Hidemasa Matsuo

Genetic regulation of the RUNX transcription factor family has antitumor effects

Runt-related transcription factor 1 (RUNX1) is generally considered to function as a tumor suppressor in the development of leukemia, but a growing body of evidence suggests that it has pro-oncogenic properties in acute myeloid leukemia (AML). Here we have demonstrated that the antileukemic effect mediated by RUNX1 depletion is highly dependent on a functional p53-mediated cell death pathway. Increased expression of other RUNX family members, including RUNX2 and RUNX3, compensated for the antitumor effect elicited by RUNX1 silencing, and simultaneous attenuation of all RUNX family members as a cluster led to a much stronger antitumor effect relative to suppression of individual RUNX members. Switching off the RUNX cluster using alkylating agent–conjugated pyrrole-imidazole (PI) polyamides, which were designed to specifically bind to consensus RUNX-binding sequences, was highly effective against AML cells and against several poor-prognosis solid tumors in a xenograft mouse model of AML without notable adverse events. Taken together, these results identify a crucial role for the RUNX cluster in the maintenance and progression of cancer cells and suggest that modulation of the RUNX cluster using the PI polyamide gene-switch technology is a potential strategy to control malignancies.

EVI1 overexpression is a poor prognostic factor in pediatric patients with mixed lineage leukemia-AF9 rearranged acute myeloid leukemia

