Abhinav Nagulapally

Molecular Guided Therapy Provides Sustained Clinical Response in Refractory Choroid Plexus Carcinoma

Choroid plexus carcinomas (CPCs) are rare, aggressive pediatric brain tumors with no established curative therapy for relapsed disease, and poor survival rates. TP53 Mutation or dysfunction correlates with poor or no survival outcome in CPCs. Here, we report the case of a 4 month-old female who presented with disseminated CPC. After initial response to tumor resection and adjuvant-chemotherapy, the tumor recurred and metastasized with no response to aggressive relapse therapy suggesting genetic predisposition. This patient was then enrolled to a Molecular Guided Therapy Clinical Trial. Genomic profiling of patient tumor and normal sample identified a TP53 germline mutation with loss of heterozygosity, somatic mutations including IDH2, and aberrant activation of biological pathways. The mutations were not targetable for therapy. However, targeting the altered biological pathways (mTOR, PDGFRB, FGF2, HDAC) guided identification of possibly beneficial treatment with a combination of sirolimus, thalidomide, sunitinib, and vorinostat. This therapy led to 92% reduction in tumor size with no serious adverse events, excellent quality of life and long term survival.

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.

Abstract 1934: Retinoic acid and DFMO induce differentiation and inhibit tumor formation in neuroblastoma

Background: A key component of high risk neuroblastoma (NB) therapy involves cis-retinoic acid (RA) for differentiation of minimal residual disease. DFMO induces differentiation and inhibition of tumor formation through the targeting of cancer stem cell (CSC) pathways via reversal of the Lin28/Let7 axis. Preventatitve DFMO therapy is currently in a phase II clinical trial at the end of therapy and in a Pilot study in combination with RA and ch14:18 antibody. We hypothesize that the combination of cis-RA and DFMO will induce greater differentiation, inhibition of tumor formation, and reduction of cell proliferation of NB. Methods: NB cell lines SMSKCNR, BE2C and CHLA90 were incubated in 96 well plates for 24 and 48 hours with low doses of DFMO (2.5 and 5 mM), RA (5 and 10 μM), and the four combinations of dosages. A Calcein AM Cell Viability Assay and BrdU Cell Proliferation Assay Kit were used to determine cell viability and cell proliferation, respectively. Western blot analysis was used to measure protein levels of CSC and differentiation markers. A neurosphere assay was used to assess inhibition of CSCs and tumor formation within wells. Cells were plated 2 cells/well in 96 well plates, drugged with single agents, cominbation, or DMSO and the percentage of wells per plate that formed neurospheres was determined after 1 and 2 weeks. IncuCyte ZOOM Live-Cell Imaging system was used for kinetic monitoring of neurite length to assess differentiation of NB cells. Results: Low dose RA and DFMO combination treatment (2.5-5mM DFMO and 5-10 μM RA) resulted in decreased cell viability as demonstrated through calcein AM. DFMO and RA combination treatments reduced cell viability by 60-71%, 75-78%, and 83-91%, in Be2C, CHLA90, and SMSR cells, respectively. BrdU incorporation demonstrated a reduction in cell proliferation at 48 hours of was 69-70.%, 60.2-64.5%, 62.7-71.1% in Be2c, CHLA90, and SMSR, respectively . Western blot analysis showed that DFMO, RA, and their combination reduced the CSC and increased the differentiation markers at 48 hours compared to control. The combination treatment also decreased tumor formation; the relative reduction in neurosphere formation at 2 weeks was 34.5% with 2.5 mM DFMO, 48% with 5 µM RA, and 73.2% with combination treatment. Lastly, differentiation was shown by neurite length increased by a factor of 1.4-1.6 and 5, in SMSR and BE2C cells, respectively with combination treatment. Conclusion: This study indicates that the combination retinoic acid and DFMO effectively cause a decrease in cell viability with a reduction in cell proliferation. Further, the combination results in differentiation of NB cells as well as targeting of CSC pathways and inhibition of tumor formation. Preventative DFMO therapy has been initiatied with RA in a pilot study for the treatment of high-risk neuroblastoma patients. Citation Format: Austin Voydanoff, Ping Zhao, Abhinav Nagulapally, Jeff Bond, Giselle L. Sholler. Retinoic acid and DFMO induce differentiation and inhibit tumor formation 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 1934. doi:10.1158/1538-7445.AM2017-1934

