Anastasija Pejkovska

Supplementary Figure 1. Summary of the somatic alterations in the 39 ckAML samples. Supplementary Figure 2. Proportion of samples expressing the top pyjacker hits, for several AML cohorts profiled with RNA-seq. Supplementary Figure 3. Example rearrangements leading to gene activation and TP53 inactivation. Supplementary Figure 4. Rearrangements leading to MECOM monoallelic expression in sample 15KM20146. Supplementary Figure 5. Validation of the translocation t(1;3)(p36;q21) in sample 16KM11270 and of the breakpoints for the CDK6 enhancer duplication next to MNX1 in sample 15PB8708 by genomic PCR. Supplementary Figure 6. Deletion between the TCR beta locus and MNX1 leading to MNX1 expression. Supplementary Figure 7. Alternative rearrangements leading to MNX1 expression. Supplementary Figure 8. Differential gene expression analysis of MNX1 status in adult and pediatric AML. Supplementary Figure 9. Gene expressions of 25 selected cancer and hematological development associated genes differentially expressed under MNX1 activation. Supplementary Figure 10. Copy number alterations on chromosome 7 for samples profiled with scRNA-seq. Supplementary Figure 11. Reciprocal 4C. Supplementary Figure 12: Knockdown of MNX1 reduces tumor load of AML PDX cells in vivo.

ST1 Methods for enhancer hijacking detection ST2 Ranked and scored hematopoietic enhancers (based on ChIP-seq data for H3K27ac and P300 from K562, MOLM1 and Kasumi1) ST3 Pyjacker results for the 10 AML cell lines ST4 Pyjacker results for the 120 medulloblastoma samples ST5 Cis-x results for the 120 medulloblastoma samples ST6 Summary information for the 39 ckAML patient samples ST7 SNVs detected in WGS data for the 39 ckAML samples ST8 SVs detected in WGS data for the 39 ckAML samples ST9 CNAs detected in WGS data for the 39 ckAML samples ST10 Fusion transcripts detected in RNAseq data for the 39 ckAML samples ST11 Pyjacker results for the 39 ckAML samples ST12 Primers for qRT-PCR and validation of breakpoints (hg19) ST13 Summary of the MNX1 expression screens ST14 Summary of the MNX1-expressing samples profiled with WGS ST15 Mutations detected in the MNX1-expressing samples profiled with WGS ST16 Results of the differential expression analysis (adult and pediatric AML) ST17 TADs derived from HiC data of HSPCs ST18 Samples IDs from the TARGET-AML cohort used for the RNA-seq analysis ST19 Clinical characteristics of the patient samples from which were derived the PDX ST20 Primers and conditions used for 4C ST21 Antibodies used

Aberrant EPO expression might cooperate with EPOR amplification in AEL. A,EPO expression in all samples in TPM, with sample 15KM18875 with EPO overexpression highlighted in green. B, Proportion of samples with nonzero EPO expression in three AEL cohorts profiled with RNA-seq (47–49). C, Copy numbers (CN) and SVs on chromosome 7 (containing EPO) and chromosome 11 in sample 15KM18875. Copy number losses are indicated in blue, and gains in red. SVs are shown at the top, with arcs connecting breakpoints or lines indicating the chromosome of the other side of the breakpoint (for C, D, and F the colors of SVs indicate the orientation: blue for deletion, red for duplication, purple for inversion, and green for interchromosomal SV). D, A 300 kb circular piece of DNA containing EPO and a putative enhancer (highlighted in orange), with P300 and H3K27ac peaks in the erythroid cell line K562. E,EPOR expression in TPM in all samples, with sample 15KM18875 highlighted in green. F, Copy numbers and SVs on chromosome 19 for sample 15KM18875. chr, chromosome.

