Pediatric Solid Tumor Cell Lines Research Articles

Evaluating antitumor activity and response determinants to trastuzumab deruxtecan in pediatric solid tumors.

10049 Background: Trastuzumab deruxtecan (T-DXd) is an anti-HER2 antibody-drug conjugate (ADC) linked to a topoisomerase inhibitor FDA-approved for several indications, including the treatment of HER2+ breast and gastric cancer. Although HER2 amplification is uncommon in pediatric cancers, recent demonstration of T-DXd efficacy in HER2-low breast cancer patients prompted us to examine the potential clinical relevance of T-DXd in pediatrics by evaluating HER2 expression and activity of T-DXd in preclinical pediatric solid tumor models. Methods: Cell viability was assessed in a panel of histologically diverse pediatric solid tumor cell lines and 2 HER2-amplified adult cancer cell lines treated with T-DXd, payload (DXd), and a control IgG-ADC. HER2 protein expression in patient-derived xenograft (PDX) tumors was evaluated by immunohistochemistry (IHC) using 5 clinically validated anti-HER2 antibodies. RNAseq was performed on clinical (n=290) and PDX (n=184) tumors to determine relative expression of ERBB2. In vivo activity of T-DXd was evaluated in osteosarcoma (OS, n=10), Wilms tumor (WT, n=1) and malignant rhabdoid tumor (MRT, n=1) PDX models and a desmoplastic small round cell tumor (DSRCT) cell line xenograft model. Differences in tumor volume and time to disease progression was assessed and compared across treatments and models. Responses were correlated with HER2 expression by IHC. Results: In vitro, HER2-amplified control cell lines demonstrated a >30-fold reduction of IC50 when comparing T-DXd to ADC control. In contrast, the ADC control IC50 was nearly identical to T-DXd across all pediatric cancer cell lines, consistent with the absence of HER2 amplification in these models. We observed focal and membranous staining of HER2 by IHC in PDXs but was quite variable across antibodies tested: WT 0-17% HER2+ cases (n=6), MRT 0-40% (n=5), DSRCT 0-33% (n=24), OS 0-7% (n=30). ERBB2 gene expression was highest in DSRCT followed by WT, OS and MRT. In vivo efficacy studies demonstrated complete and partial responses in OS, WT, and DSRCT and improved disease control rates (OS: 67%, p=0.006, Mann-Whitney; WT: 100%; DSRCT: 100%). However, T-DXd and ADC control demonstrated similar activity in all tumor types with no consistent correlation between in vivo response and HER2 expression. Consistent with these preclinical studies, 4 patients with progressive DSRCT were treated with T-DXd via compassionate/off-label access with signs of clinical and radiographic responses. Conclusions: T-DXd shows significant preclinical antitumor activity across multiple pediatric solid tumors but little correlation with HER2 expression suggesting a mechanism of action similar to the clinical activity observed in HER2-low breast cancer. Xenograft efficacy studies and preliminary clinical experience with T-DXd in DSRCT patients warrant formal clinical trial investigation in this largely incurable disease.

SYST-22 INTEGRATING GENE DEPENDENCY INTO A PEDIATRIC-FOCUSED CANCER DATA PORTAL FOR TARGETED THERAPY AND BIOMARKER DISCOVERY

Abstract Pediatric solid cancers, particularly those affecting the central nervous system (CNS), are the leading cause of disease-related deaths in children. Despite significant efforts to sequence pediatric solid tumors, they exhibit a "quiescent genome" with a considerably lower mutational burden than adult cancers. This leads to fewer actionable drug targets and poor response to cancer immunotherapy agents, including checkpoint inhibitors. As a result, it is imperative to move beyond genetic alterations and adopt a combination of functional and integrative approaches to develop novel, targeted treatments that are specifically designed for pediatric cancers. Over the past five years, the Next-Gen Precision Medicine Program team has devoted its efforts to establishing the Childhood Cancer Model Atlas (CCMA) (Sun, Daniel et al. Cancer Cell 2023). The CCMA is the largest collection of high-risk pediatric solid tumor cell lines globally, presenting a tremendous opportunity to integrate molecular features with functional dependencies using state-of-the-art computational approaches. However, the analysis of -omics level data is often limited to laboratories with specialized bioinformatics capabilities. Therefore, the establishment of the CCMA data portal with user-friendly analytical and visualization capabilities is essential to accelerate data-driven discovery and translational research in pediatric cancer on a global scale. The data portal is designed to simplify reporting, automate manual processes, and enhance the quality, usability, and longevity of the CCMA dataset. Its highlighted features concentrate on customized analysis, informative results, and user-friendly data visualization. Furthermore, by connecting the established models and their original tumor with clinical and molecular endpoints, the data portal enables clinical integration within the Australian molecular tumor board framework. This analytical data portal will enable the generation of new research hypotheses, prioritizes therapeutic opportunities, and supports international collaborative efforts in pediatric cancer.

