Cancer drugs with high repositioning potential for Alzheimer's disease.

INTRODUCTION: Despite the recent full FDA approval of lecanemab, there is currently no disease modifying therapy (DMT) that can efficiently slow down the progression of Alzheimer's disease (AD) in the general population. This statement emphasizes the need to identify novel DMTs in the shortest time possible to prevent a global epidemic of AD cases as the world population experiences an increase in lifespan. AREAS COVERED: Here, we review several classes of anti-cancer drugs that have been or are being investigated in Phase II/III clinical trials for AD, including immunomodulatory drugs, RXR agonists, sex hormone therapies, tyrosine kinase inhibitors, and monoclonal antibodies. EXPERT OPINION: Given the overall course of brain pathologies during the progression of AD, we express a great enthusiasm for the repositioning of anti-cancer drugs as possible AD DMTs. We anticipate an increasing number of combinatorial therapy strategies to tackle AD symptoms and their underlying pathologies. However, we strongly encourage improvements in clinical trial study designs to better assess target engagement and possible efficacy over sufficient periods of drug exposure.

Novel insight into the role of clusterin on intraocular pressure regulation by modifying actin polymerization and extracellular matrix remodeling in the trabecular meshwork.

This study provides comprehensive mechanistic evidence for the role of clusterin, a stress-response secretory chaperone protein, in the modulation of intraocular pressure (IOP) by regulating the trabecular meshwork (TM) actin cytoskeleton and the extracellular matrix (ECM). The pathological stressors on TM known to elevate IOP significantly lowered clusterin protein levels indicating stress-related clusterin function loss. Small interfering RNA-mediated clusterin loss in human TM cells in vitro induced actin polymerization and stabilization via protein kinase D1, serine/threonine-protein kinase N2 (PRK2), and LIM kinase 1 (LIMK1), and the recruitment and activation of adhesome proteins including paxillin, vinculin, and integrin αV and ß5. A complete loss of clusterin as seen in clusterin knockout mice (Clu-/- ) led to significant IOP elevation at postnatal Day 70. Contrarily, constitutive clusterin expression using adenovirus (AdCLU) in HTM cells resulted in the loss of actin polymerization via decreased PRK2, and LIMK1 and negative regulation of integrin αV and ß5. Furthermore, we found that AdCLU treatment in HTM cells significantly decreased the ECM protein expression and distribution by significantly increasing matrix metalloprotease 2 (MMP2) activity and lowering the levels of pro-fibrotic proteins such as transforming growth factor-ß2 (TGFß2), thrombospondin-1 (TSP-1), and plasminogen activator inhibitor-1 (PAI-1). Finally, we found that HTM cells supplemented with recombinant human clusterin attenuated the pro-fibrotic effects of TGFß2. For the first time this study demonstrates the importance of clusterin in the regulation of TM actin cytoskeleton - ECM interactions and the maintenance of IOP, thus making clusterin an interesting target to reverse elevated IOP.

Development of cell-based high throughput luminescence assay for drug discovery in inhibiting OCT4/DNA-PKcs and OCT4-MK2 interactions.

Amplification-independent c-MYC overexpression is suggested in multiple cancers. Targeting c-MYC activity has therapeutic potential, but efforts thus far have been mostly unsuccessful. To find a druggable target to modulate c-MYC activity in cancer, we identified two kinases, MAPKAPK2 (MK2) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which phosphorylate the Ser111 and the Ser93 residues of OCT4, respectively, to transcriptionally activate c-MYC. Using these observations, we present here a novel cell-based luminescence assay to identify compounds that inhibit the interaction between these kinases and OCT4. After screening approximately 80,000 compounds, we identified 56 compounds ("hits") that inhibited the luminescence reaction between DNA-PKcs and OCT4, and 65 hits inhibiting the MK2-OCT4 interaction. Using custom antibodies specific for pOCT4S93 and pOCT4S111 , the "hits" were validated for their effect on OCT4 phosphorylation and activation. Using a two-step method for validation, we identified two candidate compounds from the DNA-PKcs assay and three from the MK2 assay. All five compounds demonstrate a significant ability to kill cancer cells in the nanomolar range. In conclusion, we developed a cell-based luminescence assay to identify novel inhibitors targeting c-MYC transcriptional activation, and have found five compounds that may function as lead compounds for further development.

