Funded Investigators

Shanghai Institute of Hematology

Shanghai, China

Sai-Juan Chen, M.D., Ph.D.
Qiuhua Huang, Ph.D. Yang Shen, Ph.D. Jiang Zhu, Ph.D.

Weili Zhao, Ph.D. Ethan Dmitrovsky, M.D. Arthur Zelent, Ph.D. Samuel Waxman, M.D.  
SIH Dartmouth Medical School Institute of Cancer Research  Mount Sinai School of Medicine
Zhu Chen, M.D., Ph.D.      

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

The full-length AML1-ETO (AE) is not sufficient to induce leukemia in animal models, suggesting that additional mutations are required for leuke~nogenesis. C-KIT mutations have been identified in nearly half of patients with AE positive acute myeloid leukemia (AML). To test the hypothesis that C-KIT mutations cooperate with AE to cause overt AML, the researchers at the Shanghai Institute of Hematology (SIH) in China generated a mouse model with both mutated C-KIT and AE. They showed that tyrosine kinase domain mutants HyC-KITN822K and D816V, as well as juxtamembrane mutants HyC-KIT571+14 and 557-558De1, could transform 32D cells to IL3-independent growth.

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 (MPD), whereas HyCKIT571+14 induces both MPD and lymphocytic leukemia. Interestingly, coexpression of AE and HyC-KITN822K led to fatal AML.

Their data has further enriched the two-hit model that abnormalities of both transcription factor and 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 AE positive AML.

Oridonin targets key pathways generating AML1-ETO cleavage product with tumor suppressor activities in t(8;21) acute myeloid leukemia

Their previous studies have demonstrated that oridonin selectively induces apoptosis of AML1-ETO (AE) positive leukemia cells with caspase-3 activation and the cleavage of AE oncoprotein with the generation of truncated AE (AAE). In this work, they show that oridonin as a sulfohydryl (SH)-group binding agent exerts these effects through modulating networks in caspase-3 activation and targeting AE. On one hand, key regulators involved in the generation of intracellular reactive oxygen species (ROS), such as GSH, NADPH oxidase (NOX) and thioredoxin1thioredoxin reductase (TrxITrxR), are affected, triggering the cascade reaction of caspase-3 activation. On the other hand, oridonin binding of SH groups on AEIAAE leads to the stabilization of AAE.

Interestingly, AAE interacts with AE via NHR2 domain and possesses dominant negative functions against major activities of AE, thus contributing to the selective action of oridonin on t(8;21) leukemia cells.

As4& targets RING-type E3 ligase c-CBL to induce degradation of BCR-ABL in chronic myelogenoiis leukemia

Arsenic, a curative agent for acute promyelocytic leukemia, induces cell apoptosis and degradation of BCR-ABL in chronic myelogenous leukemia (CML). The SIH group demonstrated that ubiquitination and degradation of BCR-ABL was mediated by c-CBL, a RING-type E3 ligase which was also shown to be involved in ubiquitination for many other receptorlprotein tyrosine kinases. Our data showed that c-CBL protein was considerably upregulated by arsenic sulfide (As4S4).

Interestingly, arsenic directly bound the RING finger domain of c-CBL to inhibit its self-ubiquitinationldegradation without interfering the enhancement of ubiquitination and subsequent proteolysis of its substrate BCR-ABL.

Degradation of BCR-ABL due to c-CBL induction as a result of arsenic treatment was also observed in vivo in CML mice. These findings provide insights into the molecular mechanisms of arsenic and further support its therapeutic applications in CML in combination with tyrosine kinase inhibitors and potentially also in other malignancies involving aberrant receptorlprotein tyrosine kinase signaling. 

DNA methyltransferase gene DNMT3A mutations in acute monocytic leukemia and AML

To gain new insights into leukemogenesis and the molecular basis underlying acute monocytic leukemia (AML-M5), using exome sequencing and subsequent Sanger sequencing, the SIH group discovered mutations in DNMT3A (encoding DNA methyltransferase 3A) in 20.5 percent of AML-M5 cases. The DNMT3A mutants showed reduced enzymatic activity or aberrant affinity to histone H3 in vifro. Notably, there were alterations of DNA methylation patterns and/or gene expression profiles (such as HOB genes) in samples with DNMT3A mutations as compared with those without such changes. Leukemias with DNMT3A mutations constituted a group of poor prognosis with elderly disease onset among AML-M5 individuals. Screening of other leukemia subtypes showed Arg882 alterations of DNMT3A in 13.6 percent of acute myelomonocytic leukemia (AML-M4) cases. Their work suggests a contribution of aberrant DNA methyltransferase activity to the pathogenesis of AML-M5 and provides a useful new biomarker for relevant cases.

