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CAF isolated from patients with NB share characteristics with MSC.

Supplemental Figure 1

Cytokines and chemokines not up-regulated by NB-MSC interaction and lack of inhibition of STAT3 activation by anti-IL-6/IL-6R

Supplementary Tables

GD2 expression levels

A: List of antibodies used in the study B: Clinical characteristics of patients from which CAF-MSC and BM-MSC were isolated.

Permission letter regarding inclusion of information of CHLA-255 C-Myc over-expression

Purpose:We determined whether elimination of CD105+ cells in the tumor microenvironment (TME) with anti-CD105 antibodies enhanced anti-disialoganglioside (GD2) antibody dinutuximab therapy of neuroblastoma when combined with activated natural killer (aNK) cells.Experimental Design:The effect of MSCs and monocytes on antibody-dependent cellular cytotoxicity (ADCC) mediated by dinutuximab with aNK cells against neuroblastoma cells was determined in vitro. ADCC with anti-CD105 mAb TRC105 and aNK cells against MSCs, monocytes, and endothelial cells, which express CD105, was evaluated. Anti-neuroblastoma activity in immunodeficient NSG mice of dinutuximab with aNK cells without or with anti-CD105 mAbs was determined using neuroblastoma cell lines and a patient-derived xenograft.Results:ADCC mediated by dinutuximab with aNK cells against neuroblastoma cells in vitro was suppressed by addition of MSCs and monocytes, and dinutuximab with aNK cells was less effective against neuroblastomas formed with coinjected MSCs and monocytes in NSG mice than against those formed by tumor cells alone. Anti-CD105 antibody TRC105 with aNK cells mediated ADCC against MSCs, monocytes, and endothelial cells. Neuroblastomas formed in NSG mice by two neuroblastoma cell lines or a patient-derived xenograft coinjected with MSCs and monocytes were most effectively treated with dinutuximab and aNK cells when anti-human (TRC105) and anti-mouse (M1043) CD105 antibodies were added, which depleted human MSCs and murine endothelial cells and macrophages from the TME.Conclusions:Immunotherapy of neuroblastoma with anti-GD2 antibody dinutuximab and aNK cells is suppressed by CD105+ cells in the TME, but suppression is overcome by adding anti-CD105 antibodies to eliminate CD105+ cells.

CAF-MSC and BM-MSC enhance cell viability of NB cells in vitro and protect tumor cells from drug-induced apoptosis in vitro.

Supplementary Figure S2 shows the results of intracellular staining of aNK cells for perforin and granzyme A, as requested by reviewer #3.

Targeting CD105+ Microenvironment cells enhances immunotherpy of Dinutuxmab with aNK cells against neuroblastoma

CAF-MSC and BM-MSC induce activation of STAT3 and ERK1/2 signaling in NB cells

Supplementary Table S1 is a list of antibodies used in the study. It has been updated to include antibodies used in experiments requested by reviewer #3.

PDF file - 1549K, Supplemental Figure 1. Cytokine and chemokine release from K562 Clone 9.mbIL21 aAPC-expanded effector cells after 24-hour incubation with neuroblastoma cell line CHLA-255-Fluc alone or with anti-GD2 antibody ch14.18. Supplemental Figure 2. Anti-tumor activity of K562 Clone 9.mbIL21 aAPC-expanded and cryopreserved effector cells from a normal donor PBMC that were thawed and cultured for 3 days or thawed and immediately injected intravenously into NOD/SCID mice beginning 7 days after they had received 106 CHLA-255-Fluc neuroblastoma cells intravenously. Supplemental Figure 3. Anti-tumor activity of dose-dense treatment with K562 Clone 9.mbIL21 aAPC-expanded and cryopreserved aNK cells derived from a patient�s PBMC. Supplemental Figure 4. Anti-tumor activity of K562 Clone 9.mbIL21 aAPC-expanded and cryopreserved aNK cells derived from a patient�s PBMC when treatment was begun 21 days (late treatment) after tumor cell injection.

Cancer-associated fibroblasts (CAF) have been suggested to originate from mesenchymal stromal cells (MSC), but their relationship with MSCs is not clear. Here, we have isolated from primary human neuroblastoma tumors a population of αFAP- and FSP-1–expressing CAFs that share phenotypic and functional characteristics with bone marrow–derived MSCs (BM-MSC). Analysis of human neuroblastoma tumors also confirmed the presence of αFAP- and FSP-1–positive cells in the tumor stroma, and their presence correlated with that of M2 tumor-associated macrophages. These cells (designated CAF-MSCs) enhanced in vitro neuroblastoma cell proliferation, survival, and resistance to chemotherapy and stimulated neuroblastoma tumor engraftment and growth in immunodeficient mice, indicating an effect independent of the immune system. The protumorigenic activity of MSCs in vitro and in xenografted mice was dependent on the coactivation of JAK2/STAT3 and MEK/ERK1/2 in neuroblastoma cells. In a mouse model of orthotopically implanted neuroblastoma cells, inhibition of JAK2/STAT3 and MEK/ERK/1/2 by ruxolitinib and trametinib potentiated tumor response to etoposide and increased overall survival. These data point to a new type of protumorigenic CAF in the tumor microenvironment of neuroblastoma and to STAT3 and ERK1/2 as mediators of their activity. Cancer Res; 77(18); 5142–57. ©2017 AACR.

