Comprehensive analysis of full-length transcripts reveals novel splicing abnormalities and oncogenic transcripts in liver cancer.

Genes generate transcripts of various functions by alternative splicing. However, in most transcriptome studies, short-reads sequencing technologies (next-generation sequencers) have been used, leaving full-length transcripts unobserved directly. Although long-reads sequencing technologies would enable the sequencing of full-length transcripts, the data analysis is difficult. In this study, we developed an analysis pipeline named SPLICE and analyzed cDNA sequences from 42 pairs of hepatocellular carcinoma (HCC) and matched non-cancerous livers with an Oxford Nanopore sequencer. Our analysis detected 46,663 transcripts from the protein-coding genes in the HCCs and the matched non-cancerous livers, of which 5,366 (11.5%) were novel. A comparison of expression levels identified 9,933 differentially expressed transcripts (DETs) in 4,744 genes. Interestingly, 746 genes with DETs, including the LINE1-MET transcript, were not found by a gene-level analysis. We also found that fusion transcripts of transposable elements and hepatitis B virus (HBV) were overexpressed in HCCs. In vitro experiments on DETs showed that LINE1-MET and HBV-human transposable elements promoted cell growth. Furthermore, fusion gene detection showed novel recurrent fusion events that were not detected in the short-reads. These results suggest the efficiency of full-length transcriptome studies and the importance of splicing variants in carcinogenesis.

Albendazole induces the terminal differentiation of acute myeloid leukaemia cells to monocytes by stimulating the Krüppel-like factor 4-dihydropyrimidinase-like 2A (KLF4-DPYSL2A) axis.

Differentiation therapy is a less toxic but still a very effective treatment for a subset of acute myeloid leukaemia (AML) cases. With the goal to identify novel compounds that can effectively and safely induce the terminal differentiation of non-acute promyelocytic leukaemia (APL) AML cells, we performed a chemical screening and identified albendazole (ABZ), a widely used anti-helminthic drug, as a promising lead compound that can differentiate non-APL AML cells by stimulating the Krüppel-like factor 4-dihydropyrimidinase-like 2A (KLF4-DPYSL2A) differentiation axis to the monocytes. Our in vitro and in vivo findings demonstrate that ABZ is an attractive candidate drug as a novel differentiation chemotherapy for patients with non-APL AML.

Pivotal role of DPYSL2A in KLF4-mediated monocytic differentiation of acute myeloid leukemia cells.

Although the biological importance of Krüppel-like factor 4 (KLF4) transcription factor in the terminal differentiation of hematopoietic cells to the monocytes has been well established, the underlying mechanisms remain elusive. To clarify the molecular basis of KLF4-mediated monocytic differentiation, we performed detailed genetic studies in acute myeloid leukemia (AML) cells. Here, we report that dihydropyrimidinase like 2 (DPYSL2), also known as CRMP2, is a novel key differentiation mediator downstream of KLF4 in AML cells. Interestingly, we discovered that KLF4-mediated monocytic differentiation is selectively dependent on one specific isoform, DPYSL2A, but not on other DPYSL family genes. Terminal differentiation to the monocytes and proliferation arrest in AML cells induced by genetic or pharmacological upregulation of KLF4 were significantly reversed by short hairpin RNA (shRNA)-mediated selective depletion of DPYSL2A. Chromatin immunoprecipitation assay revealed that KLF4 associates with the proximal gene promoter of DPYSL2A and directly transactivates its expression. Together with the unique expression patterns of KLF4 and DPYSL2 limited to the differentiated monocytes in the hematopoietic system both in human and mouse, the identified KLF4-DPYSL2 axis in leukemia cells may serve as a potential therapeutic target for the development of novel differentiation therapies for patients with AML.

RUNX transcription factors potentially control E-selectin expression in the bone marrow vascular niche in mice.

Although the function of Runt-related (RUNX) transcription factors has been well characterized in leukemogenesis and regarded as an ideal target in antileukemia strategies, the effect of RUNX-inhibition therapy on bone marrow niche cells andr its impact on the engraftment of acute myeloid leukemia (AML) cells have largely been unknown. Here, we provide evidence suggesting the possible involvement of RUNX transcription factors in the transactivation of E-selectin, a member of selectin family of cell adhesion molecules, on the vascular endothelial cells of the mice bone marrow niche. In our experiments, gene switch-mediated silencing of RUNX downregulated E-selectin expression in the vascular niche and negatively controlled the engraftment of AML cells in the bone marrow, extending the overall survival of leukemic mice. Our work identified the novel role of RUNX family genes in the vascular niche and showed that the vascular niche, a home for AML cells, could be strategically targeted with RUNX-silencing antileukemia therapies. Considering the excellent efficacy of RUNX-inhibition therapy on AML cells themselves as we have previously reported, this strategy potentially targets AML cells both directly and indirectly, thus providing a better chance of cure for poor-prognostic AML patients.

