Supplementary Figure Legends Supplementary Figure 1 827 induces proliferation arrest, expression of differentiation markers and polyploidization of a human erythroleukemia cell line with the activating JAK2V617F mutation. Human HEL cells were treated with DMSO, 827 () or ruxolitinib (Rux). (a) Western blot for AURKA, STAT5 and STAT in extracts of HEL cells treated with DMSO, 827 () or ruxolitinib. (b) Dose response curve for determination of the IC5s of 827 and ruxolitinib. Growth of the drug treated cells was normalized to cells treated with DMSO. (c e) Flow cytometry plots of the effect of 827 and ruxolitinib on the expression of CD41 (c), CD42 (d), and polyploidization (e) of HEL cells after 72 hrs. Data are representative of two independent experiments. Supplementary Figure 2 827 induces proliferation arrest, expression of differentiation markers and polyploidization of a mouse megakaryocytic cell line overexpressing MPLW515L. Murine G1ME cells stably transduced with MPLW515L (G1ME-MPLW515L) were treated with DMSO, 827 () or ruxolitinib (Rux). (a) Dose response curve for determination of the IC5s of 827 and ruxolitinib. Growth of the drug treated cells was normalized to cells treated with DMSO. (b d) Flow cytometry plots of the effect of 827 and ruxolitinib on the expression of CD41 (b), CD42 (c), and polyploidization (d) of G1ME/MPLW515L cells after 72 hrs. Data are representative of two independent experiments. Supplementary Figure Nature Medicine: doi:1.18/nm.995 1
827 induces proliferation arrest, polyploidization, differentiation and apoptosis of SET-2 cells. SET-2 cells were treated with DMSO, 827 () or ruxolitinib (Rux). (a) Dose response curve for determination of the IC5s of 827 and ruxolitinib in SET-2 cells. (b e) Flow cytometry measurements of DNA content (b), expression of CD41 (c) and CD42 (d), and annexin V staining (e) 72 hrs after addition of drug. Bar graphs depict mean ± SD of two independent experiments conducted in duplicate. p.5, p.1, compared to DMSO by the unpaired two-sided Student s t test. (f,g) Western blots to assess the activation and expression of AURKA and STAT5 with increasing doses of 827 () or ruxolitinib (rux) at 6 hours (f) and 48 hours (g). Data are representative of two independent experiments. Supplementary Figure 4 Calreticulin type 1 and type 2 mutants also upregulate AURKA in murine bone marrow cells. Bone marrow Lin - cells were transduced with Migr1 vector, or vectors expressing calreticulin type 1 (CALR T1) or calreticulin type 2 (CALR T2) mutants. Cell lysates were probed with the indicated antibodies. GRB2 is shown as a loading control. Data are representative of two independent experiments. Supplementary Figure 5 Ruxolitinib and 827 differentially affect signaling in MPLW515L overexpressing cells. Bone marrow cells of mice transplanted with human MPLW515L expressing cells were treated with DMSO, 827 (), or ruxolitinib (Rux) for 24 hours. Cell lysates were probed for activation of Aurka and JAK-STAT signaling pathways. Data are representative of two independent experiments. Supplementary Figure 6 Nature Medicine: doi:1.18/nm.995 2