Purpose: Adoptive transfer of natural killer (NK) cells combined with tumor-specific monoclonal antibodies (mAb) has therapeutic potential for malignancies. We determined if large numbers of activated NK (aNK) cells can be grown ex vivo from peripheral blood mononuclear cells (PBMC) of children with high-risk neuroblastoma using artificial antigen-presenting cells (aAPC).Experimental Design: Irradiated K562-derived Clone 9.mbIL21 aAPC were cocultured with PBMC, and propagated NK cells were characterized with flow cytometry, cytotoxicity assays, Luminex multicytokine assays, and a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of disseminated neuroblastoma.Results: Coculturing patient PBMC with aAPC for 14 days induced 2,363- ± 443-fold expansion of CD56+CD3−CD14− NK cells with 83% ± 3% purity (n = 10). Results were similar to PBMC from normal donors (n = 5). Expression of DNAM-1, NKG2D, FcγRIII/CD16, and CD56 increased 6- ± 3-, 10- ± 2-, 21- ± 20-, and 18- ± 3-fold, respectively, on day 14 compared with day 0, showing activation of NK cells. In vitro, aNK cells were highly cytotoxic against neuroblastoma cell lines and killing was enhanced with GD2-specific mAb ch14.18. When mediating cytotoxicity with ch14.18, release of TNF-α, granulocyte macrophage colony-stimulating factor, IFN-γ, sCD40L, CCL2/MCP-1, CXCL9/MIG, and CXCL11/I-TAC by aNK cells increased 4-, 5-, 6-, 15-, 265-, 917-, and 363-fold (151–9,121 pg/mL), respectively, compared with aNK cells alone. Survival of NOD/SCID mice bearing disseminated neuroblastoma improved when treated with thawed and immediately intravenously infused cryopreserved aNK cells compared with untreated mice and was further improved when ch14.18 was added.Conclusion: Propagation of large numbers of aNK cells that maintain potent antineuroblastoma activities when cryopreserved supports clinical testing of adoptive cell therapy with ch14.18. Clin Cancer Res; 19(8); 2132–43. ©2013 AACR.

Purpose:Immunotherapy of neuroblastoma that remains after myeloablative chemotherapy with anti-GD2 antibody dinutuximab has increased the two-year event-free and overall survival of high-risk neuroblastoma patients; however, 40% of patients develop recurrent disease during or after this treatment. To determine the potential of such antibody-based immunotherapy earlier in treatment, a mouse model was developed in which surgical resection of the primary tumor was followed by therapy of residual disease with dinutuximab combined with ex vivo–activated human natural killer (aNK) cells.Experimental Design:The effect of combining dinutuximab with human aNK cells was determined in vitro with cellular cytotoxicity and Matrigel invasion assays. The in vivo efficacy of dinutuximab and aNK cells against neuroblastoma was assessed following resection of primary tumors formed by two cell lines or a patient-derived xenograft (PDX) in immunodeficient NOD-scid gamma mice.Results:In vitro, the combination of aNK cells and dinutuximab caused cytotoxicity and decreased invasiveness of three human neuroblastoma cell lines. Treatment of mice with dinutuximab combined with aNK cells after surgical resection of primary intrarenal tumors formed by two cell lines or a PDX decreased tumor cells in liver and bone marrow as evaluated by histopathology and bioluminescence imaging. Survival of mice after resection of these tumors was most significantly increased by treatment with dinutuximab combined with aNK cells compared with that of untreated mice.Conclusions:The combination of dinutuximab and adoptively transferred human aNK cells following surgical resection of primary neuroblastomas significantly improves survival of immunodeficient mice.

PDF file - 89K, Supplemental Table 1. List of antibodies used in this study. Supplemental table 2. Cytokines/chemokines excluded from the Luminex� panels.

Combination of ruxolitinib and trametinib inhibits the growth of subcutaneous NB tumors, cell proliferation and STAT3 and ERK1/2 activation in vivo.

Legends to supplementary figures - PDF file 87K, Legends to supplementary figures 1 to 4

Supplementary Figure 3 - PDF file 455K, A. CHLA-255 cells were cultures alone or in the presence of sIL-6R (25ng/mL) or IL-6 (10 ng/mL) before being treated with etoposide as indicated in Figure 3. After 24 hours, cells were examined for Annexin V expression by Flow cytometry. The data represents the mean percent of Annexin V positive cells from triplicate samples. B. CHLA-255 and human monocytes were co-cultured as described in figure 6E. Cells were then separated by flow cytometry using anti-GD2 antibody (CHLA-255) and an anti-CD14 (monocytes) and examined for nuclear pSTAT3 by flow cytometry. C. Increased expression of in bone marrow samples from patients with metastatic neuroblastoma. Paraffin-embedded sections of bone marrow biopsies stained by immunohistochemistry of patients without (Left panel) and with (Right panel) tumor cells (scale bar = 50 �m). Increased expression of pSTAT3 and survivin (top) and Bcl-xL (bottom) in bone marrow samples from patients with metastatic neuroblastoma. Left panel, Representative paraffin-embedded sections of bone marrow biopsies from patients, stained by immunohistochemistry for pSTAT3 and survivin of Bcl-xL as indicated in Materials and Methods (scale bar = 50 �m). Right panel, The graph represents the mean number (�SD) of pSTAT3 and survivin positive cells in five 40x fields examined in bone marrow without (n=5) and with (n=5) tumor cells

