Vidyalakshmi Chandramohan

Reciprocal Control of Forkhead box O 3a and c-Myc via the Phosphatidylinositol 3-Kinase Pathway Coordinately Regulates p27Kip1 Levels

B cell receptor (BCR) engagement of murine WEHI 231 immature B lymphoma cells leads sequentially to a drop in NF-κB and c-Myc, and induction of the p27Kip1 cyclin-dependent kinase inhibitor, which promotes growth arrest and apoptosis. BCR engagement was recently shown to induce a drop in phosphatidylinositol 3-kinase (PI3K)/Akt signaling, preceding the increase in p27. As induction of p27 is due to an increase in gene transcription, we investigated the role of the Forkhead box O (FOXO) transcription factor family, which has been shown to potently induce p27 promoter activity. We demonstrate that pharmacologic inhibitors of PI3K or BCR engagement lead to decreased inactive cytoplasmic levels and increased active functional nuclear FOXO3a. In contrast, inhibition of PI3K/Akt signaling decreased the levels of NF-κB and c-Myc, which has been shown to repress p27 promoter activity. To test the effects of ectopic c-Myc on endogenous p27 levels, WEHI 231 cells stably expressing c-Myc or empty vector DNA were prepared. Ectopic c-Myc blocked the induction of p27 expression upon either inhibition of PI3K or BCR engagement. Thus, p27Kip1 is coordinately regulated via two arms of a signaling pathway that are inversely controlled upon inhibition of PI3K: induction of the activator FOXO3a and down-regulation of the repressor c-Myc.

Evaluation of anti-podoplanin rat monoclonal antibody NZ-1 for targeting malignant gliomas ☆

INTRODUCTION Podoplanin/aggrus is a mucin-like sialoglycoprotein that is highly expressed in malignant gliomas. Podoplanin has been reported to be a novel marker to enrich tumor-initiating cells, which are thought to resist conventional therapies and to be responsible for cancer relapse. The purpose of this study was to determine whether an anti-podoplanin antibody is suitable to target radionuclides to malignant gliomas. METHODS The binding affinity of an anti-podoplanin antibody, NZ-1 (rat IgG(2a)), was determined by surface plasmon resonance and Scatchard analysis. NZ-1 was radioiodinated with (125)I using Iodogen [(125)I-NZ-1(Iodogen)] or N-succinimidyl 4-guanidinomethyl 3-[(131)I]iodobenzoate ([(131)I]SGMIB-NZ-1), and paired-label internalization assays of NZ-1 were performed. The tissue distribution of (125)I-NZ-1(Iodogen) and that of [(131)I]SGMIB-NZ-1 were then compared in athymic mice bearing glioblastoma xenografts. RESULTS The dissociation constant (K(D)) of NZ-1 was determined to be 1.2 × 10(-10) M by surface plasmon resonance and 9.8 × 10(-10) M for D397MG glioblastoma cells by Scatchard analysis. Paired-label internalization assays in LN319 glioblastoma cells indicated that [(131)I]SGMIB-NZ-1 resulted in higher intracellular retention of radioactivity (26.3 ± 0.8% of initially bound radioactivity at 8 h) compared to that from the (125)I-NZ-1(Iodogen) (10.0 ± 0.1% of initially bound radioactivity at 8 h). Likewise, tumor uptake of [(131)I]SGMIB-NZ-1 (39.9 ± 8.8 %ID/g at 24 h) in athymic mice bearing D2159MG xenografts in vivo was significantly higher than that of (125)I-NZ-1(Iodogen) (29.7 ± 6.1 %ID/g at 24 h). CONCLUSIONS The overall results suggest that an anti-podoplanin antibody NZ-1 warrants further evaluation for antibody-based therapy against glioblastoma.