Effect of dual properties of regorafenib in osteosarcoma on tumor progression and bone parameters in mouse preclinical models.

e23501 Background: Osteosarcoma (OS) constitutes the most frequent malignant primary bone tumor in children and young adults. Actual treatments in OS are based in a multimodal therapy that combines pre- and post-surgery polychemotherapies including cisplatin, doxorubicin, ifosfamide and/or methotrexate. Regardless the use of this polychemotherapeutic approach, the five years overall survival for patients has not improved during the last four decades and there is a clinical need to identify new active drugs in OS. An interesting strategy implies the use of compounds that showed therapeutic benefits in other solid tumors. Regorafenib, a small molecule with multi-target tyrosine kinase inhibitory activity against FGFR-1 and -2, KIT, PDGFR, RAF, RET, TIE2 and VEGFR-1, -2 and -3, showed an anti-angiogenic/oncogenic properties in various solid tumors that includes metastatic colorectal cancer, hepatocarcinoma and breast cancer. In addition, regorafenib also inhibits in vitro the proliferation of several pediatric solid tumor cell lines such as rhabdomyosarcoma, Ewing sarcoma and OS. Methods: The present work aims to decipher the in vitro and in vivo effects of regorafenib in OS preclinical models. Results: The treatment of MNNG-HOS, KHOS and 143B osteosarcoma cell lines with Regorafenib resulted in the inhibition of cell proliferation in a dose dependent manner. The gene expression profiles of MNNG-HOS and 143B cells treated or not with regorafenib and analyzed by NanoString technology revealed a specific regulation of molecular pathways by regorafenib in both cell lines. The in vivo effect of regorafenib was then analyzed in immunocompetent (mouse MOS-J cells inoculated in C57Bl/6 mice) and immunodeficient (human MNNG-HOS and 143B OS cells injected in Nude mice) murine osteosarcoma models. In all the cases, a significant reduction of primary tumor growth was observed as well as a significant decrease of lung metastases compared to untreated mice. Interestingly, the cortical and trabecular bone mineral density were significantly increased in regorafenib treated mice compared to untreated groups as demonstrated by X ray microtomodensitometry in all models. Conclusions: Our results support the use of regorafenib as an alternative treatment in metastatic OS and demonstrated for the first time the its bone protective effect.

Anacolosins A-F and Corymbulosins X and Y, Clerodane Diterpenes from Anacolosa clarkii Exhibiting Cytotoxicity toward Pediatric Cancer Cell Lines.

An extract of the plant Anacolosa clarkii was obtained from the NCI Natural Products Repository, and it showed cytotoxic activity toward several types of pediatric solid tumor cell lines. Bioassay-guided fractionation led to the purification of eight new clerodane diterpenes [anacolosins A-F (1-6) and corymbulosins X and Y (7 and 8)] and two known compounds (9 and 10) that contained an isozuelanin skeleton. The structures of the new natural products were determined using 1D and 2D NMR and HRESIMS data, while the relative and absolute configurations of the compounds were assessed using a combination of 1H NMR coupling constant data, ROESY experiments, ECD (electronic circular dichroism) and VCD (vibrational circular dichroism) spectroscopy, chemical methods (including Mosher and 2-naphthacyl esterification), and chiral HPLC analyses. The purified natural products exhibited a range of cytotoxic activities against cell lines representing four pediatric cancer types (i.e., rhabdomyosarcoma, Ewing sarcoma, medulloblastoma, and hepatoblastoma) with total growth inhibitory (TGI) values in the range 0.2-4.1 μM. The rhabdomyosarcoma and medulloblastoma cell lines showed higher sensitivity to compounds 1-4, which are the first compounds reported to contain an isozuelanin skeleton and feature keto carbonyl groups at the C-6 positions. In contrast, the hepatoblastoma cell line was modestly more sensitive to 7-10, which contained a C-6 hydroxy group moiety.

