Loss of CD20 expression as a mechanism of resistance to mosunetuzumab in relapsed/refractory B-cell lymphomas.

ABSTRACT: CD20 is an established therapeutic target in B-cell malignancies. The CD20 × CD3 bispecific antibody mosunetuzumab has significant efficacy in B-cell non-Hodgkin lymphomas (NHLs). Because target antigen loss is a recognized mechanism of resistance, we evaluated CD20 expression relative to clinical response in patients with relapsed and/or refractory NHL in the phase 1/2 GO29781 trial investigating mosunetuzumab monotherapy. CD20 was studied using immunohistochemistry (IHC), RNA sequencing, and whole-exome sequencing performed centrally in biopsy specimens collected before treatment at predose, during treatment, or upon progression. Before treatment, most patients exhibited a high proportion of tumor cells expressing CD20; however, in 16 of 293 patients (5.5%) the proportion was <10%. Analyses of paired biopsy specimens from patients on treatment revealed that CD20 levels were maintained in 29 of 30 patients (97%) vs at progression, where CD20 loss was observed in 11 of 32 patients (34%). Reduced transcription or acquisition of truncating mutations explained most but not all cases of CD20 loss. In vitro modeling confirmed the effects of CD20 variants identified in clinical samples on reduction of CD20 expression and missense mutations in the extracellular domain that could block mosunetuzumab binding. This study expands the knowledge about the occurrence of target antigen loss after anti-CD20 therapeutics to include CD20-targeting bispecific antibodies and elucidates mechanisms of reduced CD20 expression at disease progression that may be generalizable to other anti-CD20 targeting agents. These results also confirm the utility of readily available IHC staining for CD20 as a tool to inform clinical decisions. This trial was registered at www.ClinicalTrials.gov as #NCT02500407.

A paracrine circuit of IL-1ß/IL-1R1 between myeloid and tumor cells drives genotype-dependent glioblastoma progression.

Monocytes and monocyte-derived macrophages (MDMs) from blood circulation infiltrate glioblastoma (GBM) and promote growth. Here, we show that PDGFB-driven GBM cells induce the expression of the potent proinflammatory cytokine IL-1ß in MDM, which engages IL-1R1 in tumor cells, activates the NF-κB pathway, and subsequently leads to induction of monocyte chemoattractant proteins (MCPs). Thus, a feedforward paracrine circuit of IL-1ß/IL-1R1 between tumors and MDM creates an interdependence driving PDGFB-driven GBM progression. Genetic loss or locally antagonizing IL-1ß/IL-1R1 leads to reduced MDM infiltration, diminished tumor growth, and reduced exhausted CD8+ T cells and thereby extends the survival of tumor-bearing mice. In contrast to IL-1ß, IL-1α exhibits antitumor effects. Genetic deletion of Il1a/b is associated with decreased recruitment of lymphoid cells and loss-of-interferon signaling in various immune populations and subsets of malignant cells and is associated with decreased survival time of PDGFB-driven tumor-bearing mice. In contrast to PDGFB-driven GBM, Nf1-silenced tumors have a constitutively active NF-κB pathway, which drives the expression of MCPs to recruit monocytes into tumors. These results indicate local antagonism of IL-1ß could be considered as an effective therapy specifically for proneural GBM.

Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma.

GD2-targeting immunotherapies have improved survival in children with neuroblastoma, yet on-target, off-tumor toxicities can occur and a subset of patients cease to respond. The majority of neuroblastoma patients who receive immunotherapy have been previously treated with cytotoxic chemotherapy, making it paramount to identify neuroblastoma-specific antigens that remain stable throughout standard treatment. Cell surface glycoproteomics performed on human-derived neuroblastoma tumors in mice following chemotherapy treatment identified protein tyrosine kinase 7 (PTK7) to be abundantly expressed. Furthermore, PTK7 shows minimal expression on pediatric-specific normal tissues. We developed an anti-PTK7 chimeric antigen receptor (CAR) and find PTK7 CAR T cells specifically target and kill PTK7-expressing neuroblastoma in vitro. In vivo, human/murine binding PTK7 CAR T cells regress aggressive neuroblastoma metastatic mouse models and prolong survival with no toxicity. Together, these data demonstrate preclinical efficacy and tolerability for targeting PTK7 and support ongoing investigations to optimize PTK7-targeting CAR T cells for neuroblastoma.

