Supplemental Information Figure S1. Negative control INY-03-112 does not degrade AKT isoforms. Related to Table S1. (A) Chemical structure of negative control compound INY-03-112, with N-methylated glutarimide circled. (B) Immunoblots for AKT1, AKT2, AKT3, pan-AKT, and Vinculin in MDA-MB- 468 cells after 12 hour treatment with DMSO or INY-03-112 at the concentrations indicated (representative of n=2). 15 Figure S2. INY-03-041-mediated degradation of IKZF1 and IKZF3. (A) Immunoblots for IKZF1, IKZF3, S6K1, pan-AKT, and b-actin in Jurkat cells after 24 hour treatment with DMSO, INY-03-041, or lenalidomide (Len) at the concentrations indicated (n=3). 16 Figure S3. INY-03-041 induces degradation of AKT isoforms in T47D cells. Related to Figure 4. (A) Immunoblots of AKT1, AKT2, AKT3, and Vinculin after treating T47D cells for 24 hours with DMSO, INY-03-041, or GDC-0068 at the concentrations indicated (n=3). 17 Table S1. Biochemical IC50 (nM) values for INY-03-041 and INY-03-112. Related to Figure 1 and Figure S1. Z’lyte kinase assays (Invitrogen) were conducted to assess IC50 values for all kinases listed in the table with the exception of RET (V804M) which was assayed using LanthaScreen assays (Invitrogen). Compound INY-03-041 INY-03-112 Kinase IC50 (nM) IC50 (nM) AKT1 1.96 1.5 AKT2 6.78 13.7 AKT3 3.51 4.45 PKG1 33.2 N/A S6K1 37.3 N/A PKN1 51.7 N/A ßMSK2 3.31 N/A Haspin 4020 N/A RET (V804M) >10000 N/A Table S2. KINOMEscan hits. Related to Figure 1. Table S3. Proteomics output from limma processing of MOLT4 cells treated for 4 hours with INY-03-041 vs DMSO control. Related to Figure 2. Table S4. Hormone receptor and mutational status of PIK3CA and PTEN in cancer cell line panel. The tissue, cancer subtype, and status of each gene (Meric-Bernstam et al., 2012; Vlietstra et al., 1998) as wild-type (wt), mutated (mut) or deleted (del) is indicated. Triple negative breast cancer (TNBC). Cell Line Tissue Cancer Subtype PIK3CA PTEN ZR-75-1 Breast Luminal A wt mut (L108R) T47D Breast Luminal A mut (H1047R) wt LNCaP Prostate Androgen-dependent wt del/mut MCF-7 Breast Luminal A mut (E545K) wt MDA-MB-468 Breast TNBC – Basal A wt del HCC1937 Breast TNBC – Basal A wt del 18 Table S5. GR values indicate anti-proliferative advantage of INY-03-041. Related to Figure 3. (A) GR values were calculated after 72 hours treatment with the compounds indicated over a range of concentrations. GR50 values represent compound potency, GRmax values measure the efficacy of the drug at high concentrations. GRAOC captures changes in potency and efficacy and is calculated by integrating GR curve over a range of concentrations. GR50 (µM) Cell Line INY-03-041 INY-03-112 GDC-0068 Lenalidomide ZR-75-1 0.01599132 0.41332816 0.2289724 inf T47D 0.17831981 1.33985401 1.52509853 inf LNCaP 0.13009989 1.38277274 1.31522936 inf MCF-7 0.14798964 1.49338161 1.71566724 inf MDA-MB-468 1.68557896 2.69522214 12.9176626 inf HCC1937 1.64527129 2.41978757 inf inf GRmax Cell Line INY-03-041 INY-03-112 GDC-0068 Lenalidomide ZR-75-1 -0.6878133 -0.5171039 -0.3841938 0.74474387 T47D -0.3208748 -0.1634936 0.05440203 0.99850631 LNCaP 0.01067184 0.21216665 0.01683696 0.82853147 MCF-7 -0.3054814 -0.3317977 0.21842009 0.90960366 MDA-MB-468 -0.8194262 -0.8318163 0.51778759 0.93954195 HCC1937 -0.8131698 -0.7255681 0.53817904 0.90453 GRAOC Cell Line INY-03-041 INY-03-112 GDC-0068 Lenalidomide ZR-75-1 1.16640547 0.32890379 0.7053818 0.08476616 T47D 0.63026903 0.19803381 0.28472386 -0.0100805 LNCaP 0.54925615 0.186322 0.32963774 0.04177353 MCF-7 0.62747921 0.31979881 0.24329199 0.02059167 MDA-MB-468 0.43042352 0.31894929 0.14038282 0.03254079 HCC1937 0.41471365 0.31312878 0.15607112 -0.0084259 19 KEY RESOURCES TABLE Antibodies Antibodies Source CAT# AKT1 Cell Signaling Technology CST2938 AKT2 Cell Signaling Technology CST3063 AKT3 Cell Signaling Technology CST8018 CST4691 Pan-AKT Cell Signaling Technology CST2920 Phospho-PRAS40 (T246) Cell Signaling Technology CST2997 Total PRAS40 Cell Signaling Technology CST2691 Vinculin Cell Signaling Technology CST13901 S6K1 Cell Signaling Technology CST2708 IKZF1 (Ikaros) Cell Signaling Technology CST14859 IKZF3 (Aiolos) Cell Signaling Technology CST15103 b-actin Cell Signaling Technology CST4970 Chemicals Chemicals Source CAT# DMSO Fisher BP231-100 Bortezomib (PS-341) Cayman Chemical 10008822 Pevonedistat (MLN4924) Selleckchem S7109 GDC-0068 Selleckchem S2808 Lenalidomide Sigma Aldrich 901558 INY-03-041 This study N/A INY-03-112 This study N/A Experimental Models: Cell Lines Cell Line Source Identifier MOLT4 ATCC CRL-1582 Jurkat ATCC TIB-152 ZR-75-1 ATCC CRL-1500 LNCaP Balk Lab T47D ATCC HTB-133 MCF7 ATCC HTB-22 MDA-MB-468 ATCC HTB-132 HCC1937 ATCC