The Novel Histone Deacetylase Inhibitor, OBP-801, Induces Apoptosis in Rhabdoid Tumors by Releasing the Silencing of NOXA.

Rhabdoid tumor is an aggressive, early childhood tumor. Biallelic inactivation of the SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1)/integrase interactor 1 (INI1) gene is the only common genetic feature in rhabdoid tumors. Loss of SMARCB1 function results in downregulation of several tumor suppressor genes including p16, p21, and NOXA The novel histone deacetylase inhibitor, OBP-801, induces p21 and has shown efficacy against various cancers. In our study, OBP-801 strongly inhibited the cell growth of all rhabdoid tumor cell lines in WST-8 assay. However, Western blotting and cell-cycle analysis revealed that OBP-801 did not activate the P21-RB pathway in some cell lines. p21 knockout indicated that p21 did not dominate the OBP-801 antitumor effect in rhabdoid tumor cell lines. We discovered that OBP-801 induced NOXA expression and caspase-dependent apoptosis in rhabdoid tumor cell lines independent of TP53. Chromatin immunoprecipitation assay showed that OBP-801 acetylated histone proteins and recruited RNA polymerase II to the transcription start site (TSS) of the NOXA promotor. Moreover, OBP-801 recruited BRG1 and BAF155, which are members of the SWI/SNF complex, to the TSS of the NOXA promotor. These results suggest that OBP-801 epigenetically releases the silencing of NOXA and induces apoptosis in rhabdoid tumors. OBP-801 strongly inhibited tumor growth in human rhabdoid tumor xenograft mouse models in vivo Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and cleaved caspase-3 were stained in tumors treated with OBP-801. In conclusion, OBP-801 induces apoptosis in rhabdoid tumor cells by epigenetically releasing the silencing of NOXA, which is a key mediator of rhabdoid tumor apoptosis. The epigenetic approach for NOXA silencing with OBP-801 is promising for rhabdoid tumor treatment.

[Erratum] OBP­801, a novel histone deacetylase inhibitor, induces M­phase arrest and apoptosis in rhabdomyosarcoma cells.

Subsequent to the publication of the above article, the authors have realized that errors were introduced into Fig. 4 at the typesetting stage. Essentially, in Fig. 4B, the P­value should have read as "P=0.13" (not as 0.013), and in Fig 4D, the labels for OBP- and OBP+ were set the wrong way around. The correct version of Fig. 4, as originally submitted, is shown opposite. The Editor apologizes to the authors for introducing these errors into their figure, and to the readership for any inconvenience caused. [the original article was published in Oncology Reports 41: 643-649, 2019; DOI: 10.3892/or.2018.6813.

OBP­801, a novel histone deacetylase inhibitor, induces M­phase arrest and apoptosis in rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer of musculoskeletal origin. Despite multidisciplinary approaches, such as surgical resection, irradiation, and intensive chemotherapy, adopted for its treatment, the prognosis of patients with high­risk RMS remains poor. Thus, molecularly targeted therapies are required to improve patient survival and minimize side effects. Histone deacetylases (HDACs) modify transcription by deacetylation of the lysine residues in chromatin histone tails and several non­histone proteins. HDAC inhibitors, classes of compounds targeted to various HDAC proteins, are being studied for their roles in several types of cancers in a rigorous manner. This study aimed to investigate the potential of a novel HDAC inhibitor, OBP­801, as a therapeutic agent for the treatment of RMS. We used 8 RMS cell lines in this study. Protein expression patterns, cell proliferation, cell cycle status, and apoptosis in RMS cells after OBP­801 treatment in vitro were investigated. We also studied the antitumor activity of OBP­801 in an in vivo xenograft mouse model. We observed cell cycle arrest at the M­phase and apoptosis in all RMS cell lines after exposure to pharmacological levels of OBP­801 for 24 h. Immunofluorescence staining revealed that OBP­801 may induce mitotic catastrophe via chromosome misalignment and reduced survivin expression, ultimately leading to apoptosis. Our results demonstrated that the novel HDAC inhibitor OBP­801 was an effective inhibitor of RMS cell line proliferation and may be a potent therapeutic option for RMS.

Copy number abnormality of acute lymphoblastic leukemia cell lines based on their genetic subtypes.

In this study, we performed genetic analysis of 83 B cell precursor acute lymphoblastic leukemia (B-ALL) cell lines. First, we performed multiplex ligation-dependent probe amplification analysis to identify copy number abnormalities (CNAs) in eight genes associated with B-ALL according to genetic subtype. In Ph+ B-ALL cell lines, the frequencies of IKZF1, CDKN2A/2B, BTG1, and PAX5 deletion were significantly higher than those in Ph- B-ALL cell lines. The frequency of CDKN2A/2B deletion in KMT2A rearranged cell lines was significantly lower than that in non-KMT2A rearranged cell lines. These findings suggest that CNAs are correlated with genetic subtype in B-ALL cell lines. In addition, we determined that three B-other ALL cell lines had IKZF1 deletions (YCUB-5, KOPN49, and KOPN75); we therefore performed comprehensive genetic analysis of these cell lines. YCUB-5, KOPN49, and KOPN75 had P2RY8-CRLF2, IgH-CRLF2, and PAX5-ETV6 fusions, respectively. Moreover, targeted capture sequencing revealed that YCUB-5 had JAK2 R683I and KRAS G12D, and KOPN49 had JAK2 R683G and KRAS G13D mutations. These data may contribute to progress in the field of leukemia research.

Anti-leukemic activity of bortezomib and carfilzomib on B-cell precursor ALL cell lines.