Vorinostat and fenretinide synergize in preclinical models of T-cell lymphoid malignancies.

T-cell lymphoid malignancies (TCLMs) are in need of novel and more effective therapies. The histone deacetylase (HDAC) inhibitors and the synthetic cytotoxic retinoid fenretinide have achieved durable clinical responses in T-cell lymphomas as single agents, and patients who failed prior HDAC inhibitor treatment have responded to fenretinide. We have previously shown fenretinide synergized with the class I HDAC inhibitor romidepsin in preclinical models of TCLMs. There exist some key differences between HDAC inhibitors. Therefore, we determined if the pan-HDAC inhibitor vorinostat synergizes with fenretinide. We demonstrated cytotoxic synergy between vorinostat and fenretinide in nine TCLM cell lines at clinically achievable concentrations that lacked cytotoxicity for non-malignant cells (fibroblasts and blood mononuclear cells). In vivo, vorinostat + fenretinide + ketoconazole (enhances fenretinide exposures by inhibiting fenretinide metabolism) showed greater activity in subcutaneous TCLM xenograft models than other groups. Fenretinide + vorinostat increased reactive oxygen species (ROS, measured by 2',7'-dichlorodihydrofluorescein diacetate dye), resulting in increased apoptosis (via transferase dUTP nick end labeling assay) and histone acetylation (by immunoblotting). The synergistic cytotoxicity, apoptosis, and histone acetylation of fenretinide + vorinostat was abrogated by the antioxidant vitamin C. Like romidepsin, vorinostat combined with fenretinide achieved synergistic cytotoxic activity and increased histone acetylation in preclinical models of TCLMs, but not in non-malignant cells. As vorinostat is an oral agent and not a P-glycoprotein substrate it may have advantages in such combination therapy. These data support conducting a clinical trial of vorinostat combined with fenretinide in relapsed and refractory TCLMs.

Tumor-Associated Macrophages as Multifaceted Regulators of Breast Tumor Growth.

Breast cancer is the most commonly occurring cancer in women of Western countries and is the leading cause of cancer-related mortality. The breast tumor microenvironment contains immune cells, fibroblasts, adipocytes, mesenchymal stem cells, and extracellular matrix. Among these cells, macrophages or tumor-associated macrophages (TAMs) are the major components of the breast cancer microenvironment. TAMs facilitate metastasis of the breast tumor and are responsible for poor clinical outcomes. High TAM density was also found liable for the poor prognosis of breast cancer. These observations make altering TAM function a potential therapeutic target to treat breast cancer. The present review summarizes the origin of TAMs, mechanisms of macrophage recruitment and polarization in the tumor, and the contributions of TAMs in tumor progression. We have also discussed our current knowledge about TAM-targeted therapies and the roles of miRNAs and exosomes in re-educating TAM function.

Deep neuromuscular blockade during spinal surgery reduces intra-operative blood loss: A randomised clinical trial.

