Herui Wang

Inhibition of protein phosphatase 2A with the small molecule LB100 overcomes cell cycle arrest in osteosarcoma after cisplatin treatment

Osteosarcoma is the most common primary malignant bone tumor and affects a significant portion of pediatric oncology patients. Although surgery and adjuvant chemotherapy confer significant survival benefits, many patients go on to develop metastatic disease, particularly to the lungs, secondary to development of drug resistance. Inhibition of protein phosphatase 2A with the small molecule, LB100, has demonstrated potent chemo- and radio-sensitizing properties in numerous pre-clinical tumor models. In this study, we showed that LB100 overcame DNA repair mechanisms in osteosarcoma cells treated with cisplatin, in vitro, and recapitulated these findings in an in vivo xenograft model. Notably, the addition of LB100 to cisplatin prevented development of pulmonary metastases in the majority of treated animals. Our data indicated the mechanism of chemo-sensitization by LB100 involved abrogation of the ATM/ATR-activated DNA damage response, leading to hyperphosphorylation of Chk proteins and persistent cyclin activity. In addition, LB100 exposure suppressed Akt signaling, leading to Mdm2-mediated proteasomal degradation of functional p53. Taken together, LB100 prevented repair of cisplatin-induced DNA damage, resulting in mitotic catastrophe and cell death.

Mutant glucocerebrosidase in Gaucher disease recruits Hsp27 to the Hsp90 chaperone complex for proteasomal degradation

Significance Gaucher disease (GD) is an inherited metabolic storage disorder characterized by mutations in the gene GBA1 encoding for glucocerebrosidase (GCase). These mutations result in protein misfolding and subsequent premature degradation. Recognition by the heat shock protein (hsp) 90 complex is crucial for targeting of mutant GCase to the proteasome, but the mechanisms governing this association are unclear. This study describes a novel recruitment of Hsp27 to the Hsp90 complex that is specific to misfolded mutant GCase. Both gene knockdown and pharmacologic inhibition of Hsp27 increased GCase levels in patient-derived fibroblasts. Reduction of Hsp27 may circumvent premature protein degradation and represents a viable potential therapeutic strategy in the treatment of protein misfolding disorders. Gaucher disease is caused by mutations of the GBA1 gene, which encodes the lysosomal anchored gluococerebrosidase (GCase). GBA1 mutations commonly result in protein misfolding, abnormal chaperone recognition, and premature degradation, but are less likely to affect catalytic activity. In the present study, we demonstrate that the Hsp90/HOP/Cdc37 complex recruits Hsp27 after recognition of GCase mutants with subsequent targeting of GCase mutant peptides to degradation mechanisms such as VCP and the 26S proteasome. Inhibition of Hsp27 not only increased the quantity of enzyme but also enhanced GCase activity in fibroblasts derived from patients with Gaucher disease. These findings provide insight into a possible therapeutic strategy for protein misfolding diseases by correcting chaperone binding and altering subsequent downstream patterns of protein degradation.

Anthracyclines suppress pheochromocytoma cell characteristics, including metastasis, through inhibition of the hypoxia signaling pathway

Pheochromocytomas (PHEOs) and paragangliomas (PGLs) are rare, neuroendocrine tumors derived from adrenal or extra-adrenal chromaffin cells, respectively. Metastases are discovered in 3-36% of patients at the time of diagnosis. Currently, only suboptimal treatment options exist. Therefore, new therapeutic compounds targeting metastatic PHEOs/PGLs are urgently needed. Here, we investigated if anthracyclines were able to suppress the progression of metastatic PHEO. We explored their effects on experimental mouse PHEO tumor cells using in vitro and in vivo models, and demonstrated that anthracyclines, particularly idarubicin (IDA), suppressed hypoxia signaling by preventing the binding of hypoxia-inducible factor 1 and 2 (HIF-1 and HIF-2) to the hypoxia response element (HRE) sites on DNA. This resulted in reduced transcriptional activation of HIF target genes, including erythropoietin (EPO), phosphoglycerate kinase 1 (PGK1), endothelin 1 (EDN1), glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and vascular endothelial growth factor (VEGFA), which consequently inhibited the growth of metastatic PHEO. Additionally, IDA downregulated hypoxia signaling by interfering with the transcriptional activation of HIF1A and HIF2A. Furthermore, our animal model demonstrated the dose-dependent suppressive effect of IDA on metastatic PHEO growth in vivo. Our results indicate that anthracyclines are prospective candidates for inclusion in metastatic PHEO/PGL therapy, especially in patients with gene mutations involved in the hypoxia signaling pathway.

Unilateral vestibular schwannoma in a patient with schwannomatosis in the absence of LZTR1 mutation.

