A distinct subpopulation of leukemia initiating cells in acute precursor B lymphoblastic leukemia: quiescent phenotype and unique transcriptomic profile.

In leukemia, a distinct subpopulation of cancer-initiating cells called leukemia stem cells (LSCs) is believed to drive population expansion and tumor growth. Failing to eliminate LSCs may result in disease relapse regardless of the amount of non-LSCs destroyed. The first step in targeting and eliminating LSCs is to identify and characterize them. Acute precursor B lymphoblastic leukemia (B-ALL) cells derived from patients were incubated with fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-1, 3-diazol-4-yl) Amino)-2-Deoxyglucose (NBDG) and sorted based on NBDG uptake. Cell subpopulations defined by glucose uptake were then serially transplanted into mice and evaluated for leukemia initiating capacity. Gene expression profiles of these cells were characterized using RNA-Sequencing (RNA-Seq). A distinct population of NBDG-low cells was identified in patient B-ALL samples. These cells are a small population (1.92% of the entire leukemia population), have lower HLA expression, and are smaller in size (4.0 to 7.0 µm) than the rest of the leukemia population. All mice transplanted with NBDG-low cells developed leukemia between 5 and 14 weeks, while those transplanted with NBDG-high cells did not develop leukemia (p ≤ 0.0001-0.002). Serial transplantation of the NBDG-low mouse model resulted in successful leukemia development. NBDG-medium (NBDG-med) populations also developed leukemia. Interestingly, comprehensive molecular characterization of NBDG-low and NBDG-med cells from patient-derived xenograft (PDX) models using RNA-Seq revealed a distinct profile of 2,162 differentially-expressed transcripts (DETs) (p<0.05) with 70.6% down-regulated in NBDG-low cells. Hierarchical clustering of DETs showed distinct segregation of NBDG-low from NBDG-med and NBDG-high groups with marked transcription expression alterations in the NBDG-low group consistent with cancer survival. In conclusion, A unique subpopulation of cells with low glucose uptake (NBDG-low) in B-ALL was discovered. These cells, despite their quiescence characteristics, once transplanted in mice, showed potent leukemia initiating capacity. Although NBDG-med cells also initiated leukemia, gene expression profiling revealed a distinct signature that clearly distinguishes NBDG-low cells from NBDG-med and the rest of the leukemia populations. These results suggest that NBDG-low cells may represent quiescent LSCs. These cells can be activated in the appropriate environment in vivo, showing leukemia initiating capacity. Our study provides insight into the biologic mechanisms of B-ALL initiation and survival.

MXD3 antisense oligonucleotide with superparamagnetic iron oxide nanoparticles: A new targeted approach for neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. The outcomes for aggressive forms of NB remain poor. The aim of this study was to develop a new molecular-targeted therapy for NB using an antisense oligonucleotide (ASO) and superparamagnetic iron oxide (SPIO) nanoparticles (NPs), as a delivery vehicle, targeting the transcription regulator MAX dimerization protein 3 (MXD3). We previously discovered that MXD3 was highly expressed in high-risk NB, acting as an anti-apoptotic factor; therefore, it can be a good therapeutic target. In this study, we developed two ASO-NP complexes using electrostatic conjugation to polyethylenimine-coated SPIO NPs and chemical conjugation to amphiphilic polymers on amine-functionalized SPIO NPs. Both ASO-NP complexes demonstrated MXD3 knockdown, which resulted in apoptosis in NB cells. ASO chemically-conjugated NP complexes have the potential to be used in the clinic as they showed great efficacy with minimum NP-associated cytotoxicity.

Ipragliflozin Ameliorates Liver Damage in Non-alcoholic Fatty Liver Disease.