AURKA is downregulated during megakaryocyte differentiation. Murine lineage negative bone marrow cells transduced with Migr1-MPLW515L were cultured in the presence of THPO. CD41 /GFP and CD41 /GFP - cells were collected at days,, and 7. Lysates from these cells were probed with the indicated antibodies. GRB2 is shown as a loading control. Data are representative of two independent experiments. Supplementary Figure 7 Downregulation of c-myc suppresses AURKA signaling and affects megakaryocyte maturation. SET-2 cells were transduced with two different c-myc shrnas or the empty plko.1 vector. (a) Western blot showing the levels of MYC, AURKA, and STAT5 with knockdown. (b e) Cell number (b) and flow cytometry measurements of CD41 (c), CD42 (d), and Annexin V staining (e) with knockdown. Western blot data are representative of independent experiments. Bar graphs depict mean ± SD of two independent experiments conducted in duplicate. p.5, p.1, compared to plko.1 vector by the unpaired two-sided Student s t test. Supplementary Figure 8 Inhibition of AKT signaling does not affect the degree of AURKA signaling. SET-2 cells were incubated with the AKT inhibitor MK-226 for 6 hrs (a) or 48 hrs (b). Lysates were probed with the indicated antibodies by western blot. Data are representative of independent experiments. Supplementary Figure 9 AURKA is upregulated in PMF patient samples. Lysates of CD4 cells from 2 different PMF patients were probed for the indicated antibodies. GRB2 is shown as a Nature Medicine: doi:1.18/nm.995
loading control. Controls are cell lysates from human cord blood CD4 cells. Data are representative of 2 technical replicates. Supplementary Figure 1 AURKA is upregulated in ET and PV patient samples. Lysates of mononuclear cells from peripheral blood of ET (a) or PV (b) patients were probed for the indicated antibodies. HSC7 is shown as a loading control. Controls are cell lysates from mononuclear cells of peripheral blood from multiple healthy donors. Supplementary Figure 11 827 does not affect hematopoiesis in healthy mice. Eight to ten week old healthy Balb/c mice were treated with 15mg/kg 827 bid for three weeks. (a) Body weight following treatment with 827 () versus vehicle. (b) WBC, hemoglobin, hematocrit, and platelet count in the peripheral blood following treatment with or vehicle. (c e) Flow cytometry analysis of the myeloid (Gr1 Mac1 ), erythroid (CD71 High Ter119 ), and megakaryocytic (CD41 ) lineages. (f-h) H&E stained sections of the bone marrow (f), spleen (g) and liver (h). Results are representative of 2 independent experiments. n=6 animals per group. Bar graphs and line graphs depict mean ± SD. No significant differences were found by the unpaired two-sided Student s t test. Supplementary Figure 12 Inhibition of AURKA reduces disease burden in the MPLW515L mouse model of myelofibrosis. MPLW515L mice were treated with 15mg/kg 827 () or 6mg/kg dimf (dimf) for weeks. (a) Hematocrit and hemoglobin levels in the peripheral blood with 827 (), dimf or vehicle treatment. vs vehicle, Nature Medicine: doi:1.18/nm.995 4
p.1; dimf vs vehicle, p.1, by two-way ANOVA. (b-e) Flow cytometry measurement of CD41 cells (b), DNA content (c) LSK cells (d) and myeloid progenitor cells (e). (f i) May-Grunwald Giemsa stained blood smears (f) and H&E stained sections of the spleen (g), lung (h), and liver (i). (j) Reticulin stained spleen sections following and dimf treatment. Results are representative of 2 independent experiments. n=6 animals per group. Bar graphs and line graphs depict mean ± SD. p.5, p.1, compared to vehicle by the unpaired two-sided Student s t test. Supplementary Figure 1 827 effectively treats MPLW515L mice at later stage of disease progression. Balb/C recipients of MPLW515L bone marrow cells were treated days post-transplant with 15mg/kg 827 bid. (a) WBC and platelet counts after treatment with 827 () or vehicle. vs vehicle, p.1, by two-way ANOVA. (b,c) Spleen (b) and liver weights (c) following treatment. (d,e) Flow