AMG 176, a Selective MCL1 Inhibitor, is Effective in ... · 9/25/2018 · 18 mouse models, RNAi,...

1

AMG 176, a Selective MCL1 Inhibitor, is Effective in Hematological 1

Cancer Models Alone and in Combination with Established Therapies 2

Sean Caenepeel,1,10 Sean P. Brown,2,10* Brian Belmontes,1,10 Gordon Moody,1,10† 3

Kathleen S. Keegan,3,11† Danny Chui,4,10† Douglas A. Whittington,5,12† Xin Huang,5,12 4

Leszek Poppe,6,10 Alan C. Cheng,7,13† Mario Cardozo,7,13† Jonathan Houze,8,12 Yunxiao Li,9,13 5

Brian Lucas,9,13† Nick A. Paras,9,13† Xianghong Wang,9,13† Joshua P. Taygerly,9,13† 6

Marc Vimolratana,9,13† Manuel Zancanella,9,13† Liusheng Zhu,9,13† Elaina Cajulis,1,10 7

Tao Osgood,1,10 Jan Sun,1,10 Leah Damon,14 Regina K. Egan,14 Patricia Greninger,14 8

Joseph D. McClanaghan,14 Jianan Gong,15,16 Donia Moujalled,17 Giovanna Pomilio,17 9

Pedro Beltran,1,10† Cyril H. Benes,14 Andrew W. Roberts,15,16,18,19 David C. Huang,15,16 10

Andrew Wei,17 Jude Canon,1,10 Angela Coxon,1,10 Paul E. Hughes1,10* 11

1Oncology Research, Amgen Inc., Thousand Oaks, CA, USA; 2Medicinal Chemistry, Amgen 12

Inc., Thousand Oaks, CA, USA; 3Oncology Research, Amgen Inc., Seattle WA, USA; 4Genome 13

Analysis Unit, Amgen Inc., Thousand Oaks, CA, USA; 5Molecular Engineering, Amgen Inc., 14

Cambridge, MA, USA; 6Discovery Attribute Sciences, Amgen Inc., Thousand Oaks, CA, USA; 15 7Molecular Engineering, Amgen Inc., South San Francisco, CA, USA; 8Medicinal Chemistry, 16

Amgen Inc., Cambridge, MA, USA; 9Medicinal Chemistry, Amgen Inc., South San Francisco, 17

CA, USA; 10Amgen Research, Amgen Inc., Thousand Oaks, CA, USA; 11Amgen Research, 18

Amgen Inc., Seattle, WA, USA; 12Amgen Research, Amgen Inc., Cambridge, MA, USA; 19 13Amgen Research, Amgen Inc., South San Francisco, CA, USA; 14Department of Medicine, 20

Massachusetts General Hospital, Boston, MA, USA; 15The Walter and Eliza Hall Institute of 21

Medical Research, Melbourne, Australia; 16Department of Medical Biology, University of 22

Melbourne, Melbourne Australia; 17Malignant Haematology and Stem Cell Transplantation 23

Service, Alfred Hospital, Melbourne, Australia and Australian Centre for Blood Diseases, 24

Monash University, Melbourne Australia; 18Department of Clinical Haematology and Bone 25

Marrow Transplantation, The Royal Melbourne Hospital, Melbourne, Australia; 19Victorian 26

Comprehensive Cancer Centre, Parkville, Australia 27

*Correspondence to: Paul Hughes and Sean Brown, Amgen Inc., One Amgen Center Drive, 28

Thousand Oaks, CA, USA 91320-1799; Tel: P.H. 1-805-447-1137, S.B. 1-805-447-6905; Email: 29

P.H. [email protected], S.B. [email protected] 30

†Affiliation at the time the research was conducted. 31

Running title: AMG 176 alone and combined in hematologic cancer models 32

Keywords: AMG 176, MCL1, apoptosis, multiple myeloma, acute myeloid leukemia, xenograft 33

Conflicts of interest: S.C., S.B., B.B., X.H., L.P., J.H., Y.L., E.C., T.O., J.S., P.B., J.C., A.C., and 34

