Kentaro Kihira

NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity

Widely used as anticancer and immunosuppressive agents, thiopurines have narrow therapeutic indices owing to frequent toxicities, partly explained by TPMT genetic polymorphisms. Recent studies identified germline NUDT15 variation as another critical determinant of thiopurine intolerance, but the underlying molecular mechanisms and the clinical implications of this pharmacogenetic association remain unknown. In 270 children enrolled in clinical trials for acute lymphoblastic leukemia in Guatemala, Singapore and Japan, we identified four NUDT15 coding variants (p.Arg139Cys, p.Arg139His, p.Val18Ile and p.Val18_Val19insGlyVal) that resulted in 74.4–100% loss of nucleotide diphosphatase activity. Loss-of-function NUDT15 diplotypes were consistently associated with thiopurine intolerance across the three cohorts (P = 0.021, 2.1 × 10−5 and 0.0054, respectively; meta-analysis P = 4.45 × 10−8, allelic effect size = −11.5). Mechanistically, NUDT15 inactivated thiopurine metabolites and decreased thiopurine cytotoxicity in vitro, and patients with defective NUDT15 alleles showed excessive levels of thiopurine active metabolites and toxicity. Taken together, these results indicate that a comprehensive pharmacogenetic model integrating NUDT15 variants may inform personalized thiopurine therapy.

Heterogeneity of neuroblastoma cell lines in insulin-like growth factor 1 receptor/Akt pathway-mediated cell proliferative responses.

Insulin‐like growth factor 1 receptor (IGF‐1R) is critical for cancer cell proliferation; however, recent clinical anti‐IGF‐1R trials did not show clear clinical benefit in cancer therapy. We hypothesized that IGF‐1R signaling‐mediated proliferative response is heterogeneous in neuroblastoma (NB) cells, and analyzed the cell growth of 31 NB cell lines cultured in three different media, including Hybridoma‐SFM medium (with insulin) and RPMI1640 with/without 10% FBS. Three growth patterns were found. In response to IGF and insulin, cell proliferation and Akt phosphorylation were upregulated in 13 cell lines, and suppressed by MK2206 (Akt inhibitor) and picropodophyllin (IGF‐1R inhibitor). Interestingly, 3 of these 13 cell lines showed Akt self‐phosphorylation and cell proliferation in RPMI1640; their proliferation was downregulated by anti‐IGF‐1 or anti‐IGF‐2 neutralizing antibody, suggesting the existence of an autocrine loop in the IGF‐1R/Akt pathway. Eighteen NB cell lines did not proliferate in RPMI1640, even though Akt phosphorylation was upregulated by IGF and insulin. Based on the heterogeneous response of the IGF‐1R/Akt pathway, the 31 NB cell lines could be classified into group 1 (autocrine IGF‐mediated), group 2 (exogenous IGF‐mediated) and group 3 (partially exogenous IGF‐mediated) NB cell lines. In addition, group 3 NB cell lines were different from group 1 and 2, in terms of serum starvation‐induced caspase 3 cleavage and picropodophyllin‐induced G2/M arrest. These results indicate that the response of the IGF‐1R/Akt pathway is an important determinant of the sensitivity to IGF‐1R antagonists in NB. To our knowledge, this is the first report describing heterogeneity in the IGF‐1R/Akt‐mediated proliferation of NB cells.

Fatal Pulmonary Lymphangiectasia Manifesting After Repeated Surgeries for Intractable Chylopericardium and Chylothorax in a 20-Month-Old Girl

A 20-month-old girl was admitted to our hospital because of progressive dyspnea on exertion and a 10-month history of failure to thrive. She had bilateral hearing impairment. The family history was unremarkable. The patient had a hemangioma on the temporal portion of the head and no edema on the legs or face. A chest radiograph (Figure 1A) showed enlargement of the cardiac silhouette with pulmonary congestion. Cardiac ultrasound and computed tomographic scanning of the chest (Figure 1B) revealed copious pericardial effusion with no structural heart diseases. Diagnostic pericardiocentesis produced 150 mL of milky fluid (Figure 1C), which confirmed a diagnosis of chylopericardium because of positive Sudan III staining, a high triglyceride concentration, and a predominance of lymphocytes (>95%). Despite a medium-chain triglyceride-enriched diet and total parenteral nutrition for more than 1 month after pericardiocentesis, the massive pericardial effusion persisted, which prompted surgical management including the clipping of the thoracic duct and creation of a left pericardial …

The effects of inherited NUDT15 polymorphisms on thiopurine active metabolites in Japanese children with acute lymphoblastic leukemia