Supplementary Figure 1 - PDF file 231K, Inhibition of STAT3 activation in CHLA-255 cells by anti-IL-6R blocking antibody. A, Expression of pSTAT3 and STAT3 in CHLA-255 cells was examined by Western blot analysis as in Fig. 1A. When indicated, cells were treated with tocilizumab (2�g/mL) for 12 hours before treatment with IL-6 with or without sIL-6R. The data are representative of 2 separate experiments showing similar results. B, Nuclear extracts from CHLA-255 treated with IL-6 and sIL-6R in the presence and absence of tocilizumab as above described were examined for STAT3 DNA-binding activity by EMSA as indicated in Materials and Methods. Lane 8 is same as lane 5, except for the addition of a �supershifting� anti-STAT3 antibody also added to lane 2 (DU145 control). Top band (labeled STAT3) represents STAT3/STAT3 homodimers bound to DNA. C, CHLA-255 transfected with a STAT3 Firefly luciferase vector and a Renilla luciferase control vector were treated with IL-6 (10ng/mL) alone or in combination with sIL-6R (25ng/mL) as shown in Fig. 1 in the absence or presence of tocilizumab (4�g/mL). After 24 hours, the activity of the STAT3 reporter was examined by Dual Luciferase assay as indicated in Materials and Methods. The data represent the mean (�SD) ratio Firefly/Renilla luciferase activity from five samples for each experimental condition

Supplementary Figure 4 - PDF file 91K, A. Representative data of the analysis on one fresh patient bone marrow sample by Flow Cytometry showing the expression of pSTAT3 in various sub-populations of bone marrow cells as indicated in Materials and Methods. B. Left panel: Percentage of the 6 sub-populations of cells identified as shown in A. The data represents the mean percentage (�SD) of all mononuclear cells isolated from 8 individual patient samples. Right panel: The data represents the mean percentage of pSTAT3 positivity in each sub-population of bone marrow cells identified in the 8 samples as shown in the left panel

Drug resistance is a major cause of treatment failure in cancer. Here, we have evaluated the role of STAT3 in environment-mediated drug resistance (EMDR) in human neuroblastoma. We determined that STAT3 was not constitutively active in most neuroblastoma cell lines but was rapidly activated upon treatment with interleukin (IL)-6 alone and in combination with the soluble IL-6 receptor (sIL-6R). Treatment of neuroblastoma cells with IL-6 protected them from drug-induced apoptosis in a STAT3-dependent manner because the protective effect of IL-6 was abrogated in the presence of a STAT3 inhibitor and upon STAT3 knockdown. STAT3 was necessary for the upregulation of several survival factors such as survivin (BIRC5) and Bcl-xL (BCL2L1) when cells were exposed to IL-6. Importantly, IL-6–mediated STAT3 activation was enhanced by sIL-6R produced by human monocytes, pointing to an important function of monocytes in promoting IL-6–mediated EMDR. Our data also point to the presence of reciprocal activation of STAT3 between tumor cells and bone marrow stromal cells including not only monocytes but also regulatory T cells (Treg) and nonmyeloid stromal cells. Thus, the data identify an IL-6/sIL-6R/STAT3 interactive pathway between neuroblastoma cells and their microenvironment that contributes to drug resistance. Cancer Res; 73(13); 3852–64. ©2013 AACR.

Neuroblastoma (NBL) accounts for a disproportionate number of deaths among childhood malignancies despite intensive multimodal therapy that includes antibody targeting disialoganglioside GD2, a NBL antigen. Unfortunately, resistance to anti-GD2 immunotherapy is frequent and we aimed to investigate mechanisms of resistance in NBL. GD2 expression was quantified by flow cytometry and anti-GD2 antibody internalization was measured using real-time microscopy in 20 human NBL cell lines. Neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) assays were performed on a subset of the cell lines (n = 12), and results were correlated with GD2 expression and antibody internalization. GD2 was expressed on 19 of 20 NBL cell lines at variable levels, and neutrophil-mediated ADCC was observed only in GD2-expressing cell lines. We found no correlation between level of GD2 expression and sensitivity to neutrophil-mediated ADCC, suggesting that GD2 expression of many cell lines was above a threshold required for maximal ADCC, such that expression level could not be used to predict subsequent cytotoxicity. Instead, anti-GD2 antibody internalization, a process that occurred universally but differentially across GD2-expressing NBL cell lines, was inversely correlated with ADCC. Treatment with endocytosis inhibitors EIPA, chlorpromazine, MBCD, and cytochalasin-D showed potential to inhibit antibody internalization; however, only MBCD resulted in significantly increased sensitivity to neutrophil-mediated ADCC in 4 of 4 cell lines in vitro. Our data suggest that antibody internalization may represent a novel mechanism of immunotherapy escape by NBL and provide proof-of-principle that targeting pathways involved in antibody internalization may improve the efficacy of anti-GD2 immunotherapies.

Purpose: Immunotherapy of neuroblastoma that remains after myeloablative chemotherapy with anti-GD2 antibody dinutuximab has increased the two-year event-free and overall survival of high-risk neuroblastoma patients; however, 40% of patients develop recurrent disease during or after this treatment. To determine the potential of such antibody-based immunotherapy earlier in treatment, a mouse model was developed in which surgical resection of the primary tumor was followed by therapy of residual disease with dinutuximab combined with ex vivo–activated human natural killer (aNK) cells. Experimental Design: The effect of combining dinutuximab with human aNK cells was determined in vitro with cellular cytotoxicity and Matrigel invasion assays. The in vivo efficacy of dinutuximab and aNK cells against neuroblastoma was assessed following resection of primary tumors formed by two cell lines or a patient-derived xenograft (PDX) in immunodeficient NOD-scid gamma mice. Results: In vitro, the combination of aNK cells and dinutuximab caused cytotoxicity and decreased invasiveness of three human neuroblastoma cell lines. Treatment of mice with dinutuximab combined with aNK cells after surgical resection of primary intrarenal tumors formed by two cell lines or a PDX decreased tumor cells in liver and bone marrow as evaluated by histopathology and bioluminescence imaging. Survival of mice after resection of these tumors was most significantly increased by treatment with dinutuximab combined with aNK cells compared with that of untreated mice. Conclusions: The combination of dinutuximab and adoptively transferred human aNK cells following surgical resection of primary neuroblastomas significantly improves survival of immunodeficient mice.