P.E.H. are employees of and own stock in Amgen Inc. G.M., D.A.W., and A.C.C. were 35

employed by Amgen at the time this research was conducted and still own stock in Amgen Inc. 36

K.K., D.C., M.C., B.L., N.A.P., Q.W., J.T., M.V., M.Z., and L.Z. were employed by and owned 37

stock in Amgen Inc. at the time this research was conducted. C.B. has received research grants 38

from Amgen Inc., Novartis, and Araxes Pharma. J.G., A.W.R., and D.C.S.H. are employees of 39

Research. on September 17, 2020. © 2018 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 25, 2018; DOI: 10.1158/2159-8290.CD-18-0387





http://cancerdiscovery.aacrjournals.org/



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Walter and Eliza Institute of Medical Research and have received research funding and 1

milestone and royalty payments related to venetoclax. A.H.W. has received research grants and 2

consulting fees from Amgen Inc., Novartis, Abbvie, Celgene, and Laboratoires Servier. L.J.D., 3

R.K.E., P.G., J.M., D.M., and G.P. have no conflicts to disclose. 4

Word count: 6268 5

Tables/figures: 7 6

References: 437




3

ABSTRACT 1

The prosurvival BCL-2 family member MCL1 is frequently dysregulated in cancer. To overcome 2

the significant challenges associated with inhibition of MCL1 protein-protein interactions, we 3

rigorously applied small-molecule conformational restriction, which culminated in the discovery 4

of AMG 176, the first selective MCL1 inhibitor to be studied in humans. We demonstrate that 5

MCL1 inhibition induces a rapid and committed step towards apoptosis in subsets of 6

hematological cancer cell lines, tumor xenograft models, and primary patient samples. With the 7

use of a human MCL1 knock-in mouse, we demonstrate that MCL1 inhibition at active doses of 8

AMG 176 is tolerated and correlates with clear pharmacodynamic effects, demonstrated by 9

reductions in B-cells, monocytes and neutrophils. Furthermore, the combination of AMG 176 10

and venetoclax is synergistic in AML tumor models and in primary patient samples at tolerated 11

doses. These results highlight the therapeutic promise of AMG 176 and the potential for 12

combinations with other BH3 mimetics. 13

Word count: 150 (limit, 150) 14




4

SIGNIFICANCE 1

AMG 176 is a potent, selective and orally bioavailable MCL1 inhibitor that induces a rapid 2

commitment to apoptosis in models of hematological malignancies. The synergistic combination 3

of AMG 176 and venetoclax demonstrates robust activity in models of AML at tolerated doses, 4

highlighting the promise of BH3-mimetic combinations in hematological cancers. 5




5

INTRODUCTION 1

The evasion of apoptosis is a hallmark of cancer, sustaining tumor growth, survival, and 2

resistance to a broad spectrum of anti-cancer therapeutics (1). Dysregulation of the B-cell 3

lymphoma-2 (BCL-2) family of proteins is frequently responsible for this circumvention of 4

programed cell death (2). The BCL-2 family of proteins is comprised of anti-apoptotic members, 5

including BCL-2, MCL1, and BCL-XL, and pro-apoptotic members which are further divided into 6

two groups: the BH3-only proteins, for example BIM and BAD and the downstream apoptotic 7

effectors BAK and BAX (3). The dynamic interplay between these proteins is integral to 8

controlling the apoptotic threshold of cells, with the BH3-domain of pro-apoptotic family 9

members binding to a hydrophobic groove on the surface of their anti-apoptotic counterparts (4). 10

Small molecule BH3-mimetics, the most advanced of which is the BCL-2 inhibitor venetoclax, 11

compete for binding to this groove, enabling endogenous BH3-only pro-apoptotic proteins to 12

activate downstream components of the pathway (5). 13

The impressive clinical activity of venetoclax in chronic lymphocytic leukemia has 14

provided validation for BH3-mimetics in cancer (6). However, more modest response rates in 15

multiple myeloma (MM) and acute myeloid leukemia (AML), suggest involvement of other anti-16

apoptotic family members in these settings (7,8). Studies involving genetically engineered 17

mouse models, RNAi, and CRISPR/Cas9 genome editing have implicated MCL1 in promoting 18

the survival of multiple hematological malignancies, including MM, AML and MYC driven 19

lymphomas, highlighting the potential for MCL1 as a therapeutic target in these indications (9-20

