Mobilization of innate and adaptive antitumor immune responses by the RNP-targeting antibody ATRC-101.

Immunotherapy approaches focusing on T cells have provided breakthroughs in treating solid tumors. However, there remains an opportunity to drive anticancer immune responses via other cell types, particularly myeloid cells. ATRC-101 was identified via a target-agnostic process evaluating antibodies produced by the plasmablast population of B cells in a patient with non-small cell lung cancer experiencing an antitumor immune response during treatment with checkpoint inhibitor therapy. Here, we describe the target, antitumor activity in preclinical models, and data supporting a mechanism of action of ATRC-101. Immunohistochemistry studies demonstrated tumor-selective binding of ATRC-101 to multiple nonautologous tumor tissues. In biochemical analyses, ATRC-101 appears to target an extracellular, tumor-specific ribonucleoprotein (RNP) complex. In syngeneic murine models, ATRC-101 demonstrated robust antitumor activity and evidence of immune memory following rechallenge of cured mice with fresh tumor cells. ATRC-101 increased the relative abundance of conventional dendritic cell (cDC) type 1 cells in the blood within 24 h of dosing, increased CD8+ T cells and natural killer cells in blood and tumor over time, decreased cDC type 2 cells in the blood, and decreased monocytic myeloid-derived suppressor cells in the tumor. Cellular stress, including that induced by chemotherapy, increased the amount of ATRC-101 target in tumor cells, and ATRC-101 combined with doxorubicin enhanced efficacy compared with either agent alone. Taken together, these data demonstrate that ATRC-101 drives tumor destruction in preclinical models by targeting a tumor-specific RNP complex leading to activation of innate and adaptive immune responses.

Non-progressing cancer patients have persistent B cell responses expressing shared antibody paratopes that target public tumor antigens.

There is significant debate regarding whether B cells and their antibodies contribute to effective anti-cancer immune responses. Here we show that patients with metastatic but non-progressing melanoma, lung adenocarcinoma, or renal cell carcinoma exhibited increased levels of blood plasmablasts. We used a cell-barcoding technology to sequence their plasmablast antibody repertoires, revealing clonal families of affinity matured B cells that exhibit progressive class switching and persistence over time. Anti-CTLA4 and other treatments were associated with further increases in somatic hypermutation and clonal family size. Recombinant antibodies from clonal families bound non-autologous tumor tissue and cell lines, and families possessing immunoglobulin paratope sequence motifs shared across patients exhibited increased rates of binding. We identified antibodies that caused regression of, and durable immunity toward, heterologous syngeneic tumors in mice. Our findings demonstrate convergent functional anti-tumor antibody responses targeting public tumor antigens, and provide an approach to identify antibodies with diagnostic or therapeutic utility.

Comparative mutational landscape analysis of patient-derived tumour xenografts.

BACKGROUND: Screening of patients for cancer-driving mutations is now used for cancer prognosis, remission scoring and treatment selection. Although recently emerged targeted next-generation sequencing-based approaches offer promising diagnostic capabilities, there are still limitations. There is a pressing clinical need for a well-validated, rapid, cost-effective mutation profiling system in patient specimens. Given their speed and cost-effectiveness, quantitative PCR mutation detection techniques are well suited for the clinical environment. The qBiomarker mutation PCR array has high sensitivity and shorter turnaround times compared with other methods. However, a direct comparison with existing viable alternatives are required to assess its true potential and limitations. METHODS: In this study, we evaluated a panel of 117 patient-derived tumour xenografts by the qBiomarker array and compared with other methods for mutation detection, including Ion AmpliSeq sequencing, whole-exome sequencing and droplet digital PCR. RESULTS: Our broad analysis demonstrates that the qBiomarker's performance is on par with that of other labour-intensive and expensive methods of cancer mutation detection of frequently altered cancer-associated genes, and provides a foundation for supporting its consideration as an option for molecular diagnostics. CONCLUSIONS: This large-scale direct comparison and validation of currently available mutation detection approaches is extremely relevant for the current scenario of precision medicine and will lead to informed choice of screening methodologies, especially in lower budget conditions or time frame limitations.