cytometry measurement of CD41 (d) and CD42 (d) cells in the bone marrow. (f j) May-Grunwald Giemsa stained blood smears (f) and H&E stained sections of bone marrow (g), spleen (h), lung (i), and liver (j). Results are representative of 2 independent experiments. n=6 animals per group. Bar graphs and line graphs depict mean ± SD. p.5, p.1, compared to vehicle by the unpaired two-sided Student s t test. Supplementary Figure 14 Deletion of one allele of Aurka normalizes hematopoiesis in MPLW515L disease mice. (a,b) Liver weight (a) and flow cytometry measurement of the percentage of Gr1 Mac1 cells in the bone marrow (b) of Aurka F/ or Aurka F/ MX1-Cre. (c-f) May- Grunwald Giemsa stained blood smears (c) and H&E stained sections of the spleen (d), lung (e) and liver (f). Nature Medicine: doi:1.18/nm.995 5
Supplementary Figure 15 Homozygous deletion of Aurka reduced the disease burden in the MPLW515L mouse model of myelofibrosis. Bone marrows from Aurka F/F or Aurka F/F MX1-Cre mice were transduced with MPLW515L and transplanted into recipient mice. Three weeks later, pi-pc was injected into recipients to induce excision of Aurka by MX1-Cre. (a) Deletion of the floxed region, excision products and MX1-Cre expression were monitored by PCR in the peripheral blood (PB), spleen (Sp) and bone marrow (BM) of recipient mice 19 days after the first injection of pi-pc. (b f) White cell count (b), hematocrit (c), hemoglobin (d) platelet count (e) and percentage of GFP cells (f) in the peripheral blood. (g-i) Spleen (g), liver (h) and body weights (i) of the two groups of mice. Results are representative of 2 independent experiments. n=5 animals per group. Bar graphs depict mean ± SD. p.5, p.1, compared to Aurka F/F mice by the unpaired twosided Student s t test. Nature Medicine: doi:1.18/nm.995 6
Cell no. CD42 CD41 Supplementary figure 1 a (μm) DMSO.1. 1 1 Rux (μm) DMSO. 1 1 b p-aurka AURKA p-stat5 STAT5 (μm) DMSO. 1 1 Rux (μm) DMSO. 1 1 Relative cell growth 1.2.9.6. Rux IC5 = 4 nm IC5 = 26.5 nm p-stat. -9-8 -7-6 -5-4 STAT Log [Conc] (M) c DMSO.1 µm 1 µm Rux.1µM Rux 1 µm 42.8 85.6 1 4. 4. SSC d 1.4.8 46.5.95 2.98 e SSC.9 8.9 8.1.6.1 DAPI Nature Medicine: doi:1.18/nm.995
Cell no. CD42 CD41 Supplementary figure 2 a 1.2.9 Relative cell growth.6. Rux IC5 = 961 nm IC5 = 9 nm b. -1-8 -6-4 Log [Conc] (M) DMSO.1 µm 1 µm Rux.1 µm Rux 1 µm.87 21.6 52..94 1.97 99.1 78.4 47.5 99 98 SSC c 1.57 1.4 7.5.45. SSC d 1.92 28.9 75.2 1.54 2.2 DAPI Nature Medicine: doi:1.18/nm.995
Supplementary figure a6 Cell no. ( 1 ).8.6.4.2 Rux IC5 = 22.6 nm IC5 = 5.5 nm b 4 8N (%) 2 1 4 2 1. c 12-9 -8-7 -6-5 -4 Log Conc (M) 12 d.1 µm 1 µm Rux.1 µm Rux 1 µm CD41 (%) 8 4 8 4 CD42 (%) 2 1 2 1 e 15.1 µm 1 µm 15 Rux.1 µm Rux 1 µm.1 µm 1 µm Rux.1 µm Rux 1 µm Annexin V (%) 1 5 1 5.1 µm 1 µm Rux.1 µm Rux 1 µm f (μm) Rux (μm) g (μm) Rux (μm) DMSO.1. 1 DMSO.1. 1 DMSO.1. 1 DMSO.1. p-aurka AURKA p-stat5 STAT5 c-myc GRB2 Nature Medicine: doi:1.18/nm.995
Supplementary figure 4 AURKA p-stat5 STAT5 GRB2 Migr1 CalR CalR vector T1 T2 Nature Medicine: doi:1.18/nm.995
Supplementary figure 5 (μm) DMSO.1. 1 1 p-aurka Rux (μm) DMSO.1. 1 1 AURKA p-stat5 STAT5 p-stat STAT GRB2 Nature Medicine: doi:1.18/nm.995
Supplementary figure 6 p-aurka AURKA p-stat5 MPLW515L MK D D D7 MPLW515L- MK D D STAT5 GRB2 Nature Medicine: doi:1.18/nm.995
Supplementary figure 7 a b c 12.9 plko.1 Sh4 Sh42 p-aurka AURKA p-stat5 STAT5 c-myc GRB2 6 Cell no. ( 1 ).6.. CD41 (%) PLKO Sh4 Sh42 PLKO Sh4 Sh42 8 4 d e 15 4 1 2 CD42 (%) 5 Annexin V (%) PLKO Sh4 Sh42 PLKO Sh4 Sh42 1 Nature Medicine: doi:1.18/nm.995
