Junli Yan

Dysregulated microRNAs affect pathways and targets of biologic relevance in nasal-type natural killer/T-cell lymphoma

We performed a comprehensive genome-wide miRNA expression profiling of extranodal nasal-type natural killer/T-cell lymphoma (NKTL) using formalin-fixed paraffin-embedded tissue (n = 30) and NK cell lines (n = 6) compared with normal NK cells, with the objective of understanding the pathogenetic role of miRNA deregulation in NKTL. Compared with normal NK cells, differentially expressed miRNAs in NKTL are predominantly down-regulated. Re-expression of down-regulated miRNAs, such as miR-101, miR-26a, miR26b, miR-28-5, and miR-363, reduced the growth of the NK cell line and modulated the expression of their predicted target genes, suggesting the potential functional role of the deregulated miRNAs in the oncogenesis of NKTL. Taken together, the predicted targets whose expression is inversely correlated with the expression of deregulated miRNA in NKTL are significantly enriched for genes involved in cell cycle-related, p53, and MAPK signaling pathways. We also performed immunohistochemical validation for selected target proteins and found overexpression of MUM1, BLIMP1, and STMN1 in NKTL, and notably, a corresponding increase in MYC expression. Because MYC is known to cause repression of miRNA expression, it is possible that MYC activation in NKTL may contribute to the suppression of the miRNAs regulating MUM1, BLIMP1, and STMN1.

EZH2 overexpression in natural killer/T-cell lymphoma confers growth advantage independently of histone methyltransferase activity

The role of enhancer of zeste homolog 2 (EZH2) in cancer is complex and may vary depending on the cellular context. We found that EZH2 is aberrantly overexpressed in the majority of natural killer/T-cell lymphoma (NKTL), an aggressive lymphoid malignancy with very poor prognosis. We show that EZH2 upregulation is mediated by MYC-induced repression of its regulatory micro RNAs and EZH2 exerts oncogenic properties in NKTL. Ectopic expression of EZH2 in both primary NK cells and NKTL cell lines leads to a significant growth advantage. Conversely, knock-down of EZH2 in NKTL cell lines results in cell growth inhibition. Intriguingly, ectopic EZH2 mutant deficient for histone methyltransferase activity is also able to confer growth advantage and rescue growth inhibition on endogenous EZH2 depletion in NKTL cells, indicating an oncogenic role of EZH2 independent of its gene-silencing activity. Mechanistically, we show that EZH2 directly promotes the transcription of cyclin D1 and this effect is independent of its enzymatic activity. Furthermore, depletion of EZH2 using a PRC2 inhibitor 3-deazaneplanocin A significantly inhibits growth of NK tumor cells. Therefore, our study uncovers an oncogenic role of EZH2 independent of its methyltransferase activity in NKTL and suggests that targeting EZH2 may have therapeutic usefulness in this lymphoma.

p53 haploinsufficiency and functional abnormalities in multiple myeloma

Hemizygous deletion of 17p13, which harbors the TP53 gene, has been identified in >10% of newly diagnosed multiple myeloma (MM) patients and is associated with poor prognosis. To date, there is no conclusive evidence that TP53 is the critical gene. Furthermore, the functional effect of TP53 haploinsufficiency is not well characterized. By utilizing human myeloma cell lines, we showed that TP53 hemizygous loss was associated with decreased basal expression level with a partially or severely inactivated p53 response upon genotoxic and non-genotoxic stress. The pathway deficiency was manifested as defective p53 transcriptional activities, together with significant resistance to apoptosis. In some cases with p53 WT/− and no p53 protein expression, the remaining allele was silenced by promoter hypermethylation. We also developed a p53 target gene signature to summarize the complexity of the p53 pathway abnormalities in MM and showed that it is strongly associated with genomic complexity and patient survival. In conclusion, this study identified TP53 as the critical gene located in 17p13, and revealed its haploinsufficiency properties in MM. Furthermore, we have elucidated that multiple mechanisms can deregulate the p53 functions and that this has important prognostic impact in MM.

EZH2 phosphorylation by JAK3 mediates a switch to noncanonical function in natural killer/T-cell lymphoma

The best-understood mechanism by which EZH2 exerts its oncogenic function is through polycomb repressive complex 2 (PRC2)-mediated gene repression, which requires its histone methyltransferase activity. However, small-molecule inhibitors of EZH2 that selectively target its enzymatic activity turn out to be potent only for lymphoma cells with EZH2-activating mutation. Intriguingly, recent discoveries, including ours, have placed EZH2 into the category of transcriptional coactivators and thus raised the possibility of noncanonical signaling pathways. However, it remains unclear how EZH2 switches to this catalytic independent function. In the current study, using natural killer/T-cell lymphoma (NKTL) as a disease model, we found that phosphorylation of EZH2 by JAK3 promotes the dissociation of the PRC2 complex leading to decreased global H3K27me3 levels, while it switches EZH2 to a transcriptional activator, conferring higher proliferative capacity of the affected cells. Gene expression data analysis also suggests that the noncanonical function of EZH2 as a transcriptional activator upregulates a set of genes involved in DNA replication, cell cycle, biosynthesis, stemness, and invasiveness. Consistently, JAK3 inhibitor was able to significantly reduce the growth of NKTL cells, in an EZH2 phosphorylation-dependent manner, whereas various compounds recently developed to inhibit EZH2 methyltransferase activity have no such effect. Thus, pharmacological inhibition of JAK3 activity may provide a promising treatment option for NKTL through the novel mechanism of suppressing noncanonical EZH2 activity.