Milk derived colloid as a novel drug delivery carrier for breast cancer.

Triple negative breast cancer has an extremely poor prognosis when chemotherapy is no longer effective. To overcome drug resistance, novel drug delivery systems based on nanoparticles have had remarkable success. We produced a novel nanoparticle component 'MDC' from milk-derived colloid. In order to evaluate the anti-cancer effect of MDC, we conducted in vitro and in vivo experiments on cancer cell lines and a primary tumor derived breast xenograft. Doxorubicin (Dox) conjugated to MDC (MDC-Dox) showed higher cancer cell growth inhibition than MDC alone especially in cell lines with high EGFR expression. In a mouse melanoma model, MDC-Dox significantly suppressed tumor growth when compared with free Dox. Moreover, in a primary tumor derived breast xenograft, one of the mice treated with MDC-Dox showed partial regression, while mice treated with free Dox failed to show any suppression of tumor growth. We have shown that a novel nanoparticle compound made of simple milk-derived colloid has the capability for drug conjugation, and serves as a tumor-specific carrier of anti-cancer drugs. Further research on its safety and ability to carry various anti-cancer drugs into multiple drug-resistant primary breast models is warranted.

5-azacytidine reduces methylation, promotes differentiation and induces tumor regression in a patient-derived IDH1 mutant glioma xenograft.

Somatic mutations in Isocitrate Dehydrogenase 1 (IDH1) are frequent in low grade and progressive gliomas and are characterized by the production of 2-hydroxyglutarate (2-HG) from α-ketoglutarate by the mutant enzyme. 2-HG is an "oncometabolite" that competitively inhibits α-KG dependent dioxygenases resulting in various widespread cellular changes including abnormal hypermethylation of genomic DNA and suppression of cellular differentiation. Despite the growing understanding of IDH mutant gliomas, the development of effective therapies has proved challenging in part due to the scarcity of endogenous mutant in vivo models. Here we report the generation of an endogenous IDH1 anaplastic astrocytoma model which rapidly grows in vivo, produces 2-HG and exhibits DNA hypermethylation. Using this model, we have demonstrated the preclinical efficacy and mechanism of action of the FDA approved demethylating drug 5-azacytidine in vivo. Long term administration of 5-azacytidine resulted in reduction of DNA methylation of promoter loci, induction of glial differentiation, reduction of cell proliferation and a significant reduction in tumor growth. Tumor regression was observed at 14 weeks and subsequently showed no signs of re-growth at 7 weeks despite discontinuation of therapy. These results have implications for clinical trials of demethylating agents for patients with IDH mutated gliomas.

Notch pathway is activated by MAPK signaling and influences papillary thyroid cancer proliferation.

Mutually exclusive genetic alterations in the RET, RAS, or BRAF genes, which result in constitutively active mitogen-activated protein kinase (MAPK) signaling, are present in about 70% of papillary thyroid carcinomas (PTCs). However, the effect of MAPK activation on other signaling pathways involved in oncogenic transformation, such as Notch, remains unclear. In this study, we tested the hypothesis that the MAPK pathway regulates Notch signaling and that Notch signaling plays a role in PTC cell proliferation. Conditional induction of MAPK signaling oncogenes RET/PTC3 or BRAF(T1799A) in normal rat thyroid cell line mediated activation of Notch signaling, upregulating Notch1 receptor and Hes1, the downstream effector of Notch pathway. Conversely, pharmacological inhibition of MAPK reduced Notch signaling in PTC cell. Thyroid tumor samples from transgenic mice expressing BRAF(T1799A) and primary human PTC samples showed high levels of Notch1 expression. Down-regulation of Notch signaling by γ-secretase inhibitor (GSI) or NOTCH1 RNA interference reduces PTC cell proliferation. Moreover, the combination of GSI with a MAPK inhibitor enhanced the growth suppression in PTC cells. This study revealed that RET/PTC and BRAF(T1799A) activate Notch signaling and promote tumor growth in thyroid follicular cell. Taken together, these data suggest that Notch signaling may be explored as an adjuvant therapy for thyroid papillary cancer.