Abstract B36: A biomarker-guided approach to combining PARP inhibitors with radiotherapy in pediatric sarcomas

Abstract Background: Ewing sarcoma (EWS) expresses high levels of Schlafen-11 (SLFN11). SLFN11 disrupts checkpoint maintenance and may serve as a biomarker to assess sensitivity to Poly (ADP-ribose) polymerase 1 and 2 inhibitors (PARPi). The goal of this study is to evaluate SLFN11 protein expression in a panel of pediatric solid tumors and correlate levels of protein with sensitivity to PARP inhibition combined with ionizing radiation (IR), a component of therapy for many pediatric sarcomas and a potent inducer of DNA damage. Methods: SLFN11 mRNA and protein levels were assessed by quantitative RT-PCR, and immunohistochemistry, Western blot, and immunofluorescence microscopy, respectively. PARPi included talazoparib (TAL), niraparib (NIR), veliparib (VEL), and olaparib (OLA). Approximately 30 minutes after addition of systemic therapy, graded doses of radiation were delivered and viability across a panel of pediatric solid tumor cell lines was measured using the ATP-based Cell TiterGlo assay and confirmed with the colony formation assay. Results: We found that SLFN11 mRNA and protein is expressed at high levels in EWS, and SLFN11 is also variably present in a subset of other pediatric sarcomas, including desmoplastic small round cell tumor, osteosarcoma, and rhabdomyosarcoma. In all tumor cells with detectable SLFN11 expression, viability was reduced by greater than 90% when exposed to 2 Gy IR and 1-10 nM TAL, whereas cells with undetectable levels of SLFN11 were 5-10 times less sensitive. Intriguingly, variation in the potentiation between specific PARPi and IR correlated with the ability to form drug-induced PARP-DNA adducts, with the strong PARP trapper TAL showing ~10-fold higher potency compared to the moderate trapper NIR, and ~300-fold more potency relative to the weak trapper VEL. Consistent with our PARPi findings, the toposiomerase 1 inhibitor irinotecan, which also forms DNA adducts, potentiated with IR similarly to TAL at concentrations <10 nM in tumor cells expressing detectable levels of SLFN11. Conclusion: SLFN11 is present in select pediatric sarcomas and may induce a DNA repair defect that is best exploited by combining low doses of TAL and irinotecan with IR. Citation Format: Anang A. Shelat, Christopher L. Tinkle, Elizabeth Stewart, Sara Michele Federico, Brandon Bianski, Marcia Mellado-Lagarde. A biomarker-guided approach to combining PARP inhibitors with radiotherapy in pediatric sarcomas [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B36.

A biomarker-guided approach to combining PARP inhibitors with radiotherapy in pediatric solid tumors.

10556 Background: Ewing sarcoma (EWS) expresses high levels of Schlafen-11 (SLFN11). SLFN11 disrupts checkpoint maintenance and may serve as a biomarker to assess sensitivity to Poly (ADP-ribose) polymerase 1 and 2 inhibitors (PARPi). The goal of this study is to evaluate SLFN11 protein expression in a panel of pediatric solid tumors and correlate levels of protein with sensitivity to PARP inhibition combined with ionizing radiation (IR), a component of therapy for many pediatric solid tumors and a potent inducer of DNA damage. Methods: SLFN11 mRNA and protein levels were assessed by quantitative RT-PCR, and immunohistochemistry, Western blot, and immunofluorescence microscopy, respectively. PARPi included: talazoparib (TAL), niraparib (NIR), veliparib (VEL), and olaparib (OLA). Approximately 30 minutes after addition of systemic therapy, graded doses of radiation were delivered and viability across a panel of pediatric solid tumor cell lines was measured using the ATP-based Cell TiterGlo assay and confirmed with the colony formation assay. Results: We found that SLFN11 mRNA and protein is expressed at high levels in EWS, and SLFN11 is also variably present in a subset of other pediatric solid tumor lines, including desmoplastic small round cell tumor, osteosarcoma, and rhabdomyosarcoma. In all tumor cells with detectable SLFN11 expression, viability was reduced by greater than 90% when exposed to 2Gy IR and 1-10nM TAL, whereas cells with undetectable levels of SLFN11 were 5-10 times less sensitive. Intriguingly, variation in the potentiation between specific PARPi and IR correlated with the ability to form drug-induced PARP-DNA adducts, with the strong PARP trapper TAL showing ~10-fold higher potency compared to the moderate trapper NIR, and ~300-fold more potency relative to the weak trapper VEL. Consistent with our PARPi findings, the toposiomerase 1 inhibitor irinotecan, which also forms DNA adducts, potentiated with IR similarly to TAL at concentrations < 10nM in tumor cells expressing detectable levels of SLFN11. Conclusions: SLFN11 is present in select pediatric solid tumors and may induce a DNA repair defect that is best exploited by combining low-doses of TAL and irinotecan with IR.