Deregulated MIR335 that targets MAPK1 is implicated in poor outcome of paediatric acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia (ALL) is the most common paediatric malignancy. Although 90% of patients are now long‐term survivors, the remaining 10% have poor outcome predominantly due to drug resistance. In this study, we carried out genome‐wide microRNA (miRNA) microarray analysis on diagnostic bone marrow samples to determine miRNA expression profiles associated with poor outcome in ALL. A reduced expression of MIR335 was identified as the most significant miRNA abnormality associated with poor outcome. It is well known that glucocorticoid (GC) resistance is one of the major reasons contributing to poor outcome. We show that exogenous expression of MIR335 in ALL cells increases sensitization to prednisolone‐mediated apoptosis. Moreover, we demonstrate that MAPK1 is a novel target of MIR335, and that MEK/ERK inhibitor treatment enhanced prednisolone‐induced cell death through the activation of BIM (BCL2L11). These results provide a possible underlying molecular mechanism to explain the association between reduced MIR335 with poor clinical outcome, and suggest that approaches to re‐introduce MIR335 expression or override MAPK1 activity may offer promising therapeutic strategies in the treatment of ALL.

Genome-wide pharmacologic unmasking identifies tumor suppressive microRNAs in multiple myeloma

Epigenetic alterations have emerged as an important cause of microRNA (miRNA) deregulation. In Multiple Myeloma (MM), a few tumor suppressive miRNAs silenced by DNA hypermethylation have been reported, but so far there are few systemic investigations on epigenetically silenced miRNAs. We conducted genome-wide screening for tumor suppressive miRNAs epigenetically silenced in MM. Four Human MM Cell lines were treated with demethylating agent 5′azacytidine (5′aza). Consistently upregulated miRNAs include miR-155, miR-198, miR-135a*, miR-200c, miR-125a-3p, miR-188-5p, miR-483-5p, miR-663, and miR-630. Methylation array analysis revealed increased methylation at or near miRNA-associated CpG islands in MM patients. Ectopic restoration of miR-155, miR-198, miR-135a*, miR-200c, miR-663 and miR-483-5p significantly repressed MM cell proliferation, migration and colony formation. Furthermore, we derived a 33-gene signature from predicted miRNA target genes that were also upregulated in MM patients and associated with patient survival in three independent myeloma datasets. In summary, we have revealed important, epigenetically silenced tumor suppressive miRNAs by pharmacologic reversal of epigenetic silencing.

RUNX3 is oncogenic in natural killer/T-cell lymphoma and is transcriptionally regulated by MYC

RUNX3, runt-domain transcription factor, is a master regulator of gene expression in major developmental pathways. It acts as a tumor suppressor in many cancers but is oncogenic in certain tumors. We observed upregulation of RUNX3 mRNA and protein expression in nasal-type extranodal natural killer (NK)/T-cell lymphoma (NKTL) patient samples and NKTL cell lines compared to normal NK cells. RUNX3 silenced NKTL cells showed increased apoptosis and reduced cell proliferation. Potential binding sites for MYC were identified in the RUNX3 enhancer region. Chromatin immunoprecipitation–quantitative PCR revealed binding activity between MYC and RUNX3. Co-transfection of the MYC expression vector with RUNX3 enhancer reporter plasmid resulted in activation of RUNX3 enhancer indicating that MYC positively regulates RUNX3 transcription in NKTL cell lines. Treatment with a small-molecule MYC inhibitor (JQ1) caused significant downregulation of MYC and RUNX3, leading to apoptosis in NKTL cells. The growth inhibition resulting from depletion of MYC by JQ1 was rescued by ectopic MYC expression. In summary, our study identified RUNX3 overexpression in NKTL with functional oncogenic properties. We further delineate that MYC may be an important upstream driver of RUNX3 upregulation and since MYC is upregulated in NKTL, further study on the employment of MYC inhibition as a therapeutic strategy is warranted.

LEE011 and ruxolitinib: a synergistic drug combination for natural killer/T-cell lymphoma (NKTCL)

Natural killer/T-cell lymphoma (NKTCL) is an aggressive non-Hodgkin lymphoma that has been facing limited success with conventional treatments, urging for the discovery of alternative strategies. Recent studies including ours have revealed that EZH2 and JAK-STAT signalling pathways are key contributors to NKTCL pathogenesis. In particular, we found that EZH2 is overexpressed and directly transcriptionally activates the CCND1 gene to confer growth advantage. CCND1 codes for cyclin D1, which complexes with CDK4/6 to promote G1 to S phase transition. Therefore in this study we investigated whether inhibiting both JAK1/2 and CDK4/6, using LEE011 and ruxolitinib respectively is effective in NKTL. We first demonstrate that separate LEE011 and ruxolitinib treatment is sufficient to cause growth inhibition of NKTCL cells. More importantly, we found that there is synergistic growth inhibitory effects on NKTCL cells with combination treatment of LEE011 and ruxolitinib. The results obtained shows that the targeting of both CDK4/6 and JAK1/2 are promising to develop better treatment alternatives for NKTCL.