<i>In silico</i> identification of putative CGIs and characterization of the primary <i>mir-142</i> transcript.

<p>(<b>A</b>) Schematic representation of putative CGIs associated with <i>mir-142</i>. Each vertical line represents an individual CpG. Black, dark grey and light grey horizontal bars; CGIs predicted with CpG Island Explorer, CpG Island Searcher and CpGcluster, respectively. The numbers indicate the position relative to the <i>mir-142</i> precursor (<i>pre-mir-142</i>) (+1 to +87). O/E, observed/expected. (<b>B</b>) Schematic representation of the genomic <i>mir-142</i> region. The approximate 5′- end of <i>mir-142</i> was mapped using qPCR with assays specific for four different regions (depicted by vertical arrowheads). Grey and black arrowheads indicate increased expression or no change, respectively. The transcription start (TSS) and polyadenylation sites were identified using rapid amplification of cDNA ends. The approximate positions of the gene-specific primers (GSPs) and associated amplicons are indicated by horizontal arrowheads and attached lines, respectively. The putative CGIs are represented by horizontal bars. (<b>C</b>) Schematic representation of the 2 transcript variants of primary <i>mir-142</i> (<i>pri-mir-142</i>).</p

Endogenous transcript levels of mature miR-142-5p/3p and primary <i>mir-142</i> in cells of mesenchymal and hematopoietic origin, and after treatment with epigenetic modifiers.

<p>(<b>A</b>) Expression levels as determined by qPCR in tumorigenic cells (MG-63, OHS, IOR/OS14, U-2 OS, IOR/OS10, IOR/SARG, IOR/MOS and HOS), non-tumorigenic mesenchymal cells (iMSC#3, hMSCs and primary osteoblasts), and hematopoietic cells (K562 and PBPCs). Expression is depicted relative to the MG-63 cells (set to 1) and the numbers above the histograms represent the expression level of primary <i>mir-142</i>. <i>RNU44</i> or Glyceraldehyde 3-phosphate dehydrogenase (<i>GAPDH</i>) were used for normalization of mature and primary transcripts, respectively. iMSC#3, immortalized bone marrow-derived stromal cells; hMSCs, primary bone marrow-derived stromal cells; K562, K562 leukemia cells; PBPCs, peripheral blood progenitor cells. (<b>B</b>) Quantification of expression using qPCR after treatment with 5-Aza-2′-deoxycytidine (5-Aza). Expression is shown relative to untreated cells (set to 1). <i>RNU44</i> or <i>GAPDH</i> were used for normalization of mature and primary transcripts, respectively. (<b>C</b>) Expression levels of miR-142-3p, primary <i>mir-142</i> and mesoderm specific transcript (<i>MEST</i>) in MG-63 cells as determined by qPCR, after treatment with 5-Aza alone, a combination of 5-Aza and trichostatin A (TSA), or TSA alone. <i>RNU44</i> or <i>GAPDH</i> were used for normalization of mature and primary transcripts (including <i>MEST</i>), respectively. The error bars in all qPCR experiments show the standard deviation of technical replicates.</p

Analyses of methylation status of CGIs associated <i>mir-142</i> and <i>in vitro</i> methylation of its upstream regulatory region.

