upplemental Figure. (A) Rounds of replating (R) of LL-AF transformed LK colonies from initiation experiments (.E. of triplicate experiments, p=.)., p<.;, p<.;, p<.; (B) pleen weights and (C) Liver weights from moribund mice that were from the primary initiation experiment, data from si f/f n= and si Δ/Δ
n= mice. Weights from spleen (D) and liver (E) from moribund mice that were from the primary maintenance experiment, si f/f n= and si Δ/Δ n= mice. p<., p<., p<.. upplemental Figure. (A) Frequency of F and CD of leukemic cells from mice of indicated genotypes si f/f n= and si Δ/Δ n= from maintenance LK experiments based on staining in Figure C. (B) Representative images of Wright-iemsa staining of cytospins from bone marrow isolated leukemia cells taken from the initiation experiment. (C) Cell cycle status based on Hoescht staining in c-kit gated cells from initiation si f/f n= and si Δ/Δ n= and maintenance experiments si f/f n= si Δ/Δ n=. (D) Apoptosis was calculated from the sub- phase from samples in (C). (E) Colony assays from initiation LK experiment si f/f n= and si Δ/Δ n= leukemic mice. (F) Cell counts from colonies analyzed in (E). () Representative flow plot of I intracellular staining showing complete and incomplete deletion of si. ean frequency of I high population in c-kit high cells of the indicated genotypes from mice from (H) LK initiation, si f/f n= and si Δ/Δ n= (I) P initiation si f/f n= and si Δ/Δ n= and (J) maintenance experiment si f/f n= and si Δ/Δ n=. upplemental Figure. urvival analysis of the secondary transplants of leukemia cells from different donors from primary transplanted mice that were combined in Figure E (A-D) LK initiation experiment and Figure F (E-H) maintenance experiment.
p<.,, p<.. (A) si f/f n= and si Δ/Δ n=. (B) si f/f n= and si Δ/Δ n=. (C) A combination of three different donors si f/f n= and si Δ/Δ n=. (D) si f/f n= and si Δ/Δ n=. (E) si f/f n= and si Δ/Δ n=. (F) si f/f n= and si Δ/Δ n=. () si f/f n= and si Δ/Δ n=. (H) Frequency of I positive cells in the c-kit high cells in upplemental Figure F obtained by intracellular analysis of I High cells in the c-kit High compartment si f/f n= and si Δ/Δ n=. upplemental Figure. si controls growth factor signaling in LCs. (A) Representative flow cytometric gating on c-kit High in leukemia cells isolated from the bone marrow. (B) Representative histograms and levels of perk gated in (A) after serum starvation and stimulation with cytokines (IL, IL and CF) for indicated time points (minutes) and then stained for intracellular phosphorylated ERK (perk). (C) Fold of perk edian fluorescence intensity (FI) after cytokine stimulation. ean and.e. from leukemia cells of genotype si f/f n= and si Δ/Δ n= mice from two independent experiments are shown. (D) Fold stimulation of p and (E) ptat after cytokine stimulation. ean and.e. from leukemia cells from si f/f n= and si Δ/Δ n= mice from two independent experiments are shown, p<.;, p<.. upplemental Figure. (A) Unsupervised clustering of the si Δ/Δ LL-AF LC signature cluster on ECO AL patients. Heat map and unsupervised clustering identifies nine
groups when () genes differentially expressed in the si f/f and si Δ/Δ LC RNA-sequencing data is overlapped with RNA profiling from AL patients (n= patients). Columns represent individual patients and rows are genes Log TP expression normalized by row means. Color bar above gene rows are displayed as follows: I expression above one standard deviation of the median in patients were labeled as High, below as Low and between as id, Cluster numbers separated by colors and mutations as shown. Cluster- patients are further subdivided with shaded grey for normal bone marrow controls. Patients in cluster- and Cluster- were isolated based on their differential I levels see Figure. (B) Cluster (C) Cluster from Figure E, Circos plots indicating relative frequency and pairwise co-occurrence of mutations in patients. The length of the arc corresponds to the frequency of mutations in the first gene, and the width of the ribbon corresponds to the percentage of patients who also had a mutation in the second gene. The number next to the gene represents the number of patients. upplemental Figure. IV generated plot showing reads on the Ikzf locus from RNA sequencing data obtained from LCs of si f/f n= and si Δ/Δ n=. ashimi plot shows the splicing of Ikzf. upplemental Figure (A) mrna stability of Ikzf, Hoxa and c-yc in control and I O/E cells. Data of two independent experiments are shown. (B) qpcr of Ikzf, Hoxa and c-yc
