The therapeutic effect of arsenic trioxide (As2O3 ) in the treatment of acute promyelocytic leukemia (APL) was evaluated among 15 APL patients at relapse after all-trans retinoic acid (ATRA) induced and chemotherapy maintained complete remission (CR). As2O3 was administered intravenously at the dose of 10 mg/d. Clinical CR was achieved in nine of 10 (90%) patients treated with As2O3 alone and in the remaining five patients treated by the combination of As2O3 and low-dose chemotherapeutic drugs or ATRA. During the treatment with As2O3 , there was no bone marrow depression and only limited side effects were encountered. Pharmacokinetic studies, which were performed in eight patients, showed that after a peak level of 5.54 μmol/L to 7.30 μmol/L, plasma arsenic was rapidly eliminated, and the continuous administration of As2O3 did not alter its pharmacokinetic behaviors. In addition, increased amounts of arsenic appeared in the urine, with a daily excretion accounting for approximately 1% to 8% of the total daily dose administered. Arsenic contents in hair and nail were increased, and the peak content of arsenic could reach 2.5 to 2.7 μg/g tissue at CR. On the other hand, a decline of the arsenic content in hair and nail was observed after withdrawal of the drug. We conclude that As2O3 treatment is an effective and relatively safe drug in APL patients refractory to ATRA and conventional chemotherapy.

To explore the genetic abnormalities that cooperate with AML1-ETO (AE) fusion gene to cause acute myeloid leukemia (AML) with t(8;21), we screened a number of candidate genes and identified 11 types of mutations in C-KIT gene (mC-KIT), including 6 previously undescribed ones among 26 of 54 (48.1%) cases with t(8;21). To address a possible chronological order between AE and mC-KIT, we showed that, among patients with AE and mC-KIT, most leukemic cells at disease presentation harbored both genetic alteration, whereas in three such cases investigated during complete remission, only AE, but not mC-KIT, could be detected by allele-specific PCR. Therefore, mC-KIT should be a subsequent event on the basis of t(8;21). Furthermore, induced expression of AE in U937-A͞E cells significantly up-regulated mRNA and protein levels of C-KIT. This may lead to an alternative way of C-KIT activation and may explain the significantly higher C-KIT expression in 81.3% of patients with t(8;21) than in patients with other leukemias. These data strongly suggest that t(8;21) AML follows a stepwise model in leukemogenesis, i.e., AE represents the first, fundamental genetic hit to initiate the disease, whereas activation of the C-KIT pathway may be a second but also crucial hit for the development of a full-blown leukemia. Additionally, Gleevec suppressed the C-KIT activity and induced proliferation inhibition and apoptosis in cells bearing C-KIT N822K mutation or overexpression, but not in cells with D816 mC-KIT. Gleevec also exerted a synergic effect in apoptosis induction with cytarabine, thus providing a potential therapeutic for t(8;21) leukemia.A bnormalities in genes that encode transcription factors (TFs) and tyrosine kinases (TKs) represent two classes of the most frequently detected genetic events in human leukemias (1-3). Disruptions of TFs, often as results of recurring chromosomal translocations where fusion genes are generated, may lead to inhibition of hematopoietic differentiation and subsequent apoptosis, whereas mutations or alterations of TKs may confer proliferative and͞or survival advantage to hematopoietic stem͞ progenitor cells. Recent evidence suggests that alterations in TFs and TKs are required to cooperate in causing full-blown leukemia (3, 4). However, whether a causal relationship or a chronological order exists between the two genetic events remains largely unknown in a clinical setting.The t(8;21)(q22;q22), where coding sequences of the AML1 gene on chromosome 21 are juxtaposed to coding sequences of the ETO gene on chromosome 8 generating an AML1-ETO (AE) fusion transcript, represents the most common chromosomal translocation in acute myeloid leukemia (AML) (5). The AE chimeric protein recruits the N-CoR-mSin3-HDAC complex (6) and represses wild-type AML1, which is a crucial TF for hematopoiesis, modifies intranuclear targets of AML1 (5, 7), and even represses genes that normally are not regulated by AML1 (8). At cellular level, the AE fusion protein transforms NIH 3T3 cells and activates TF AP-1 (9), maintai...