<p>(<b>A</b>) Methylation-specific PCR (MSP) analyses of the CGIs, before and after treatment with 5-Aza-2′-deoxycytidine (5-Aza). U and M, unmethylated and methylated products. Both standard gel images and 3D densitograms of the signal intensities are shown. (<b>B</b>) Schematic representation of the <i>mir-142</i> locus. Locations of MSP amplicon #1 and #2 are indicated by arrows. 18 CpGs (region #1) and 14 CpGs (region #2) were subjected to bisulfite sequencing. CpG-containing transcription factor binding motifs are enclosed by boxes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079231#pone-0079231-g003" target="_blank">Figure 3B</a> for more information). E-box, enhancer box. Numbers indicate the position relative to <i>pre-miR-142</i> (+1 to +87). (<b>C</b>) Bisulfite sequencing of DNA from MG-63 cells before and after treatment with 5-Aza for 72 hours, and untreated IOR/OS14 cells. The Wilcoxon signed rank test was used to test for statistical differences between treated and untreated MG-63 cells, as described in more details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079231#s2" target="_blank">Materials and Methods</a>. A <i>P</i> value ≤0.05 was considered as significant. (<b>D</b>) Bisulfite sequencing of immortalized bone marrow-derived stromal cells (iMSC#3) and primary osteoblasts. (<b>E</b>) Bisulfite sequencing of K562 leukemia- and peripheral blood progenitor cells. Black and white circles represent methylated and unmethylated CpGs, respectively, and each row represents a single clone. Grey circles, not determined. Ten clones were sequenced (n = 10), with the exception of MG-63 cells (region #1, n = 13; region #2, n = 12). (<b>F</b>) The 2,031 bp upstream region of <i>pre-mir-142</i> was cloned into the promoter-less luciferase reporter construct pCpGL-basic and <i>in vitro</i> methylated with M.SssI (pCpGL/2031_M.SssI) or mock-methylated (pCpGL/2031_mock). All three constructs were individually transfected into U-2 OS cells along with a <i>Renilla</i> reporter construct. The luciferase activity was measured after 48 hours and calculated relative to that of pCpGL-basic (set to 1). Each histogram shows the average relative luciferase activity, and the error bars show the standard deviation of biological experiments (n≥5). The Wilcoxon signed rank test was used to test for statistical differences and the <i>P</i> values are shown above the histograms. A <i>P</i> value ≤0.05 was considered as significant.</p

Preclinical Evaluation of the Pan-FGFR Inhibitor LY2874455 in FRS2-Amplified Liposarcoma

Background: FGFR inhibition has been proposed as treatment for dedifferentiated liposarcoma (DDLPS) with amplified FRS2, but we previously only demonstrated transient cytostatic effects when treating FRS2-amplified DDLPS cells with NVP-BGJ398. Methods: Effects of the more potent FGFR inhibitor LY2874455 were investigated in three DDLPS cell lines by measuring effects on cell growth and apoptosis in vitro and also testing efficacy in vivo. Genome, transcriptome and protein analyses were performed to characterize the signaling components in the FGFR pathway. Results: LY2874455 induced a stronger, longer-lasting growth inhibitory effect and moderate level of apoptosis for two cell lines. The third cell line, did not respond to FGFR inhibition, suggesting that FRS2 amplification alone is not sufficient to predict response. Importantly, efficacy of LY2874455 was confirmed in vivo, using an independent FRS2-amplified DDLPS xenograft model. Expression of FRS2 was similar in the responding and non-responding cell lines and we could not find any major difference in downstream FGFR signaling. The only FGF expressed by unstimulated non-responding cells was the intracellular ligand FGF11, whereas the responding cell lines expressed extracellular ligand FGF2. Conclusion: Our study supports LY2874455 as a better therapy than NVP-BGJ398 for FRS2-amplified liposarcoma, and a clinical trial is warranted

Preclinical Evaluation of the Pan-FGFR Inhibitor LY2874455 in FRS2-Amplified Liposarcoma

