Changhong Yin

MS-1020 is a novel small molecule that selectively inhibits JAK3 activity

In order to identify Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling inhibitors, a cell‐based high throughput screening was performed using a plant extract library that identified Nb‐(α‐hydroxynaphthoyl)serotonin called MS‐1020 as a novel JAK3 inhibitor. MS‐1020 potently inhibited persistently‐active STAT3 in a cell type‐specific manner. Further examination showed that MS‐1020 selectively blocked constitutively‐active JAK3 and consistently suppressed interleukin‐2‐induced JAK3/STAT5 signalling but not prolactin‐induced JAK2/STAT5 signalling. Furthermore, MS‐1020 affected cell viability only in cancer cells harbouring persistently‐active JAK3/STATs, and in vitro kinase assays showed MS‐1020 binds directly with JAK3, blocking its catalytic activity. Therefore, the present study suggested that this reagent selectively inhibits JAK3 and subsequently leads to a block in STAT signalling. Finally, MS‐1020 decreased cell survival by inducing apoptosis via down‐regulation of anti‐apoptotic gene expression. These results suggest that MS‐1020 may have therapeutic potential in the treatment of cancers harbouring aberrant JAK3 signalling.

A small-molecule compound identified through a cell-based screening inhibits JAK/STAT pathway signaling in human cancer cells

Inappropriate activation of JAK/STAT signaling occurs with high frequency in human cancers and is associated with cancer cell survival and proliferation. Therefore, the development of pharmacologic STAT signaling inhibitors has therapeutic potential in the treatment of human cancers. Here, we report 2-[(3,5-bis-trifluoromethyl-phenyl)-hydroxy-methyl]-1-(4-nitro-phenylamino)-6-phenyl-1,2,4a,7a-tetrahydro-pyrrolo[3,4-b]-pyridine-5,7-dione (AUH-6-96) as a novel small-molecule inhibitor of JAK/STAT signaling that we initially identified through a cell-based high-throughput screening using cultured Drosophila cells. Treatment of Drosophila cells with AUH-6-96 resulted in a reduction of Unpaired-induced transcriptional activity and tyrosine phosphorylation of STAT92E, the sole Drosophila STAT homologue. In human cancer cell lines, AUH-6-96 inhibited both constitutive and interleukin-6-induced STAT3 phosphorylation. Specifically, in Hodgkin lymphoma L540 cells, treatment with AUH-6-96 resulted in reduced levels of tyrosine phosphorylated STAT3 and of the STAT3 downstream target gene SOCS3 in a dose- and time-dependent manner. In addition, AUH-6-96-treated L540 cells showed decreased expression of persistently activated JAK3, suggesting that AUH-6-96 inhibits the JAK/STAT pathway signaling in L540 cells by affecting JAK3 activity and subsequently blocking STAT3 signaling. Importantly, AUH-6-96 selectively affected cell viability only of cancer cells harboring aberrant JAK/STAT signaling. In support of the specificity of AUH-6-96 for inhibition of JAK/STAT signaling, treatment with AUH-6-96 decreased cancer cell survival by inducing programmed cell death by down-regulating the expression of STAT3 downstream target antiapoptotic genes, such as Bcl-xL. In summary, this study shows that AUH-6-96 is a novel small-molecule inhibitor of JAK/STAT signaling and may have therapeutic potential in the treatment of human cancers harboring aberrant JAK/STAT signaling. [Mol Cancer Ther 2008;7(9):2672–80]

Detection of central nervous system leukemia in children with acute lymphoblastic leukemia by real-time polymerase chain reaction.

Accurate detection of central nervous system (CNS) involvement in children with newly diagnosed acute lymphoblastic leukemia (ALL) could have profound prognostic and therapeutic implications. We examined various cerebrospinal fluid (CSF) preservation methods to yield adequate DNA stability for polymerase chain reaction (PCR) analysis and developed a quantitative real-time PCR assay to detect occult CNS leukemia. Sixty CSF specimens were maintained in several storage conditions for varying amounts of time, and we found that preserving CSF in 1:1 serum-free RPMI tissue culture medium offers the best stability of DNA for PCR analysis. Sixty CSF samples (30 at diagnosis and 30 at the end of induction therapy) from 30 children with ALL were tested for CNS leukemic involvement by real-time PCR using patient-specific antigen receptor gene rearrangement primers. Six of thirty patient diagnosis samples were PCR-positive at levels ranging from 0.5 to 66% leukemic blasts in the CSF. Four of these patients had no clinical or cytomorphological evidence of CNS leukemia involvement at that time. All 30 CSF samples drawn at the end of induction therapy were PCR-negative. The data indicate that real-time PCR analysis of CSF is an excellent tool to assess occult CNS leukemia involvement in patients with ALL and can possibly be used to refine CNS status classification.