in c-kit high cells of indicated genotypes si f/f n= and si Δ/Δ n=. (C) Immunofluorescence staining of c-yc in sorted si f/f and si Δ/Δ LCs. Cells were stained with c-yc specific Ab (left) and DAPI (middle); a merged image is shown (right). Bars, µm. Fluorescence intensity was quantified and normalized to the control (three independent experiments; and cells were quantified per group in total). eans and E are shown. (D) Representative flow cytometric plot for intracellular staining of I and IKZF in c-kit High cells (left panel). Frequency of IKZF High cells in LL-AF leukemic cells averaged from si f/f n= and si Δ/Δ n= (right panel). eans and.e. for B- D,, p<.;, p<.;, p<.. (E) Immunoblot of lysates from leukemic bone marrow cells from moribund mice of indicated genotypes si f/f n= and si Δ/Δ n=. ean frequency of (F) I, () IKZF and (H) yc positive cells after acute deletion of si at hrs after n -OHT treatment in si f/f Cre-ER - /+ leukemic cells. Data shown are from intracellular staining of si f/f Cre- n= and si f/f Cre+ n=. eans and.e., p<.;, p<.;, p<.. upplemental Figure (A) qpcr of Ikzf in leukemic cells from si f/f n= and si Δ/Δ n= from the maintenance secondary transplant # where all si Δ/Δ mice formed leukemia. (B) Representative flow plot showing IKZF high cells by intracellular staining. ean frequency of IKZF high cells in si f/f n= and si Δ/Δ n= from secondary maintenance transplants (upplemental Figure D and F) where all si Δ/Δ formed leukemia. (C) ean frequency of IKZF high cells in si f/f n= and si Δ/Δ n= from secondary maintenance transplants (upplemental Figure
E and ) where majority of si Δ/Δ did not form leukemia. upplemental Table. HIT-CLIP fold change rank list. K UV-cross linked and RNA-IP rank list is a fold change between FLA-I overexpression and empty vector transduced cells. Negative Fold change represents background binding of transcripts that have a FKP> read on the RNA-seq. upplemental Table. ummary of deletion status of primary si deficient leukemia. amples that had more than % deletion in the gated FP+ leukemic cells were considered deleted based on intracellular flow cytometry and FKP> read from the RNA-seq. upplemental Table. ummary of differentially expressed genes in si f/f and si Δ/Δ LCs and si LC signature. List of downregulated genes and upregulated genes ranked on log-fold change= >., p<., FDR<.. List of differentially expressed genes that have human homologs > mean log intensity). upplemental Table. ene sets enrichment analysis in si Δ/Δ LC that are downregulated. upplemental Table. ene sets enrichment analysis in si Δ/Δ LC that are upregulated. upplemental Table. si deficient LK and LC gene sets enrichment analysis that overlaps with the HIT-CLIP gene sets (Figure B).
upplemental Table. List of Top HIT-CLIP I bound genes and LL-AF previously annotated gene sets. upplemental Table. List of the overlap of HIT-CLIP I bound genes with LL-AF transcriptional targets for RNA-IP prioritization.
A B C # Colonies si f/f si Δ/Δ pleen Weight (g)..... si f/f si Δ/Δ Liver Weight (g) si f/f si Δ/Δ D pleen Weight (g) pleen weight...... Plating # R R R R R ree+ Leukemic aintenance Liver Weight (g) E..... Liver weight e- + si f/f si Δ/Δ Leukemic aintenance. Leukemic Initiation Leukemic Initiation upplemental Figure.
A B Differentiation aintenance E No of colonies si f/f si Δ/Δ Colony B H I I % positive of c-kit High si f/f No of cells (X cells) F si Δ/Δ Colony cell number e- e+ si f/f si Δ/Δ I Initiation LK Initiation P aintenance LK I % positive of c-kit High ns si f/f! si Δ/Δ si f/f! si Δ/Δ si f/f! si Δ/Δ J I % positive of c-kit High C % of Kit High cells FP Initiation si f/f si Δ/Δ Cell cycle % of Kit High cells / / T aintenance O T O si f/f si Δ/Δ T O D ub-(%) si f/f! si Δ/Δ si Δ/Δ Deleted (% deletion) Apoptosis Init. aint. Undeleted (% deletion) upplemental Figure.