CRL-2336 20 Critical Commercial Assays Assay Source Identifier Z’ Lyte (AKT1) Invitrogen Assay ID 247 Z’ Lyte (AKT2) Invitrogen Assay ID 250 Z’ Lyte (AKT3) Invitrogen Assay ID 253 Z’ Lyte (PKG1) Invitrogen Assay ID 757 Z’ Lyte (S6K1) Invitrogen Assay ID 810 Z’ Lyte (PKN1) Invitrogen Assay ID 703 Z’ Lyte (βMSK2) Invitrogen Assay ID 801 Z’ Lyte (Haspin) Invitrogen Assay ID 1115 Lanthascreen (RET Invitrogen Assay ID 1847 (V804M)) Deposited Data Pride: PXD015207 Software and Algorithms Software Source CAT# GraphPad GraphPad www.graphpad.com/ Prism Software, Inc. Columbus PerkinElmer http://www.perkinelmer.com/product/image-data-storage- image Informatics and-analysis-system-columbus storage and analysis Adobe Adobe https://www.adobe.com/creativecloud.html Illustrator Creative Cloud Proteome Thermo https://www.thermofisher.com/order/catalog/product/OPTON- Discoverer Fisher 30795 2.2 Scientific R Team RCR: http://www.R-project.org/ Framework A Language and Environment for Statistical Computing 21 METHODS DETAILS Cell Culture and Reagents MOLT4, Jurkat, ZR-75-1, LNCaP, T47D, MCF-7, MDA-MB-468, and HCC1937 cells were cultured in RPMI media (Wisent Bioproducts, Cat #350000CL) supplemented with 10% heat inactivated fetal bovine serum (Thermo Fisher Scientific) at 37°C in the presence of 5% CO2. Drug Treatment Experiments Cells were plated at 250,000 cells per mL (MDA-MB-468) or 200,000 cells per mL (T47D) in 2 mL per well RPMI media with 10% serum in 6-well treated tissue culture plates (Greiner, Cat # TCG-657160) or 60 mm treated tissue culture plates (Corning, Cat # 430166) and incubated overnight. The next day, cells were treated with the indicated compounds at the appropriate concentration and protein lysates were harvested at the times specified. Immunoblotting Cells were washed once in 1x PBS then lysed in RIPA buffer (150 mM Tris-HCl, 150 mM NaCl, 0.5% (w/v) sodium deoxycholate, 1% (v/v) NP-40, pH 7.5) containing 0.1% (w/v) sodium dodecyl sulfate, 1 mM sodium pyrophosphate, 20 mM sodium fluoride, 50 nM calyculin, and 0.5% (v/v) protease inhibitor cocktail (Sigma-Aldrich®) for 15 minutes. Cell extracts were precleared by centrifugation at 14,000 rpm for 10 minutes at 4°C. The Bio-Rad DC protein assay was used to assess protein concentration, and sample concentration was normalized using SDS sample buffer. Lysates were resolved on acrylamide gels by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membrane (BioRad) at 100 volts for 90 minutes. Membranes were blocked in 5% (w/v) nonfat dry milk in tris-buffered saline (TBS) buffer for 1 hour then incubated with specific primary antibodies diluted in 5% (w/v) nonfat dry milk in TBS-T (TBS with 0.05% Tween-20) at 4°C overnight, shaking. The next day, membranes were washed with TBS-T then incubated for 1 hour at room temperature with fluorophore-conjugated secondary antibodies (LI-COR Biosciences). The membrane was washed again with TBS-T then imaged with a LI-COR Odyssey CLx Imaging System (LI-COR Biosciences). Growth Rate Assay and Analysis Cell lines were plated at densities ranging from 500 to 2000 cells per well in a 384-well plate using a Matrix WellMate Reagent Dispenser (Thermo Fisher) and allowed 24 hours to adhere to plate prior to treatment. A D300 Digital Dispenser (Hewlett-Packard) was used to treat cells with dilution series of compounds as indicated. At the time of treatment and after 72 hours of treatment, cells were stained and fixed for subsequent analysis. Cells were stained with LIVE/DEAD Far Red Dead Cell Stain (LDR) (Thermo Fisher Scientific) at 1:2000 for one hour at 37°C. Cells were fixed for 30 minutes at room temperature in 4% formaldehyde (Sigma Aldrich) then permeabilized with 0.5% Triton X-100 in PBS. Cells were blocked for one hour using Odyssey Blocking Buffer (LI-COR Biosciences) and stained overnight at 4°C with 2 µg/ml Hoechst 33342 (Sigma Aldrich). An Operetta microscope was used to image fixed cells, and data was stored and analyzed using Columbus software (PerkinElmer). Nuclei were segmented by Hoechst signal using the Columbus system (Perkin Elmer). The average LDR and Hoechst 22 intensities were determined within the nuclear area. Dead cells were classified by LDR signal. Experiments were performed in technical triplicate. In Vitro Kinase Assays Z’-LYTE assays were conducted for AKT1, AKT2, AKT3, PKG1, S6K1, PKN1, ßMSK2, and Haspin at Life Technologies in a 10-point dose response using Km ATP concentrations. LanthaScreen assays were conducted for RET (V804M) in a 10-point dose response at Life Technologies. TMT LC-MS Sample Preparation MOLT4 cells were treated with DMSO, 250 nM INY-03-041-01 for 4 hours in biological triplicates.