Abstract 3197: BKM120 promotes apoptosis and suppresses tumor growth in medulloblastoma by targeting the phosphoinositide 3-kinase pathway

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Background: Medulloblastoma (MB) is the most common malignant brain tumor in children with poor survival outcome. New treatment strategies are needed for control of MB. 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. This pathway is often constitutively activated in human tumor cells, providing unique opportunities for anticancer therapeutic intervention. BKM120 (Buparlisib) is an oral pan-class I PI3K inhibitor that targets all 4 isoforms of class I PI3K. BKM120 is currently being clinically evaluated for the treatment of different adult cancers including breast cancer, glioblastoma, prostate cancer, advanced non-small cell lung cancer, and colorectal cancer. In this study, we screened our MB established and patient primary cell lines by genomic profiling analysis, and validated the targeted therapy both in vitro and in vivo in xenograft mouse model. Methods: RNA expression profiling analysis was performed with Affymetrix GeneChip U133 Plus 2.0 genome wide expression cDNA microarray. Analysis was done using R/Bioconductor packages and Partek Genomics Suite. Eleven MB cell lines were treated with increasing concentrations (0-4 μM) of BKM120 for 48 hours. CellTiter-Glo Luminescent Cell Viability Assay was used to determine cell viability. IC50 values were calculated with a four-parameter variable-slope dose response curve using GraphPad Prism v.5 software. IncuCyte ZOOM Live-Cell Imaging system was used for kinetic monitoring of cytotoxicity of BKM120 and apoptosis in MB cells. Western blot analysis was used to measure phospho-Akt, phospho-mTOR, and cleaved caspase 3 protein levels. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with CellTiter-Glo luminescent cell viability assay. Xenograft study was performed with DAOY cells implanted in the flank of nude mice and treated with vehicle, BKM120 at 30 mg/kg and 60 mg/kg via oral gavage daily. Results: BKM120 exhibited cytotoxicity in MB cells in a dose-dependent manner by inhibiting activation of downstream signaling molecules Akt and mTOR, and activating apoptotic pathways and inducing cell death in the eleven cell lines tested. IC50s of BKM120 in the MB cell lines ranged from 0.456 to 2.9 μM determined by cell viability assay. Furthermore, BKM120 decreased cellular glycolytic metabolic activity in MB cell lines in a dose-dependent manner. In MB xenograft mouse study, BKM120 significantly suppressed tumor growth and prolonged mouse survival at 30 mg/kg and 60 mg/kg. Conclusion: This study indicates that BKM120 promotes apoptosis and suppresses medulloblastoma tumor growth both in vitro and in vivo. Additional investigation of BKM120 for the treatment of pediatric medulloblastoma is warranted. Citation Format: Ping Zhao, Jacob Hall, Austin Voydanoff, Mary Durston, Elizabeth VanSickle, Abhinav B. Nagulapally, Jeffery Bond, Giselle Saulnier Sholler. BKM120 promotes apoptosis and suppresses tumor growth in medulloblastoma by targeting the phosphoinositide 3-kinase 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 3197.