Supplementary figure 8 a p-aurka MK-226 (μm) DMSO. 1 1 b MK-226 (μm) DMSO. 1 1 AURKA p-akt AKT HSC7 Nature Medicine: doi:1.18/nm.995
Supplementary figure 9 p-aurka Ctrl PMF Ctrl PMF4 AURKA GRB2 Nature Medicine: doi:1.18/nm.995
Supplementary figure 1 a Ctrl ET1 ET2 Ctrl ET ET4 p-aurka AURKA HSC7 b Ctrl PV1 PV2 Ctrl PV PV4 p-stat5 Ctrl ET1 ET2 Ctrl ET ET4 Ctrl PV1 PV2 Ctrl PV PV4 STAT5 HSC7 Ctrl ET1 ET2 Ctrl ET Ctrl PV1 PV2 Ctrl PV PV4 c-myc HSC7 Nature Medicine: doi:1.18/nm.995
High Supplementary figure 11 a Body weight (g) 25 2 15 1 5 b 1 8 WBC ( 1 /µl) 6 4 2 Hematocrit (%) 8 6 4 2 Platelets ( 1 /µl) 1, 8 6 4 2 c 1 1 2 Time after treatment (weeks) d 4 e 2.5 Gr1 /Mac1 (%) 8 6 4 2 CD71 /Ter119 (%) 2 1 CD41 (%) 2. 1.5 1..5 f. g h Nature Medicine: doi:1.18/nm.995
Supplementary figure 12 a Hematocrit (%) 1 8 6 4 2 Hemoglobin (g/dl) 2 15 1 5 b dimf CD41 (%) 6 4 2 c 4 1 2 Time after treatment (weeks) d.5 1 2 Time after treatment (weeks) e 5 dimf vs, p=.7 8N (%) 2 1 LSK (%).4..2.1 Myeloid progenitors (%) 4 2 1 f. dimf dimf dimf dimf g h i j Nature Medicine: doi:1.18/nm.995
Supplementary figure 1 a WBC ( 1 /µl) 2 1 Platelets ( 1 /µl) 4,, 2, 1, b 1.5 Spleen weight (g) 1..5 c -15 15 45 Time after treatment (days) d 6-15 15 45 Time after treatment (days) e Liver weight (g) 2 1 CD41 (%) 4 2 CD42 (%) 2 1 f g h i j Nature Medicine: doi:1.18/nm.995
Supplementary figure 14 a Liver weight (g) 2 1 b Gr1 /Mac1 (%) 8 6 4 2 c Aurka F/ MX1-Cre Aurka F/ Aurka F/ MX1-Cre Aurka F/ d Aurka F/ Aurka F/ MX1-Cre Aurka F/ Aurka F/ MX1-Cre e f Nature Medicine: doi:1.18/nm.995
Supplementary figure 15 a F/F F/F Aurka MX1-Cre Aurka F/F Aurka MX1-Cre Aurka F/F F/F Aurka MX1-Cre Aurka F/F Flox WT Excision MX1-Cre PB Sp BM b c d 15 8 25 WBC ( 1 /µl) 1 5 Aurka F/F MX1-Cre Aurka F/F Hematocrit (%) 6 4 2 e f g 2, 1 Aurka F/F MX1-Cre Aurka F/F Hemoglobin (g/dl) 2 15 1 5 1. Aurka F/F MX1-Cre Aurka F/F Platelets ( 1 /µl) 1,5 1, 5 GFP (%) 8 6 4 2 Spleen weight (g).8.6.4.2 h 4 Aurka F/F MX1-Cre Aurka F/F ibody Weight (g) 25 Aurka F/F MX1-Cre Aurka F/F. Aurka F/F MX1-Cre Aurka F/F Liver weight (g) 2 2 15 1 1 5 Aurka F/F MX1-Cre Aurka F/F Aurka F/F MX1-Cre Aurka F/F Nature Medicine: doi:1.18/nm.995
Table S1 PMF patient information Patient # Gender Age Ethnicity Mutations DIPSS- plus Risk score category 1 Female 7 Caucasian MPL W515 2 int- 2 2 Female 6 African American JAK2V617F 2 int- 2 Male 6 Native Hawaiian/ Pacific Islander CALR (22bp del) 2 int- 1 4 Male 71 Caucasian JAK2V617F 6 high risk 5 Female 62 Caucasian CALR(5bp ins) 6 high risk 6 Female 42 African American CALR (52bp del) 6 high risk 7 Male 7 Caucasian JAK2V617F int- 2 8 Female 51 Caucasian JAK2V617F low risk 9 Female 66 Caucasian JAK2V617F 1 int- 1 1 Male 69 Caucasian CALR (E79fs47; 115-1147del) int- 2 11 Male 69 Caucasian JAK2V617F 1 int- 1 12 Male 61 Caucasian Triple negative int- 2 1 Male 6 Caucasian JAK2V617F int- 2 14 Male 65 Caucasian CALR (K75fs52; 1124-11del) int- 2 Nature Medicine: doi:1.18/nm.995
Table S2 ET and PV patient information Patient # Gender Age Ethnicity Mutations ET1 Female 42 Caucasian JAK2V617F ET2 Female 59 Caucasian JAK2V617F ET Female 41 Caucasian CALR (52bp del) ET4 Male 64 Caucasian JAK2V617F PV1 Male 6 Caucasian JAK2V617F PV2 Female 19 Caucasian JAK2V617F PV Female 68 Caucasian JAK2V617F PV4 Female 52 Caucasian JAK2V617F Nature Medicine: doi:1.18/nm.995
Table S Synergy between 827 and Ruxolitinib Multiples of IC5 Conc (μm) Rux Conc (μm) Combination incex.5 9.7 11.4.54 1 19.4 22.7.28 2 8.7 45.4.25 58.1 68.1.1 4 77.5 9.8.15 8 155. 181.5 1E- 15 Nature Medicine: doi:1.18/nm.995