The ecotropic viral integration site-1 gene (EVI1) encodes a zinc finger protein that functions as a transcriptional regulator of hematopoietic stem cell self-renewal and long-term multilineage repopulating activity.1,2 The mixed lineage leukemia gene (MLL) rearrangements [i.e. t(11q23)] occur at high frequency in pediatric acute myeloid leukemia (AML) patients with EVI1 overexpression,3 and EVI1 is a transcriptional target of MLL oncoproteins.4 EVI1 overexpression has been reported in up to 10% of patients with AML and is associated with an adverse prognosis. However, the prognostic value of EVI1 overexpression has been studied mostly in adult AML.5–9 Only two studies have examined EVI1 overexpression in pediatric AML, but a detailed analysis according to the type of leukemia was not performed because of the small sample size.3,10 Recent data from an international consortium, including those from our group, suggest that pediatric MLL-rearranged AML can be divided into certain risk groups on the basis of different translocation partners.11 However, clinical outcome data leading to risk stratification of the MLL-rearranged subgroups are still scarce and further investigation is necessary to identify new prognostic factors. Here, we retrospectively examined EVI1 expression levels and clinical outcomes of pediatric MLL-rearranged AML patients treated in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 study. After excluding patients with acute promyelocytic leukemia, Down syndrome, secondary AML, myeloid/natural killer cell leukemia and myeloid sarcoma, 485 AML patients were enrolled in the AML-05 study. Overall, 42 patients were excluded, mainly because of misdiagnosis. Details of the treatment schedules and risk stratification were described in previous publication.12 This study was conducted in accordance with the principles set down in the Declaration of Helsinki and was approved by the Ethics Committees of all participating institutions. All patients, or the patients’ parents/guardians, provided written informed consent. RNA obtained from diagnostic bone marrow samples was used to analyze the expression of EVI1 using a previously established EVI1 quantitative real-time polymerase chain reaction assay that covers the various EVI1 splice variants.7 Event-free survival (EFS) was defined as the time from the diagnosis of AML to the last follow up or the first event (failure to achieve remission, relapse, secondary malignancy, or any cause of death). In this study, most of the events were relapses (n=23) and the rest were deaths with sepsis (n=1) and acute respiratory distress syndrome (n=1). Overall survival (OS) was defined as the time from the diagnosis of AML to any cause of death. All tests were two-tailed and P<0.05 was considered statistically significant. Among 443 eligible AML patients, 69 were diagnosed as MLL-rearranged AML and diagnostic samples from 50 patients were analyzed for EVI1 mRNA expression. No significant differences in the characteristics and clinical outcomes were observed between these 50 patients and the 19 patients who did not have EVI1 data [EFS (P=0.20), OS (P=0.45)]. EVI1 expression levels were dichotomized based on a cut off of 0.1 relative to SKOV3, an ovarian carcinoma cell line overexpressing EVI1: values higher than 0.1 were defined as EVI1+ and those lower than 0.1 or undetectable were defined as EVI1−, as described in a previous study.7 EVI1+ was present in 18 patients (36%). EVI1 expression levels in different MLL translocation partners relative to that in SKOV3 cells are shown in Online Supplementary Figure S1. The clinical features of EVI1+ and EVI1− patients are summarized in Table 1. EVI1+ patients were significantly older (P=0.03) and had a higher WBC count (P=0.01) at the time of diagnosis than EVI1− patients. Most of the MLL-rearranged AML cases were classified as FAB-M5 or FAB-M4. Specifically, most EVI1− patients (84%) presented with FAB-M5 morphology, which was less frequent in EVI1+ patients (22%), consistent with the findings of a previous study.8 EVI1+ was not correlated with sex or MLL translocation partners. The frequency of FLT3-ITD was significantly higher in EVI1+ patients (P=0.04). We also analyzed CEBPA and NPM1 mutations, which are established favorable prognostic factors; however, none of the patients harbored these mutations, except for one EVI1− patient harboring double CEBPA mutations. Table 1. Characteristics of patients categorized according to EVI1 expression status. Next, clinical outcomes were compared between EVI1+ patients and EVI1− patients (Figure 1). In the MLL-rearranged AML cohort (n=50), EVI1+ patients had a significantly worse EFS than EVI1− patients (P<0.0001) (Figure 1A). However, OS did not differ significantly between the two groups (P=0.054) (Figure 1B). Among several types of MLL-rearrangements, MLL-AF9 was the most common translocation (n=29, 58%) (Table 1). Therefore, clinical outcomes in the cohort of MLL-AF9 positive patients were compared between EVI1+ patients (n=11) and EVI1−patients (n=18). The results showed significant differences in EFS (P<0.0001) and OS (P=0.0008) (Figure 1C and D). By contrast, no differences in EFS (P=0.36) or OS (P=0.57) were observed among patients with MLL-rearranged AML after excluding MLL-AF9 positive patients (Figure 1E and F). The clinical outcomes associated with each type of MLL-rearrangement could not be analyzed because of the small sample size. Multivariate Cox regression analysis, including FLT3-ITD, WBC count, and age identified EVI1+ as the only prognostic factor predicting poor EFS in the total cohort of MLL-rearranged AML (hazard ratio (HR), 4.94; P<0.01) and in the MLL-AF9 positive cohort (HR, 33.81; P<0.01), but not OS (Online Supplementary Table S1). Figure 1. Kaplan-Meier survival curves of event-free survival (EFS) and overall survival (OS) from the time of diagnosis according to EVI1 expression status. (A) Kaplan-Meier estimates of EFS in the cohort of MLL-rearranged AML in EVI1+ and EVI1− patients. ... These results suggest that EVI1 overexpression is an independent adverse prognostic factor because of its association with reduced remission duration in pediatric patients with MLL-rearranged AML, especially in patients harboring MLL-AF9. A recent large study identified several novel prognostic MLL-rearranged subgroups, including a favorable-risk MLL-AF1q positive subgroup and a poor-risk MLL-AF6 positive subgroup.11 However, MLL-AF9 positive patients are categorized as an intermediate risk group, and this subgroup may be dichotomized as a favorable and poor-risk subgroup based on EVI1 expression levels. Pretreatment screening for EVI1 expression should be considered in patients with MLL-rearranged AML to enable better risk assessment and alternative consolidation therapies to be considered. Our results need to be confirmed in larger studies because of the limited case numbers. From a biological viewpoint, the ‘evil’-like adverse effects of EVI1 in patients with MLL-AF9-positive AML were partially elucidated in a recent study in which EVI1 positive cells harboring MLL-AF9 showed distinct morphological, molecular, and mechanistic differences from EVI1 negative cells.13 Moreover, EVI1 overexpression has been linked to CD52 overexpression, which could be a therapeutic target for monoclonal antibody treatment.14 Further investigation is required to identify novel prognostic factors in the various subgroups of MLL-rearranged AML and to develop therapeutic strategies effective for patients with EVI1 overexpression.