BACKGROUND: Spinal surgery is usually performed in the prone position using a posterior approach. However, the prone position may cause venous engorgement in the back and thus increase surgical bleeding with interruption of surgery. The prone position also affects cardiac output since large vessels are compressed decreasing venous return to the heart. OBJECTIVE: We hypothesised that deep neuromuscular blockade would be associated with less surgical bleeding during spinal surgery in the prone position. DESIGN: Randomised, single blinded trial. SETTING: University teaching hospital. PARTICIPANTS: Eighty-eight patients in two groups. INTERVENTIONS: Patients were randomly assigned to moderate neuromuscular blockade or deep neuromuscular blockade. In the moderate neuromuscular blockade group, administration of rocuronium was adjusted such that the train-of-four count was one to two. In the deep neuromuscular blockade group, rocuronium administration was adjusted such that the train-of-four count was zero with a posttetanic count 2 or less. MAIN OUTCOME MEASURES: The primary outcome was the volume of intra-operative surgical bleeding. The surgeon's satisfaction with operating conditions, haemodynamic and respiratory status, and postoperative pain scores were evaluated. RESULTS: The median [IQR] volume of intra-operative surgical bleeding was significantly less in the deep neuromuscular blockade group than in the moderate neuromuscular blockade group; 300 ml [200 to 494] vs. 415 ml [240 to 601]; difference: 117 ml (95% CI, 9 to 244; P = 0.044). The mean ±â€ŠSD surgeon's satisfaction with the intra-operative surgical conditions was greater in the deep neuromuscular blockade group than in the moderate neuromuscular blockade group; 3.5 ±â€Š1.0 vs. 2.9 ±â€Š0.9 (P = 0.004). In intergroup comparisons of respiratory variables, peak inspiratory pressure was lower in the deep neuromuscular blockade group overall (P < 0.001). The median [IQR] postoperative pain score was lower in the deep neuromuscular blockade group than the moderate neuromuscular blockade group; 50 [36 to 60] vs. 60 [50 to 70], (P = 0.023). CONCLUSION: Deep neuromuscular blockade reduced intra-operative surgical bleeding in patients undergoing spinal surgery. This may be related to greater relaxation in the back muscles and lower intra-operative peak inspiratory pressure when compared with moderate neuromuscular blockade. TRIAL REGISTRATION: KCT0001264 (http://cris.nih.go.kr).

Cytotoxicity and molecular activity of fenretinide and metabolites in T-cell lymphoid malignancy, neuroblastoma, and ovarian cancer cell lines in physiological hypoxia.

OBJECTIVE: All-trans-N-(4-hydroxyphenyl)retinamide or fenretinide (4-HPR) acts by reactive oxygen species (ROS) and dihydroceramides (DHCers). In early-phase clinical trials 4-HPR has achieved complete responses in T-cell lymphomas (TCL) and neuroblastoma (NB) and signals of activity in ovarian cancer (OV). We defined the activity of 4-HPR metabolites in N-(4-methoxyphenyl)retinamide (MPR), 4-oxo-N-(4-hydroxyphenyl)retinamide (oxoHPR), and the 4-HPR isomer 13-cis-fenretinide (cis-HPR) in NB, OV, and TCL cell lines cultured in physiological hypoxia. METHODS: We compared the effect of 4-HPR, cis-HPR, oxoHPR, and MPR on cytotoxicity, ROS, and DHCers in a panel of TCL, NB, and OV cell lines cultured in bone marrow level physiological hypoxia (5% O2), utilizing a fluorescence-based cytotoxicity assay (DIMSCAN), flow cytometry, and quantitative mass spectrometry. RESULTS: 4-HPR (10 µmol/l) achieved more than three logs of cell kill in nine of 15 cell lines. Cytotoxicity of 4-HPR and oxoHPR was comparable; in some cell lines, cis-HPR cytotoxicity was lower than 4-HPR, but additive when combined with 4-HPR. MPR was not cytotoxic. ROS and DHCers were equivalently increased by 4-HPR and oxoHPR in all cell lines (P<0.01), to a lesser extent by cis-HPR (P<0.01), and not increased in response to MPR (P>0.05). Mitochondrial membrane depolarization, caspase-3 cleavage, and apoptosis (TUNEL) were all significantly increased by 4-HPR and oxoHPR (P<0.01). CONCLUSION: Cytotoxic and pharmacodynamic activity was comparable with 4-HPR and oxoHPR, lower with cis-HPR, and MPR was inactive. Neither MPR or cis-HPR antagonized 4-HPR activity. These data support focusing on achieving high 4-HPR exposures for maximizing antineoplastic activity.

Cytotoxic activity of difluoromethylornithine compared with fenretinide in neuroblastoma cell lines.

