Shu-Min Xiong

Arsenic trioxide-induced apoptosis and differentiation are associated respectively with mitochondrial transmembrane potential collapse and retinoic acid signaling pathways in acute promyelocytic leukemia

Recent studies showed that arsenic trioxide (As2O3) could induce apoptosis and partial differentiation of leukemic promyelocytes. Here, we addressed the possible mechanisms underlying these two different effects. 1.0 μM As2O3-induced apoptosis was associated with condensation of the mitochondrial matrix, disruption of mitochondrial transmembrane potentials (ΔΨm) 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 As2O3-induced apoptosis. Surprisingly, 1.0 μM As2O3 did not induce the ΔΨm collapse and apoptosis, while 0.1 μM As2O3 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 As2O3-induced NB4 cell differentiation, and 0.10.5 μM As2O3 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 0.10.5 μM As2O3 could also induce differentiation-related changes in ATRA-sensitive HL60 cells. However, the differentiation-inducing effect could not be seen in ATRA-resistant HL60 sublines with RARα mutation. Moreover, low-dose As2O3 and ATRA yielded similar gene expression profiles in APL cells. These results encouraged us to hypothesize that As2O3 induces APL cell differentiation through direct or indirect activation of retinoic acid receptor-related signaling pathway(s), while ΔΨm collapse is the common mechanism of As2O3-induced apoptosis.

Studies on the clinical efficacy and pharmacokinetics of low-dose arsenic trioxide in the treatment of relapsed acute promyelocytic leukemia: a comparison with conventional dosage

Twenty cases of patients with relapsed acute promyelocytic leukemia (APL) were entered into this study for evaluating the clinical efficacy and pharmacokinetics of low-dose arsenic trioxide (As2O3). As2O3 was given at a daily dose of 0.08 mg/kg intravenously for 28 days. Pharmacokinetic study was carried out in eight patients. 16/20 (80%) patients achieved CR. The occurrence of some toxic events including gastrointestinal disturbance, facial edema and cardiac toxicity seemed reduced in the low-dose group than those in the standard-dose group. Differentiation changes were observed in peripheral blood, as well as in bone marrow (BM). Pharmacokinetic study showed that the plasma concentration increased soon after administration of As2O3 with the peak values of 1.535–3.424 μmol/l. After infusion, the plasma concentration was around 0.1–0.5 μmol/l. The arsenic concentration of the plasma of BM aspirates 24 h after administration in five patients was close to the level needed for differentiation-inducing effect. The estimated 2-year OS and RFS were 61.55 ± 15.79% and 49.11 ± 15.09% respectively, with no difference as compared with those in patients treated with conventional dose (P = 0.2865 and 0.7146, respectively). In conclusion, we demonstrated that low-dose As2O3 had the same effect as the conventional dosage and the mechanism of low-dose arsenic seemed to primarily induce differentiation of APL cells.

NOTCH1 mutations in T-cell acute lymphoblastic leukemia: prognostic significance and implication in multifactorial leukemogenesis.

Purpose: NOTCH signaling pathway is essential in T-cell development and NOTCH1 mutations are frequently present in T-cell acute lymphoblastic leukemia (T-ALL). To gain insight into its clinical significance, NOTCH1 mutation was investigated in 77 patients with T-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 (≤18 years), possibly due to the relatively good overall response of childhood T-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.

Clinical and cytogenetic features of 508 Chinese patients with myelodysplastic syndrome and comparison with those in Western countries

Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder characterized by ineffective hematopoiesis and leukemia progression. Racial differences may exist on clinical pictures and the molecular events leading to MDS, which are heterogeneous. To better define the clinical and cytogenetic features in Chinese patients, a retrospective multicentric study was performed in 508 MDS cases. Compared with Western countries, Chinese patients showed younger age (median: 49 vs 65–73 years), lower percentages of RARS (2.8 vs 6.6–15.3%), and CMML (5.2 vs 11.7–30.6%). Cytogenetically, among 367 cases with evaluable data, abnormal karyotypes were found in 136 cases, including 56 numerical and 80 structural changes. Incidences of single chromosome 5 and 7 abnormalities were lower than those in Western countries (2.2 vs 17.8–42.5%). However, complex cytogenetic aberrations and chromosome translocations were frequently observed and related to poor prognosis. Both multiple chromosome deletions and translocations were detected in advanced subtypes (RAEB and RAEB-T). Analysis of 200 cases revealed a higher incidence of hepatitis-B-virus infection than that in non-MDS population (21.00 vs 9.75%). This study further confirmed: (1) different genetic/environmental backgrounds between Asian and Western MDS populations; (2) a strong predictive value of cytogenetic abnormalities on disease outcome and involvement of genomic instability in leukemia clone development.

C-KIT mutation cooperates with full-length AML1-ETO to induce acute myeloid leukemia in mice

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 two-hit 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.

Molecular cytogenetic characterization and clinical relevance of additional, complex and/or variant chromosome abnormalities in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by typical morphological manifestation, t(15;17) translocation and active response to all-trans retinoic acid (ATRA) in the great majority of patients. However, a subset of APL cases may present atypical phenotypic, cytogenetic or molecular features at different stages of the disease. The biological and clinical significance of these features sometimes remains obscure. In this study, 284 APL patients were cytogenetically analyzed and precise diagnosis was performed according to the molecular cytogenetic results. Twenty-six APL patients were identified as having additional, complex and/or variant chromosomal abnormalities at diagnosis or at relapse, 16 of them being further analyzed using fluorescence in situ hybridization (FISH) or chromosome painting (CP). Interestingly, some of these chromosomal aberrations were found to be associated with atypical morphology and/or drug response, indicating a genotype–phenotype correlation. Analysis of the complex karyotype may also allow a better understanding of the levels of cellular origin of the leukemogenesis. Examination of the remission induction and survival data showed that the presence of the additional/complex chromosome abnormalities was related to the prognosis in both primarily diagnosed and relapsed patients in this series.

