1. A B C D Figure S1 BCR-ABL+ cell lines Initial Transformation
Unstained
Cdk6+/+
Cdk6-/-
Initial Transformation
CFU/5x106 cells 10 20 30 40
Cdk6+/+
(n=6)
Cdk6-/-
94.3±2.7%
87.4±2.1%
CD19
B220
93.6±7.6%
98.8±0.5%
CD19
CD43 C
Day after BCR-ABL transduction 10 13 16 19 22
Cdk6+/+
7.2±2.3%
8±4.6%
6.8±2.5%
70.7±22.9%
8±3.2%
56.2±9.7%
10.8±6%
52.1±9.3%
5.5±1.3%
23.9±17%
Annexin V
80.8±7.5%
3.9±1.6%
18.4±22.4%
4.1±0.5%
30.6±7.1%
5.2±1.6%
30±9.9%
7.1±1.7%
67±18.5%
3.6±0.5%
Cdk6-/-
2.3±0.4%
12.4±3.5%
7±1.5%
55.3±21.3%
5±3.6%
70.2±10.7%
5.6±1.5%
70.8±7%
0.5±0.2%
93.5±3.6%
80.4±4.5%
5±1.2%
34.5±22%
3.2±0.3%
21.6±8.1%
3.3±0.7%
19.7±6.6%
3.9±1%
2.6±2.4%
3.3±1.1%
7-AAD D
BCR-ABL+ cell lines
Relative MFI
0.0
0.5
1.0
1.5 *
Cdk6+/+
(n=6)
Cdk6-/-
(n=6)
Figure S1

2. A B C D Figure S2 pre-pro-B cells ex vivo gating strategy CD19 Counts
Cdk6+/+
Cdk6-/- UT
5 Gy UT
5 Gy
1.9±1%
86.1±1.2%
3.4±0.7%
8.6±0.9%
5.2±2.6%
48.6±2%
5.5±2.5%
40.6±3.3%
2±0.3%
80.1±0.7%
3.7±0.4%
14.3±0.9%
3.7±1.1%
42±3.2%
3.6±0.4%
50.7±3%
Annexin V
7-AAD C
Cdk6+/+
Thymocytes ex vivo
Cdk6-/- UT
2.5 Gy UT
2.5 Gy
1.2±0.5%
46.9±11.6%
0.5±0.2%
15.1±0.2%
16.7±4.4%
0.4±0.2%
39.3±3.6%
1.6±0.6%
Annexin V
82.9±4.6%
0.1±0%
51.5±12.2%
0.4±0.2%
84.4±0.4%
0.1±0%
58.8±4.2%
0.2±0.1%
7-AAD D
pre-pro-B cells in vitro
Cdk6+/+
Cdk6-/- UT
NCS UT
NCS
102
103
104
105
6.3±3%
3.2±3.5%
102
103
104
105
15.3±0.3%
38.4±12.4%
105
4.9±0.3%
1.6±0.6%
102
103
104
105
15.2±2.2%
42.3±7.3%
104
Annexin V
103
102
102
103
104
105
102
104
105
103
102
104
105
103
102
104
105
103
89.8±7.3%
0.8±0.7%
45.8±12.2%
0.6±0.1%
93.4±1%
0.3±0.1%
42.1±5.3%
0.5±0.1%
7-AAD
Figure S2