15). 21

The shallow BH3-domain binding pocket in MCL1, along with the high-affinity 22

interactions with its binding partners, have made the development of potent and selective MCL1 23

inhibitors with suitable drug-like properties challenging. However, recent success has been 24

reported with the small molecule S63845 (16). 25




6

We report the discovery of AMG 176, a first-in-class orally bioavailable MCL1 inhibitor in 1

clinical development for hematological malignancies (ClinicalTrials.gov, NCT02675452). Using 2

structure-based design, we optimized a series of spiro-macrocyclic molecules integrating 3

conformational restriction as a guiding principle throughout the optimization process (17). In 4

hematological cancer cell lines, this class of MCL1 inhibitors induced a rapid commitment 5

toward apoptosis at nanomolar concentrations following exposure to drug for as little as 30 6

minutes. Discontinuous oral administration of AMG 176 inhibited the growth of human AML and 7

MM tumor xenografts at tolerated doses. Furthermore, in contrast to published reports with 8

S63845, where little to no effect was observed on white blood cell counts following intravenous 9

administration of S63845 at a maximally tolerated dose, oral administration of AMG 176 10

resulted in dose dependent reductions in B-cells, monocytes, and neutrophils, highlighting their 11

potential as pharmacodynamic endpoints of MCL1 inhibition (16). Finally, we show that the 12

combination of AMG 176 and venetoclax was synergistic in primary AML patient samples and 13

demonstrated robust activity in AML xenograft models at tolerated doses. These data warrant 14

further evaluation of AMG 176 in the clinical setting and highlight the promise of combined 15

MCL1 and BCL-2 inhibition as a means of achieving maximal clinical benefit with BH3-mimetics 16

in hematological cancers.17




7

RESULTS 1

AMG 176 is a Potent and Selective MCL1 Inhibitor 2

A successful strategy for inhibiting protein-protein interactions (PPIs) is to identify weakly bound 3

small molecule fragments and improve their affinity by increasing their size to gain additional 4

interactions with the target protein (18). Although this approach improves affinity, it often results 5

in molecules with poor selectivity and pharmacokinetic properties, such as low oral 6

bioavailability, presumably due to their large size and high flexibility (19). Guided by x-ray 7

structure and small molecule conformational analysis, we approached the optimization of 8

inhibitors of MCL1 with the strategy of conformational restriction to reduce non-binding 9

conformations (17). Based on the hypothesis that high levels of non-binding conformations 10

increase the likelihood of poor selectivity and pharmacokinetic properties, we successfully used 11

conformational restriction as a guiding principle for the optimization of these PPI inhibitors. 12

Compound 1 was identified as a racemate (IC50=3.4 µM) from a screen of a 248,090-13

compound library for disruption of the MCL1/BIM interaction (Fig. 1A). Separation of the 14

enantiomers (2 and 3) and expansion of the central 6-membered heterocyclic ring to a 7-15

membered heterocycle provided compound 4 (IC50=0.3 µM). Co-crystallization of 4 with MCL1 16

revealed a cryptic binding pocket not present in the co-crystal structure of MCL1 when bound to 17

the BIM peptide (Fig. 1A,B,C; video; PDB validation reports 9100015620 and 9100015541; 18

Supplemental Table 1) (20). Examination of the MCL1/4 structure revealed the near co-19

planarity of the stereogenic hydrogen and the ortho-chlorine, prompting an exchange of these 20

atoms for an ethylene unit that could better fill the cryptic pocket and engender conformational 21

restriction by constraining the rotation of the aryl ring (Fig. 1A). Gratifyingly, spirocycle 5 22

showed improved biochemical potency (IC50=0.04 µM). In an effort to maintain the ionic 23

interaction between the carboxylic acid of 2 and Arg 263 of MCL1 while introducing an 24




