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Supplementary Material

Peptide synthesis and purification

Solid phase peptide syntheses were performed using Fmoc chemistry (ABI 433A peptide

synthesizer; Applied Biosystems). Preloaded 4-Hydroxylmethylphenoxy (HMP) resin (AnaSpec)

was used for all syntheses. 2-(1H-9-Azabenzotriazole-1-yl)-1,1,3,3-tetramethyl-aminium

hexafluorophosphate (HBTU)/N-Hydroxybenzotriazole (HOBt) in DMF was used as the coupling

reagent (AnaSpec). The 5-TAMRA labeled peptide was prepared by reacting with 5-TAMRA

succinimidyl ester (AnaSpec). Following completion of peptide chain assembly, peptide-resins

were washed with CH2Cl2, and then cleaved with 95%TFA/2.5% H2O/2.5% triiosopropylsilyl

(TIPS) for 1.5 hours at RT. Ethyl ether extraction was carried out three times for the peptide-

TFA solution to remove TFA and scavengers. The resultant white precipitate was re-dissolved in

aqueous solution, and a crude sample was taken for analytic HPLC before purification. The

synthetic peptides were purified with a Varian HPLC system (Phenomenex, Inc) using a semi-

preparative reverse-phase column (Gemini C18, 10 x 250 mm, 10 µm. Purification conditions

were as follows: buffer A, 0.1% TFA in H2O; buffer B, 0.08% TFA in CH3CN; gradient, 10% to

50% B over 60 minutes, or 0% to 50% B over 50 minutes; flow rate, 6 mL/min; UV detector, 230

nm. The purified peptide was verified by LC-MS using an Agilent 1100 HPLC system (Agilent

Technologies) with an API 150EX mass spectrometer (Applied Biosystems) over a C18 reverse

phase column (Agilent, ZORBAX 300SB-C18, 2.1 x 150 mm; gradient 0% to 50% B over 10

min; flow rate, 0.4 mL/min).

Conformational energy calculations

The initial small-molecule conformation for compound 8 (AM-2910025) was taken from the co-

crystal structure of MCL1 with 8. The starting conformation for compound 9 was modeled based

on the conformation of 8. We further prepared the models of 8 and 9 by adding hydrogens,

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deprotonating the acyclsulfonamide at the nitrogen, and performing a restrained energy

minimization. The minimization, and all molecular mechanics calculations, were performed in

MOE (Chemical Computing Group, Montreal) using the MMFF94x force-field with electrostatic

cutoffs disabled and a Born solvation model with an interior dielectric of 1.8 and an exterior

dielectric of 80. To generate the conformational ensemble for each molecule, we used the low-

mode MD search algorithm in MOE with a high rejection limit of 300 to ensure

completeness. The energy window was limited to 5 kcal/mol, and the heavy atoms of the core

spiro ring system (consisting of the four connected rings) were restrained with tethers

(weight=10, [L,U]=[0,0.25]) to help ensure completeness of the search. Results are from MOE

v2016. We have also performed the same calculations with MOE v2012 and obtained

essentially identical results. To generate more accurate calculated relative conformational

energies, we recalculated the energies for each conformation using quantum chemistry

calculations in Gaussia09 at the B3LYP/6-31+G*//B3LYP/6-31+G* level, where we performed

geometry optimization with inclusion of IEFPCM solvation in order to accurately represent the

formal negative charge.

Time-resolved fluorescence resonance energy transfer binding assays

Recombinant 6XHis-tagged human MCL1 (171-327), dog MCL1 (171-327, C286S), mouse

MCL1 (152-308), and BCL-XL (1-196) were produced at Amgen. All proteins were expressed in

E. coli and purified using metal ion affinity chromatography followed by size-exclusion

chromatography. Recombinant 10XHis-tagged human BCL-2 (2-211) was purchased from R&D

Systems. Human Biotin-Bim BH3 peptide (Biotin-DMRPEIWIAQELRRIGDEFNAYYARR) and

mouse Biotin-Bim BH3 peptide (Biotin-DLRPEIRIAQELRRIGDEFNETYTRR) were custom

synthesized by CPC Scientific. Streptavidin-XLent was obtained from Cisbio US. LANCE® Eu-

W1024 Anti-6xHis, 384-well OptiPlate, and EnVision™ Multilabel Reader were purchased from

PerkinElmer.