3. E F G H Figure S2 Figure S2 Untransformed pre–pro-B cells Cdk6+/+
PUMA
NOXA
4x10-1
Cdk6+/+
Cdk6-/-
2.5Gy UT
5.0x10-4
1.0x10-3
1.5x10-3
2.0x10-3 * * **
Cdk6+/+
Cdk6-/-
2.5Gy UT
1x10-1
5x10-2
1x10-2
5x10-3
2x10-1
***
4x10-2 **
Relative mRNA levels
2x10-2 UT
2.5Gy UT
2.5Gy
Cdk6+/+
Cdk6-/- F
Untransformed Thymocytes
4x10-1
p21
PUMA
NOXA
3x10-1
5.0x10-4
1.0x10-3
1.5x10-3
2.0x10-3
Cdk6+/+
Cdk6-/-
2.5Gy UT *
2x10-1
Relative mRNA levels
6x10-2
2x10-2
4x10-2
6x10-2
8x10-2
1x10-1
Cdk6+/+
Cdk6-/-
2.5Gy UT **
4x10-2
2x10-2 UT
2.5Gy UT
2.5Gy
Cdk6+/+
Cdk6-/- G
BCR-ABL+ cell lines
p21
2.0x10-3
PUMA
NOXA
1x10-1
2x10-1
3x10-1
4x10-1 *
1x10-1 ** *
1.0x10-3
8x10-2
6.0x10-5
Relative mRNA levels
6x10-2
4.0x10-5
4x10-2
2.0x10-5
2x10-2 UT
NCS UT
NCS UT
NCS UT
NCS UT
NCS UT
NCS
Cdk6+/+
Cdk6-/-
Cdk6+/+
Cdk6-/-
Cdk6+/+
Cdk6-/- H
BCR-ABL+ cell lines
p21
PUMA
NOXA
4x10-1 **
2.0x10-5
4.0x10-5
1.0x10-3
1.5x10-3
2.0x10-3
1x10-1
3x10-1
8x10-2
Relative mRNA levels *
2x10-1
6x10-2 *
1x10-1
2x10-2
1x10-2 UT
Eto UT
Eto UT
Eto UT
Eto UT
Eto UT
Eto
Cdk6+/+
Cdk6-/-
Cdk6+/+
Cdk6-/-
Cdk6+/+
Cdk6-/-
Figure S2
Figure S2

4. I J Figure S2 Cdk6+/+ Cdk6-/- Annexin V Annexin V Cdk6+/+ Cdk6-/-
BCR-ABL+ cell lines
Cdk6+/+
Cdk6-/- UT
2.5 Gy UT
2.5 Gy
76.6±1.6%
0.4±0.3%
90±2.6%
5.5±3.4%
4.2±1.1%
1.1±0.3%
6±4.3%
16.4±6.2%
0.2±0.1%
94.4±0.7%
1.9±0.3%
3.6±0.5%
0.3±0.1%
90.1±5.3%
2.4±0.1%
7.2±5.2%
8 hours
Annexin V
7-AAD
86.4±1.6%
56.8±2.5%
0.4±0%
1.4±0.4%
92.3±3.1%
0.2±0.2%
87.8±6.5%
0.3±0.2%
2.6±0.4%
4.9±2.9%
3.1±1.5%
8.9±5.2%
5.8±1.6%
7.4±0%
18.6±3.9%
23.3±1.8%
24 hours
Annexin V
7-AAD J
BCR-ABL+ cell lines
Cdk6+/+
Cdk6-/- UT
NCS UT
NCS
1.8±1.4%
94.3±3%
2.5±1.3%
1.5±0.5%
3.6±0.4%
43±12.4%
8.3±1%
45±11.8%
1.8±1.9%
95.2±1%
1.8±0.4%
1.1±0.7%
7.9±3.2%
3.3±1.3%
Annexin V
86.1±3.3%
2.6±1.6%
7-AAD
Figure S2

5. A B C Figure S3 BCR-ABL+ cell lines UT Cdk6+/+ UT Cdk6-/- Untreated 5050
100
% cells
4h NCS
24h NCS
Sub G1
G0/G1 S
G2/M
Counts
NCS
Cdk6+/+
NCS
Cdk6-/-
Cdk6
+/+
-/-
+/+
-/-
+/+
-/- PI B
Annexin V
Etoposide
DMSO
Cdk6+/+
Cdk6-/-
0.9±0.4%
95.5±0.8%
3.2±0.9%
61.3±10.5%
0.5±0.1%
95.1±1.2%
1.4±0.5%
92±2.1%
2.2±0.5%
1.3±0.2%
25.7±7.2%
9.8±3.1%
3.2±0.6%
1.1±0.6%
4.6±1.3%
2.1±1.1%
BCR-ABL+ cell lines
7-AAD C
Sub-G1
G0/G1 S
G2/M
Sub-G1
G0/G1 S
G2/M
BCR-ABL+ cell lines
Cdk6+/+
Cdk6-/-
DMSO
DMSO
Sub G1
G0/G1 S
G2/M
DMSO
Etoposide
Counts
100
% cells
Etoposide
Cdk6+/+
Etoposide
Cdk6-/- 50
Cdk6
+/+
-/-
+/+
-/- PI
Figure S3

