Kazuhiro Maki

Role of the RUNX1-EVI1 fusion gene in leukemogenesis

RUNX1‐EVI1 is a chimeric gene generated by t(3;21)(q26;q22) observed in patients with aggressive transformation of myelodysplastic syndrome or chronic myelogenous leukemia. RUNX1‐EVI1 has oncogenic potentials through dominant‐negative effect over wild‐type RUNX1, inhibition of Jun kinase (JNK) pathway, stimulation of cell growth via AP‐1, suppression of TGF‐β‐mediated growth inhibition and repression of C/EBPα. Runx1‐EVI1 heterozygous knock‐in mice die in uteri due to central nervous system (CNS) hemorrhage and severe defects in definitive hematopoiesis as Runx1–/– mice do, indicating that RUNX1‐EVI1 dominantly suppresses functions of wild‐type RUNX1 in vivo. Acute myelogenous leukemia is induced in mice transplanted with bone marrow cells expressing RUNX1‐EVI1, and a Runx1‐EVI1 knock‐in chimera mouse developed acute megakaryoblastic leukemia. These results suggest that RUNX1‐EVI1 plays indispensable roles in leukemogenesis of t(3;21)‐positive leukemia. Major leukemogenic effect of RUNX1‐EVI1 is mainly through histone deacetyltransferase recruitment via C‐terminal binding protein. Histone deacetyltransferase could be a target in molecular therapy of RUNX1‐EVI1‐expressing leukemia. (Cancer Sci 2008; 99: 1878–1883)

Chronic idiopathic myelofibrosis expressing a novel type of TEL - PDGFRB chimaera responded to imatinib mesylate therapy

Chronic idiopathic myelofibrosis expressing a novel type of TEL - PDGFRB chimaera responded to imatinib mesylate therapy

RUNX1/EVI1, which blocks myeloid differentiation, inhibits CCAAT–enhancer binding protein α function

The RUNX1/EVI1 chimeric transcription factor produced by t(3;21) causes leukemic transformation in hematopoietic stem cell tumors, possibly through a differentiation block of malignant myeloid progenitors. A dominant negative effect over wild‐type RUNX1 has been shown to constitute one of the underlying molecular mechanisms. We introduced RUNX1/EVI1 cDNA into LG‐3 cells that differentiate along the myeloid lineage upon exposure to granulocyte colony stimulating factor, and confirmed that RUNX1/EVI1 suppressed the differentiation. To further investigate the molecular mechanisms of RUNX1/EVI1‐mediated differentiation block, we analyzed RUNX1/EVI1s effect on the functions of CCAAT–enhancer binding protein α (C/EBPα), a key transcriptional regulator that induces granulocytic differentiation. RUNX1/EVI1 was found to associate with C/EBPα. By using a reporter assay with the CEBPA promoter, we observed a dominant negative effect of RUNX1/EVI1 over C/EBPα‐mediated transcriptional activation via the carboxyl terminal‐binding protein (CtBP)‐binding site in the EVI1 portion. In a gel‐shift assay, RUNX1/EVI1 downregulated the DNA‐binding activity of C/EBPα. Therefore, recruitment of histone deacetylase via CtBP and disruption of DNA binding could be likely scenarios for the RUNX1/EVI1‐induced dominant repression on C/EBPα. Importantly, coexpression of C/EBPα restored the differentiation ability of the RUNX1/EVI1‐expressing LG‐3 cells. All of these data argue that inhibition of C/EBPα function may be causatively related to the leukemogenic potential of RUNX1/EVI1. (Cancer Sci 2007; 98: 1752–1757)

Identification of a novel fusion gene, TTL , fused to ETV6 in acute lymphoblastic leukemia with t(12;13)(p13;q14), and its implication in leukemogenesis