A B C D urvival (%) Initiation econdary # Initiation econdary # Initiation econdary # urvival (%) urvival (%) urvival (%) Initiation econdary # si f/f si Δ/Δ E Time Days post transplant aintenance econdary # F Days post transplant aintenance econdary # Time Days post transplant aintenance econdary # Cre-ER H Days post transplant Deletion of (F) si pos cells urvival (%) Days post transplant urvival (%) Days post transplant urvival (%) Days Days post transplant si f/f si Δ/Δ I % positive of c-kit High si f/f! si /Δ upplemental Figure.
A D ac- Fold stimulation (min) d si f/f! c-kit High c-kit p c-kit High LL-AF - - d si f/f si Δ/Δ si Δ/Δ c-kit High E Fold stimulation (min) d B d c-kit High perk ptat c-kit High LL-AF ns - - si f/f si Δ/Δ IL- IL CF: - - si f/f si Δ/Δ C Fold stimulation (min) perk c-kit High LL-AF d - - si f/f d ns si Δ/Δ upplemental Figure.
NB_ NB_ NB_ NB_ NB_ NB_ NB_ NB_ NB_ NB_ NB_ NB_ NB_ NB_ NB_ I text High LOW ID I text High LOW ID LL splitll LLPTDLL splinp tll LLPTD FLT_TKD utations: LL splflt_inp ittlld LL FLT_TKD LLPTDFLTLL splflt_inp ithtllerd NPDNTA_ot LLPTD FLT_TKD FLT DNTA_R FLT FLT_INPhTerD DNTA DNTA_ot DNTA FLT_TKD High Clusters DNTA_R clustflter.id DNTA_ot IIDNTA tflt_iefltxt.tehtxterd id I status DNTA_R cluster.id DNTA_ot DNTA I.tehxter Low DNTA_R cluster.id IDNTA.text cluster.id I.text row min A upplemental Figure. relative row min row max relative row max I text Id I text Id Id Id ITONPRd OX CEACA HDB LCA FCRA EP PKIB HNT RNAE RNAE LCA ILR CKAP ILEC ILEC FCRT BCLA TRE CYBB CAR A A Corf IRF LN PTK ERPINF NRBP TPINP FRAT FRAT NDR KLF CDKNA KLF TKC VILL PLCR PRA CFP APOBR RARP CFR CDLF PLEKH -ep EN REPIN FAD NOTCH CLTN PN IFIT IFIT A IFIT TCF BIN COLAPR BNT LCAIX OX EPHA COLA TON ALPI ALPI LCA PR ALPPLEPHA COLA PTPRU PTPRU OX ALPPL LCA ITON X ALDHL EPHA PRALPP ALPI BNT COLADC ALPPL ALDHL LCA OX PKDL PTPRU HIA EPHA TON IALPP X NATL DC ALPIPRCDLD CK PKDL ALPPL COLAACOT BNT HIPTPRU ACDKNA LCA ALDHL NATL ALPPIEPHAX PHLDA EPL ALPI RCOR DCACOT CDLD BNT ALPPLKICNAAL PKDL PTPRUA CXCR HICKALDHL CDKNA ALPP IX TU NATL PHLDA DC OC ACOT Corf EPL PKDL BNTCEACA CDLD NA RCOR AERPI CKHIALDHL P KINATL ALPPAALP CDKNA P CN ACOT DC PHLDA CXCR CDLD PKDLP EPL CLIC TU CKHIACEACA RCOR CDKNA NATL KIOC AALCEACA LCA PHLDA ACOTfIL CNCor LEC CEACA EPL CDLDIPPPRA CXCR ERPI RCOR CK NFRZBA TU P KICDKNA AALCDCBPA OC P PHLDAfCEACA CN Cor CL P EPLKLK CXCR CEACA P RCORNARBP TU ERPI FEZ KIACAL PCLIOC C CN fffar Cor PCEACA CEACA CXCR CEACA P CAP CD TUNRVI ERPI A PLCA CLEC CXCR OC CLIIP IP L fltfdact Cor CEACA CEACA CEACAPLXNA P CEACA KLK ERPINDWD P LCA BAIA CLIPLECC PEAR IRBP FEZ P TAP ILCEACA NLN CL P LCN CEACA CEACA P LCAEL KLKEL ICLICLEC YCP RBPLCN XD ICEACA L ILEC FEZPPPRA FRZB CEACACNN CEACA CL LAC LCAFTL KLK ELCDCBPA ILECCorf RBP LCNCAP T CD FEZIL HPA PPPRA CEACA