P300/CBP inhibition sensitizes mantle cell lymphoma to PI3Kδ inhibitor idelalisib.

Mantle cell lymphoma (MCL) is a lymphoproliferative disorder lacking reliable therapies. PI3K pathway contributes to the pathogenesis of MCL, serving as a potential target. However, idelalisib, an FDA-approved drug targeting PI3Kδ, has shown intrinsic resistance in MCL treatment. Here we report that a p300/CBP inhibitor, A-485, could overcome resistance to idelalisib in MCL cells in vitro and in vivo. A-485 was discovered in a combinational drug screening from an epigenetic compound library containing 45 small molecule modulators. We found that A-485, the highly selective catalytic inhibitor of p300 and CBP, was the most potent compound that enhanced the sensitivity of MCL cell line Z-138 to idelalisib. Combination of A-485 and idelalisib remarkably decreased the viability of three MCL cell lines tested. Co-treatment with A-485 and idelalisib in Maver-1 and Z-138 MCL cell xenograft mice for 3 weeks dramatically suppressed the tumor growth by reversing the unsustained inhibition in PI3K downstream signaling. We further demonstrated that p300/CBP inhibition decreased histone acetylation at RTKs gene promoters and reduced transcriptional upregulation of RTKs, thereby inhibiting the downstream persistent activation of MAPK/ERK signaling, which also contributed to the pathogenesis of MCL. Therefore, additional inhibition of p300/CBP blocked MAPK/ERK signaling, which rendered maintaining activation to PI3K-mTOR downstream signals p-S6 and p-4E-BP1, thus leading to suppression of cell growth and tumor progression and eliminating the intrinsic resistance to idelalisib ultimately. Our results provide a promising combination therapy for MCL and highlight the potential use of epigenetic inhibitors targeting p300/CBP to reverse drug resistance in tumor.

Therapeutic targeting miR130b counteracts diffuse large B-cell lymphoma progression via OX40/OX40L-mediated interaction with Th17 cells.

MicroRNAs (miRNAs) are involved in lymphoma progression by regulating the tumor microenvironment. Serum miR130b is overexpressed in diffuse large B-cell lymphoma (DLBCL), inducing Th17 cell alterations. To further illustrate its biological significance and therapeutic rationale, miR130b was detected by quantitative real-time PCR in the serum samples of 532 newly diagnosed DLBCL patients. The mechanism of miR130b on lymphoma progression and the tumor microenvironment was investigated both in vitro and in vivo. Therapeutic targeting miR130b was also evaluated, including OX40 agonistic antibody and lipid nanoparticles (LNPs)-miR130b antagomir. The results showed that serum miR130b significantly correlated with tumor miR130b and serum interleukin-17, indicating lymphoma relapse and inferior survival of DLBCL patients. MiR130b overexpression altered tumor microenvironment signaling pathways and increased Th17 cell activity. As mechanism of action, miR130b downregulated tumor OX40L expression by directly targeting IFNAR1/p-STAT1 axis, recruiting Th17 cells via OX40/OX40L interaction, thereby promoting immunosuppressive function of Th17 cells. In co-culture systems of B-lymphoma cells with immune cells, miR130b inhibited lymphoma cell autophagy, which could be counteracted by OX40 agonistic antibody and LNPs-miR130b antagomir. In murine xenograft model established with subcutaneous injection of A20 cells, both OX40 agonistic antibody and LNPs-miR130b antagomir remarkably inhibited Th17 cells and retarded miR130b-overexpressing tumor growth. In conclusion, as an oncogenic biomarker of DLBCL, miR130b was related to lymphoma progression through modulating OX40/OX40L-mediated lymphoma cell interaction with Th17 cells, attributing to B-cell lymphoma sensitivity towards OX40 agonistic antibody. Targeting miR130b using LNPs-miR130b antagomir could also be a potential immunotherapeutic strategy in treating OX40-altered lymphoid malignancies.

CREBBP/EP300 mutations promoted tumor progression in diffuse large B-cell lymphoma through altering tumor-associated macrophage polarization via FBXW7-NOTCH-CCL2/CSF1 axis.

