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A B D E F I H J G C K Figure S1 Figure S1 A B 4-month-old mice 20 2000 ** WT * ** Asxl1+/- 15 1500 Nf1+/- Asxl1+/-;Nf1+/- ** 10 1000 *** Leukemia RBC (K/µl) MPN 5 Platelet (K/µl) 500 MDS Hemoglobin (g/dl) MDS/MPN Relative mRNA expression Relative mRNA Asxl1 Nf1 0 0 WT Asxl1+/-;Nf1+/- C DE Diseased mice ** ) ** 7 ** * ** WT Asxl1+/- Nf1+/- Asxl1+/-;Nf1+/- *** *** Body weight (g) weight Body Relative mRNA expression Relative mRNA Asxl1 Nf1 spleen (x10 no. per Cell F G H Asxl1+/-;Nf1+/- WT WT 1 2 3 4 WT Asxl1+/-;Nf1+/- ** % survival +/- ;Nf1 +/- Asxl1 Days 100 μm 10 μm I J 36.9 40.8 Spleen weight (g) Recipient No. Splenomegaly (%) Latency (weeks) mean ± s.d. no ab (log) no ab cKit (log) n = 9 100% 0.369 ± 0.052 <6 21.7 0.55 no ab (log) Mac1 (log) K Normal Leukemia#1 Ratio #1 Leukemia#2 Ratio #2 Asxl1 Nf1 Figure S1. Development of myeloid leukemia in Asxl1+/-;Nf1+/- mice. (A) qPCR results showing the mRNA levels of Asxl1 and Nf1 in WT and Asxl1+/-;Nf1+/- mice. Top, BM cKit+ cells from 4-month-old Asxl1+/-;Nf1+/- mice (n = 4 per group); bottom, spleen cells from diseased Asxl1+/-;Nf1+/- mice (n = 7 per group, two with myeloid leukemia and five with MDS/MPN in Asxl1+/-;Nf1+/- mice). (B) Parameters of PB were summarized from WT, Asxl1+/-, Nf1+/-, and Asxl1+/-;Nf1+/- mice at the time of sacrifice (n = 11 to 12 per group). (C) Spleen cellularity (n = 10 to 11 mice per group). (D) The gross morphologies of the liver from each genotype of mice. Tumors infiltrating liver in Asxl1+/-;Nf1+/- mice. (E) Body weight of WT, Asxl1+/-, Nf1+/- and Asxl1+/-;Nf1+/- mice at the time of sacrifice (n = 10 to 11 per group). (F) Representative H&E stained sections from the liver of WT and Asxl1+/-;Nf1+/- mice are shown. Scale bar: left, 100 μm; right, 10 μm. (G) Kaplan-Meier survival curve of secondary recipient mice transplanted with Asxl1+/-;Nf1+/- spleen cells (WT, n = 5 and Asxl1+/-;Nf1+/- n = 9). A log-rank test was used for survival statistics. (H) Representative photographs of bone and spleen from secondary recipient mice transplanted with Asxl1+/-;Nf1+/- spleen cells at 6-week after transplantation. (I) Flow cytometric analysis of BM cells revealed that the recipients had the similar Mac1+/cKit+ population with the primary mouse. (J) Enlargement of spleens from secondary recipient mice transplanted with Asxl1+/-;Nf1+/- spleen cells at 6-week after transplantation. (K) Asxl1 and Nf1 gene coverages and read depth ratios obtained through whole exome sequencing of Asxl1+/-;Nf1+/- leukemia cells (n = 2) and matched normal tissue. Data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; unpaired Student’s t-test (A) or one-way ANOVA with Tukey’s multiple comparisons test (B-C, and E). Figure S2 AB C BMT WT WT AKO +/- +/- Asxl1 ;Nf1 10 μm NKO DKO * 96.1 % survival % survival 98.5 CD45.2 Gr1 Days CD45.1 Mac1 Days D *** EFG *** *** *** *** *** *** *** *** *** *** ** WT AKO NKO DKO RBC (K/µl) WBC (K/µl) Platelet (K/µl) Hemoglobin (g/dl) HIJ PB BM Spleen 3 ** 2 *** in BM (%) + 1 cKit Spleen weight (g) Spleen weight 10 μm 0 WT DKO WT DKO K BM