Supplemental Materials and Methods

Table S1. MS Supplemental Table_AML.

Genotyping Primer Sequence Fbxo9 5’ F 5’ – GTC TCT TCG AGG CAG AAC GTA TC – 3’ Fbxo9 5’ R 5’ – GCA ACG GGA TCA GAT GTC CAA TG – 3’ Fbxo9 3’ F 5’ – GCC TGA GAC TGA AAG ATC CTT GC – 3’ Fbxo9 3’ R 5’ – CAG GCT TAC AAA GAG GGC CTA CG – 3’ Hygro F 5’ – CCA TCG TCG AGA TCC AGA CAT – 3’ Hygro R 5’ – GTA TAT GCT CCG CAT TGG TCT TG – 3’ Generic Cre F 5’ – GCG GTC TGG CAG TAA AAA CTA TC – 3’ Generic Cre R 5’ – GTG AAA CAG CAT TGC TGT CAC TT – 3’ Inv Post F 5’ – CCC CCG TAC TGT GTG TGT CT – 3’ Inv Post R 5’ – GCC AGA CGG GTC AAC AAT AC – 3’

qRT-PCR Primer Sequence Gapdh F 5’ – CAT GGC CTT CCG TGT TCC TA – 3’ Gapdh R 5’ – CTG GTC CTC AGT GTA GCC CAA – 3’ Fbxo9 Exon 4 F 5’ – AGA AGC TCT ATG CTG AAA GCA G – 3’ Fbxo9 Exon 4 R 5’ – CAT CGC CCT ACG GTA GAA CT – 3’ Fbxo9 Exon 2-3 F 5’ – ATG AGA GTC CGG CTG AGA GA – 3’ Fbxo9 Exon 2-3 R 5’ – AGA GCT TCT TCC TGC TCT GC – 3’

Western Blot Antibodies: Antibody Company Ref# ARF-1 Proteintech 10790-1-AP Β-actin-HRP Santa Cruz sc-47778 FLAG-HRP Proteintech 12926-1-AP PSMA2 Bethyl A303-816A PSMB7 Bethyl A303-848A PSMD11 Cell Signaling 14303S

Flow Cytometry Antibodies Antibody Fluorochrome Clone Company 7-AAD n/a n/a eBioscience Annexin V PE n/a BioLegend B220 PacBlue, BV510, APCcy7, APC, Biotin, PE RA3-6B2 BioLegend CD4 APCcy7, APC, Biotin, PE CK1.5 BioLegend CD8 APCcy7, APC, Biotin, PE 53-6.7 BioLegend, eBioscience CD11b FITC, APCcy7, APC, Biotin, PE M1/70 BioLegend CD16/32 FITC, APC 93 BioLegend CD34 APC, PE HM34 BioLegend CD48 FITC HM48-1 BioLegend CD131 PE n/a BD Pharmingen CD150 BV510, BV785 TC15-12F12.2 BioLegend cKit APC, BV421, PacBlue 2B8 BioLegend Gr-1 APCcy7, APC, Biotin, PE, BV421 RB6-8C5 BioLegend Ki67 FITC 16A8 BioLegend Sca-1 PE, PEcy7 D7 BioLegend, eBioscience Strep FITC n/a BioLegend Ter-119 APCcy7, PacBlue, APC, Biotin, PE TER-119 BioLegend

Hynes-Smith et al. Fbxo9 in acute myeloid leukemia Class MILE Study Diagnosis No of Samples HBM Non-leukemia and healthy bone marrow 74 MDS Myelodisplastic syndrome 206 Normal AML with normal karyotype 351 Complex AML with complex karyotype 48 inv(16) AML with inv(16)/t(16;16) 28 t(15;17) AML with t(15;17) 37 t(8;21) AML with t(8;21) 40 MLL AML with t(11q23)/MLL 38 CML Chronic myeloid leukemia 76 Total 898

Class TARGET Study Diagnosis No of Samples HBM Non-leukemia and healthy bone marrow 5 Unknown AML with unknown diagnosis 10 Normal AML with normal karyotype 53 inv(16) AML with inv(16)/t(16;16) 36 t(8;21) AML with t(8;21) 45 MLL AML with t(11q23)/MLL 41 Other AML with other diagnoses 47 Total 237

FAB Subtype TCGA Study Diagnosis No of Samples AML M0 AML with minimal maturation 15 AML M1 AML without maturation 41 AML M2 AML with maturation 42 AML M3 Acute promyelocytic leukemia 15 AML M4 Acute myelomonocytic leukemia 37 AML M5 Acute monoblastic or monocytic leukemia 22 AML M6 Acute erythroid leukemia 2 AML M7 Acute megakaryoblastic leukemia 3 Other Other subtype 2 Total 179

Mass Spectrometry Method:

Samples were loaded onto trap column Acclaim PepMap 100 75 µm x 2 cm C18 LC Columns (Thermo ScientificTM) at flow rate of 5 µl/min then separated with a Thermo RSLC Ultimate 3000 (Thermo ScientificTM) from 5-20% solvent B (0.1% FA in 80% ACN) from 10-98 minutes at 300 nL/min and 50 ºC with a 120 minutes total run time for fractions one and two. For fractions three to six, solvent B was used at 5-45% for the same duration. Eluted peptides were analyzed by a Thermo Orbitrap Fusion Lumos Tribrid (Thermo ScientificTM) mass spectrometer in a data dependent acquisition mode using synchronous precursor selection method. A survey full scan MS (from m/z 375-1500) was acquired in the Orbitrap with a resolution of 120000. The AGC target for MS2 in iontrap was set as 1 x 104 and ion filling time set as 150ms and fragmented using CID fragmentation with 35% normalized collision energy. The AGC target for MS3 in orbitrap was set as 1 x 105 and ion filling time set as 200 ms with a scan range of 100-500 and fragmented using HCD with 65% normalized collision energy. identification was performed using proteome discoverer software version 2.2 (Thermo Fisher Scientific) by searching MS/MS data against the UniProt mouse protein database. The search was set up for full tryptic peptides with a maximum of 2 missed cleavage sites. Oxidation, TMT6plex of the amino terminus, GG and GGQ ubiquitination, phosphorylation, and acetylation were included as variable modifications and carbamidomethylation and TMT6plex of the amino terminus were set as fixed modifications. The precursor mass tolerance threshold was set at 10 ppm for a maximum fragment mass error of 0.6 Da with a minimum peptide length of 6 and a maximum peptide length of 144. The significance threshold of the ion score was calculated based on a false discovery rate calculated using the percolator node. Protein accessions were put into Ingenuity Pathway Analysis (QIAGEN Inc.) to identify symbols and localizations. pathway analysis was performed using DAVID Bioinformatics Database 6.8 using the functional annotation tool.

A FBXO9 Translation Exon 3 Exon 4 Exon 5 Normal . . . QELAKEEKAREL . . . GALYEAIKFYRRA . . . Mutant . . . QELAKEEK______PSSSTVGR . . .

B

ΔF-box ΔTPR ΔExon4 FLAG-tagged- kDa UntreatedFBXO9 FBXO9 FBXO9 FBXO9 70 55 FLAG 35 55 β-Actin

Supplementary Figure S2 Deletion of Fbxo9 exon 4 results in a frame shift and premature stop. (A) translation of sequenced cDNA from Fbxo9+/+ and Fbxo9-/- mice. (B) Western blot expression of overex- pressed flag-tagged FBXO9 with various deletion mutations. A 80 Fbxo9+/+ B 100 Fbxo9+/+ +/- +/-

n n Fbxo9 96 Fbxo9

o

o

i i -/- -/-

t t 60

i i Fbxo9 92 Fbxo9

s

s

o o 88

p p 40

m 12

m

o

o

C C 8 20

% % 4 0 0 Myeloid B cell T cell Lin- cKit+ LSK Lin- cKit+ LSK Live Dead D 100 +/+

n Fbxo9

o

i -/- t Fbxo9

c 75

+/+ +/- -/- e C Fbxo9 Fbxo9 Fbxo9 j

n

I

4.61 3.7 2.64 l 50

a

i

t

i

n I 25

% 0 0 1 2 3 4

FSC Lin+ Gated on single cells Month Fbxo9+/+ Fbxo9+/- Fbxo9-/- Fbxo9+/+ Fbxo9+/- Fbxo9-/- 22.4 1.18 28.8 1.47 30.2 2.59 59.1 10.4 67.4 5.73 56.2 13.0

22.6 7.83 21.6 5.29 21.8 9.06

cKit

CD48 Sca-1 Gated on Lin - cells CD150 Gated on LSK cells Fbxo9+/+ Fbxo9+/- Fbxo9-/- Fbxo9+/+ Fbxo9+/- Fbxo9-/-

74.0 68.5 60.8 16.3 19.1 18.6

12.5 12.2 14.6 11.0 12.4 26.2 69.5 66.9 64.6

CD16/32

Ki67 CD34 Gated on cKit + cells DAPI Gated on LSK cells Fbxo9+/+ Fbxo9+/- Fbxo9-/- Supplementary Figure S3 Loss of Fbxo9 does not 56.9 56.9 54.7 affect hematopoietic cell populations. (A) Bar graph (mean ±standard deviation) of BM cell percentages of mature lineages following treatment with Poly(I:C). (B) Bar graph (mean ±standard deviation) of live and dead 5.15 6.14 9.41 cells. (C) Flow cytometry gating strategy for isolating + Ki67 LSK and cKit populations from total BM and analyzing DAPI Gated on cKit + cells for stage. (D) Competitive repopulation cell +/+ -/- composition of Fbxo9 and Fbxo9 cells.