6. D E F G Figure S3 -/- -/- BCR-ABL+ cell lines Cdk6+/+ Cdk6-/- p53
30µM
p53 -
10µM
HSC-70
p21
Nutlin-3 E
DMSO PI
Cdk6+/+
Cdk6-/-
BCR-ABL+ cell lines
Nutlin-3
DMSO
100
Sub-G1
G0/G1 S
G2/M
% cells 50
-/-
Counts
Cdk6
+/+
Nutlin-3
100
% cells 50
Cdk6
+/+
-/- F
BCR-ABL+ cell lines
Cdk6+/+
Cdk6-/-
DMSO
Nutlin-3
DMSO
Nutlin-3
1.4±0.1%
2.4±0.5%
12.1±2.4%
65.9±11.1%
1.1±0.6%
1.6±0.5%
2.9±0.9%
10.4±2.2%
Annexin V
95.6±0.8%
0.6±0.3%
20.7±13.3%
1.3±0.2%
96.9±1%
0.4±0.1%
86±2.6%
0.7±0.2%
7-AAD G
BCR-ABL+ cell lines
p21
Relative mRNA levels 5 10 15 20
Nutlin-3 + -
Cdk6+/+
Cdk6-/-
****
Figure S3

7. H I J Figure S3 BCR-ABL+ cell lines Cdk6+/+ Cdk6-/- Cdk6-/- CDK6 p53
+Vector Ctrl.
+CDK6-K43M
+CDK6
CDK6
p53
ß-Actin I
BCR-ABL+ cell lines
Cdk6-/- + Ctrl. Vector
Cdk6-/- + CDK6-K43M
Cdk6-/- + CDK6 UT
NCS UT
NCS UT
NCS
1.1±0.9%
2.2±0.9%
3±1.5%
8.6±1.4%
0.8±0.5%
1.7±0.7%
1.9±0.3%
7.3±1.6%
0.7±0.5%
1.9±0.5%
2.1±0.4%
9.6±3.2%
Annexin V
94.5±1.4%
2.3±1.6%
85.1±3.2%
3.3±1.8%
95.2±1%
2.1±1.5%
87.4±0.7%
3.4±1.2%
96.5±1%
1±0.2%
85.8±4.5%
2.6±1.2%
7-AAD J
BCR-ABL+ cell lines
p21
Relative mRNA levels 5 10 15 20 *
NCS - +
Cdk6+/+
Cdk6-/-
+Vector Ctrl.
+CDK6K43M
+CDK6
Figure S3

8. A B C D Figure S4 Cdk6 +/+ -/- +/+ -/- +/+ -/- +/+ -/-
BCR-ABL+ cell lines B
BCR-ABL+ cell lines
AZD1152 (log10 µM)
100
150 -3 -2 -1 1 2 50
6-Mercaptopurine (log10 µM)
Viability (% DMSO)
Etoposide (log10 µM)
5-Azacytidine (log10 µM) -5 -4
Cdk6+/+
Cdk6-/- C
BCR-ABL+ cell lines
Cdk6+/+
Cdk6-/-
7-AAD
Annexin V
MMC
DMSO
93.4±1,9%
37.8±28.4%
0.8±0.4%
1.1±0.1%
94.7±2.4%
0.5±0.2%
80.5±8.4%
1.4±0.4%
2.3±0.8%
3.5±0.9%
11.7±2.1%
49.4±26.2%
7.5±4%
2.3±1.4%
2.5±0.9%
10.6±4.5% D
DMSO
Cdk6+/+
DMSO
Cdk6-/-
BCR-ABL+ cell lines
Counts
MMC
Cdk6+/+
MMC
Cdk6-/- PI
DMSO 24h
MMC 24h
DMSO 48h
MMC 48h 50
100 50
100 50
100 50
100
Sub G1
G0/G1 S
% cells
G2/M
Cdk6
+/+
-/-
+/+
-/-
+/+
-/-
+/+
-/-
Figure S4