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The inhibition of the interaction between Biotin-Bim BH3 peptide and MCL1, BCL-2, or

BCL-XL was measured using the time-resolved fluorescence resonance energy transfer (TR-

FRET) assays. The assays were conducted in the 384-well white OptiPlates with a total volume

of 40 μL per well in the binding buffer of 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.016 mM

Brij35, and 1 mM DTT. Serially diluted test compounds were pre-incubated with Biotin-Bim BH3

peptide and protein (MCL1, BCL-2, or BCL-XL) for 60 min before addition of the detection

mixture (LANCE® Eu-W1024 Anti-6xHis and Streptavidin-XLent). The reaction plates were

further incubated overnight and then were read on an EnVision™ Multilabel Reader. Human

and dog MCL1 assays used the same assay conditions and contained 0.1 nM human or dog

MCL1, 0.05 nM human Biotin-Bim BH3 peptide, 0.05 nM LANCE® Eu-W1024 Anti-6xHis, and

0.072 nM Streptavidin-XLent. Mouse MCL1 assay contained 0.3 nM mouse MCL1, 0.35 nM

mouse Biotin-Bim BH3 peptide, 0.05 nM LANCE® Eu-W1024 Anti-6xHis, and 0.3 nM

Streptavidin-XLent. Human BCL-2 assays contained 0.2 nM human BCL-2, 0.37 nM human

Biotin-Bim BH3 peptide, 0.05 nM LANCE® Eu-W1024 Anti-6xHis, and 0.3 nM Streptavidin-

XLent. BCL-XL assays contained 1 nM human BCL-XL, 1 nM human Biotin-Bim BH3 peptide,

0.1 nM LANCE® Eu-W1024 Anti-6xHis, and 0.4 nM Streptavidin-XLent. In all the assays

mentioned above, the concentration of Biotin-Bim BH3 peptide was chosen to be at 1X KD to

allow for a meaningful comparison of affinity based on the IC50 value. Fluorescence signals

were measured at 620 nm and 665 nm with a 300 µs delay after excitation at 320 nm. The

signal ratio at 665/620 nm corresponded to the interaction between Biotin-Bim BH3 peptide and

MCL1, BCL-2, or BCL-XL and was used in all data analyses. The IC50 values of test compounds

were determined by fitting concentration-response data with a four-parameter sigmoidal model

in Genedata Screener (Genedata, Basel, Switzerland).

To determine the equilibrium dissociation constant (KI) of test compounds toward human

MCL, a similar TR-FRET assay procedure was used. The concentration of human Biotin-Bim

BH3 peptide was raised to 100X KD to enable accurate determination of highly potent inhibitors.

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The reaction mixture contained 0.1 nM human MCL1, 5 nM human Biotin-Bim BH3 peptide,

0.05 nM LANCE® Eu-W1024 Anti-6xHis, 2.5 nM Streptavidin-XLent, and serially diluted test

compounds. The KI values of test compounds were determined by fitting concentration-

response data to the Morrison equation implemented in Genedata Screener.

MCL1:BIM complex immunoassay 

A427 cells were seeded an optimized seeding density in 6-well tissue culture plates and

incubated overnight at 37°C in 5% CO2. Cells were treated with AM-8621 at the indicated

concentrations for 0.5, 1, 4, 12, or 24 hours. Cells were washed with ice-cold PBS and lysed in

Meso-Scale Discovery® (MSD) lysis buffer. Lysates were subjected to BCA protein assays to

determine protein concentrations. Custom MCL1 capture plates were developed using an MCL1

antibody (BD Biosciences, 559027). Capture plates were blocked in MSD blocking solution A for

1 hour at room temperature (RT) with shaking. Plates were then washed 3 times followed by the

addition of 40 µg/well of lysate. Lysates were incubated with shaking for 1 hour at RT. Plates

were then washed 3 times followed by the addition of BIM detection antibody (Cell Signaling

Technologies, 2819) and shaken for 1 hour at RT. Three additional washes were performed

followed by the addition of MSD read buffer. Plates were read on an MSD SI6000 plate reader.