A +/56M +/+ +/56M +/- +/56M -/- B 2.5 Cbfb+/56M

Cbfb Fbxo9 Cbfb Fbxo9 Cbfb Fbxo9 )

g +/+ ( 2.0 Fbxo9 p = 0.3428 t +/56M

92.9 99.8 h Cbfb

g i 1.5 Fbxo9+/-

e +/56M

W Cbfb p = 0.0988 1.0 n Fbxo9-/-

e

e l 0.5

p

S cKit 0.0 SSC Spleen 27.5 11.8 5.64

Gr1 CD11b Gated on cKit+ cells

Supplementary Figure S4 Mice with inv(16) AML and Fbxo9 cKO have a more aggressive and homoge- nous tumor. (A) Representative FACS plots of tumor cells isolated from the BM of mice at time of sacrifice. Cells are first gated on total live singlets and subsequently on cKit + cells which are further analyzed for expression of Gr1/CD11b. (B) Bar graph (mean ± standard deviation) of spleen weight of mice at time of sacrifice.

A ns B ns ns ns ns ns ns ns +/56M +/+ 60 Cbfb Fbxo9 100 +/56M +/+ **** +/56M +/- Cbfb Fbxo9 50 **** Cbfb Fbxo9 Cbfb+/56MFbxo9+/- B +/56M -/- Cbfb Fbxo9 +/56M -/- P 75 40 Cbfb Fbxo9

t

n

i

i

K

0

30 c 50

2

2

%

B 20 25 % 10 0 0 3 5 BM Sp PB Weeks post-transplant At Sacrifice

C Spleen D Liver Supplementary Figure S5 Mice with trans- 80 planted inv(16) tumor cells show a more 100 aggressive phenotype for cells lacking n n 60 o o Fbxo9 expression. (A) Bar graph of B cell i 80 i *

t t

a a (B220) expression in the PB following trans-

r r

t t

l 60 l 40

i i

f f plantation (mean ± standard deviation). (B)

n n + I 40 I Bar graph of percentage of cKit tumor cells in 20 % % the BM, spleen (Sp), and PB at time of sacri- 20 ** fice (mean ± standard deviation, **** p < 0 0 0.0001). (C-D) Bar graph of (C) spleen (Cbf- -/- +/- -/- +/56M +/- +/56M +/+ +/- +/+ b Fbxo9 , p = 0.0890 and Cbfb F- 9 9 9 9 9 9 o o o o o x o x x bxo9-/-, p = 0.1356) and (D) liver infiltration x x x b b b b b b F F F F F F where each datapoint represents an individu- +/56M +/56M +/56M +/56M +/56M +/56M al mouse (* p < 0.05, ** p < 0.01).

Cbfb Cbfb Cbfb Cbfb Cbfb Cbfb

A B C 25 130 250 15 100 130 70 100 70 55 ARF-1 55 35 PSMB7 actin D 35 E F 25 250 130 15 100 100 70 70 55 55

PSMD11 SKAP2

G H 250 250 130 130 100 100 70 70 55 55 35 35 25 25 15 15

FLAG actin

A B C

D E F ARF1 12 ns ns ns ns ns n 11 ** ns ns

o

i

s 10

s

e

r 9

p x 8

E

2 7

g

o 6

L 5 l x ) ) ) L L M S a e 6 7 1 L B D m l (1 ;1 ;2 M H M r p v 5 8 M C o m n 1 t( N o I t( C G PSMD11 H PSMA2 I PSMB7 ns ns ns 12 9 ns 11 ** *** ns *

n ** ns n n ns ns ns ** ** ns **** ns ns *

o

o 11

o

i

i i ns ****

8 s s *** s 10

s

s s **** 10

e

e

e

r 7 r

r

p 9

p p 9

x

x x 6

E

E E 8 8

2

2

2 5

g

g

g

o 7 o

o 7

L 4 L

L 6 6 l l l ) ) ) S a x ) ) ) L L S a x ) ) ) L L M S a x L L M le 6 7 1 L M le 6 7 1 L le 6 7 1 L B D m (1 ;1 ;2 M B D m p (1 ;1 ;2 M B D m p (1 ;1 ;2 M H M r p v 5 8 M C H M r v 5 8 M C H M r v 5 8 M C o m n 1 t( o m n 1 t( o m n 1 t( N o I t( N o I t( N o I t( C C C J SKAP2 12 ns ** ** Figure S7 RNA expression correlation of UPS- **** ****

n 11 ns **** associated . (A-E) FBXO9 has no correla-

o i **** s 10 tion with (A) ARF1, (B) PSMD11, (C) PSMA2, or (D)

s

e 9 r PSMB7, but has a marginal positive correlation with p 8 x (E) SKAP2. (F-J) Violin plots of RNA expression for E 7

2 (F) ARF1, (G) PSMD11, (H) PSMA2, (I) PSMB7, and

g 6

o

L 5 (J) SKAP2. 4 l x ) ) ) L L M S a e 6 7 1 L B D m l 1 ;1 ;2 M H M r p v( 5 8 M C o m n 1 t( N o I t( C