BACKGROUND: There are few effective medications for non-alcoholic steatohepatitis (NASH). We investigated the efficacy of ipragliflozin (selective sodium-glucose cotransporter-2 inhibitor [SGLT2I]) for the treatment of patients with type 2 diabetes mellitus (T2DM) complicated by non-alcoholic fatty liver disease (NAFLD). METHODS: We prospectively enrolled patients with T2DM complicated by NAFLD treated at our institutions from January 2015 to December 2016. Patients received oral ipragliflozin (50 mg/day) once daily for 24 weeks. Body composition was evaluated using an InBody720 analyzer. We used transient elastography to measure liver stiffness and the controlled attenuation parameter for the quantification of liver steatosis in patients with NASH. RESULTS: Forty-three patients with T2DM and NAFLD were enrolled (12 with biopsy-proven NASH and 31 with NAFLD diagnosed by ultrasonography). After 24 weeks, body weight, hemoglobin A1c (HbA1c), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase, body fat mass, and steatosis were significantly decreased compared to baseline measurements in patients with NASH. However, muscle mass was not reduced, and liver stiffness showed a statistically insignificant tendency to decrease. NAFLD patients also showed a significant reduction in body weight, HbA1c, AST, and ALT compared to baseline measurements. CONCLUSION: Ipragliflozin may be effective in patients with T2DM complicated by NAFLD.

Low Urine pH Is Associated with Non-alcoholic Fatty Liver Disease: A Community-based Cross-sectional Study.

Objective Low urine pH is associated with several metabolic diseases, such as dyslipidemia, diabetes, and metabolic syndrome. However, the association between low urine pH and non-alcoholic fatty liver disease (NAFLD) remains unknown. Therefore, we conducted a community-based cross-sectional study to investigate this association. Methods Between April 2013 and March 2014, the records of 4,945 Japanese subjects who had undergone annual health checkups were reviewed to identify subjects who met the diagnostic criteria for NAFLD. Patients Based on urine pH, the participants were classified into four groups; a low urine pH was defined as ≤5.5. Of the 3,411 subjects who qualified for enrollment, 1,028 met the diagnostic criteria for NAFLD. Results The prevalence of NAFLD was significantly increased with decreasing urine pH in both men and women (p<0.01 and p=0.02, respectively). A multivariate analysis, including adjustments for age, metabolic markers, and the renal function, showed a significant association between low urine pH and NAFLD in men and women (odds ratio, 1.37; 95% confidence interval, 1.01-1.85, p=0.04 and odds ratio, 1.73; 95% confidence interval, 1.15-2.62, p<0.01, respectively). Conclusion Our study indicates that NAFLD is associated with a low urine pH in both sexes, findings that might help clinicians identify patients at high risk for NAFLD.

Relationship between urine pH and abnormal glucose tolerance in a community-based study.

AIMS/INTRODUCTION: The association between urine pH and abnormal glucose tolerance in men and women is unclear; therefore, we carried out a community-based, cross-sectional study to investigate sex-specific associations between these values, possible indicators of prediabetes and type 2 diabetes. MATERIALS AND METHODS: We enrolled 4,945 Japanese individuals (2,490 men and 2,455 women), who had undergone annual health checkups. To investigate the relationship between low urine pH and abnormal glucose tolerance, participants were divided into three groups based on their fasting plasma glucose levels (<6.11 mmol/L, 6.11-6.99 mmol/L and ≥6.99 mmol/L), and three groups based on their glycated hemoglobin levels (≤44.3 mmol/mol, 44.3-47.5 mmol/mol and ≥47.5 mmol/mol). To examine the effects of urine pH on abnormal glucose tolerance, participants were categorized into five groups based on their urine pH (5.0, 5.5, 6.0, 6.5 and ≥7.0). RESULTS: Multivariate analysis adjusted for age, body mass index, systolic blood pressure, triglycerides, high-density lipoprotein cholesterol, uric acid, creatinine and antidiabetic agent use showed significant associations between low urine pH and both high fasting plasma glucose and high glycated hemoglobin levels (P for trend = 0.0260, 0.0075) in men. Furthermore, after the same adjustments, prevalence rates of abnormal glucose tolerance (≥6.11 mmol/L and ≥6.99 mmol/L), increased significantly as urine pH levels decreased (P for trend = 0.0483, 0.0181) in men. In women, a similar trend was observed without a significant difference. CONCLUSIONS: Low urine pH is significantly associated with abnormal glucose tolerance; therefore, measuring urine pH might prove useful for identifying patients at high risk for diabetes.

