Guijie Guo

A long noncoding RNA critically regulates Bcr-Abl-mediated cellular transformation by acting as a competitive endogenous RNA

Aberrant expression of long noncoding RNAs (lncRNAs) is associated with various human cancers. However, the role of lncRNAs in Bcr-Abl-mediated chronic myeloid leukemia (CML) is unknown. In this study, we performed a comprehensive analysis of lncRNAs in human CML cells using an lncRNA cDNA microarray and identified an lncRNA termed lncRNA-BGL3 that acted as a key regulator of Bcr-Abl-mediated cellular transformation. Notably, we observed that lncRNA-BGL3 was highly induced in response to disruption of Bcr-Abl expression or by inhibiting Bcr-Abl kinase activity in K562 cells and leukemic cells derived from CML patients. Ectopic expression of lncRNA-BGL3 sensitized leukemic cells to undergo apoptosis and inhibited Bcr-Abl-induced tumorigenesis. Furthermore, transgenic (TG) mice expressing lncRNA-BGL3 were generated. We found that TG expression of lncRNA-BGL3 alone in mice was sufficient to impair primary bone marrow transformation by Bcr-Abl. Interestingly, we identified that lncRNA-BGL3 was a target of miR-17, miR-93, miR-20a, miR-20b, miR-106a and miR-106b, microRNAs that repress mRNA of phosphatase and tensin homolog (PTEN). Further experiments demonstrated that lncRNA-BGL3 functioned as a competitive endogenous RNA for binding these microRNAs to cross-regulate PTEN expression. Additionally, our experiments have begun to address the mechanism of how lncRNA-BGL3 is regulated in the leukemic cells and showed that Bcr-Abl repressed lncRNA-BGL3 expression through c-Myc-dependent DNA methylation. Taken together, these results reveal that Bcr-Abl-mediated cellular transformation critically requires silence of tumor-suppressor lncRNA-BGL3 and suggest a potential strategy for the treatment of Bcr-Abl-positive leukemia.

eIF4B phosphorylation by Pim kinases plays a critical role in cellular transformation by Abl oncogenes

Alterations in translation occur in cancer cells, but the precise pathogenic processes and mechanistic underpinnings are not well understood. In this study, we report that interactions between Pim family kinases and the translation initiation factor eIF4B are critical for Abl oncogenicity. Pim kinases, Pim-1 and Pim-2, both directly phosphorylated eIF4B on Ser406 and Ser422. Phosphorylation of eIF4B on Ser422 was highly sensitive to pharmacologic or RNA interference-mediated inhibition of Pim kinases. Expression and phosphorylation of eIF4B relied upon Abl kinase activity in both v-Abl- and Bcr-Abl-expressing leukemic cells based on their blockade by the Abl kinase inhibitor imatinib. Ectopic expression of phosphomimetic mutants of eIF4B conferred resistance to apoptosis by the Pim kinase inhibitor SMI-4a in Abl-transformed cells. In contrast, silencing eIF4B sensitized Abl-transformed cells to imatinib-induced apoptosis and also inhibited their growth as engrafted tumors in nude mice. Extending these observations, we found that primary bone marrow cells derived from eIF4B-knockdown transgenic mice were less susceptible to Abl transformation, relative to cells from wild-type mice. Taken together, our results identify eIF4B as a critical substrate of Pim kinases in mediating the activity of Abl oncogenes, and they highlight eIF4B as a candidate therapeutic target for treatment of Abl-induced cancers.