Recombinant anti-podoplanin (NZ-1) immunotoxin for the treatment of malignant brain tumors

Our study demonstrates the glioma tumor antigen podoplanin to be present at very high levels (>90%) in both glioblastoma (D2159MG, D08‐0308MG and D08‐0493MG) and medulloblastoma (D283MED, D425MED and DAOY) xenografts and cell line. We constructed a novel recombinant single‐chain antibody variable region fragment (scFv), NZ‐1, specific for podoplanin from the NZ‐1 hybridoma. NZ‐1‐scFv was then fused to Pseudomonas exotoxin A, carrying a C‐terminal KDEL peptide (NZ‐1‐PE38KDEL). The immunotoxin (IT) was further stabilized by a disulfide (ds) bond between the heavy‐chain and light‐chain variable regions as the construct NZ‐1‐(scdsFv)‐PE38KDEL. NZ‐1‐(scdsFv)‐PE38KDEL exhibited significant reactivity to glioblastoma and medulloblastoma cells. The affinity of NZ‐1‐(scdsFv), NZ‐1‐(scdsFv)‐PE38KDEL and NZ‐1 antibody for podoplanin peptide was 2.1 × 10−8 M, 8.0 × 10−8 M and 3.9 × 10−10 M, respectively. In a protein stability assay, NZ‐1‐(scdsFv)‐PE38KDEL retained 33–98% of its activity, whereas that of NZ‐1‐PE38KDEL declined to 13% of its initial levels after incubation at 37°C for 3 days. In vitro cytotoxicity of the NZ‐1‐(scdsFv)‐PE38KDEL was measured in cells isolated from glioblastoma xenografts, D2159MG, D08‐0308MG and D08‐0493MG, and in the medulloblastoma D283MED, D425MED and DOAY xenografts and cell line. The NZ‐1‐(scdsFv)‐PE38KDEL IT was highly cytotoxic, with an 50% inhibitory concentration in the range of 1.6–29 ng/ml. Significantly, NZ‐1‐(scdsFv)‐PE38KDEL demonstrated tumor growth delay, averaging 24 days (p < 0.001) and 21 days (p < 0.001) in D2159MG and D283MED in vivo tumor models, respectively. Crucially, in the D425MED intracranial tumor model, NZ‐1‐(scdsFv)‐PE38KDEL caused a 41% increase in survival (p ≤ 0.001). In preclinical studies, NZ‐1‐(scdsFv)‐PE38KDEL exhibited significant potential as a targeting agent for malignant brain tumors.

c‐Myc represses FOXO3a‐mediated transcription of the gene encoding the p27Kip1 cyclin dependent kinase inhibitor

The p27Kip1 (p27) cyclin‐dependent kinase inhibitor and c‐Myc oncoprotein play essential roles in control of cell cycle progression and apoptosis. Induction of p27 (CDKN1B) gene transcription by Forkhead box O proteins such as FOXO3a leads to growth arrest and apoptosis. Previously, we observed that B cell receptor (surface IgM) engagement of WEHI 231 immature B lymphoma cells with an anti‐IgM antibody results in activation of FOXO3a, growth arrest and apoptosis. As ectopic c‐Myc expression in these cells prevented anti‐IgM induction of p27 and cell death, we hypothesized that c‐Myc represses FOXO3a‐mediated transcription. Here we show that c‐Myc inhibits FOXO3a‐mediated activation of the p27 promoter in multiple cell lines. The mechanism of this repression was explored using a combination of co‐immunoprecipitation, oligonucleotide precipitation, and chromatin immunoprecipitation experiments. The studies demonstrate a functional association of FOXO3a and c‐Myc on a proximal Forkhead binding element in the p27 promoter. This association involves the Myc box II domain of c‐Myc and the N‐terminal DNA‐binding portion of FOXO3a. Analysis of publicly available microarray datasets showed an inverse pattern of c‐MYC and p27 RNA expression in primary acute myeloid leukemia, prostate cancer and tongue squamous cell carcinoma samples. The inhibition of FOXO3a‐mediated activation of the p27 gene by the high aberrant expression of c‐Myc in many tumor cells likely contributes to their uncontrolled proliferation and invasive phenotype. J. Cell. Biochem. 104: 2091–2106, 2008.