Immune checkpoint therapy has resulted in minimal clinical response in many pediatric cancers. We sought to understand the influence of immune checkpoint inhibition using anti-PD-1 and anti-CTLA-4 antibodies individually, in combination, and after chemotherapy on immune responses in minimal and established murine neuroblastoma models. We also sought to understand the role of the tumor microenvironment (TME) and PD-L1 expression and their alteration post-chemotherapy in our models and human tissues. PD-L1 expression was enriched in human tumor-associated macrophages and up-regulated after chemotherapy. In a murine minimal disease model, single and dual immune checkpoint blockade promoted tumor rejection, improved survival, and established immune memory with long-term anti-tumor immunity against re-challenge. In an established tumor model, only dual immune checkpoint blockade showed efficacy. Interestingly, dual immune checkpoint therapy distinctly influenced adaptive and innate immune responses, with significant increase in CD8+CD28+PD-1+ T cells and inflammatory macrophages (CD11bhiCD11c−F4/80+Ly6Chi) in tumor-draining lymph nodes. Adding chemotherapy before immunotherapy provided significant survival benefit for mice with established tumors receiving anti-PD-1 or dual immune checkpoint blockade. Our findings demonstrate anti-PD-1 and anti-CTLA-4 therapy induces a novel subset of effector T cells, and support administration of induction chemotherapy immediately prior to immune checkpoint blockade in children with high-risk neuroblastoma.

BackgroundImmunotherapy with anti-disialoganglioside dinutuximab has improved survival for children with high-risk neuroblastoma (NB) when given after induction chemotherapy and surgery. However, disease recurrence and resistance persist. Dinutuximab efficacy has not been evaluated when initiated before primary tumor removal. Using a surgical mouse model of human NB, we examined if initiating dinutuximab plus ex vivo-activated natural killer (aNK) cells before resection of the primary tumor improves survival.MethodsIn vitro, human NB cells (SMS-KCNR-Fluc, CHLA-255-Fluc) were treated with dinutuximab and/or aNK cells and cytotoxicity was measured. In vivo, NB cells (SMS-KCNR-Fluc, CHLA-255-Fluc, or COG-N-415x PDX) were injected into the kidney of NOD-scid gamma mice. Mice received eight intravenous infusions of aNK cells plus dinutuximab beginning either 12 days before or 2 days after resection of primary tumors. Tumors in control mice were treated by resection alone or with immunotherapy alone. Disease was quantified by bioluminescent imaging and survival was monitored. aNK cell infiltration into primary tumors was quantified by flow cytometry and immunohistochemistry at varying timepoints.ResultsIn vitro, aNK cells and dinutuximab were more cytotoxic than either treatment alone. In vivo, treatment with aNK cells plus dinutuximab prior to resection of the primary tumor was most effective in limiting metastatic disease and prolonging survival. aNK cell infiltration into xenograft tumors was observed after 1 day and peaked at 5 days following injection.ConclusionDinutuximab plus aNK cell immunotherapy initiated before resection of primary tumors decreases disease burden and prolongs survival in an experimental mouse model of NB. These findings support the clinical investigation of this treatment strategy during induction therapy in patients with high-risk NB.

Neuroblastoma (NBL) accounts for a disproportionate number of deaths among childhood malignancies despite intensive multimodal therapy that includes antibody targeting disialoganglioside GD2, a NBL antigen. Unfortunately, resistance to anti-GD2 immunotherapy is frequent and we aimed to investigate mechanisms of resistance in NBL. GD2 expression was quantified by flow cytometry and anti-GD2 antibody internalization was measured using real-time microscopy in 20 human NBL cell lines. Neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) assays were performed on a subset of the cell lines (n = 12), and results were correlated with GD2 expression and antibody internalization. GD2 was expressed on 19 of 20 NBL cell lines at variable levels, and neutrophil-mediated ADCC was observed only in GD2-expressing cell lines. We found no correlation between level of GD2 expression and sensitivity to neutrophil-mediated ADCC, suggesting that GD2 expression of many cell lines was above a threshold required for maximal ADCC, such that expression level could not be used to predict subsequent cytotoxicity. Instead, anti-GD2 antibody internalization, a process that occurred universally but differentially across GD2-expressing NBL cell lines, was inversely correlated with ADCC. Treatment with endocytosis inhibitors EIPA, chlorpromazine, MBCD, and cytochalasin-D showed potential to inhibit antibody internalization; however, only MBCD resulted in significantly increased sensitivity to neutrophil-mediated ADCC in 4 of 4 cell lines in vitro. Our data suggest that antibody internalization may represent a novel mechanism of immunotherapy escape by NBL and provide proof-of-principle that targeting pathways involved in antibody internalization may improve the efficacy of anti-GD2 immunotherapies.

