Abstract
Background
Previously, the chromosomal translocation t(12;15)(p13;q25) has been found to recurrently occur in both solid tumors and leukemias. This translocation leads to ETV6-NTRK3 (EN) gene fusions resulting in ectopic expression of the NTRK3 neurotropic tyrosine receptor kinase moiety as well as oligomerization through the donated ETV6-sterile alpha motif domain. As yet, no in vitro cell line model carrying this anomaly is available. Here we genetically characterized the acute promyelocytic leukemia (APL) cell line AP-1060 and, by doing so, revealed the presence of a t(12;15)(p13;q25). Subsequently, we evaluated its suitability as a model for this important clinical entity.
Methods
Spectral karyotyping, fluorescence in situ hybridization (FISH), and genomic and transcriptomic microarray-based profiling were used to screen for the presence of EN fusions. qRT-PCR was used for quantitative expression analyses. Responses to AZ-23 (NTRK) and wortmannin (PI3K) inhibitors, as well as to arsenic trioxide (ATO), were assessed using colorimetric assays. An AZ-23 microarray screen was used to define the EN targetome, which was parsed bioinformatically. MAPK1 and MALAT1 activation were assayed using Western blotting and RNA-FISH, respectively, whereas an AML patient cohort was used to assess the clinical occurrence of MALAT1 activation.
Results
An EN fusion was detected in AP1060 cells which, accordingly, turned out to be hypersensitive to AZ-23. We also found that AZ-23 can potentiate the effect of ATO and inhibit the phosphorylation of its canonical target MAPK1. The AZ-23 microarray screen highlighted a novel EN target, MALAT1, which also proved sensitive to wortmannin. Finally, we found that MALAT1 was massively up-regulated in a subset of AML patients.
Conclusions
From our data we conclude that AP-1060 may serve as a first publicly available preclinical model for EN. In addition, we conclude that these EN-positive cells are sensitive to the NTRK inhibitor AZ-23 and that this inhibitor may potentiate the therapeutic efficacy of ATO. Our data also highlight a novel AML EN target, MALAT1, which was so far only conspicuous in solid tumors.
Electronic supplementary material
The online version of this article (10.1007/s13402-017-0356-2) contains supplementary material, which is available to authorized users.
Keywords: APL, ETV6, MALAT1, MAPK1, NTRK3
Introduction
The ETV6 (ETS variant gene 6) gene located at 12p13 has been found to be involved in various translocations in human malignancies. Through a recurrent t(12;15)(p13;q25) the N-terminal sterile alpha motif (SAM) dimerization domain of ETV6 fuses to the C-terminal protein tyrosine kinase (PTK) domain of the neurotrophin-3 transmembrane receptor NTRK3. This fusion (EN) has been encountered in both solid and hematologic malignancies, including fibrosarcoma, secretory breast carcinoma and its salivary gland analogue, colorectal carcinoma, congenital mesoblastic nephroma, ductal carcinoma, glioblastoma, papillary thyroid carcinoma and radiation-induced thyroid cancer, as well as in acute myeloid leukemia (AML) [1–7]. The ETV6 SAM domain promotes oligomerization, NTRK3 auto-phosphorylation and ligand-independent activation [4, 8, 9], leading to constitutive activation of two major signaling pathways, i.e., the RAS/MAPK [10] and the PI3K/AKT [11] pathways, both affecting cellular growth and survival [3].
Exclusion of a single ETV6 exon distinguishes AML from solid tumor fusion products. In EN transgenic mouse models, it has been found that the AML form (lacking exon 5) enhances hematopoietic stem cell renewal and initiates leukemia, while the solid tumor form (including exon 5) results in embryonic lethality with impaired angiogenesis and hematopoiesis [12]. While leukemic EN fusions have hitherto been found to be confined to adult AML-M0/M2 cases [7, 13], we here report the occurrence of a similar rearrangement in an acute promyelocytic leukemia (APL)-derived cell line, AP-1060. In addition, we characterize its putative signaling networks using a selective NTRK inhibitor, AZ-23 [14] and identify MALAT1 [15, 16] as a new EN target. We also show that this long non-coding RNA may incidentally be over-expressed in primary AML cases, thereby highlighting NTRK3 involvement.
Materials and methods
Cell culture
All cell lines used were drawn from the DSMZ repositories, authenticated by DNA STR profiling and cytogenetics and cultured according to standard DSMZ protocols (www.dsmz.de). The AP-1060 and NB-4 cell lines, both harboring a t(15;17)(q22;q21) and a PML-RARA fusion, have been described elsewhere [17].
Patients
In total 70 de novo AML patients, diagnosed and classified according to the WHO criteria in the First Affiliated Hospital of Soochow University (FAHSU, China) from May 2012 to June 2014, were enrolled into this study, together with 11 healthy control bone marrow donors. This study was approved by the ethics committee of FAHSU in accordance with the Declaration of Helsinki.
Cell proliferation assays
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma, Taufkirchen, Germany) cell proliferation assays were performed. To this end, micro-well cultures (100 μl) were incubated with 10 μl MTT solution (5 mg/ml in PBS) and stopped after 4 h by the addition of 120 μl DMSO/isopropanol. After this, the absorbance was measured at 570 nm using an ELISA reader (Thermo Electron, Vantaa, Finland). Cell viabilities were measured using a standard Trypan Blue exclusion assay. Cells were treated with ATO (Sigma) dissolved in 1 M NaOH neutralized with 1 M HCl, AZ-23 [14] dissolved in 0.1% DMSO, wortmannin (Sigma) dissolved in 0.1% DMSO and IL-32α (R&D Systems, Abingdon, UK) dissolved in PBS.