NY-ESO-1 expression in meningioma suggests a rationale for new immunotherapeutic approaches.

Meningiomas are the most common primary intracranial tumors. Surgical resection remains the treatment of choice for these tumors. However, a significant number of tumors are not surgically accessible, recur, or become malignant, necessitating the repetition of surgery and sometimes radiation. Chemotherapy is rarely used and is generally not recognized as an effective treatment. Cancer/testis (CT) genes represent a unique class of genes, which are expressed by germ cells, normally silenced in somatic cells, but activated in various cancers. CT proteins can elicit spontaneous immune responses in patients with cancer and this feature makes them attractive targets for immunotherapy-based approaches. We analyzed mRNA expression of 37 testis-restricted CT genes in a discovery set of 18 meningiomas by reverse transcription PCR. The overall frequency of expression of CT genes ranged from 5.6% to 27.8%. The most frequently expressed was NY-ESO-1, in 5 patients (27.8%). We subsequently analyzed NY-ESO-1 protein expression in a larger set of meningiomas by immunohistochemistry and found expression in 108 of 110 cases. In some cases, NY-ESO-1 expression was diffused and homogenous, but in most instances it was heterogeneous. Importantly, NY-ESO-1 expression was positively correlated with higher grade and patients presenting with higher levels of NY-ESO-1 staining had significantly worse disease-free and overall survival. We have also shown that NY-ESO-1 expression may lead to humoral immune response in patients with meningioma. Considering the limited treatment options for patients with meningioma, the potential of NY-ESO-1-based immunotherapy should be explored.

Yes-associated protein 1 is activated and functions as an oncogene in meningiomas.

The Hippo signaling pathway is functionally conserved in Drosophila melanogaster and mammals, and its proposed function is to control tissue homeostasis by regulating cell proliferation and apoptosis. The core components are composed of a kinase cascade that culminates with the phosphorylation and inhibition of Yes-associated protein 1 (YAP1). Phospho-YAP1 is retained in the cytoplasm. In the absence of Hippo signaling, YAP1 translocates to the nucleus, associates with co-activators TEAD1-4, and functions as a transcriptional factor promoting the expression of key target genes. Components of the Hippo pathway are mutated in human cancers, and deregulation of this pathway plays a role in tumorigenesis. Loss of the NF2 tumor suppressor gene is the most common genetic alteration in meningiomas, and the NF2 gene product, Merlin, acts upstream of the Hippo pathway. Here, we show that primary meningioma tumors have high nuclear expression of YAP1. In meningioma cells, Merlin expression is associated with phosphorylation of YAP1. Using an siRNA transient knockdown of YAP1 in NF2-mutant meningioma cells, we show that suppression of YAP1 impaired cell proliferation and migration. Conversely, YAP1 overexpression led to a strong augment of cell proliferation and anchorage-independent growth and restriction of cisplatin-induced apoptosis. In addition, expression of YAP1 in nontransformed arachnoidal cells led to the development of tumors in nude mice. Together, these findings suggest that in meningiomas, deregulation of the Hippo pathway is largely observed in primary tumors and that YAP1 functions as an oncogene promoting meningioma tumorigenesis.

Human replication protein A-Rad52-single-stranded DNA complex: stoichiometry and evidence for strand transfer regulation by phosphorylation.

The eukaryotic single-stranded DNA-binding protein, replication protein A (RPA), is essential in DNA metabolism and is phosphorylated in response to DNA-damaging agents. Rad52 and RPA participate in the repair of double-stranded DNA breaks (DSBs). It is known that human RPA and Rad52 form a complex, but the molecular mass, stoichiometry, and exact role of this complex in DSB repair are unclear. In this study, absolute molecular masses of individual proteins and complexes were measured in solution using analytical size-exclusion chromatography coupled with multiangle light scattering, the protein species present in each purified fraction were verified via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western analyses, and the presence of biotinylated ssDNA in the complexes was verified by chemiluminescence detection. Then, employing UV cross-linking, the protein partner holding the ssDNA was identified. These data show that phosphorylated RPA promoted formation of a complex with monomeric Rad52 and caused the transfer of ssDNA from RPA to Rad52. This suggests that RPA phosphorylation may regulate the first steps of DSB repair and is necessary for the mediator function of Rad52.

