High STAP1 expression in DUX4-rearranged cases is not suitable as therapeutic target in pediatric B-cell precursor acute lymphoblastic leukemia

Approximately 25% of the pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cases are genetically unclassified. More thorough elucidation of the pathobiology of these genetically unclassified (‘B-other’) cases may identify novel treatment options. We analyzed gene expression profiles of 572 pediatric BCP-ALL cases, representing all major ALL subtypes. High expression of STAP1, an adaptor protein downstream of the B-cell receptor (BCR), was identified in BCR-ABL1-like and non-BCR-ABL1-like B-other cases. Limma analysis revealed an association between high expression of STAP1 and BCR signaling genes. However, STAP1 expression and pre-BCR signaling were not causally related: cytoplasmic Igμ levels were not abnormal in cases with high levels of STAP1 and stimulation of pre-BCR signaling did not induce STAP1 expression. To elucidate the role of STAP1 in BCP-ALL survival, expression was silenced in two human BCP-ALL cell lines. Knockdown of STAP1 did not reduce the proliferation rate or viability of these cells, suggesting that STAP1 is not a likely candidate for precision medicines. Moreover, high expression of STAP1 was not predictive for an unfavorable prognosis of BCR-ABL1-like and non-BCR-ABL1-like B-other cases. Remarkably, DUX4-rearrangements and intragenic ERG deletions, were enriched in cases harboring high expression of STAP1.

for precision medicine, for the remaining group of BCR-ABL1-like and non-BCR-ABL1-like B-other cases, no druggable targets have yet been identified.
BCP-ALL cells are immature B-cells in which differentiation is arrested at early immature stages. The precursor B-cell receptor (pre-BCR), an immature immunoglubulin (Igµ) heavy chain with 'surrogate' light-chain components, is involved in the expansion and maturation of pre-B cells. For a short period of time, in-frame rearranged V H DJ H gene segments are expressed to pass the pre-BCR checkpoint. In absence of this expression, pre-B cells are eliminated by programmed cell death 11,12 . Malignant pre-B cells can evade this pre-B cell checkpoint via activation of alternative pathways 12 . In mature B-cells PI3K-AKT signaling can rescue BCR deficient cells 13 . Signaling of pre-BCR and mature BCR are largely similar 14 . Targeting BCR signaling is an attractive treatment strategy in mature B-cell malignancies and is also being explored in BCP-ALL [15][16][17][18][19][20] .
Advanced understanding of the pathobiology of genetically unclassified BCP-ALL cases may identify novel treatment options. To identify dysregulated genes in these cases, we analyzed gene expression profiles in leukemia cells of cohort of BCP-ALL patients at initial diagnosis 21 . High expression levels of the adapter protein signal transducing adaptor family member 1 (STAP1) were identified in a subgroup of BCR-ABL1-like and non-BCR-ABL1-like B-other patients. STAP1 is a relatively unknown protein, consisting out of Peckstrin homology (PH) domains and unique Src homology 2 (SH2) domains, suggesting that STAP1 recruits signaling proteins to receptor tyrosine kinases 22,23 . STAP1 is reported to be a docking protein downstream of Tec protein tyrosine kinase (TEC), which is involved in BCR signaling 24,25 . In addition, reports show enriched expression in hematopoietic stem cells and a potential role of STAP1 in microglia activation has been suggested 26,27 . The current study aimed to establish the therapeutic potential of inhibiting STAP1 in the subset of BCP-ALL cases that express high levels of STAP1.

Discriminative expression profile of STAP1 in BCR-ABL1-like and B-other cases.