Abstract 2816: Evaluation of Cdk5 inhibitors in neural crest tumors

Abstract Cyclin-dependent kinase 5 (Cdk5) is a ubiquitously expressed proline-directed serine threonine kinase active mainly in post-mitotic neurons due to abundant expression of its obligate activating partners p35 and/or p39. Cdk5 has been viewed narrowly as a neuron-specific kinase and as an essential regulator of neuronal function and migration. Recently, Cdk5 activity has been implicated in mediating tumor invasion, progression, and metastasis. We have found invariably high expression and activity of Cdk5/p35 in a series of pediatric tumor cell lines including neuroblastoma. To evaluate the potential role of Cdk5 inhibitors in the treatment of neural crest tumors, we evaluated the in vitro kinase activity and growth inhibition of 4 Cdk5 inhibitors (AT7519, dinaciclib, flavopiridol, and seliciclib). We compared the in vitro activity against Cdk2/cyclin E, Cdk5/p25, Cdk5/p35, Cdk7/cyclin H, and Cdk9/cyclin K. Using a DNA-based proliferation assay, we assessed the in vitro growth inhibition in a panel of 10 neural crest-derived tumors (melanoma, neuroblastoma, Ewing sarcoma). All Cdk5 inhibitors demonstrated enzyme and growth inhibition at clinically achievable concentrations. Dinaciclib was the most potent inhibitor of Cdk5/p35 with an enzyme IC50 of 0.0002 μM, compared to 0.003 μM for AT7519, 0.021 μM for flavopiridol, and 0.097 μM for seliciclib. Dinaciclib was also the most potent inhibitor of Cdk2. However, flavopiridol and AT7519 were the most potent inhibitors of Cdk7 and Cdk9, respectively. Across all cell lines, dinaciclib was the most potent inhibitor of growth. The median (range) growth inhibition IC50 for dinaciclib was 0.006 μM (0.004 - 0.14), compared to 0.079 μM (0.045-0.16) for flavopiridol, 0.52 μM (0.14 - >15) for AT7519, and 13.2 μM (7.8 - 22.1) for seliciclib. Interestingly, within the Ewing sarcoma cell lines, there was disparate sensitivity to the 2 most potent Cdk5 inhibitors, dinaciclib and AT7519. Based on the activity of these Cdk inhibitors in neural-crest tumors, we expanded our evaluation of AT7519 in 6 pediatric tumor cell lines (medulloblastoma, rhabdomyosarcoma, osteosarcoma) and found similar in vitro potency with a median (range) IC50 of 0.43 μM (0.20 - 1.26). AT7519, dinaciclib, flavopiridol, and seliciclib demonstrated in vitro growth inhibition in a panel of neural crest and pediatric solid tumor cell lines at clinically achievable concentrations. Further in vivo evaluations and in vitro combination studies including this novel class of agents in models of neural crest and select pediatric cancers are warranted. Citation Format: Robin E. Norris, John J. Pink, Tej Pareek, John L. Letterio. Evaluation of Cdk5 inhibitors in neural crest 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 2816.

Abstract B36: Enhancement of histone deacetylase inhibitor's efficacy with hydroxychloroquine via autophagy inhibition in pediatric tumor cell lines