The presence of vestibular schwannomas has long been considered an exclusion criterion for the diagnosis of schwannomatosis. Recently, 2 cases of vestibular schwannoma were reported in patients with schwannomatosis, leading to a revision of the diagnostic criteria for this genetic disorder. Overall, the relative infrequency of vestibular schwannomas in schwannomatosis is unexplained, and the genetics of this uncommon phenomenon have not been described. The authors report on a family with clinical manifestations consistent with schwannomatosis, including 4 affected members, that was identified as having an affected member harboring a unilateral cerebellopontine angle mass with extension into the internal auditory canal. Radiologically, this mass was consistent with a vestibular schwannoma and resulted in a symptomatic change in ipsilateral hearing (word recognition 86% at 52 dB) and increased latency of the wave I-V interval on auditory brainstem response testing. The patient was found to be negative for a germline mutation of NF2 and LZTR1, and her affected mother was found to harbor neither NF2 nor SMARCB1 mutations on genetic testing. Although vestibular schwannomas have been classically considered to not occur in the setting of schwannomatosis, this patient with schwannomatosis and a vestibular schwannoma further confirms that schwannomas can occur on the vestibular nerve in this syndrome. Further, this is the first such case found to be negative for a mutation on the LZTR1 gene.

Vorinostat suppresses hypoxia signaling by modulating nuclear translocation of hypoxia inducible factor 1 alpha

Histone deacetylase inhibitors (HDACis) are a potent class of tumor-suppressive agents traditionally believed to exert their effects through loosening tightly-wound chromatin resulting in de-inhibition of various tumor suppressive genes. Recent literature however has shown altered intratumoral hypoxia signaling with HDACi administration not attributable to changes in chromatin structure. We sought to determine the precise mechanism of HDACi-mediated hypoxia signaling attenuation using vorinostat (SAHA), an FDA-approved class I/IIb/IV HDACi. Through an in-vitro and in-vivo approach utilizing cell lines for hepatocellular carcinoma (HCC), osteosarcoma (OS), and glioblastoma (GBM), we demonstrate that SAHA potently inhibits HIF-a nuclear translocation via direct acetylation of its associated chaperone, heat shock protein 90 (Hsp90). In the presence of SAHA we found elevated levels of acetyl-Hsp90, decreased interaction between acetyl-Hsp90 and HIF-a, decreased nuclear/cytoplasmic HIF-α expression, absent HIF-α association with its nuclear karyopharyin Importin, and markedly decreased HIF-a transcriptional activity. These changes were associated with downregulation of downstream hypoxia molecules such as endothelin 1, erythropoietin, glucose transporter 1, and vascular endothelial growth factor. Findings were replicated in an in-vivo Hep3B HRE-Luc expressing xenograft, and were associated with significant decreases in xenograft tumor size. Altogether, this study highlights a novel mechanism of action of an important class of chemotherapeutic.Histone deacetylase inhibitors (HDACis) are a potent class of tumor-suppressive agents traditionally believed to exert their effects through loosening tightly-wound chromatin resulting in de-inhibition of various tumor suppressive genes. Recent literature however has shown altered intratumoral hypoxia signaling with HDACi administration not attributable to changes in chromatin structure. We sought to determine the precise mechanism of HDACi-mediated hypoxia signaling attenuation using vorinostat (SAHA), an FDA-approved class I/IIb/IV HDACi. Through an in-vitro and in-vivo approach utilizing cell lines for hepatocellular carcinoma (HCC), osteosarcoma (OS), and glioblastoma (GBM), we demonstrate that SAHA potently inhibits HIF-a nuclear translocation via direct acetylation of its associated chaperone, heat shock protein 90 (Hsp90). In the presence of SAHA we found elevated levels of acetyl-Hsp90, decreased interaction between acetyl-Hsp90 and HIF-a, decreased nuclear/cytoplasmic HIF-α expression, absent HIF-α association with its nuclear karyopharyin Importin, and markedly decreased HIF-a transcriptional activity. These changes were associated with downregulation of downstream hypoxia molecules such as endothelin 1, erythropoietin, glucose transporter 1, and vascular endothelial growth factor. Findings were replicated in an in-vivo Hep3B HRE-Luc expressing xenograft, and were associated with significant decreases in xenograft tumor size. Altogether, this study highlights a novel mechanism of action of an important class of chemotherapeutic.