BAX-/-BAK-/- cell lines viability assay

BAX-/-BAK-/- AMO1, H929, and OPM-2 cells were generated using CRISPR/Cas9 and reported

previously (1). To determine the sensitivity of wild-type and BAX-/-BAK-/- cells to AM-8621, cells

were seeded into 96-well plates at 3,000 cells/well in duplicate and treated with serially diluted

concentrations of AM-8621 (0–10 μM, 1:8 dilution for 5 points). Cell viability following 24h of

treatment was determined by CellTiter-Glo Assay (Promega) according to the manufacturer’s

instructions and the plates were read using an EnVision™ Multilabel plate reader (Perkin

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Elmer). Results were normalized to the viability of cells treated with DMSO. Data of three

independent experiments were presented.

Immunoblot analysis

Treated cells were washed with PBS and lysed in radioimmunoprecipitation assay (RIPA) lysis

buffer containing appropriate phosphatase and protease inhibitors (Roche). Lysates were then

pre-cleared via centrifugation at 4°C, followed by analysis by DCTM protein assay (Bio-Rad) to

determine protein concentration. Lysates were resolved on NuPAGE® gels (Life Technologies)

per manufacturer’s recommendation and transferred to PVDF membranes (Life Technologies),

which were blocked in TBST + 5% milk and probed with indicated primary antibodies against

MCL1 (Cell Signaling Technologies, 4572), BCL-XL (Cell Signaling Technologies, 2764), BCL-2

(BD Biosciences, 610538), BIM (Cell Signaling Technologies, 2819), cleaved PARP (Cell

Signaling Technologies, 9541), and β-actin (Abcam, ab8227). Membranes were then incubated

with secondary anti-species antibodies conjugated to HRP (GE Healthcare). SuperSignal™

West Pico Chemiluminescent Substrate (ThermoFisher Scientific) was used to develop HRP

signal which was captured with a ChemiDoc imaging system (Bio-Rad).

Tumor cell line profiling screens

For the 952–cell line screen, high-throughput drug profiling and sensitivity modeling (curve fitting

and IC50 estimation) were performed as previously described (2). Briefly, cells were grown in

RPMI or DMEM/F12 medium supplemented with 5% FBS and penicillin/streptomycin, and

maintained at 37°C in a humidified atmosphere at 5% CO2. Cell lines were propagated in these

two media to minimize the potential effect of varying the media on sensitivity to therapeutic

compounds in the assay and to facilitate high-throughput screening. To exclude cross-

contaminated or synonymous lines, a panel of 92 SNPs was profiled for each cell line

(Sequenom) and a pair-wise comparison score calculated. In addition, short tandem repeat

(STR) analysis (AmpFlSTR Identifiler, Applied Biosystems) was performed and matched to an

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existing STR profile generated by the providing repository. Cells were seeded in 384-well plates

at variable density to ensure optimal proliferation during the assay. AM-8621 was added to the

cells the day after seeding for adherent cell lines and the day of seeding for suspension cell

lines. For tumor subtypes containing both adherent and suspension cells, all lines were treated

with AM-8621 on the same day. A series of nine doses of AM-8621 with a 2-fold dilution factor

was used, for a total concentration range of 256 fold. Viability was determined using resazurin

after 5 days of AM-8621 exposure.

For the focused screen of MM, AML, and DLBCL cell lines, cells were seeded at an

optimized density in 96-well tissue culture plates followed by overnight incubation at 37°C and

5% CO2. Cells were then treated with a 9-point serial dilution of AM-8621, using a top

concentration of 20 µM, 1:3 serial dilution steps and a DMSO-only control. Cells were incubated

in the presence of drug for 24 hours. Effects on cell viability were measured with the CellTiter-

Glo® Luminescent Cell Viability Assay (Promega) per manufacturer’s recommendation. Assay

plates were read on an EnVision™ Multilabel Reader using the luminescence setting. POC

values were calculated as follows: POC = 100 * (treatment / vehicle). Mean POC values were

then calculated for each treatment condition and used to fit dose response curves applying a

four-parameter logistic fit model in XLfit 4.0.