ETS variant gene 6 (ETV6)/translocation, ETS, leukemia (TEL)-involving chromosomal translocations are frequently observed in various hematologic neoplasms. We describe here a novel ETV6-involving translocation, t(12;13)(p13;q14), found in the case of acute lymphoblastic leukemia, in which ETV6 fused with a previously unknown gene, named Twelve-thirteen Translocation Leukemia gene (TTL), at 13q14. TTL was weakly but ubiquitously expressed in normal human tissues as detected by reverse transcribed-PCR. Three TTL splicing forms were identified, TTL-T from a human testis cDNA library, with an open-reading frame of 402u2009bp encoding 133 amino acids (aa), and TTL-B1 and -B2 from a human brain cDNA library. These proteins have no homology to known proteins. In leukemic cells from the patient, both reciprocal fusion transcripts, ETV6/TTL and TTL/ETV6, were expressed. The predominant fusion transcript, TTL/ETV6-1, encodes a predicted 530 aa fusion protein containing 89 aa of the N-terminal TTL fusing to the helix–loop–helix domain and ETS-binding domain of ETV6. Although the function of TTL is yet to be elucidated, our findings will provide another insight into the molecular pathogenesis of leukemia having ETV6-involving translocations.

Aberrant expression of MIR9 indicates poor prognosis in acute myeloid leukaemia.

microRNAs (miRNA) play vital roles in each step of normal haematopoiesis starting at the level of haematopoietic stem cell and continuing during the differentiation process of both myeloid and lymphoid lineages (Bhagavathi & Czader, 2010; Havelange & Garzon, 2010). On the other hand, dysregulated expression of miRNA results in the development of haematological malignancies including leukaemia, lymphoma and myeloma. Considering that inactivation of RUNX1 is one of the major roles in the development of acute leukaemia, we screened expression levels of several RUNX1-inhibiting miRNAs in leukaemic bone marrow samples for preliminary study. Among them, only the amount of mature MIR9, transcribed from either MIR9-1, MIR9-2 or MIR9-3 genes, was extraordinarily increased in a certain fraction of patients (data not shown). We thus conducted a further study on mature MIR9 expression by real-time polymerase chain reaction (PCR) methods with a total of 124 bone marrow samples from 101 adult acute myeloid leukaemia (AML) patients at diagnosis and 23 controls, all of whom gave written informed consent for this study. Expression of MIR9 was normalized to the mean expression of three control small nuclear RNAs (SNORD7, RNU6-2 and SNORA74A). The patient cohort consisted of 59 men and 42 women with a median age of 62 years (range; 20–85 years). Eight, 11, 35, 17, 10, 13, 6 and 1 patients were diagnosed as AML-M0, 1, 2, 3, 4, 5, 6 and 7, respectively. Thirty-eight had normal karyotype. Twelve patients were classified as core binding factor (CBF) leukaemia including eight with t (8;21) and four with inv(16)/t(16;16). All of the 17 AMLM3 (acute promyelocytic leukaemia; APL) patients carried t (15;17). Notably, MIR9 expression was hardly detected in normal bone marrow cells. In contrast, some fraction of bone marrow samples from AML patients showed significantly increased levels of MIR9. Thus, we set a cut-off value at the highest level of MIR9 observed in normal bone marrow cells, and divided the AML patients into two groups; MIR9 (+) and MIR9 ( ) groups. Nineteen patients (19%) belonged to the positive group and 82 (81%) to the negative group. Demographic and laboratory characteristics were not significantly different between these two groups. On the other hand, the distribution of French-AmericanBritish subtypes (P = 0·008) and chromosomal findings (P = 0·017) was statistically different between the two groups. No patients in the MIR9 (+) group were diagnosed as AML-M3/t(15;17) or CBF leukaemia/t(8;21) or inv(16)/t (16;16), while three of them had 11q23 abnormalities. We observed FLT3, NPM1 and CEBPA mutations at a frequency of 19%, 8% and 33% in the entire patient cohort. There was no association with aberrant expression of MIR9 and FLT3, NPM1 or CEBPA mutations (P = 0·353, P = 0·641 or P = 0·082). Although patients received various kinds of induction chemo and/or all-trans retinoic acid (ATRA) therapy depending on the subtype of leukaemia and their age, differences in treatment regimens between the MIR9 (+) and ( ) groups in the entire and non-APL cohort were not statistically significant (P = 0·173 and P = 0·699, respectively). Patients with aberrant MIR9 expression had a lower rate of complete remission (44%) compared with those without the expression (59%), but this difference was not statistically significant (P = 0·246). We then compared overall survival (OS) and relapse-free survival (RFS) between the MIR9 (+) and MIR9 ( ) groups by Kaplan–Meier method and LogRank test. The median observation period was 513 d. The MIR9 (+) group was associated with significantly inferior OS (P = 0·005: Fig 1A) and RFS (P < 0·001: Fig 1B) compared with the MIR9 ( ) group. Even when excluding those APL patients who showed a better prognosis with ATRA therapy, the presence of MIR9 expression was significantly associated with unfavourable OS (P = 0·028:Fig 1C) and RFS (P = 0·009: Fig 1D). We performed univariate Cox regression analyses to evaluate the status of aberrant MIR9 expression as well as known prognostic factors as predictors of OS and RFS. As a result, age over 65 years (hazard ratio [HR]=1·992, P=0·035), white blood cell count greater than 50 9 10/l (HR = 2·434, P = 0·011), lactate dehydrogenase (LDH) greater than 600 iu/l (HR = 2·606, P = 0·004), and the presence of aberrant MIR9 expression (HR = 2·602, P = 0·006) were found to be significant predictors for poor OS (data not shown). On the other hand, the presence of aberrant MIR9 expression was the sole predictor of increased risk of relapse in the same group (HR = 3·560, P = 0·001). When the analyses were restricted to non-APL patients, LDH greater than 600 iu/l (HR = 2·088, P = 0·034), the presence of FLT3-internal tandem duplication mutation (HR = 3·397, P = 0·003) and MIR9 expression (HR = 2·153, P = 0·032)