Novel Targeting of Transcription and Metabolism in Glioblastoma

Purpose: Glioblastoma (GBM) is highly resistant to treatment, largely due to disease heterogeneity and resistance mechanisms. We sought to investigate a promising drug that can inhibit multiple aspects of cancer cell survival mechanisms and become an effective therapeutic for GBM patients. Experimental Design: To investigate TG02, an agent with known penetration of the blood–brain barrier, we examined the effects as single agent and in combination with temozolomide, a commonly used chemotherapy in GBM. We used human GBM cells and a syngeneic mouse orthotopic GBM model, evaluating survival and the pharmacodynamics of TG02. Mechanistic studies included TG02-induced transcriptional regulation, apoptosis, and RNA sequencing in treated GBM cells as well as the investigation of mitochondrial and glycolytic function assays. Results: We demonstrated that TG02 inhibited cell proliferation, induced cell death, and synergized with temozolomide in GBM cells with different genetic background but not in astrocytes. TG02-induced cytotoxicity was blocked by the overexpression of phosphorylated CDK9, suggesting a CDK9-dependent cell killing. TG02 suppressed transcriptional progression of antiapoptotic proteins and induced apoptosis in GBM cells. We further demonstrated that TG02 caused mitochondrial dysfunction and glycolytic suppression and ultimately ATP depletion in GBM. A prolonged survival was observed in GBM mice receiving combined treatment of TG02 and temozolomide. The TG02-induced decrease of CDK9 phosphorylation was confirmed in the brain tumor tissue. Conclusions: TG02 inhibits multiple survival mechanisms and synergistically decreases energy production with temozolomide, representing a promising therapeutic strategy in GBM, currently under investigation in an ongoing clinical trial. Clin Cancer Res; 24(5); 1124–37. ©2017 AACR.

CHCHD10 is involved in the development of Parkinson’s disease caused by CHCHD2 loss-of-function mutation p.T61I

Previously we identified the p.Thr61Ile mutation in coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) in a Chinese family with autosomal dominant Parkinsons disease. But the mechanism is still unclear. In this study, we explored the effects of CHCHD2 p.Thr61Ile mutation in cells and its association with coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10). We found that overexpression of Parkinsons disease-associated T61I mutant CHCHD2 did not produce mitochondrial dysfunction. Rather, its protective effect from stress was abrogated. And, the level of the CHCHD2 protein and mRNA in patient fibroblasts was not significantly different from control. In addition, CHCHD2 T61I mutation caused increased interaction with CHCHD10 and reduced CHCHD10 level. The mitochondrial ultrastructural alterations in CHCHD2 T61I mutant patient fibroblasts are similar to that of CHCHD10 mutations. We therefore propose that CHCHD10 is involved in the development of Parkinsons disease caused by CHCHD2 loss-of-function mutation p.T61I.

Inhibition of Protein Phosphatase 2A Sensitizes Mucoepidermoid Carcinoma to Chemotherapy via the PI3K-AKT Pathway in Response to Insulin Stimulus

Background/Aims: Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase that mediates cell cycle regulation and metabolism. Mounting evidence has indicated that PP2A inhibition exhibits considerable anticancer potency in multiple types of human cancers. However, the efficacy of PP2A inhibition remains unexplored in mucoepidermoid carcinoma (MEC), especially in locally advanced and metastatic cases with limited systemic treatment. In this study, we demonstrated the therapeutic potency of LB100 in mucoepidermoid carcinoma. Methods: In this study, the expression of PP2A was evaluated using immunohistochemical (IHC) staining. The effects associated with LB100 alone and in combination with cisplatin for the treatment of mucoepidermoid carcinoma were investigated both in vitro, regarding metabolism, proliferation, and migration, and in vivo in a mucoepidermoid carcinoma xenograft model. In addition, with LB100 treatment and in response to an insulin stimulus, the expression levels and phosphorylation levels of targets in the PI3K-AKT pathway were determined using western blot analysis and immunoblotting. Results: The expression of protein phosphatase 2A was significantly upregulated in the clinical specimens of high-grade MECs compared with those of low-/medium-grade MECs and normal controls. In this article, we report that a small molecule PP2A inhibitor, LB100, decreased cellular viability and glycolytic activity and induced G2/M cell cycle arrest. Importantly, LB100 enhanced the efficacy of cisplatin in mucoepidermoid carcinoma cells both in vitro and in vivo. PP2A inhibition by LB100 increased the phosphorylation of insulin receptor substrate 1(IRS-1) on serine residues, downregulated the expression of phosphatidylinositol 3-kinase (PI3K) p110 alpha subunit and dephosphorylated AKT at Ser473 and Thr308 in mucoepidermoid carcinoma cells in response to insulin stimulus. Conclusion: These results highlight the translational potential of PP2A inhibition to synergize with cisplatin in mucoepidermoid carcinoma treatment.