Abstract Background: Histone deacetylase (HDAC) inhibitors, such as vorinostat, have been of interest in adult and pediatric cancers. Preclinical in vitro data has shown an effect in many different cancer cell types, but response has not correlated well in xenograft models. A concern is the effect of the HDAC inhibitors being compensated by autophagy. Autophagy is induced by multiple anticancer agents, as a tumor survival-promoting mechanism. Hydroxychloroquine, an autophagy inhibitor, treatment leads to the accumulation of autophagosomes, and accelerates tumor cell death. The hypothesis of this study is that by combining a HDAC inhibitor with hydroxychloroquine, there will be an improved anti-tumor response compared to HDAC inhibitor alone in pediatric cancer cell lines. Objectives: To determine if there is an improved efficacy of vorinostat when combined with hydroxychloroquine in pediatric solid tumor cell lines. Methods: Three pediatric cancer cell lines: Hepatoblastoma (Hep293TT), Ewing's Sarcoma (SKES1), and Medulloblastoma (D283) were treated with different concentrations of vorinostat or hydroxychloroquine, and cell viability was measured by CellTiter Glo assay. Dose-response curves were plotted to determine IC50 (inhibitory concentration 50%). The expression of proliferation and autophagy markers was determined by Western blot analysis. Results: The IC50 of vorinostat and hydroxychloroquine was in the rage of 0.5-1.25μM and 25-50μM, respectively. To evaluate the functional consequences, cells were treated with vorinostat and hydroxychloroquine alone and in combination for 72 hours. The combination treatment resulted in a dramatic decrease in cell viability, with these two drugs cooperating in a dose-dependent manner. Overall, in all three cell lines there was an improved response when vorinostat was combined with hydroxychloroquine. LC3, a mammalian homolog of yeast Atg8, is a reliable marker of autophagosomes, and tracking the conversion of LC3-I to LC3-II is indicative of autophagy blockade. Hydroxychloroquine treatment resulted in profound conversion of LC3-I to LC3-II, and this effect was greater in cells treated with both vorinostat and hydroxychloroquine alone. Therefore, hydroxychloroquine blocked autophagic flux, which was more pronounced in the setting of autophagy induction by vorinostat. Finally, exposure to the combination of vorinostat and hydroxychloroquine resulted in decreased levels of phospho-ERK compared to single-agent treatment, indicating reduced cell proliferation. Taken together, our data demonstrate that when autophagy was inhibited by hydroxychloroquine in the presence of vorinostat, autophagosomes accumulated and cell viability/proliferation reduced. Conclusion: Preclinical studies in the different pediatric cancer cell lines show a clear and improved response when vorinostat is combined with hydroxychloroquine compared to vorinostat alone. The increased LC3-II/I ratio indicative of efficient autophagy inhibition while the decrease in the phospho-ERK signifies a decrease in proliferation. Furthermore the IC50 concentration of vorinostat, about 200ng/ml, necessary for effect is well within the achievable clinical range of 150-500ng/ml. The next step would be to validate these results in a xenograft model with one of the pediatric cancer tumors, and then hopefully continue forward to build a phase 1 clinical trial in recurrent pediatric solid tumors. This combination of drugs has yet to be explored in pediatrics but has the potential to enhance the effect of an established chemotherapy agent with a well-known and easily accessible non-cancer drug. Citation Format: Abigail Joy Cruz, Hima Bansal, Gail Tomlinson, Sanjay Bansal, Steven Weitman. Enhancement of histone deacetylase inhibitor's efficacy with hydroxychloroquine via autophagy inhibition in pediatric tumor cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B36.

Nab-Paclitaxel Is an Active Drug in Preclinical Model of Pediatric Solid Tumors

To investigate the antitumor effect of nab-paclitaxel, an albumin-stabilized nanoparticle formulation of paclitaxel, on pediatric solid tumor models. A panel of three rhabdomyosarcoma, one osteosarcoma and seven neuroblastoma cell lines were exposed to increasing concentrations of nab-paclitaxel in vitro. Cell viability was evaluated using the Alamar Blue Assay. Antitumor effect was further assessed in vivo in NOD/SCID xenograft and metastatic neuroblastoma mouse models. Tumor sections were analyzed by immunohistochemistry for cleaved caspase-3 and phospho-histone H3. Plasma and intratumoral paclitaxel concentrations were measured by liquid chromatography-mass spectrometry. Ratio of intratumoral and plasma concentration was compared between nab-paclitaxel and paclitaxel treatment groups. Nab-paclitaxel displayed significant cytotoxicity against most pediatric solid tumor cell lines in vitro in a dose-dependent manner. In vivo, nab-paclitaxel showed antitumor activity in both rhabdomyosarcoma (RH4 and RD) and neuroblastoma [SK-N-BE(2) and CHLA-20] xenograft models. In the SK-N-BE(2) metastatic model, nab-paclitaxel treatment significantly extended animal survival compared with control (P < 0.01). Nab-paclitaxel treatment induced tumor cell-cycle arrest and apoptosis in vivo. In the RH4 model, increased local relapse-free intervals were observed with nab-paclitaxel treatment (37.7 ± 3.2 days) comparing with paclitaxel (13.6 ± 2.07 days). Local relapsed tumors following paclitaxel treatment proved to be paclitaxel-resistant and remained responsive to nab-paclitaxel. Mechanistically, a higher tumor/plasma paclitaxel drug ratio in favor of nab-paclitaxel was observed. Nab-paclitaxel showed significant antitumor activity against all pediatric solid tumors associated with an enhanced drug intratumor delivery. Furthermore, testing of nab-paclitaxel in pediatric solid-tumor patient population is under development.