Prognostic implications of CEBPA mutations in pediatric acute myeloid leukemia: a report from the Japanese Pediatric Leukemia/Lymphoma Study Group

CCAAT/enhancer-binding protein alpha (CEBPA) mutations are a favorable prognostic factor in adult acute myeloid leukemia (AML) patients; however, few studies have examined their significance in pediatric AML patients. Here we examined the CEBPA mutation status and clinical outcomes of pediatric AML patients treated in the AML-05 study. We found that 47 (14.9%) of the 315 evaluable patients harbored mutations in CEBPA; 26 cases (8.3%) harbored a single mutation (CEBPA-single) and 21 (6.7%) harbored double or triple mutations (CEBPA-double). After excluding core-binding factor-AML cases, patients harboring CEBPA mutations showed better overall survival (OS; P=0.048), but not event-free survival (EFS; P=0.051), than wild-type patients. Multivariate analysis identified CEBPA-single and CEBPA-double as independent favorable prognostic factors for EFS in the total cohort (hazard ratio (HR): 0.47 and 0.33; P=0.02 and 0.01, respectively). CEBPA-double was also an independent favorable prognostic factor for OS (HR: 0.30; P=0.04). CEBPA-double remained an independent favorable factor for EFS (HR: 0.28; P=0.04) in the normal karyotype cohort. These results suggest that CEBPA mutations, particularly CEBPA-double, are an independent favorable prognostic factor in pediatric AML patients, which will have important implications for risk-stratified therapy.

Enhancement of neutrophil autophagy by an IVIG preparation against multidrug-resistant bacteria as well as drug-sensitive strains

Autophagy occurs in human neutrophils after the phagocytosis of multidrug‐resistant bacteria and drug‐sensitive strains, including Escherichia coli and Pseudomonas aeruginosa. The present study detected autophagy by immunoblot analysis of LC3B conversion, by confocal scanning microscopic examination of LC3B aggregate formation and by transmission electron microscopic examination of bacteria‐containing autophagosomes. Patients with severe bacterial infections are often treated with IVIG alongside antimicrobial agents. Here, we showed that IVIG induced neutrophil‐mediated phagocytosis of multidrug‐resistant strains. Compared with untreated neutrophils, neutrophils exposed to IVIG showed increased levels of bacterial cell killing, phagocytosis, O2− release, MPO release, and NET formation. IVIG also increased autophagy in these cells. Inhibiting the late phase of autophagy (fusion of lysosomes with autophagosomes) with bafilomycin A1‐reduced, neutrophil‐mediated bactericidal activity. These findings indicate that autophagy plays a critical role in the bactericidal activity mediated by human neutrophils. Furthermore, the autophagosomes within the neutrophils contained bacteria only and their organelles only, or both bacteria and their organelles, a previously undocumented observation. Taken together, these results suggest that the contents of neutrophil autophagosomes may be derived from specific autophagic systems, which provide the neutrophil with an advantage. Thus, IVIG promotes the neutrophil‐mediated killing of multidrug‐resistant bacteria as well as drug‐sensitive strains.

The subtype-specific features of EVI1 and PRDM16 in acute myeloid leukemia

We read with interest the response “The closely related rare and severe acute myeloid leukemias carrying EVI1 or PRDM16 mutations share singular biological features” by Eveillard et al .[1][1] to our recent publication.[2][2] EVI1 and PRDM16 belong to the Prdm family, which is characterized by

Acute myeloid leukaemia with myelodysplastic features in children: a report of Japanese Paediatric Leukaemia/Lymphoma Study Group.

