The miR-223 host non-coding transcript linc-223 induces IRF4 expression in acute myeloid leukemia by acting as a competing endogenous RNA

Alterations in genetic programs required for terminal myeloid differentiation and aberrant proliferation characterize acute myeloid leukemia (AML) cells. Here, we identify the host transcript of miR-223, linc-223, as a novel functional long non-coding RNA (lncRNA) in AML. We show that from the primary nuclear transcript, the alternative production of miR-223 and linc-223 is finely regulated during monocytic differentiation. Moreover, linc-223 expression inhibits cell cycle progression and promotes monocytic differentiation of AML cells. We also demonstrate that endogenous linc-223 localizes in the cytoplasm and acts as a competing endogenous RNA for miR-125-5p, an oncogenic microRNA in leukemia. In particular, we show that linc-223 directly binds to miR-125-5p and that its knockdown increases the repressing activity of miR-125-5p resulting in the downregulation of its target interferon regulatory factor 4 (IRF4), which it was previously shown to inhibit the oncogenic activity of miR-125-5p in vivo. Furthermore, data from primary AML samples show significant downregulation of linc-223 in different AML subtypes. Therein, these findings indicate that the newly identified lncRNA linc-223 may have an important role in myeloid differentiation and leukemogenesis, at least in part, by cross-talking with IRF4 mRNA

METTL3 regulates WTAP protein homeostasis

The Wilms tumor 1 (WT1)-associated protein (WTAP) is upregulated in many tumors, including, acute myeloid leukemia (AML), where it plays an oncogenic role by interacting with different proteins involved in RNA processing and cell proliferation. In addition, WTAP is also a regulator of the nuclear complex required for the deposition of N6-methyladenosine (m6A) into mRNAs, containing the METTL3 methyltransferase. However, it is not clear if WTAP may have m6A-independent regulatory functions that might contribute to its oncogenic role. Here, we show that both knockdown and overexpression of METTL3 protein results in WTAP protein upregulation, indicating that METTL3 levels are critical for WTAP protein homeostasis. However, we show that WTAP upregulation is not sufficient to promote cell proliferation in the absence of a functional METTL3. Therein, these data indicate that the reported oncogenic function of WTAP is strictly connected to a functional m6A methylation complex

Argonaute 2 drives miR-145-5p-dependent gene expression program in breast cancer cells

To perform their regulatory functions, microRNAs (miRNAs) must assemble with any of the four mammalian Argonaute (Ago) family of proteins, Ago1–4, into an effector complex known as the RNA-induced silencing complex (RISC). While the mature miRNA guides the RISC complex to its target mRNA, the Ago protein represses mRNA translation. The specific roles of the various Ago members in mediating miRNAs activity, however, haven’t been clearly established. In this study, we investigated the contribution of Ago2, the only human Ago protein endowed with nuclease activity, to the function of tumor-suppressor miR-145-5p in breast cancer (BC). We show that miR-145-5p and Ago2 protein are concomitantly downregulated in BC tissues and that restoration of miR-145-5p expression in BC cells leads to Ago2 protein induction through the loosening of Ago2 mRNA translational repression. Functionally, miR-145-5p exerts its inhibitory activity on cell migration only in presence of Ago2, while, upon Ago2 depletion, we observed increased miR-145/Ago1 complex and enhanced cell motility. Profiling by microarray of miR-145-5p target mRNAs, in BC cells depleted or not of Ago2, revealed that miR-145-5p drives Ago2-dependent and -independent activities. Our results highlight that the Ago2 protein in cancer cells strictly dictates miR-145-5p tumor suppressor activity

Effect of miR-204&211 and RUNX2 control on the fate of human mesenchymal stromal cells

MiR-204 and 211 enforced expression in murine mesenchymal stromal cells (MSCs) has been shown to induce adipogenesis and impair osteogenesis, through RUNX2 down-modulation. This mechanism has been suggested to play a role in osteoporosis associated with obesity. However, two further fundamental MSC functions, chondrogenesis and hematopoietic supporting activity, have not yet been explored. To this end, we transduced, by a lenti-viral vector, miR-204 and 211 in a model primary human MSC line, opportunely chosen among our MSC collection for displaying all properties of canonical bone marrow MSCs, except adipogenesis. Enforced expression of miR-204&211 in these cells, rescued adipogenesis, and inhibited osteogenesis, as previously reported in murine MSCs, but, surprisingly, also damaged cartilage formation and hematopoietic supporting activity, which were never explored before. RUNX2 has been previously indicated as the target of miR-204&211, whose down modulation is responsible for the switch from osteogenesis to adipogenesis. However, the additional disruption of chondrogenesis and hematopoietic supporting activity, which we report here, might depend on diverse miR-204&211 targets. To investigate this hypothesis, permanent RUNX2 knock-down was performed. Sh-RUNX2 fully reproduced the phenotypes induced by miR-204&211, confirming that RUNX2 down modulation is the major event leading to the reported functional modification on our MSCs. It seems thus apparent that RUNX2, a recognized master gene for osteogenesis, might rule all four MSC commitment and differentiation processes. Hence, the formerly reported role of miR204&211 and RUNX2 in osteoporosis and obesity, coupled with our novel observation showing inhibition of cartilage differentiation and hematopoietic support, strikingly resemble the clinical traits of metabolic syndrome, where osteoarthritis, osteoporosis, anaemia and obesity occur together. Our observations, corroborating and extending previous observations, suggest that miR-204&211-RUNX2 axis in human MSCs is possibly involved in the pathogenesis of this rapidly growing disease in industrialized countries, for possible therapeutic intervention to regenerate former homeostasis