Cytogenetics
Conventional cytogenetic analyses, fluorescence in situ hybridization (FISH) and Spectral Karyotyping (SKY) were performed as described previously [18–20]. Slides were mounted in Vectashield (Vector Labs, Burlinghame, CA, USA) containing 0.01% DAPI (4′,6-diamidino-2-phenylindole) (Sigma) and images were recorded using a Zeiss Axioimager microscope (Zeiss, Oberkochen, Germany) equipped with a HiSKY imaging system (Applied Spectral Imaging, Neckarhausen, Germany).
qRT-PCR
qRT-PCR analyses were performed as reported before [21]. Briefly, total RNA was extracted using TRIzol reagent (Invitrogen/Thermo Fischer, Darmstadt, Germany). cDNA was synthesized from 5 μg RNA by random priming using Superscript II (Invitrogen). Subsequent qRT-PCR analyses were performed using a 7500 Real-time System with commercial buffer and primer sets (Applied Biosystems, Life Technologies, Darmstadt, Germany). For expression level normalization the TBP and ABL1 genes were used. All quantitative analyses were performed in triplicate. Standard deviations are presented in the figures as error bars. Statistical significance was assessed by Student’s t-test and the calculated p-values are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant). Relative quantitative expression analyses were performed using the Applied Biosystems software tool (ΔΔCt).
Rapid amplification of cDNA ends (RACE) PCR
RACE PCR analyses [22] were performed according to the manufacturer’s protocol (Invitrogen). Primer sequences are available from the authors.
RNA sequencing
mRNA was prepared using a TruSeq RNA Preparation Kit (Illumina, San Diego, CA, USA). Libraries were prepared and sequenced on an Illumina HiSeq 2500 system. Paired-end reads were aligned to the human grch37-lite reference sequence, Refseq, and combined sequence files using the Burrows Wheeler Aligner (0.5.10).
RNA FISH
The RNA FISH probe for MALAT1 was purchased from Biosearch Technologies (Novato, CA, USA) and used to visualize RNA in nuclear speckles. Hybridizations were performed according to the manufacturer’s instructions. Briefly, slides were hybridized overnight with 50 pmol probe in 20 μl hybridization buffer, washed in RNAse-free PBS, dehydrated in ethanol, post-fixed in 1% paraformaldehyde (Life Technologies, Darmstadt, Germany) in PBS, and treated for 2 min with 0.1% Tritox-X100 (Serva, Heidelberg, Germany). The resulting slides were mounted and counterstained as described above for DNA FISH.
Western blotting
Proteins were extracted from cell lysates using a SIGMAFast protease inhibitor cocktail (Sigma), separated by polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder in standard phosphate-buffered saline buffer. For protein detection, the following antibodies were used: anti-alpha-Tubulin (Sigma), anti-MAPK1 and anti-pMAPK1 (both Santa Cruz Biotechnology, Heidelberg, Germany). As a loading control, the Western blots were reversibly stained with Poinceau (Sigma). Secondary antibodies were linked to peroxidase for ECL detection (Perkin Elmer, Waltham, MA, USA). For imaging a ChemoStar Imager (INTAS, Göttingen, Germany) was used.
Results and discussion
Next to the previously reported 46,XY,t(3;14)(p21;q11.2),der(15)t(15;17)(q22;q21) karyotype of AP-1060 cells [17], we detected a cryptic t(12;15)(p13;q25) (Fig. 1a, b) using spectral karyotyping (SKY). By subsequently using the BIOMED-1 reverse transcriptase (RT)-PCR protocol [23] a standard L-form PML-RARA (but not RARA-PML) fusion mRNA was detected (data not shown). FISH analysis (Fig. S1A/B) revealed a 4-break rearrangement involving 12p13, 15q22, 15q25 and 17q21, by which clone G248P82470G7 was split through the 12p13 break within the ETV6 gene between exons 4 and 5 (Fig. 1b), implying juxtaposition to a fusion partner at 15q25. To identify this putative fusion partner, 3′-RACE was performed. The resulting sequence products revealed an in-frame fusion between exon 2 of ETV6 and exon 18 of NTRK3. Nested RT-PCR using ETV6- and NTRK3-specific primers revealed two products, of which the respective sequences showed an in-frame fusion of exon 4 of ETV6 to exon 14 of NTRK3, and an out-of-frame fusion of exon 2 of ETV6 to exon 18 of NTRK3, the former predominating (Fig. 1c). The AP-1060 cells were found to be negative for the expression of reciprocal NTRK3-ETV6 transcripts, as revealed by nested RT-PCR, consistent with the cytogenetic data which place the 15q25 breakpoint near exon 13 of NTRK3. Using aCGH, we found that both the NTRK3 and PML genes bear micro-deletions at/near their breakpoints (Fig. S2). Taken together, we conclude that AP-1060 cells carry a t(12;15)(p13,q25) resulting in an ETV6-NTRK3 gene fusion.
Fig. 1.