Fifty-eight acute promyelocytic leukemia (APL) patients (11 newly diagnosed and 47 relapsed) were studied for arsenic trioxide (As2O3) treatment. Clinical complete remission (CR) was obtained in 8 of 11 (72.7%) newly diagnosed cases. However, As2O3 treatment resulted in hepatic toxicity in 7 cases including 2 deaths, in contrast to the mild liver dysfunction in one third of the relapsed patients. Forty of forty-seven (85.1%) relapsed patients achieved CR. Two of three nonresponders showed clonal evolution at relapse, with disappearance of t(15;17) and PML-RAR fusion gene in 1 and shift to a dominant AML-1-ETO population in another, suggesting a correlation between PML-RAR expression and therapeutic response. In a follow-up of 33 relapsed cases over 7 to 48 months, the estimated disease-free survival (DFS) rates for 1 and 2 years were 63.6% and 41.6%, respectively, and the actual median DFS was 17 months. Patients with white blood cell (WBC) count below 10 × 109/L at relapse had better survival than those with WBC count over 10 × 109/L (P = .038). The duration of As2O3-induced CR was related to postremission therapy, because there was only 2 of 11 relapses in patients treated with As2O3 combined with chemotherapy, compared with 12 of 18 relapses with As2O3 alone (P = .01). Reverse transcription polymerase chain reaction (RT-PCR) analysis in both newly diagnosed and relapsed groups showed long-term use of As2O3 could lead to a molecular remission in some patients. We thus recommend that ATRA be used as first choice for remission induction in newly diagnosed APL cases, whereas As2O3 can be either used as a rescue for relapsed cases or included into multidrug consolidation/maintenance clinical trials.

Recent studies showed that arsenic trioxide (As 2 O 3 ) could induce apoptosis and partial differentiation of leukemic promyelocytes. Here, we addressed the possible mechanisms underlying these two different effects. 1.0 M As 2 O 3 -induced apoptosis was associated with condensation of the mitochondrial matrix, disruption of mitochondrial transmembrane potentials (⌬⌿m) and activation of caspase-3 in acute promyelocytic leukemia (APL) cells regardless of their sensitivity to all-trans retinoic acid (ATRA). All these effects were inhibited by dithiothreitol (DTT) and enhanced by buthionine sulfoximine (BSO). Furthermore, BSO could also render HL60 and U937 cells, which had the higher cellular catalase activity, sensitive to As 2 O 3 -induced apoptosis. Surprisingly, 1.0 M As 2 O 3 did not induce the ⌬⌿m collapse and apoptosis, while 0.1 M As 2 O 3 induced partial differentiation of fresh BM cells from a de novo APL patient. In this study, we also showed that 0.2 mM DTT did not block low-dose As 2 O 3 -induced NB4 cell differentiation, and 0.1ෂ0.5 M As 2 O 3 did not induce differentiation of ATRA-resistant NB4-derived sublines, which were confirmed by cytomorphology, expression of CD11b, CD33 and CD14 as well as NBT reduction. Another interesting finding was that 0.1ෂ0.5 M As 2 O 3 could also induce differentiation-related changes in ATRA-sensitive HL60 cells. However, the differentiationinducing effect could not be seen in ATRA-resistant HL60 sublines with RAR␣ mutation. Moreover, low-dose As 2 O 3 and ATRA yielded similar gene expression profiles in APL cells. These results encouraged us to hypothesize that As 2 O 3 induces APL cell differentiation through direct or indirect activation of retinoic acid receptor-related signaling pathway(s), while ⌬⌿m collapse is the common mechanism of As 2 O 3 -induced apoptosis. Leukemia (2000) 14, 262-270.

