Non-Coding Variants Connect Enhancer Dysregulation with Nuclear Receptor Signaling in Hematopoietic Malignancies Kailong Li1,2,5, Yuannyu Zhang1,2,5, Xin Liu1,2,5, Yuxuan Liu1,2,5, Zhimin Gu1,2, Hui Cao1,2, Kathryn E. Dickerson1,2, Mingyi Chen3, Weina Chen3, Zhen Shao4, Min Ni1, Jian Xu1,2,* 1Children’s Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA 2Department of Pediatrics, Harold C. Simmons Comprehensive Cancer Center, and Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA 3Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA 4Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 5These authors contributed equally *Corresponding Author: [email protected] (J.X.) Running Title: Non-Coding Variants in Hematopoietic Malignancies Figure S1. Workflow for the identification of non-coding variants in human leukemia (A) Schematic of the major steps in targeted resequencing and functional analysis of non-coding alterations. The detailed information about the samples, identified mutations and variant-associated CREs are shown on the right. (B) Flowchart is shown for the annotation of blood CRE repertories. (C) Flowchart is shown for the mutation discovery pipeline. Figure S2. Identification of non-coding variants and recurrently mutated CREs (A) Schematic of the mutation discovery pipeline for identifying high-confidence (Tier I) somatic SNVs and INDELs in tumor/normal paired AML samples. (B) Flowchart is shown for the identification of Tier II somatic SNVs and INDELs in other leukemia and lymphoma samples and cell lines. (C) Variant allele frequency (VAF) of the identified SNVs and INDELs in targeted sequencing. (D) Comparisons of gene mutations in this study and recently published WGS studies of AML samples from TCGA (1), ICGC (http://icgc.org), and Beat AML (3 batches of samples) (http://www.vizome.org) (2) cohorts. Both Tier I and II somatic gene mutations identified in targeted CRE sequencing are shown. (E) Flowchart is shown for the identification of recurrently mutated CREs. Figure S3. Analysis of coding and non-coding variants in human leukemia (A) Heatmap shows the recurrent protein-coding gene mutations identified by targeted resequencing in human leukemia. The gene symbols are shown on the left. The cumulative numbers of mutated samples for each gene are shown on the right. The numbers of mutated genes in each sample are shown on the top. Samples are categorized by disease types (AML or MDS, lymphoma, ALL and normal) and sources (patient and cell line) with each sample ID shown on the bottom. (B) Comparison of coding and non-coding mutation frequency between patient samples and cell lines. (C) Comparison of coding and non-coding mutation frequency between different leukemia types (AML, lymphoma and ALL) and normal controls. Results are means ± SD and analyzed by a one-way ANOVA. ***P < 0.001, n.s. not significant. (D) Comparison of coding and non-coding mutation frequency between different sites (BM vs PB), genders (male vs female), and FAB classification of AML subtypes (M0 to M4). Results are means ± SD and analyzed by a student’s t-test or one-way ANOVA. n.s. not significant. (E) Lack of correlation between mutation frequency and ages. All leukemia samples (purple) or AML samples (blue) are shown. Pearson correlation coefficient (R) and P values are shown. Figure S4. Development of the CRE perturbation screening systems (A) Flowchart is shown for the design of the sgRNA pool for CRE perturbation screening. (B) Validation of CRE perturbation screens by independent replicate experiments. CRE-specific sgRNAs (black) and non-targeting control sgRNAs (red) are shown. The x- and y-axis denote the log2 fold changes of sgRNA enrichment or dropout in T28 relative to T0 samples. The Pearson correlation coefficient (R) values are shown. (C) Comparisons of replicate experiments of enCRISPRa screening at different time points. The x- and y- axis of each graph represent the normalized sgRNA counts in enCRISPRa screens at day0 (T0) and day28 (T28). Spearman correlation value is shown for each comparison. (D) Comparisons of replicate experiments of enCRISPRi screening at different time points. (E) enCRISPRa-mediated activation of candidate oncogenic or tumor suppressive CREs resulted in significant enrichment or dropout of CRE-targeting sgRNAs, respectively. Box plots are shown for sgRNAs targeting oncogenic, tumor suppressive or other CREs, or non-targeting controls (x-axis) by the log2 fold changes of sgRNA enrichment or dropout in T28 relative to T0 samples (y-axis). Boxes show median of the data and quantiles, and whiskers extend to 1.5x of the interquartile