Key genetic alterations in AP-1060 cells. (a) Spectral karyotyping (SKY) showing genomic rearrangements present in AP-1060 cells (G-banding, raw and pseudocolored images) with rearranged chromosomes indicated by arrows. Note a four-break rearrangement involving chromosomes 12, 15 and 17 resulting in ETV6-NTRK3 (EN) and PML-RARA fusions: der(12)t(12;15)(p13;q25), der(15)t(15;17)(q22;q21), der(17)t(15;17)(q22;q21)t(15;12)(q25;p13). (b) BAC clones and fosmids used for mapping the t(12;15)(p13;q25), and a labeling scheme adopted for FISH. Arrows mark the breakpoints. (c) RT-PCR detection of alternately spliced EN fusion transcripts (upper image, arrows): type I, 1151 bp and type II, 303 bp in AP-1060 cells. NB-4 cells tested negative for these transcripts. RT-PCR detection of fusions between exon 4 of ETV6 and exon 14 of NTRK3 and between exon 2 of ETV6 and exon 18 of NTRK3. (lower image)
Through aCGH, additional non-polymorphic deletions affecting multiple loci were detected, i.e., at Xq24 (bi-allelic loss of exons 6–8 of IL13RA1), 2q36 (mono-allelic losses of AGFG1, COL4A4, IRS1), 3q26 (mono-allelic loss of MECOM), 5p15 (mono-allelic loss of TERT), 6p22 (mono-allelic loss of HLA), 11p13 (bi-allelic loss of WT1/WT1as) and 11p15 (mono-allelic loss of TRIM22). To assess their putative relevance, microarray-based expression data of the AP-1060 cells were compared with those of another PML-RARA-positive APL cell line (NB-4) and a panel of AML cell lines lacking either PML-RARA or ETV6-NTRK3 rearrangements (Fig. S3). We found that the expression levels of the genes within most loci remained unaltered, except WT1, which was silenced by a homozygous deletion, and MECOM which was affected by an intronic micro-deletion, surpassing even MUTZ-3 cells carrying an inv(3)(q21q26) [24]. Although tested normal by FISH (Fig. S4), it should be mentioned that cryptic MECOM rearrangement(s) cannot be excluded, including those affecting regulatory motifs (https://genome.ucsc.edu/). Finally, it should be noted that among the AML samples tested NTKR3 activation was unique to AP-1060 cells (Fig. S3), which can directly be attributed to its fusion with ETV6 which, in turn, is ubiquitously expressed in hematologic malignancies.
To gauge a potential therapeutic relevance of the NTRK3 rearrangement, AP-1060, NB-4 and a panel of other leukemic cell lines were treated with AZ-23, a selective NTRK inhibitor [14]. A subsequent proliferation assay confirmed an anticipated increased sensitivity of AP-1060 cells among the panel of cell lines covering various leukemic entities (Fig. 2a), indicating both its growth-dependence on EN (addiction) and its potential druggability. Given the reported resistance of AP-1060 cells to arsenic trioxide (ATO) [17], we next set out to assess the contribution of NTRK3 activation to this resistance. To this end, the responses of AP-1060 and NB-4 cells to ATO treatment in combination with AZ-23 were compared (Fig. 2b). We did not observe an increased ATO resistance in AP-1060 cells, since the ATO responses in both cell lines overlapped. Interestingly, however, we found that exposure to low amounts of AZ-23 significantly increased the toxicity of ATO in only AP-1060 cells. From these results we conclude that AZ-23 may potentiate the inhibitory effects of ATO in EN-positive cells, a finding that may be of potential clinical relevance. The observed potentiation of ATO by AZ-23 may reflect its inhibition of the PI3K/AKT/NF-κB axis [25], or its effect on p38 MAPK/AP-1 signaling [26]. The putative therapeutic implications of ATO potentiation by selective EN inhibition warrants confirmation in other experimental and/or pre-clinical systems.
Fig. 2.
Cell proliferation assays. (a) MTT responses to AZ-23 show a conspicuous sensitivity of ETV6-NTRK3 (EN)-positive AP-1060 cells, while both normal control and EN-negative leukemic cell lines showed lower levels of inhibition at the doses tested. For clarity, the error bars of the controls are omitted. The cells tested were: AML, SKNO-1; APL, AP-1060, NB-4; B-cell neoplasia, FARAGE, HG-3, KARPAS-1106P, U-2940; T-cell neoplasia, T8ML-1, TLBR-2; and normal B−/T- lymphoblastoid cells NC-NC and 39-HVS. (b) Data showing overlapping sensitivities of AP-1060 and NB-4 cells to ATO (solid lines). Simultaneous administration of ATO is accompanied by increases in growth inhibition by AZ-23 (broken lines) in EN-positive cells. Error bars are omitted for clarity. Abbreviation: vc, vehicle control
In order to more broadly investigate the EN targetome, genome-wide microarray-based expression data were compared between AP-1060 and NB-4 cells, as well as other AML-derived cell lines lacking PML-RARA gene fusions (Fig. S5). We found that, in addition to the MECOM and NTRK3 genes discussed above, in AP-1060 cells several genes with a known/potential leukemic relevance were over-expressed, i.e., ALOX5, BAALC, BRAF, CCR6, CD200, HEG1, IGLL1/CD179B, IL1R1, IL32, OSMR, SELL, SLITRK4 and SOCS3.
Since oncogenic signaling in leukemic cells by EN may involve the MAPK1/ERK pathway [4], we set out to investigate whether MAPK1 activation in EN cells is sensitive to AZ-23 treatment. We found that AZ-23 dose-responsively impaired MAPK1 phosphorylation in AP-1060 cells, but not its expression. In control HG-3 cells lacking EN, we found that the MAPK1/pMAPK1 ratio remained impervious to AZ-23 treatment at both endpoints (Fig. 3a), similar to NB-4 cells (data not shown). Thus, consistent with canonical EN signaling driven by gene fusion, MAPK1 signaling requires intact NTRK3 activity in AP-1060 cells.
Fig. 3.