// Michael D. Hadjidaniel 1, * , Sakunthala Muthugounder 1, * , Long T. Hung 1, * , Michael A. Sheard 1 , Soheila Shirinbak 1 , Randall Y. Chan 1, 3 , Rie Nakata 1 , Lucia Borriello 1 , Jemily Malvar 1 , Rebekah J. Kennedy 1 , Hiroshi Iwakura 2 , Takashi Akamizu 2 , Richard Sposto 1, 3 , Hiroyuki Shimada 1, 3 , Yves A. DeClerck 1, 3 and Shahab Asgharzadeh 1, 3 1 Children's Hospital Los Angeles, Children's Center for Cancer and Blood Diseases, Division of Hematology, Oncology and Blood & Marrow Transplantation, and The Saban Research Institute, Los Angeles, CA, USA 2 The First Department of Medicine, Wakayama Medical University, Wakayama, Japan 3 Keck School of Medicine, University of Southern California, Los Angeles, CA, USA * These authors have contributed equally to this work Correspondence to: Shahab Asgharzadeh, email: sasgharzadeh@chla.usc.edu Keywords: neuroblastoma, tumor-associated macrophages, tumor microenvironment, STAT3, MYC Received: March 17, 2017 Accepted: July 19, 2017 Published: September 16, 2017 ABSTRACT Tumor-associated macrophages (TAMs) are strongly associated with poor survival in neuroblastomas that lack MYCN amplification. To study TAM action in neuroblastomas, we used a novel murine model of spontaneous neuroblastoma lacking MYCN amplification, and observed recruitment and polarization of TAMs, which in turn enhanced neuroblastoma proliferation and growth. In both murine and human neuroblastoma cells, we found that TAMs increased STAT3 activation in neuroblastoma cells and transcriptionally up-regulated the MYC oncogene. Analysis of human neuroblastoma tumor specimens revealed that MYC up-regulation correlates with markers of TAM infiltration. In an IL6 ko neuroblastoma model, the absence of IL-6 protein had no effect on tumor development and prevented neither STAT3 activation nor MYC up-regulation. In contrast, inhibition of JAK-STAT activation using AZD1480 or the clinically admissible inhibitor ruxolitinib significantly reduced TAM-mediated growth of neuroblastomas implanted subcutaneously in NOD scid gamma mice. Our results point to a unique mechanism in which TAMs promote tumor cells that lack amplification of an oncogene common to the malignancy by up-regulating transcriptional expression of a distinct oncogene from the same gene family, and underscore the role of IL-6-independent activation of STAT3 in this mechanism. Amplification of MYCN or constitutive up-regulation of MYC protein is observed in approximately half of high-risk tumors; our findings indicate a novel role of TAMs as inducers of MYC expression in neuroblastomas lacking independent oncogene activation.

Cancer-associated fibroblasts (CAF) have been suggested to originate from mesenchymal stromal cells (MSC), but their relationship with MSCs is not clear. Here, we have isolated from primary human neuroblastoma tumors a population of αFAP- and FSP-1–expressing CAFs that share phenotypic and functional characteristics with bone marrow–derived MSCs (BM-MSC). Analysis of human neuroblastoma tumors also confirmed the presence of αFAP- and FSP-1–positive cells in the tumor stroma, and their presence correlated with that of M2 tumor-associated macrophages. These cells (designated CAF-MSCs) enhanced in vitro neuroblastoma cell proliferation, survival, and resistance to chemotherapy and stimulated neuroblastoma tumor engraftment and growth in immunodeficient mice, indicating an effect independent of the immune system. The protumorigenic activity of MSCs in vitro and in xenografted mice was dependent on the coactivation of JAK2/STAT3 and MEK/ERK1/2 in neuroblastoma cells. In a mouse model of orthotopically implanted neuroblastoma cells, inhibition of JAK2/STAT3 and MEK/ERK/1/2 by ruxolitinib and trametinib potentiated tumor response to etoposide and increased overall survival. These data point to a new type of protumorigenic CAF in the tumor microenvironment of neuroblastoma and to STAT3 and ERK1/2 as mediators of their activity. Cancer Res; 77(18); 5142–57. ©2017 AACR.

Introduction: CD19-directed chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia (ALL), inducing complete remission in 70-90% of patients. Importantly, 40% of patients receiving CD19 CAR T cell therapy develop immune effector cell-associated neurotoxicity syndrome (ICANS), which manifests clinically as confusion, delirium, aphasia and seizures. The neurologic and immune mechanisms of ICANS remain poorly understood, and identifying biomarkers of ICANS are critical to develop strategies to mitigate or prevent this serious side effect. Methods: In an ongoing prospective, longitudinal study, we report on 11 patients with relapsed ALL (ages 6-19 years, 7 males) who received CD19 CAR T cell therapy and underwent serial comprehensive assessments including multimodal magnetic resonance imaging (MRI) of the brain (anatomical, perfusion, diffusion tensor imaging and MR spectroscopy) and neuropsychological testing. Extensive neuropsychological testing of a wide range of cognitive functions was performed. Time points for data collection included: 1) at baseline, prior to lymphodepleting (LD) chemotherapy, consisting of fludarabine and cyclophosphamide, and post-CD19 CAR T cell infusion, 2) on day 10, at peak ICANS (if applicable), and 3) at day 28, upon resolution of cytokine release syndrome (CRS) and ICANS. In addition, multimodal MRI and neuropsychological tests were acquired in 14 healthy controls (ages 5-20 years, 5 males), at one time point as baseline. The MRI data were acquired on a 3T Philips MR scanner using a 32 channel head coil without the use of sedatives or contrast agents. All MRI data were processed blinded to the participant diagnosis and the order of data acquisition. Results: In our cohort of 11 patients, 7 developed cytokine release syndrome and 4 developed ICANS (Table 1). At baseline, patients relative to controls had thinner cortices across the entire brain (Fig.1A). Patients who developed ICANS, compared to those who did not, had additional cortical thinning in the frontal, anterior cingulate, anterior temporal, dorsal parietal, and posterior cingulate cortices (Fig.1B), which together constitute the dorsal attentional network. Consistent with the thinning of attentional cortices, baseline neuropsychological assessments of patients who developed ICANS relative to those who did not revealed worse scores in the measures of inattention (p=0.025), ADHD (p=0.038), and panic disorder (p=0.016), as well as impaired working memory represented by poorer performance on the backward digit span (p=0.038) and digit total (p=0.01) tasks. Regions in the dorsal attentional network demonstrated further cortical thinning on day 10 post-infusion and rebounded to baseline values by day 28 post-infusion in most, but not all patients (Fig.1B). Conclusions: MRI findings revealed that all patients demonstrated global thinning of the cortex at baseline, likely secondary to prior chemotherapy and/or radiation therapy for ALL. These findings were more pronounced in patients who later developed ICANS and were concentrated in the area of the dorsal attentional network of the brain. This was recapitulated in baseline neuropsychological assessments which revealed deficits in attention and working memory in patients who later developed ICANS. In addition, patients with ICANS had additional cortical thinning by day 10 post-CAR T cell infusion, yet had the least cortical reserve to withstand such changes. Although most patients returned to baseline cortical thickness by day 28, which was coincident with ICANS resolution, a subset of patients failed to return to baseline cortical thickness, and we predict that these may be the patients at highest risk for long term brain changes and associated cognitive disturbances. We anticipate that the combination of MRI changes in cortical thickness, especially in the dorsal attentional network, and neuropsychologic impairments at baseline could serve as predictive biomarkers for patients who develop ICANS which in turn could allow for future interventional trials designed to mitigate or prevent ICANS in at-risk individuals. Disclosures Pulsipher: Novartis: Honoraria; Jasper: Honoraria; Bellicum: Honoraria; Mesoblast: Honoraria; Miltenyi: Honoraria, Research Funding; Adaptive: Research Funding. Wayne:Kite, a Gilead Company: Research Funding; Servier: Research Funding.

