viability was measured using Calcein AM fluorescent assay at BKM120 doses 0.2uM, 0.5uM, and 1.0uM. Western blot analysis was used to measure PI3K pathway members including pAKT, p-mTOR, mTOR, and apoptosis markers including Cleaved Caspase 3, Caspase 3, PARP, and cPARP. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with the Cell Titer GLO luminescent cell viability assay. IncuCyte imaging of sytox and kinetic caspase-3 reagent was used to measure apoptosis in NB cells treated with BKM120. Results: BKM120 is cytotoxic to NB cell lines with IC509s ranging from 0.9-5.5 uM. BKM120 increases protein expression levels of Cleaved Caspase 3 and cleaved PARP by inducing apoptosis and decreases MTOR, pAKT and MYCN at increasing drug doses. BKM120 decreases ATP/cell related to glycolytic metabolism activity in NB cell lines. Increased expression of MTOR and PI3KCD correlate with increased resistance to BKM120. Conclusion: This study indicates that BKM120 targets the MTOR/PI3K/AKT pathway and may play a role in MYCN driven tumors. In addition, BKM120 inhibits NB cell proliferation and induces caspase-mediated apoptosis in vitro in NB. Given the current lack of effective treatments and the high incidence of relapse and metastatic disease for patients, further assessment in clinical trial setting is needed to assess BKM1209s therapeutic activity for children with NB. Citation Format: Monica M. Pomaville, Ping Zhao, Sarah DeCou, Abhinav B. Nagulapally, Jeffrey Bond, Giselle L. Saulnier Sholler. BKM120 is cytotoxic in neuroblastoma targeting the PI3K pathway. [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 3199.

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 (750 ± 250 mg/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 1941: Cell surface vimentin is a novel marker for CTC detection in neuroblastoma

Among children in the United States, brain cancers now account for most number of cancer-related deaths. Neuroblastomas (NB) and sarcomas account for 13% of all childhood cancers in the United States. Despite significant progress yielding increased 5-year survival, 1250 deaths are expected this year from childhood cancers and cancer incidence has been steadily increasing. One of the key contributors to cancer mortality is tumor metastasis. It is generally believed that CTCs are shed into the circulation from primary tumors and contain unique driver mutations enabling their aggressive phenotype. However, detection of CTCs from NB is difficult as there is no direct method. Our lab has developed a monoclonal antibody targeting vimentin on the cells’ surface. Cell surface vimentin (CSV) is only observed in tumor cells; it remains intracellular in normal cells. We have published data demonstrating the superior specificity and sensitivity of our approach in breast, colon, and prostate cancer. Here, we report direct CTC detection in NB using our CSV mAb. As part of our collaborative effort analyzing NB blood samples from a multicenter Phase II trial, we present novel discoveries regarding treatment of NB with difluoromethylornithine (DFMO) and its effects on CTC release into the circulation in patients under remission. Among our observations, approximately ~1/3 patients have low or no CTCs while ~1/4 with high numbers of CTC are responding to DFMO treatment. Remaining patients show oscillating CTC numbers. Further, treatment with DFMO showed a statistically significant decline in CTCs after the beginning of therapy. Ongoing work including whole genome analysis can possibly reveal new insight into CTCs’ entering the circulation. These data demonstrate that our CTC capture using CSV is a novel and unique approach for detection in NB. Citation Format: Izhar S. Batth, Heming Li, Giselle Saulnier Sholler, Shulin Li. Cell surface vimentin is a novel marker for CTC detection in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1941. doi:10.1158/1538-7445.AM2017-1941

Abstract 581: Bone marrow-derived B-cell hybridomas from neuroblastoma patients generate antibodies that bind to patients’ own tumors

Today antibody therapy is considered to be one of the most important and successful strategies to treat a variety of cancers. For example, the addition of antibodies such as Herceptin and Avastin to a chemotherapy regimen has shown improved survival in the treatment of breast cancer and colorectal cancer, respectively. Main problems with this kind of therapy are that many patients are not candidates because their tumors do not overexpress the drug target and that patient develop resistance to the targeted drug. A method to rapidly develop different sets of therapeutic antibodies would greatly contribute to the field of targeted anticancer therapy. This work evaluated the feasibility of using residual clinical material from pediatric neuroblastoma patients to generate antibodies to autologous tumor. Neuroblastoma is the most common extracranial solid tumor in children, accounting for 8-10% of all childhood cancers. Most patients with neuroblastoma are young and commonly present with metastatic disease. Bone marrow aspirate from neuroblatoma patients was the source material for the mononuclear cells and the tumor cells used in present study. Tumor cells were cultured and xenograft tumors were produced in mice. Hybridomas were generated by electrofusion of stimulated bone marrow mononuclear cells with plasmacytoma P3×63.Ag8.653 under hypo-osmolar condition using Eppendorf Multiporator/Helix chamber. Following hypoxanthine-aminopterin-thymidine (HAT) selection and monoclonal distribution, the culture supernatants were assayed for immunoglobulin secretion by ELISA. The supernatants from the positive clones were evaluated by immunofluorescence microscopy for binding to cultured neuroblastoma cells and neuroblastoma xenograft tissue sections derived from the same patient from which the hybridomas were generated. The results demonstrated that multiple hybridomas of bone marrow mononuclear cells secreted monoclonal antibodies that bound autologous neuroblastoma cells. Further evaluation of the tumor-binding antibodies on a panel of normal human tissues showed no binding to most of the tissues in the panel. Successful outcome of these experiments demonstrate the feasibility of generating human monoclonal antibodies from residual marrow specimens that bind autologous neuroblastoma cells. However, it remains to be determined whether these antibodies are bioactive and whether this approach will be generally applicable in more patients with neuroblastoma. It may be concluded that the strategy described here, which exploits the cancer patient9s own immune repertoire, has a great potential for neuroblastoma target discovery and developing antibodies with possible therapeutic and/or diagnostic utility in cancer. Citation Format: Girja S. Shukla, Giselle S. Sholler, Yujing Sun, Stephanie C. Pero, Chelsea L. Carman, Ping Zhao, David N. Krag. Bone marrow-derived B-cell hybridomas from neuroblastoma patients generate antibodies that bind to patients’ own tumors. [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 581.