Epigenetic alterations play an important role in tumor progression of diffuse large B-cell lymphoma (DLBCL). However, the biological relevance of epigenetic gene mutations on tumor microenvironment remains to be determined. The core set of genes relating to histone methylation (KMT2D, KMT2C, EZH2), histone acetylation (CREBBP, EP300), DNA methylation (TET2), and chromatin remodeling (ARID1A) were detected in the training cohort of 316 patients by whole-genome/exome sequencing (WGS/WES) and in the validation cohort of 303 patients with newly diagnosed DLBCL by targeted sequencing. Their correlation with peripheral blood immune cells and clinical outcomes were assessed. Underlying mechanisms on tumor microenvironment were investigated both in vitro and in vivo. Among all 619 DLBCL patients, somatic mutations in KMT2D (19.5%) were most frequently observed, followed by mutations in ARID1A (8.7%), CREBBP (8.4%), KMT2C (8.2%), TET2 (7.8%), EP300 (6.8%), and EZH2 (2.9%). Among them, CREBBP/EP300 mutations were significantly associated with decreased peripheral blood absolute lymphocyte-to-monocyte ratios, as well as inferior progression-free and overall survival. In B-lymphoma cells, the mutation or knockdown of CREBBP or EP300 inhibited H3K27 acetylation, downregulated FBXW7 expression, activated the NOTCH pathway, and downstream CCL2/CSF1 expression, resulting in tumor-associated macrophage polarization to M2 phenotype and tumor cell proliferation. In B-lymphoma murine models, xenografted tumors bearing CREBBP/EP300 mutation presented lower H3K27 acetylation, higher M2 macrophage recruitment, and more rapid tumor growth than those with CREBBP/EP300 wild-type control via FBXW7-NOTCH-CCL2/CSF1 axis. Our work thus contributed to the understanding of aberrant histone acetylation regulation on tumor microenvironment as an alternative mechanism of tumor progression in DLBCL.

Anti-tumor effect of beta-elemene in glioblastoma cells depends on p38 MAPK activation.

beta-Elemene, a natural plant drug extracted from Curcuma wenyujin, has been used as an antitumor drug for different tumors, including glioblastoma. However, the mechanism of its anti-tumor effect is largely unknown. Here we report that anti-proliferation of glioblastoma cells induced by beta-elemene was dependent on p38 MAPK activation. Treatment of glioblastoma cell lines with beta-elemene, led to phosphorylation of p38 MAPK, cell-cycle arrest in G0/G1 phase and inhibition of proliferation of these cells. Inhibition of p38 MAPK reversed beta-elemene-mediated anti-proliferation effect. Furthermore, the growth of glioblastoma cell-transplanted tumors in nude mice was inhibited by intraperitoneal injection of beta-elemene. Taken together, our findings indicate that activation of p38 MAPK is critical for the anti-proliferation effect of beta-elemene and that p38 MAPK might be a putative pharmacological target for glioblastoma therapy.

[A genome-wide screening for pathological myopia suggests a novel locus on chromosome 15q12 - 13].

OBJECTIVE: Pathological myopia has a genetic background. Previous studies have mapped six loci at 18p11.31, 12q21-23, 7q36, 17q21-22, 4q22-q27 and 2q37.1 in autosomal dominant (AD) pathological myopia. The aim of the present study was to map the mutate gene associated with this disorder in Chinese population. METHODS: A family with AD pathological myopia including 12 individuals, of which 7 members were affected, consented to participate our study. Three hundred and thirty pairs of highly heterozygous microsatellite marker primers were selected for a genome-wide screening. Two-point linkage was calculated by LINKAGE package in an autosomal dominant mode with full penetrance at gene frequency of 0.0133. Multipoint LOD scores were calcu1ated by use of GENEHUNTER program. Genetic distance between marker loci examined was determined on the basis of Genethon linkage map. Haplotype analysis was performed by software of Cyrillic 2.0 based on the lowest recombination principle. RESULTS: Evidence of significant linkage was found on chromosome 15q in the family by two-point linkage analysis. The maximum LOD score was 1.76 with the markers D15S1010, D15S1007 and D15S1042 at a recombination fraction of 0.00. Multipoint linkage analysis also supported existence of linkage on this region with NPL score 5.16. Haplotype analysis refined this myopia locus to a 12 cM interval between D15S1019 and D15S146 on 15q12 - 13. No evidence of linkage was found at any known myopia loci, including AD pathological myopia loci on 18p11.31, 12q21 - 23, 7q36, 17q21 - 22, 4q22 - q27 and 2q37.1, and syndromic myopia loci on 15q15-21, 12q13.11-13.2, 6p21.3, 1q21-31, 1p21 and 21q22.3. CONCLUSIONS: Our study indicates a novel myopia locus on 15q12 - 13. There are 94 known genes locate on this region, screening for sequence of candidate genes within this region will be helpful to find the mutant gene. This study also provides additional support for genetic heterogeneity of this disorder.

Recurrent gain-of-function USP8 mutations in Cushing's disease.