Abstract 2474: DFMO targets cancer stem cells in neuroblastoma

Background: Difluoromethylornithine (DFMO) has shown promise in recent clinical trials as a therapeutic agent in treating neuroblastoma (NB). High-risk NB patients have an approximately 50% relapse rate, after which survival rate drops to lower than 10%. By targeting the cancer stem cells (CSC) we could decrease relapse rate in high-risk patients and improve long-term survival. DFMO is known to inhibit ornithine decarboxylase (ODC) impeding key polyamine biosynthesis as well as the LIN28/Let7 pathway and disrupting the glycolytic metabolism pathways both of which have been shown to be important in CSC models. We propose that DFMO may be an effective treatment against neuroblastoma CSCs. Methods: BE(2)C and SMS-KCNR NB cell lines were studied. Limiting dilution assays of xenograft studies were performed. Untreated, 10-day 5mM DFMO-pretreated, or 20-day 5mM DFMO-pretreated cells were injected into mice between 10-5000 cells/mouse and followed for tumor formation. Xenograft Be2C mice were treated with either vehicle or 2%DFMO in drinking water and harvested at 7-14 days for tumor analysis. Tumors and cell cultures of untreated and DFMO-treated cells were analyzed by RT-qPCR, Western Blot analysis, and GeneChip gene expression analysis measuring levels of ODC1, LIN28B, mirLet7, MYCN, SLC2A4 (a glucose transporter), and CSC markers such as CXCR4, POU5F1, NANOG, SOX2, and KLF4. Riboflavin induced autofluorescence (AF) was used to identify CSC. This method detected cells after a 24-hour bath in 30μM riboflavin and excitement at 488nm in a MoFlo cell sorter at the University of Michigan Flow Core, after which cells are sorted into AF- and AF+ sub-populations for imaging, neurosphere assays with or without DFMO, RT-qPCR and Western Blot analysis. Knock-down of ODC1 is achieved by siRNA transfection via Lipofectamine in cells and analyzed by RT-qPCR, Western Blot, and neurosphere assays with or without DFMO. Results: Pretreatment with DFMO in BE(2)C and SMS-KCNR cells in vitro and in vivo inhibits tumor formation in neurosphere assays and in limiting dilution xenograft models respectively. Tumors harvested from xenografts show a decrease in expression of CSC biomarkers and neurosphere formation capability in those treated with DFMO. AF+ cells showed an increase in stem cell marker expression and increase neurosphere formation relative to AF- cells, identifying them as cancer stem cells. In addition, AF+ cells are more sensitive to DFMO treatment with a greater decrease in neurosphere formation. Cells transfected with ODC siRNA or subjected to DFMO show a reduction in CSC biomarker RNA and protein and a decrease in neurospheres formation. Conclusion: These results suggest that DFMO targets the CSC subpopulation in neuroblastoma, disrupting cell growth pathways and inhibiting tumor formation. This concept is under further investigation in a Phase II clinical trial to prevent relapse in high risk neuroblastoma patients. Citation Format: Tracey Avequin, Ping Zhao, Abhinav Nagulapally, Jeffrey Bond, Ebrahim Azizi, Max Wicha, Giselle L. Saulnier Sholler. DFMO targets cancer stem cells in neuroblastoma. [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 2474.

Abstract B01: Sustained clinical response to treatment directed by genomic expression profiling suggests mTOR signaling is an effective target in choroid plexus carcinoma

Background: Choroid plexus tumors comprise 10-20% of intracranial tumors in children less than one year of age, the majority of these are benign papillomas, however some are highly aggressive carcinomas. Choroid Plexus Carcinoma (CPC) is derived from the choroid plexus which is neuroepithelial tissue that normally produces CSF and lines the intracranial ventricles. The prognosis of choroid plexus carcinomas is poor with 5 year event free survival rates varying between 10%-50% while relapsed or metastatic CPC is usually fatal. A patient with relapsed CPC who had progressed on 5 previous salvage chemotherapy combinations was enrolled on Neuroblastoma Medulloblastoma Translational Research Consortium (NMTRC) Molecular-Guided Therapy Trial for the Treatment of Patients with Relapsed or Refractory Childhood Cancer in which we performed genomic profiling allowing a targeted approach to therapeutic decision making. Methodology: The patient with multiply relapsed metastatic CPC enrolled on NMTRC Molecular Guided Therapy Clinical Trial after obtaining written informed consent. A tumor biopsy was sent to the Clinical Reference Laboratory (CRL) for mRNA expression analysis using U133 2.0 Plus GeneChip, and to Translational Genomics Research Institute (TGen) for high-performance RNA-seq analysis and DNA exome analysis. The differential expression data was interpreted in the context of systems biology annotation. Analysis of RNA expression results were discussed in a NMTRC tumor board leading to a therapeutic plan using study targeted treatments. Patient response was determined by clinical examination with serial MRI of the brain. Results: In this CPC specimen the PI3K/Akt/mTOR pathway was found to be highly expressed as well as PDGF, FGF2 and HDAC3 which were chosen by the tumor board for targeted therapy. Exomes analysis confirms mutation in RPTOR involved in mTOR activation. Treatment with sirolimus (mTOR), thalidomide (FGF2), Sunitinib (PDGF), and vorinostat (HDAC3) resulted in a 68% tumor reduction and the patient maintains a continuous response after 11 months while continuing to receive this therapy. This treatment combination has been well tolerated with no serious adverse events and excellent quality of life. Conclusion: Genomic Profiling analyses revealed activation of the mTOR pathway in this chemo-resistant Choroid Plexus Carcinoma. Targeted therapy has demonstrated clinical benefit suggesting a novel therapeutic approach. Further investigation of the mTOR pathway as a therapeutic target in CPC warranted. Citation Format: Albert S. Cornelius, Jessica Foley, Deanna Mitchell, Abhinav Nagulapally, Jeff Bond, Matthew Huntelman, Jason Corneveaux, Jeff Trent, Giselle Sholler. Sustained clinical response to treatment directed by genomic expression profiling suggests mTOR signaling is an effective target in choroid plexus carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B01.