YAP-Mediated Repression of HRK Regulates Tumor Growth, Therapy Response, and Survival Under Tumor Environmental Stress in Neuroblastoma.

Following chemotherapy and relapse, high-risk neuroblastoma tumors harbor more genomic alterations than at diagnosis, including increased transcriptional activity of the Yes-associated protein (YAP), a key downstream component of the Hippo signaling network. Although YAP has been implicated in many cancer types, its functional role in the aggressive pediatric cancer neuroblastoma is not well-characterized. In this study, we performed genetic manipulation of YAP in human-derived neuroblastoma cell lines to investigate YAP function in key aspects of the malignant phenotype, including mesenchymal properties, tumor growth, chemotherapy response, and MEK inhibitor response. Standard cytotoxic therapy induced YAP expression and transcriptional activity in patient-derived xenografts treated in vivo, which may contribute to neuroblastoma recurrence. Moreover, YAP promoted a mesenchymal phenotype in high-risk neuroblastoma that modulated tumor growth and therapy resistance in vivo. Finally, the BH3-only protein, Harakiri (HRK), was identified as a novel target inhibited by YAP, which, when suppressed, prevented apoptosis in response to nutrient deprivation in vitro and promoted tumor aggression, chemotherapy resistance, and MEK inhibitor resistance in vivo. Collectively, these findings suggest that YAP inhibition may improve chemotherapy response in patients with neuroblastoma via its regulation of HRK, thus providing a critical strategic complement to MEK inhibitor therapy. SIGNIFICANCE: This study identifies HRK as a novel tumor suppressor in neuroblastoma and suggests dual MEK and YAP inhibition as a potential therapeutic strategy in RAS-hyperactivated neuroblastomas.

Tumour-associated macrophage-derived interleukin-1 mediates glioblastoma-associated cerebral oedema.

Glioblastoma is the most common and uncompromising primary brain tumour and is characterized by a dismal prognosis despite aggressive treatment regimens. At the cellular level, these tumours are composed of a mixture of neoplastic cells and non-neoplastic cells, including tumour-associated macrophages and endothelial cells. Cerebral oedema is a near-universal occurrence in patients afflicted with glioblastoma and it is almost exclusively managed with the corticosteroid dexamethasone despite significant drawbacks associated with its use. Here, we demonstrate that dexamethasone blocks interleukin-1 production in both bone marrow-derived and brain resident macrophage populations following stimulation with lipopolysaccharide and interferon gamma. Additionally, dexamethasone is shown to inhibit downstream effectors of interleukin-1 signalling in both macrophage populations. Co-culture of bone marrow-derived macrophages with organotypic tumour slices results in an upregulation of interleukin-1 cytokines, an effect that is absent in co-cultured microglia. Genetic ablation of interleukin-1 ligands or receptor in mice bearing RCAS/tv-a-induced platelet-derived growth factor B-overexpressing glioblastoma results in reduced oedema and partial restoration of the integrity of the blood-brain barrier, respectively; similar to results obtained with vascular endothelial growth factor neutralization. We establish that tumours from dexamethasone-treated mice exhibit reduced infiltration of cells of the myeloid and lymphoid compartments, an effect that should be considered during clinical trials for immunotherapy in glioblastoma patients. Additionally, we emphasize that caution should be used when immune profiling and single-cell RNA sequencing data are interpreted from fresh glioblastoma patient samples, as nearly all patients receive dexamethasone after diagnosis. Collectively, this evidence suggests that interleukin-1 signalling inhibition and dexamethasone treatment share therapeutic efficacies and establishes interleukin-1 signalling as an attractive and specific therapeutic target for the management of glioblastoma-associated cerebral oedema.

Upregulation of the chromatin remodeler HELLS is mediated by YAP1 in Sonic Hedgehog Medulloblastoma.