Cushing's disease, also known as adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas (PAs) that cause excess cortisol production, accounts for up to 85% of corticotrophin-dependent Cushing's syndrome cases. However, the genetic alterations in this disease are unclear. Here, we performed whole-exome sequencing of DNA derived from 12 ACTH-secreting PAs and matched blood samples, which revealed three types of somatic mutations in a candidate gene, USP8 (encoding ubiquitin-specific protease 8), exclusively in exon 14 in 8 of 12 ACTH-secreting PAs. We further evaluated somatic USP8 mutations in additional 258 PAs by Sanger sequencing. Targeted sequencing further identified a total of 17 types of USP8 variants in 67 of 108 ACTH-secreting PAs (62.04%). However, none of these mutations was detected in other types of PAs (n = 150). These mutations aggregate within the 14-3-3 binding motif of USP8 and disrupt the interaction between USP8 and 14-3-3 protein, resulting in an elevated capacity to protect EGFR from lysosomal degradation. Accordingly, PAs with mutated USP8 display a higher incidence of EGFR expression, elevated EGFR protein abundance and mRNA expression levels of POMC, which encodes the precursor of ACTH. PAs with mutated USP8 are significantly smaller in size and have higher ACTH production than wild-type PAs. In surgically resected primary USP8-mutated tumor cells, USP8 knockdown or blocking EGFR effectively attenuates ACTH secretion. Taken together, somatic gain-of-function USP8 mutations are common and contribute to ACTH overproduction in Cushing's disease. Inhibition of USP8 or EGFR is promising for treating USP8-mutated corticotrophin adenoma. Our study highlights the potentially functional mutated gene in Cushing's disease and provides insights into the therapeutics of this disease.

Expression of Exogenous E-cadherin Regulates Anchorage-independent Growth in Human Lung Adenocarcinoma Cells.

To explore the suppressive effects of E-cadherin on aggregation and anchorage-independent growth of human lung adenocarcinoma cells, a mammalian expression vector containing full E-cadherin cDNA was constructed and transfected into a metastatic human lung adenocarcinoma cell line A549. RT-PCR and Western blot were used to analyse expression levels of E-cadherin. Cells aggregation and anchorage-independent growth of the tumor cells before and after gene transfection were assessed respectively. The results indicated that stable transfectants showed markedly increased levels of expressed E-cadherin compared with the corresponding sham transfectants. The transfections showed more intense cellular aggregation and anchorage-independent growth.

[Identification of a novel inheritable CCM1 gene mutation of 671del AT in a Chinese family with cerebral cavernous malformation].

OBJECTIVE: To investigate the hereditary characters of familial cerebral cavernous malformation (FCCM) and the novel gene mutation in a Chinese family. METHODS: Head MRI examination and clinical neurological check were performed on a Chinese family with one proband of FCCM, female, 27 years old, and 16 family members, 9 males and 12 females, and 19 controls, including patients with sporadic CCM and other diseases and healthy persons. DNA was extracted from the white blood cells of the peripheral blood of the subjects. PCR and DNA direct sequencing were used to detect the mutation in CCM1 gene. RESULTS: Head MRI found 11 FCCM patients in the 16 family members of the proband (69%), the youngest one being 4 years old, including multiple intracranial lesions in 7 patients and single lesion in 4. Relevant clinical manifestations were found in 6 out of the 11 family members. Nucleotide sequence analysis of the proband and other affected family members revealed a deletion frameshift mutation of A and T at nucleotides (nt) 671 and 672 in exon 13 of the CCM1 gene, resulting in truncated encoding KRIT1 protein. No mutation was detected in the healthy family members and the controls. CONCLUSION: A novel inheritable CCM1 gene mutation of 671del AT has been found in patients with FCCM.

A novel gene mutation (1292 deletion) in a Chinese family with cerebral cavernous malformations.

OBJECTIVE: Hereditary cerebral cavernous malformations (CCMs) are characterized by focal abnormalities of small blood vessels in the brain and consequent hemorrhage and seizures. Previous studies of this type of CCM have mainly reported on this disorder in Hispanic and Caucasian cases. Here, we report on hereditary CCM in a Chinese family further characterized by a novel CCM1 gene mutation. METHODS: We investigated a family of 21 members, of whom 3 died and 16 of the survivors became the subjects of this study by brain magnetic resonance imaging. RESULTS: Brain magnetic resonance imaging demonstrated abnormal results in 11 members (69% penetrance), including multiple intracranial lesions in seven cases and single lesions in four cases. The clinical manifestation of CCM was found in these cases. The youngest patient was 4 years old. The remaining 5 members were normal. Nucleotide sequencing analysis of the family member representing the index case and other affected members revealed a deletion frameshift mutation of A and T at nucleotides 1292 and 1293 in exon 13 of the CCM1 gene, which resulted in truncated encoding Krev interaction trapped-1 protein. CONCLUSION: Our results indicated a novel hereditary CCM1 gene mutation of 1292delAT, a finding that may contribute to the clarification of the mechanism of the disease.