Autonomous feedback loop of RUNX1-p53-CBFB in acute myeloid leukemia cells.

Although runt-related transcription factor 1 (RUNX1) and its associating core binding factor-ß (CBFB) play pivotal roles in leukemogenesis, and inhibition of RUNX1 has now been widely recognized as a novel strategy for anti-leukemic therapies, it has been elusive how leukemic cells could acquire the serious resistance against RUNX1-inhibition therapies and also whether CBFB could participate in this process. Here, we show evidence that p53 (TP53) and CBFB are sequentially up-regulated in response to RUNX1 depletion, and their mutual interaction causes the physiological resistance against chemotherapy for acute myeloid leukemia (AML) cells. Mechanistically, p53 induced by RUNX1 gene silencing directly binds to CBFB promoter and stimulates its transcription as well as its translation, which in turn acts as a platform for the stabilization of RUNX1, thereby creating a compensative RUNX1-p53-CBFB feedback loop. Indeed, AML cells derived from relapsed cases exhibited higher CBFB expression levels compared to those from primary AML cells at diagnosis, and these CBFB expressions were positively correlated to those of p53. Our present results underscore the importance of RUNX1-p53-CBFB regulatory loop in the development and/or maintenance of AML cells, which could be targeted at any sides of this triangle in strategizing anti-leukemia therapies.

Genetic regulation of the RUNX transcription factor family has antitumor effects.

Runt-related transcription factor 1 (RUNX1) is generally considered to function as a tumor suppressor in the development of leukemia, but a growing body of evidence suggests that it has pro-oncogenic properties in acute myeloid leukemia (AML). Here we have demonstrated that the antileukemic effect mediated by RUNX1 depletion is highly dependent on a functional p53-mediated cell death pathway. Increased expression of other RUNX family members, including RUNX2 and RUNX3, compensated for the antitumor effect elicited by RUNX1 silencing, and simultaneous attenuation of all RUNX family members as a cluster led to a much stronger antitumor effect relative to suppression of individual RUNX members. Switching off the RUNX cluster using alkylating agent-conjugated pyrrole-imidazole (PI) polyamides, which were designed to specifically bind to consensus RUNX-binding sequences, was highly effective against AML cells and against several poor-prognosis solid tumors in a xenograft mouse model of AML without notable adverse events. Taken together, these results identify a crucial role for the RUNX cluster in the maintenance and progression of cancer cells and suggest that modulation of the RUNX cluster using the PI polyamide gene-switch technology is a potential strategy to control malignancies.

Paradoxical enhancement of leukemogenesis in acute myeloid leukemia with moderately attenuated RUNX1 expressions.

Besides being a classical tumor suppressor, runt-related transcription factor 1 (RUNX1) is now widely recognized for its oncogenic role in the development of acute myeloid leukemia (AML). Here we report that this bidirectional function of RUNX1 possibly arises from the total level of RUNX family expressions. Indeed, analysis of clinical data revealed that intermediate-level gene expression of RUNX1 marked the poorest-prognostic cohort in relation to AML patients with high- or low-level RUNX1 expressions. Through a series of RUNX1 knockdown experiments with various RUNX1 attenuation potentials, we found that moderate attenuation of RUNX1 contributed to the enhanced propagation of AML cells through accelerated cell-cycle progression, whereas profound RUNX1 depletion led to cell-cycle arrest and apoptosis. In these RUNX1-silenced tumors, amounts of compensative upregulation of RUNX2 and RUNX3 expressions were roughly equivalent and created an absolute elevation of total RUNX (RUNX1 + RUNX2 + RUNX3) expression levels in RUNX1 moderately attenuated AML cells. This elevation resulted in enhanced transactivation of glutathione S-transferase α 2 (GSTA2) expression, a vital enzyme handling the catabolization of intracellular reactive oxygen species (ROS) as well as advancing the cell-cycle progressions, and thus ultimately led to the acquisition of proliferative advantage in RUNX1 moderately attenuated AML cells. Besides, treatment with ethacrynic acid, which is known for its GSTA inhibiting property, actually prolonged the survival of AML mice in vivo. Collectively, our findings indicate that moderately attenuated RUNX1 expressions paradoxically enhance leukemogenesis in AML cells through intracellular environmental change via GSTA2, which could be a novel therapeutic target in antileukemia strategy.