Treatment for a primary multidrug-resistant B-cell acute lymphoblastic leukemia patient carrying a SSBP2-CSF1R fusion gene: a case report.

SSBP2-CSF1R is an important biomarker for clinical diagnosis and prognosis of Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL). This case report presents a pediatric Ph-like ALL patient carrying the SSBP2-CSF1R fusion gene. The patient was resistant to most conventional chemotherapy regimens and to dasatinib, an inhibitor that has been reported to have a therapeutic effect on SSBP2-CSF1R fusion Ph-like ALL, as she remained minimal residual disease (MRD) positive (detection by flow cytometry) and SSBP2-CSF1R fusion gene (detection by RT-PCR) positive after five rounds of such regimens. We thus conducted a large-scale in vitro screening to assess the sensitivity of the patient's leukemic cells to anti-cancer drugs. Based on the susceptibility results, we chose to combine cytarabine, homoharringtonine, dexamethasone, fludarabine, vindesine, and epirubicin for treatment. Clinical results showed that after a course of treatment, both MRD and SSBP2-CSF1R fusion gene turned negative, and there was no recurrence during an 18-month follow-up. In conclusion, our study suggests that the SSBP2-CSF1R fusion gene may be an important biomarker of primary drug resistance in Ph-like ALL, and indicate that the combination of cytarabine, homoharringtonine, dexamethasone, fludarabine, vindesine, and epirubicin can achieve optimal therapeutic results in this category of patients.

Screening and Activity Evaluation of Novel BCR-ABL/T315I Tyrosine Kinase Inhibitors.

INTRODUCTION: Chronic myeloid leukemia (CML) is a kind of malignant tumor formed by the clonal proliferation of bone marrow hematopoietic stem cells. BCR-ABL fusion protein, found in more than 90% of patients, is a vital target for discovering anti-CML drugs. Up to date, imatinib is the first BCR-ABL tyrosine kinase inhibitor (TKI) approved by the FDA for treating CML. However, the drug resistance problems appeared for many reasons, especially the T135I mutation, a "gatekeeper" of BCR-ABL. Currently, there is no long-term effective and low side effect drug in clinical. METHODS: This study intends to find novel TKIs targeting BCR-ABL with high inhibitory activity against T315I mutant protein by combining artificial intelligence technology and cell growth curve, cytotoxicity, flow cytometry and western blot experiments. RESULTS: The obtained compound was found to kill leukemia cells, which had good inhibitory efficacy in BaF3/T315I cells. Compound No 4 could induce cell cycle arrest, cause autophagy and apoptosis, and inhibit the phosphorylation of BCR-ABL tyrosine kinase, STAT5 and Crkl proteins. CONCLUSION: The results indicated that the screened compound could be used as a lead compound for further research to discover ideal chronic myeloid leukemia therapeutic drugs.

Structural Optimization of Cannabidiol as Multifunctional Cosmetic Raw Materials.

Cannabidiol (CBD), derived from the plant cannabis, can be used in the cosmetics industry for its antioxidant, anti-inflammatory, anti-wrinkle and whitening effects. However, CBD is purified from the hemp plant extract, its source is very limited and under strict control. So in this study, computational and experimental methods were combined to search for novel CBD substitutes with high biology potencies. The action mode between CBD and target protein cannabidiol receptor 1 was studied to find the key skeleton, which was used to virtually screen a natural products database to search for compounds with 70% similarity. The hit compounds with high docking scores were selected for the ABTS and DPPH free radical scavenging experiments for antioxidant evaluation. The effects on the expressions of nitric oxide (NO), interleukin-6 (IL-6), COX-2 and iNOS in RAW264.7 cell line were detected to demonstrate their anti-inflammatory abilities. The effect of anti-wrinkle ability were evaluated by detecting the extracellular matrix, such as collagen, elastin, fibronectin and reactive oxygen species (ROS) in HFF-1. The effects on melanin production and tyrosinase activity in Bb16F10 were also detected. As a result, two compounds were found to be superior to cannabidiol, in terms of antioxidant, anti-wrinkle and whitening efficacy with a lower cytotoxicity.

Treatment for a B-cell acute lymphoblastic leukemia patient carrying a rare TP53 c.C275T mutation: A case report.

