Next-Generation Sequencing of Minimal Residual Disease for Predicting Relapse after Tisagenlecleucel in Children and Young Adults with Acute Lymphoblastic Leukemia.

We assessed minimal residual disease (MRD) detection and B-cell aplasia after tisagenlecleucel therapy for acute lymphoblastic leukemia (ALL) to define biomarkers predictive of relapse (N = 143). Next-generation sequencing (NGS) MRD detection >0 in bone marrow (BM) was highly associated with relapse. B-cell recovery [signifying loss of functional chimeric antigen receptor (CAR) T cells] within the first year of treatment was associated with a hazard ratio (HR) for relapse of 4.5 [95% confidence interval (CI), 2.03-9.97; P < 0.001]. Multivariate analysis at day 28 showed independent associations of BMNGS-MRD >0 (HR = 4.87; 95% CI, 2.18-10.8; P < 0.001) and B-cell recovery (HR = 3.33; 95% CI, 1.44-7.69; P = 0.005) with relapse. By 3 months, the BMNGS-MRD HR increased to 12 (95% CI, 2.87-50; P < 0.001), whereas B-cell recovery was not independently predictive (HR = 1.27; 95% CI, 0.33-4.79; P = 0.7). Relapses occurring with persistence of B-cell aplasia were largely CD19- (23/25: 88%). Detectable BMNGS-MRD reliably predicts risk with sufficient time to consider approaches to relapse prevention such as hematopoietic cell transplantation (HCT) or second CAR-T cell infusion. SIGNIFICANCE: Detectable disease by BMNGS-MRD with or without B-cell aplasia is highly predictive of relapse after tisagenlecleucel therapy for ALL. Clonotypic rearrangements used to follow NGS-MRD did not change after loss of CD19 or lineage switch. High-risk patients identified by these biomarkers may benefit from HCT or investigational cell therapies.See related commentary by Ghorashian and Bartram, p. 2.This article is highlighted in the In This Issue feature, p. 1.

Antitumor Activity Associated with Prolonged Persistence of Adoptively Transferred NY-ESO-1 c259T Cells in Synovial Sarcoma.

We evaluated the safety and activity of autologous T cells expressing NY-ESO-1c259, an affinity-enhanced T-cell receptor (TCR) recognizing an HLA-A2-restricted NY-ESO-1/LAGE1a-derived peptide, in patients with metastatic synovial sarcoma (NY-ESO-1c259T cells). Confirmed antitumor responses occurred in 50% of patients (6/12) and were characterized by tumor shrinkage over several months. Circulating NY-ESO-1c259T cells were present postinfusion in all patients and persisted for at least 6 months in all responders. Most of the infused NY-ESO-1c259T cells exhibited an effector memory phenotype following ex vivo expansion, but the persisting pools comprised largely central memory and stem-cell memory subsets, which remained polyfunctional and showed no evidence of T-cell exhaustion despite persistent tumor burdens. Next-generation sequencing of endogenous TCRs in CD8+ NY-ESO-1c259T cells revealed clonal diversity without contraction over time. These data suggest that regenerative pools of NY-ESO-1c259T cells produced a continuing supply of effector cells to mediate sustained, clinically meaningful antitumor effects.Significance: Metastatic synovial sarcoma is incurable with standard therapy. We employed engineered T cells targeting NY-ESO-1, and the data suggest that robust, self-regenerating pools of CD8+ NY-ESO-1c259T cells produce a continuing supply of effector cells over several months that mediate clinically meaningful antitumor effects despite prolonged exposure to antigen. Cancer Discov; 8(8); 944-57. ©2018 AACR.See related commentary by Keung and Tawbi, p. 914This article is highlighted in the In This Issue feature, p. 899.

A novel preclinical strategy for identifying cardiotoxic kinase inhibitors and mechanisms of cardiotoxicity.