High expression of long non‐coding RNA H19 is required for efficient tumorigenesis induced by Bcr‐Abl oncogene

Dysregulation of non‐coding RNA H19 has been observed in various tumors. However, it remains unknown whether H19 is involved in Bcr‐Abl‐induced leukemia. Here, we demonstrate a critical requirement for H19 in Bcr‐Abl‐mediated tumorigenesis. H19 was highly expressed in Bcr‐Abl‐transformed cell lines and primary cells derived from patients in a Bcr‐Abl kinase‐dependent manner. Silencing H19 expression sensitized leukemic cells to undergo imatinib‐induced apoptosis and inhibited Bcr‐Abl‐induced tumor growth. Furthermore, H19 was shown to be regulated by c‐Myc in Bcr‐Abl‐expressing cells. These results reveal an important role H19 plays in Bcr‐Abl‐mediated transformation and provide novel insights into complex mechanisms underlying Bcr‐Abl‐induced cancers.

Oncogenic E17K mutation in the pleckstrin homology domain of AKT1 promotes v-Abl-mediated pre-B-cell transformation and survival of Pim-deficient cells

Abl-mediated transformation requires the activation of multiple pathways involved in the cellular proliferation and survival, including PI3K/AKT and JAK/STAT-dependent Pim kinases. Recently, the E17K mutation in the AKT1 has been associated with multiple human malignancies and leukemia in mice. However, this mutation has not been identified in Abl-transformed cells. We investigated the presence of the AKT1(E17K) mutation in v-Abl-transformed cell clones. AKT1(E17K) was detected in 3 (2.6%) of 116 specimens examined. To show the involvement of AKT1(E17K) directly in v-Abl-mediated tumorigenesis, we infected bone marrow cells from mice with bicistronic retroviruses encoding v-Abl and either wild-type or the mutant AKT1. Interestingly, we found that E17K mutant greatly increased the v-Abl transformation efficiency as compared with wild-type AKT1. Ectopic expression of E17K mutant increased the expression levels of antiapoptotic protein BCL2 and phosphorylation levels of proapoptotic protein BAD. This correlated with an increased protection from imatinib-induced apoptosis in Abl transformants. Furthermore, AKT1(E17K) promotes survival of the Pim-deficient cells, indicating a functional link between AKT and Pim in v-Abl transformation. In addition, AKT1(E17K) delays loss of Pim-1 and Pim-2 protein levels on v-Abl inactivation, which suggests that there exists reciprocal signaling between AKT and Pim in v-Abl transformants.

Robust expression of vault RNAs induced by influenza A virus plays a critical role in suppression of PKR-mediated innate immunity

Protein kinase R (PKR) is a vital component of host innate immunity against viral infection. However, the mechanism underlying inactivation of PKR by influenza A virus (IAV) remains elusive. Here, we found that vault RNAs (vtRNAs) were greatly induced in A549 cells and mouse lungs after infection with IAV. The viral NS1 protein was shown to be the inducer triggering the upregulation of vtRNAs. Importantly, silencing vtRNA in A549 cells significantly inhibited IAV replication, whereas overexpression of vtRNAs markedly promoted the viral replication. Furthermore, in vivo studies showed that disrupting vtRNA expression in mice significantly decreased IAV replication in infected lungs. The vtRNA knockdown animals exhibited significantly enhanced resistance to IAV infection, as evidenced by attenuated acute lung injury and spleen atrophy and consequently increased survival rates. Interestingly, vtRNAs promoted viral replication through repressing the activation of PKR and the subsequent antiviral interferon response. In addition, increased expression of vtRNAs was required for efficient suppression of PKR by NS1 during IAV infection. Moreover, vtRNAs were also significantly upregulated by infections of several other viruses and involved in the inactivation of PKR signaling by these viruses. These results reveal a novel mechanism by which some viruses circumvent PKR-mediated innate immunity.