The homeobox genes GSX2 and MNX1 can be activated by atypical mechanisms. A,GSX2 expression in all samples in TPM, with sample 16PB5693 with GSX2 expression highlighted in green. B,MNX1 expression in all samples in TPM, with sample 15PB8708 with MNX1 overexpression highlighted in green. C, Variant allele frequencies in WGS and RNA-seq for an SNP in MNX1 in sample 15PB8708 (major allele frequency in blue and minor allele frequency in red). D, Circos plot showing CNAs and SVs in sample 16PB5693 for the chromosomes involved in a chromothripsis event. Copy number (CN) losses are indicated in blue, and gains in red. SVs are shown as arcs at the center, with interchromosomal breakpoints in green, duplications in red, deletions in blue, and inversion in purple. E, HiC data from HSPCs (19) and ChIP-seq data from myeloid cell lines in the region around GSX2. The putative enhancer is highlighted in orange, and the region in gray is deleted in sample 16PB5693. F, CNs and breakpoints on chromosome 7 for sample 15PB8708. In the chromosome ideogram, regions highlighted in red and teal correspond to the regions shown in (G), with matching colors. G, ChIP-seq tracks for P300 and H3K27ac in the myeloid cell lines MOLM-1 and Kasumi-1 in the region around MNX1 on the rearranged chromosome of sample 15PB8708. Enhancers of the CDK6 region are highlighted in orange. chr, chromosome.

ST1 Methods for enhancer hijacking detection ST2 Ranked and scored hematopoietic enhancers (based on ChIP-seq data for H3K27ac and P300 from K562, MOLM1 and Kasumi1) ST3 Pyjacker results for the 10 AML cell lines ST4 Pyjacker results for the 120 medulloblastoma samples ST5 Cis-x results for the 120 medulloblastoma samples ST6 Summary information for the 39 ckAML patient samples ST7 SNVs detected in WGS data for the 39 ckAML samples ST8 SVs detected in WGS data for the 39 ckAML samples ST9 CNAs detected in WGS data for the 39 ckAML samples ST10 Fusion transcripts detected in RNAseq data for the 39 ckAML samples ST11 Pyjacker results for the 39 ckAML samples ST12 Primers for qRT-PCR and validation of breakpoints (hg19) ST13 Summary of the MNX1 expression screens ST14 Summary of the MNX1-expressing samples profiled with WGS ST15 Mutations detected in the MNX1-expressing samples profiled with WGS ST16 Results of the differential expression analysis (adult and pediatric AML) ST17 TADs derived from HiC data of HSPCs ST18 Samples IDs from the TARGET-AML cohort used for the RNA-seq analysis ST19 Clinical characteristics of the patient samples from which were derived the PDX ST20 Primers and conditions used for 4C ST21 Antibodies used

Activation of MECOM and its homolog PRDM16 by a GATA2 enhancer. A, Expression of MECOM in all samples in TPM, ranked by expression of MECOM, in which samples 15PB19457 and 15KM20146 with breakpoints near MECOM are highlighted in green. B, Variant allele frequencies in WGS (DNA) and RNA-seq for SNPs in MECOM for sample 15PB19457 (major allele frequencies in blue and minor allele frequencies in red). C, Copy numbers (CN) and SVs on chromosome 3 for sample 15PB19457. Copy number losses are indicated in blue, and gains in red. SVs are shown as arcs at the top, in which the color indicates the orientation of the breakpoint: blue for deletion, red for duplication, and purple for inversion. In the chromosome ideogram, the three regions that are displayed with a zoom-in in (D) are highlighted in colors, with colors matching the arrows in (D). D, ChIP-seq tracks for P300 and H3K27ac in the myeloid cell lines MOLM-1 and Kasumi-1 in the region around MECOM for the rearranged chromosome of sample 15PB19457. The putative enhancer is highlighted in orange. E, Expression of PRDM16 in all samples, ranked by PRDM16 expression, in which sample 16KM11270 with a breakpoint near PRDM16 is highlighted in green. F, Variant allele frequencies in WGS (DNA) and RNA-seq for SNPs in PRDM16 in TPM for sample 16KM11270 (major allele frequency in blue and minor allele frequency in red). G, ChIP-seq tracks for P300 and H3K27ac in the myeloid cell lines MOLM-1 and Kasumi-1 in the region around PRDM16 on the rearranged chromosome of sample 16KM11270. The putative enhancer is highlighted in orange. chr, chromosome.