Responses of AP-1060 cells to AZ-23: congruence with the NTRK3 targetome. (a) MAPK1 activation requires intact ETV6-NTRK3 (EN) signaling in AP-1060 cells. Western blot showing a dose-response dependent reduction in MAPK1 activation (phosphorylation) in AP-1060 cells after sub-toxic AZ-23 treatment, while MAPK1 protein expression remains unaltered. Note the unperturbed pMAPK1/MAPK1 ratios in control HG-3 cells. Alpha-tubulin is included as a loading control. Data from three experiments are shown. (b) Expression of MALAT1 in EN-positive cells is sensitive to NTRK (AZ-23) and PI3K (wortmannin) inhibition, respectively. qRT-PCR was performed using oligos matching with two 3′-MALAT1 transcripts highlighted by the microarray data and other reports [27, 28] (sequences are available from the authors upon request). (c) RNA FISH to validate the targeting of MALAT1 by AZ-23. DAPI, FISH and composite images of MALAT1 RNA in nuclei of AP-1060 and NB-4 cells untreated (top), vehicle control treated (middle) and AZ-23 treated (bottom; 1.25 μM for 72 h) are shown. Note weaker diffuse MALAT1 staining in NB-4 cells (including higher background) impervious to AZ-23, compared to fine homogeneous granular staining by stronger, but fewer, marginal signals in AP-1060 cells. (d) MALAT1 expression in normal control and AML patient groups, t(15;17)-positive, t(8;21)-positive, inv(16)-positive and others. Note MALAT1 activation in AML patients only, in particular in three from each of the main cytogenetic groups. The cytogenetic categories and numbers of patients were as follows: inv(16)(p13.1q22) /t(16;16)(p13.1;q22), n = 8; t(8;21)(q22;q22), n = 12; t(15;17)(q22;q12) n = 10; others, including, t(9;11)(p22;q23), t(6;9)(p23;q34), inv(3)(q21q26.2) or t(3;3)(q21;q26.2), t(1;22)(p13;q13), t(9;22)(q34;q11), n = 40. The median age of the cases was 39 years (range 14–80 years)
In order to select bona fide EN targets, an AZ-23 screen was devised. Accordingly, the transcriptomes of AP-1060 and NB-4 cells were compared after sub-toxic AZ-23 treatment. The genes that were most exclusively deregulated in AP-1060 cells were subsequently ranked and the 300 top/bottom outliers were selected for gene-set analysis. The top/bottom 100 are depicted in Fig. S6. From the top AZ-23 targets, this exercise highlighted MALAT1, a long non-coding RNA that has been widely implicated in solid tumor development [15, 16, 27, 28]. Weaker inhibitory candidates included ESR1 and MLL (not shown). We also found that NTRK3 expression per se was unaffected, which is consistent with its post-transcriptional mode of activation [14]. The same (transcription-led) negative gene-set (i.e., candidates for NTRK3 support) when seeded with NTRK3 and MAPK1, respectively, convincingly self-assembled a network centered on MAPK1 using STRING algorithms (Fig. S7A). This gene-set evidenced networking enrichment (p = 0.003) highlighted multiple GO terms, including, “sequence-specific DNA binding by a transcription factor” (FDR = 0.022) and KEGG-Pathway “proteoglycans in cancer” (FDR = 0.0416) (Table S1A/B). Subsequent bioinformatic analysis at PathwayNet using nodal genes (ESR1, MAPK1, SMAD3, SRF) yielded interactograms for linkage at high probabilities via phosphorylation using post-translational and transcriptional algorithms (Fig. S7B), thus highlighting hypothetical gene networks for experimental validation. In short, we found that MAPK1 may be nodal to NTRK3 signaling.
Validation of the microarray-based expression data was performed using qRT-PCR. While MALAT1 expression in NB-4 (and HG-3) cells (both lacking EN) showed no significant perturbations, in AP-1060 cells MALAT1 expression was found to be significantly reduced after AZ-23 treatment (Fig. 3b upper panels). To investigate whether MALAT1 is directly regulated by EN via PI3K signaling, its expression was measured in cells treated with the PI3K inhibitor wortmannin (Fig. 3b lower panels). Similar to AZ-23, we found that wortmannin inhibited MALAT1 expression in AP-1060 cells but not in NB-4 cells, implicating an involvement of PI3K signaling in MALAT1 activation in EN-positive cells. Collectively, these findings indicate that MALAT1 may be subject to canonical NTRK regulation. Since MALAT1 expression is intra-nuclear, RNA FISH was used to subsequently assess the potential impact of AZ-23 treatment on its nuclear expression in AP-1060 cells. We found that, while equivalent AZ-23 treatment elicited weaker paraspeckling in NB-4 cells, nuclear MALAT1 RNA signals in AP-1060 cells, though paradoxically stronger, became sparse and marginalized to nuclear lamina zones (Fig. 3c). Thus, we conclude that EN influences intra-nuclear speckling of MALAT1. We also found that the pMAPK1 immunofluorescence signals were suppressed by AZ-23 in AP-1060 cells only (Fig. S8). Although nuclear PML speckling (bioinformatically invoked as MAPK1 activation target [Fig. S7A]) is ipso facto diminished in AP-1060 cells, this diminished speckling remained un-restored by AZ-23 treatment. Since its cytoplasmic expression excludes co-localization with MALAT1 (Fig. S8), direct interaction at this level can be excluded. Collectively, these findings indicate that nuclear expression of MAPKI is inhibited by AZ-23 in EN-positive cells.
Since little is known regarding its regulation in hematopoietic malignancies, we set out to assess MALAT1 expression in bone marrow mononuclear cells derived from 70 AML patients at presentation compared to healthy donors. While MALAT1 expression was found to be low in the control samples, in all AML subgroups included (inv(16)-, t(8;21)- and t(15;17)-positive, and those bearing other changes) MALAT1 was found to be moderately expressed in a significant proportion of the patients, in the last group trending to significance (Fig. 3d). Moreover, we found that in each of the three main cytogenetic subgroups one patient showed a strikingly high MALAT1 expression level. From two of these three MALAT1 HIGH patients sufficient material was available for quantitative gene expression analysis. By doing so, we found that the NTRK3 level was dramatically raised in one of the MALAT1 HIGH patients, whereas in the MALAT1 LOW patients NTRK3 expression was low (Fig. S9). The NTRK3 HIGH/MALAT1 HIGH patient carried a t(15;17), whereas the MALAT1 HIGH/NTRK3 LOW patient carried a t(8;21). Subsequent RNA-sequencing of the NTRK3 HIGH/MALAT1 HIGH sample confirmed the presence of a PML-RARA fusion, whereas no ETV6-NTRK3 fusion was detected, indicating that the NTRK3 activation in this sample must have been brought about by an alternative mechanism (Table S2). Thus, we found that MALAT1 is activated in a subset of AML patients and that NTRK3 activation may serve as a contributory factor. Longitudinal studies are underway of the MALAT1 HIGHAML patients described here to evaluate a putative significance of MALAT1 as prognostic biomarker, as well as its possible association with NTRK3 activation.