PPIC CL FRZBRVI CXCR KLK CLECA EL CDCBPA RE DACT RBP PO LCN CAP OX TON Id Id OX TON PR BNT IX COLA ALPI LCA EPHA PTPRU ALPPL ALDHL ALPP DC PKDL HIA NATL CDLD CK ACOT CDKNA PHLDA EPL RCOR KIAAL CN CXCR TU OC Corf CEACA ERPINA P P P P CLIC CEACA CEACA LCA IL ILEC OX PPPRA FRZB TON CDCBPA PR CEACA BNT CL IX KLK COLA RBP ALPI FEZ LCA C EPHA FFAR CAP PTPRU CD ALPPL RVI ALDHL CXCR LTF ALPP DACT DC PLXNA PKDL DWD HIA BAI PEAR NATL TAP CDLD NLN CK LCN ACOT EL CDKNA YCP PHLDA XD EPL ILEC RCOR CNN LAC KIAAL FTL CN Corf CXCR T TU HPA OC PPIC Corf CLECA CEACA RE ERPINA PO IKZF P TPAN P PRAP P TTCB P YO CLIC TOL CEACA PRICKLE CEACA FNBPL LCA NPHP APH IL CTP ILEC PHACTR PPPRA FYB FRZB LCA CDCBPA DYNCLI CEACA ERINC CL ITPR KLHDC KLK KLHL RBP CAP FEZ ARNTL C Corf FFAR HEBP CAP LCA CD I RVI ABLI ORL CXCR TPINP LTF TFPI DACT ITA PLXNA PAQR DWD LYPLAL BAI HEBP PEAR HDDC TAP PNP TRAIP NLN NTDC LCN PR EL UP YCP CEACA XD HDB ILEC LCA CNN FCRA LAC EP PKIB FTL HNT Corf RNAE T RNAE HPA LCA PPIC ILR CLECA CKAP RE ILEC PO ILEC FCRT IKZF BCLA TPAN TRE PRAP CYBB TTCB CAR YO TOL A PRICKLE A FNBPL Corf IRF NPHP LN APH PTK CTP ERPINF PHACTR NRBP FYB TPINP LCA FRAT DYNCLI FRAT ERINC NDR KLF ITPR CDKNA KLHDC KLF KLHL TKC CAP VILL ARNTL PLCR Corf PRA HEBP CFP LCA APOBR RARP I CFR ABLI CDLF ORL PLEKH TPINP -ep TFPI EN ITA REPIN PAQR FAD LYPLAL NOTCH CLTN HEBP PN HDDC IFIT PNP IFIT TRAIP A NTDC IFIT PR TCF UP BIN
IDH () N/KRA () B C Cluster Cluster WT () LL () None () DNTA () None () DNTA () TET () N/KRA () FLT () LL () FLT () WT () NP () TET () NP () IDH () upplemental Figure.
RNA-seq LL-AF LCs (Ikzf) Reads: - si f/f! Ikzf - si Δ/Δ upplemental Figure.
A C si f/f si Δ/Δ Normalized expression... Ikzf. mrna stability... YC in LL-AF LCs c-kit High c-yc DAPI erged Hoxa. Relative expression Time (h) si f/f D I si Δ/Δ... yc Cntrl W HI O/E Hoxa. ated FP + c-kit High si f/f! si Δ/Δ IKZF.... IKZF High cells % IKZF High cells B Relative expression/si f/f si f/f!si /Δ t o qpcr in LL-AF c-kit High si f/f si Δ/Δ HoxA E Ikzf yc LL-AF pleen cells si f/f si Δ/Δ IKZF& HOXA& YC& I& F H % Positive cells (FP) upplemental Figure. CreER- TA I Intracellular staining CreER+ CreER+ TA CreER- CreER- CreER- TA CreER+ CreER+ TA CreER- CreER- TA IKZF Intracellular staining CreER+ CreER+ TA CreER- CreER- TA CreER+ CreER+ TA CreER- CreER- TA YC Intracellular staining ated c-kit High c-kit Low ated c-kit High c-kit Low ated c-kit High c-kit Low CreER+ CreER+ TA CreER- CreER- TA si f/f Cre-ER- CreER+ CreER+ TA ACTIN -OHT si f/f Cre-ER- si f/f Cre-ER+ -OHT si f/f Cre-ER+
A B C FOLD Ikzf qpcr si f/f! si Δ/Δ I upplemental Figure D and F ated FP + c-kit High si f/f! IKZF si Δ/Δ % IKZF High cells IKZF High si f/f! si Δ/Δ upplemental Figure D and F % IKZF High cells IKZF High si f/f! si Δ/Δ upplemental Figure E and upplemental Fig.