A critical role for the RNA m6A methylation complex in myeloid leukemia

N6-methyladenosine (m6A) is a well-known RNA modification that can affect mRNA splicing, stability and translation (D. Dominissini et al., 2012; X. Wang et al. 2015; Y. Wang et al. 2014; J. Zhou et al. 2015; J. Choi et al. 2016), and the processing of miRNA precursors (C.R. Alarcon et al. 2015). In mammals, the m6A writer is a multicomponent complex composed of the two methyltransferases METTL3 and METTL14 (J. Liu et al. 2014), and the regulatory protein WTAP (XL. Ping et al. 2014). Post-transcriptional m6A RNA modification is indispensable for cell viability and development, yet its role in cell differentiation and disease are still poorly understood. Notably, WTAP protein is an oncogene overexpressed in Acute Myeloid Leukemia (AML) compared to healthy control cells (H. Bansal et al., 2014). Moreover, METTL3, METTL14, and RBM15 are highly expressed in AML compared with other cancers (S.R. Jaffrey and M G. Kharas, 2017). We analysed the expression of the m6A RNA methylation complex components in AML observing that both METTL3 and METTL14 mRNAs are upregulated in AML samples (TCGA) respect to fully differentiated myeloid cells (GeoDatasets). Conversely, WTAP mRNA has lower levels in AML, even if the protein is upregulated. We showed that in AML and Chronic Myeloid Leukemia (CML) cell lines METTL3 is localized in both nucleus and cytoplasm. In cytoplasm METTL3 binds WTAP mRNA enhancing its translation independently from its catalytic activity, possibly explaining, at least partially, WTAP protein increased levels observed in AML cells. Moreover, we investigated the role of m6A RNA methylation complex components in myeloid leukemia cells, confirming that WTAP affects proliferation and differentiation of AML cells acting as an oncogene and demonstrating that METTL3 and METTL14 are essential proteins in these cells. Indeed, depletion of the two methyltransferases leads to a marked cellular mortality of AML cells. Notably, METTL3 affects viability and proper myeloid differentiation also in hematopoietic stem cells CD34+ derived from healthy umbilical cord. In CML K562 cells METTL3 and METTL14 depletion does not lead to apoptosis but could affect proper ribosome biogenesis, leading to an evident slowdown of cellular proliferation. Notably, decreased levels of METTL3 and METTL14 in K562 cells cause a downregulation of MYC protein, as recently reported in AML cells (L.P. Vu et al., 2017). Analysing ENCODE MYC ChIP- seq data we observed strong MYC peaks on the promoter of all the genes critical for ribosome biogenesis that are affected by METTL3 and METTL14 depletion, suggesting a MYC-mediated effect. In conclusion, we analysed some functional aspects of m6A RNA chemical modification in both acute and chronic myeloid leukemia cells, identifying differential m6A-regulated pathways between these two kinds of pathologies. Moreover, we also highlighted a more complex and double role for METTL3 protein in these cells, being one of the two essential m6A methyltransferase in the nucleus, and an RNA binding protein promoting translation in cytoplasm. Altogether, these data indicate a link between m6A factors and leukemogenesis and pave the way to possible future therapies targeting the RNA m6a methylation complex components

A positive feed-forward regulatory loop between METTL3 and WTAP sustains the oncogenic role of the m6A methylation complex in myeloid leukemia

The Wilms tumor 1 (WT1)-associated protein (WTAP) is upregulated in many tumours, including, acute myeloid leukemia (AML), where it plays an oncogenic role by interacting with different proteins involved in RNA processing and cell proliferation. In addition, WTAP is also a regulator of the nuclear complex required for the deposition of N6-Methyladenosine (m6A) into mRNAs, containing the METTL3 methyltransferase. However, it is not clear if WTAP may have m6A-independent regulatory functions that might contribute to its oncogenic role. Here, we show that both knockdown and overexpression of METTL3 protein results in WTAP protein upregulation, indicating that METTL3 levels are critical for WTAP protein homeostasis. However, we show that WTAP upregulation is not sufficient to promote cell proliferation in the absence of a functional METTL3. Our results indicate the existence of a positive feedforward regulatory loop, where METTL3 upregulates WTAP, which is relevant to increase WTAP expression concomitantly to the METTL3/METTL14 core m6A methylation complex and sustain the oncogenic role reported for the m6A modification complex in leukemia

The METTL3/METTL14 m6A methylation complex plays a crucial role in Chronic Myeloid Leukemia survival by regulating MYC expression.