While this is the first report linking NTRK3 to MALAT1 expression, public Boolean Gene Regulatory Network data (which include elusive L-shaped correlations [29, 30]) indicate a positive transcriptional correlation between NTRK3 and MALAT1 in cancer (http://crookneck.stanford.edu/microarray/interactionsU133Plus-new/), i.e., at low MALAT1 levels NTRK3 was mostly found be silent, while increased NTRK3 expression was found to be accompanied by MALAT1 up-regulation at supra-median levels, consistent with a threshold effect (Fig. S10). Overall, these data indicate high-level NTRK3 and MALAT1 co-expression in cancer patients.
Since MALAT1 binds to active chromatin, its intra-nuclear relocation may affect transcription in general [28], via MAPK1 signaling [25] or via TP53 signaling [26] as revealed bioinformatically. An emerging role for MALAT1 in leukemia/lymphoma development has been substantiated by its epigenetic association with EZH2 in mantle cell lymphoma [31], by maintaining proliferation potential during early hematopoiesis [26], and by overcoming p53-dependent G1/S DNA damage checkpoint control in multiple myeloma [32], supporting the AZ-23 signature described here.
In summary, we re-mapped the genomic landscape of the APL cell line AP-1060 and, by doing so, detected an ETV6-NTRK3 (EN) gene fusion. This is the first publicly available in vitro model for this key cytogenetic entity, which may uniquely affect both solid and hematologic malignancies. Next, we found that AP-1060 cells are hypersensitive to a selective NTRK inhibitor (AZ-23), confirming both the importance of NTRK3 signaling in these cells and the potential druggability of EN-positive cases by selective small molecule inhibitors. We also found that AZ-23 potentiated the in vitro cytostatic activity of the standard APL therapeutic agent ATO, a finding that may be of potential clinical relevance. The transcriptional responses of the cells to AZ-23 and wortmannin were consistent with canonical MAPK1 and PI3K signaling, respectively, which are both confirmed downstream targets of NTRK3 [11]. The same inhibitors unveiled a potential new NTRK3 target, MALAT1, whose role is hitherto best known in solid tumors. Publicly available data have already indicated that high MALAT1 expression is correlated with NTRK3 activation. Importantly, we found that MALAT1 was highly up-regulated in 3/70 AML patients investigated, in one together with NTRK3 activation. Taken together, we conclude that AP-1060 may serve as the first publicly available EN model, rendering this cell line into a suitable preclinical tool for investigating the underlying biology and therapeutic options for this important, yet puzzling, malignant entity.
Electronic supplementary material
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FISH of ETV6 and NTKR3. FISH for ETV6 and PML (RP11-96B23) (A) and NTRK3 (B) yield breakpoint data shown in Fig 1b. Bacterial artificial chromosome (BAC) and fosmid clones were obtained from BACPAC Resources, (Children’s Hospital, Oakland, CA/USA) and labelled by nick translation with dUTP fluors (Dyomics, Jena/Germany). Slides were mounted in Vectashield (Vector Labs, Burlinghame, CA/USA) containing 0.01% DAPI (4′,6-diamidino-2-phenylindole) (Sigma) and images recorded using a Zeiss Axioimager microscope (Zeiss, Oberkochen/Germany) configured to a HiSKY imaging system (Applied Spectral Imaging, Neckarhausen/Germany) (GIF 384 kb)
Copy number variations (CNV) and zygosity in AP-1060 cells. CNV were investigated with high density combined oligonucleotide SNP arrays. Images depict the following 12 mainly exonic gene regions: 3q26/MECOM (conspicuously high); 4q12/FIP1L1 (inconspicuous); 5p15/TERT; 5q21/translocation breakpoint (extragenic); 8q12/LYN (underexpression); 9p24/JAK2 (underexpression); 10q26/DMBT1 (inconspicuous); 11p13 biallelic WT1 (conspicuously silent); 11p15.4/TRIM5 (inconspicuous) and TRIM22 (overexpressed); 11p15.5/MRPL23 (underexpressed); 11q14/GAB2 (overexpressed); 12p12/BCAT1-intronic (conspicuously silent); 12q22/NUDT4 (underexpressed); 15q24/PML (breakpoint region); 15q25/NTRK3 (breakpoint region); 17q21/ATP5G1 translocation breakpoint (inconspicuous); 17q25/3′-ACOX1 (overexpressed); Xq24/IL13RA1 (splicing?). Cytoscan images show (top-to-bottom): genomic copy number (violet), losses of heterozygosity (plum); Database of Genomic Variation (DGV) showing recurrent losses (red) and gains (blue); BAC clones (yellow); Genbank genes with exons (pink); Online Mendelian Inheritance in Man (OMIM) genes (green and grey), and at the bottom genome coordinates (GRCh37/hg19). CytoScan High Density Arrays combining oligonucleotide and single nucleotide polymorphism (SNP) probes (Affymetrix, High Wycombe/UK) were employed to analyse genomic copy number alterations (CNA), loss of heterozygosity (LOH) and unbalanced chromosome translocation breakpoints as described recently [20]. Briefly, DNA was extracted using the Qiagen Gentra Puregene Kit (Hilden/Germany). Labeling, hybridization, washing and CytoScan HD arrays were performed per manufacturer’s recommendations and quality control criteria. Data were analysed using the Chromosome Analysis Suite software version 2.0.1.2 (Affymetrix) which links to the Database of Genomic Variants for identification of polymorphic CNV (http://dgv.tcag.ca/dgv/app/home). (GIF 126 kb)