Novel targeted therapy for neuroblastoma: silencing the MXD3 gene using siRNA.

BackgroundNeuroblastoma is the second most common extracranial cancer in children. Current therapies for neuroblastoma, which use a combination of chemotherapy drugs, have limitations for high-risk subtypes and can cause significant long-term adverse effects in young patients. Therefore, a new therapy is needed. In this study, we investigated the transcription factor MXD3 as a potential therapeutic target in neuroblastoma.MethodsMXD3 expression was analyzed in five neuroblastoma cell lines by immunocytochemistry and quantitative real-time reverse transcription PCR, and in 18 primary patient tumor samples by immunohistochemistry. We developed nanocomplexes using siRNA and superparamagnetic iron oxide nanoparticles to target MXD3 in neuroblastoma cell lines in vitro as a single-agent therapeutic and in combination with doxorubicin, vincristine, cisplatin, or maphosphamide-common drugs used in current neuroblastoma treatment.ResultsMXD3 was highly expressed in neuroblastoma cell lines and in patient tumors that had high-risk features. Neuroblastoma cells treated in vitro with the MXD3 siRNA nanocomplexes showed MXD3 protein knockdown and resulted in cell apoptosis. Furthermore, on combining MXD3 siRNA nanocomplexes with each of the four drugs, all showed additive efficacy.ConclusionThese results indicate that MXD3 is a potential new target and that the use of MXD3 siRNA nanocomplexes is a novel therapeutic approach for neuroblastoma.

Novel Targeted Therapy for Precursor B Cell Acute Lymphoblastic Leukemia: anti-CD22 Antibody-MXD3 Antisense Oligonucleotide Conjugate.

The exponential rise in molecular and genomic data has generated a vast array of therapeutic targets. Oligonucleotide-based technologies to down regulate these molecular targets have promising therapeutic efficacy. However, there is relatively limited success in translating this into effective in vivo cancer therapeutics. The primary challenge is the lack of effective cancer cell-targeted delivery methods, particularly for a systemic disease such as leukemia. We developed a novel leukemia-targeting compound composed of a monoclonal antibody directly conjugated to an antisense oligonucleotide (ASO). Our compound uses an ASO that specifically targets the transcription factor MAX dimerization protein 3 (MXD3), which was previously identified to be critical for precursor B cell (preB) acute lymphoblastic leukemia (ALL) cell survival. The MXD3 ASO was conjugated to an anti-CD22 antibody (αCD22 Ab) that specifically targets most preB ALL. We demonstrated that the αCD22 Ab-ASO conjugate treatment showed MXD3 protein knockdown and leukemia cell apoptosis in vitro. We also demonstrated that the conjugate treatment showed cytotoxicity in normal B cells, but not in other hematopoietic cells, including hematopoietic stem cells. Furthermore, the conjugate treatment at the lowest dose tested (0.2mg/kg Ab for 6 doses - twice a week for 3 weeks) more than doubled the mouse survival time in both Reh (median survival time 20.5 vs. 42.5 days, p<0.001) and primary preB ALL (median survival time 29.3 vs. 63 days, p<0.001) xenograft models. Our conjugate that uses αCD22 Ab to target the novel molecule MXD3, which is highly expressed in preB ALL cells, appears to be a promising novel therapeutic approach.

Alveolar rhabdomyosarcoma after treatment of osteosarcoma.

Secondary rhabdomyosarcoma (RMS) after treatment of osteosarcoma (OS) is rare. Reported here is the case of a metachronous RMS in the nasal cavity, developing 12 years after successful treatment of non-metastatic OS. The patient was diagnosed as having OS of the femur at 2 years of age. Chemotherapy for OS included doxorubicin (cumulative dose, 488 mg/m(2) ). No radiotherapy was given. There was no family history suggestive of cancer predisposition syndrome. At 14 years of age, alveolar RMS was diagnosed on histopathology. PAX3-FKHR fusion transcripts were detected on reverse transcription-polymerase chain reaction. Germline TP53 mutation was not seen on standard DNA sequencing. The occurrence of secondary sarcomas, in the Children's Cancer Survivor study conducted in North America, has been associated with high cumulative doses of anthracyclines, which may also have played a role in the development of RMS in the present case. In the future, novel molecular technologies might uncover genetic cancer predisposition in patients with metachronous cancers.