Elastic net model

We applied the approach of Zou and Hastie’s elastic net (3), a multivariate variable selection

technique with a penalization approach. Genomic data including mutation status of 428 genes

obtained through WES (whole exome sequencing) and filtered for their appearance in tumor

samples (COSMIC), continuous copy number data for 1789 genes selected for their correlation

with mRNA expression changes, and 18,535 transcript levels, as well as tissue classification in

19 types, were employed. This represented a total of 20,771 features across all cell lines. The

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elastic net was used to select which of these features were associated with drug response as

measured by IC50 across the cell line panel.

Let 𝑋 be a 𝑝 𝑛 matrix of input features (where 𝑝 is the number of features and 𝑛 is the number

of cell lines) and 𝑦 be a vector of drug sensitivities of length 𝑛. For any non-negative and

𝐿 𝜆 , 𝜆 , 𝛽 |𝑦 𝑋𝛽| 𝜆 ∑ 𝛽 𝜆 ∑ 𝛽 .(1)

Let 𝛽 be the naïve elastic net estimator. Then 𝛽 argmin 𝐿 𝜆 , 𝜆 , 𝛽 . A scaling factor of

1 𝜆 was added to the naïve elastic net to prevent double shrinking.

𝛽 elastic net 1 𝜆 𝛽 naïve elastic net .(2)

To determine the optimal 𝜆 and 𝜆 we let 𝛼 .

Then 1 𝛼 ∑ 𝛽 𝛼 ∑ 𝛽 is the elastic net penalty.

10-fold cross validation was performed to optimize 𝜆 and 𝜆 in equation (2), denoted as 𝜆 and

𝜆 respectively. To find the variables that are associated with drug response, 𝜆 , 𝜆 , X, and y

were input into equation (2) to solve for vector 𝛽. The variables with nonzero 𝛽s were

determined to be features associated with drug response.

This procedure was repeated 100 times for each drug in order to assess the stability of

the features when applying the 10-fold cross validation procedure. For each of the 100 runs, a

feature list was built comprised of genes, transcripts, and tissues with weights assigned to each.

The final signature of markers for a given drug consisted of all features which appeared in any

of the 100 runs along with the statistics on the frequency with which the feature appears and the

average weight given to that feature over the 100 runs.

1 2

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The weights 𝛽 were used to assess effect sizes of features in a drug’s marker

signature. The effect size of a feature was calculated by multiplying the feature’s weight by its

standard deviation across the cell line panel. The effect size is therefore a normalization of the

feature’s weight to account for the different scales used to measure the different genomic

features. Features with higher stability of correlation in cross-validation 𝑓 were considered of

the highest confidence of truly being associated with drug response. The most significant

features associated with drug response are those with both large frequency and effect size.

In vitro combination studies

In vitro combination studies were carried out as previously described (4). Briefly, cells were

seeded in 384-well tissue culture plates at optimized seeding densities, followed by incubation

overnight at 37°C in 5% CO2. Compounds were added to the culture plates in a 10x10 (10-point

titrations) matrixed format in multiple replicates, with one agent titrated along the x-axis and the

second agent along the y-axis. CellTiter-Glo® Luminescent Cell Viability assay kits were used to

determine the numbers of viable cells. Luminescence was measured with an EnVision™

Multilabel Reader for each cell line at time zero (V0) before the addition of compounds, as well

as after 72 hours of compound treatment. Growth inhibition (GI) was calculated on a 200-point

scale according to the following equations, where V72 was luminescence of DMSO control at 72

hours and T72 was luminescence of the compound-treated sample: if T72 > V0, then GI = 100 x

(1 – ((T72-V0) / (V72 – V0))); if T72 < V0, then GI = 100 x (1 – ((T72-V0) / V0)). Data were analyzed

for synergistic interactions using the Chalice™ Analyzer software (Zalicus, Cambridge, MA),

which generated synergy scores based on the Loewe Additivity model. Individual heterologous

combinations (AxB) were evaluated by comparing their respective synergy scores to that of their

component self-crosses (AxA or BxB). Heterologous combinations were considered synergistic

only when their synergy score exceeded three times that of either component self-cross synergy

score.

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Generation of human MCL1 knock-in mouse

Human MCL1 knock-in mice were created by targeting C57Bl/6 embryonic stem cells (The

Jackson Laboratory) with a targeting vector containing the full human MCL1 genomic locus

flanked by homologous mouse sequences upstream and downstream of the mouse MCL1

genomic locus. Correctly targeted ES cells were confirmed by genomic Southern blot and

microinjected into albino C57Bl/6 (Taconic Biosciences) blastocysts to generate chimeric mice.