TP53 mutations are associated with poor prognosis in the vast majority of cancers. In this study, we present a pediatric B-cell acute lymphoblastic leukemia (B-ALL) patient carrying a rare TP53 c.C275T mutation. This extremely rare mutation affects an amino acid residue located between the TAD domain and the DNA-binding domain of p53. The patient was resistant to most conventional chemotherapy regimens and remained minimal residual disease (MRD)-positive after five rounds of such regimens. We tested the sensitivity of the patient's leukemic cells to 21 anti-cancer drugs by performing in vitro drug sensitivity assays. The results showed that bortezomib had a very strong killing effect on the patient's leukemic cells. Therefore, we subsequently treated the patient with bortezomib combined with vindesine, cytarabine, and fludarabine. After one course of treatment, the patient became MRD-negative, and there was no recurrence during a 9-month follow-up. In conclusion, our report suggests that the TP53 c.C275T mutation is associated with poor prognosis in B-ALL. Fortunately, bortezomib combined with chemotherapy could achieve a better therapeutic effect than conventional regimens in this type of ALL.

Identification of two downregulated circRNAs in patients with acute B-lymphocytic leukemia.

Acute B-lymphocytic leukemia (B-ALL) is associated with a high mortality rate, with no effective treatment strategies available. The identification of diagnostic and prognostic biomarkers of B-ALL can contribute to the development of novel therapeutic methods and drugs, which can improve the survival outcomes of patients with B-ALL. The present study aimed to identify downregulated circular RNAs (circRNAs) in patients with B-ALL. RNA sequencing was performed to construct the circRNA expression profiles in B-ALL cells and normal human lymphoblasts. The Database for Annotation, Visualization and Integrated Discovery was used to perform Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses. In addition, reverse transcription-quantitative (RT-q)PCR analysis was performed to detect the expression levels of the downregulated circRNAs. A total of 263 differentially expressed circRNAs were identified, including 76 upregulated and 187 downregulated circRNAs, respectively. The upregulated circRNAs were mainly enriched in 'macromolecule modification', 'protein modification' and 'cellular protein modification processes', while the downregulated circRNAs were mainly enriched in the 'negative regulation of RNA biosynthetic processes', 'natural killer cell-mediated cytotoxicity' and 'viral carcinogenesis'. RT-qPCR analysis demonstrated that two of the downregulated circRNAs (hsa_circ_0000745 and chr15:87949594-87966067-), identified during microarray analysis were also significantly downregulated in Ball-1 cells and B-ALL bone marrow samples. Thus, these circRNAs may serve as biomarkers for patients with B-ALL.

miR-377-3p-Mediated EGR1 Downregulation Promotes B[a]P-Induced Lung Tumorigenesis by Wnt/Beta-Catenin Transduction.

Polycyclic aromatic hydrocarbons (PAHs), particularly benzo[a]pyrene (B[a]P), found in cigarette smoke and air pollution, is an important carcinogen. Nevertheless, early molecular events and related regulatory effects of B[a]P-mediated cell transformation and tumor initiation remain unclear. This study found that EGR1 was significantly downregulated during human bronchial epithelial cell transformation and mice lung carcinogenesis upon exposure to B[a]P and its active form BPDE, respectively. In contrast, overexpression of EGR1 inhibited the BPDE-induced cell malignant transformation. Moreover, miR-377-3p was strongly enhanced by BPDE/B[a]P exposure and crucial for the inhibition of EGR1 expression by targeting the 3'UTR of EGR1. MiR-377-3p antagomir reversed the effect of EGR1 downregulation in cell malignant transformation and tumor initiation models. Furthermore, the B[a]P-induced molecular changes were evaluated by IHC in clinical lung cancer tissues and examined with a clinic database. Mechanistically, EGR1 inhibition was also involved in the regulation of Wnt/ß-catenin transduction, promoting lung tumorigenesis following B[a]P/BPDE exposure. Taken together, the results demonstrated that bBenzo[a]pyrene exposure might induce lung tumorigenesis through miR-377-3p-mediated reduction of EGR1 expression, suggesting an important role of EGR1 in PAHs-induced lung carcinogenesis.