Heatmap shows expression at genomically rearranged loci. Note conspicuous upregulation of three genes: MECOM which, though lacking detectible chromosome rearrangement at 3q26 bears a short intronic deletion (Supplementary Fig. S2); NTRK3 fused with ETV6 via t(12;15); and WT1 which showed conspicuous silencing attributable to biallelic deletions which were embedded within a ca. 1.8 Mbp LOH tract, one of several covering most of the 11p region, consistent with uniparental disomy (Supplementary Fig. S2). Cell lines were as follows AP-1060 and NB-4 (APL) and F-36P, GDM-1, GF-D8, HEL, KG-1, MB-02, MEG-01, MOLM-17, MUTZ-3, MUTZ-8, MV4–11, OCI-M1, OCI-M2, PLB-985 (HL-60 subclone), SAML-2, SET-2, THP-1, UKE-1, and UOC-M1.. Approximately 7.5 μg RNA prepared as for RQPCR was biotinylated using the 3′ IVT Express Kit (Affymetrix). cDNA was fragmented and placed in a cocktail along with hybridization controls (BioB, BioC, BioD, and Cre) per manufacturer’s recommendations. Samples were hybridized to Affymetrix GeneChip HG-U133 2.0 Plus for 16 h at 45 °C. Washing and staining were performed using a fluidics station 450 according to the recommended FS450 protocol. Image analysis was performed on GCS3000 Scanner and GCOS1.2 Software Suite (Affymetrix). Comparison datasets were kindly supplied by Prof. Andreas Rosenwald (University of Würzburg, Germany) or downloaded from the BROAD Institute (www.broadinstitute.org). For heatmaps we used CLUSTER 2.11 and TREEVIEW 1.60 (http://rana.lbl.gov/EisenSoftware.htm). Array data were parsed using the Broad/GSEA (http://www.broadinstitute.org/gsea/index.jsp), and STRING (http://string-db.org/) platforms. Signaling pathway models were cross-checked using PathwayNet software (http://pathwaynet.princeton.edu/). (GIF 220 kb)
FISH of MECOM . Image shows FISH analysis of MECOM locus using tilepath BAC clones. Note wild type configuration of both MECOM alleles at 3q26 remote from t(3;14) breakpoint at 3p14. Centromeric (RP11-94 J18) and telomeric (RP11-101 L8) were labeled and analysed as described above. (GIF 314 kb)
Comparative transcriptomics of AP-1060 and NB-4. Shows microarray expression of genes conspicuously upregulated (upper figure) or downregulated (lower) in AP-1060 with respect to NB-4 cells, together with unsupervised data from other cell lines. As well as singular activation of NTRK3 and MECOM commented above, consistent upregulation of several genes with known or potential leukemic activity: BAALC, BRAF, CCR6, CLEC7A, F2RL1, FRMD6, IL1R1, IL32, LOC100507645/MALAT1, OSMR, RGL4, SELL, SLITRK4, SOCS3 and VSTM1 (red arrows). In addition to WT1, top downregulated genes included: ANXA5, BCAT1, E2F1, HNRPLL, PON2, RAB13, TPD52, TRIM4 and ZAK (green arrows). Transcriptional profiling and heatmapping were performed as described. (GIF 301 kb)
Comparative transcriptomics of AP-1060 and NB-4 highlight MALAT1. To define transcriptional responses of AP-1060 cells to AZ-23, expression array data were substractively compared with NB-4 cells lacking EN. Top inhibitory and stimulatory targets were MALAT1 (upper figure) and MIR4680 (lower), respectively Lanes were (top-to-bottom): 1/2 untreated AP-1060 /NB-4; 3/4 AP-1060/NB-4 untreated/vehicle; 5/6 AP-1060/NB-4 + AZ-23. Note singular inhibition of MALAT1 restricted to AP-1060 highlighted as potential EN target (arrows). (GIF 399 kb)
Gene networks of NTRK3 signaling. A) STRING database of known and predicted protein-protein interactions was used to assemble a gene network of the top 300 genes inhibited by NTRK3 in AP-1060 cells. Note central, tightly interconnected network including NTRK3, MAPK1 and ESR1 (red). B) High confidence PathWay Net inference of “post-translational regulation” shows network linking seeding input genes ESR1, IL32, NTRK3 and TP53 (red lines). Note unprompted inclusion of MAPK1 and PML, contrasted with boycotting of IL32 from close partner network. Minimum confidence = 0.94. (MALAT1 is ipso facto excluded from protein based analyses.) (GIF 262 kb)
Immunofluorescence of MAPK1 and PML expression. Immunofluoresence shows inhibition by AZ-23 of activation (phosphorylation) of MAPK1 while sparing expression. Neither endpoint is affected in HG-23, paralleling western blot data (Fig. 3a). PML (bottom row), in contrast, was unaffected by inhibiting NTRK3 showing both nuclear and cytoplasmic staining in HG-3, while expression was mainly cytoplasmic in AP-1060 reflecting depletion of nuclear bodies therein. Microscope slides bearing cyto-centrifuged cells from log-phase cultures were fixed in methanol for 15 min at room temperature (RT), air dried and incubated in blocking buffer (5% BSA/PBS) for 30 min under soft plastic coverslips (Grace Bio-Labs, Bend, OR/USA) in a humidified atmosphere at RT. Slides were then incubated with primary antibodies (as detailed above for Westerns), together with anti-PML (Santa Cruz). MAPK1/pMAPK1 and PML antibodies were detected using anti-rabbit (SC-2359) and anti-mouse (SC-1030) FITC-labelled secondary antibodies (Santa Cruz). All procedures followed manufacturers` protocols. After ethanol dehydration and air-drying slides were mounted and as described for DNA FISH. (GIF 217 kb)
NTRK3 expression in high vs low MALAT1 expressing AML patients. Shows RqPCR expression data for exons-14/15/16 (retained in NTRK3 fusion mRNA). Note NTRK3 upregulation in (patient)-S1 [t(15;17)] absent from S2 [t(8;21)] and all MALAT1 LOW patients tested. (GIF 512 kb)
Co-expression of NTRK3 and MALAT1 in Cancer Patients. Public gene regulatory network data show positive association between NTRK3 and MALAT1 expression. Public National Cancer Institute data (https://gdc-portal.nci.nih.gov/projects/TCGA-HNSC) were mined to reveal Boolean relationships between expression of gene pairs. Variable thresholds are shown as blue lines which partition variables into low or high levels. Green and purple lines are, respectively, −0.5 and +0.5 standard deviations from X axis thresholds. Samples between the green/purple vertical lines (X-axis) and yellow/blue horizontal lines (Y-axis) were ignored. Each point represents a tumor sample (n = 528). Boolean implications are generated between variables when one quadrant is sparsely inhabited, thus detecting L-shaped relationships which may be overlooked by linear methods. Here, peak MALAT1 and NTRK3 expression appear cohabitory implying thresholding. (GIF 153 kb)
Acknowledgements
The authors thank the cell line donors, and Poul Sorensen for critical reading of the manuscript and useful suggestions.