Germline transmission of the human MCL1 knock-in allele as well as removal of any selectable

markers flanked by loxP sites was accomplished through breeding with systemic cre expressing

transgenic mice (CMV-cre; The Jackson Laboratory). Homozygous human MCL1 knock-in mice

were created by the breeding of heterozygous human MCL1 knock-in mice that lacked the Cre

transgene and selectable markers. The full length human MCL1 genomic locus was synthesized

with flanking mouse sequences just upstream and downstream of the mouse MCL1 genomic

locus. Positive (neomycin) and negative (Herpes simplex virus thymidine kinase) selectable

markers were incorporated into the targeting vector for selection during homologous

recombination in mouse embryonic stem (ES) cells. Primer sets 6213-06/6213-07 and 6000-

36/6213-04 were used to detect correctly targeted mouse ES cells. Clones that survived

neomycin and ganclycovir drug selection were expanded, and DNA was obtained for analysis

using PCR. Primers 6213-06 (external to the targeting vector) and 6213-07 (targeting specific)

were used to detect homologous recombination of the 5’ targeting arm; 31 putative targeted

clones were identified. To detect homologous recombination on the 3’ side, primers 6213-04

(external to the targeting vector) and 6000-36 (targeting specific) were used; 31 putative clones

were detected for the 3’ homologous recombination. Only 20 clones were positive for

homologous recombination for both the 5’ and 3’ regions; 13 of 20 clones were moved forward

for genomic Southern analysis. DNA from PCR positive targeted human MCL1 knock-in ES

clones was digested with restriction enzymes SacI and Cla1, resolved on an agarose gel and

probed with a radioactive DNA probe corresponding to a region outside the targeting vector and

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within the mouse genomic MCL1 locus. Clones exhibiting a 9.2 kb targeted band and a 4.1 wild

type non-targeted band were selected for chimeric mouse production. Removal of the loxP

flanked Neo selection marker was performed by breeding germ line heterozygous human MCL1

knock-in mice to a transgenic CMV-cre line (The Jackson Laboratory). Homozygous human

MCL1 knock-in mice were created by breeding heterozygous MCL1 knock-in (neo removed and

CMV-cre negative) mice.

Ex vivo analysis of mouse splenocytes

Spleens from human MCL1 knock-in and WT mice were harvested into 5 mL RPMI 1640 media.

Spleens were mashed gently and passaged through 100 micron cell strainers. Cell suspensions

were pelleted at 1500 RPM for 5 minutes, followed by resuspension in 4 mL of ammonium-

chloride-potassium (ACK) buffer. Samples were again pelleted and resuspended in RPMI 1640

media containing 10% FBS. Cells were counted and seeded in 96-well tissue culture plates at a

density of 2 × 105 cells / well. Cells were then treated with a 7-step, 3-fold serial dilution of AM-

8621, ranging from 3.3 µM to 4.5 nM for 6 hours, pelleted via centrifugation, and washed with 1x

PBS. Cells were then stained with LIVE/DEAD™ blue dye (Invitrogen) for 30 minutes in 1x PBS

to assess cytotoxicity, followed by staining with B220-Alexa647 (Beckton Dickinson, BD557683)

and CD3-PE (Beckton Dickinson, 553240) antibodies at a dilution of 1:100 in PBS + 1% FBS.

Samples were then analyzed on a LSRII flow cytometer (BD Biosciences).

Flow cytometry based BAK activation assay

OPM-2 cells were treated with 1 µM AM-8621 for 2 hours at 37°C in 5% CO2. Cells were fixed

and permeabilized in Cytofix/Cytoperm™ solution (BD Biosciences) for 30 minutes at room

temperature. Perm/Wash buffer (BD Biosciences) was used to wash cells followed by staining

with primary antibody (2 µg/mL) recognizing activated BAK (CalBiochem/EMD Millipore, AMO3)

for 1 hour at RT. Cells were then washed three times in Perm/Wash buffer followed by

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incubation with secondary anti-mouse Alexa Fluor 647 conjugated antibody (0.5 µg/mL) for 30

minutes at RT. Cells were then washed two times with Perm/Wash buffer and analyzed on an

LSRII flow cytometer (BD Biosciences).