They further analyzed the prognostic value of genetic mutations for 1,178 AML patients, including the newly found DNMT3A mutations. They revealed 13 novel DNMT3A mutations and the DNMT3A mutations obviously had an higher incidence in M4 and M5 subtypes of AML. The use of four molecular markers allowed us to subdivide the AML patients without recognizable karyotype abnormalities into three distinct prognostic groups with potential clinical relevance. DNMT3A and MIL mutations as independent factors predicted an inferior overall survival (0s) and time to treatment failure (TTF), while CEBPA mutations and NPM1 mutant but DNMT3A negative conferred a better OS and TTF, and patients without detectable gene mutations corresponded to an intermediate status.

Histone H3 lysine 36 methyltransferase and other epigenetic targets

Hypb homozygous knockout mice die at embryonic stage with severe defects in blood vessel development, but heterozygous mice are viable.

To investigate the potential role of this H3K36-specific methyltransferase in hematopoesis, back cross mating was done to deplete the influence of blending inheritance. During this process, some of Hypb+/- mice at the age of 4 months to 5 months started to show a series of abnormal phenotype including body weight loss, splenomegalia and shorten survival. Immunophenotyping analysis of BM showed the increased granulocytes and decreased lymphocytes, especially in the subpopulations of pro-B and pre-B cells. Immunophenotyping analysis of spleen also showed the increased granulocytes and decreased lymphocytes.

Quantitative analysis of hematopoietic stemlprogenitor compartments showed 2 fold of increased Lin-Scal+C-Kit+ (LSK) population. Serial BM transplantation was done to assess the role of Hypb in HSC repopulating ability. Our results showed ~ ~ p bmi^ce had impaired long-term hematopoietic repopulating ability in comparision to wide-type mice. To assess the effect of Hypb in hematopoietic recovery after ~nyelosuppressive treatment, we injected the same dose of 5FU to wild-type and ~ ~micep. ~ b~mi~ce ~sho webd a sig'nifica nt delay in the HSC recovery. These data suggest that Hypb might play an important role in hematopoiesis.  

Rig-I induction inhibits leukemia cell proliferation through enhancing Stat1 phosphorylaion

The SIH group made progress in two related projects. One concerns how retinoic acid inducible gene I (Rig-I) induction contributes to the RAIIFN-induced differentiation and growth inhibition of AML cells, including U937 and acute promyelocytic leukemia (APL) cells, while the other is, at a cellular level, to better understand the heterogeneity of APL cells as well as the relevant molecular mechanisms that maintain the leukemia-initiating ability of two different leukemia-initiating cell (LIC) subsets isolated in an in-vim setting. For the first project, based on the previous observation that Rig-I deficient myeloid cells showed the reduced expression of three IFN-stimulatory genes (ICSBP, KLF-4 and TRAIL), we hypothesized that rather than simply acting as a type I IFN generation inducer in innate immunity, Rig-I may possess an additional role in promoting IFN intracellular signaling. In line with this assumption, by using experimental models such as Rig-I inducible expression, Rig-I knockdown in leukemia cells as well as Rig-I deficient mice, we were able to demonstrate that Rig-I induction can promote the activation of Statl, and that this newly discovered pathway contributes to Rig-I induction-triggered growth inhibition of leukemia cells in vitro and a significant reduction in LIC ability of mouse APL cells. This work was actually highlighted by SciBX on Jan. 27, 2011, once published, for a potential interest to be exploited to better leukemia treatment in the future.

On the APL LIC story, from the realization that mouse APL cells maintained in vivo comprise at least two different LIC subsets and one non-LIC subset, we were trying to figure out what is the major inside factor or pathway other than PML-RARE that plays a pivotal role in imparting the APL cells with the leukemia-initiating ability. The researchers have identified a few potential candidates, now are testing in vivo model, through induction and siRNA assays, which of them plays a real function role.