8

additional vector for derivatization, compound 6 was synthesized. An NMR structure of 6 1

complexed with MCL1 showed a binding conformation of the benzyl acyl sulfonamide where the 2

phenyl ring was in close proximity to the four-membered ring suggesting conformational 3

restriction could again be employed to form a macrocyclic ring (PDB validation report 4

9100015681). Combination of the conformationally restricted macrocyclic and spirocyclic rings 5

provided the high-affinity MCL1 inhibitor, compound 7 (IC50=0.01 µM). The binding 6

conformations of compounds 5 and 7, when complexed with MCL1, suggested fusion of a trans-7

cyclobutane onto the macrocycle would generate additional hydrophobic contacts and further 8

conformationally restrict the macrocycle (PDB validation reports 9100015557 and 9

9100015544; Supplementary Table S1). Following this hypothesis resulted in the synthesis of 10

compound 8, which showed a significant increase in potency (Ki=0.00014 µM). The x-ray crystal 11

structure of compound 8 complexed with MCL1 along with molecular modeling revealed that the 12

conformation of 8 observed in complex with MCL1 was the fifth most populated conformation, 13

representing only 12% of the total conformational ensemble (Fig. 1D; PDB validation report 14

9100015543; Supplementary Table S1). Analysis of 8 provided an opportunity to further 15

conformationally restrict the macrocycle by installation of a trans-olefin between carbons 7 and 16

8, yielding compounds 9 and 10 (Ki=0.00004 µM and 0.00005 µM, respectively). Compound 10 17

(AM-8621), had a suitable potency and selectivity profile to serve as a tool MCL1 inhibitor for 18

characterizing the mechanism of action of MCL1 inhibition in vitro. The improvement in potency 19

of compound 9 was presumably due to conformational restriction, where now the observed 20

conformation of 9 in complex with MCL1 was the most abundant conformation (45%; Fig. 1E; 21

PDB validation report 9100015542). Moreover, installation of the olefin reduced the number of 22

energetically accessible conformations within 3 kcal/mol from 22 (compound 8) to 8 (compound 23

9). Compounds 9 and 10 had short half-lives and poor oral bioavailability (Fig. 1F). Simple 24

methylation of the alcohol led to a series of compounds with improved pharmacokinetic profiles, 25

including increased oral bioavailability, which ultimately provided compound 11 or AMG 176, the 26




9

first selective, orally bioavailable MCL1 inhibitor to advance into human clinical trials, validating 1

our strategy of optimization guided by conformational restriction (video; ClinicalTrials.gov, 2

NCT02675452). 3

Disruption of MCL1 Interactions Induces Apoptosis 4

AMG 176 and the related analog AM-8621 exhibit picomolar affinity for human MCL1, ~1000-5

fold reduced affinity towards murine MCL1 and minimal binding affinity towards BCL-2, and 6

BCL-XL (Fig. 1A). The ability of the tool compound, AM-8621, to disrupt the interaction between 7

MCL1 and BAK was evaluated in HEK293M cells using a split-luciferase complementation 8

assay, (IC50, 43 nM; Fig. 2A) (21). AM-8621 also disrupted the interaction between MCL1 and 9

BIM in a co-complex immunoassay, exhibiting dose- and time-dependent inhibition in the 10

NSCLC cell line A427 (Fig. 2B). A427 cells were selected for these studies because of their 11

appreciable MCL1:BIM co-complex levels and relative insensitivity to AM-8621 treatment. 12

We next evaluated the effect of AM-8621 on MCL1 protein levels (22). The AM-8621 13

insensitive MM cell line, U266B1, was selected for these experiments so that changes in MCL1 14

protein levels would not be influenced by treatment-induced effects on cell viability. A dose-15

dependent induction of MCL1 protein was observed following compound treatment (Fig. 2C). 16

Although independent of changes in transcription (Supplementary Fig. S1), this increase was 17

at least partially driven by an extended protein half-life (Fig 2D). Consistent with rapid target 18

engagement, elevated MCL1 protein was detected within 15 minutes of treatment initiation. 19