In recent years, neutrophils have attracted increasing attention because of their cancer-promoting effects. An elevated neutrophil-to-lymphocyte ratio is considered a prognostic indicator for patients with cancer. Neutrophils are no longer regarded as innate immune cells with a single function, let alone bystanders in the pathological process of cancer. Their diversity and plasticity are being increasingly recognized. This review summarizes previous studies assessing the roles and mechanisms of neutrophils in cancer initiation, progression, metastasis and relapse. Although the findings are controversial, the fact that neutrophils play a dual role in promoting and suppressing cancer is undeniable. The plasticity of neutrophils allows them to adapt to different cancer microenvironments and exert different effects on cancer. Given the findings from our own research, we propose a reasonable hypothesis that neutrophils may be reprogrammed into a cancer-promoting state in the cancer microenvironment. This new perspective indicates that neutrophil reprogramming in the course of cancer treatment is a problem worthy of attention. Preventing or reversing the reprogramming of neutrophils may be a potential strategy for adjuvant cancer therapy.

The full-length AML1-ETO (AE) fusion gene resulting from t(8;21) (q22;q22) in human acute myeloid leukemia (AML) is not sufficient to induce leukemia in animals, suggesting that additional mutations are required for leukemogenesis. We and others have identified activating mutations of C-KIT in nearly half of patients with t (8;21) AML. To test the hypothesis that activating C-KIT mutations cooperate with AE to cause overt AML, we generated a murine transduction and transplantation model with both mutated C-KIT and AE. To overcome the intracellular transport block of human C-KIT in murine cells, we engineered hybrid C-KIT (HyC-KIT) by fusing the extracellular and transmembrane domains of the murine c-Kit in-frame to the intracellular signaling domain of human C-KIT. We showed that tyrosine kinase domain mutants HyC-KIT N822K and D816V, as well as juxtamembrane mutants HyC-KIT 571+14 and 557-558Del, could transform murine 32D cells to cytokine-independent growth. The protein tyrosine kinase inhibitor dasatinib inhibited the proliferation of 32D cells expressing these C-KIT mutants, with potency in the low nanomolar range. In mice, HyC-KIT N822K induced a myeloproliferative disease, whereas HyC-KIT 571+14 induces both myeloproliferative disease and lymphocytic leukemia. Interestingly, coexpression of AE and HyC-KIT N822K led to fatal AML. Our data have further enriched the twohit model that abnormalities of both transcription factor and membrane/cytosolic signaling molecule are required in AML pathogenesis. Furthermore, dasatinib prolonged lifespan of mice bearing AE and HyC-KIT N822K-coexpressing leukemic cells and exerted synergic effects while combined with cytarabine, thus providing a potential therapeutic for t(8;21) leukemia.stem cell factor | mouse model | targeted therapy | combinatorial therapy | interleukin-3 T he t(8;21)(q22;q22) translocation, which generates AML1-ETO (AE) fusion gene, is one of the most common chromosomal abnormalities detected in acute myeloid leukemia (AML) (1, 2). A high-level expression of full-length AE can be detected in all patients with t(8;21) and is thus considered to play a fundamental role in this type of leukemia. Studies of several murine models have demonstrated, nevertheless, that AE alone is not sufficient to induce AML, in that no leukemia development was found in mice carrying an AE knockin allele (3, 4) in AE-transgenic mice (5, 6) or in wild-type (WT) mice that received a transplant of bone marrow (BM) cells transduced retrovirally with AE (7). Of note, in the presence of additional mutations [e.g., mutagen N-ethyl-N-nitrosourea (6), mutations of FLT3 (8), or the TEL-PDGFRβ fusion gene (9)], AE induces an AML phenotype. Moreover, ectopically expression of AE9a (a C-terminally truncated variant of AE) can induce AML in mice (10). These results indicate that full-length AE may need cooperation with other molecular events in initiating AML.Genetic abnormalities affecting transcription factors and mutations affecting genes involved in signal transduction represent two ...