Chronic Myeloid Leukemia (CML) is a malignant myeloproliferative disease caused by a chromosomal translocation that produces the constitutively activated tyrosine kinase BCR-ABL1 fusion protein. Tyrosine kinase inhibitors (TKIs) are the first-choice treatment. However, resistance to TKIs remains a challenge for curing CML patients. To gain insight into the role of m6A modification in CML, we analyze the role of the METTL3/METTL14 m6A writing complex in the BCR-ABL1+ K562 CML cellular model. We show that knockdown of METTL3 and METTL14 strongly impaired proliferation of both TKI-sensitive and TKI-resistant cells. Furthermore, we demonstrate that MYC oncogenes is highly m6A methylated in CML and that m6A marks are required for its efficient expression. Therefore, our findings demonstrate an important role for m6A methylation in CML and show that targeting the METTL3/METTL14 complex may represent a promising therapeutic strategy for TKIs resistant CML cells

Argonaute 2 drives miR-145-dependent gene expression program influencing epithelial to mesenchymal transition in breast cancer cells

To perform their regulatory functions, microRNAs (miRNAs) must assemble with any of the four mammalian Argonaute (Ago) family of proteins, Ago1–4, into an effector complex known as the RNA-induced silencing complex (RISC). While the mature miRNA guides the RISC complex to its target mRNA, the Ago protein represses mRNA translation. The specific roles of the various Ago members in mediating miRNAs activity, however, haven’t been clearly established. In this study, we investigated the contribution of Ago2, the only human Ago protein endowed with nuclease activity, to the function of tumor-suppressor miR-145-5p in breast cancer (BC). We show that miR-145-5p and Ago2 protein are concomitantly downregulated in BC tissues and that restoration of miR-145-5p expression in BC cells leads to Ago2 protein induction through the loosening of Ago2 mRNA translational repression. Functionally, miR-145-5p exerts its inhibitory activity on cell migration only in presence of Ago2, while, upon Ago2 depletion, we observed increased miR-145/Ago1 complex and enhanced cell motility. Profiling by microarray of miR-145-5p target mRNAs, in BC cells depleted or not of Ago2, revealed that miR-145-5p drives Ago2-dependent and –independent activities. Our results highlight that the Ago2 protein in cancer cells strictly dictates miR-145-5p tumor suppressor activity

Effect of miR-204&211 and RUNX2 control on the fate of human mesenchymal stromal cells

MiR-204 and 211 enforced expression in murine mesenchymal stromal cells (MSCs) has been shown to induce adipogenesis and impair osteogenesis, through RUNX2 down-modulation. This mechanism has been suggested to play a role in osteoporosis associated with obesity. However, two further fundamental MSC functions, chondrogenesis and hematopoietic supporting activity, have not yet been explored. To this end, we transduced, by a lenti-viral vector, miR-204 and 211 in a model primary human MSC line, opportunely chosen among our MSC collection for displaying all properties of canonical bone marrow MSCs, except adipogenesis. Enforced expression of miR-204&211 in these cells, rescued adipogenesis, and inhibited osteogenesis, as previously reported in murine MSCs, but, surprisingly, also damaged cartilage formation and hematopoietic supporting activity, which were never explored before. RUNX2 has been previously indicated as the target of miR-204&211, whose down modulation is responsible for the switch from osteogenesis to adipogenesis. However, the additional disruption of chondrogenesis and hematopoietic supporting activity, which we report here, might depend on diverse miR-204&211 targets. To investigate this hypothesis, permanent RUNX2 knock-down was performed. Sh-RUNX2 fully reproduced the phenotypes induced by miR-204&211, confirming that RUNX2 down modulation is the major event leading to the reported functional modification on our MSCs. It seems thus apparent that RUNX2, a recognized master gene for osteogenesis, might rule all four MSC commitment and differentiation processes. Hence, the formerly reported role of miR204&211 and RUNX2 in osteoporosis and obesity, coupled with our novel observation showing inhibition of cartilage differentiation and hematopoietic support, strikingly resemble the clinical traits of metabolic syndrome, where osteoarthritis, osteoporosis, anaemia and obesity occur together. Our observations, corroborating and extending previous observations, suggest that miR-204&211–RUNX2 axis in human MSCs is possibly involved in the pathogenesis of this rapidly growing disease in industrialized countries, for possible therapeutic intervention to regenerate former homeostasis