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1 Supporting Information Rampal et al /pnas 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

2 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 of8

3 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

4 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

5 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 < of8

6 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 F Der (5;17), trisomy 9, 13q None 4 ET > MF 9 56 M No mitotic cells recovered 02/16/2010 None 6 ET 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 INCB 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 INCB PMF 2 68 M Hyperdip -7 CC PMF 2 59 M Pseudodip i(17q), del(20q) SB PMF M Diploid None 56 ET > MF 1 69 M Hyperdip del(7); pseudodip -9,+mar INCB 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 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 INCB ,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

7 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

8 Table S3. Variants identified by whole-exome sequencing comparing tertiary and primary murine leukemia Chromosome Position Reference Alternate Gene Protein effect Effect Variant frequency Chr G A C130026I21Rik p.a108v Nonsynonymous coding Chr T C Zfp534 Splice site acceptor 0.8 Chr G T Chd5 p.r1298l Nonsynonymous coding Chr G C Akap9 p.g3306a Nonsynonymous coding Chr C A Arap2 p.g230w Nonsynonymous coding Chr C A Mkrn1 p.m293i Nonsynonymous coding Chr T A Ptpro p.v1035d Nonsynonymous coding Chr T A Unc45a p.d185v Nonsynonymous coding Chr C T Gas6 p.g422s Nonsynonymous coding Chr C A Aga p.h195n Nonsynonymous coding Chr T C Wdr17 p.i546v Nonsynonymous coding Chr C T Wdr17 p.g32e Nonsynonymous coding Chr C T Gm17293 p.r150c Nonsynonymous coding Chr G A Gm17293 p.r150h Nonsynonymous coding Chr C T BC Splice site region Chr T C Fbxo8 Splice site region Chr G A Hmgb2 Splice site region 1 Chr C G Gm11167 p.s22c Nonsynonymous coding 0.3 Chr A T L09Rik Splice site region Chr C A Gm6996 p.t97k Nonsynonymous coding Chr G A Sfi1 Start gained Chr G T Myh8 p.e1858* Start gained Chr G A Stard3 Splice site region Chr C A Acly p.g96v Nonsynonymous coding Chr G A G18Rik Splice site donor Chr C T Jph4 p.g253s Nonsynonymous coding Chr C T Brd1 p.v917i Nonsynonymous coding Chr A T Atp13a4 Splice site region Chrx A C Gm3701 p.y390* Stop gained 0.15 Chr GT G Rprd1b Intragenic Chr CT C Fam83d Deletion Exon Chr GC G Slc12a5 Intragenic 0.5 Chr GAC G Cdh7 Intragenic Chr ACTTT A Cep170 Deletion Frame shift Chr TG T Scaf1 Deletion Exon Chr TG T Vps37a Splice site donor Chr GAA G Spcs3 Deletion Exon Chr TC T D230002A01Rik Deletion Exon Chr TCC T Tcp11l2 Deletion Exon Chr TG T Ifi27l1 Deletion Exon Chr GC G D22Rik Deletion FRAME_SHIFT Chr CA C Myh7 Deletion Exon Chr TA T Igf2r Intragenic Chr CT C P10Rik Deletion EXON Chrx GAT G Prdx4 Intragenic Chr A AG Hjurp Intragenic Chr C CTT Glra3 Intragenic Chr T TGGC Dip2b Intragenic of8