BACKGROUND: Maintenance therapy with 13-cis-retinoic acid and immunotherapy (given after completion of intensive cytotoxic therapy) improves outcome for high-risk neuroblastoma patients. The synthetic retinoid fenretinide (4-HPR) achieved multiple complete responses in relapse/refractory neuroblastoma in early-phase clinical trials, has low systemic toxicity, and has been considered for maintenance therapy clinical trials. Difluoromethylornithine (DFMO, an irreversible inhibitor of ornithine decarboxylase with minimal single-agent clinical response data) is being used for maintenance therapy of neuroblastoma. We evaluated the cytotoxic activity of DFMO and fenretinide in neuroblastoma cell lines. PROCEDURE: We tested 16 neuroblastoma cell lines in bone marrow-level hypoxia (5% O2 ) using the DIMSCAN cytotoxicity assay. Polyamines were measured by HPLC-mass spectrometry and apoptosis by transferase dUTP nick end labeling (TUNEL) using flow cytometry. RESULTS: At clinically achievable levels (100 µM), DFMO significantly decreased (P < 0.05) polyamine putrescine and achieved modest cytotoxicity (<1 log (90% cytotoxicity). Prolonged exposures (7 days) or culture in 2% and 20% O2 did not enhance DFMO cytotoxicity. However, fenretinide (10 µM) even at a concentration lower than clinically achievable in neuroblastoma patients (20 µM) induced ≥ 1 log cell kill in 14 cell lines. The average IC90 and IC99 of fenretinide was 4.7 ± 1 µM and 9.9 ± 1.8 µM, respectively. DFMO did not induce a significant increase (P > 0.05) in apoptosis (TUNEL assay). Apoptosis by fenretinide was significantly higher (P < 0.001) compared with DFMO or controls. CONCLUSIONS: DFMO as a single agent has minimal cytotoxic activity for neuroblastoma cell lines.

Phase I study of vorinostat in combination with isotretinoin in patients with refractory/recurrent neuroblastoma: A new approaches to Neuroblastoma Therapy (NANT) trial.

BACKGROUND: Vorinostat combined with retinoids produces additive antitumor effects in preclinical studies of neuroblastoma. Higher systemic exposures of vorinostat than achieved in pediatric phase I trials with continuous daily dosing are necessary for in vivo increased histone acetylation and cytotoxic activity. We conducted a phase I trial in children with relapsed/refractory neuroblastoma to determine the maximum tolerated dose (MTD) of vorinostat on an interrupted schedule, escalating beyond the previously identified pediatric MTD. METHODS: Isotretinoin (cis-13-retinoic acid) 80 mg/m2 /dose was administered by mouth twice daily on days 1-14 in combination with escalating doses of daily vorinostat up to 430 mg/m2 /dose (days 1-4; 8-11) in each 28-day cycle using the standard 3 + 3 design. Vorinostat pharmacokinetic testing and histone acetylation assays were performed. RESULTS: Twenty-nine patients with refractory or relapsed neuroblastoma were enrolled and 28 were evaluable for dose escalation decisions. Median number of cycles completed was two (range 1-15); 11 patients received four or more cycles. Three patients experienced cycle 1 dose-limiting toxicities. A total of 18 patients experienced grade 3/4 toxicities related to study therapy. The maximum intended dose of vorinostat (430 mg/m2 /day, days 1-4; 8-11) was tolerable and led to increased histone acetylation in surrogate tissues when compared to lower doses of vorinostat (P = 0.009). No objective responses were seen. CONCLUSIONS: Increased dose vorinostat (430 mg/m2 /day) on an interrupted schedule is tolerable in combination with isotretinoin. This dose led to increased vorinostat exposures and demonstrated increased histone acetylation. Prolonged stable disease in patients with minimal residual disease warrants further investigation.

P450 inhibitor ketoconazole increased the intratumor drug levels and antitumor activity of fenretinide in human neuroblastoma xenograft models.