Disseminated BCG infection mimicking metastatic nasopharyngeal carcinoma in an immunodeficient child with a novel hypomorphic NEMO mutation.

BACKGROUND: Nuclear factor-κB essential modulator (NEMO) deficiency is a developmental and immunological disorder. The genetic and phenotypic correlation has been described. METHODS: We report a unique clinical presentation and the identification of a novel missense mutation in the NEMO gene in a 3-year-old boy with bacillus Calmette-Guerin (BCG) infection. RESULTS: The patient presented with fever, cervical lymphadenopathy, and abnormal anti-Epstein-Barr virus (EBV) antibody titers, suggestive of EBV-related diseases including chronic active EBV infection, X-linked lymphoproliferative syndrome, or nasopharyngeal carcinoma. Although the biopsy specimen from a nasopharyngeal lesion was initially diagnosed as squamous cell carcinoma, this was changed to disseminated BCG infection involving the nasopharynx, multiple systemic lymph nodes, and brain. A novel mutation (designated D311E) in the NEMO gene, located in the NEMO ubiquitin-binding (NUB) domain, was identified as the underlying cause of the immunodeficiency. Impaired immune responses which are characteristic of patients with NEMO deficiency were demonstrated. The patient underwent successful unrelated bone marrow transplantation at 4.9 years of age. CONCLUSION: This study suggests the importance of the NUB domain in host defense against mycobacteria. The unique presenting features in our patient indicate that a hypomorphic NEMO mutation can be associated with atypical pathological findings of the epithelial tissues in patients with BCG infection.

Reactive tonsillar enlargement with strong 18F-FDG uptake after chemotherapy for tonsillar diffuse large B-cell lymphoma.

We describe a 14-year-old boy who exhibited left palatine tonsillar enlargement after 6 cycles of aggressive chemotherapy for diffuse large B-cell lymphoma of the right palatine tonsil. The cervical computed tomography scan at 4 months after completion of chemotherapy revealed enlargement of the left palatine tonsil in addition to the thymus without any clinical symptoms. The F-fluorodeoxyglucose positron emission tomography indicated focal areas of strong F-fluorodeoxyglucose uptake in the left palatine tonsil. Histologic examination confirmed tonsillar hyperplasia with no evidence of recurrence. Reactive tonsillar hyperplasia after chemotherapy is rarely reported.

Human T-cell leukemia virus type 1 Tax induces an aberrant clustering of the tumor suppressor Scribble through the PDZ domain-binding motif dependent and independent interaction.

Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia. HTLV-1 Tax1 transforming protein interacts with several PDZ domain-containing proteins, and the interaction is associated with the transforming activities of Tax1 as well as persistent HTLV-1 infection. In this study, we show that Tax1 interacts with the tumor suppressor Scribble containing PDZ domains. Unlike other Tax1-interacting PDZ domain proteins, the PDZ domain-binding motif (PBM) of Tax1 was not required for the interaction with transiently expressed Scribble in 293T cells, but it was essential for the interaction with endogenous Scribble. Endogenous Scribble in 293T cells was primarily localized at the plasma membrane and colocalized with Tax1 but not Tax1C lacking PBM, whereas transiently expressed Scribble was localized in the cytoplasm and colocalized with Tax1C as well as Tax1, thus suggesting that Tax1 is recruited to the site of endogenous Scribble, such as the plasma membrane, in a PBM-dependent manner, and thereafter it interacts with Scribble in a PBM-independent and PBM-dependent manner. Endogenous Scribble was diffusely localized at the plasma membrane of HTLV-1-uninfected T-cell lines, whereas it colocalized with Tax1 as small and large aggregate at the plasma membranes. These results suggest that Tax1 through two binding sites induce aberrant clustering of Scribble, thereby altering the functions in HTLV-1-infected cells, which may thus play a role in persistent HTLV-1 infection and the pathogenesis.