MCL1 levels returned to baseline 4 hours after washout of AM-8621, confirming the reversibility 20

of compound binding (Fig. 2E). This induction of MCL1 protein following AM-8621 treatment 21

was not limited to U266B1 cells; similar observations were made in several additional cell lines 22

(including A427, MV-4-11, and NCI-H1568; Supplementary Fig. S2). 23

AM-8621 was then tested for its ability to activate the intrinsic apoptosis pathway. 24

Treatment with AM-8621 increased activated BAK levels, a proximal downstream effector of 25




10

MCL1, in a panel of AM-8621–sensitive cell lines (OPM-2, MM; MV-4-11, AML; MOLM13, AML; 1

and Ramos, Burkitt’s lymphoma) but not in the AM-8621–insensitive cell line U266B1 (MM; Fig. 2

3A). Together with the liberation of BH3-domain-containing proteins, BAK activation represents 3

a key prelude to the induction of downstream components of the intrinsic apoptosis pathway, 4

including Caspase 3 and 7 (5). Highly AM-8621–sensitive cell lines (OPM-2, MV-4-11, 5

MOLM13, and Ramos) exhibited Caspase 3 and 7 activation within 1–4 hours of treatment. 6

Reductions in cell viability were observed shortly thereafter, demonstrating a near maximal 7

response within 8 hours of treatment initiation (Fig. 3B). AM-8621 washout studies revealed a 8

further dependence on MCL1 in these lines. Although a limited effect on OPM-2 viability was 9

observed 1 hour after treatment initiation, subsequent washout of AM-8621, followed by 10

incubation in the absence of drug for 23 hours, resulted in >80% reduction in viability 11

approaching that achieved with 24 hours of continuous treatment. Similar observations were 12

made in Ramos cells following 30 minutes of treatment, and to a lesser extent, in MV-4-11 and 13

MOLM13 cells following 2 hours of treatment (Fig. 3C). To confirm the on mechanism activity of 14

this class of MCL1 inhibitors, BAX-/-BAK-/- AMO1, H929, and OPM-2 cells were treated with AM-15

8621 and compared with parental cell lines (9). In contrast to the parental cell lines, no effect on 16

viability was observed with any of the BAX-/-BAK-/- lines, providing compelling evidence for the 17

on target MCL1 mediated activity of these compounds (Fig. 3D). 18

Hematological Cancer Cell Lines Are Sensitive to MCL1 Inhibition 19

To identify those tumor types with greatest sensitivity to MCL1 inhibition, AM-8621 was profiled 20

against a panel of 952 tumor cell lines (23) (Fig. 4A). Cell lines derived from hematological 21

malignancies exhibited greater sensitivity to AM-8621 than did solid tumor lines (P<1×10−13, 22

Fisher Exact Test). The hematological indications exhibiting greatest sensitivity included MM 23

(p=0.006), AML (p=1×10−5), and B-cell lymphoma (p<1×10−6), with subsets of ALL and Burkitt’s 24

lymphoma also exhibiting dependency on MCL1 (Fig. 4B, Supplementary Table S2). Among 25




11

solid tumor cell lines, breast cancer lines demonstrated the greatest sensitivity to AM-8621 1

(p=0.001). 2

To understand the dependency of hematological cancer cell lines on specific pro-survival 3

BCL-2 family members, expanded panels of MM, AML, and DLBCL cell lines were assessed for 4

sensitivity to AM-8621 and the BCL-2 selective inhibitor venetoclax. In agreement with results 5

from the initial screen, dependency on MCL1 was observed in a subset of cell lines (Fig. 4C). 6

Most notably, MM cell lines relied predominantly on MCL1 for survival, whereas AML and 7

DLBCL cell lines were more heterogeneous, exhibiting sensitivity to selective inhibition of MCL1, 8