To identify differentially expressed genes in BCR-ABL1-like and non-BCR-ABL1-like B-other cases, microarrays were performed of a representative pediatric BCP-ALL cohort of 572 cases at initial diagnosis 21 . Limma analyses revealed STAP1 to be the probeset with the highest fold-change in BCR-ABL1-like and non-BCR-ABL1-like B-other patients compared to remaining BCP-ALL cases (fold change = 2.88, adjusted p-value < 0.0001; Supplementary  Table S1). Strikingly, this elevated expression was characteristic for a subset of patients reflecting about 20% of BCR-ABL1-like and non-BCR-ABL1-like B-other cases (Fig. 1a, henceforth: STAP1-high cases). The remaining BCR-ABL1-like and non-BCR-ABL1-like B-other cases (henceforth: STAP1-low) had STAP1 expression levels comparable to those observed in other ALL subtypes and mononuclear bone marrow cells of healthy controls ( Supplementary Fig. S1). Microarray results were validated using RT-qPCR (Fig. 1b, Supplementary Fig. S1). Subsequently, we analyzed which genes were associated with high expression levels of STAP1. Limma analyses revealed 8894 probesets to be differentially expressed between STAP1-high and STAP1-low cases (adjusted p-value < 0.05; Supplementary Table S2). Ingenuity pathway analysis was performed to identify pathways associated with these differentially expressed genes. EIF2 signaling, mTOR signaling, regulation of eIF4 and p70S6K signaling, B cell receptor signaling (BCR), and AMPK signaling included the top five differentially regulated canonical pathways ( Supplementary Fig. S1). Identification of the BCR signaling pathway was a striking observation, as STAP1 is reported to be a docking protein downstream of TEC in this pathway 24,25 . Since the signaling pathways activated by pre-BCR and mature BCR are highly overlapping 14 , we hypothesized a similar function for STAP1 in the downstream signaling cascade of the pre-BCR. Taken together, the association between STAP1 and the BCR signaling cascade may suggest an activated pre-BCR signaling pathway in a subset of BCR-ABL1-like and non-BCR-ABL1-like B-other cases. This hypothesis was further explored since it may offer a targeted treatment strategy for these cases 17-20 . High levels of STAP1 and pre-BCR signaling are not causally connected. We studied whether high levels of STAP1 associated with high expression of the pre-BCR complex. To determine in-frame rearrangements of immunoglobulin heavy chain V H DJ H gene segments (indicative for a functional pre-BCR), cytoplasmic Igµ (CyIgµ) levels were measured in 142 BCR-ABL1-like and non-BCR-ABL1-like B-other cases. 14 of the 32 (44%) STAP1-high cases showed positivity (>30% CyIgµ + cells) compared with 31 of the 110 (28%) STAP1-low cases (Fisher exact test. p = 0.13). This result suggests that STAP1-high and STAP1-low cases do not differ in the number of pre-BCR positive cells. In addition, we examined whether STAP1 expression itself may be induced by pre-BCR signaling. Therefore, the BCP-ALL cell lines Nalm6 and Kasumi-2 (high and low STAP1 expression, respectively) were stimulated for 4 days with 1 µg anti-IgM antibody. To confirm activation of pre-BCR signaling by the anti-IgM antibody, phosphorylation levels of AKT Ser473 were analyzed ( Supplementary Fig. S2). Stimulation of the pre-BCR did not increase STAP1 expression levels in the STAP1-high cell line Nalm6. In the STAP1-low cell line Kasumi-2 only a slight increase was detected in time (~1.3-1.6 fold; Fig. 2). Taken together, these results suggest that high levels of STAP1 and pre-BCR signaling are not causally connected.
Silencing of STAP1 does not affect cell viability and drug sensitivity. To elucidate the role of STAP1 in BCP-ALL survival, STAP1 expression was silenced by four different shRNAs in the BCP-ALL cell lines Nalm6 and Kasumi-2. The knockdown efficiency at mRNA level ranged from 50-80% for both cell lines and was confirmed on protein level (Fig. 3a, Supplementary Fig. S3,4). Three out of four shRNA constructs did not alter the proliferation rate and viability of Nalm6 cells, despite effective knockdown. Only shRNA-2 reduced those parameters in Nalm6 cells, but not in Kasumi-2 cells (Fig. 3b). Seven days after transduction, the majority of leukemic cells remained alive in both cell lines and in all knockdown conditions (Fig. 3c). These data suggest that STAP1 is not essential for the survival of BCP-ALL cells.
Next, we studied whether silencing of STAP1 affected sensitivity for inhibitors of pre-BCR (ibrutinib) and mTOR signaling (rapamycin), and the ALL spearhead drug prednisolone. Silencing of STAP1 did not alter the sensitivity to these compounds in the Nalm6 or Kasumi-2 cell lines (Fig. 4). To elucidate in which other signaling pathway STAP1 may be involved, the phosphorylation status of 17 proteins covering multi-signaling pathways was examined (Fig. 5). Silencing STAP1 did not alter the phosphorylated levels/ activation of Src-family, PI3K, Ras, Stat, JNK, p38 and NFκB kinase signaling members, nor of the reported STAP1 target in Ramos cells, i.e. phosphorylated level of CREB Ser13325 . Taken together, STAP1 inhibition did not affect the survival and proliferation of the BCP-ALL cell lines Nalm6 and Kasumi-2 or the phosphorylated levels of downstream signaling molecules, nor did knockdown result into sensitization for prednisolone or signaling inhibitors (ibrutinib and rapamycin).