Inhibition of the signalling kinase JAK3 alleviates inflammation in monoarthritic rats

BACKGROUND AND PURPOSE Many cytokines associated with autoimmune disorders and inflammation have been shown to activate the signalling kinase JAK3, implying that JAK3 plays key roles in the pathogenesis of these diseases. Therefore, investigating the alterations of JAK3 activity and the efficacy of selective JAK3 antagonists in animal models of such disorders is essential to a better understanding of the biology of JAK3 and to assess the potential clinical benefits of JAK3 inhibitors.

NSC114792, a novel small molecule identified through structure-based computational database screening, selectively inhibits JAK3.

BackgroundHuman or animals lacking either JAK3 or the common gamma chain (γc) expression display severe combined immunodeficiency disease, indicating the crucial role of JAK3 in T-cell development and the homeostasis of the immune system. JAK3 has also been suggested to contribute to the pathogenesis of tumorigenesis. Recent studies identified activating JAK3 mutations in patients with various hematopoietic malignancies, including acute megakaryoblastic leukemia. Importantly, functional analyses of some of those JAK3 mutations have been shown to cause lethal hematopoietic malignancies in animal models. These observations make JAK3 an ideal therapeutic target for the treatment of various human diseases. To identify novel small molecule inhibitors of JAK3, we performed structure-based virtual screen using the 3D structure of JAK3 kinase domain and the NCI diversity set of compounds.ResultsWe identified NSC114792 as a lead compound. This compound directly blocked the catalytic activity of JAK3 but not that of other JAK family members in vitro. In addition, treatment of 32D/IL-2Rβ cells with the compound led to a block in IL-2-dependent activation of JAK3/STAT5 but not IL-3-dependent activation of JAK2/STAT5. Consistent with the specificity of NSC114792 for JAK3, it selectively inhibited persistently-activated JAK3, but failed to affect the activity of other JAK family members and other oncogenic kinases in various cancer cell lines. Finally, we showed that NSC114792 decreases cell viability by inducing apoptosis through down-regulating anti-apoptotic gene expression only in cancer cells harboring persistently-active JAK3.ConclusionsNSC114792 is a lead compound that selectively inhibits JAK3 activity. Therefore, our study suggests that this small molecule inhibitor of JAK3 can be used as a starting point to develop a new class of drugs targeting JAK3 activity, and may have therapeutic potential in various diseases that are caused by aberrant JAK3 activity.

The effects of DLEU1 gene expression in Burkitt lymphoma (BL): Potential mechanism of chemoimmunotherapy resistance in BL

Following a multivariant analysis we demonstrated that children and adolescents with Burkitt lymphoma (BL) and a 13q14.3 deletion have a significant decrease in event free survival (EFS) despite identical short intensive multi-agent chemotherapy. However, how this deletion in the 13q14.3 region is associated with a significant decrease in EFS in children and adolescents with BL is largely unknown. The gene Deleted in Lymphocytic Leukemia 1 (DLEU1) is located in the region of 13q14.3. Here, we report that DLEU1 expression is implicated in the regulation of BL programmed cell death, cell proliferation, and expression of apoptotic genes in transcription activator-like effector nuclease (TALEN)s-induced DLEU1 knockdown and DLEU1 overexpressing BL cell lines. Furthermore, NSG mice xenografted with DLEU1 knockdown BL cells had significantly shortened survival (p < 0.05 and p < 0.005), whereas those xenografted with DLEU1 overexpressing BL cells had significantly improved survival (p < 0.05 and p < 0.0001), following treatment with rituximab and/or cyclophosphamide. These data suggest that DLEU1 may in part function as a tumor suppressor gene and confer chemoimmunotherapy resistance in children and adolescents with BL.

Identification of mutant alleles of JAK3 in pediatric patients with acute lymphoblastic leukemia

Abstract Children with acute lymphoblastic leukemia (ALL) have an 80% chance of long-term survival. Despite the high rate of cure, children relapse, and recurrent ALL is difficult to cure with chemotherapeutic regimens. Therefore, improved biological understanding of ALL and the development of rationally designed therapeutics targeting molecules associated with the pathogenesis of ALL are essential. We identified missense and synonymous JAK3 mutations in 16 of 91 pediatric patients with ALL. The expression of JAK3V722I mutant caused the cytokine-independent activation of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling and conferred the factor-independent growth of murine interleukin-3 (IL-3)-dependent pro-B Ba/F3 cells. Importantly, inhibition of JAK3 by the known JAK3 inhibitor CP-690 550 converted the Ba/F3-JAK3V722I cells back to factor-dependent growth. These observations suggest that JAK3 may contribute to the pathogenesis of pediatric ALL and serve as an important therapeutic target which can be leveraged to improve outcomes for pediatric patients with ALL.