Background: FGFR inhibition has been proposed as treatment for dedifferentiated liposarcoma (DDLPS) with amplified FRS2, but we previously only demonstrated transient cytostatic effects when treating FRS2-amplified DDLPS cells with NVP-BGJ398. Methods: Effects of the more potent FGFR inhibitor LY2874455 were investigated in three DDLPS cell lines by measuring effects on cell growth and apoptosis in vitro and also testing efficacy in vivo. Genome, transcriptome and protein analyses were performed to characterize the signaling components in the FGFR pathway. Results: LY2874455 induced a stronger, longer-lasting growth inhibitory effect and moderate level of apoptosis for two cell lines. The third cell line, did not respond to FGFR inhibition, suggesting that FRS2 amplification alone is not sufficient to predict response. Importantly, efficacy of LY2874455 was confirmed in vivo, using an independent FRS2-amplified DDLPS xenograft model. Expression of FRS2 was similar in the responding and non-responding cell lines and we could not find any major difference in downstream FGFR signaling. The only FGF expressed by unstimulated non-responding cells was the intracellular ligand FGF11, whereas the responding cell lines expressed extracellular ligand FGF2. Conclusion: Our study supports LY2874455 as a better therapy than NVP-BGJ398 for FRS2-amplified liposarcoma, and a clinical trial is warranted

A PML/Slit Axis Controls Physiological Cell Migration and Cancer Invasion in the CNS

Cell migration through the brain parenchyma underpins neurogenesis and glioblastoma (GBM) development. Since GBM cells and neuroblasts use the same migratory routes, mechanisms underlying migration during neurogenesis and brain cancer pathogenesis may be similar. Here, we identify a common pathway controlling cell migration in normal and neoplastic cells in the CNS. The nuclear scaffold protein promyelocytic leukemia (PML), a regulator of forebrain development, promotes neural progenitor/stem cell (NPC) and neuroblast migration in the adult mouse brain. The PML pro-migratory role is active also in transformed mouse NPCs and in human primary GBM cells. In both normal and neoplastic settings, PML controls cell migration via Polycomb repressive complex 2 (PRC2)-mediated repression of Slits, key regulators of axon guidance. Finally, a PML/SLIT1 axis regulates sensitivity to the PML-targeting drug arsenic trioxide in primary GBM cells. Taken together, these findings uncover a drug-targetable molecular axis controlling cell migration in both normal and neoplastic cells

Disruption Of PML Nuclear Bodies Cooperates In The Pathogenesis Of Acute Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) is characterised by the t(15;17)(q22;q21) leading to fusion of PML to the gene encoding the myeloid transcription factor Retinoic Acid Receptor α (RARα). Chromosomal translocations such as the t(15;17) are considered to be initiating events in leukemogenesis; however, sequencing of APL genomes has provided further evidence that the PML-RARα fusion is insufficient to induce leukemia, which depends upon the acquisition of cooperating mutations. The PML-RARα oncoprotein exerts a profound effect on nuclear architecture, disrupting multiprotein structures known as PML nuclear bodies (NBs). The function of these structures remains an enigma; however, their disruption in PML-RARα+ APL and acute lymphoblastic leukemia with the t(9;15)(p13;q24)/PAX5-PML fusion is associated with delocalisation of a number of component proteins including