Compliance with ethical standards
Conflict of interest
The authors have no conflicting interests to disclose.
Footnotes
Stefan Nagel and Bjoern Schneider contributed equally to this work.
References
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
(XLSX 57 kb)
(XLSX 10 kb)
FISH of ETV6 and NTKR3. FISH for ETV6 and PML (RP11-96B23) (A) and NTRK3 (B) yield breakpoint data shown in Fig 1b. Bacterial artificial chromosome (BAC) and fosmid clones were obtained from BACPAC Resources, (Children’s Hospital, Oakland, CA/USA) and labelled by nick translation with dUTP fluors (Dyomics, Jena/Germany). Slides were mounted in Vectashield (Vector Labs, Burlinghame, CA/USA) containing 0.01% DAPI (4′,6-diamidino-2-phenylindole) (Sigma) and images recorded using a Zeiss Axioimager microscope (Zeiss, Oberkochen/Germany) configured to a HiSKY imaging system (Applied Spectral Imaging, Neckarhausen/Germany) (GIF 384 kb)
Copy number variations (CNV) and zygosity in AP-1060 cells. CNV were investigated with high density combined oligonucleotide SNP arrays. Images depict the following 12 mainly exonic gene regions: 3q26/MECOM (conspicuously high); 4q12/FIP1L1 (inconspicuous); 5p15/TERT; 5q21/translocation breakpoint (extragenic); 8q12/LYN (underexpression); 9p24/JAK2 (underexpression); 10q26/DMBT1 (inconspicuous); 11p13 biallelic WT1 (conspicuously silent); 11p15.4/TRIM5 (inconspicuous) and TRIM22 (overexpressed); 11p15.5/MRPL23 (underexpressed); 11q14/GAB2 (overexpressed); 12p12/BCAT1-intronic (conspicuously silent); 12q22/NUDT4 (underexpressed); 15q24/PML (breakpoint region); 15q25/NTRK3 (breakpoint region); 17q21/ATP5G1 translocation breakpoint (inconspicuous); 17q25/3′-ACOX1 (overexpressed); Xq24/IL13RA1 (splicing?). Cytoscan images show (top-to-bottom): genomic copy number (violet), losses of heterozygosity (plum); Database of Genomic Variation (DGV) showing recurrent losses (red) and gains (blue); BAC clones (yellow); Genbank genes with exons (pink); Online Mendelian Inheritance in Man (OMIM) genes (green and grey), and at the bottom genome coordinates (GRCh37/hg19). CytoScan High Density Arrays combining oligonucleotide and single nucleotide polymorphism (SNP) probes (Affymetrix, High Wycombe/UK) were employed to analyse genomic copy number alterations (CNA), loss of heterozygosity (LOH) and unbalanced chromosome translocation breakpoints as described recently [20]. Briefly, DNA was extracted using the Qiagen Gentra Puregene Kit (Hilden/Germany). Labeling, hybridization, washing and CytoScan HD arrays were performed per manufacturer’s recommendations and quality control criteria. Data were analysed using the Chromosome Analysis Suite software version 2.0.1.2 (Affymetrix) which links to the Database of Genomic Variants for identification of polymorphic CNV (http://dgv.tcag.ca/dgv/app/home). (GIF 126 kb)
Heatmap shows expression at genomically rearranged loci. Note conspicuous upregulation of three genes: MECOM which, though lacking detectible chromosome rearrangement at 3q26 bears a short intronic deletion (Supplementary Fig. S2); NTRK3 fused with ETV6 via t(12;15); and WT1 which showed conspicuous silencing attributable to biallelic deletions which were embedded within a ca. 1.8 Mbp LOH tract, one of several covering most of the 11p region, consistent with uniparental disomy (Supplementary Fig. S2). Cell lines were as follows AP-1060 and NB-4 (APL) and F-36P, GDM-1, GF-D8, HEL, KG-1, MB-02, MEG-01, MOLM-17, MUTZ-3, MUTZ-8, MV4–11, OCI-M1, OCI-M2, PLB-985 (HL-60 subclone), SAML-2, SET-2, THP-1, UKE-1, and UOC-M1.. Approximately 7.5 μg RNA prepared as for RQPCR was biotinylated using the 3′ IVT Express Kit (Affymetrix). cDNA was fragmented and placed in a cocktail along with hybridization controls (BioB, BioC, BioD, and Cre) per manufacturer’s recommendations. Samples were hybridized to Affymetrix GeneChip HG-U133 2.0 Plus for 16 h at 45 °C. Washing and staining were performed using a fluidics station 450 according to the recommended FS450 protocol. Image analysis was performed on GCS3000 Scanner and GCOS1.2 Software Suite (Affymetrix). Comparison datasets were kindly supplied by Prof. Andreas Rosenwald (University of Würzburg, Germany) or downloaded from the BROAD Institute (www.broadinstitute.org). For heatmaps we used CLUSTER 2.11 and TREEVIEW 1.60 (http://rana.lbl.gov/EisenSoftware.htm). Array data were parsed using the Broad/GSEA (http://www.broadinstitute.org/gsea/index.jsp), and STRING (http://string-db.org/) platforms. Signaling pathway models were cross-checked using PathwayNet software (http://pathwaynet.princeton.edu/). (GIF 220 kb)