Construction of an Immunotoxin, D2C7-(scdsFv)-PE38KDEL, Targeting EGFRwt and EGFRvIII for Brain Tumor Therapy

Purpose: The EGF receptor gene (EGFR) is most frequently amplified and overexpressed, along with its deletion mutant, EGFRvIII, in glioblastoma. We tested the preclinical efficacy of the recombinant immunotoxin, D2C7-(scdsFv)-PE38KDEL, which is reactive with a 55-amino acid (AA) region present in the extracellular domain of both EGFRwt (583-637 AAs) and EGFRvIII (292-346 AAs) proteins. Experimental Design: The binding affinity and specificity of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII were measured by surface-plasmon resonance and flow cytometry. In vitro cytotoxicity of D2C7-(scdsFv)-PE38KDEL was measured by inhibition of protein synthesis in human EGFRwt-transfected NR6 (NR6W), human EGFRvIII-transfected NR6 (NR6M), EGFRwt-overexpressing A431-epidermoid-carcinoma, and glioblastoma xenograft cells (43, D08-0493MG, D2159MG, and D270MG). In vivo antitumor efficacy of D2C7-(scdsFv)-PE38KDEL was evaluated using 43, NR6M, and D270MG orthotopic tumor models. Results: The KD of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII was 1.6 × 10−9 mol/L and 1.3 × 10−9 mol/L, respectively. Flow cytometry with NR6W and NR6M cells confirmed the specificity of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII. The D2C7-(scdsFv)-PE38KDEL IC50 was 0.18 to 2.5 ng/mL on cells expressing EGFRwt (NR6W, A431, 43, and D08-0493MG). The D2C7-(scdsFv)-PE38KDEL IC50 was approximately 0.25 ng/mL on EGFRvIII-expressing cells (NR6M) and on EGFRwt- and EGFRvIII-expressing glioblastoma xenograft cells (D2159MG and D270MG). Significantly, in intracranial tumor models of 43, NR6M, and D270MG, treatment with D2C7-(scdsFv)-PE38KDEL by convection-enhanced delivery prolonged survival by 310% (P = 0.006), 28% (P = 0.002), and 166% (P = 0.001), respectively. Conclusions: In preclinical studies, the D2C7-(scdsFv)-PE38KDEL immunotoxin exhibited significant potential for treating brain tumors expressing EGFRwt, EGFRvIII, or both. Clin Cancer Res; 19(17); 4717–27. ©2013 AACR.

CAR T Cells Targeting Podoplanin Reduce Orthotopic Glioblastomas in Mouse Brains.

The glioblastoma subtypes with the worst prognoses often bear podoplanin. T cells expressing a chimeric antigen receptor that targets podoplanin were specific and effective against PDPN-positive glioblastoma cells in vitro and increased survival time in a mouse model. Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in adults with a 5-year overall survival rate of less than 10%. Podoplanin (PDPN) is a type I transmembrane mucin-like glycoprotein, expressed in the lymphatic endothelium. Several solid tumors overexpress PDPN, including the mesenchymal type of GBM, which has been reported to present the worst prognosis among GBM subtypes. Chimeric antigen receptor (CAR)–transduced T cells can recognize predefined tumor surface antigens independent of MHC restriction, which is often downregulated in gliomas. We constructed a lentiviral vector expressing a third-generation CAR comprising a PDPN-specific antibody (NZ-1–based single-chain variable fragment) with CD28, 4-1BB, and CD3ζ intracellular domains. CAR-transduced peripheral blood monocytes were immunologically evaluated by calcein-mediated cytotoxic assay, ELISA, tumor size, and overall survival. The generated CAR T cells were specific and effective against PDPN-positive GBM cells in vitro. Systemic injection of the CAR T cells into an immunodeficient mouse model inhibited the growth of intracranial glioma xenografts in vivo. CAR T-cell therapy that targets PDPN would be a promising adoptive immunotherapy to treat mesenchymal GBM. Cancer Immunol Res; 4(3); 259–68. ©2016 AACR.