Aberrations in B-cell development genes and clinical characteristics. Genes involved in B-cell
development are frequently altered in BCR-ABL1-like patients 2,3 . Therefore, frequencies of lesions in genes involved in lymphoid differentiation, proliferation, cell cycle and transcription were studied in STAP1-high and STAP1-low BCR-ABL1-like and non-BCR-ABL1-like B-other cases. Copy number alterations affecting EBF1, PAR1, and ETV6 were virtually absent in STAP1-high cases compared to a frequency ranging from 10.9-22.4% in STAP1-low cases (Fisher exact test, p < 0.05; Table 1). More strikingly, high expression of STAP1 was associated with intragenic deletion of the ETS transcription factor ERG: 27.6% (8/29) in STAP1-high cases compared to 0.8% (1/133) in STAP1-low cases (p < 0.0001, odds ratio = 47.7; Table 1). Deletions of ERG are associated with a favorable prognosis in genetically unclassified high-risk pediatric BCP-ALL [28][29][30] . The five-year CIR and EFS of STAP1-high and STAP1-low BCR-ABL1-like and non-BCR-ABL1-like B-other cases did not significantly differ in our cohort, although STAP1-high cases showed a trend for a more favorable outcome (Fig. 6a,b). STAP1-high cases had a higher median age (Mann-Whitney U test; p = 0.013; 9 years, range 2-16) than STAP1-low cases (6  Table S3). In addition, ex vivo cytotoxicity of prednisolone, vincristine, daunorubicin, l-asparaginase, 6-thioguanine, 6-mercaptopurine was measured in primary leukemic cells. No difference in cytotoxicity of these compounds was observed in STAP1-high and STAP1-low BCR-ABL1-like and non-BCR-ABL1-like B-other cells ( Fig. 6c-h).
Interestingly, ERG deletions were very recently shown to be a hallmark of a newly identified BCP-ALL subtype, involving rearrangements of the double homeobox transcription factor DUX4 [8][9][10]31 . The association between high STAP1 levels and ERG deletions prompted us to investigate the presence of DUX4 rearrangements in our cases. To this aim, we screened an independent BCP-ALL cohort (n = 2 KMT2A-rearranged, n = 1 BCR-ABL1, n = 21 B-other, n = 17 ETV6-RUNX1, n = 21 high hyperdiploid) for DUX4-rearrangements and ERG deletions. Four DUX4-rearranged cases were detected in the B-other group, of which two showed high expression levels of STAP1 (Fig. 7). None of these DUX4-rearranged cases or remaining B-other cases harbored an ERG deletion. In addition, we analyzed the association between STAP1, ERG, and DUX4 using a publically available dataset of 304 BCP-ALL cases 10,32 . Similar to the observation made in our own patients, DUX4-rearranged cases displayed higher levels of STAP1 mRNA ( Supplementary Fig. S6), which was independent of intragenic ERG deletions. Taken together, high expression of STAP1 is not associated with (long-term) clinical outcome parameters or ex vivo drug resistance. However, the STAP1-high group is enriched for DUX4-rearrangements and intragenic ERG deletions.

Discussion
The aim of the current study was to assess the potential of STAP1 as a therapeutic target in BCP-ALL cases. High expression of STAP1 was detected in a subset of BCR-ABL1-like and non-BCR-ABL1-like B-other patients. Interference with STAP1 did not induce cell death or block proliferation nor did it make cells more sensitive to BTK (ibrutinib) and mTOR (rapamycin) inhibitors. In addition, high expression levels of STAP1 were not associated with a poor five-year EFS, high MRD levels, or ex vivo resistance to chemotherapeutic drugs that are traditionally used in the treatment of BCP-ALL. Taken together, these results suggest that STAP1 is not a likely target for precision medicines in childhood ALL.