Abstract 1621: Tolcapone, a catechol-O-methyltransferase inhibitor, alone and in combination with oxaliplatin induces cell death in neuroblastoma

Background: Neuroblastoma (NB) is an aggressive childhood cancer that arises from neural crest cells of the sympathetic nervous system. These cells contain cathecholamines which may be a therapeutic target. Catechol-O-methyltransferase (COMT) metabolizes catechol-containing compounds, including dopamine. COMT inhibitors, including tolcapone, are used as an adjunctive treatment for Parkinson9s disease, as they increase dopamine within cells. Increase in dopamine and other catecholamines within cells may result in cell toxicity. Methods: Cell viability was measured using Calcein AM fluorescent assay at tolcapone doses 1.5625 μM - 200 μM, both alone and in combination with oxaliplatin at doses 1.5 μM - 6 μM. Western blot analysis was used to measure cleaved poly ADP ribose polymerase (PARP) and cleaved and full caspase-3 levels. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with the Cell Titer GLO luminescent cell viability assay. Expression of COMT was confirmed using microarray analysis and immunofluorescence. Reactive oxygen species (ROS) levels were measured using DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit. IncuCyte ZOOM machine was used in conjugation with SYTOX Green dye to measure cell death and CellPlayer™ Kinetic Caspase-3/7 Apoptosis Assay Reagent to measure apoptosis over 48 hours. Microarray analysis using U133+ RNA expression profiles were used to evaluate gene expression changes after cells were treated with tolcapone for 12 hours. Results: Tolcapone IC50 values ranged from 13.33 μM to 156 μM in seven different NB cell lines. Concentrations of 25 μM and 50 μM increased levels of cleaved PARP and cleaved caspase-3 over 48, 72, and 96 hours treated. Intracellular ATP decreased significantly with concentrations ranging from 12.5-200 μM treated over 48 hours and ROS levels increased significantly with concentrations ranging from 12.5-200 μM treated over 24 hours. IncuCyte Zoom analysis displayed dose dependent levels of cell death and presence of cleaved caspase-3 and 7 with increasing concentrations ranging from 1.5625 μM -200 μM. The combination of tolcapone at 25 μM and oxaliplatin at 3.5 μM show synergy in cell viability assays. Conclusion: There is no curative therapy for relapsed/refractory NB patients. The preclinical evidence suggests that patients with overexpression of COMT may respond to tolcapone via catecholamine induced ROS and cytotoxicity, especially when combined with oxaliplatin. Therefore, tolcapone and oxaliplatin may be a potential new therapy for children with NB with plans to be evaluated in a Phase I/II trial. Citation Format: David E. Hayes, Ping Zhao, Austin Voydanoff, Abhinav Nagulapally, Jeff Bond, Giselle Sholler. Tolcapone, a catechol-O-methyltransferase inhibitor, alone and in combination with oxaliplatin induces cell death in neuroblastoma. [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 1621. doi:10.1158/1538-7445.AM2015-1621