In Situ Follicular Lymphoma Associated With Progressive Transformation of Germinal Centers

The authors report here a case of in situ follicular lymphoma (FL) associated with progressive transformation of the germinal center (PTGC). A 39-year-old Japanese male developed a mass in the right cervical region. Biopsy of the enlarged lymph node led to a diagnosis of PTGC. Then, 5 years later, the lymphadenopathy recurred. The second biopsy specimens contained numerous germinal centers, including PTGC. Although most follicles were cytologically reactive, a few GCs appeared to be somewhat monotonous, composed predominantly of centrocytes and lacking mitotic figures and tangible body macrophages. Immunohistochemical studies demonstrated that these atypical GCs were CD10+, CD20+, and bcl-2+, with λ-light-chain restriction. A previous report emphasized the differential diagnostic problem between PTGC and the floral variant of FL. However, the present case indicated that in situ FL should be added to the list of differential diagnoses for PTGC.

Leukemia-related transcription factor TEL/ETV6 expands erythroid precursors and stimulates hemoglobin synthesis

TEL/ETV6 located at chromosome 12p13 encodes a member of the E26 transformation‐specific family of transcription factors. TEL is known to be rearranged in a variety of leukemias and solid tumors resulting in the formation of oncogenic chimeric protein. Tel is essential for maintaining hematopoietic stem cells in the bone marrow. To understand the role of TEL in erythropoiesis, we generated transgenic mice expressing human TEL under the control of Gata1 promoter that is activated during the course of the erythroid‐lineage differentiation (GATA1‐TEL transgenic mice). Although GATA1‐TEL transgenic mice appeared healthy up to 18 months of age, the level of hemoglobin was higher in transgenic mice compared to non‐transgenic littermates. In addition, CD71+/TER119+ and c‐kit+/CD41+ populations proliferated with a higher frequency in transgenic mice when bone marrow cells were cultured in the presence of erythropoietin and thrombopoietin, respectively. In transgenic mice, enhanced expression of Alas‐e and β‐major globin genes was observed in erythroid‐committed cells. When embryonic stem cells expressing human TEL under the same Gata1 promoter were differentiated into hematopoietic cells, immature erythroid precursor increased better compared to controls as judged from the numbers of burst‐forming unit of erythrocytes. Our findings suggest some roles of TEL in expanding erythroid precursors and accumulating hemoglobin. (Cancer Sci 2009; 100: 689–697)