AML1-ETO9a is correlated with C-KIT overexpression/mutations and indicates poor disease outcome in t(8;21) acute myeloid leukemia-M2.

AML1-ETO fusion gene is generated from chromosomal translocation t(8;21) mainly in acute myeloid leukemia M2 subtype (AML-M2). Its spliced variant transcript, AML1-ETO9a, rapidly induces leukemia in murine model. To evaluate its clinical significance, AML1-ETO9a expression was assessed in 118 patients with t(8;21) AML-M2, using qualitative and nested quantitative reverse transcriptase (RT)–PCR methods. These cases were accordingly divided into the AML1-ETO9a-H group (n=86, positive for qualitative RT–PCR, with higher level of AML1-ETO9a by quantitative RT–PCR) and the AML1-ETO9a-L group (n=32, negative for qualitative RT–PCR, with lower but still detectable level of AML1-ETO9a by quantitative RT–PCR). C-KIT expression was significantly increased in the AML1-ETO9a-H group, as compared with the AML1-ETO9a-L group. Of the 36 patients harboring C-KIT mutations, 32 patients overexpressed AML1-ETO9a (P=0.0209). Clinically, AML1-ETO9a-H patients exhibited significantly elevated white blood cells count, less bone marrow aberrant myelocytes, increased CD56 but decreased CD19 expression (P=0.0451, P=0.0479, P=0.0149 and P=0.0298, respectively). Moreover, AML1-ETO9a overexpression was related to short event-free and overall survival time (P=0.0072 and P=0.0076, respectively). Taken together, these data suggest that AML1-ETO9a is correlated with C-KIT overexpression/mutations and indicates poor disease outcome in t(8;21) AML-M2.

DNMT3A Arg882 mutation drives chronic myelomonocytic leukemia through disturbing gene expression/DNA methylation in hematopoietic cells

Significance Epigenetic modifications are required for the regulation of hematopoiesis. DNA methyltransferase 3A (DNMT3A), a critical epigenetic modifier responsible for de novo DNA methylation, was reported recently to be a frequently mutated gene in hematopoietic malignancies. However, the role of mutated DNMT3A in hematopoiesis remains largely unknown. Here we show that the Arg882 (R882) mutation of DNMT3A disrupts the normal function of this enzyme and results in chronic myelomonocytic leukemia (CMML) in mice. Meanwhile, the gene expression, DNA methylation, and protein–protein interaction assays suggest that DNMT3A R882 mutation drives CMML by disturbing the transcriptional expression/DNA methylation program and cell-cycle regulation of hematopoietic cells. This study may shed light on the function of DNMT3A mutant in myeloid leukemogenesis. 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.

Newly diagnosed acute lymphoblastic leukemia in China (II): prognosis related to genetic abnormalities in a series of 1091 cases

The molecular characterization of cytogenetic abnormalities has not only provided insights into the mechanisms of leukemogenesis but also led to the establishment of new treatment strategies targeting these abnormalities and thereby further improve the prognosis of patients. We analyzed the prognosis of 1091 Chinese patients with newly diagnosed acute lymphoblastic leukemia (ALL) and explored the prognostic impacts of a large number of cytogenetic/molecular abnormalities. It was demonstrated that, in both B- and T-ALL settings, the prognosis was negatively correlated to the age as reported to date. For childhood T-ALL patients, it was also documented that the HOX11 expression represented a favorable prognostic factor as it was in adult ones. We identified CRLF2 overexpression as an intermediate-risk marker and Ik6 variant of IKZF1 gene as a high-risk one when stratifying pediatric B-ALL cases according to cytogenetic/molecular risks. We also found that Ik6 variant and CRLF2 overexpression had an important role in dictating the prognosis of Ph-negative patients, which may be useful markers in guiding the treatment of ALL in the future, with tyrosine kinase inhibitors on the other hand reversing the fate of Ph-positive ALL patients.

Newly diagnosed acute lymphoblastic leukemia in China (I): abnormal genetic patterns in 1346 childhood and adult cases and their comparison with the reports from Western countries

It has been generally acknowledged that the diagnosis, treatment and prognosis evaluation of leukemia largely rely on an adequate identification of genetic abnormalities. A systemic analysis of genetic aberrations was performed in a cohort of 1346 patients with newly diagnosed acute lymphoblastic leukemia (ALL) in China. The pediatric patients had higher incidence of hyperdiploidy and t(12;21) (p13;q22)/ETV6–RUNX1 than adults (P<0.0001); in contrast, the occurrence of Ph and Ik6 variant of IKZF1 gene was much more frequent in adult patients (all P<0.0001). In B-ALL, the existence of Ik6 and that of BCR–ABL were statistically correlated (P<0.0001). In comparison with Western cohorts, the incidence of t(9;22) (q34;q11)/BCR–ABL (14.60%) in B-ALL and HOX11 expression in T-ALL (25.24%) seemed to be much higher in our group, while the incidence of t(12;21) (p13;q22)/ETV6–RUNX1 (15.34%) seemed to be lower in Chinese pediatric patients. The occurrence of hyperdiploidy was much lower either in pediatric (10.61% vs 20–38%) or adult patients (2.36% vs 6.77–12%) in our study than in Western reports. In addition, the frequencies of HOX11L2 in adult patients were much higher in our cohort than in Western countries (20.69% vs 4–11%). In general, it seems that Chinese ALL patients bear more adverse prognostic factors than their Western counterparts do.