Supporting Information Rampal et al. 10.1073/pnas.1407792111 Fig. S1. Genetic events in leukemic transformation of chronic-phase MPNs. (A) Survival of post-mpn AML patients according to mutational status (JAK2- mutant, CALR-mutant, or wild type for both). No significant differences were observed (log-rank test). (B) Relative frequency of mutations in chronic-phase MPN and post-mpn AML in paired samples. (C) VAF of TP53 mutations in all chronic-phase MPN and AML samples in this cohort. (D) Analysis of clonal architecture by comparison of variant alleles and frequency in paired chronic-phase and post-mpn AML samples. Red arrow represents an increase in allele burden. (E) Representative analysis of VAF of mutations occurring at chronic MPN phase and AML stage from a single patient. 1of8
Fig. S2. Blood counts of mice with loss of Tp53 alone and in combination with JAK2V617F overexpression. (A) HCT is significantly elevated in Tp53-KO/ JAK2V617F mice compared with control arms at day 100 posttransplantation (P < 0.05 for each comparison, t test). (B) PLT counts demonstrating increases in JAK2V617F- and Tp53-KO/JAK2V617F-transplanted mice relative to controls at day 100. 2of8
Fig. S3. Representative FACS analysis of differentiation markers displayed in Fig. 3. (A) Representative analysis of erythroid differentiation markers. (B and C) Representative analysis of bone marrow (B) and spleen cells (C) demonstrates increase in the MEP population in Tp53-KO/JAK2V617F mice. 3of8
Fig. S4. Pharmacologic inhibition of JAK2 or degradation of JAK2 promotes hematopoietic differentiation in Tp53-KO/JAK2V617F mice. Representative FACS analysis plots of differentiated myeloid and lymphoid cells from (A) peripheral blood, (B) bone marrow, and (C) spleen cells of Tp53-KO/JAK2V617F mice treated with vehicle, ruxolitinib, and PU-H71. 4of8
Fig. S5. Effect of inhibitor therapy on survival, peripheral blood counts, and LSK/myeloid progenitors in Tp53-KO/JAK2V617F leukemic mice (also see Fig. 5). (A) Serial peripheral blood counts of placebo- and inhibitor-treated mice. (B) Representative FACS analysis plots from bone marrow of mice treated with placebo or inhibitors. *P < 0.01. 5of8
Table S1. Sample ID Clinical characteristics of post-mpn AML patients MPN Time from MPN to AML, years Age at MPN diagnosis, years Sex CG karyotype Therapy received 1 PV 15 48 F Der (5;17), trisomy 9, 13q None 4 ET > MF 9 56 M No mitotic cells recovered 02/16/2010 None 6 ET 34 25 F Diploid None 9 PMF 3 61 M Diploid None 10 PMF 4 52 F Der(6)t(1;6)(Q23;P25) Thalidomide 12 PMF 3 58 M Insufficient metaphases for analysis None 18 PMF UNK UNK M Diploid None 21 PMF 6 51 F Diploid None 23 PMF 4 73 M Trisomy 13 INCB18424 25 PMF 9 57 F Diploid None 