STAT3 activates MSK1-mediated histone H3 phosphorylation to promote NFAT signaling in gastric carcinogenesis.

Epigenetic abnormalities contribute significantly to the development and progression of gastric cancer. However, the underlying regulatory networks from oncogenic signaling pathway to epigenetic dysregulation remain largely unclear. Here we showed that STAT3 signaling, one of the critical links between inflammation and cancer, acted as a control pathway in gastric carcinogenesis. STAT3 aberrantly transactivates the epigenetic kinase mitogen- and stress-activated protein kinase 1 (MSK1), thereby phosphorylating histone H3 serine10 (H3S10) and STAT3 itself during carcinogen-induced gastric tumorigenesis. We further identified the calcium pathway transcription factor NFATc2 as a novel downstream target of the STAT3-MSK1 positive-regulating loop. STAT3 forms a functional complex with MSK1 at the promoter of NFATc2 to promote its transcription in a H3S10 phosphorylation-dependent way, thus affecting NFATc2-related inflammatory pathways in gastric carcinogenesis. Inhibiting the STAT3/MSK1/NFATc2 signaling axis significantly suppressed gastric cancer cell proliferation and xenograft tumor growth, which provides a potential novel approach for gastric carcinogenesis intervention by regulating aberrant epigenetic and transcriptional mechanisms.

De Novo Mutations Activating Germline TP53 in an Inherited Bone-Marrow-Failure Syndrome.

Inherited bone-marrow-failure syndromes (IBMFSs) include heterogeneous genetic disorders characterized by bone-marrow failure, congenital anomalies, and an increased risk of malignancy. Many lines of evidence have suggested that p53 activation might be central to the pathogenesis of IBMFSs, including Diamond-Blackfan anemia (DBA) and dyskeratosis congenita (DC). However, the exact role of p53 activation in each clinical feature remains unknown. Here, we report unique de novo TP53 germline variants found in two individuals with an IBMFS accompanied by hypogammaglobulinemia, growth retardation, and microcephaly mimicking DBA and DC. TP53 is a tumor-suppressor gene most frequently mutated in human cancers, and occasional germline variants occur in Li-Fraumeni cancer-predisposition syndrome. Most of these mutations affect the core DNA-binding domain, leading to compromised transcriptional activities. In contrast, the variants found in the two individuals studied here caused the same truncation of the protein, resulting in the loss of 32 residues from the C-terminal domain (CTD). Unexpectedly, the p53 mutant had augmented transcriptional activities, an observation not previously described in humans. When we expressed this mutant in zebrafish and human-induced pluripotent stem cells, we observed impaired erythrocyte production. These findings together with close similarities to published knock-in mouse models of TP53 lacking the CTD demonstrate that the CTD-truncation mutations of TP53 cause IBMFS, providing important insights into the previously postulated connection between p53 and IBMFSs.

Loss of function mutations in RPL27 and RPS27 identified by whole-exome sequencing in Diamond-Blackfan anaemia.

Diamond-Blackfan anaemia is a congenital bone marrow failure syndrome that is characterized by red blood cell aplasia. The disease has been associated with mutations or large deletions in 11 ribosomal protein genes including RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPS29, RPL5, RPL11, RPL26 and RPL35A as well as GATA1 in more than 50% of patients. However, the molecular aetiology of many Diamond-Blackfan anaemia cases remains to be uncovered. To identify new mutations responsible for Diamond-Blackfan anaemia, we performed whole-exome sequencing analysis of 48 patients with no documented mutations/deletions involving known Diamond-Blackfan anaemia genes except for RPS7, RPL26, RPS29 and GATA1. Here, we identified a de novo splicing error mutation in RPL27 and frameshift deletion in RPS27 in sporadic patients with Diamond-Blackfan anaemia. In vitro knockdown of gene expression disturbed pre-ribosomal RNA processing. Zebrafish models of rpl27 and rps27 mutations showed impairments of erythrocyte production and tail and/or brain development. Additional novel mutations were found in eight patients, including RPL3L, RPL6, RPL7L1T, RPL8, RPL13, RPL14, RPL18A and RPL31. In conclusion, we identified novel germline mutations of two ribosomal protein genes responsible for Diamond-Blackfan anaemia, further confirming the concept that mutations in ribosomal protein genes lead to Diamond-Blackfan anaemia.