PML, which have been implicated in growth control and neoplastic transformation. It is now established that the PML moiety contributes to APL pathogenesis by conferring via the translocation a novel dimerisation capacity to RARα, but it has been unclear whether deregulation of PML and other NB components cooperates in leukemic transformation or impacts the response to differentiating agents. To address these questions, we generated a knock-in mouse model with targeted NB disruption achieved through mutation of key zinc-binding cysteine residues in the amino-terminal RING domain of Pml. Homozygous Pml RING mutant mice are viable, with no overt developmental defect; however, analysis of the bone marrow revealed significant expansion of the Lin(-)Sca-1(+)c-Kit(+) (LSK) population compared to wild type (WT) controls (p<0.01), accompanied by increased LSK cell proliferation (p<0.0001) as evaluated by in vivo labelling through incorporation of 5-ethynyl-2'-deoxyuridine (EdU). In addition, hematopoietic cells derived from homozygous Pml RING mutant mice exhibited markedly elevated levels of DNA damage compared to WT cells from age-matched controls, as evidenced by increased numbers of γH2AX foci (p=0.009). This was associated with significantly delayed DNA damage repair responses in Pml RING mutant cells following γ-irradiation (p=0.005). Accordingly, expression of PML-RARα in human hematopoietic cells, which led to disruption of NBs, also induced a significant increase in γH2AX foci (p=0.0023). While no leukemias arose in homozygous Pml RING mutant mice, they developed an excess of T- and B-cell lymphomas (p=0.03), consistent with the proposed tumour suppressor function of PML and the NBs. Since a key property conferred by the PML moiety required for leukemogenicity of the PML-RARα oncoprotein is the capacity to dimerise, we evaluated whether Pml NB disruption could cooperate with forced RARα homodimerisation (mediated artificially by linking RARα to the p50 dimerisation motif of NFκB). While Pml NB disruption or p50-RARA expressed under the control of the MRP8 promoter in murine hematopoietic stem/progenitor cells conferred limited replating capacity, in combination they exhibited marked cooperativity, with a significant increase in third round colonies (p=0.03). Moreover, NB disruption was found to cooperate with forced RARα homodimerisation in vivo with a doubling in the rate of leukemia development in p50-RARα mice with mutated Pml (p<0.0001), leading to a penetrance comparable to that observed in previously published PML-RARα transgenic models. Moreover, the latency to onset of leukemia was significantly shorter in p50-RARα mice with the Pml RING mutation, occurring from 213 days of age vs 310 days with WT Pml (p=0.008). While Pml NB disruption did not affect engraftment of p50-RARα leukemias in serial transplantation, the in vitro differentiation response of p50-RARα leukemias to All transretinoic acid (ATRA) as determined by nitroblue tetrazolium assay was significantly impaired in the context of NB disruption (p<0.05). Moreover, prolongation of survival following ATRA treatment in mice transplanted with p50-RARα leukemic blasts was dependent upon Pml NB integrity (p=0.03). Overall, these data suggest that the NB disruption mediated by the PML-RARα oncoprotein plays a key role in APL pathogenesis contributing to expansion of the LSK population and defective DNA repair predisposing to the acquisition of cooperating mutations, but also implicate NBs in the response to differentiating agents