29 PMF 3 56 M Diploid Fludarabine, cytarabine, Gemtuzumab-ozogamicin 34 PMF 8 56 F Pseudodiploid inv(12), der (6) Unknown 35 ET 4 65 F Minus 5/5q; -7/7q Decitabine 38 MDS/MPN 1 65 M Hyperdip +13; i(17q) Gimatecan 39 PV 4 67 M Plus 8 Decitabine and valproic acid 40 PMF 3 68 M Diploid Azacytidine 43 ET 1 62 M Del(5q); +15 Azacytidine 46 ET > MF 3 60 F Not done INCB18424 50 PMF 2 68 M Hyperdip -7 CC-4047 52 PMF 2 59 M Pseudodip i(17q), del(20q) SB1518 53 PMF 26 44 M Diploid None 56 ET > MF 1 69 M Hyperdip del(7); pseudodip -9,+mar INCB18424 58 PMF 1 62 F Hyperdip +8, +22 Bevacizumab 59 PV 5 62 M Hyperdip del(9p); +21 None 62 PMF 1 67 F Insufficient metaphases for analysis Gemtuzumab-ozogamicin 63 PMF 5 70 F Hyperdip +6; +8; +21 None 64 PV > MF 15 67 M Diploid None 17,365 ET 6 63 M Trisomy 9 None 8,486 MPN NOS 3 51 M Diploid Hydrea 5,946 PMF 3 69 F Trisomy 8 INCB18424 10,626 PV UNK UNK F Trisomy 11q23. Daunorubicin and cytarabine 30,526 MPN NOS 1 53 F Inv(16) Daunorubicin and cytarabine 8,365 ET 4 72 M Del(5q), rearrangement of 11q, amplification of MLL(11q23) gene and the p53 gene by FISH. None MDS, myelodysplastic syndrome; MF, myelofibrosis; NOS, not otherwise specified; UNK, unknown. 6of8
Table S2. Genomic variants identified in MPN and paired post-mpn AML samples Sample ID Somatic variants Rearrangements Amplification Homozygous deletions MPN-4 IDH2 R140Q, SF3B1 K700E None None JARID2 loss (0, exons 18 of 19) AML-4 IDH2 R140Q, RUNX1 A149P, SF3B1 K700E, ASXL1 G646fs* MPN-12 CRBN E30K, NOTCH3 P21S, NTRK1 G18E, REL L331S, SETD2 P2054L, TNFRSF11A D427N AML-12 U2AF1 Q157P MPN-21 IDH2 R140W, JAK2 V617F, RUNX1 L98fs*24 AML-21 none MPN-23 ASXL1 G646fs*, IDH2 R140Q, JAK2 V617F, PTCH1 T778M, PTPN11 P491L, U2AF1 Q157R AML-23 IDH2 R140Q, KRAS I24N, PTCH1 T778M, PTPN11 P491L, U2AF1 Q157R, JAK2 V617F, ASXL1 G646fs* MPN-25 JAK2 V617F, MLL2 R3087L, PARP4 I1039T, TP53 C275Y AML-25 IDH2 R172K, MLL2 R3087L, TP53 C275Y), JAK2 V617F MPN-29 IDH1 R132H, MPL W515L, SRSF2 R94_S101del AML-29 IDH1 R132H, MPL W515L, ASXL1 G646fs*12 MPN-38 NRAS G12S, SRSF2 P95R None None ETV6 loss, CDKN1B loss AML-38 NRAS G12S, SRSF2 P95R None None ETV6 loss AML-43 PTPN11 D61V, TP53 Y234* MPN-43 PTPN11 D61V, TP53 Y234* MPN-46 JAK2 V617F, U2AF1 Q157P AML-46 ASXL1 R693*, TP53 E258A, TP53 R248Q, U2AF1 Q157P, JAK2 V617F MPN-50 ARID1A R1950Q, SETBP1 G870S, SETBP1 I871T, U2AF1 Q157P AML-50 ARID1A R1950Q, ASXL1 R965*, NRAS Q61R, NRAS G13R, NRAS G12D, U2AF1 Q157P, SETBP1 G870S MPN-52 JAK2 V617F AML-52 NRAS G12A, SETBP1 G870S MPN-56 IDH2 R140Q, JAK2 V617F AML-56 IDH2 R140Q, TP53 R248Q, JAK2 V617F None Jak2 amplification None (7, exons 23 of 23) MPN-58 ASXL1 G646fs*12, EZH2 R690H AML-58 EZH2 R690H, NRAS G13D, ASXL1 G646fs*12 7of8