Supplementary Figure 1. Summary of the somatic alterations in the 39 ckAML samples. Supplementary Figure 2. Proportion of samples expressing the top pyjacker hits, for several AML cohorts profiled with RNA-seq. Supplementary Figure 3. Example rearrangements leading to gene activation and TP53 inactivation. Supplementary Figure 4. Rearrangements leading to MECOM monoallelic expression in sample 15KM20146. Supplementary Figure 5. Validation of the translocation t(1;3)(p36;q21) in sample 16KM11270 and of the breakpoints for the CDK6 enhancer duplication next to MNX1 in sample 15PB8708 by genomic PCR. Supplementary Figure 6. Deletion between the TCR beta locus and MNX1 leading to MNX1 expression. Supplementary Figure 7. Alternative rearrangements leading to MNX1 expression. Supplementary Figure 8. Differential gene expression analysis of MNX1 status in adult and pediatric AML. Supplementary Figure 9. Gene expressions of 25 selected cancer and hematological development associated genes differentially expressed under MNX1 activation. Supplementary Figure 10. Copy number alterations on chromosome 7 for samples profiled with scRNA-seq. Supplementary Figure 11. Reciprocal 4C. Supplementary Figure 12: Knockdown of MNX1 reduces tumor load of AML PDX cells in vivo.

Detection of enhancer hijacking in 39 ckAML samples. A, Schematic representation of the main sources of information used by Pyjacker: breakpoints, overexpression, monoallelic expression, and enhancers. B, Scatter plot of genes identified by Pyjacker in 39 ckAML samples as being potentially activated by genomic rearrangements in one or more samples, in which the x-axis shows the genomic location of the genes, and the y-axis shows the FDR. Gene names for the enhancer hijacking candidates are written in bold, and if a fusion transcript is detected, the fusion partner is named. chr, chromosome.

MNX1 is expressed in 1.4% of all AML cases, often with del(7)(q22q36). A, qRT-PCR screen for MNX1 expression in three AML cohorts (Rotterdam, Ulm, and Jena). B, Fifteen MNX1-expressing samples with del(7)(q22q36) profiled with WGS, with a zoom-in around the breakpoints (hg19 reference). The blue rectangles indicate the genomic regions that are retained, and dashed lines represent breaks. C, Percentage of samples with mutations in frequently mutated genes for MNX1+ samples with breakpoints near MNX1, MNX1+ samples without breakpoints, and TCGA-LAML samples. D, scRNA-seq analysis for MNX1+ and control del(7q) AML samples. Left, UMAP showing cell type labels of 53,479 cells integrated across eight patients. Right, UMAP highlighting MNX1 expression (top) and the presence of a del(7q) (bottom) as predicted for patients with del(7q) (n = 4) and patients with del(7q) and MNX1 activation (n = 4). chr, chromosome; UMAP, Uniform Manifold Approximation and Projection.

Knockdown of MNX1 reduces tumor load of AML PDX cells in vivo. A, Scheme depicting the experimental setup of the in vivo constitutive experiment. AML-661 PDX cells expressing the cassettes for both CRE-ERT2 and the shRNA addressing MNX1 or a control gene were amplified in mice. Fresh PDX cells were stimulated with TAM (single dose, 200 nmol/L, 72 hours) to induce the knockdown in vitro. Cells with knockdown were enriched by flow cytometry gating on the respective fluorochrome markers GFP (knockdown of MNX1) and T-Sapphire (control knockdown). The two populations were mixed at a 1:1 ratio and injected into mice. The ratio between both populations was measured at advanced leukemic disease in different organs (more than 60% hCD33+ cells in PB). B, The results of the experiment described in (A) using five mice. C, Scheme depicting the experimental setup of the in vivo inducible experiment. The cell populations described in (A) were mixed in a 1:1 ratio and injected into 13 mice. Fourteen days after injection, three mice were sacrificed (N = 3) to quality control the 1:1 ratio of the two cell populations by flow cytometry. TAM (50 mg/kg) was orally administered to the 10 remaining mice. Five mice were sacrificed 3 days later to measure the rate of shRNA induction by TAM. At an advanced stage of leukemia, the remaining five mice were sacrificed to determine the ratio between the control vs. MNX1 knockdown populations. D, The results of the experiment described in (C). P values determined by a one-tailed unpaired t test. **, P < 0.01; ****, P < 0.0001.