range. P values were calculated by the Welch’s t-test. (F) enCRISPRi-mediated repression of candidate oncogenic or tumor suppressive CREs resulted in significant enrichment or dropout of CRE-targeting sgRNAs, respectively. A Tumor Suppressive Promoters Oncogenic Promoters CRE Chromosome Nearest CRE923 CRE19872 CRE18011 CRE210 CRE Chromosome Nearest ID coordinates genes E/P (BCAR3;MIR760) CRE9782 (TOX) (ZNF253) (SPEN;FLJ37453) ID coordinates genes E/P CRE1161 chr1:146696609-146697700 FMO5 P CRE2191 chr10:6622165-6623239 PRKCQ-AS1;PRKCQ P 2 CRE2191 (BSG) CRE15272 CRE13414 chr21:36261257-36262671 RUNX1 P CRE923 chr1:94312038-94313684 MIR760;BCAR3 P (PRKCQ-AS1; (LPP;LPP-AS2) CRE18973 chr7:75367805-75369511 HIP1 P CRE16157 chr4:183837553-183839385 DCTD P PRKC1Q) CRE676 chr1:47696820-47697746 TAL1 P CRE9782 chr19:571003-572971 BSG P CRE16157 CRE18528 chr7:6098255-6099259 EIF2AK1 P CRE13645 chr22:20747916-20748772 ZNF74 P (DCTD) CRE18532 chr7:6387722-6389229 FAM220A P CRE19872 chr8:60031112-60032935 TOX P CRE11749 chr2:85132395-85133594 TMSB10 P CRE18372 chr6:159289942-159291725 C6orf99 P CRE13645 CRE3461 chr11:58345090-58346561 ZFP91;ZFP91-CNTF;LPXN P CRE10392 chr19:19976653-19977762 ZNF253 P (ZNF74) CRE2336 chr10:27541273-27542255 LRRC37A6P P CRE15272 chr3:187871255-187872821 LPP;LPP-AS2 P 0 CRE19440 chr7:149157199-149158530 ZNF777 P CRE9362 chr17:80022771-80024344 DUS1L P CRE11619 chr2:65454438-65455887 ACTR2 P CRE15386 chr4:925395-926355 TMEM175;GAK P CRE14603 chr3:57741420-57742382 SLMAP P CRE3575 chr11:64084943-64085746 PRDX5;TRMT112 P CRE11193 chr2:9695628-9696614 ADAM17 P CRE10913 chr19:50379407-50381413 TBC1D17;AKT1S1 P CRE8469 chr17:18163661-18164706 MIEF2;FLII P CRE1243 chr1:151118617-151119720 SEMA6C P CRE1592 chr1:179334231-179335393 AXDND1 P CRE21622 chrX:51636112-51637065 MAGED1 P CRE6194 chr14:73393076-73394163 DCAF4 P CRE6288 chr14:81687251-81687975 GTF2A1 P CRE19307 chr7:130080230-130081447 CEP41 P CRE20879 chr9:104159977-104161656 ZNF189;MRPL50 P CRE4519 chr12:48099412-48100456 RPAP3 P -2 CRE2030 chr1:236030212-236031340 LYST P CRE8291 chr17:4699228-4700114 PSMB6 P CRE12525 chr2:220042083-220043315 FAM134A;CNPPD1 P CRE850 chr1:85039161-85040173 CTBS P CRE16028 chr4:141677432-141678382 TBC1D9 P CRE21853 chrX:102941837-102942975 MORF4L2;MORF4L2-AS1 P CRE9489 chr18:12750264-12751100 LOC100996324 P CRE20513 chr9:34611607-34612582 RPP25L P CRE8971 CRE1245 chr1:151137838-151139325 SCNM1;LYSMD1 P chr1:220219140-220220593 EPRS P CRE12502 chr2:218990178-218990892 CXCR2 P CRE1857 (MSI2) CRE1225 chr1:150265803-150266742 MRPS21 P CRE19253 chr7:117823550-117824621 LSM8 P CRE3624 chr11:65292339-65293467 SCYL1 P CRE726 chr1:54355079-54355938 YIPF1 P CRE16100 chr4:159593272-159594085 ETFDH;C4orf46 P -4 CRE2044 chr1:236958591-236959468 MTR P CRE829 chr1:78244796-78245875 FAM73A P CRE18528 CRE12149 chr2:153574105-153575086 ARL6IP6;PRPF40A P CRE210 chr1:16174350-16175665 SPEN;FLJ37453 P (EIF1AK1) CRE18532 CRE4969 chr12:104609311-104610168 TXNRD1 P CRE7300 chr16:685892-687101 C16orf13 P (FAM220A) CRE3651 chr11:65728616-65729612 SART1 P CRE9953 chr19:4638110-4640206 TNFAIP8L1 P CRE17909 chr6:74170579-74172329 MTO1 P CRE18581 chr7:16460306-16461220 ISPD P CRE676 CRE10400 chr19:20747733-20748908 ZNF737 P CRE296 chr1:24126114-24128109 GALE P (TAL1) CRE11736 chr2:75185312-75186150 POLE4 P CRE4612 chr12:53614787-53615630 RARG P CRE17233 chr6:2875435-2876320 SERPINB9P1 P CRE16355 chr5:40678875-40679834 PTGER4 P CRE18973 CRE20707 chr9:80911164-80912545 PSAT1 P CRE15384 chr4:774868-776706 LOC100129917 P -6 (HIP1) CRE8971 chr17:55334065-55335008 MSI2 P enCRISPRi, log2 Enrichment (T28/T0) CRE153 chr1:10269924-10271441 KIF1B P CRE20172 chr8:124428099-124429820 WDYHV1 P CRE11583 chr2:64067994-64068916 UGP2 P sgRNAs targeting: CRE4244 chr12:6930058-6931215 GPR162 P CRE15745 chr4:76911162-76912330 SDAD1 P CRE13414 CRE20486 chr9:32551244-32552657 TOPORS-AS1;TOPORS P chr19:12163235-12164016 ZNF878 P CRE11314 chr2:27631829-27633103 PPM1G P CRE10147 All other promoters (RUNX1) CRE7302 chr16:698362-699516 WDR90 P CRE15960 chr4:120987496-120988961 MAD2L1 P CRE17302 chr6:8064039-8065222 BLOC1S5 P CRE7905 chr16:67193183-67194371 FBXL8;TRADD P CRE11386 chr2:37898839-37899616 CDC42EP3 P Non-targeting controls CRE175 chr1:11865359-11866764 CLCN6;MTHFR P CRE8561 chr17:27139090-27140525 FAM222B P -8 CRE7304 chr16:729283-730989 STUB1 P CRE17861 chr6:53211832-53213718 ELOVL5 P Tumor suppressive promoters CRE19753 chr8:37552261-37553387 ZNF703 P CRE19118 chr7:100181388-100182161 FBXO24 P CRE1161 CRE13663 chr22:21983495-21984551 YDJC P CRE11652 chr2:69870283-69871432 AAK1 P Oncogenic promoters