The neurofibromatosis 2 tumor suppressor gene product, merlin, regulates human meningioma cell growth by signaling through YAP.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder characterized by the occurrence of schwannomas and meningiomas. Several studies have examined the ability of the NF2 gene product, merlin, to function as a tumor suppressor in diverse cell types; however, little is known about merlin growth regulation in meningiomas. In Drosophila, merlin controls cell proliferation and apoptosis by signaling through the Hippo pathway to inhibit the function of the transcriptional coactivator Yorkie. The Hippo pathway is conserved in mammals. On the basis of these observations, we developed human meningioma cell lines matched for merlin expression to evaluate merlin growth regulation and investigate the relationship between NF2 status and Yes-associated protein (YAP), the mammalian homolog of Yorkie. NF2 loss in meningioma cells was associated with loss of contact-dependent growth inhibition, enhanced anchorage-independent growth and increased cell proliferation due to increased S-phase entry. In addition, merlin loss in both meningioma cell lines and primary tumors resulted in increased YAP expression and nuclear localization. Finally, siRNA-mediated reduction of YAP in NF2-deficient meningioma cells rescued the effects of merlin loss on cell proliferation and S-phase entry. Collectively, these results represent the first demonstration that merlin regulates cell growth in human cancer cells by suppressing YAP.

Notch activation is associated with tetraploidy and enhanced chromosomal instability in meningiomas.

The Notch signaling cascade is deregulated in diverse cancer types. Specific Notch function in cancer is dependent on the cellular context, the particular homologs expressed, and cross-talk with other signaling pathways. We have previously shown that components of the Notch signaling pathway are deregulated in meningiomas. However, the functional consequence of abnormal Notch signaling to meningiomas is unknown. Here, we report that exogenous expression of the Notch pathway effector, HES1, is associated with tetraploid cells in meningioma cell lines. Activated Notch1 and Notch2 receptors induced endogenous HES1 expression and were associated with tetraploidy in meningiomas. Tetraploid meningioma cells exhibited nuclear features of chromosomal instability and increased frequency of nuclear atypia, such as multipolar mitotic spindles and accumulation of cells with large nuclei. FACS-sorted tetraploid cells are viable but have higher rates of spontaneous apoptosis when compared with diploid cells. We have used spectral karyotyping to show that, in contrast to diploid cells, tetraploid cells develop a higher number of both numerical and structural chromosomal abnormalities. Our findings identify a novel function for the Notch signaling pathway in generating tetraploidy and contributing to chromosomal instability. We speculate that abnormal Notch signaling pathway is an initiating genetic mechanism for meningioma and potentially promotes tumor development.

An orthotopic skull base model of malignant meningioma.

Meningioma tumor growth involves the subarachnoid space that contains the cerebrospinal fluid. Modeling tumor growth in this microenvironment has been associated with widespread leptomeningeal dissemination, which is uncharacteristic of human meningiomas. Consequently, survival times and tumor properties are varied, limiting their utility in testing experimental therapies. We report the development and characterization of a reproducible orthotopic skull-base meningioma model in athymic mice using the IOMM-Lee cell line. Localized tumor growth was obtained by using optimal cell densities and matrigel as the implantation medium. Survival times were within a narrow range of 17-21 days. The xenografts grew locally compressing surrounding brain tissue. These tumors had histopathologic characteristics of anaplastic meningiomas including high cellularity, nuclear pleomorphism, cellular pattern loss, necrosis and conspicuous mitosis. Similar to human meningiomas, considerable invasion of the dura and skull and some invasion of adjacent brain along perivascular tracts were observed. The pattern of hypoxia was also similar to human malignant meningiomas. We use bioluminescent imaging to non-invasively monitor the growth of the xenografts and determine the survival benefit from temozolomide treatment. Thus, we describe a malignant meningioma model system that will be useful for investigating the biology of meningiomas and for preclinical assessment of therapeutic agents.