GRAM domain-containing protein 1B (GRAMD1B), a novel component of the JAK/STAT signaling pathway, functions in gastric carcinogenesis

Dysregulated JAK/STAT signaling has been implicated in the molecular pathogenesis of gastric cancer. However, downstream effectors of STAT signaling that facilitate gastric carcinogenesis remain to be explored. We previously identified the Drosophila ortholog of human GRAMD1B in our genome-wide RNAi screen to identify novel components of the JAK/STAT signaling pathway in Drosophila. Here, we examined the involvement of GRAMD1B in JAK/STAT-associated gastric carcinogenesis. We found that GRAMD1B expression is positively regulated by JAK/STAT signaling and GRAMD1B inhibition decreases STAT3 levels, suggesting the existence of a positive feedback loop. Consistently, GRAMD1B and JAK/STAT signaling acted synergistically to promote gastric cancer cell survival by upregulating the expression of the anti-apoptotic molecule Bcl-xL. Interestingly, our immunohistochemical analysis for GRAMD1B revealed a gradual loss of cytoplasmic staining but an increase in the nuclear accumulation of GRAMD1B, as gastric tissue becomes malignant. GRAMD1B expression levels were also found to be significantly associated with clinicopathological features of the gastric cancer patients, particularly the tumor grades and lymph node status. Moreover, GRAMD1B and pSTAT3 (Tyr705) showed a positive correlation in gastric tissues, thereby confirming the existence of a close link between these two signaling molecules in vivo. This new knowledge about JAK/STAT-GRAMD1B regulation deepens our understanding of JAK/STAT signaling in gastric carcinogenesis and provides a foundation for the development of novel biomarkers in gastric cancer.

Development of a High-Throughput Cell-Based Reporter Assay for Screening of JAK3 Inhibitors

JAK3 is an ideal target for the treatment of immune-related diseases and the prevention of organ allograft rejection. Several JAK3 inhibitors have been identified by biochemical enzymatic assays, but the majority display significant off-target effects on JAK2. Therefore, there is a need to develop new experimental approaches to identify compounds that specifically inhibit JAK3. Here, we show that in 32D/IL-2Rβ cells, STAT5 becomes phosphorylated by an IL-3/JAK2- or IL-2/JAK3-dependent pathway. Importantly, the selective JAK3 inhibitor CP-690,550 blocked the phosphorylation and the nuclear translocation of STAT5 following treatment of cells with IL-2 but not with IL-3. In an attempt to use the cells for large-scale chemical screens to identify JAK3 inhibitors, we established a cell line, 32D/IL-2Rβ/6xSTAT5, stably expressing a STAT5 reporter gene. Treatment of this cell line with IL-2 or IL-3 dramatically increased the reporter activity in a high-throughput format. As expected, CP-690,550 selectively inhibited the activity of the 6xSTAT5 reporter following treatment with IL-2. By contrast, the pan-JAK inhibitor curcumin inhibited the activity of this reporter following treatment with either IL-2 or IL-3. Thus, this study indicates that the STAT5 reporter cell line can be used as an efficacious cellular model for chemical screens to identify selective JAK3 inhibitors.

Ruxolitinib significantly enhances in vitro apoptosis in Hodgkin lymphoma and primary mediastinal B-cell lymphoma and survival in a lymphoma xenograft murine model

Hodgkin lymphoma (HL) and primary mediastinal B-cell lymphoma (PMBL) share similar molecular features by gene expression profiling. Frequent gains of chromosome 9p exhibit higher Janus Kinase 2 (JAK2) transcript levels with increased JAK2 activity, suggesting aberrant activity of JAK2 and STAT pathways. This signaling pathway alteration may in part play an important role in the pathogenesis and/or chemoradiotherapy resistance in HL and PMBL. Ruxolitinib is a potent and selective JAK1/JAK2 inhibitor, with activity against myeloproliferative neoplasms (MPNs) including those harboring the JAK2V617F mutation. We investigated the in vitro and in vivo efficacy of ruxolitinib and changes in downstream signaling pathways in HL and PMBL. We demonstrated that ruxolitinib significantly inhibited STAT signaling in both HL and PMBL with constitutively active JAK2 signaling. We also observed that ruxolitinib significantly induced in vitro anti-proliferative effects (p < 0.05) and increased programmed cell death (p < 0.05) against both HL and PMBL cells. Importantly, ruxolitinib significantly inhibited tumor progression by bioluminescence (p < 0.05) and significantly improved survival in HL (p = 0.0001) and PMBL (p < 0.0001) xenograft NSG mice. Taken altogether, these studies suggest that ruxolitinib may be a potential adjuvant targeted agent in the therapeutic approach in patients with high risk HL and PMBL.