Purpose: We determined whether elimination of CD105+ cells in the tumor microenvironment (TME) with anti-CD105 antibodies enhanced anti-disialoganglioside (GD2) antibody dinutuximab therapy of neuroblastoma when combined with activated natural killer (aNK) cells. Experimental Design: The effect of MSCs and monocytes on antibody-dependent cellular cytotoxicity (ADCC) mediated by dinutuximab with aNK cells against neuroblastoma cells was determined in vitro. ADCC with anti-CD105 mAb TRC105 and aNK cells against MSCs, monocytes, and endothelial cells, which express CD105, was evaluated. Anti-neuroblastoma activity in immunodeficient NSG mice of dinutuximab with aNK cells without or with anti-CD105 mAbs was determined using neuroblastoma cell lines and a patient-derived xenograft. Results: ADCC mediated by dinutuximab with aNK cells against neuroblastoma cells in vitro was suppressed by addition of MSCs and monocytes, and dinutuximab with aNK cells was less effective against neuroblastomas formed with coinjected MSCs and monocytes in NSG mice than against those formed by tumor cells alone. Anti-CD105 antibody TRC105 with aNK cells mediated ADCC against MSCs, monocytes, and endothelial cells. Neuroblastomas formed in NSG mice by two neuroblastoma cell lines or a patient-derived xenograft coinjected with MSCs and monocytes were most effectively treated with dinutuximab and aNK cells when anti-human (TRC105) and anti-mouse (M1043) CD105 antibodies were added, which depleted human MSCs and murine endothelial cells and macrophages from the TME. Conclusions: Immunotherapy of neuroblastoma with anti-GD2 antibody dinutuximab and aNK cells is suppressed by CD105+ cells in the TME, but suppression is overcome by adding anti-CD105 antibodies to eliminate CD105+ cells.

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.

Tumor-associated macrophages can promote growth of cancers. In neuroblastoma, tumor-associated macrophages have greater frequency in metastatic versus loco-regional tumors, and higher expression of genes associated with macrophages helps to predict poor prognosis in the 60% of high-risk patients who have MYCN-non-amplified disease. The contribution of cytotoxic T-lymphocytes to anti-neuroblastoma immune responses may be limited by low MHC class I expression and low exonic mutation frequency. Therefore, we modelled human neuroblastoma in T-cell deficient mice to examine whether depletion of monocytes/macrophages from the neuroblastoma microenvironment by blockade of CSF-1R can improve the response to chemotherapy. In vitro, CSF-1 was released by neuroblastoma cells, and topotecan increased this release. In vivo, neuroblastomas formed by subcutaneous co-injection of human neuroblastoma cells and human monocytes into immunodeficient NOD/SCID mice had fewer human CD14(+) and CD163(+) cells and mouse F4/80(+) cells following CSF-1R blockade. In subcutaneous or intra-renal models in immunodeficient NSG or NOD/SCID mice, CSF-1R blockade alone did not affect tumor growth or mouse survival. However, when combined with cyclophosphamide plus topotecan, the CSF-1R inhibitor BLZ945, either without or with anti-human and anti-mouse CSF-1 mAbs, inhibited neuroblastoma growth and synergistically improved mouse survival. These findings indicate that depletion of tumor-associated macrophages from neuroblastomas can be associated with increased chemotherapeutic efficacy without requiring a contribution from T-lymphocytes, suggesting the possibility that combination of CSF-1R blockade with chemotherapy might be effective in patients who have limited anti-tumor T-cell responses.