Thiopurines [e.g. mercaptopurine (MP)] are widely used as chemotherapeutic agents in the treatment of pediatric acute lymphoblastic leukemia with dose-limiting hematopoietic toxicity. Recently, germline variants in NUDT15 have been identified as a major genetic cause for MP-related bone marrow suppression, and there is increasing interest in the clinical implementation of NUDT15 genotype-guided MP dose individualization. Therefore, we sought to evaluate the effects of NUDT15 on thiopurine metabolism and identify pharmacologic markers to inform NUDT15 genotype-guided MP dosing. In 55 Japanese children with acute lymphoblastic leukemia, we simultaneously measured both thioguanine nucleotides (TGN) in red blood cells and DNA-incorporated thioguanine (DNA-TG) in white blood cells. TGN levels were significantly lower in patients with NUDT15 deficiency, likely because of toxicity-related MP dose reduction. In contrast, when exposed to the same dose of MP, DNA-TG accumulated more efficiently in vivo with increasing number of risk alleles in NUDT15 (P=4.0×10−9). Cytosolic TGN and nuclear DNA-TG were correlated positively with each other across genotype groups (P=6.5×10−4), but the ratio of DNA-TG to TGN was significantly higher in NUDT15-deficient patients (P=3.6×10−9), consistent with excessive MP activation. In conclusion, our results suggest that DNA-TG is a more relevant MP metabolite than TGN to inform NUDT15 genotype-guided dose adjustments.

Mesenchymal Stromal Cell Secretome Up- Regulates 47 kDa CXCR4 Expression, and Induce Invasiveness in Neuroblastoma Cell Lines

Neuroblastoma accounts for 15% of childhood cancer deaths and presents with metastatic disease of the bone and the bone marrow at diagnosis in 70% of the cases. Previous studies have shown that the Mesenchymal Stromal Cell (MSC) secretome, triggers metastases in several cancer types such as breast and prostate cancer, but the specific role of the MSC factors in neuroblastoma metastasis is unclear. To better understand the effect of MSC secretome on chemokine receptors in neuroblastoma, and its role in metastasis, we studied a panel of 20 neuroblastoma cell lines, and compared their invasive potential towards MSC-conditioned-RPMI (mRPMI) and their cytokine receptor expression profiles. Western blot analysis revealed the expression of multiple CXCR4 isoforms in neuroblastoma cells. Among the five major isoforms, the expression of the 47 kDa isoform showed significant correlation with high invasiveness. Pretreatment with mRPMI up-regulated the expression of the 47 kDa CXCR4 isoform and also increased MMP-9 secretion, expression of integrin α3 and integrin β1, and the invasive potential of the cell; while blocking CXCR4 either with AMD 3100, a CXCR4 antagonist, or with an anti-47 kDa CXCR4 neutralizing antibody decreased the secretion of MMP-9, the expression of integrin α3 and integrin β1, and the invasive potential of the cell. Pretreatment with mRPMI also protected the 47 kDa CXCR4 isoform from ubiquitination and subsequent degradation. Our data suggest a modulatory role of the MSC secretome on the expression of the 47 kDa CXCR4 isoform and invasion potential of the neuroblastoma cells to the bone marrow.

Erratum to: PDK1-mTOR signaling pathway inhibitors reduce cell proliferation in MK2206 resistant neuroblastoma cells

[This corrects the article DOI: 10.1186/s12935-015-0239-4.].

Disease Recurrence After Early Discontinuation of Eculizumab in a Patient With Atypical Hemolytic Uremic Syndrome With Complement C3 I1157T Mutation.

Eculizumab, terminal complement inhibitor, has become the frontline treatment for atypical hemolytic uremic syndrome (aHUS). However, the optimal treatment schedule has not yet been established. We describe here an aHUS patient with a mutation of C3 I1157T who achieved remission with eculizumab and suffered a recurrence after eculizumab discontinuation, a clinical situation that has not been previously described in patients with C3 mutation. A 9-year-old male experienced an onset of aHUS after viral gastroenteritis and was treated with hemodialysis. At 13 years of age he developed bacterial enterocolitis due to Campylobacter jejuni and experienced a recurrence of aHUS. Eculizumab was initiated on day 4 after disease onset resulting in recovering laboratory parameters. The patient received eculizumab for 5 months before its discontinuation. Second relapse induced by bacterial pharyngitis was confirmed 4 months after eculizumab discontinuation and prompt eculizumab reinitiation resulted in rapid remission. The patients carrying mutations in CFH or C3 have a high frequency of relapse and worse prognosis. More than 50% of aHUS relapses occurred during the first year after the onset. Therefore, long-term treatment with eculizumab is appropriate in patients with aHUS who have experienced a relapse or have mutations associated with poor prognosis.