AMG 176 formulation

For all in vivo experiments AMG 176 was formulated in 25% 2-hydroxypropyl-beta-cyclodextrin

and 75% water at pH 9. AMG 176 was formulated at a dose volume of 10 ml/kg and each

mouse was dosed by bodyweight. AMG 176 was dosed in solution. Mice were dosed orally

using an oral gavage needle.

Flow cytometry analysis of monocytes, B cells and neutrophils from human MCL1 knock-

in mice

Human MCL1 knock-in mice were treated with AMG 176 as a single agent or in combination

with venetoclax for 9 days. AMG 176 was dosed twice weekly (days 1, 2, 8 and 9) and

venetoclax was administered daily (days 1–9). Peripheral blood was harvested 24 hours

following the second dose of AMG 176 on cycle 1 (day 3). Peripheral blood and bone marrow

were harvested 24 hours following the second dose of AMG 176 on cycle 2 (day 10).

Lymphocytes were stained with anti-CD45.2 (clone 104), anti-CD11b (M1/70), anti-Ly6G (1A8),

anti-Ly6C (HK1.4), anti-CD19 (1D3) (BD Biosciences, Biolegend). Cells were analyzed on a

LSRII flow cytometer (BD Biosciences) (Supplementary Fig. S7).

Complete blood count analysis in human MCL1 Knock-In mice

Human MCL1 knock-in mice were treated with AMG 176 at doses of 30 and 60 mg/kg.

AMG 176 was dosed orally once daily for two days. Peripheral blood was harvested 24 hours

following the second dose (day 3). EDTA-anticoagulated blood was shipped overnight to IDEXX

and complete blood count analysis was performed on an Adiva 1200 hematology analyzer.

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Pharmacodynamic evaluation of active BAK, cleaved PARP and cleaved Caspase 3 in

subcutaneous human tumor models 

Tumors were harvested and snap frozen in liquid nitrogen from 2–24 hours post treatment.

Tumors were then pulverized and lysed in complete lysis buffer (0.05% NP-40, 1% CHAPS,

142.5 mM KCl, 5 mM MgCl2, 1 mM EGTA, 20mM HEPES pH 7.5, 1X Halt Protease and

Phosphatase Inhibitor [ThermoFisher]) followed by determination of protein concentration with a

BCA assay (Pierce). Active BAK was detected with the use of a custom immunoassay (MSD).

Capture plates, coated with an antibody specific to the N-terminal active BAK epitope (R&D

Systems, MAB8161), were blocked in MSD blocking solution A for 1 hour at RT with shaking.

Plates were then washed 3 times, followed by the addition of 12.5 µg/well of lysate. Lysates

were incubated with shaking for 1 hour at RT. Plates were then washed 3 times followed by the

addition of a total BAK detection antibody (Abcam, ab53153). Plates were incubated at RT for 1

hour with shaking. Plates were then washed 3 times followed by the addition of MSD read

buffer. Plates were read on an MSD SI6000 plate reader. Cleaved PARP and cleaved Caspase

3 immunoassays were performed per manufacturer’s (MSD) protocol (cleaved Caspase 3,

N451CFB-1; cleaved PARP, N450DEB-1).

Immunohistochemistry

Tissue sections (4 µm FFPE) were depariffinized in xylene and hydrated through gradient

ethanols to deionized water. The sections were then subjected to heat induced epitope retrieval

with DIVA (citrate-based, pH 6.2; Biocare) and endogenous peroxidase, and endogenous

proteins were blocked with 3% hydrogen peroxide/PBS and serum-free casein (Invitrogen),

respectively, for 20 minutes each. The slides were rinsed in PBST 3 times, and the primary

cleaved Caspase 3 antibody (Cell Signaling Technologies, Asp175a) was applied (1:200) and

incubated for 60 minutes. The slides were rinsed again and a goat anti-rabbit HRP-conjugated

secondary antibody (EnVision™+ HRP DAKO) was applied and incubated for 30 minutes. After

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rinsing with TBST, the diaminobenzidine (DAB+, DAKO) chromogenic substrate was applied for

5 minutes, followed by a light hematoxylin counterstain. The slides were stained on an itelliPath

autostainer (Biocare) at RT.