BACKGROUND: Single agent studies targeting the tumor microenvironment in central nervous system (CNS) tumors have largely been disappointing. Combination therapies targeting various pathways and cell types may be a more effective strategy. In this phase I study, we evaluated the combination of dasatinib, lenalidomide, and temozolomide in children with relapsed or refractory primary CNS tumors. METHODS: Patients 1–21 years old with relapsed or refractory CNS tumors were eligible. Starting doses of dasatinib and lenalidomide were 65 mg/m(2)/dose twice daily and 55 mg/m(2) once daily, respectively, while temozolomide was constant at 75 mg/m(2) daily. The study followed a 3+3 phase I design, with a 4-week dose-limiting toxicity (DLT) evaluation period. Serial peripheral blood lymphocyte subsets were evaluated in consenting patients. RESULTS: Fifteen patients were enrolled and thirteen were DLT-evaluable. DLTs occurred in 5 patients, including somnolence and confusion (1 patient), hypokalemia (1 patient) and thrombocytopenia (3 patients). The maximum tolerated dose for the combination was dasatinib 65 mg/m(2) twice daily, lenalidomide 40 mg/m(2) daily, and temozolomide 75 mg/m(2) daily, for 21 days followed by 7 days rest in repeating 28-day cycles. Transient increases in natural killer effector cells and cytotoxic T-cells were seen after 1 week of treatment. One out of 6 response-evaluable patients showed a partial response. CONCLUSIONS: The combination was feasible and relatively well tolerated in this heavily pre-treated population. The most common toxicities were hematologic. Preliminary evidence of clinical benefit was seen.

Introduction: CD19-directed chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment for B-cell acute lymphoblastic leukemia (ALL), inducing complete remission in 70-90% of highly refractory patients. Unfortunately, CAR-T cell therapy is associated with immune effector cell-associated neurotoxicity syndrome (ICANS) in 40% of patients, which presents as confusion, memory impairment, aphasia, altered level of consciousness and/or delirium. The neurobiological and immune bases of ICANS remain poorly understood, and identifying them will help develop strategies for early intervention to mitigate ICANS. Methods: From an ongoing prospective, longitudinal study, we report on the first 7 patients with relapsed ALL (age 5-19 years, 5 males) who underwent comprehensive assessments at baseline before lympho-depleting (LD) chemotherapy (fludarabine and cyclophosphamide) and infusion of CD19-directed CAR T-cells, and post-infusion, on days 10 and 28. These assessments included multimodal magnetic resonance imaging (MRI) of the brain (anatomical, perfusion, diffusion tensor imaging and MR spectroscopy), neuropsychological testing, serum measures of blood-brain barrier permeability and cytokines, and immune cell profiles in peripheral blood using mass cytometry by time-of-flight (CyTOF). We also acquired multimodal MRI and neuropsychological testing in 9 healthy controls (age 5-20 years, 7 males), once, at baseline. MRI data were acquired on a 3T Philips MR scanner using a 32 channel head coil without the use of sedatives or contrast agents and were processed blind to the diagnosis and order of data acquisition. Results: In our cohort of 7 patients, 5 developed cytokine release syndrome and 4 developed ICANS. Brain imaging findings are demonstrated in figures 1 (cortical thickness) and 2 (blood perfusion). Prior to CAR T-cell infusion patients relative to healthy controls had thinner cortex bilaterally across large portions of the brain. By post-infusion day 10, cortical thickness decreased in the postcentral gyrus (PoG) and posterior temporal lobe (pTL). Subsequently, by post-infusion day 28, cortex thickened in the PoG but continued thinning in the pTL. Patients relative to controls had lower regional cerebral blood flow (rCBF) in the thalamus, insular cortex, and cingulate gyrus, and higher rCBF in the genu of the corpus callosum (gCC). By post-infusion day 10, rCBF increased in the thalamus and decreased in the putamen and gCC. By post-infusion day 28, rCBF in the thalamus decreased and reverted towards baseline values, whereas rCBF in gCC continued to decrease away from baseline values. CyTOF analyses, as expected, showed expansion of monocytes and myeloid-derived suppressor cells by post-infusion day 10. Furthermore, in these immune cells, phosphorylation of key signaling proteins (e.g. extracellular-signal regulated kinase, ERK1/2) increased in response to external stimulation. Conclusions: Patients had widespread brain abnormalities at baseline compared to controls, likely because of prior therapies and/or underlying ALL. Post LD chemotherapy and CAR T-cell infusion, several brain abnormalities worsened by day 10 and then reverted toward baseline values by day 28. These temporal changes are generally consistent with clinically reported development of ICANS by day 10, which largely resolves by day 28. However, in certain regions brain measures progressively deviated from baseline and control values through day 28, suggesting some abnormalities associated with CAR T therapy may persist. Preliminary immune cell profiling implicates monocytes as a possible source of elevated cytokines following CAR T-cell infusion. Analysis for correlation of these findings with symptomatic ICANS and neuropsychological measures is ongoing. Patients on this study will also be followed at 6 months and 1 year to evaluate for long term neurological effects of CAR T-cell therapy. Figure 1: Cortical Thickness abnormalities in patients relative to healthy controls (1st panel); within-patient changes post-infusion (2nd, 3rd, & 4th panel). Blue & violet show decreases and orange & red show increases in cortical thickness. Figure 2: Blood perfusion abnormalities in patients relative to healthy controls (1st panel); within patient changes post infusion (2nd, 3rd, & 4th panel). Blue & violet show decreases and orange & red show increases in regional cerebral blood flow. Disclosures Pulsipher: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Other: Education for employees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Miltenyi: Research Funding; Bellicum: Consultancy; Amgen: Other: Lecture; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Medac: Honoraria. Wayne:Spectrum Pharmaceuticals: Consultancy, Research Funding; AbbVie: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding; Servier: Consultancy.