BCL-2, or both (Fig. 4D). 9

Low BCL-XL and High BAK Expression Predict for Sensitivity to MCL1 Inhibition 10

To identify predictive biomarkers of response to AM-8621, we assessed the relationship 11

between sensitivity and genomic features within the 952 tumor cell lines. A multivariate linear 12

regression analysis (elastic net) was used to capture determinants of response to AM-8621 13

(23). Among all genomic features included in the model, expression or copy number variation of 14

a small number of genes (n=165) was predictive of response across all cell lines 15

(Supplementary Table S3). Strikingly, high BCL-XL expression was the strongest predictor of 16

resistance, while conversely, high expression of BAK was the strongest predictor of sensitivity. 17

MCL1 expression was not predictive. The identification of BCL-XL expression as a resistance 18

feature was confirmed by the strong correlations observed between AM-8621 sensitivity and 19

BCL-XL transcript and protein expression in the expanded panel of MM cell lines, with high BCL-20

XL expression again being associated with resistance to AM-8621 (Fig. 4E, Supplementary 21

Fig. S3). The identification of BCL-XL and BAK, both members of the BCL-2 protein family, as 22

the strongest predictors of resistance/response provides further evidence supporting the on 23

mechanism activity of AM-8621. 24




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AMG 176 is Efficacious in MM and AML xenograft models and Well Tolerated in Human 1

MCL1 Knock-in Mice 2

To characterize the kinetics of apoptosis induction in vivo, we used the clinical-stage molecule 3

AMG 176 given its superior pharmacokinetic properties over AM-8621. Dose-dependent 4

activation of the intrinsic apoptosis pathway in OPM-2 xenografts, as measured by activated 5

BAK, cleaved Caspase 3, and cleaved Poly (ADP-ribose) polymerase (PARP), was detected as 6

early as 2 hours after oral administration of AMG 176 (Fig. 5A), with sustained cleaved PARP 7

and activated BAK detectable through 12 hours and cleaved Caspase 3 through 24 hours. 8

Immunohistochemistry analysis revealed a similar dose-dependent increase in cleaved 9

Caspase 3 (Supplementary Figure S4). 10

The ability of AMG 176 to rapidly induce apoptosis in pharmacodynamic assays 11

suggested that it may be efficacious when administered using a discontinuous schedule. To test 12

this hypothesis, mice harboring subcutaneous OPM-2 xenografts were treated with AMG 176 13

twice-weekly and compared with mice treated daily. Discontinuous doses of 30 and 60 mg/kg 14

were selected based on their ability to induce apoptotic markers in pharmacodynamic assays. 15

Twice-weekly oral administration of AMG 176 at a dose of 30 mg/kg achieved 54% tumor 16

growth inhibition (TGI) relative to vehicle, while a dose of 60 mg/kg achieved 21% tumor 17

regression relative to initial tumor volume. Daily administration of AMG 176 achieved 84% TGI 18

and 100% regression, respectively (Fig. 5B). Similarly, once-weekly oral administration of 19

AMG 176 at doses of 50 and 100 mg/kg achieved 97% TGI and 70% regression, respectively 20

(Fig. 5C). We next assessed the activity of AMG 176 in the MOLM13 luciferase-labeled 21

orthotopic model of AML, in which tumor cells have engrafted in the bone marrow of mice. 22

Twice-weekly oral administration of AMG 176 at 30 or 60 mg/kg resulted in significant dose-23

dependent inhibition of tumor burden as assessed by whole body luminescence (28% and 69% 24

reduction in bioluminescence imaging [BLI], respectively; Fig. 5D). 25




13

Several conditional knock-out studies have documented the dependency of B-cells, 1

monocytes, neutrophils and/or their progenitors on MCL1 for survival, suggesting these cell 2

types may serve as pharmacodynamic markers of MCL1 inhibition (24-27). Furthermore, the 3

failure of AMG 176 to inhibit mouse MCL1 limits our understanding of the relationship between 4

tolerability and effects on normal cell types at efficacious doses. To further investigate these 5

relationships, we generated a human MCL1 knock-in mouse, replacing the Mcl1 gene with its 6

human ortholog (Supplementary Fig. S5). Ex-vivo treatment of splenocytes with AM-8621 for 6 7

hours resulted in Caspase 3 activation and reduced viability in B-cells derived from human 8