Primary patient samples

Bone marrow samples from patients with AML were collected after informed consent in

accordance with guidelines approved by The Alfred and Royal Melbourne Hospital human

research ethics committees. Mononuclear cells were isolated by Ficoll-Paque (GE Healthcare)

density-gradient centrifugation, followed by red cell depletion in ammonium chloride (NH4Cl)

lysis buffer at 37°C for 10 minutes. Cells were then resuspended in PBS containing 2% FBS

(Sigma). Mononuclear cells were suspended in RPMI-1640 (Gibco) medium containing penicillin

and streptomycin (Gibco) and 15% heat-inactivated FBS (Sigma).

Ex vivo drug testing of primary AML patient samples

Freshly purified AML cells were plated in RPMI and 15% FBS at 2.5×105 cells/mL, and 100 µl of

cell suspensions were aliquoted per well into 96-well plates (Sigma). Cells were then treated

with AM-8621, Ara-C (Pfizer), venetoclax (Active Biochem) or idarubicin (Sigma) over a 6 log

concentration range from 1 nM to 10 μM, (10nM to 100 μM Ara-C) for 48 h and incubated at

37°C and 5% CO2. Cells were then stained with the Sytox blue nucleic acid stain (Invitrogen)

and fluorescence measured by flow cytometric analysis using a LSR-II Fortessa machine

(Becton Dickinson). FACSDiva® software was used for data collection, and FlowJo® software for

data analysis. Blast cells were gated using forward and side light scatter properties. Viable cells

excluding Sytox blue were determined at six concentrations for each drug and the 50% lethal

concentration (LC50, in μM) was calculated using nonlinear regression algorithms in Prism

software (GraphPad®).

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Supplementary Figure S1. Increases in MCL1 protein levels observed following AM-8621 treatment are independent of changes in MCL1 transcript. Quantitative PCR analysis of MCL1 transcript levels in U266B1 cells treated with AM-8621 for 24 hours.

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Supplementary Figure S2. Treatment with AM-8621 induces MCL1 protein levels in tumor cell lines beyond U266B1. A, Immunoblot characterization of AM-8621–mediated increase in MCL1 protein in A427 cells. For left panels, cells were treated with 750 nM AM-8621 for indicated duration. For right panels, cells were treated with AM-8621 (750 nM) for 6 hours, followed by drug washout for indicated durations. B, Immunoblot characterization of AM-8621–mediated increase in MCL1 protein in MV-4-11 cells treated with AM-8621 (1 µM) for 1 hour. C, Immunoblot characterization of AM-8621–mediated increase in MCL1 protein in NCI-H1568 cells treated with AM-8621 (4 µM) for indicated durations.

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Supplementary Figure S3. Low BCL-XL protein expression is predictive of sensitivity to AM-8621 in MM cell lines. Relationship between BCL-XL protein expression and sensitivity to AM-8621 across a MM cell line panel. BCL-XL protein expression levels for MM cell lines were obtained with a custom BCL-XL immunoassay and reported relative to a standard curve of AGS cell lysates, a MM cell line with elevated BCL-XL expression.

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Supplementary Figure S4. AMG 176 induces apoptosis in vivo. Immunohistochemistry analysis of cleaved Caspase 3 in established OPM-2 luc tumors assessed 6 and 24 hours after administration of a 50 mg/kg or 100 mg/kg dose of AMG 176.

18  

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Supplementary Figure S5. Strategy for generating human MCL1 knock-in mouse. A, Human MCL1 knock-in mice were created via targeted homologous recombination of the human MCL1 genomic locus with the equivalent mouse locus in C57BL/6 embryonic stem (ES) cells. B and C, Correctly targeted ES cells were identified by confirmatory PCR using primer pairs recognizing 5’ and 3’ ends of the targeting sequence. Individual primer pairs recognized sequence external and internal to the targeting vector. D, Southern blot confirmation of heterozygous human MCL1 knock-in ES cells.