Medulloblastoma is a malignant pediatric tumor that arises from neural progenitors in the cerebellum. Despite a five-year survival rate of ~70%, nearly all patients incur adverse side effects from current treatment strategies that drastically impact quality of life. Roughly one-third of medulloblastoma are driven by aberrant activation of the Sonic Hedgehog (SHH) signaling pathway. However, the scarcity of genetic mutations in medulloblastoma has led to investigation of other mechanisms contributing to cancer pathogenicity including epigenetic regulation of gene expression. Here, we show that Helicase, Lymphoid Specific (HELLS), a chromatin remodeler with epigenetic functions including DNA methylation and histone modification, is induced by Sonic Hedgehog (SHH) in SHH-dependent cerebellar progenitor cells and the developing murine cerebella. HELLS is also up-regulated in mouse and human SHH medulloblastoma. Others have shown that HELLS activity generally results in a repressive chromatin state. Our results demonstrate that increased expression of HELLS in our experimental systems is regulated by the oncogenic transcriptional regulator YAP1 downstream of Smoothened, the positive transducer of SHH signaling. Elucidation of HELLS as one of the downstream effectors of the SHH pathway may lead to novel targets for precision therapeutics with the promise of better outcomes for SHH medulloblastoma patients.

Tumour-associated macrophages exhibit anti-tumoural properties in Sonic Hedgehog medulloblastoma.

Medulloblastoma, which is the most common malignant paediatric brain tumour, has a 70% survival rate, but standard treatments often lead to devastating life-long side effects and recurrence is fatal. One of the emerging strategies in the search for treatments is to determine the roles of tumour microenvironment cells in the growth and maintenance of tumours. The most attractive target is tumour-associated macrophages (TAMs), which are abundantly present in the Sonic Hedgehog (SHH) subgroup of medulloblastoma. Here, we report an unexpected beneficial role of TAMs in SHH medulloblastoma. In human patients, decreased macrophage number is correlated with significantly poorer outcome. We confirm macrophage anti-tumoural behaviour in both ex vivo and in vivo murine models of SHH medulloblastoma. Taken together, our findings suggest that macrophages play a positive role by impairing tumour growth in medulloblastoma, in contrast to the pro-tumoural role played by TAMs in glioblastoma, another common brain tumour.

Reactive Oxygen Species Signaling Promotes Hypoxia-Inducible Factor 1α Stabilization in Sonic Hedgehog-Driven Cerebellar Progenitor Cell Proliferation.

Cerebellar development is a highly regulated process involving numerous factors acting with high specificity, both temporally and by location. Part of this process involves extensive proliferation of cerebellar granule neuron precursors (CGNPs) induced by Sonic Hedgehog (SHH) signaling, but downstream effectors of mitogenic signaling are still being elucidated. Using primary CGNP cultures, a well-established model for SHH-driven proliferation, we show that SHH-treated CGNPs feature high levels of hypoxia-inducible factor 1α (HIF1α), which is known to promote glycolysis, stemness, and angiogenesis. In CGNPs cultured under normoxic conditions, HIF1α is posttranslationally stabilized in a manner dependent upon reactive oxygen species (ROS) and NADPH oxidase (NOX), both of which are also upregulated in these cells. Inhibition of NOX activity resulted in HIF1α destabilization and reduced levels of cyclin D2, a marker of CGNP proliferation. As CGNPs are the putative cells of origin for the SHH subtype of medulloblastoma and aberrant SHH signaling is implicated in other neoplasms, these studies may also have future relevance in the context of cancer. Taken together, our findings suggest that a better understanding of nonhypoxic HIF1α stabilization through NOX-induced ROS generation can provide insights into normal cell proliferation in cerebellar development and SHH-driven cell proliferation in cancers with aberrant SHH signaling.

p53 Function Is Compromised by Inhibitor 2 of Phosphatase 2A in Sonic Hedgehog Medulloblastoma.

Medulloblastomas, the most common malignant pediatric brain tumors, have been genetically defined into four subclasses, namely WNT-activated, Sonic Hedgehog (SHH)-activated, Group 3, and Group 4. Approximately 30% of medulloblastomas have aberrant SHH signaling and thus are referred to as SHH-activated medulloblastoma. The tumor suppressor gene TP53 has been recently recognized as a prognostic marker for patients with SHH-activated medulloblastoma; patients with mutant TP53 have a significantly worse outcome than those with wild-type TP53. It remains unknown whether p53 activity is impaired in SHH-activated, wild-type TP53 medulloblastoma, which is about 80% of the SHH-activated medulloblastomas. Utilizing the homozygous NeuroD2:SmoA1 mouse model with wild-type Trp53, which recapitulates human SHH-activated medulloblastoma, it was discovered that the endogenous Inhibitor 2 of Protein Phosphatase 2A (SET/I2PP2A) suppresses p53 function by promoting accumulation of phospho-MDM2 (S166), an active form of MDM2 that negatively regulates p53. Knockdown of I2PP2A in SmoA1 primary medulloblastoma cells reduced viability and proliferation in a p53-dependent manner, indicating the oncogenic role of I2PP2A. Importantly, this mechanism is conserved in the human medulloblastoma cell line ONS76 with wild-type TP53. Taken together, these findings indicate that p53 activity is inhibited by I2PP2A upstream of PP2A in SHH-activated and TP53-wildtype medulloblastomas. IMPLICATIONS: This study suggests that I2PP2A represents a novel therapeutic option and its targeting could improve the effectiveness of current therapeutic regimens for SHH-activated or other subclasses of medulloblastoma with wild-type TP53.