A Critical Role of CDKN3 in Bcr-Abl-Mediated Tumorigenesis

CDKN3 (cyclin-dependent kinase inhibitor 3), a dual specificity protein phosphatase, dephosphorylates cyclin-dependent kinases (CDKs) and thus functions as a key negative regulator of cell cycle progression. Deregulation or mutations of CDNK3 have been implicated in various cancers. However, the role of CDKN3 in Bcr-Abl-mediated chronic myelogenous leukemia (CML) remains unknown. Here we found that CDKN3 acts as a tumor suppressor in Bcr-Abl-mediated leukemogenesis. Overexpression of CDKN3 sensitized the K562 leukemic cells to imanitib-induced apoptosis and dramatically inhibited K562 xenografted tumor growth in nude mouse model. Ectopic expression of CDKN3 significantly reduced the efficiency of Bcr-Abl-mediated transformation of FDCP1 cells to growth factor independence. In contrast, depletion of CDKN3 expression conferred resistance to imatinib-induced apoptosis in the leukemic cells and accelerated the growth of xenograph leukemia in mice. In addition, we found that CDKN3 mutant (CDKN3-C140S) devoid of the phosphatase activity failed to affect the K562 leukemic cell survival and xenografted tumor growth, suggesting that the phosphatase of CDKN3 was required for its tumor suppressor function. Furthermore, we observed that overexpression of CDKN3 reduced the leukemic cell survival by dephosphorylating CDK2, thereby inhibiting CDK2-dependent XIAP expression. Moreover, overexpression of CDKN3 delayed G1/S transition in K562 leukemic cells. Our results highlight the importance of CDKN3 in Bcr-Abl-mediated leukemogenesis, and provide new insights into diagnostics and therapeutics of the leukemia.

Noncoding RNAs and their functional involvement in regulation of chronic myeloid leukemia

Noncoding RNAs (ncRNAs) comprise multiple classes of transcripts that have no protein-coding ability but play critical roles as RNA regulators in various cellular processes. To date, the well-studied ncRNAs are microRNAs (miRs) that generally act as regulators of gene expression through binding to target mRNAs. Recent advances in high-throughput sequencing technologies have led to the discovery of thousands of unannotated noncoding transcripts, especially long noncoding RNAs (lncRNAs). These lncRNAs are being increasingly recognized as key regulators in diverse biological processes via a variety of mechanisms. Aberrant expression of miRs and lncRNAs has been shown to be associated with many human diseases and cancers. Increasing ncRNAs have been identified as biomarkers for patient prognosis and potential therapeutic agents for cancers. Furthermore, it is worth noting that progresses have been made in understanding the functional involvement of ncRNAs in Bcr-Abl-induced chronic myeloid leukemia (CML). Here, we highlight the pathogenesis of CML, functional significance of miRs and lncRNAs in regulation of CML development and involved mechanisms underlying their action.

Abstract 11: A requirement for SOCS-1 and SOCS-3 phosphorylation in BCR-ABL-mediated transformation

Previous studies have demonstrated that constitutive activation of JAK/STAT signaling is required for efficient transformation by the Abelson murine leukemia virus (A-MuLV), which expresses v-Abl oncoprotein. Importantly, there is increasing evidence that deregulation of the JAK or STAT activity plays a critical role in BCR-ABL-induced malignant transformation. SOCS-1 and SOCS-3 are inhibitors of JAK-STAT pathway and as such are involved in a negative feedback loop of cytokine signaling. However, the mechanism by which Abl oncogene bypasses SOCS inhibitory regulation remains poorly defined. Here, we found that co-expression of BCR-ABL with SOCS-1 or SOCS-3 resulted in tyrosine phosphorylation of these SOCS proteins. Interestingly, tyrosine-phosphorylated SOCS-1 was detected in three of five primary CML samples which express BCR-ABL. BCR-ABL-dependent phosphorylation of SOCS-1 and SOCS-3 occurred mainly on Tyr 155 and Tyr 204 residues of SOCS-1, and Tyr 221 residue of SOCS-3. Phosphorylation of these SOCS proteins was associated with its binding to BCR-ABL, and this interaction was disrupted by mutation of the sites of SOCS phosphorylation. We demonstrated that BCR-ABL-dependent phosphorylation of SOCS-1 and SOCS-3 alters their inhibitory effects on the activation of JAK1 and JAK2 and thereby enhances JAK-STAT signaling. Moreover, disrupting the tyrosine phosphorylation of SOCS-1 or SOCS-3 promotes K562 leukemic cells to undergo apoptosis and blocks tumor formation in mouse model. Together, these results reveal a requirement for tyrosine phosphorylation of SOCS-1 and SCOCS-3 in BCR-ABL-mediated transformation in the presence of these SOCS proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 11. doi:1538-7445.AM2012-11