Molecular signatures define two main classes of meningiomas.

BACKGROUND: Meningiomas are common brain tumors that are classified into three World Health Organization grades (benign, atypical and malignant) and are molecularly ill-defined tumors. The purpose of this study was identify molecular signatures unique to the different grades of meningiomas and to unravel underlying molecular mechanisms driving meningioma tumorigenesis. RESULTS: We have used a combination of gene expression microarrays and array comparative genomic hybridization (aCGH) to show that meningiomas of all three grades fall into two main molecular groups designated 'low-proliferative' and 'high-proliferative' meningiomas. While all benign meningiomas fall into the low-proliferative group and all malignant meningiomas fall into the high-proliferative group, atypical meningiomas distribute into either one of these groups. High-proliferative atypical meningiomas had an elevated median MIB-1 labeling index and a greater frequency of copy number aberrations (CNAs) compared to low-proliferative atypical meningiomas. Additionally, losses on chromosome 6q, 9p, 13 and 14 were found exclusively in the high-proliferative meningiomas. We have identified genes that distinguish benign low-proliferative meningiomas from malignant high-proliferative meningiomas and have found that gain of cell-proliferation markers and loss of components of the transforming growth factor-beta signaling pathway were the major molecular mechanisms that distinguish these two groups. CONCLUSION: Collectively, our data suggests that atypical meningiomas are not a molecularly distinct group but are similar to either benign or malignant meningiomas. It is anticipated that identified molecular and CNA markers will potentially be more accurate prognostic markers of meningiomas.

Expression of the hypoxia marker carbonic anhydrase 9 is associated with anaplastic phenotypes in meningiomas.

PURPOSE: Hypoxia in the tumor microenvironment triggers a variety of genetic and adaptive responses that regulate tumor growth. Tumor hypoxia is often associated with more malignant phenotypes, resistance to therapy, and poor survival. The purpose of this study was to evaluate the prevalence of hypoxia in meningiomas using the endogenous hypoxia marker carbonic anhydrase 9 (CA9) and to relate the expression of CA9 to tumor vascularity, histopathologic grade, and clinical variables, such as recurrent tumor status. EXPERIMENTAL DESIGN: Expression of CA9 and CD34, an endothelial cell marker, was examined in serial paraffin-embedded sections by immunohistochemistry in 25 grade 1, 17 grade 2, and 20 grade 3 meningiomas. Areas of immunoreactivity were semiquantitatively scored and correlated to clinical variables using Statistical Analysis System statistical software. RESULTS: Approximately 50% (29 of 62) of all meningiomas contained regions of hypoxia as judged by expression of CA9, and this expression was significantly associated with higher-grade histology (P = 0.001). In contrast, vascularity, as assessed by the percentage of vascular hotspots, was inversely associated with tumor grade (P = 0.023) and was not associated with CA9 expression. Among lower-grade meningiomas, CA9 expression tended to be more common in recurrent tumors. CONCLUSIONS: Tumor hypoxia is an endogenous feature of meningiomas, and therapeutic regimens should include strategies to target the subpopulation of hypoxic as well as the normoxic cells within the tumor. Hypoxia in meningiomas is associated with an aggressive phenotype. Further studies to define the contribution of hypoxia to meningioma pathophysiology are warranted.

A genetic strategy to overcome the senescence of primary meningioma cell cultures.

Even though meningiomas are the second most common brain tumor in adults, little is known about the molecular basis of their growth and development. The lack of suitable cell culture model systems is an impediment to this understanding. Most studies on meningiomas rely on primary, early passage cell lines that eventually senesce or a few established cell lines that have been derived from aggressive variants of meningiomas. We have isolated three primary meningioma cell lines that are negative for telomerase activity. We can overcome the senescence of a Grade III derived meningioma cell line by expressing the telomerase catalytic subunit (hTERT), whereas Grade I meningioma cell lines require the expression of the human papillomavirus E6 and E7 oncogenes in conjunction with hTERT. Meningioma cell lines, immortalized in this manner, maintain their pre-transfection morphology and form colonies in vitro. We have confirmed the meningothelial origin of these cell lines by assessing expression of vimentin and desmoplakin, characteristic markers for meningiomas. Additionally, we have karyotyped these cell lines using array CGH and shown that they represent a spectrum of the genetic diversity seen in primary meningiomas. Thus, these cell lines represent novel cellular reagents for investigating the molecular oncogenesis of meningiomas.