Spleen WT DKO 100 μm 100 μm Figure S2. Deletion of Asxl1 and Nf1 accelerates the development of myeloid leukemia in vivo. (A) Kaplan-Meier curve representing percent survival of recipient mice transplanted with BM cells from WT and young Asxl1+/-;Nf1+/- mice (n = 5 per group). No lethality was observed for recipients of WT BM cells. A log-rank test was used for survival statistics. (B) The appearance of one Asxl1+/-;Nf1+/- transplanted mouse (n = 5) with a large mass in the stomach (left panel). Representative microphotograph of H&E stained section of the mass (right panel). Below is the flow cytometric analysis of the cells within the large mass. (C) Kaplan-Meier survival curve showing the percent survival of WT (n = 10), Asxl1∆/∆ (AKO, n = 5), Nf1∆/∆ (NKO, n = 10) and DKO (n = 18) mice over time. (D-G) Peripheral WBC count (D), RBC count (E), Hemoglobin (F), and Platelets (G) for each genotype of mice (n = 8 to 21 per group) at time of sacrifice (3-6 months after pIpC injection). (H) May-Grunwald-Giemsa- stained blood smear and cytospin preparations of BM and spleen cells from WT and DKO mice. Scale bar: 10 µm. (I) Flow cytometric analysis of the BM cells revealed a significantly increased cKit+ population in DKO mice (n = 5 per group). (J) Spleen weight from WT and DKO mice is shown (n = 8 to 9 per group). (K) Representative H&E stained sections from femur and spleen from WT and DKO mice are shown. Scale bar: 100 μm. Data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; one-way ANOVA with Tukey’s multiple comparisons test (D-G) or unpaired Student’s t-test (I and J). Figure S3 AB50 WT Asxl1+/- Nf1+/- Asxl1+/-;Nf1+/- upregulated downregulated 40 Ccnd1 3 all Myc 30 0 (fold change) 20 2 -3 Log FPKM (RNA-seq) 10 -6 6 0 0481216 Stat1 Usp18 Myc Nrp1 Ccnd1 Irf9 Log2(average expression) C VALK_AML_CLUSTER DAWSON_BRD4_CORE_SIGNATURE HOHMANN_AML_BRD9_KD_DN 0.8 0.6 0.4 NES = 1.51 NES = 2.28 NES = 1.69 P = 0.003 P = 0 P = 0.001 FDR = 0.191 FDR = 0.060 0.0 0.0 FDR = 0 0.0 Enrichment score Enrichment score Enrichment score Asxl1+/-;Nf1+/- Control Asxl1+/-;Nf1+/- Control Asxl1+/-;Nf1+/- Control PERNA_MYC_SERUM_RESPONSE TAKEDA_NUP98_HOXA9_TARGETS_UP HOELZEL_NF1_TARGETS_DN 0.5 0.4 0.5 NES = 1.58 NES = 1.93 NES = 1.65 P = 0 P = 0 P = 0.001 FDR = 0.132 FDR = 0.002 FDR = 0.083 0.0 0.0 Enrichment score 0.0 Enrichment score Enrichment score +/- +/- Asxl1 ;Nf1 Control Asxl1+/-;Nf1+/- Control Asxl1+/-;Nf1+/- Control SCHRAETS_MLL_TARGETS_UP XU_MLL_KNOCKOUT_DN PLASARI_TGFβ1_TARGETS_UP 0.6 0.4 0.0 NES = 1.69 NES = 1.50 NES = -2.71 P = 0.001 P = 0 P = 0 FDR = 0.058 FDR = 0.195 0.0 0.0 -0.7 FDR = 0 Enrichment score Enrichment score Enrichment score Asxl1+/-;Nf1+/- Control Asxl1+/-;Nf1+/- Control Asxl1+/-;Nf1+/- Control D E WT Asxl1+/-;Nf1+/- Myc DAPI Merge MYC β-ACTIN WT F WT Asxl1+/-;Nf1+/- Asxl1+/-;Nf1+/- 30 25 10 μm +-+-GDP -+-+GTPγS G AML_TCGA AML_GSE42064 AML_GSE84359 AML_TCGA 0.35 0.25 0.35 0.4 NES = 1.41 NES = 1.63 P = 0 NES = 1.96 P = 0 NES = 1.37 FDR = 0 P = 0 FDR = 0 P = 0 FDR = 0 0.0 0.00 FDR = 0 Enrichment score 0.00 -0.10 ASXL1 mut ASXL1 wt ASXL1 mut ASXL1 wt ASXL1 mut ASXL1 