Interaction of Abl Tyrosine Kinases with SOCS3 Impairs Its Suppressor Function in Tumorigenesis

Suppressor of cytokine signaling 3 (SOCS3) is involved in Bcr-Abl–induced tumorigenesis. However, how SOCS3 interacts with Bcr-Abl and is regulated by Abl kinases remains largely unknown. Since c-Abl plays a critical role in tumorigenesis, we asked whether SOCS3 is regulated by c-Abl–dependent phosphorylation. Here, we found that SOCS3 interacted with all three Abl kinases (Bcr-Abl, v-Abl, and c-Abl), and SH1 domain of the Abl kinases was critically required for such interaction. Furthermore, the SH2 domain of SOCS3 was sufficient to pull down the SH1 domain but not the full length of Bcr-Abl. Importantly, SOCS3 was highly tyrosine phosphorylated by c-Abl, leading to impairment of its ability to suppress JAK8+72 activity. In addition, disrupting the tyrosine phosphorylation of SOCS3 promoted apoptosis of c-Abl–expressing cells and impeded xenograft growth of these tumor cells in nude mice. The results demonstrate that SOCS3 is highly tyrosine phosphorylated by c-Abl and that tyrosine phosphorylation of SOCS3 is required for the survival and tumorigenesis of certain cells. Our findings provide novel insights into complicated mechanisms underlying the oncogenic function of Abl kinases.

Abstract 4976: Inhibition of eIF4B suppresses the growth and induces the apoptosis of Bcr-Abl positive cells

Alterations in translation are associated with various human malignancies, but the precise pathogenic processes and mechanistic underpinnings remain to be better understood. The oncogenic tyrosin kinase Bcr-Abl, which is believed to induce chronic myelogenous leukemia (CML), could cause the dysregulation of mRNA translation initiation by activating a series of signal pathways. Eukaryotic initiation factor 4B (eIF4B), a critical element in the assembly of translation initiation complex, has been shown to anticipate in Bcr-Abl-mediated tumorigenesis. eIF4B is activated through Ser422 phosphorylation by several pathways including JAK/STAT/Pim and PI3K/AKT/mTOR. Such redundant activation provides mechanistic bases for the less potent antitumor consequences under single-pathway targeted therapy. Interestingly, profound synergy was observed against cell survival and tumor growth and greater decrease of eIF4B Ser422 phosphorylation was detected upon the combination of Pim and mTOR inhibitors. Knockdown of eIF4B would increase the dependence of Bcr-Abl positive cells on Pim and mTOR signaling and then sensitize cells to the synergistical effect mediated by the combined inhibition. In addition, inhibiting mTOR pathway by shRNA targeting AKT or mTOR renders cells dependent on Pim pathway and knockdown Pim1 would sensitize cells to mTOR inhibitor. Thus, eIF4B acts as a prominent role in Bcr-Abl-driven pathogenesis by playing as the convergent point of these pathways which are active in Bcr-Abl positive tumors. Our results reveal a novel mechanism by which Bcr-Abl-induced cellular transformation can be blokced and provides a rationale for combination therapy targeting the dysregulation of translation for CML. Note: This abstract was not presented at the meeting. Citation Format: Ke Chen, Jian-Ling Yang, Guijie Guo, Jilong Chen. Inhibition of eIF4B suppresses the growth and induces the apoptosis of Bcr-Abl positive cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4976. doi:10.1158/1538-7445.AM2015-4976