A transcript finishing initiative for closing gaps in the human transcriptome.

We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experimental validation was designated a transcript finishing unit (TFU). A total of 489 TFUs were selected for validation, and an overall efficiency of 43.1% was achieved. We generated a total of 59,975 bp of transcribed sequences organized into 432 exons, contributing to the definition of the structure of 211 human transcripts. The structure of several transcripts reported here was confirmed during the course of this project, through the generation of their corresponding full-length cDNA sequences. Nevertheless, for 21% of the validated TFUs, a full-length cDNA sequence is not yet available in public databases, and the structure of 69.2% of these TFUs was not correctly predicted by computer programs. The TF strategy provides a significant contribution to the definition of the complete catalog of human genes and transcripts, because it appears to be particularly useful for identification of low abundance transcripts expressed in a restricted set of tissues as well as for the delineation of gene boundaries and alternatively spliced isoforms.

Rede ONSA e o projeto genoma humano do câncer: contribuiçäo ao genoma humano / ONSA network and the human cancer genome project: contribution to the human genome

A contribuiçäo maior da ciência brasileira ao genoma humano foi trazida pelo Projeto Genoma Humano do Câncer (Human Genome Cancer Project- HCGP) uma parceria da FAPESP e do Ludwig Institute for Cancer Research e desenvolvido por 29 diferentes laboratórios de seqüenciamento e um centro de bioinformática. Foram seqüenciados mais de 1 milhäo de fragmentos génicos expressos (expressed sequences tags, ESTs), provenientes de diferentes tumores humanos. Grande parte destes dados é de acesso público através da website do Gene Bank (www.ncbi.nim.nig.gov), mantido pelo NCBI - National Center for Biotechnology Information. Atualmente, diversos projetos estäo em desenvolvimento utilizando informações geradas no HCGP e abrangem observar a expressäo diferenciada dos genes em diferentes tumores, caracterizaçäo completa de genes específicos, assim como o estudo funcional e estrutural dos produtos protéicos. É promissora a perspectiva de que num futuro próximo, diferentes resultados provenientes destas investigações possam trazer benefícios preventivos, prognósticos e clínicos em câncer e outras doenças.

Inheritance of Anthracnose Resistance in Common Bean Differential Cultivar AB 136.

Inheritance of anthracnose resistance of the common bean (Phaseolus vulgaris L.) differential cultivar AB 136 to races 89, 64, and 73 (binary system designation) was studied in crosses with the susceptible differential cultivars Michelite (race 89), Mexico 222 (race 64), and Cornell 49-242 (race 73). In each cross two progenitors, the F1, F2, and backcross-derived plants were inoculated with the respective race under environmentally controlled greenhouse conditions. The results indicate that single dominant gene(s) control resistance to races 89 and 64, giving a segregation ratio of 3:1 in the F2, 1:0 in the backcrosses to AB 136, and 1:1 in the backcross to Michelite (race 89), and to Mexico 222 (race 64). For race 73, the following segregation ratios between resistant and susceptible plants were observed: 13:3 in the F2, 1:0 in the backcross to AB 136, and 1:1 in the backcross to Cornell 49-242. Such results suggest that two independent genes may determine resistance of AB 136 to race 73, one dominant (Co-6) and one recessive that is proposed to be assigned co-8. Genotypes Co-6_ or co-8 co-8 would condition resistance, whereas susceptibility would be present in genotypes co-6 co-6 Co-8_. Given the dominant nature of anthracnose resistance genes present in line AB 136 and its resistance to 25 races of Colletotrichum lindemuthianum identified in Brazil by other researchers, we included this cultivar as one of the donor parents in our molecular marker-assisted backcross breeding program, to develop common bean cultivars resistant to anthracnose and adapted to Central Brazil.