Neuroblastoma cells have been reported to be resistant to death induced by soluble, recombinant forms of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (CD253/TNFSF10) because of low or absent expression of caspase-8 and/or TRAIL-receptor 2 (TRAIL-R2/DR5/CD262/TNFRSF10b). However, their sensitivity to membrane-bound TRAIL on natural killer (NK) cells is not known. Comparing microarray gene expression and response to NK cell-mediated cytotoxicity, we observed a correlation between TRAIL-R2 expression and the sensitivity of 14 neuroblastoma cell lines to the cytotoxicity of NK cells activated with interleukin (IL)-2 plus IL-15. Even though most NK cytotoxicity was dependent upon perforin, the cytotoxicity was supplemented by TRAIL in 14 of 17 (82%) neuroblastoma cell lines as demonstrated using an anti-TRAIL neutralizing antibody. Similarly, a recently developed NK cell expansion system employing IL-2 plus lethally irradiated K562 feeder cells constitutively expressing membrane-bound IL-21 (K562 clone 9.mbIL21) resulted in activated NK cells derived from normal healthy donors and neuroblastoma patients that also utilized TRAIL to supplement cytotoxicity. Exogenous interferon-γ upregulated expression of caspase-8 in 3 of 4 neuroblastoma cell lines and increased the contribution of TRAIL to NK cytotoxicity against 2 of the 3 lines; however, relatively little inhibition of cytotoxicity was observed when activated NK cells were treated with an anti-interferon-γ neutralizing antibody. Constraining the binding of anti-TRAIL neutralizing antibody to membrane-bound TRAIL but not soluble TRAIL indicated that membrane-bound TRAIL alone was responsible for essentially all of the supplemental cytotoxicity. Together, these findings support a role for membrane-bound TRAIL in the cytotoxicity of NK cells against neuroblastoma cells.

Purpose: Immunotherapy of high-risk neuroblastoma using the anti-GD2 antibody dinutuximab induces antibody-dependent cell-mediated cytotoxicity (ADCC). Galunisertib, an inhibitor of TGFβR1, was examined for its ability to enhance the efficacy of dinutuximab in combination with human ex vivo activated NK (aNK) cells against neuroblastoma. Experimental Design: TGFB1 and TGFBR1 mRNA expression was determined for 249 primary neuroblastoma tumors by microarray analysis. The ability of galunisertib to inhibit SMAD activity induced by neuroblastoma patient blood and bone marrow plasmas in neuroblastoma cells was tested. The impact of galunisertib on TGFβ1-induced inhibition of aNK cytotoxicity and ADCC in vitro and on anti-neuroblastoma activity in NOD-scid gamma (NSG) mice was determined. Results: Neuroblastomas express TGFB1 and TGFBR1 mRNA. Galunisertib suppressed SMAD activation in neuroblastoma cells induced by exogenous TGFβ1 or by patient blood and bone marrow plasma, and suppressed SMAD2 phosphorylation in human neuroblastoma cells growing in NSG mice. In NK cells treated in vitro with exogenous TGFβ1, galunisertib suppressed SMAD2 phosphorylation and restored the expression of DNAM-1, NKp30, and NKG2D cytotoxicity receptors and the TRAIL death ligand, the release of perforin and granzyme A, and the direct cytotoxicity and ADCC of aNK cells against neuroblastoma cells. Addition of galunisertib to adoptive cell therapy with aNK cells plus dinutuximab reduced tumor growth and increased survival of mice injected with two neuroblastoma cell lines or a patient-derived xenograft. Conclusions: Galunisertib suppresses activation of SMAD2 in neuroblastomas and aNK cells, restores NK cytotoxic mechanisms, and increases the efficacy of dinutuximab with aNK cells against neuroblastoma tumors. Clin Cancer Res; 23(3); 804–13. ©2016 AACR. See related commentary by Zenarruzabeitia et al., p. 615

As p53 loss of function (LOF) confers high-level drug resistance in neuroblastoma, p53-independent therapies might have superior activity in recurrent neuroblastoma. We tested the activity of vorinostat, a histone deacetylase inhibitor, and flavopiridol, a pan-Cdk inhibitor, in a panel of multidrug-resistant neuroblastoma cell lines that included lines with wild-type (wt) and transcriptionally active TP53 (n = 3), mutated (mt), and LOF TP53 (n = 4) or p14ARF deletion (n = 1). The combination of vorinostat and flavopiridol was synergistic and significantly more cytotoxic (P < 0.001) in cell lines with p53-LOF and in the clones stably transfected with dominant-negative p53 plasmids. Cell cycle analysis by flow cytometry showed prominent cell-cycle arrest in G2/M (37%) for a cell line with wt TP53 (SK-N-RA) at 16 to 20 hours, while cells with mt TP53 (CHLA-90) slipped into sub-G1 at 6 to 24 hours (25%–40% specific cell death). The morphological hallmarks of mitotic cell death, including defective spindle formation and abnormal cytokinesis, were detected by confocal microscopy after the treatment with vorinostat + flavopiridol combination in CHLA-90. The combination caused reduction in the expression of G2/M proteins (cyclin B1, Mad2, MPM2) in 2 cell lines with mt TP53 but not in those with wt TP53. Plk1 expression was reduced in all treated lines. Small interfering RNA knockdown of Mad2 and cyclin B1 or Plk1 synergistically reduced the clonogenicity of CHLA-90 cells. The combination of HDAC inhibitor and flavopiridol may be a unique approach to treating neuroblastomas with p53 LOF, one that evokes induction of mitotic failure. Mol Cancer Ther; 9(12); 3289–301. ©2010 AACR.

10522Background: Ch14.18 (dinutuximab) increases event free and overall survival in patients with high-risk NB when given in a regimen with GM-CSF/IL-2. However, this therapy has significant toxici...