Prognosis of childhood acute lymphoblastic leukemia (ALL) has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ), a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated in vitro BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+) ALL, IKZF1 deletion, and biallelic loss of CDKN2A were associated with favorable response. Even in Ph-negative ALL cell lines, IKZF1 deletion and bilallelic loss of CDKN2A were independently associated with higher BTZ sensitivity. BTZ showed only marginal cross-resistance to four representative chemotherapeutic agents (vincristine, dexamethasone, l-asparaginase, and daunorubicin) in B-cell precursor-ALL cell lines. To improve the efficacy and safety of proteasome inhibitor combination chemotherapy, we also analyzed the anti-leukemic activity of carfilzomib (CFZ), a second-generation proteasome inhibitor, as a substitute for BTZ. CFZ showed significantly higher activity than BTZ in the majority of ALL cell lines except for the P-glycoprotein-positive t(17;19) ALL cell lines, and IKZF1 deletion was also associated with a favorable response to CFZ treatment. P-glycoprotein inhibitors effectively restored the sensitivity to CFZ, but not BTZ, in P-glycoprotein-positive t(17;19) ALL cell lines. P-glycoprotein overexpressing ALL cell line showed a CFZ-specific resistance, while knockout of P-glycoprotein by genome editing with a CRISPR/Cas9 system sensitized P-glycoprotein-positive t(17;19) ALL cell line to CFZ. These observations suggested that IKZF1 deletion could be a useful biomarker to predict good sensitivity to CFZ and BTZ, and that CFZ combination chemotherapy may be a new therapeutic option with higher anti-leukemic activity for refractory ALL that contain P-glycoprotein-negative leukemia cells.

Monitoring of minimal residual disease after HLA-mismatched allogeneic hematopoietic stem cell transplantation using six-color based HLA-Flow method in pediatric acute leukemia.

HLA-Flow is a flow cytometry-based method using anti-HLA antibodies against mismatched HLA alleles combined with the antibodies against antigens expressed on leukemic cells. It is a sensitive assay to determine minimal residual disease (MRD) after HLA mismatched hematopoietic stem cell transplantation (HSCT). In this study, we report the results of our HLA-Flow using six-color based multicolor fluorescence-activated cell sorting for HLA-mismatched HSCT. We performed HLA-Flow monitoring after HLA mismatched HSCT from July 2013 to July 2016 in nine patients (three with acute lymphoblastic leukemia, five with acute myeloid leukemia, and one with therapy-related acute myeloid leukemia) for MRD monitoring. We detected a relapse after HSCT in three of the nine patients, two of them at MRD levels. HLA-Flow is a sensitive, fast, and inexpensive method for the detection of MRD in patients with HLA-mismatched HSCT.

Development of acute lymphoblastic leukemia with IgH-EPOR in a patient with secondary erythrocytosis.

We report the first patient to develop ALL with a fusion gene of the erythropoietin receptor (EPOR) with immunoglobulin heavy chain (IgH) 22 years after a diagnosis of secondary erythrocytosis with unknown etiology. The IgH-EPOR rearrangement is known to induce increased expression of EPOR, and activates EPO-associated signal pathways by exogenous EPO stimulation, resulting in the increased proliferation and survival of IgH-EPOR-positive leukemic cells. Interestingly, this case may provide supporting the possibility that IgH-EPOR-positive ALL has a growth advantage under sustained high concentrations of EPO.

Traumatic herniation of the buccal fat pad.

Traumatic herniation of the buccal fat pad is a rare traumatic disease. Treatment consists of either excision or replacement. We herein report the first case in which a traumatic herniation of the buccal fat pad healed naturally. It was necessary to differentiate the disease from lipoblastoma. A 17-month-old boy was admitted to a clinic with an intraoral tumor that had suddenly increased in size. The tumor was diagnosed as herniation of the buccal fat pad on pathology of a biopsy specimen. In the present case, the escaped buccal fat body returned naturally and engrafted without dysfunction or facial defects. Given that young children may easily fall down with various objects in their mouth, care is required to prevent traumatic accidents. Traumatic herniation of the buccal fat pad should be considered in the differentiation of tumors of the oral cavity in young children.

BCL2 Inhibitor (ABT-737): A Restorer of Prednisolone Sensitivity in Early T-Cell Precursor-Acute Lymphoblastic Leukemia with High MEF2C Expression?

Early T-cell precursor-acute lymphoblastic leukemia (ETP-ALL) has been identified as a high-risk subtype of pediatric T-cell acute lymphoblastic leukemia (T-ALL). Conventional chemotherapy is not fully effective for this subtype of leukemia; therefore, potential therapeutic targets need to be explored. Analysis of the gene expression patterns of the transcription factors in pediatric T-ALL revealed that MEF2C and FLT3 were expressed at higher levels in ETP-ALL than typical T-ALL. Using human T-ALL and BaF3 cell lines with high expression levels of MEF2C, the present study tested whether the BCL2 inhibitor (ABT-737) restores the sensitivity to prednisolone (PSL), because MEF2C causes PSL resistance, possibly by augmenting the anti-apoptotic activity of BCL2. Treatment with PSL and ABT-737 caused a significant reduction in the IC50 of PSL in the MEF2C-expressing LOUCY cells, in addition to the MEF2C-transduced BaF3 cells, but not in the non-MEF2C-expressing Jurkat cells. The combination treatment significantly accelerated the killing of primary leukemic blast cells of ETP-ALL with high expression levels of MEF2C, which were co-cultured with murine stromal cells. These findings suggest that BCL2 inhibitors may be a therapeutic candidate in vivo for patients with ETP-ALL with high expression levels of MEF2C.