Preclinical evaluation of the PARP inhibitor, olaparib, in combination with cytotoxic chemotherapy in pediatric solid tumors

Poly(ADP-ribose) polymerase (PARP) signals DNA damage and facilitates DNA repair. PARP inhibitors are being evaluated in cancers with defective DNA repair mechanisms or in combination with cytotoxic therapy or radiation. We evaluated the PARP inhibitor, olaparib, in combination with chemotherapy using in vitro and in vivo pediatric solid tumor models. The IC50 of olaparib alone and in combination with cytotoxic agents was determined in 10 pediatric solid tumor cell lines. Synergy was assessed using the combination index of Chou-Talalay. Olaparib alone and in combination with topotecan/cyclophosphamide was evaluated in xenograft models of Ewing sarcoma (RD-ES) and neuroblastoma (NGP). PAR activity was evaluated in cell lines and tumor lysates. Olaparib induced growth inhibition, median (range) IC50 = 3.6 (1-33.8) µM, and inhibited PAR activity in pediatric solid tumor cell lines. The addition of olaparib to DNA damaging agents resulted in additive to synergistic interactions. In RD-ES and NGP xenografts, olaparib inhibited PAR activity by 88-100% as a single agent and 100% when administered with cyclophosphamide/topotecan. Although the addition of olaparib did not antagonize the activity of cyclophosphamide/topotecan, clear evidence of synergy could not be demonstrated. In pediatric solid tumor cell lines, clinically achievable concentrations of single agent olaparib caused growth inhibition. Although the in vitro data demonstrated synergistic efficacy of olaparib when added to the camptothecins and alkylating agents, synergy was not discernible in vivo. Clinical trials of PARP inhibitors in combination DNA damaging agents are necessary to establish the role of PARP inhibitors in childhood cancer.

Natural killer cells can exert a graft-vs-tumor effect in haploidentical stem cell transplantation for pediatric solid tumors

Little progress has been made with regard to the survival of children with metastatic and refractory solid tumors. Preliminary data from haploidentical stem cell transplantation (haplo-SCT) suggested a clinically beneficial allograft-vs-tumor effect associated with natural killer cell (NK) donor-recipient mismatch. We hypothesized that interaction between activatory receptors on NK cells and their ligands on tumor cells could be also important. To evaluate the NK-cell-mediated allograft-vs-tumor effect, we conducted a pilot study of haplo-SCT on six children with refractory solid tumors. Our specific goal for this study was NKG2D-major histocompatibility complex class I-related chain A interaction. Tasks include specific immunoassays that support haplo-SCT in refractory solid tumors. Patients suffered from neuroblastoma (n = 1), Ewing sarcoma (n = 2), a desmoplastic tumor (n = 1), nasopharyngeal carcinoma (n = 1), and embryonal rhabdomyosarcoma (n = 1). Pretransplantation disease status showed progressive disease in 2 patients, partial remission in 2 patients, and complete remission in 2 patients. NK-cell mismatch was present in three donor-recipients. Ligands for NKG2D receptors, major histocompatibility complex class I-related chain A and UL16 binding protein 2 were overexpressed in six of six and four of six tumors, respectively. NK cells led early immune reconstitution. After haplo-SCT, three patients were in complete remission, one patient showed partial remission, and two patients were in stable disease. With a median follow-up of 14 months, three patients were alive and in complete remission, and three patients had died; two due to progressive disease and one of transplant-related toxicity. Blocking NKG2D-major histocompatibility complex class I-related chain A interaction in vitro reduced NK-cell cytotoxicity. Our preliminary results suggest a beneficial effect from haplo-SCT in refractory solid tumors.

Preclinical evaluation of pemetrexed in pediatric solid tumors

Renewed interest in antifols for the treatment of childhood cancers has resulted from identification of novel antifols with broad spectrums of anti-cancer activity. We evaluated the in vitro cytotoxicity of methotrexate and pemetrexed in a panel of 12 pediatric solid tumor cell lines using the sulforhodamine-B assay. The Ewing sarcoma (ES) cell lines demonstrated the greatest sensitivity to both methotrexate and pemetrexed. Expression of folate pathway genes (DHFR, TS, GARFT, GGH) did not correlate with in vitro drug sensitivity. Further evaluation of pemetrexed for the treatment of ES is warranted.