We previously reported that concurrent ketoconazole, an oral anti-fungal agent and P450 enzyme inhibitor, increased plasma levels of the cytotoxic retinoid, fenretinide (4-HPR) in mice. We have now determined the effects of concurrent ketoconazole on 4-HPR cytotoxic dose-response in four neuroblastoma (NB) cell lines in vitro and on 4-HPR activity against two cell line-derived, subcutaneous NB xenografts (CDX) and three patient-derived NB xenografts (PDX). Cytotoxicity in vitro was assessed by DIMSCAN assay. Xenografted animals were treated with 4-HPR/LXS (240 mg/kg/day) + ketoconazole (38 mg/kg/day) in divided oral doses in cycles of five continuous days a week. In one model, intratumoral levels of 4-HPR and metabolites were assessed by HPLC assay, and in two models intratumoral apoptosis was assessed by TUNEL assay, on Day 5 of the first cycle. Antitumor activity was assessed by Kaplan-Meier event-free survival (EFS). The in vitro cytotoxicity of 4-HPR was not affected by ketoconazole (p ≥ 0.06). Ketoconazole increased intratumoral levels of 4-HPR (p = 0.02), of the active 4-oxo-4-HPR metabolite (p = 0.04), and intratumoral apoptosis (p ≤ 0.0006), compared to 4-HPR/LXS-alone. Concurrent ketoconazole increased EFS in both CDX models compared to 4-HPR/LXS-alone (p ≤ 0.008). 4-HPR + ketoconazole also increased EFS in PDX models compared to controls (p ≤ 0.03). Thus, concurrent ketoconazole decreased 4-HPR metabolism with resultant increases of plasma and intratumoral drug levels and antitumor effects in neuroblastoma murine xenografts. These results support the clinical testing of concurrent ketoconazole and oral fenretinide in neuroblastoma.

Initial testing (stage 1) of the curaxin CBL0137 by the pediatric preclinical testing program.

BACKGROUND: CBL0137 is a novel drug that modulates FAcilitates Chromatin Transcription (FACT), resulting in simultaneous nuclear factor-κB suppression, heat shock factor 1 suppression and p53 activation. CBL0137 has demonstrated antitumor effects in animal models of several adult cancers and neuroblastoma. PROCEDURES: CBL0137 was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro cell line panel at concentrations ranging from 1.0 nM to 10.0 µM and against the PPTP in vivo solid tumor xenograft and acute lymphocytic leukemia (ALL) panels at 50 mg/kg administered intravenously weekly for 4 weeks. RESULTS: The median relative IC50 (rIC50 ) value for the PPTP cell lines was 0.28 µM (range: 0.13-0.80 µM). There were no significant differences in rIC50 values by histotype. CBL0137 induced significant differences in event-free survival (EFS) distribution compared to control in 10 of 31 (32%) evaluable solid tumor xenografts and in eight of eight (100%) evaluable ALL xenografts. Significance differences in EFS distribution were observed in four of six osteosarcoma lines, three of three rhabdoid tumor lines and two of six rhabdomyosarcoma lines. No objective responses were observed among the solid tumor xenografts. For the ALL panel, one xenograft achieved complete response and four achieved partial response. CONCLUSIONS: The most consistent in vivo activity for CBL0137 was observed against ALL xenografts, with some solid tumor xenograft lines showing tumor growth delay. It will be important to relate the drug levels in mice at 50 mg/kg to those in humans at the recommended phase 2 dose.

Pharmacodynamic and genomic markers associated with response to the XPO1/CRM1 inhibitor selinexor (KPT-330): A report from the pediatric preclinical testing program.

BACKGROUND: Selinexor (KPT-330) is an inhibitor of the major nuclear export receptor, exportin 1 (XPO1, also termed chromosome region maintenance 1, CRM1) that has demonstrated activity in preclinical models and clinical activity against several solid and hematological cancers. PROCEDURES: Selinexor was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro cell line panel at concentrations from 1.0 nM to 10 µM and against the PPTP in vivo xenograft panels administered orally at a dose of 10 mg/kg thrice weekly for 4 weeks. RESULTS: Selinexor demonstrated cytotoxic activity in vitro, with a median relative IC50 value of 123 nM (range 13.0 nM to >10 µM). Selinexor induced significant differences in event-free survival (EFS) distribution in 29 of 38 (76%) of the evaluable solid tumor xenografts and in five of eight (63%) of the evaluable ALL xenografts. Objective responses (partial or complete responses, PR/CR) were observed for 4 of 38 solid tumor xenografts including Wilms tumor, medulloblastoma (n = 2), and ependymoma models. For the ALL panel, two of eight (25%) xenografts achieved either CR or maintained CR. Two responding xenografts had FBXW7 mutations at R465 and two had SMARCA4 mutations. Selinexor induced p53, p21, and cleaved PARP in several solid tumor models. CONCLUSIONS: Selinexor induced regression against several solid tumor and ALL xenografts and slowed tumor growth in a larger number of models. Pharmacodynamic effects for XPO1 inhibition were noted. Defining the relationship between selinexor systemic exposures in mice and humans will be important in assessing the clinical relevance of these results.