RATIONALE: Despite intense interest in strategies to predict which kinase inhibitor (KI) cancer therapeutics may be associated with cardiotoxicity, current approaches are inadequate. Sorafenib is a KI of concern because it inhibits growth factor receptors and Raf-1/B-Raf, kinases that are upstream of extracellular signal-regulated kinases (ERKs) and signal cardiomyocyte survival in the setting of stress. OBJECTIVES: To explore the potential use of zebrafish as a preclinical model to predict cardiotoxicity and to determine whether sorafenib has associated cardiotoxicity, and, if so, define the mechanisms. METHODS AND RESULTS: We find that the zebrafish model is readily able to discriminate a KI with little or no cardiotoxicity (gefitinib) from one with demonstrated cardiotoxicity (sunitinib). Sorafenib, like sunitinib, leads to cardiomyocyte apoptosis, a reduction in total myocyte number per heart, contractile dysfunction, and ventricular dilatation in zebrafish. In cultured rat cardiomyocytes, sorafenib induces cell death. This can be rescued by adenovirus-mediated gene transfer of constitutively active MEK1, which restores ERK activity even in the presence of sorafenib. Whereas growth factor-induced activation of ERKs requires Raf, α-adrenergic agonist-induced activation of ERKs does not require it. Consequently, activation of α-adrenergic signaling markedly decreases sorafenib-induced cell death. Consistent with these in vitro data, inhibition of α-adrenergic signaling with the receptor antagonist prazosin worsens sorafenib-induced cardiomyopathy in zebrafish. CONCLUSIONS: Zebrafish may be a valuable preclinical tool to predict cardiotoxicity. The α-adrenergic signaling pathway is an important modulator of sorafenib cardiotoxicity in vitro and in vivo and appears to act through a here-to-fore unrecognized signaling pathway downstream of α-adrenergic activation that bypasses Raf to activate ERKs.

Nuclear factor kappaB inhibitors alleviate and the proteasome inhibitor PS-341 exacerbates radiation toxicity in zebrafish embryos.

Inflammatory changes are a major component of the normal tissue response to ionizing radiation, and increased nuclear factor kappaB (NF-kappaB) activity is an important mediator of inflammatory responses. Here, we used zebrafish embryos to assess the capacity of two different classes of pharmacologic agents known to target NF-kappaB to modify radiation toxicity in the vertebrate organism. These were proteasome inhibitors, including lactacystin, MG132, and PS-341 (Bortezomib/VELCADE), and direct inhibitors of NF-kappaB activity, including ethyl pyruvate (EP) and the synthetic triterpenoid CDDO-TFEA (RTA401), among others. The proteasome inhibitors either did not significantly affect radiation sensitivity of zebrafish embryos (MG132, lactacystin) or rendered zebrafish embryos more sensitive to the lethal effects of ionizing radiation (PS-341). Radiosensitization by PS-341 was reduced in fish with impaired p53 expression or function but not associated with enhanced expression of select p53 target genes. In contrast, the direct NF-kappaB inhibitors EP and CDDO-TFEA significantly improved overall survival of lethally irradiated zebrafish embryos. In addition, direct NF-kappaB inhibition reduced radiation-induced apoptosis in the central nervous system, abrogated aberrations in body axis development, restored metabolization and secretion of a reporter lipid through the gastrointestinal system, and improved renal clearance compromised by radiation. In contrast to amifostine, EP and CDDO-TFEA not only protected against but also mitigated radiation toxicity when given 1 to 2 hours postexposure. Finally, four additional IkappaB kinase inhibitors with distinct mechanisms of action similarly improved overall survival of lethally irradiated zebrafish embryos. In conclusion, inhibitors of canonical pathways to NF-kappaB activation may be useful in alleviating radiation toxicity in patients.

Interaction with LC8 is required for Pak1 nuclear import and is indispensable for zebrafish development.

Pak1 (p21 activated kinase 1) is a serine/threonine kinase implicated in regulation of cell motility and survival and in malignant transformation of mammary epithelial cells. In addition, the dynein light chain, LC8, has been described to cooperate with Pak1 in malignant transformation of breast cancer cells. Pak1 itself may aid breast cancer development by phosphorylating nuclear proteins, including estrogen receptor alpha. Recently, we showed that the LC8 binding site on Pak1 is adjacent to the nuclear localization sequence (NLS) required for Pak1 nuclear import. Here, we demonstrate that the LC8-Pak1 interaction is necessary for epidermal growth factor (EGF)-induced nuclear import of Pak1 in MCF-7 cells, and that this event is contingent upon LC8-mediated Pak1 dimerization. In contrast, Pak2, which lacks an LC8 binding site but contains a nuclear localization sequence identical to that in Pak1, remains cytoplasmic upon EGF stimulation of MCF-7 cells. Furthermore, we show that severe developmental defects in zebrafish embryos caused by morpholino injections targeting Pak are partially rescued by co-injection of wild-type human Pak1, but not by co-injection of mutant Pak1 mRNA disrupting either the LC8 binding or the NLS site. Collectively, these results suggest that LC8 facilitates nuclear import of Pak1 and that this function is indispensable during vertebrate development.

Inhibition of p73 function by Pifithrin-alpha as revealed by studies in zebrafish embryos.