Abstract 5357: Pediatric preclinical evaluation of the PARP inhibitor olaparib

Abstract Purpose: Olaparib (AZD2281) is an oral poly(ADP)-ribose polymerase (PARP) inhibitor in clinical development as a single agent and in combination with cytotoxic therapy in adults with solid malignancies. In BRCA-1 deficient tumors, olaparib concentrations of 30 nM result in significant PARP inhibition (Rottenberg, PNAS, 2008). Adult patients appear to tolerate olaparib doses associated with a Cmax of 10 µM (Fong, NEJM, 2009). We evaluated the interaction of olaparib with commonly used cytotoxic drugs in a panel of pediatric solid tumor cell lines. Methods: In vitro growth inhibition was assessed using the sulforhodamine B assay in pediatric solid tumor cell lines including: osteosarcoma (MG-63), neuroblastoma (NGP), Ewings sarcoma (RD-ES), medulloblastoma (Daoy), and rhabdomyosarcoma (RD). IC50's were determined following a 120 h exposure to vehicle, olaparib, cytotoxic agent (carboplatin, melphalan, irinotecan, doxorubicin, vincristine) or olaparib in combination with each cytotoxic agent. The combination index (CI) of Chou-Talalay was used to test for synergy of combinations. Results: As a single agent, olaparib was cytotoxic in all cell lines studied, with a median (range) IC50 of 1.7 (0.3-5.3) μM. The Ewings sarcoma cell line was the most sensitive line with a mean (SD) IC50 of 0.7 (0.3) μM. The combination of olaparib with melphalan or irinotecan was synergistic (CI &amp;lt; 1), while the addition of olaparib to doxorubicin or carboplatin appeared additive to synergistic (CI &amp;lt; 1). The combination of olaparib and vincristine trended toward antagonism (CI &amp;gt; 1). Conclusions: The addition of olaparib to DNA damaging agents resulted in additive or synergistic growth inhibition in a panel of pediatric solid tumor cell lines. Further preclinical and clinical evaluation to determine if olaparib can enhance the efficacy of current pediatric chemotherapy regimens is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5357. doi:10.1158/1538-7445.AM2011-5357

Human neuroblastoma cells rapidly enter cell cycle arrest and apoptosis following exposure to C-28 derivatives of the synthetic triterpenoid CDDO

Synthetic triterpenoids, such as 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivatives, are an extremely potent class of new anti-cancer therapeutic agents, characterized by high anti-tumor potency and low toxicity to normal tissues. This report is the first to investigate the effects of C-28 derivatives of CDDO on 22 pediatric solid tumor cell lines, including neuroblastoma, rhabdomyosarcoma, osteosarcoma, and Ewing's sarcoma. We determined IC50s in the range of 5 – 170 nM for inhibition of colony formation and DNA synthesis, and 110 – 630 nM for metabolic cell death and decrease in cell number, using the C-28 CDDO analogs, CDDO methyl ester (CDDO-Me), CDDO imidazolide (CDDO-Im), CDDO ethyl amide (CDDO-EA), CDDO trifluoroethyl amide (CDDO-TFEA), and CDDO diethylamide (CDDO-DE). After treatment of human neuroblastoma cells with CDDO-Me, cell cycle studies show depletion of the S-phase, while apoptosis studies show conformational activation and mitochondrial translocation of Bax protein, as well as activation of caspases -3 and -8. These data demonstrate the potential utility of CDDO analogs as promising novel therapeutic agents for high-risk pediatric solid tumors.

Identification of side population cells (stem-like cell population) in pediatric solid tumor cell lines

Recent evidence has supported the cancer stem cell theory that cancer contains a small number of cancer stem cells (CSC) as a reservoir of cancer cells. Only the CSC, but not most of the remaining constituent cancer cells, are thought to be responsible for tumorigenesis, progression, and metastasis as well as cancer relapse, suggesting that the CSC should be targeted to eradicate the cancer. Side population (SP) cells isolated by fluorescence-activated cell sorting (FACS) using Hoechst dye are known to be enriched in stem cells in various normal tissues as well as cancers. The authors investigated whether such stem-like SP cells may exist in pediatric solid tumors (PSTs). Sixteen pediatric tumor cell lines including 7 neuroblastomas, 4 rhabdomyosarcomas, and 5 Ewing's sarcomas were used for FACS analysis. Analysis of SP cells based on the exclusion of the DNA binding dye, Hoechst 33342, with and without verapamil using FACS was performed. One Ewing's sarcoma cell line did not show an SP fraction, and only a small fraction of SP cells (0.12%-14.6%) was detected in the other 15 cell lines. These SP cells were all sensitive to verapamil. This study suggested that most PSTs would contain a small fraction of SP cells (possible stem-like population). Targeting the CSC will provide a novel treatment strategy to eradicate refractory PSTs.

Aberrations of the CHK2 gene are rare in pediatric solid tumors

In pediatric solid tumors, such as neuroblastoma (NB), it has been reported that the frequency of TP53 gene alterations is lower than that in adult tumors, suggesting that other tumor suppressor genes may play more important roles in the development of pediatric solid tumors. The CHK2 gene, whose product is a checkpoint kinase that plays a central role in DNA damage response and acts upstream of TP53, has been found to be mutated in a subset of Li-Fraumeni syndrome without mutations of TP53 and in some other sporadic human tumors, earmarking this serine/threonine kinase as a candidate tumor suppressor gene. Thus, we analyzed the CHK2 gene to address whether it is a candidate tumor suppressor gene for pediatric solid tumors. We screened for mutations of the CHK2 gene in 25 NB, 8 rhbdomyosarcoma, 12 Ewing sarcoma, and 26 other pediatric solid tumor cell lines as well as 77 fresh tumors including two cases of multiple cancers. Using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis and reverse transcriptase (RT)-PCR-SSCP followed by direct sequence analysis, we detected only one missense mutation (S505T) in one NB cell line and two silent mutations in one NB cell line and one NB fresh tumor, respectively. Through RT-PCR and subcloning analysis, we detected a similar expression of the CHK2 gene in all of the NB cell lines and fresh tumors; however, we identified at least three isoforms of the CHK2 gene, two of which have not been reported previously. These results suggest that aberrations of the CHK2 gene are rare in pediatric solid tumors.