9. E F G H Figure S4 Cdk6+/+ Cdk6-/- Cdk6+/+ Cdk6-/- DMSO 5-FU DMSO 5-FU
BCR-ABL+ cell lines
Cdk6-/-
DMSO
Etoposide
DMSO
Etoposide
1.3±0.2%
2.2±0.5%
9.8±3.1%
25.7±7.2%
1.1±0.6%
3.2±0.6%
2.1±1.1%
4.6±1.3%
Annexin V
95.5±0.8%
0.9±0.4%
61.3±10.5%
3.2±0.9%
95.1±1.2%
0.5±0.1%
92±2.1%
1.4±0.5%
7-AAD F
BCR-ABL+ cell lines
Sub-G1
G0/G1 S
G2/M
Sub-G1
G0/G1 S
G2/M
Sub G1
G0/G1 S
G2/M
DMSO
DMSO
Cdk6+/+
DMSO
Cdk6-/-
100
% cells 50
Cdk6
+/+
-/- PI PI
Counts
Etoposide
Etoposide
Cdk6+/+
Etoposide
Cdk6-/-
100
% cells 50 PI PI
Cdk6
+/+
-/- G
Cdk6+/+
BCR-ABL+ cell lines
Cdk6-/-
DMSO
5-FU
DMSO
5-FU
1.8±0.4%
3.5±0.4%
19.2±2.7%
19.3±2.3%
1.9±1.1%
4.1±1%
4.8±3.2%
7.5±1.7%
Annexin V
93.1±1.9%
1.6±1.2%
59.1±4%
2.3±1.8%
92±0.5%
2±1.5%
86.3±4.7%
1.4±0.5%
7-AAD H
BCR-ABL+ cell lines
Sub G1
G0/G1
DMSO
Sub-G1
G0/G1 S
G2/M
Sub-G1
G0/G1 S
G2/M S
G2/M 50
100
DMSO
Cdk6+/+
DMSO
Cdk6-/-
% cells
Cdk6
+/+
-/- PI PI
Counts
5-FU
5-FU
5-FU
Cdk6+/+
Cdk6-/- 50
100
% cells PI PI
Figure S4
Cdk6
+/+
-/-

10. p53 mutation Human homologue
Genotype
p53 mutation Human homologue
Domain
Citation
Cdk6+/+
 C135R
DNA binding
Aranda et al., 2007
Cdk6-/-
V172M
Forbes et al., 2008
T211I
Hsiao et al., 2011
R248H
Asher et al., 2003
A159D
Robinson et al., 2015
C176Y
Cabelguenne et al., 2000
R181C
Chang et al., 2005
Cdk6K43M
C176R
Agrawal et al., 2012
 Q144P 
A138V
Cuddihy et al., 2008 B
Cdk6-/- BCR-ABL+ cell lines
1µM PRIMA-Met
30µM PRIMA-Met
1µM PRIMA-Met
+1µM Etoposide
30µM PRIMA-Met
+1µM Etoposide
1.3±0.5%
1.7±0.2%
4.1±1.1%
7.8±4.9%
8.2±1.4%
16.6±5.5%
7.4±3.4%
74.4±22.2%
Annexin V
95.9±0.6%
1.1±0.5%
86.8±6.2%
1.3±0.7%
73±4.8%
2.3±1.1%
15.7±18.2%
2.6±1.6%
7-AAD
Figure S5

11. A B C D E Figure S6 Cdk6+/+ Cdk6K43M Cdk6-/- H2O 100 50
Density plof of Intensity (Before Normalization)
Density plof of Intensity (After Normalization)
Density (scaled)
Density (scaled)
Intensity
Expression B C
Volcano plot of p-value of Cdk6+/+ - Cdk6-/-
Cdk6+/+
Cdk6-/-
Cell
line
Cell
line
Day 10 16 19 10 16 19
pS780RB
PRMT5
-log10(p-value)
BCL2
pY207Crkl
CDK6
CDK4
GAPDH D
log2(fold change)
Initial Transformation **
Cdk6+/+
Cdk6K43M
Cdk6-/-
RRM2
Relative mRNA levels
0.0
0.5
1.0
1.5
2.0
2.5
MDM2
Relative mRNA levels
0.0
0.5
1.0
1.5
2.0
2.5
Initial
Transformation
Cell
lines
Initial
Transformation
Cell
lines
Cdk6+/+
Cdk6-/- E
MDM4
MDM4 Skipped Exon
Cdk6+/+
Cdk6K43M
Cdk6-/-
H2O
100 bp
200 bp
300 bp 50
100
MDM4S
MDM4 mRNA levels
Percent of total
Figure S6