STAP1 is a relatively unknown gene. A potential role for the protein in neuronal apoptosis and degeneration has been suggested 26 . However, involvement of the protein in cancer thus far remains elusive. We studied the gene expression profiles of STAP1-high BCP-ALL cases. The observed association between high expression of STAP1 (a-c) The efficacy of prednisolone (a), ibrutinib (b), and rapamycin (c) was tested in Nalm6 and Kasumi-2 cells, in which STAP1 was silenced. Three days after transduction, Nalm6 and Kasumi-2 cells were exposed to a concentration range of the indicated compounds. After four days, viability was quantified using an MTS assay. Values represent mean ± SEM of four independent experiments of Nalm6 and three independent experiments of Kasumi-2. Fluorescent intensity values relative to GAPDH are depicted. Expression was measured three and seven days after transduction. Infection and selection for stably transduced cells required three days. Subsequently, proliferation, viability and MTS assays were initiated, which took four days. mRNA and protein expression levels were examined after selection (day 3) and at the end of these functional experiments (day 7). At day 7, not enough material was available of Nalm6 cells, which were transduced with shRNA-2. Values represent mean ± SEM of four independent experiments at three days after transduction and three independent experiments at seven days after transduction. and BCR signaling is in concordance with the few reports suggesting that STAP1 is a docking protein downstream of the BCR 24,25 . Targeting the BCR pathway is an effective treatment strategy for mature B-cell malignancies and this approach is also being explored in BCP-ALL [15][16][17][18][19][20] . The overlap in downstream molecules involved in pre-BCR and BCR signaling 14 , prompted us to investigate the connection between STAP1 expression and the pre-BCR pathway. However, CyIgµ expression (indicative for a functional pre-BCR) was not enriched in STAP1-high cases, and stimulation of leukemic cell lines with IgM only marginally induced expression STAP1 in one cell line. In addition, silencing of STAP1 did not reduce the proliferation rate and viability of leukemic cells in the majority of shRNAs tested. Strikingly, knockdown of STAP1 did also not affect phosphorylation levels of marker proteins involved in pre-BCR signaling (SRC) or other signaling pathway (PI3K, Ras, Stat, JNK, p38, NFκB), which are important for proliferation and survival of cells. In addition, silencing of STAP1 did not affect the phosphorylated levels of CREB, which is a downstream target of STAP1 in the Ramos/Burkitt lymphoma cell line 25 . The question remains regarding the signaling pathway in which STAP1 is involved. Nevertheless, our results imply that STAP1 is not an oncogenic driver and that the high expression is most likely a consequence of another transforming event.
Interestingly, we observed intragenic deletions of the transcription factor ERG in a part of the STAP1-high cases. The proto-oncogene ERG is a regulator of hematopoiesis, including B-cell development 33,34 , and is implicated in the pathogenesis of Ewing sarcoma, prostate cancer, and acute myeloid leukemia [35][36][37] . Recently, ERG deletions were shown to be a hallmark of a novel identified subtype of BCP-ALL, i.e. DUX4-rearranged ALL [8][9][10] . DUX4 encodes a double homeobox transcription factor and is located within the D4Z4 macrosatellite repeat array on 4q35 and 10q26. This pro-apoptotic gene is normally expressed in germline and testis cells, but epigenetically silenced in somatic cells 38,39 . DUX4-fusion proteins, but not wildtype DUX4, were shown to have oncogenic potential in NIH3T3 fibroblasts 9 . The pathway induced by DUX4-rearrangements is yet unknown. In the present work we show that high expression of STAP1 is characteristic for DUX4-rearranged cases (Fig. 7, Supplementary  Fig. S6). The association between STAP1 and DUX4 was independent of ERG deletions. Therefore, upregulation of STAP1 may be a consequence of the oncogenic signaling pathway induced in DUX4-rearranged cases which is independent of concomitant ERG deletions. Further unraveling of the signaling pathway downstream of DUX4 will be crucial for the development of targeted treatment strategies for this genetic BCP-ALL subtype.

Methods
A detailed description of all methods can be found in the supplement.