The landscape of somatic mutations in Down syndrome-related myeloid disorders.

Transient abnormal myelopoiesis (TAM) is a myeloid proliferation resembling acute megakaryoblastic leukemia (AMKL), mostly affecting perinatal infants with Down syndrome. Although self-limiting in a majority of cases, TAM may evolve as non-self-limiting AMKL after spontaneous remission (DS-AMKL). Pathogenesis of these Down syndrome-related myeloid disorders is poorly understood, except for GATA1 mutations found in most cases. Here we report genomic profiling of 41 TAM, 49 DS-AMKL and 19 non-DS-AMKL samples, including whole-genome and/or whole-exome sequencing of 15 TAM and 14 DS-AMKL samples. TAM appears to be caused by a single GATA1 mutation and constitutive trisomy 21. Subsequent AMKL evolves from a pre-existing TAM clone through the acquisition of additional mutations, with major mutational targets including multiple cohesin components (53%), CTCF (20%), and EZH2, KANSL1 and other epigenetic regulators (45%), as well as common signaling pathways, such as the JAK family kinases, MPL, SH2B3 (LNK) and multiple RAS pathway genes (47%).

Clonal selection in xenografted TAM recapitulates the evolutionary process of myeloid leukemia in Down syndrome.

Transient abnormal myelopoiesis (TAM) is a clonal preleukemic disorder that progresses to myeloid leukemia of Down syndrome (ML-DS) through the accumulation of genetic alterations. To investigate the mechanism of leukemogenesis in this disorder, a xenograft model of TAM was established using NOD/Shi-scid, interleukin (IL)-2Rγ(null) mice. Serial engraftment after transplantation of cells from a TAM patient who developed ML-DS a year later demonstrated their self-renewal capacity. A GATA1 mutation and no copy number alterations (CNAs) were detected in the primary patient sample by conventional genomic sequencing and CNA profiling. However, in serial transplantations, engrafted TAM-derived cells showed the emergence of divergent subclones with another GATA1 mutation and various CNAs, including a 16q deletion and 1q gain, which are clinically associated with ML-DS. Detailed genomic analysis identified minor subclones with a 16q deletion or this distinct GATA1 mutation in the primary patient sample. These results suggest that genetically heterogeneous subclones with varying leukemia-initiating potential already exist in the neonatal TAM phase, and ML-DS may develop from a pool of such minor clones through clonal selection. Our xenograft model of TAM may provide unique insight into the evolutionary process of leukemia.

Naturally occurring oncogenic GATA1 mutants with internal deletions in transient abnormal myelopoiesis in Down syndrome.

Children with Down syndrome have an increased incidence of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia. The majority of these cases harbor somatic mutations in the GATA1 gene, which results in the loss of full-length GATA1. Only a truncated isoform of GATA1 that lacks the N-terminal 83 amino acids (GATA1-S) remains. We found through genetic studies of 106 patients with TAM that internally deleted GATA1 proteins (GATA1-IDs) lacking amino acid residues 77-119 or 74-88 (created by splicing mutations) contributed to the genesis of TAM in 6 patients. Analyses of GATA1-deficient embryonic megakaryocytic progenitors revealed that the GATA1 function in growth restriction was disrupted in GATA1-IDs. In contrast, GATA1-S promoted megakaryocyte proliferation more profoundly than that induced by GATA1 deficiency. These results indicate that the internally deleted regions play important roles in megakaryocyte proliferation and that perturbation of this mechanism is involved in the pathogenesis of TAM.

A novel mutation of ribosomal protein S10 gene in a Japanese patient with diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow disease. The condition is characterized by anemia that usually presents during infancy or early childhood and congenital malformation. Several reports show that DBA is associated with mutations in the ribosomal protein (RP) genes, RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, and RPS7. Recently, 5 and 12 patients with mutations in RPS10 and RPS26, respectively, were identified in a cohort of 117 DBA probands. Therefore, we screened the DBA patients who were negative for mutations in these DBA genes for mutations in RPS10 and RPS26. The present case report describes the identification of the first Japanese DBA patient with a novel mutation in RPS10.