12. F Figure S6 Initial Transformation CDK6 PRMT5 MDM4
Relative mRNA levels 10 20 30 5 15
**** *
Cdk6-/-
Cdk6-/-
Cdk6-/-
Cdk6-/-
Cdk6-/-
Cdk6-/-
+pMSCV-CDK6
+pMSCV-CDK6
+pMSCV-CDK6
Figure S6

13. Numbers of ChIP-Seq peaks
Initial Transformation
Cell lines
Biological adhesion
Biological regulation
Cell killing
Cellular component organization
Cellular process
Developmental proc.
Immune system proc.
Localization
Locomotion
Metabolic process
Multicellular organismal process
Reproduction
Response to stimulus
Rhytmic proc.
IκB kinase/
NF-κB cascade
JAK-STAT cascade
MAPK casc.
Calcium-mediated signaling
Cell communication
Cell cycle
Cell proliferation
Cell recognition
Cellular component movement
Chromosome segregation
Cytokinesis
Cell growth
Chromatin organization
Chromatin remodeling
Cytoskeleton organization
Mitochondrion organization
Chromatin Assembly
Behavior
Cellular defense response
Defense response to bacterium
Immune response
Response to abiotic stimulus
Response to biotic stim.
Response to endogenous stim.
Response to external stim.
Response to stress
Response to toxic substance
Biosynthetic process
Catabolic process
Coenzyme metabolic process
Generation of precursor metabolites and energy
Nitrogen compound metabolic process
Phosphate-containing compound metabolic process
Primary metabolic process
Secondary metabolic process
Sulfur compound metabolic process
Vitamin metabolic process B C
H2O
Cd19 Input
Vegfa Input
Prmt5 Input
Mdm4 Input
Mdm4 ChIP
Prmt5 ChIP
Vegfa ChIP (PC)
Cd19 ChIP (NC)
Enrichment over NC NC
Vegf-A
Prmt5
Mdm4 10 20 30
ChIP
Input
Ppm1d D
Numbers of ChIP-Seq peaks HA
Antibody
CDK6 Antibody
7883
13813
5741
Figure S7

14. E Figure S7 ChIP-Seq Overlay Initial Transformation Cell Lines
Intersect
Behavior
Cellular defense response
Defense response to bacterium
Immune response
Response to abiotic stimulus
Response to biotic stim.
Response to endogenous stim.
Response to external stim.
Response to stress
Response to toxic substance
Biological adhesion
Biological regulation
Cell killing
Cellular component organization
Cellular process
Developmental proc.
Immune system proc.
Localization
Locomotion
Metabolic process
Multicellular organismal process
Reproduction
Response to stimulus
Rhytmic proc.
IκB kinase/
NF-κB cascade
JAK-STAT cascade
MAPK casc.
Calcium-mediated signaling
Cell communication
Cell cycle
Cell proliferation
Cell recognition
Cellular component movement
Chromosome segregation
Cytokinesis
Cell growth
Chromatin organization
Chromatin remodeling
Cytoskeleton organization
Mitochondrion organization
Chromatin Assembly
Biosynthetic process
Catabolic process
Coenzyme metabolic process
Generation of precursor metabolites and energy
Nitrogen compound metabolic process
Phosphate-containing compound metabolic process
Primary metabolic process
Secondary metabolic process
Sulfur compound metabolic process
Vitamin metabolic process
Figure S7