Disruption Of PML Nuclear Bodies Cooperates In The Pathogenesis Of Acute Promyelocytic Leukemia

Abstract Acute promyelocytic leukemia (APL) is characterised by the t(15;17)(q22;q21) leading to fusion of PML to the gene encoding the myeloid transcription factor Retinoic Acid Receptor α (RARα). Chromosomal translocations such as the t(15;17) are considered to be initiating events in leukemogenesis; however, sequencing of APL genomes has provided further evidence that the PML-RARα fusion is insufficient to induce leukemia, which depends upon the acquisition of cooperating mutations. The PML-RARα oncoprotein exerts a profound effect on nuclear architecture, disrupting multiprotein structures known as PML nuclear bodies (NBs). The function of these structures remains an enigma; however, their disruption in PML-RARα+ APL and acute lymphoblastic leukemia with the t(9;15)(p13;q24)/PAX5-PML fusion is associated with delocalisation of a number of component proteins including PML, which have been implicated in growth control and neoplastic transformation. It is now established that the PML moiety contributes to APL pathogenesis by conferring via the translocation a novel dimerisation capacity to RARα, but it has been unclear whether deregulation of PML and other NB components cooperates in leukemic transformation or impacts the response to differentiating agents. To address these questions, we generated a knock-in mouse model with targeted NB disruption achieved through mutation of key zinc-binding cysteine residues in the amino-terminal RING domain of Pml. Homozygous Pml RING mutant mice are viable, with no overt developmental defect; however, analysis of the bone marrow revealed significant expansion of the Lin(-)Sca-1(+)c-Kit(+) (LSK) population compared to wild type (WT) controls (p&lt;0.01), accompanied by increased LSK cell proliferation (p&lt;0.0001) as evaluated by in vivo labelling through incorporation of 5-ethynyl-2'-deoxyuridine (EdU). In addition, hematopoietic cells derived from homozygous Pml RING mutant mice exhibited markedly elevated levels of DNA damage compared to WT cells from age-matched controls, as evidenced by increased numbers of γH2AX foci (p=0.009). This was associated with significantly delayed DNA damage repair responses in Pml RING mutant cells following γ-irradiation (p=0.005). Accordingly, expression of PML-RARα in human hematopoietic cells, which led to disruption of NBs, also induced a significant increase in γH2AX foci (p=0.0023). While no leukemias arose in homozygous Pml RING mutant mice, they developed an excess of T- and B-cell lymphomas (p=0.03), consistent with the proposed tumour suppressor function of PML and the NBs. Since a key property conferred by the PML moiety required for leukemogenicity of the PML-RARα oncoprotein is the capacity to dimerise, we evaluated whether Pml NB disruption could cooperate with forced RARα homodimerisation (mediated artificially by linking RARα to the p50 dimerisation motif of NFκB). While Pml NB disruption or p50-RARA expressed under the control of the MRP8 promoter in murine hematopoietic stem/progenitor cells conferred limited replating capacity, in combination they exhibited marked cooperativity, with a significant increase in third round colonies (p=0.03). Moreover, NB disruption was found to cooperate with forced RARα homodimerisation in vivo with a doubling in the rate of leukemia development in p50-RARα mice with mutated Pml (p&lt;0.0001), leading to a penetrance comparable to that observed in previously published PML-RARα transgenic models. Moreover, the latency to onset of leukemia was significantly shorter in p50-RARα mice with the Pml RING mutation, occurring from 213 days of age vs 310 days with WT Pml (p=0.008). While Pml NB disruption did not affect engraftment of p50-RARα leukemias in serial transplantation, the in vitro differentiation response of p50-RARα leukemias to All transretinoic acid (ATRA) as determined by nitroblue tetrazolium assay was significantly impaired in the context of NB disruption (p&lt;0.05). Moreover, prolongation of survival following ATRA treatment in mice transplanted with p50-RARα leukemic blasts was dependent upon Pml NB integrity (p=0.03). Overall, these data suggest that the NB disruption mediated by the PML-RARα oncoprotein plays a key role in APL pathogenesis contributing to expansion of the LSK population and defective DNA repair predisposing to the acquisition of cooperating mutations, but also implicate NBs in the response to differentiating agents. Disclosures: No relevant conflicts of interest to declare

HSP90 inhibition blocks ERBB3 and RET phosphorylation in myxoid/round cell liposarcoma and causes massive cell death in vitro and in vivo

Myxoid sarcoma (MLS) is one of the most common types of malignant soft tissue tumors. MLS is characterized by the FUS-DDIT3 or EWSR1-DDIT3 fusion oncogenes that encode abnormal transcription factors. The receptor tyrosine kinase (RTK) encoding RET was previously identified as a putative downstream target gene to FUS-DDIT3 and here we show that cultured MLS cells expressed phosphorylated RET together with its ligand Persephin. Treatment with RET specific kinase inhibitor Vandetanib failed to reduce RET phosphorylation and inhibit cell growth, suggesting that other RTKs may phosphorylate RET. A screening pointed out EGFR and ERBB3 as the strongest expressed phosphorylated RTKs in MLS cells. We show that ERBB3 formed nuclear and cytoplasmic complexes with RET and both RTKs were previously reported to form complexes with EGFR. The formation of RTK hetero complexes could explain the observed Vandetanib resistence in MLS. EGFR and ERBB3 are clients of HSP90 that help complex formation and RTK activation. Treatment of cultured MLS cells with HSP90 inhibitor 17-DMAG, caused loss of RET and ERBB3 phosphorylation and lead to rapid cell death. Treatment of MLS xenograft carrying Nude mice resulted in massive necrosis, rupture of capillaries and hemorrhages in tumor tissues. We conclude that complex formation between RET and other RTKs may cause RTK inhibitor resistance. HSP90 inhibitors can overcome this resistance and are thus promising drugs for treatment of MLS/RCLS.status: publishe