The clinical characteristics and prognostic relevance of acute myeloid leukaemia (AML) with myelodysplastic features remains to be clarified in children. We prospectively examined 443 newly diagnosed patients in a multicentre clinical trial for paediatric de novo AML, and found ‘AML with myelodysplasia‐related changes’ (AML‐MRC) according to the 2008 World Health Organization classification in 93 (21·0%), in whom 59 were diagnosed from myelodysplasia‐related cytogenetics alone, 28 from multilineage dysplasia alone and six from a combination of both. Compared with 111 patients with ‘AML, not otherwise specified’ (AML‐NOS), patients with ‘AML‐MRC’ presented at a younger age, with a lower white blood cell count, higher incidence of 20–30% bone marrow blasts, unfavourable cytogenetics and a lower frequency of Fms‐like tyrosine kinase 3 internal tandem duplication (FLT3‐ITD), NPM1 and CEBPA mutations. Complete remission rate and 3‐year probability of event‐free survival were significantly worse in ‘AML‐MRC’ patients (67·7 vs. 85·6%, P < 0·01, 37·1% vs. 53·8%, P = 0·02, respectively), but 3‐year overall survival and relapse‐free survival were comparable with ‘AML‐NOS’ patients. By multivariate analysis, FLT3‐ITD was solely associated with worse overall survival. These results support the distinctive features of the category ‘AML‐MRC’ even in children.

CXCR4 Overexpression is a Poor Prognostic Factor in Pediatric Acute Myeloid Leukemia With Low Risk: A Report From the Japanese Pediatric Leukemia/Lymphoma Study Group

Overexpression of CXC chemokine receptor 4 (CXCR4+) is a poor prognostic factor in adult acute myeloid leukemia (AML); however, its prognostic significance in pediatric AML is unclear.

Multiplex fusion gene testing in pediatric acute myeloid leukemia

Gene abnormalities, particularly chromosome rearrangements generating gene fusion, are associated with clinical characteristics and prognosis in pediatric acute myeloid leukemia (AML). Karyotyping is generally performed to enable risk stratification, but the results are not always consistent with those of reverse transcription–polymerase chain reaction (RT‐PCR), and more accurate and rapid methods are required.

Autonomous feedback loop of RUNX1-p53-CBFB in acute myeloid leukemia cells

Although runt-related transcription factor 1 (RUNX1) and its associating core binding factor-β (CBFB) play pivotal roles in leukemogenesis, and inhibition of RUNX1 has now been widely recognized as a novel strategy for anti-leukemic therapies, it has been elusive how leukemic cells could acquire the serious resistance against RUNX1-inhibition therapies and also whether CBFB could participate in this process. Here, we show evidence that p53 (TP53) and CBFB are sequentially up-regulated in response to RUNX1 depletion, and their mutual interaction causes the physiological resistance against chemotherapy for acute myeloid leukemia (AML) cells. Mechanistically, p53 induced by RUNX1 gene silencing directly binds to CBFB promoter and stimulates its transcription as well as its translation, which in turn acts as a platform for the stabilization of RUNX1, thereby creating a compensative RUNX1-p53-CBFB feedback loop. Indeed, AML cells derived from relapsed cases exhibited higher CBFB expression levels compared to those from primary AML cells at diagnosis, and these CBFB expressions were positively correlated to those of p53. Our present results underscore the importance of RUNX1-p53-CBFB regulatory loop in the development and/or maintenance of AML cells, which could be targeted at any sides of this triangle in strategizing anti-leukemia therapies.

Monitoring of fusion gene transcripts to predict relapse in pediatric acute myeloid leukemia

In acute myeloid leukemia (AML), accurate detection of minimal residual disease (MRD) enables better risk‐stratified therapy. There are few studies, however, on the monitoring of multiple fusion transcripts and evaluation of their accuracy as indicators of MRD at multiple time points.