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

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

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

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

A B C Figure S3 BCR-ABL+ cell lines UT Cdk6+/+ UT Cdk6-/- Untreated 50 50 100 % 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

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

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

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

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 +/+ -/-

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

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

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

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

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

A B C D Figure S8 Protein Abundance Ratio: Cdk6-/- / Cdk6+/+ Abundances Grouped (Cdk6+/+) Abundances Grouped (Cdk6-/-) MDM4 0.833 109.025 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

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

A B C Figure S9 Cdk6-/- + HA-CDK6 Cdk6-/- + HA-CDK6K43M IP: HA-CDK6 In 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

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

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

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

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