Induction of Viral, 7-Methyl-Guanosine Cap-Independent Translation and Oncolysis by Mitogen-Activated Protein Kinase-Interacting Kinase-Mediated Effects on the Serine/Arginine-Rich Protein Kinase

ABSTRACT Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m7G) “cap”-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNKs known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. IMPORTANCE We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells.

Toxin-Based Targeted Therapy for Malignant Brain Tumors

Despite advances in conventional treatment modalities for malignant brain tumors—surgery, radiotherapy, and chemotherapy—the prognosis for patients with high-grade astrocytic tumor remains dismal. The highly heterogeneous and diffuse nature of astrocytic tumors calls for the development of novel therapies. Advances in genomic and proteomic research indicate that treatment of brain tumor patients can be increasingly personalized according to the characteristics of the targeted tumor and its environment. Consequently, during the last two decades, a novel class of investigative drug candidates for the treatment of central nervous system neoplasia has emerged: recombinant fusion protein conjugates armed with cytotoxic agents targeting tumor-specific antigens. The clinical applicability of the tumor-antigen-directed cytotoxic proteins as a safe and viable therapy for brain tumors is being investigated. Thus far, results from ongoing clinical trials are encouraging, as disease stabilization and patient survival prolongation have been observed in at least 109 cases. This paper summarizes the major findings pertaining to treatment with the different antiglioma cytotoxins at the preclinical and clinical stages.

Antibody, T-cell and dendritic cell immunotherapy for malignant brain tumors

Modest improvement in brain tumor patient survival has been achieved through advances in surgical, adjuvant radiation and chemotherapeutic strategies. However, these traditional approaches have been unsuccessful in permanently controlling these aggressive tumors, with recurrence being quite common. Hence, there is a need for novel therapeutic approaches that specifically target the molecularly diverse brain tumor cell population. The ability of the immune system to recognize altered tumor cells while avoiding surrounding normal cells offers an enormous advantage over the nonspecific nature of the conventional treatment schemes. Therefore, immunotherapy represents a promising approach that may supplement the standard therapies in eliminating the residual brain tumor cells. This review summarizes different immunotherapeutic approaches currently being tested for malignant brain tumor treatment.

T-Cell Exhaustion Signatures Vary with Tumor Type and Are Severe in Glioblastoma

Purpose: T-cell dysfunction is a hallmark of glioblastoma (GBM). Although anergy and tolerance have been well characterized, T-cell exhaustion remains relatively unexplored. Exhaustion, characterized in part by the upregulation of multiple immune checkpoints, is a known contributor to failures amid immune checkpoint blockade, a strategy that has lacked success thus far in GBM. This study is among the first to examine, and credential as bona fide, exhaustion among T cells infiltrating human and murine GBM. Experimental Design: Tumor-infiltrating and peripheral blood lymphocytes (TILs and PBLs) were isolated from patients with GBM. Levels of exhaustion-associated inhibitory receptors and poststimulation levels of the cytokines IFNγ, TNFα, and IL2 were assessed by flow cytometry. T-cell receptor Vβ chain expansion was also assessed in TILs and PBLs. Similar analysis was extended to TILs isolated from intracranial and subcutaneous immunocompetent murine models of glioma, breast, lung, and melanoma cancers. Results: Our data reveal that GBM elicits a particularly severe T-cell exhaustion signature among infiltrating T cells characterized by: (1) prominent upregulation of multiple immune checkpoints; (2) stereotyped T-cell transcriptional programs matching classical virus-induced exhaustion; and (3) notable T-cell hyporesponsiveness in tumor-specific T cells. Exhaustion signatures differ predictably with tumor identity, but remain stable across manipulated tumor locations. Conclusions: Distinct cancers possess similarly distinct mechanisms for exhausting T cells. The poor TIL function and severe exhaustion observed in GBM highlight the need to better understand this tumor-imposed mode of T-cell dysfunction in order to formulate effective immunotherapeutic strategies targeting GBM. Clin Cancer Res; 24(17); 4175–86. ©2018 AACR. See related commentary by Jackson and Lim, p. 4059