Development of megakaryoblastic leukaemia in Runx1-evi1 knock-in chimaeric mouse

(Figure 1c). The lower rates of relapse in allogeneic transplantation compared with autologous bone marrow transplantation, the striking clinical benefit of donor-lymphocyte infusions as well as the finding that human T cells can destroy chemotherapyresistant cell lines from chronic myeloid leukemia and multiple myeloma, have prompted development of immunotherapeutic strategies against hematological cancers. Among these approaches, active specific immunization or vaccination is emerging as a valuable tool to boost the adaptive immune system against malignant cells. In this regard, the identification of leukemia-associated antigens is crucial. However, very few antigens are characterized in a conceptual framework in which the biology, microenvironment, and conventional disease management have been taken into consideration. Myeloid cell factor-1 (Mcl-1) is a death-inhibiting member of the Bcl-2 family that is expressed in early monocyte differentiation. Elevated levels of Mcl-1 have been reported for a number of solid and hematopoitic cancers, for example, B-cell chronic lymphocytic leukemia (B-CLL) and in AML and ALL upon relapse. In B-CLL patients, higher levels of Mcl-1 are strongly correlated with failure to achieve complete remission after single-agent therapy. In multiple myeloma, Mcl-1 plays an important role in the survival of malignant cells. We have reported that spontanous Mcl-1-specific T-cell responses are frequent in cancer patients and that these T-cells are highly cytotoxic against cancer cells. Hence, Mcl-1 appears to be a very attactrive target for anticancer immunotherapy both in hematopoetic and solid cancers.

Overexpression of MIR9 indicates poor prognosis in acute lymphoblastic leukemia

Abstract Aberrant expression of MIR9 predicts a poor prognosis in acute myelogenous leukemia. To evaluate its clinical significance in acute lymphoblastic leukemia, we analyzed expression levels of MIR9 in bone marrow samples from patients with acute lymphoblastic leukemia and compared them to those in normal bone marrow cells. Approximately 20% of them showed higher expression compared with controls. There was a tendency that patients who showed overexpression of MIR9 underwent worse clinical courses, but without statistical significance. However, when the analyses were restricted to patients who did not receive a stem cell transplant, overexpression of MIR9 was significantly associated with worse overall survival. Interestingly, exaggerated MIR9 expression and higher white blood cell count at presentation were independent unfavorable prognostic factors in all patients for overall survival by multivariate analysis. The presence of higher MIR9 expression could be a useful indicator for treatment stratification.

Acute myeloid leukemia with t(7;21)(q11.2;q22) expresses a novel, reversed-sequence RUNX1―DTX2 chimera

The RUNX1 gene is frequently rearranged in acute leukemia. We cloned a novel RUNX1 chimeric gene generated by t(7;21)(q11.2;q22) in a patient with acute myeloid leukemia. 3′-rapid amplification of cDNA ends analysis showed a tail-to-tail fusion between RUNX1 on 21q22 and DTX2 on 7q11.2, with an insertion of short complementary sequence from UPK3B adjacent to DTX2. DTX2 encodes a putative E3-ubiquitin ligase with no known biological function. There are two possible functions of RUNX1-reversed UPK3B–DTX2: one from aberrant RUNX1 chimeric protein and the other from the reversed sequence of DTX2. The predicted aberrant protein expressed under the RUNX1 promoter was highly structurally similar to RUNX1a. In a reporter assay, the aberrant protein inhibited the trans-activation function of RUNX1 in a dominant-negative manner, similar to RUNX1a. In contrast, the DTX2 reversed sequence may degrade wild-type DTX2 transcript or suppress its translation. In conclusion, we identified a novel fusion RUNX1 partner, DTX2, which chimerize in a reverse direction. This is the first example of RUNX1 chimera in an opposing direction generated by chromosomal translocation in leukemia. In addition to the aberrantly truncated RUNX1 protein, the DTX2 antisense sequence may play some role in the development of leukemia carrying the t(7;21) translocation.