FISH of MECOM . Image shows FISH analysis of MECOM locus using tilepath BAC clones. Note wild type configuration of both MECOM alleles at 3q26 remote from t(3;14) breakpoint at 3p14. Centromeric (RP11-94 J18) and telomeric (RP11-101 L8) were labeled and analysed as described above. (GIF 314 kb)
Comparative transcriptomics of AP-1060 and NB-4. Shows microarray expression of genes conspicuously upregulated (upper figure) or downregulated (lower) in AP-1060 with respect to NB-4 cells, together with unsupervised data from other cell lines. As well as singular activation of NTRK3 and MECOM commented above, consistent upregulation of several genes with known or potential leukemic activity: BAALC, BRAF, CCR6, CLEC7A, F2RL1, FRMD6, IL1R1, IL32, LOC100507645/MALAT1, OSMR, RGL4, SELL, SLITRK4, SOCS3 and VSTM1 (red arrows). In addition to WT1, top downregulated genes included: ANXA5, BCAT1, E2F1, HNRPLL, PON2, RAB13, TPD52, TRIM4 and ZAK (green arrows). Transcriptional profiling and heatmapping were performed as described. (GIF 301 kb)
Comparative transcriptomics of AP-1060 and NB-4 highlight MALAT1. To define transcriptional responses of AP-1060 cells to AZ-23, expression array data were substractively compared with NB-4 cells lacking EN. Top inhibitory and stimulatory targets were MALAT1 (upper figure) and MIR4680 (lower), respectively Lanes were (top-to-bottom): 1/2 untreated AP-1060 /NB-4; 3/4 AP-1060/NB-4 untreated/vehicle; 5/6 AP-1060/NB-4 + AZ-23. Note singular inhibition of MALAT1 restricted to AP-1060 highlighted as potential EN target (arrows). (GIF 399 kb)
Gene networks of NTRK3 signaling. A) STRING database of known and predicted protein-protein interactions was used to assemble a gene network of the top 300 genes inhibited by NTRK3 in AP-1060 cells. Note central, tightly interconnected network including NTRK3, MAPK1 and ESR1 (red). B) High confidence PathWay Net inference of “post-translational regulation” shows network linking seeding input genes ESR1, IL32, NTRK3 and TP53 (red lines). Note unprompted inclusion of MAPK1 and PML, contrasted with boycotting of IL32 from close partner network. Minimum confidence = 0.94. (MALAT1 is ipso facto excluded from protein based analyses.) (GIF 262 kb)
Immunofluorescence of MAPK1 and PML expression. Immunofluoresence shows inhibition by AZ-23 of activation (phosphorylation) of MAPK1 while sparing expression. Neither endpoint is affected in HG-23, paralleling western blot data (Fig. 3a). PML (bottom row), in contrast, was unaffected by inhibiting NTRK3 showing both nuclear and cytoplasmic staining in HG-3, while expression was mainly cytoplasmic in AP-1060 reflecting depletion of nuclear bodies therein. Microscope slides bearing cyto-centrifuged cells from log-phase cultures were fixed in methanol for 15 min at room temperature (RT), air dried and incubated in blocking buffer (5% BSA/PBS) for 30 min under soft plastic coverslips (Grace Bio-Labs, Bend, OR/USA) in a humidified atmosphere at RT. Slides were then incubated with primary antibodies (as detailed above for Westerns), together with anti-PML (Santa Cruz). MAPK1/pMAPK1 and PML antibodies were detected using anti-rabbit (SC-2359) and anti-mouse (SC-1030) FITC-labelled secondary antibodies (Santa Cruz). All procedures followed manufacturers` protocols. After ethanol dehydration and air-drying slides were mounted and as described for DNA FISH. (GIF 217 kb)
NTRK3 expression in high vs low MALAT1 expressing AML patients. Shows RqPCR expression data for exons-14/15/16 (retained in NTRK3 fusion mRNA). Note NTRK3 upregulation in (patient)-S1 [t(15;17)] absent from S2 [t(8;21)] and all MALAT1 LOW patients tested. (GIF 512 kb)
Co-expression of NTRK3 and MALAT1 in Cancer Patients. Public gene regulatory network data show positive association between NTRK3 and MALAT1 expression. Public National Cancer Institute data (https://gdc-portal.nci.nih.gov/projects/TCGA-HNSC) were mined to reveal Boolean relationships between expression of gene pairs. Variable thresholds are shown as blue lines which partition variables into low or high levels. Green and purple lines are, respectively, −0.5 and +0.5 standard deviations from X axis thresholds. Samples between the green/purple vertical lines (X-axis) and yellow/blue horizontal lines (Y-axis) were ignored. Each point represents a tumor sample (n = 528). Boolean implications are generated between variables when one quadrant is sparsely inhabited, thus detecting L-shaped relationships which may be overlooked by linear methods. Here, peak MALAT1 and NTRK3 expression appear cohabitory implying thresholding. (GIF 153 kb)