The p53 family of proteins contains two members that have been implicated in sensitization of cells and organisms to genotoxic stress, i.e., p53 itself and p73. In vitro, lack of either p53 or p73 can protect certain cell types in the adult organism against death upon exposure to DNA damaging agents. The present study was designed to assess the relative contribution of p53 to radiation resistance of an emerging vertebrate model organism, i.e., zebrafish embryos. Consistent with previous reports, suppressing p53 protein expression using antisense morpholino oligonucleotides (MOs) increased survival and reduced gross morphological alterations in zebrafish embryos exposed to ionizing radiation. By contrast, a pharmacological inhibitor of p53 function [Pifithrin-alpha(PFTalpha)] caused developmental abnormalities affecting the head, brain, eyes and kidney function and did not protect against lethal effects of ionizing radiation when administered at 3 hours post fertilization (hpf). The phenotypic abnormalities associated with PFTalpha treatment were similar to those caused by antisense MO knock down (kd) used to reduce p73 expression. PFTalpha also inhibited p73-dependent transcription of a reporter gene construct containing canonical p53-responsive promoter sequences. Notably, when administered at later stages of development (23 hpf), PFTalpha did not cause overt developmental defects but exerted radioprotective effects in zebrafish embryos. In summary, this study highlights off-target effects of the pharmacological p53 inhibitor PFTalpha related to inhibition of p73 function and essential roles of p73 at early but not later stages of zebrafish development.

In vivo radioprotection by the fullerene nanoparticle DF-1 as assessed in a zebrafish model.

PURPOSE: We have previously shown that zebrafish (Danio rerio) embryos can be used as an in vivo model to validate modifiers of the radiation response. Here, we evaluated the radioprotective effect of the nanoparticle DF-1, a fullerene with antioxidant properties, in zebrafish embryos. EXPERIMENTAL DESIGN: Zebrafish embryos were exposed to different doses of ionizing radiation ranging from 20 to 80 Gy in the presence and absence of DF-1. Toxicity and radioprotective effects were assessed by monitoring overall survival and morphology as well as organ functions by employing assays to measure kidney excretory function and development of sensory nerve cells (neuromasts). Antioxidant properties of DF-1 were assessed in whole fish. RESULTS: DF-1 had no apparent adverse effects on normal zebrafish morphology or viability throughout the concentration range tested (1-1,000 micromol/L). Ionizing radiation (10-40 Gy) caused time-dependent and dose-dependent perturbations of normal zebrafish morphology and physiology, notably defective midline development resulting in dorsal curvature of the body axis ("curly-up"), neurotoxicity, impaired excretory function, and decreased survival of the exposed embryos. DF-1 (100 micromol/L) markedly attenuated overall and organ-specific radiation-induced toxicity when given within 3 hours before or up to 15 minutes after radiation exposure. By contrast, DF-1 afforded no protection when given 30 minutes after ionizing radiation. The degree of radioprotection provided by DF-1 was comparable with that provided by the Food and Drug Administration-approved radioprotector amifostine (4 mmol/L). Protection against radiation-associated toxicity using DF-1 in zebrafish embryos was associated with marked reduction of radiation-induced reactive oxygen species. CONCLUSION: The fullerene DF-1 protects zebrafish embryos against deleterious effects of ionizing radiation due, in part, to its antioxidant properties.

Antisense inhibition of cyclin D1 expression is equivalent to flavopiridol for radiosensitization of zebrafish embryos.

PURPOSE: Flavopiridol, a small molecule pan-cyclin inhibitor, has been shown to enhance the radiation response of tumor cells both in vitro and in vivo. The clinical utility of flavopiridol, however, is limited by toxicity, previously attributed to pleiotropic inhibitory effects on several targets affecting multiple signal transduction pathways. Here we used zebrafish embryos to investigate radiosensitizing effects of flavopiridol in normal tissues. METHODS AND MATERIALS: Zebrafish embryos at the 1- to 4-cell stage were treated with 500 nM flavopiridol or injected with 0.5 pmol antisense hydroxylprolyl-phosphono nucleic acid oligomers to reduce cyclin D1 expression, then subjected to ionizing radiation (IR) or no radiation. RESULTS: Flavopiridol-treated embryos demonstrated a twofold increase in mortality after exposure to 40 Gy by 96 hpf and developed distinct radiation-induced defects in midline development (designated as the "curly up" phenotype) at higher rates when compared with embryos receiving IR only. Cyclin D1-deficient embryos had virtually identical IR sensitivity profiles when compared with embryos treated with flavopiridol. This was particularly evident for the IR-induced curly up phenotype, which was greatly exacerbated by both flavopriridol and cyclin D1 downregulation. CONCLUSIONS: Treatment of zebrafish embryos with flavopiridol enhanced radiation sensitivity of zebrafish embryos to a degree that was very similar to that associated with downregulation of cyclin D1 expression. These results are consistent with the hypothesis that inhibition of cyclin D1 is sufficient to account for the radiosensitizing action of flavopiridol in the zebrafish embryo vertebrate model.