15. A B C D Figure S8 Protein Abundance Ratio: Cdk6-/- / Cdk6+/+
Abundances Grouped (Cdk6+/+)
Abundances Grouped (Cdk6-/-)
MDM4
0.833
92.05
PPM1D
0.868
313.5
286.5
PRMT5
0.893
304.8
295.2 B
ZBTB7A
2.0x106
1.5x106
1.0x106
0.5x106
ZBTB7A
3x106
2x106
(Arbitraty Units)
RFU
(Arbitraty Units)
RFU
1x106 1 10
100
1000
CDK6 + - + + +
ng CDK6
Palbociclib - - - + -
ATP - + + + +
Substrate + + + + -
EBF1
EBF1
4x105
1x105
2x105
3x105
4x105
3x105
(Arbitraty Units)
RFU
(Arbitraty Units)
RFU
2x105
1x105 1 10
100
1000
CDK6 + - + + +
ng CDK6
Palbociclib - - - + -
ATP - + + + +
Substrate + + + + - C D
NFYA Immunoprecipitation
BCR-ABL+ cell lines
Enrichment over NC
CD19
Prmt5
Sp1
Cyclin G1
Cdk1 2 4 6 8
BCR-ABL+ cell lines
CDK6 ChIP
Cdk6-/-
Cdk6+/+ In SN IP In SN IP
HSC-70
NFYA
CDK6
Figure S8

16. E F Figure S8 Motif Targets p-value Score ETV1(ETS) 37.56% 1e-573
Initial Transformation
Cdk6+/+;p53-/-
shCtrl
sh CDK6
sh NFYA 1
sh NFYA 2
shSP1 1
shSP1 2
shSP1 3 50
100
Cdk6+/+;p53+/+
% BCR-ABL+ cells
Early Apoptosis
Late Apoptosis
Necrotic Cells/
Debris
Living F
Motif
Targets
p-value
Score
ETV1(ETS)
37.56%
1e-573
Homer(0.952)
ELK1(ETS)
54.65%
1E-167
Homer(0.751)
SP1
25.00%
1E-150
Homer(0.912)
NRF
9.22%
1E-117
Homer(0.972)
RUNX2
5.91%
1E-98
Jaspar(0.955)
CRE(bZIP)
12.89%
1E-93
Homer(0.893)
GFY
2.83%
1E-78
Homer(0.992)
NFY
9.57%
1E-63
Homer(0.940)
CTCF
3.62%
1E-59
Homer(0.908)
CEBPG
28.33%
1E-56
Jaspar(0.667)
Figure S8

17. A B C Figure S9 Cdk6-/- + HA-CDK6 Cdk6-/- + HA-CDK6K43M IP: HA-CDK6In SN IP In SN IP In SN IP In SN IP
p53 HA
IP: CDK6
Cdk6+/+
Cdk6-/-
p53-/- In SN IP In SN IP In SN IP
p53
CDK6 B
Cdk6-/- +
HA-CDK6
Cdk6-/- +
HA-CDK6K43M
Cdk6+/+
Cdk6-/-
p53-/-
IP: p53 In SN IP In SN IP In SN IP In IP In IP
p53
CDK6 C 4
PUMA 4
NOXA 3 3
Relative mRNA levels 2
Relative mRNA levels 2 1 1
Cdk6+/+
Cdk6K43M
Cdk6-/-
Cdk6+/+
Cdk6K43M
Cdk6-/-
Figure S9

18. A B Figure S10 ALL (BCR-ABL+) ALL (BCR-ABL+) MDS MDS
ALL (BCR-ABL-) MILE
ALL (BCR-ABL-) MILE
ALL (BCR-ABL-) Target
ALL (BCR-ABL-) Target
Figure S10

19. A B Figure S11 BM Ctrl. (MILE) BCR-ABL+ (MILE) BCR-ABL- (MILE)
BCR-ABL- (Target)
MDS (MILE) 1 -1
-0.5
0.5
Correlation coefficient
Common Set
Common Set + ChIP Peak B
BCR-ABL- (Target)
BCR-ABL+ (MILE)
MDS (MILE)
BCR-ABL- (MILE)
Figure S11

20. C D E F Figure S11 Correlated in all leukemia datasets
Correlated in at least one leukemia datasets
Healthy
Leukemia
Healthy
Leukemia E F
Thymoma
Glioblastoma
Leukemia
10000
7.5
5.5
1000 5
Normalized CDK6 expression 5
RPKM (x1000)
Normalized CDK6 expression
4.5
2.5
100 4 10
p53
mut WT
mut WT
-7/del(7q)
Yes No
Figure S11

21. Figure S12 p19ARF Relative mRNA levels * ** 5 10 15 Cdk6+/+ Cdk6-/-5 10 15
Cdk6+/+
Cdk6-/- * **
Initial Transformation
Cell lines
Figure S12