Extensive gene deletions in Japanese patients with Diamond-Blackfan anemia.

Fifty percent of Diamond-Blackfan anemia (DBA) patients possess mutations in genes coding for ribosomal proteins (RPs). To identify new mutations, we investigated large deletions in the RP genes RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26. We developed an easy method based on quantitative-PCR in which the threshold cycle correlates to gene copy number. Using this approach, we were able to diagnose 7 of 27 Japanese patients (25.9%) possessing mutations that were not detected by sequencing. Among these large deletions, similar results were obtained with 6 of 7 patients screened with a single nucleotide polymorphism array. We found an extensive intragenic deletion in RPS19, including exons 1-3. We also found 1 proband with an RPL5 deletion, 1 patient with an RPL35A deletion, 3 with RPS17 deletions, and 1 with an RPS19 deletion. In particular, the large deletions in the RPL5 and RPS17 alleles are novel. All patients with a large deletion had a growth retardation phenotype. Our data suggest that large deletions in RP genes comprise a sizable fraction of DBA patients in Japan. In addition, our novel approach may become a useful tool for screening gene copy numbers of known DBA genes.

Down syndrome and GATA1 mutations in transient abnormal myeloproliferative disorder: mutation classes correlate with progression to myeloid leukemia.

Twenty percent to 30% of transient abnormal myelopoiesis (TAM) observed in newborns with Down syndrome (DS) develop myeloid leukemia of DS (ML-DS). Most cases of TAM carry somatic GATA1 mutations resulting in the exclusive expression of a truncated protein (GATA1s). However, there are no reports on the expression levels of GATA1s in TAM blasts, and the risk factors for the progression to ML-DS are unidentified. To test whether the spectrum of transcripts derived from the mutant GATA1 genes affects the expression levels, we classified the mutations according to the types of transcripts, and investigated the modalities of expression by in vitro transfection experiments using GATA1 expression constructs harboring mutations. We show here that the mutations affected the amount of mutant protein. Based on our estimates of GATA1s protein expression, the mutations were classified into GATA1s high and low groups. Phenotypic analyses of 66 TAM patients with GATA1 mutations revealed that GATA1s low mutations were significantly associated with a risk of progression to ML-DS (P < .001) and lower white blood cell counts (P = .004). Our study indicates that quantitative differences in mutant protein levels have significant effects on the phenotype of TAM and warrants further investigation in a prospective study.

Mutations in the ribosomal protein genes in Japanese patients with Diamond-Blackfan anemia.

BACKGROUND: Diamond-Blackfan anemia is a rare, clinically heterogeneous, congenital red cell aplasia: 40% of patients have congenital abnormalities. Recent studies have shown that in western countries, the disease is associated with heterozygous mutations in the ribosomal protein (RP) genes in about 50% of patients. There have been no studies to determine the incidence of these mutations in Asian patients with Diamond-Blackfan anemia. DESIGN AND METHODS: We screened 49 Japanese patients with Diamond-Blackfan anemia (45 probands) for mutations in the six known genes associated with Diamond-Blackfan anemia: RPS19, RPS24, RPS17, RPL5, RPL11, and RPL35A. RPS14 was also examined due to its implied involvement in 5q- syndrome. RESULTS: Mutations in RPS19, RPL5, RPL11 and RPS17 were identified in five, four, two and one of the probands, respectively. In total, 12 (27%) of the Japanese Diamond-Blackfan anemia patients had mutations in ribosomal protein genes. No mutations were detected in RPS14, RPS24 or RPL35A. All patients with RPS19 and RPL5 mutations had physical abnormalities. Remarkably, cleft palate was seen in two patients with RPL5 mutations, and thumb anomalies were seen in six patients with an RPS19 or RPL5 mutation. In contrast, a small-for-date phenotype was seen in five patients without an RPL5 mutation. CONCLUSIONS: We observed a slightly lower frequency of mutations in the ribosomal protein genes in patients with Diamond-Blackfan anemia compared to the frequency reported in western countries. Genotype-phenotype data suggest an association between anomalies and RPS19 mutations, and a negative association between small-for-date phenotype and RPL5 mutations.