CD15 CD16 CD14 CD33 HLA-‐DR CD71 CD235 CD34 CD38 CD3

CD15 CD16 CD14 CD33 HLA-‐DR CD71 CD235 CD34 CD38 CD3

CD3 CD7 CD45 CD56 CD10 CD19 CD20 CD11b CD15 CD16 CD14 CD33 HLA-­‐DR CD71 CD235 CD38 CD34 Supplementary Figure 1: SPADE plots of normal bone marrow sample #6. SPADE clustering was performed on all samples (normal and AML) simultaneously to generate a single tree structure for all samples. All of the cell events from each sample were then mapped to the common tree structure. Each node of the SPADE tree is colored for the median expression of the indicated markers from low (blue) to high (red). The size of each node is correlated to the fracTon of cells mapping to the node; however, a minimum size was enforced for most nodes to allow visualizaTon of node color. Immunophenotypic grouping of nodes was performed manually on the basis of the median marker expression level of each node, and based on analysis of the relevant biaxial plots (e.g., CD38 vs. CD34). H
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L Supplementary Figure 2: The frequency of cells in the indicated (manually-­‐gated) popula:ons. Colored boxes group immunophenotypic popula:ons. HSPC, hematopoie:c stem and progenitor cells; B, blasts (immunophenotypic); Mono, = monocyte lineage cells; Gran, granulocyte lineage; RBC, red blood cell lineage; B-­‐
Cell, B cell lineage. Error bars indicate standard errors. Nl#5 – A Normal Nl#5 – B Normal Nl#3 – A Normal Nl#3 – B Normal Nl#4 – A Normal Nl#4 – B Normal Nl#6 – A Normal Nl#6 – B Normal Nl#1 – A Normal Nl#1 – B Normal CR#2 NK – FLT3wt CR#6 NK – FLT3wt CR#3 FLT3-­‐ITD+ AML#25 PRK – Mono 7 APL#3 t(15;17) – FLT3-­‐ITD APL#4 t(15;17) – FLT3-­‐ITD AML#36 NK – FLT3wt AML#42 NK – FLT3wt CR#5 NK – FLT3wt AML13 NK – FLT3wt AML8 FLT3 -­‐ TKD AML#41 PRK – t(1;3) AML#14 NK – FLT3wt AML#10 CBF – inv(16) AML#12 Myeloid Sarcoma CR#7 del(q13) AML#19 PRK – Mono 7 AML#20 FLT3-­‐ITD * AML#18 PRK – Complex AML#33 NK – FLT3wt AML#4 PRK -­‐ Complex AML#5 CBF – t(8;21) AML#15 NK – FLT3wt AML#26 CBF – t(8;21) AML#35 CBF – t(8;21) AML#32 CBF – t(16;16) AML#22 MLL – t(10;11) AML#7 NK – FLT3-­‐ITD AML#39 NK – FLT3-­‐ITD AML#23 NK – FLT3-­‐ITD AML#6 NK – FLT3-­‐TKD AML#40 NK – FLT3-­‐ITD AML#27 NK – FLT3-­‐ITD AML#38 NK – FLT3-­‐ITD AML#9 NK – FLT3-­‐ITD AML#21 NK – FLT3-­‐ITD AML#30 NK – FLT3-­‐ITD AML#31 NK – FLT3-­‐TKD APL#5 t(15;17) – FLT3-­‐ITD APL#1 t(15;17) – FLT3-­‐ITD AML#37 NK – FLT3-­‐ITD AML#29 PRK -­‐ Complex Supplementary Figure 3: Mul:-­‐dimensional binning analysis of the CD34+CD38low subset confirmed that dis:nct karyotype and genotype-­‐specific immunophenotypic pacerns are observed in high-­‐dimensional space. Hierarchical grouping of normal and AML samples based on the pairwise correla:on of the distribu:on of cells across the 100 mul:-­‐dimensional bins are shown. A Decreased Increased H
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L Supplementary Figure 4: AML subtypes are characterized by disTnct phosphoprotein signaling paZerns. Log fold change in phosphoprotein staining relaTve to normal cells within each gated immunophenotypic populaTon. Measured phosphoproteins are: A) pSTAT5, B) pMAPKAPK2, C) pERK, and D) p4E-­‐BP1. Colored boxes group immunophenotypic populaTons: HSPC, hematopoieTc stem and progenitor cells; B, blasts (immunophenotypic); Mono, monocyte lineage cells; Gran, granulocyte lineage; RBC, red blood cell lineage; B-­‐Cell, B cell lineage. Error bars indicate standard errors. Supplementary Figure 5A Normal 1 Normal 3 AML26 – t(8;21) AML35 – t(8;21) Normal 4 Normal 5 Normal 6 AML22 – t(10;11) APL4 – FLT3-­‐ITD+ APL5 – FLT3-­‐ITD+ tSNE-­‐2 AML9 – FLT3-­‐TID+ AML21 – FLT3-­‐ITD+ AML23 – FLT3-­‐ITD+ AML27 – FLT3-­‐ITD+ AML14 – NK-­‐AML tSNE-­‐1 z = p-­‐STAT5 Supplementary Figure 5B Normal 1 Normal 3 AML26 – t(8;21) AML35 – t(8;21) Normal 4 Normal 5 Normal 6 AML22 – t(10;11) APL4 – FLT3-­‐ITD+ APL5 – FLT3-­‐ITD+ tSNE-­‐2 AML9 – FLT3-­‐TID+ AML21 – FLT3-­‐ITD+ AML23 – FLT3-­‐ITD+ AML27 – FLT3-­‐ITD+ AML14 – NK-­‐AML tSNE-­‐1 z = p-­‐MAPKAPK2 Supplementary Figure 5: viSNE analysis of CD34+CD38low subset demonstrates signaling aberrancy is most pronounced in cells outside the viSNE space occupied by normal cells. Samples were analyzed across 19 surface marker dimensions (as in Figure 5) and then each cell event was colored for the phospho-­‐epitope staining of A) pSTAT5 or B) pMAPKAPK2. Blue cell events have the lowest levels of staining while red cell events exhibit the highest levels. The AML subtype is indicated for each sample. Supplementary Figure 6 A Normal t(8;21) inv(16) APL (FLT3+) FLT3-­‐ITD+ FLT3-­‐TKD+ FLT3wt NK-­‐AML MLL t(10;11) Adverse-­‐Risk CR / CRi pSTAT5 Medians (CD34+CD38low) Supplementary Figure 6 B Normal t(8;21) inv(16) APL (FLT3+) FLT3-­‐ITD+ FLT3-­‐TKD+ FLT3wt NK-­‐AML MLL t(10;11) Adverse-­‐Risk CR / CRi pMAPKAPK2 Medians (CD34+CD38low) Supplementary Figure 6 C Normal t(8;21) inv(16) APL (FLT3+) FLT3-­‐ITD+ FLT3-­‐TKD+ FLT3wt NK-­‐AML MLL t(10;11) Adverse-­‐Risk CR / CRi pERK Medians (CD34+CD38low) Supplementary Figure 6 D Normal t(8;21) inv(16) APL (FLT3+) FLT3-­‐ITD+ FLT3-­‐TKD+ FLT3wt NK-­‐AML MLL t(10;11) Adverse-­‐Risk CR / CRi p4E-­‐BP1 Medians (CD34+CD38low) Supplementary Figure 6 E Normal t(8;21) inv(16) APL (FLT3+) FLT3-­‐ITD+ FLT3-­‐TKD+ FLT3wt NK-­‐AML MLL t(10;11) Adverse-­‐Risk CR / CRi pRP-­‐S6 Medians (CD34+CD38low) Supplementary Figure 6 F Normal t(8;21) inv(16) APL (FLT3+) FLT3-­‐ITD+ FLT3-­‐TKD+ FLT3wt NK-­‐AML MLL t(10;11) Adverse-­‐Risk CR / CRi pAKT Medians (CD34+CD38low) Supplementary Figure 6 G Normal t(8;21) inv(16) APL (FLT3+) FLT3-­‐ITD+ FLT3-­‐TKD+ FLT3wt NK-­‐AML MLL t(10;11) Adverse-­‐Risk CR / CRi H3K9ac Medians (CD34+CD38low) Supplementary Figure 6 H Normal t(8;21) inv(16) APL (FLT3+) FLT3-­‐ITD+ FLT3-­‐TKD+ FLT3wt NK-­‐AML MLL t(10;11) Adverse-­‐Risk CR / CRi pATM Medians (CD34+CD38low) Supplementary Figure 6 I Normal t(8;21) inv(16) APL (FLT3+) FLT3-­‐ITD+ FLT3-­‐TKD+ FLT3wt NK-­‐AML MLL t(10;11) Adverse-­‐Risk CR / CRi pCREB Medians (CD34+CD38low) Supplementary Figure 6: Median basal intracellular signaling of CD34+CD38low cells from AML samples of each subtype. Median expression level of the indicated markers in the total CD34+CD38low popula:on, each data point represents the median expression level of one pa:ent sample or one of the 14 sample aliquots from the five healthy donors. Each phospho-­‐epitope is indicated. H
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L Supplementary Figure 7: HU treatment does not increase the frac:on of apopto:c cells measured by cPARP levels. Nega:ve values reflect the very low cPARP signal that is further reduced by noise subtrac:on. Colored boxes group immunophenotypic popula:ons. HSPC, hematopoie:c stem and progenitor cells; B, blasts (immunophenotypic); Mono, monocyte lineage cells; Gran, granulocyte lineage; RBC, red blood cell lineage; B-­‐Cell, B cell lineage. Error bars indicate standard errors. Supplementary Figure 8: Histone 3 lysine 9 acetyla:on decreases with immunophenotypic differen:a:on in both normal and AML pa:ent samples. Median an:body staining for H3K9ac in cells of the indicated (manually-­‐gated) popula:ons. Error bars indicate standard errors.