Initial Testing (Stage 1) of MK-8242-A Novel MDM2 Inhibitor-by the Pediatric Preclinical Testing Program.

BACKGROUND: MK-8242 is an inhibitor of MDM2 that stabilizes the tumor suppressor TP53 and induces growth arrest or apoptosis downstream of TP53 induction. PROCEDURES: MK-8242 was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro cell line panel at concentrations from 1.0 nM to 10.0 µM and against the PPTP in vivo xenograft panels using oral gavage on Days 1-5 and Day 15-19 at a dose of 125 mg/kg (solid tumors) or 75 mg/kg (acute lymphoblastic leukemia [ALL] models). RESULTS: The median IC50 for MK-8242 was 0.07 µM for TP53 wild-type cell lines versus >10 µM for TP53 mutant cell lines. MK-8242 induced a twofold or greater delay in time to event in 10 of 17 (59%) of TP53 wild-type solid tumor xenografts, excluding osteosarcoma xenografts that have very low TP53 expression. Objective responses were observed in seven solid tumor xenografts representing multiple histotypes. For the systemic-disease ALL panel, among eight xenografts there were two complete responses (CRs) and six partial responses (PRs). Two additional MLL-rearranged xenografts (MV4;11 and RS4;11) grown subcutaneously showed maintained CR and PR, respectively. The expected pharmacodynamic responses to TP53 activation were observed in TP53 wild-type models treated with MK-8242. Pharmacokinetic analysis showed that MK-8242 drug exposure in SCID mice appears to exceed that was observed in adult phase 1 trials. CONCLUSIONS: MK-8242-induced tumor regressions across multiple solid tumor histotypes and induced CRs or PRs for most ALL xenografts. This activity was observed at MK-8242 drug exposures that appear to exceed those observed in human phase 1 trials.

Initial Testing of NSC 750854, a Novel Purine Analog, Against Pediatric Tumor Models by the Pediatric Preclinical Testing Program.

BACKGROUND: NSC 750854 is a purine analog with an antitumor activity profile distinctive from that of other anticancer purines. It has shown significant activity against adult cancer preclinical models. PROCEDURE: NSC 750854 was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro cell line panel at concentrations from 1.0 nM to 10 µM and against the PPTP in vivo xenograft panels administered intraperitoneally at a dose of 5 mg/kg daily for 5 days repeated at day 15. RESULTS: The median relative IC50 (rIC50 ) value for the PPTP cell lines was 32 nM (range from 11 to 124 nM), with consistent cytotoxicity across all cell lines. Acute lymphoblastic leukemia (ALL) cell lines were more sensitive to NSC 750854 than non-ALL cell lines. NSC 750854 induced significant differences in EFS distribution compared to control in 31 of 35 (89%) solid tumor xenografts. It induced tumor growth inhibition meeting criteria for intermediate or high event free survival (EFS) T/C activity in 17 of 32 (53%) evaluable solid tumor xenografts (most consistently in the rhabdomyosarcoma panel). Objective responses were observed in 15 of 37 (41%) solid tumor xenografts and in all eight leukemia models with complete response (CR) or maintained complete response (MCR) in seven of eight leukemia models. CONCLUSIONS: NSC 750854 has a unique spectrum of antitumor activity compared with other agents tested by the PPTP as it induces regression in tumor models with limited sensitivity to most agents tested to date. Given the promising level of activity observed for NSC 750854 against PPTP preclinical models, further exploration of its mechanism of action is warranted.

A Phase I New Approaches to Neuroblastoma Therapy Study of Buthionine Sulfoximine and Melphalan With Autologous Stem Cells for Recurrent/Refractory High-Risk Neuroblastoma.