Table S3. Variants identified by whole-exome sequencing comparing tertiary and primary murine leukemia Chromosome Position Reference Alternate Gene Protein effect Effect Variant frequency Chr1 85256314 G A C130026I21Rik p.a108v Nonsynonymous coding 0.104 Chr4 147048438 T C Zfp534 Splice site acceptor 0.8 Chr4 151751234 G T Chd5 p.r1298l Nonsynonymous coding 0.533 Chr5 4064639 G C Akap9 p.g3306a Nonsynonymous coding 0.395 Chr5 63140226 C A Arap2 p.g230w Nonsynonymous coding 0.444 Chr6 39351358 C A Mkrn1 p.m293i Nonsynonymous coding 0.053 Chr6 137392115 T A Ptpro p.v1035d Nonsynonymous coding 0.993 Chr7 87481325 T A Unc45a p.d185v Nonsynonymous coding 0.067 Chr8 13471484 C T Gas6 p.g422s Nonsynonymous coding 0.056 Chr8 54603183 C A Aga p.h195n Nonsynonymous coding 0.421 Chr8 55750449 T C Wdr17 p.i546v Nonsynonymous coding 0.282 Chr8 55781722 C T Wdr17 p.g32e Nonsynonymous coding 0.556 Chr8 57987987 C T Gm17293 p.r150c Nonsynonymous coding 0.111 Chr8 57987988 G A Gm17293 p.r150h Nonsynonymous coding 0.333 Chr8 59026329 C T BC088983 Splice site region 0.511 Chr8 59068849 T C Fbxo8 Splice site region 0.532 Chr8 59991551 G A Hmgb2 Splice site region 1 Chr9 3020219 C G Gm11167 p.s22c Nonsynonymous coding 0.3 Chr10 23530587 A T 1110021L09Rik Splice site region 0.205 Chr10 52248615 C A Gm6996 p.t97k Nonsynonymous coding 0.062 Chr11 3093446 G A Sfi1 Start gained 0.108 Chr11 67120382 G T Myh8 p.e1858* Start gained 0.053 Chr11 98239537 G A Stard3 Splice site region 0.431 Chr11 100381663 C A Acly p.g96v Nonsynonymous coding 0.077 Chr14 55272291 G A 4930579G18Rik Splice site donor 0.042 Chr14 55732638 C T Jph4 p.g253s Nonsynonymous coding 0.048 Chr15 88531311 C T Brd1 p.v917i Nonsynonymous coding 0.068 Chr16 29454971 A T Atp13a4 Splice site region 0.069 Chrx 3666183 A C Gm3701 p.y390* Stop gained 0.15 Chr2 157954507 GT G Rprd1b Intragenic 0.3077 Chr2 158608831 CT C Fam83d Deletion Exon 0.2414 Chr2 164813139 GC G Slc12a5 Intragenic 0.5 Chr1 112035975 GAC G Cdh7 Intragenic 0.4615 Chr1 178686113 ACTTT A Cep170 Deletion Frame shift 0.3542 Chr7 52258470 TG T Scaf1 Deletion Exon 0.5714 Chr8 41626500 TG T Vps37a Splice site donor 0.2121 Chr8 55608522 GAA G Spcs3 Deletion Exon 0.2857 Chr8 94328716 TC T D230002A01Rik Deletion Exon 0.4375 Chr10 84047564 TCC T Tcp11l2 Deletion Exon 0.4444 Chr12 104675836 TG T Ifi27l1 Deletion Exon 0.2045 Chr13 59792959 GC G 4921517D22Rik Deletion FRAME_SHIFT 0.3962 Chr14 55605790 CA C Myh7 Deletion Exon 0.2188 Chr17 12932256 TA T Igf2r Intragenic 0.5556 Chr19 53526115 CT C 5830416P10Rik Deletion EXON 0.3333 Chrx 151775143 GAT G Prdx4 Intragenic 0.3333 Chr1 90156864 A AG Hjurp Intragenic 0.2727 Chr8 58483901 C CTT Glra3 Intragenic 0.3092 Chr15 99869096 T TGGC Dip2b Intragenic 0.8182 8of8