Processing of primary patient material. Bone marrow and/or peripheral blood samples were obtained from children (1-18 years) with newly diagnosed ALL. Written informed consent was obtained from parents or guardians to use excess of diagnostic material for research purposes, as approved by the Medical Ethics Committee of the Erasmus Medical Center, The Netherlands. Studies were conducted in accordance with the Declaration of Helsinki. Mononuclear cells were isolated using Lymphoprep gradient separation and the leukemic blast percentage was determined microscopically by May-Grünwald Giemsa stained cytospin preparations, as described previously 40 . Samples were enriched to >90% leukemic cells by depleting normal cells, using anti-CD marker coated magnetic beads, i.e. anti-CD3, anti-CD14, anti-CD15, anti-CD33, and/or H1 beads (IQ Products, Groningen, The Netherlands) combined with pan mouse IgG dynabeads (Invitrogen, Bleiswijk, Netherlands). Primary leukemic cells were maintained in RPMI-1640 Dutch modification (Life Technologies, Breda, Netherlands) supplemented with 20% fetal calf serum (Integro, Zaandam, Netherlands), with 0.1% insulin-transferrin-sodium selenite (Sigma-Aldrich, Zwijndrecht, Netherlands), 0.4 mM glutamine (Invitrogen), 0.25 μg/ml gentamycine (Thermo Scientific, Breda, Netherlands), 100 IU/ml penicillin (Thermo Scientific), 100 μg/ml streptomycin (Thermo Scientific), 0.125 μg/ml fungizone (Thermo Scientific).

Transfection
Multiplexed fluorescent bead-based immunoassay (Luminex). Cells were lysed in RIPA buffer (ThermoFisher Scientific) with freshly added protease and phosphatase inhibitors. Protein concentration was determined using the BCA method (Thermo Scientific). Expression of 17 proteins was determined in 10 µg lysate, using a fluorescent bead-based immunoassay (multi-pathway magnetic bead 9-plex and the 8-plex human Src family kinase kit; Merck Millipore, Amsterdam, Netherlands) according to the manufacturer's protocol. GAPDH beads (Merck Millipore) were used as internal reference for each sample.
Genome-wide DNA copy number arrays (array-CGH). To identify ERG deletions, genome-wide DNA copy number arrays were performed as described previously 4 . Briefly, Agilent SurePrint G3 Hmn 4 × 180 K arrays (Agilent Technologies, Amstelveen, the Netherlands) were co-hybridized with 1 μg patient DNA labeled with ULS-Cy5 and 1 μg reference genomic DNA male pool (G147A, Promega, Leiden, the Netherlands) labeled with ULS-Cy3 (Agilent Genomic DNA ULS Labeling Kit). Using median log ratios, data were normalized using the CGHcall 47 version 2.14.0, centralized using CGHnormaliter 48 version 1.8.0, and segmented and called using CGHcall default settings (−1 for loss, 0 for diploid, 1 for gain and 2 for amplification) in R version 2.14.1.

Clinical characteristics and statistics.
To identify whether copy number alterations (CNAs), clinical characteristics or CyIgμ expression were depleted or enriched in cases harboring high expression levels of STAP1, the Fisher's exact test in R (version 3.2.1) was applied. Cumulative incidence of relapse (CIR) was estimated using a competing risk model and significance was determined using the Gray's test. Relapse and non-response (counted at day 79 of therapy) were considered as events and death as competing event. Event-free survival (EFS) probabilities were estimated using cox regression and compared using the Wald test. Relapse, non-response, secondary malignancies and death were counted as events. Outcome analyses were performed in R (version 3.2.1), using the packages cmprsk version 2.2-7 49  SNP arrays. Genome-wide human SNP arrays 6.0 (Affymetrix) were performed according to the manufacturer's protocol. Raw probe values were extracted from CEL files and processed with the R package aroma.affymetrix version 3.1.0. Samples were compared to reference values, which was the average of 53 diploid BCP-ALL and T-ALL samples. To correct for bias introduced by differences in GC content of DNA fragments, the R package ArrayTV version 1.12.0 was used. To compare the logR values of the copy numbers between samples a centralization step was performed, using the R package CGHnormaliter version 1.28.0. Data were called using CGHcall version 2.36.0 default settings (−2 for double loss, −1 for loss, 0 for diploid, 1 for gain and 2 for amplification) in R version 3.3.3.
RNAseq. mRNA was extracted from total RNA and amplified using random hexamer primers. Further Library construction was done using a strand-specific protocol. Sequencing was performed on a HiSeq. 2000 producing 151 bp paired-end reads with a median library size of 50 million read pairs per sample. Fastq-files with paired-end data were aligned to the GRCh37 reference sequence using STAR 2.5.0b. Read counts were determined with HTSeq-count version 0.6.1p1. To determine DUX4 expression, the fastq-files were aligned to the DNA sequence of the gene ENSG00000259128.1 plus 200 bp down-and upstream. DUX4-partner genes were identified using NCBI blast, in which unknown sequences after the breakpoint were aligned 52 .
Data availability. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.