BACKGROUND: Myeloablative therapy for high-risk neuroblastoma commonly includes melphalan. Increased cellular glutathione (GSH) can mediate melphalan resistance. Buthionine sulfoximine (BSO), a GSH synthesis inhibitor, enhances melphalan activity against neuroblastoma cell lines, providing the rationale for a Phase 1 trial of BSO-melphalan. PROCEDURES: Patients with recurrent/resistant high-risk neuroblastoma received BSO (3 gram/m(2) bolus, then 24 grams/m(2) /day infusion days -4 to -2), with escalating doses of intravenous melphalan (20-125 mg/m(2) ) days -3 and -2, and autologous stem cells day 0 using 3 + 3 dose escalation. RESULTS: Among 28 patients evaluable for dose escalation, one dose-limiting toxicity occurred at 20 mg/m(2) melphalan (grade 3 aspartate aminotransferase/alanine aminotransferase) and one at 80 mg/m(2) (streptococcal bacteremia, grade 4 hypotension/pulmonary/hypocalcemia) without sequelae. Among 25 patients evaluable for response, there was one partial response (PR) and two mixed responses (MRs) among eight patients with prior melphalan exposure; one PR and three MRs among 16 patients without prior melphalan; one stable disease with unknown melphalan history. Melphalan pharmacokinetics with BSO were similar to reports for melphalan alone. Melphalan Cmax for most patients was below the 10 µM concentration that showed neuroblastoma preclinical activity with BSO. CONCLUSIONS: BSO (75 gram/m(2) ) with melphalan (125 mg/m(2) ) is tolerable with stem cell support and active in recurrent/refractory neuroblastoma. Further dose escalation is feasible and may increase responses.

Preservation of high glycolytic phenotype by establishing new acute lymphoblastic leukemia cell lines at physiologic oxygen concentration.

Cancer cells typically exhibit increased glycolysis and decreased mitochondrial oxidative phosphorylation, and they continue to exhibit some elevation in glycolysis even under aerobic conditions. However, it is unclear whether cancer cell lines employ a high level of glycolysis comparable to that of the original cancers from which they were derived, even if their culture conditions are changed to physiologically relevant oxygen concentrations. From three childhood acute lymphoblastic leukemia (ALL) patients we established three new pairs of cell lines in both atmospheric (20%) and physiologic (bone marrow level, 5%) oxygen concentrations. Cell lines established in 20% oxygen exhibited lower proliferation, survival, expression of glycolysis genes, glucose consumption, and lactate production. Interestingly, the effects of oxygen concentration used during cell line initiation were only partially reversible when established cell cultures were switched from one oxygen concentration to another for eight weeks. These observations indicate that ALL cell lines established at atmospheric oxygen concentration can exhibit relatively low levels of glycolysis and these levels are semi-permanent, suggesting that physiologic oxygen concentrations may be needed from the time of cell line initiation to preserve the high level of glycolysis commonly exhibited by leukemias in vivo.

Initial testing (stage 1) of the anti-microtubule agents cabazitaxel and docetaxel, by the pediatric preclinical testing program.

BACKGROUND: Although microtubule-destabilizing agents (principally vincristine) are in common use in pediatric oncology, the microtubule-stabilizing taxanes are uncommonly used to treat childhood cancers. Cabazitaxel has been reported to have activity superior to that of docetaxel in preclinical models of multidrug-resistant adult cancers, and it was active in patients who had progressed on or after docetaxel. The PPTP conducted a comparison of these two agents against the PPTP in vitro panel and against a limited panel of solid tumor xenografts. PROCEDURES: Cabazitaxel and docetaxel were tested against the PPTP in vitro cell line panel at concentrations from 0.01 to 0.1 µM and in vivo against a subset of the PPTP solid tumor xenograft models at a dose of 10 or 7.5 mg/kg on an every 4 days × 3 I.V. schedule. RESULTS: In vitro, both cabazitaxel and docetaxel had similar potency (median rIC50 0.47 nM and 0.88 nM, respectively) and a similar activity profile, with Ewing sarcoma cells being significantly more sensitive to both agents. In vitro sensitivity to docetaxel inversely correlated with mRNA expression for ABCB1, but the correlation with ABCB1 expression was weaker for cabazitaxel. In vivo cabazitaxel demonstrated significantly greater activity than docetaxel in five of 12 tumor models, inducing regressions in six models compared with three models for docetaxel. CONCLUSIONS: Cabazitaxel demonstrated superior activity compared to docetaxel. The lower cabazitaxel systemic exposure tolerated in humans compared to mice needs to be considered when extrapolating these results to the clinical setting.