Nanoparticles for targeted delivery of antioxidant enzymes to the brain after cerebral ischemia and reperfusion injury.

Stroke is one of the major causes of death and disability in the United States. After cerebral ischemia and reperfusion injury, the generation of reactive oxygen species (ROS) and reactive nitrogen species may contribute to the disease process through alterations in the structure of DNA, RNA, proteins, and lipids. We generated various nanoparticles (liposomes, polybutylcyanoacrylate (PBCA), or poly(lactide-co-glycolide) (PLGA)) that contained active superoxide dismutase (SOD) enzyme (4,000 to 20,000 U/kg) in the mouse model of cerebral ischemia and reperfusion injury to determine the impact of these molecules. In addition, the nanoparticles were untagged or tagged with nonselective antibodies or antibodies directed against the N-methyl-D-aspartate (NMDA) receptor 1. The nanoparticles containing SOD protected primary neurons in vitro from oxygen-glucose deprivation (OGD) and limited the extent of apoptosis. The nanoparticles showed protection against ischemia and reperfusion injury when applied after injury with a 50% to 60% reduction in infarct volume, reduced inflammatory markers, and improved behavior in vivo. The targeted nanoparticles not only showed enhanced protection but also showed localization to the CA regions of the hippocampus. Nanoparticles alone were not effective in reducing infarct volume. These studies show that targeted nanoparticles containing protective factors may be viable candidates for the treatment of stroke.

Enzyme-nanoparticle conjugates for biomedical applications.

Enzymes hold a great promise as therapeutic agents because of their unique specificity and high level of activity. Yet, clinically important enzyme drugs are for less common than conventional low molecular weight drugs due to a number of disadvantages. Most important among these are poor stability, potential immunogenicity, and potential systemic toxicity. Recent developments in synthesis and characterization of nanoparticles and exciting novel properties of some classes of nanomaterials have boosted interest in the potential use of nanoparticles as carriers of enzyme drugs. In certain cases, use of enzymes attached to nanoparticles can help to overcome some of the above problems and improve the prospects of clinical applications of enzyme drugs. Here, we review recent data on the use of nanoparticles as carriers for several clinically important enzyme drugs and discuss advantages and potential limitations of such constructs. While promising preliminary results were obtained with regard to their performance in vitro and in some animal models, further investigations and clinical trials, as well as addressing regulatory issues, are warranted to make these delivery systems suitable for clinical applications.

Proteins conjugated to poly(butyl cyanoacrylate) nanoparticles as potential neuroprotective agents.

Poly(butyl cyanoacrylate) (PBCA) nanoparticles (NPs) can penetrate blood-brain barrier providing the means for drug delivery to the central nervous system. Here, we study attachment of superoxide dismutase (SOD) and anti-glutamate N-methyl D-aspartate receptor 1 (NR1) antibody to PBCA NPs with the ultimate goal to design neuroprotective therapeutics for treatment of secondary spinal cord injury. Synthesis of monodispersed, ∼200 nm-diameter PBCA NPs was performed using polymerization at pH 2.0 with Dextran 70,000 as the stabilizer. Sulfo-HSAB spacers were used to covalently attach SOD and NR1 antibodies to the dextran-coated NPs. The prepared protein-NP conjugates possessed SOD activity and were capable of binding to rat cerebellar neurons. Thus, SOD and NR1 antibodies may be simultaneously attached to PBCA NPs while retaining at least a fraction of enzymatic activity and receptor-binding ability. The conjugates showed neuroprotective efficacy in vitro with rat cerebellar cell cultures challenged by superoxide.