Genomic structure and mutational analysis of the human KIF1Bα gene located at 1p36.2 in neuroblastoma

KIF1a is a member of the kinesin superfamily proteins that are microtubule-dependent molecular motors involved in important intracellular functions such as organelle transport and cell division. We previously determined the structure of the human KIF1Bbeta gene, which was found to be a homologue of the murine Kif1bbeta, and demonstrated that the human KIF1Bbeta is a causative gene of Charcot-Marie-Tooth disease type 2A although we did not prove that it is a tumor suppressor gene of neuroblastoma. Here, we identified another isoform of the human KIF1B gene, KIF1Balpha. The KIF1Balpha and KIF1Bbeta are alternative splicing products of the KIF1B gene located on 1p36.2. The KIF1Balpha is distinct from KIF1Bbeta in the C-terminal cargo-binding domain; however, they have the same N-terminal motor domain. We found that the transcript of approximately 7.8 kb of KIF1Balpha was expressed in several tissues, especially in skeletal muscle, by Northern blot analysis. To determine whether this gene is one of the candidate tumor suppressor genes for neuroblastoma (NB) or other pediatric solid tumors, we performed mutational screening of KIF1Balpha in 25 NB, 9 rhabdomyosarcoma, 12 Ewing sarcoma and 24 other pediatric solid tumor cell lines. Using RT-PCR single-strand conformation polymorphism analysis and direct sequencing we detected a missense mutation (M807I) in 1 NB cell line (SK-N-SH), 3 silent mutations in 2 NB cell lines and 1 primitive neuroectodermal tumor cell line, respectively. RT-PCR analysis revealed that KIF1Balpha was obviously expressed in almost all of the tumor cell lines examined except NB-1. Furthermore, real-time quantitative RT-PCR showed that there was no significant difference in KIF1Balpha expression between 14 early-stage (stage I and II) and 14 advanced-stage (stage III and IV) NB fresh tumor specimens. These results suggest that KIF1Ba in addition to KIF1Bbeta may not be a candidate tumor suppressor gene for NB.

MGI 114: augmentation of antitumor activity when combined with topotecan.

6-Hydroxymethylacylfulvene (HMAF; MGI 114; Irofulven) is a semisynthetic analogue of the mushroom toxin illudin S that has been shown to be a potent cytotoxic agent with an improved therapeutic index compared with its parent compound. The studies were conducted to evaluate the antitumor activity of MGI 114 as a single agent and in combination with topotecan against pediatric solid tumor cell lines and xenograft models. In vitro studies were designed to determine the cytotoxic potential of MGI 114 using the MTT assay and 13 pediatric tumor cell lines. In addition, combination in vitro studies were performed with MGI 114 and topotecan to generate isoeffect plots. Single agent and combination in vivo studies were also performed using MGI 114 against rhabdomyosarcoma and neuroblastoma xenograft models. After a 1-hour exposure to MGI 114, the mean IC50 (+/-standard error of mean) for medulloblastoma, neuroblastoma, Ewing sarcoma/primitive neuroectodermal tumor, and rhabdomyosarcoma cell lines were 1.58+/-0.51, 1.60+/-0.82, 1.18+/-0.08, and 3.99+/-1.69 microg/mL, respectively. When tumor cells were exposed concurrently to MGI 114 and topotecan, evidence of synergy was observed in 10 of 12 (83%) cell lines. Single agent and combination in vivo studies with MGI 114 showed that this agent had substantial, and at times curative, antitumor activity against rhabdomyosarcoma and neuroblastoma xenograft tumors. These data suggest that MGI 114 has significant efficacy as a single agent in preclinical studies against pediatric tumors. In addition, based on previous reports and the results presented here, combining MGI 114 with topotecan appears to be an attractive approach to the treatment of pediatric malignancies. After completion of the pediatric phase I studies of MGI 114, consideration should be given to phase II single agent and phase I combination studies with a topoisomerase I inhibitor such as topotecan or irinotecan.