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Supplementary Figure S6. Treatment of splenocytes from human MCL1 knock-in mice with AM-8621 induces cleaved Caspase 3 and loss of viability in B cells. Effects of ex-vivo AM-8621 treatment (6 hours) on cytotoxicity and cleaved Caspase 3 of B220+ B cells from human MCL1 knock-in and wild-type (WT) mice. Experiments were performed on two individual knock-in and WT mice.

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Supplementary Figure S7. Gating scheme for flow cytometry analysis of B cells, monocytes and neutrophils from human MCL1 knock-in and WT mice.

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Supplementary Figure S8. Complete blood count analysis of peripheral blood from human MCL1 knock-in mice treated with AMG 176. AMG 176 was dosed orally once daily for two days. Peripheral blood was harvested 24 hours following the second dose (day 3). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (One-way ANOVA with Dunnett’s post hoc).

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Supplementary Figure S9. AMG 176 was well tolerated in human MCL1 knock-in mice. Observed body weights in wild-type (WT) and human MCL1 knock-in mice following twice-weekly oral administration of AMG 176 at doses of 30 and 60 mg/kg.

.

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Supplementary Figure S10. Combined AM-8621 + dexamethasone treatment exhibits synergy in MM cell lines. Synergy analysis of AM-8621 + dexamethasone combination in OPM-2, KMS11, MM.1S, and MM.1R cell lines. Effects on viability were captured following treatment with a 10x10 dose matrix combination of AM-8621 and dexamethasone at indicated doses for 72 hours. The MM.1R cell line is resistant to dexamethasone and was included as negative control for detection of synergy. Synergy scores are reported for the 2-way combination of AM-8621 + dexamethasone and self-cross combinations of dexamethasone + dexamethasone and AM-8621 + AM-8621, respectively.

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Supplementary Figure S11. Limited synergistic interaction detected between AM-8621 and carfilzomib in MM cell lines in vitro. Synergy analysis of AM-8621 + carfilzomib combination in U266B1 and NCI-H929 cell lines. Effects on viability were captured following treatment with a 10 × 10 dose matrix combination of AM-8621 and carfilzomib at indicated doses for 72 hours. Synergy scores are reported for the 2-way combination of AM-8621 + carfilzomib and self-cross combinations of carfilzomib + carfilzomib and AM-8621 + AM-8621, respectively.

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Supplementary Figure S12. AM-8621 exhibits synergy with standard of care therapeutics and venetoclax in AML cell lines. Synergy analysis of AM-8621 in combination with cytarabine, decitabine, doxorubicin, and ABT 199 in EOL-1 (A), GDM-1 (B), MOLM13 (C), and MV-4-11 (D) cell lines. Effects on viability were captured following treatment with a 10 × 10 dose matrix combination of AM-8621 and indicated therapies at indicated doses for 72 hours. Synergy scores are reported for the various 2-way combinations and self-cross combinations of indicated SOC + SOC and AM-8621 + AM-8621, respectively.

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Supplementary Figure S13. Treatment with venetoclax reduces tumor burden in the MOLM13 orthotopic AML model. Change in the bioluminescence of mice bearing orthotopic MOLM13 Luc xenografts following administration of vehicle or venetoclax at doses of 10, 30, or 100 mg/kg on a once-daily dosing schedule. Depicted are representative day 17 bioluminescence images.

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References for Supplement

1. Gong JN, Khong T, Segal D, Yao Y, Riffkin CD, Garnier JM, et al. Hierarchy for targeting

prosurvival BCL2 family proteins in multiple myeloma: pivotal role of MCL1. Blood

2016;128(14):1834-44 doi 10.1182/blood-2016-03-704908.

2. Garnett MJ, Edelman EJ, Heidorn SJ, Greenman CD, Dastur A, Lau KW, et al.

Systematic identification of genomic markers of drug sensitivity in cancer cells. Nature

2012;483(7391):570-5 doi 10.1038/nature11005.

3. Zou H, Hastie T. Regularization and variable selection via the elastic net. J R Statistic

Soc B 2005;67(2):301-20.

4. Saiki AY, Caenepeel S, Yu D, Lofgren JA, Osgood T, Robertson R, et al. MDM2

antagonists synergize broadly and robustly with compounds targeting fundamental

oncogenic signaling pathways. Oncotarget 2014;5(8):2030-43 doi

10.18632/oncotarget.1918.