wt RAS pathway mut RAS pathway wt H AML_TCGA AML_TCGA AML_GSE42064 AML_GSE84359 (RAS pathway mut vs (ASXL1 mut vs ASXL1 wt) (ASXL1 mut vs ASXL1 wt) (ASXL1 mut vs ASXL1 wt) RAS pathway wt) mouse mouse mouse mouse human human human human 793 38 453 824 757 817 14 129 810 21 305 P = 1.2e-4 P = 7.2e-3 P = 2.3e-4 P = 0.026 USP18, NRP1, IRF7, IRF8, CXCL12, CXCL12, GJA1, CDC42BPA, LGALS3BP, UGCG, ACSL6, ABCB6, USP18, OAF, SAMD14, MYL9, MX1, TNC, UGCG, MX1, IFI27, LGMN, OAF, FAM213A, MMP8, PLSCR4, SPARC IFI27, EPB42, ABCG2, RHAG, PLS1, OAS3, ISG15, SIGLEC1, NRIP1, GP6, CARD11, LPL, SIGLEC1, ZBP1, HEMGN, GYPA, GCLM, TMCC2, CXCR3, IFIT1, PPBP, IFI27, EPSTI1, AMIGO2, ISG20, GSTM5, ISG15, TMEM51 CD22, COL5A1, NCR1, ALDH1A1, COL1A2, FSTL1, CXCR3, SLC25A21, WBSCR27, CLEC9A TGFBI, ACHE, EPSTI1, FABP4, IGFBP5, ABCG2, GJA1, PKDCC, IL10RA, NINL, FCRL5, IFIT1, SEMA6D, CD22, OAS2 I Piezo2 Penk Nop16 Expression Cmas Il6 Ngfrap1 Zfp637 Mphosph10 Z-score Adcy2 Tubb2a Nolc1 Klf10 Il6st Ddx18 Stxbp6 Ak1 1.5 Arhgap22 Ipo4 Smad7 Pdgfd Il1rn Ppan Pls1 Tcof1 Wnt9a Gas2 Crip2 Trak2 Gja1 Supv3l1 Ryr3 Srm Hes1 Pros1 Nqo1 Cdk4 Rap1gap Nr4a1 Gp1ba Adcy2 Wdr74 Net1 BC094916 Bysl Csrnp1 0 Pccb Grwd1 Aim1 Irgm2 Bhlhe40 Sod1 Stat1 Pa2g4 Blvra Myc Fcgrt Mnda Serpine1 Wbp5 Wdr43 Tnik Ifi35 Plk1 Egr2 Serpinb8 Usp18 Pprc1 Tpm1 -1.5 Urod Ifih1 Slc29a2 Ptgs2 Optn Cfh Mybbp1a Slc6a9 Hspe1 Egr3 Pdzk1ip1 Ifi205 Hspd1 Slc25a37 Lif Arhgef12 Aim1 Rrp9 Aldh1a1 Nop2 Sptb Isg15 Slc20a1 Clcn3 AI607873 Phb Rhd Noc4l Cdk5r1 Snca Mmp13 Mcm4 Trim10 Cdc42bpa Pltp Npm1 Id1 Ank1 Pydc3 Pes1 Fech Aimp2 Ier3 Gypa Ifi204 Myh10 Mgp Las1l Rasl11b Tns1 Slc19a1 Icam4 Tap1 Selenbp1 Nop56 Gadd45b Selenbp2 Ercc5 Map3k6 Minpp1 Pon3 Rcl1 Spata13 Tal1 Utp20 Tfr2 Pydc4 Ier5 Fads2 B2m Pus1 Gata1 Sord Rhag Pyhin1 Gadd45g Kel Gnl3 Klf1 Mndal Ung Junb VALK_AML_CLUSTER CASTELLANO_NRAS_TARGETS_UP HALLMARK_MYC_TARGETS_V2 PLASARI_TGFβ1_TARGETS_UP J Myc Card11 Upregulation Cd226 Cxcl12 Eif2ak2 No change Gbp2 Ifi205 Downregulation Ifih1 Il10ra Irf7 Expression difference Mmp14 Stat1 Tlr3 Usp18 Il6st Irf8 Cd63 Dusp1 Ebf1 Gfra2 Gnai3 Hspa1b Id3 Irs2 Plaur Prkce Tnfaip3 Tsc22d3 Vav1 Zc3h12a Figure S3. Cooperative effect of Asxl1 and Nf1 loss on the induction of a MYC- driven transcription signature. (A) Fragments per kilobase of transcript per million (FPKM) values of DEGs in RNA-seq are shown. Data represent the mean of two biological replicates. (B) MA plot showing the dysregulated genes in Asxl1+/-;Nf1+/- cKit+ cells compared with WT and single mutant cells. Myc and Ccnd1 are highlighted in black. (C) GSEA showing expression differences of representative transcription signatures associated with AML, MYC, and MYC upregulators BRD4/9, NUP98- HOXA9, NF1, MLL, and TGFβ target genes in Asxl1+/-;Nf1+/- cKit+ cells compared with control cells. (D) Immunofluorescence assay shows the MYC protein accumulation (Green) in Asxl1+/-;Nf1+/- BMMNCs compared with WT cells. Nuclei were visualized with DAPI (Blue). Scale bars: 10 μm. (E) Western blot showing the MYC protein levels in Asxl1+/-;Nf1+/- and WT BMMNCs.
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