Initial testing (stage 1) of BAL101553, a novel tubulin binding agent, by the pediatric preclinical testing program.

BAL101553 is a highly water soluble prodrug of BAL27862 that arrests tumor cell proliferation and induces cell death in cancer cells through disruption of the microtubule network. In vitro BAL27862 demonstrated potent activity, with the median relative IC50 (rIC50 ) of 13.8 nM (range 5.4-25.2 nM). The in vitro activity of BAL27862 against the PPTP cell lines is distinctive from that previously described for vincristine. BAL101553 induced significant differences in EFS distribution compared to control in 16 of 30 (53%) solid tumor xenografts and in two of four (67%) of the evaluable ALL xenografts. No objective responses were observed.

Initial testing (stage 1) of the PARP inhibitor BMN 673 by the pediatric preclinical testing program: PALB2 mutation predicts exceptional in vivo response to BMN 673.

BACKGROUND: BMN 673 is a potent inhibitor of poly-ADP ribose polymerase (PARP) that is in clinical testing with a primary focus on BRCA-mutated cancers. BMN 673 is active both through inhibiting PARP catalytic activity and by tightly trapping PARP to DNA at sites of single strand breaks. PROCEDURE: BMN 673 was tested in vitro at concentrations ranging from 0.1 nM to 1 µM and in vivo at a daily dose of 0.33 mg/kg administered orally twice daily (Mon-Fri) and once daily on weekends (solid tumors) for 28 days. RESULTS: The median relative IC50 (rIC50 ) concentration against the PPTP cell lines was 25.8 nM. The median rIC50 for the Ewing cell lines was lower than for the remaining cell lines (6.4 vs. 31.1 nM, respectively). In vivo BMN 673 induced statistically significant differences in EFS distribution in 17/43 (39.5%) xenograft models. Three objective regressions were observed: a complete response (CR) in a medulloblastoma line (BT-45), a maintained CR in a Wilms tumor line (KT-10), and a maintained CR in an ependymoma line (BT-41). BMN 673 maintained its high level of activity against KT-10 with a threefold reduction in dose. KT-10 possesses a truncating mutation in PALB2 analogous to PALB2 mutations associated with hereditary breast and ovarian cancer that abrogate homologous recombination (HR) repair. CONCLUSIONS: The PPTP results suggest that single agent BMN 673 may have limited clinical activity against pediatric cancers. Single agent activity is more likely for patients whose tumors have defects in HR repair.

Sectoral variations in the distribution of axonal cytoskeleton proteins in the human optic nerve head.

Axonal cytoskeleton proteins are intrinsically related to retinal ganglion cell (RGC) health and disease. This study quantifies and documents the sectoral distribution of axonal cytoskeleton proteins in the different laminar compartments of the normal human optic nerve head (ONH). Nine human eyes were used (mean age of 46.9 ± 3.7 years). Coronal sections of the prelaminar, anterior lamina cribrosa, posterior lamina cribrosa and retrolaminar regions were examined using immunohistochemical and confocal microscopy techniques. The axonal cytoskeleton was studied using antibodies to neurofilament light (NFL), neurofilament medium (NFM), neurofilament heavy (NFH), phosphorylated NFH (NFHp), microtubule associated protein-1 (MAP-1) and tubulin. Axonal cytoskeleton intensity was quantified in a standardized manner and comparisons were made within and between laminar regions. The intensity of NFM, NFH and NFHp is significantly greater in the nasal and peripheral sectors of the prelaminar, anterior lamina cribrosa and posterior lamina cribrosa regions. There is no significant difference in the distribution of NFL, tubulin and MAP-1 proteins between sectors in any laminar region. The intensity of all axonal cytoskeleton proteins in unmyelinated laminar regions is greater than the retrolaminar region. The distribution of all cytoskeleton proteins in the myelinated, retrolaminar region of the optic nerve appears to be relatively even. The distribution of axonal cytoskeleton proteins may have relevance for understanding the vulnerability of distinct ONH sectors to intraocular pressure- and ischaemia-mediated damage. The findings in this study may be important for understanding patho-physiological mechanisms involved in glaucomatous and ischaemic optic neuropathy.