Purpose: NOTCH signaling pathway is essential in T-cell development and NOTCH1 mutations are frequently present inT-cell acute lymphoblastic leukemia (T-ALL). To gain insight into its clinical significance, NOTCH1 mutation was investigated in 77 patients withT-ALL. Experimental Design: Detection of NOTCH1 mutation was done using reverse transcription-PCR amplification and direct sequencing, and thereby compared according to the clinical/ biological data of the patients. Results: Thirty-two mutations were identified in 29 patients (with dual mutations in 3 cases), involving not only the heterodimerization and proline/glutamic acid/serine/threonine domains as previously reported but also the transcription activation and ankyrin repeat domains revealed for the first time. These mutations were significantly associated with elevated WBC count at diagnosis and independently linked to short survival time. Interestingly, the statistically significant difference of survival according to NOTCH1 mutations was only observed in adult patients (>18 years) but not in pediatric patients (V18 years), possibly due to the relatively good overall response of childhoodT-ALL to the current chemotherapy. NOTCH1 mutations could coexist with HOX11, HOX11L2, or SIL-TAL1 expression. The negative effect of NOTCH1 mutation on prognosis was potentiated by HOX11L2 but was attenuated by HOX11. Conclusion: NOTCH1 mutation is an important prognostic marker in T-ALL and its predictive value could be even further increased if coevaluated with other T-cell-related regulatory genes. NOTCH pathway thus acts combinatorially with oncogenic transcriptional factors on T-ALL pathogenesis.

The gene encoding DNA methyltransferase 3A (DNMT3A) is mutated in ∼20% of acute myeloid leukemia cases, with Arg882 (R882) as the hotspot. Here, we addressed the transformation ability of the DNMT3A-Arg882His (R882H) mutant by using a retroviral transduction and bone marrow transplantation (BMT) approach and found that the mutant gene can induce aberrant proliferation of hematopoietic stem/progenitor cells. At 12 mo post-BMT, all mice developed chronic myelomonocytic leukemia with thrombocytosis. RNA microarray analysis revealed abnormal expressions of some hematopoiesis-related genes, and the DNA methylation assay identified corresponding changes in methylation patterns in gene body regions. Moreover, DNMT3A-R882H increased the CDK1 protein level and enhanced cell-cycle activity, thereby contributing to leukemogenesis.genomic variation | epigenetic abnormality | leukemogenic effect D NA methylation represents one of the major epigenetic modifications and plays a key role in a number of regulatory mechanisms of life processes (1-3). In mammals, the executors of genome methylation are members of the DNA methyltransferase (DNMT) family, including DNMT1, DNMT3A and DNMT3B. It has been well established that DNMT3A forms complex with DNMT3L to catalyze the de novo DNA methylation (4, 5). Both DNMT3A and DNMT3B show high expression levels at the early embryogenesis, and their expressions are down-regulated along with the embryonic development and cell differentiation (6, 7).It is well known that all blood-cell lineages originate from the multipotent hematopoietic stem cells (HSCs). A number of regulations are involved in directing HSCs activities, and the epigenetic modifications are of great importance (8,9). It has been shown that loss of Dnmt3a in a Dnmt3a-conditional knockout mouse results in progressive impairment of HSCs differentiation and expansion (10). Notably, DNMT3A recently has been reported to be mutated in up to 20% of cases of acute myeloid leukemia (AML), mostly in cases with monocytic lineage (AML-M5 or -M4) and clinical features including old age, normal karyotype, leukocytosis and thrombocytosis, and poor prognosis (11,12). Although a variety of DNMT3A mutations have been identified, the majority (∼50%) affect Arg882 (R882) located at the catalytic domain, and the most common substitution is Arg882His (R882H) (11,13,14). It also has been suggested that R882 mutation may interfere with oligomerization of DNMT3A and thereby exert an aberrant effect on its enzymatic function (15). Evidence has been obtained supporting DNMT3A mutations as the fundamental genetic event at the initiation stage of AML pathogenesis (16,17). However, the in vivo transformation power of DNMT3A mutations needs to be addressed, and the relevant molecular and cellular mechanisms of these mutations in AML pathogenesis remain obscure.In the present work, using retroviral transduction and bone marrow transplantation (BMT) technology, we were able to investigate the in vivo effect of the DNMT3A-R882H mutation on the transforming po...