Knockdown of synapse-associated protein Dlg1 reduces syncytium formation induced by human T-cell leukemia virus type 1.

Human T-cell leukemia virus type 1 (HTLV-1) spreads through cell-to-cell contact by forming a virological synapse. Based on the finding that HTLV-1 envelope glycoprotein (Env) binds to a PDZ domain containing scaffold protein Dlg1, whose function has been implicated in the organization of neuronal and immunological synapses, we examined the role of Dlg1 in the cell-cell infection by HTLV-1. The coculture of an HTLV-1-infected T-cell line MT-2 with an uninfected MOLT-4 induced syncytium, a marker of cell-cell HTLV-1 infection, but an RNA interference-mediated knockdown of Dlg1 in both cells cooperatively reduced the syncytium formation. In HTLV-1-uninfected 293T cells, Dlg1 induced the clustering of GLUT1, a cellular receptor for HTLV-1, but such clustering was abrogated by a deletion of the PDZ domain binding motif of GLUT1 (GLUT1DeltaC). GLUT1 expression in MDBK cells induced HTLV-1-mediated syncytium formation, and the activity was much greater than that of GLUT1DeltaC. These results suggest that Dlg1, through the interaction with GLUT1 as well as Env, plays a positive role in the syncytium formation induced by HTLV-1.

Cooperation of NF-kappaB2/p100 activation and the PDZ domain binding motif signal in human T-cell leukemia virus type 1 (HTLV-1) Tax1 but not HTLV-2 Tax2 is crucial for interleukin-2-independent growth transformation of a T-cell line.

Human T-cell leukemia virus type 1 (HTLV-1) but not HTLV-2 is associated with adult T-cell leukemia, and the distinct pathogenicity of these two closely related viruses is thought to stem from the distinct biological functions of the respective transforming proteins, HTLV-1 Tax1 and HTLV-2 Tax2. In this study, we demonstrate that Tax1 but not Tax2 interacts with NF-kappaB2/p100 and activates it by inducing the cleavage of p100 into the active transcription factor p52. Using RNA interference methods, we further show that NF-kappaB2/p100 is required for the transformation induced by Tax1, as determined by the ability to convert a T-cell line (CTLL-2) from interleukin-2 (IL-2)-dependent to -independent growth. While Tax2 shows a reduced transforming activity relative to Tax1, Tax2 fused with a PDZ domain binding motif (PBM) present only in Tax1 shows transforming activity equivalent to that of Tax1 in CTLL-2 cells expressing an inducer of p52 processing. These results reveal that the activation of NF-kappaB2/p100 plays a crucial role in the Tax1-mediated transformation of T cells and that NF-kappaB2/p100 activation and PBM function are both responsible for the augmented transforming activity of Tax1 relative to Tax2, thus suggesting that these Tax1-specific functions play crucial roles in HTLV-1 leukemogenesis.

Inactivation of tumor suppressor Dlg1 augments transformation of a T-cell line induced by human T-cell leukemia virus type 1 Tax protein.

BACKGROUND: The interaction of human T-cell leukemia virus type 1 (HTLV-1) Tax1 protein with the tumor suppressor Dlg1 is correlated with cellular transformation. RESULTS: Here, we show that Dlg1 knockdown by RNA interference increases the ability of Tax1 to transform a mouse T-cell line (CTLL-2), as measured interleukin (IL)-2-independent growth. A Tax1 mutant defective for the Dlg1 interaction showed reduced transformation of CTLL-2 compared to wild type Tax1, but the transformation was minimally affected by Dlg1 reduction. The few Tax1DeltaC-transduced CTLL-2 cells that became transformed expressed less Dlg1 than parental cells, suggesting that Dlg1-low cells were selectively transformed by Tax1DeltaC. Moreover, all human T-cell lines immortalized by HTLV-1, including the recombinant HTLV-1-containing Tax1DeltaC, expressed less Dlg1 than control T-cell lines. CONCLUSION: These results suggest that inactivation of Dlg1 augments Tax1-mediated transformation of CTLL-2, and PDZ protein(s) other than Dlg1 are critically involved in the transformation.