Preclinical evaluation of NUDT15-guided thiopurine therapy and its effects on toxicity and antileukemic efficacy

Thiopurines (eg, 6-mercaptopurine [MP]) are highly efficacious antileukemic agents, but they are also associated with dose-limiting toxicities. Recent studies by us and others have identified inherited NUDT15 deficiency as a novel genetic cause of thiopurine toxicity, and there is a strong rationale for NUDT15-guided dose individualization to preemptively mitigate adverse effects of these drugs. Using CRISPR-Cas9 genome editing, we established a Nudt15-/- mouse model to evaluate the effectiveness of this strategy in vivo. Across MP dosages, Nudt15-/- mice experienced severe leukopenia, rapid weight loss, earlier death resulting from toxicity, and more bone marrow hypocellularity compared with wild-type mice. Nudt15-/- mice also showed excessive accumulation of a thiopurine active metabolite (ie, DNA-incorporated thioguanine nucleotides [DNA-TG]) in an MP dose-dependent fashion, as a plausible cause of increased toxicity. MP dose reduction effectively normalized systemic exposure to DNA-TG in Nudt15-/- mice and largely eliminated Nudt15 deficiency-mediated toxicity. In 95 children with acute lymphoblastic leukemia, MP dose adjustment also directly led to alteration in DNA-TG levels, the effects of which were proportional to the degree of NUDT15 deficiency. Using leukemia-bearing mice with concordant Nudt15 genotype in leukemia and host, we also confirmed that therapeutic efficacy was preserved in Nudt15-/- mice receiving a reduced MP dose compared with Nudt15+/+ counterparts exposed to a standard dose. In conclusion, we demonstrated that NUDT15 genotype-guided MP dose individualization can preemptively mitigate toxicity without compromising therapeutic efficacy.

Low Incidence of Osteonecrosis in Childhood Acute Lymphoblastic Leukemia Treated With ALL-97 and ALL-02 Study of Japan Association of Childhood Leukemia Study Group

Purpose Osteonecrosis (ON) is a serious complication of the treatment of childhood acute lymphoblastic leukemia (ALL); however, data relating to ON in Asian pediatric patients with ALL are scarce. Therefore, we performed a retrospective analysis of cohorts of Japanese patients with ALL to clarify the incidence, clinical characteristics, and risk factors of ON. Patients and Methods The incidence and characteristics of ON were determined in patients with ALL (n = 1,662) enrolled in two studies from the Japan Association of Childhood Leukemia Study (JACLS) group (n = 635 and n = 1,027 patients treated with the ALL-97 and ALL-02 protocols, respectively). Results In total, 24 of 1,662 patients suffered from ON, of which 12 of 635 and 12 of 1,027 patients were treated with the ALL-97 and the ALL-02 protocol, respectively. Of the 24 patients, 23 were older than 10 years. In multivariate analysis, age (≥ 10 years) was the sole significant risk factor for ON ( P < .001). Separate evaluation of patients ≥ 10 years of age indicated a 5-year cumulative incidence of ON of 7.2% (95% CI, 4.0% to 12.6%) and 5.9% (95% CI, 3.3% to 10.4%) in the ALL-97 and the ALL-02 protocol, respectively, which was lower than reported previously, despite an administration of dexamethasone (DEX) similar to that in comparable studies; however, concomitant administration of DEX and l-asparaginase was reduced in the JACLS protocols. Conclusion We identified a low frequency of ON in the JACLS ALL-97 and ALL-02 studies. Although the sole risk factor for ON was age (≥ 10 years), even among high-risk patients, ON incidence was significantly lower than that reported in previous studies. These results suggest that, not only the total amount of DEX, but also how DEX and l-asparaginase are administered, which affects the clearance of DEX, may be associated with ON incidence in patients with ALL.

Abstract 41: 47 kda CXCR4: A marker for highly invasive neuroblastoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Neuroblastoma; a pediatric cancer of the sympathetic nervous system, which accounts for 15% of childhood cancer deaths, presents with metastatic disease of the bone and the bone marrow at diagnosis in 70% of the cases. Although studies have shown that the CXCL12 chemokine receptor CXCR4 mediates the process of metastasis in many cancer types, its role in the bone marrow metastasis of neuroblastoma is unclear. In an effort to better understand the role of the CXCR4- CXCL12 axis in bone marrow metastasis of neuroblsatoma, we screened 20 neuroblastoma cell lines for their invasive potential, and expression of CXCR4, and sought to identify a correlation between tumor invasiveness and CXCR4 expression. Western blot analyses of cell lysates revealed the presence of multiple CXCR4 isoforms with 3 prominent species at about 35 kda, 47 kda and 62 kda. Cell lines expressing the 47 kda isoform showed higher secretion of MMP9 in gelatin zymography and higher migration and invasion towards mesenchymal stem cells (MSCs) and MSC-conditioned-RPMI, in transwell migration/invasion studies. Far western blot analysis with immuno-precipitated CXCR4, showed that the 47kda protein bound with CXCL12 at a higher rate. Blocking CXCR4 with AMD3100 or a neutralizing antibody against the 47 Kda isoform decreased the secretion of MMP9, the expression of integrin α3 and integrin β1 and the invasive potential of the cell. These data suggest that the 47 kda CXCR4 plays a prominent role in the bone marrow metastasis of neuroblastoma and could possibly be used as a diagnostic marker for highly invasive neuroblastoma and the development of minimal residual disease. Citation Format: Vipin Shankar, Lei Qi, Kentaro Kihira, Yoshihiro Komada, Hiroki Hori. 47 kda CXCR4: A marker for highly invasive neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 41. doi:10.1158/1538-7445.AM2014-41