A novel splicing site IRP1 somatic mutation in a patient with pheochromocytoma and JAK2 V617F positive polycythemia vera: a case report

BackgroundThe role of the hypoxia signaling pathway in the pathogenesis of pheochromocytoma/paraganglioma (PPGL)-polycythemia syndrome has been elucidated. Novel somatic mutations in hypoxia-inducible factor type 2A (HIF2A) and germline mutations in prolyl hydroxylase type 1 and type 2 (PHD1 and PHD2) have been identified to cause upregulation of the hypoxia signaling pathway and its target genes including erythropoietin (EPO) and its receptor (EPOR). However, in a minority of patients presenting with this syndrome, the genetics and molecular pathogenesis remain unexplained. The aim of the present study was to uncover novel genetic causes of PPGL-polycythemia syndrome.Case presentationA female presented with a history of JAK2V617F positive PV, diagnosed in 2007, and right adrenal pheochromocytoma diagnosed and resected in 2011. Her polycythemia symptoms and hematocrit levels continued to worsen from 2007 to 2011, with an increased frequency of phlebotomies. Postoperatively, until early 2013, her hematocrit levels remained normalized. Following this, the hematocrit levels ranged between 46.4 and 48.9% [35–45%]. Tumor tissue from the patient was further tested for mutations in genes related to upregulation of the hypoxia signaling pathway including iron regulatory protein 1 (IRP1), which is a known regulator of HIF-2α mRNA translation. Functional studies were performed to investigate the consequences of these mutations, especially their effect on the HIF signaling pathway and EPO. Indel mutations (c.267-1_267delGGinsTA) were discovered at the exon 3 splicing site of IRP1. Minigene construct and splicing site analysis showed that the mutation led to a new splicing site and a frameshift mutation of IRP1, which caused a truncated protein. Fluorescence in situ hybridization analysis demonstrated heterozygous IRP1 deletions in tumor cells. Immunohistochemistry results confirmed the truncated IRP1 and overexpressed HIF-2α, EPO and EPOR in tumor cells.ConclusionsThis is the first report which provides direct molecular genetic evidence of association between a somatic IRP1 loss-of-function mutation and PHEO and secondary polycythemia. In patients diagnosed with PHEO/PGL and polycythemia with negative genetic testing for mutations in HIF2A, PHD1/2, and VHL, IRP1 should be considered as a candidate gene.

Abstract LB-193: Protein phosphatase 2A inhibition,with a novel small molecule inhibitor, LB-100, achieves durable immune-mediated antitumor activity when combined with PD1 blockade in a preclinical model

LB-100 is a novel, first-in-class, small molecule inhibitor of protein phosphatase 2A (PP2A) recently shown in a Phase I trial to be well-tolerated at doses associated with stabilization of progressive solid tumors (Chung. Clin Cancer Res. 2017). PP2A has been implicated in mediating Akt signaling downstream of CTLA-4 (Parry. MolCell Biol. 2005). An in vivo pooled short hairpin RNA screen identified PP2A as a key regulator in suppressing T-cell proliferation in the tumor microenvironment (Zhou. Nature. 2014) and to be essential for regulatory-T-cell (Treg) function (Apostolidis. Nat Immunol. 2014). We hypothesized that pharmacologic inhibition of PP2A could enhance cancer immunity. We assessed the effect of LB-100 on T-cells in human allogenic mixed lymphocyte reactions, in which CD8+ or CD4+ T cells were co-cultured with monocyte-derived dendritic cells. We found a dose dependent increase in T cell proliferation in CD8+ and CD4+ cells and an increase in IFNγ secretion in CD4+ T cells. We investigated the effect of LB-100 plus anti-PD-1 antibody on CD4+ T cells in the same assay. The combination enhanced proliferation and IFNγ production compared to anti-PD-1 alone. For in vivo syngeneic studies, BALB/c mice were implanted subcutaneously in the right flank with CT26 colon carcinoma cells (5 x10^5 cells). 12 days after implantation, mice with tumors between 30-100 mm3 were randomized into four treatment groups (placebo, LB-100 - 0.16 mg/kg, anti-PD1- 10 mg/kg, and combination). Treatment was given every 2 days up to 28 days. 10 days after treatment, there was a significant decrease in tumor growth in the combination group compared to placebo or anti-PD-1 alone. 7/11 (63.6%) of mice treated with the combination and none in the other treatment groups achieved complete remission (CR). FACS analysis of the tumor infiltrating lymphocytes (TIL) after 10 days of treatment demonstrated enhanced IFNγ production in CD8+ T cells in the combination group compared to either treatment alone. There was also a marked decrease in FoxP3+ Treg cells in LB-100 treated group compared to placebo (3% vs 12% of CD4+ T cells, p Citation Format: Winson S. Ho, Herui Wang, John S. Kovach, Rongze Lu, Zhengping Zhuang. Protein phosphatase 2A inhibition,with a novel small molecule inhibitor, LB-100, achieves durable immune-mediated antitumor activity when combined with PD1 blockade in a preclinical model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-193. doi:10.1158/1538-7445.AM2017-LB-193