MCL1 knock-in, but not wild-type, mice (Supplementary Fig. S6). These effects translated in 9

vivo where oral administration of AMG 176 at 30 and 60 mg/kg resulted in dose-dependent 10

decreases in B-cells, monocytes and neutrophils in the blood (Fig. 5E; Supplementary Fig. 11

S7). Similar reductions were observed in bone marrow, highlighting the ability of AMG 176 to 12

distribute into tissues. Broader complete blood count (CBC) analysis revealed additional effects 13

on eosinophils, basophils, and reticulocytes (Supplementary Fig. S8). No evidence of overt 14

systemic toxicity was observed in either AMG 176 treatment group as determined by changes in 15

body weight (Supplementary Fig. S9). 16

AMG 176 and AM-8621 Exhibit Activity in Combination with Clinically Relevant Agents 17

That Target Hematological Malignancies 18

Improved long-term survival in MM has been achieved through combination therapy with 19

proteasome inhibitors (PI), immunomodulatory drugs (IMiDs), and corticosteroids (eg, 20

dexamethasone) (28). To investigate the opportunity for combining an MCL1 inhibitor with 21

corticosteroids, we evaluated the effect of AM-8621 combined with dexamethasone in a panel of 22

MM cell lines (OPM-2, KMS-11, MM.1S) (29). AM-8621 further potentiated the cytotoxic effects 23

observed with dexamethasone alone, exhibiting a synergistic interaction in each line 24

(Supplementary Fig. S10). We also evaluated the synergistic potential of AM-8621 combined 25




14

with the PI carfilzomib. Although a limited synergistic interaction was detected in U266B1 cells, 1

the profound single agent activity of AM-8621 and carfilzomib in other tested cell lines limited 2

the opportunity for detecting synergy (Supplementary Fig. S11). To further elucidate the 3

therapeutic potential of this combination, we tested AMG 176 and carfilzomib in an orthotopic 4

OPM-2 luciferase-labeled model. In mice treated once-daily with AMG 176 (20 mg/kg) and 5

twice-weekly with carfilzomib (3 mg/kg), the combination achieved significant inhibition of tumor 6

burden (99% reduction in BLI), exceeding the effect achieved with either single agent (85% and 7

82% reduction in BLI with AMG 176 and carfilzomib, respectively; Fig. 6A). 8

Combination therapy is a mainstay of AML treatment regimens as well, with induction 9

therapy including nucleoside analogs, (eg, cytarabine), hypomethylating agents (eg, decitabine), 10

and anthracyclines (eg, doxorubicin) (30). Our observation that AML cell lines exhibit a range of 11

sensitivities to AM-8621 (Fig. 4C) suggested that combined therapy with standard-of-care 12

(SOC) agents may further sensitize AML tumors to treatment with an MCL1 inhibitor. Thus, we 13

characterized the effects of AM-8621 combined with cytarabine, decitabine, and doxorubicin on 14

four AML cell lines (EOL-1, GDM-1, MOLM13, and MV-4-11). A synergistic interaction was 15

observed with all three combinations across the cell line panel (Fig. 6B, Supplementary Fig. 16

S12), highlighting the potential for combining MCL1 inhibitors with SOC agents in AML. 17

Cell line profiling studies with AM-8621 and venetoclax demonstrated sensitivity to either 18

MCL1 inhibition alone, BCL-2 inhibition alone, or both MCL1 and BCL-2 inhibition in many AML 19

lines (Fig. 4D), suggesting that combination therapy may provide benefit beyond selective 20

inhibition of either protein alone. To test this hypothesis, we profiled the same panel of four AML 21

cell lines with the combination of AM-8621 and venetoclax. A synergistic interaction was 22

detected in each cell line (Fig. 6B, Supplementary Fig. S12), highlighting their codependence 23

on MCL1 and BCL-2. Next, we tested the combination of AMG 176 and venetoclax in the 24

MOLM13 orthotopic model. Single-agent dose-finding studies revealed significant activity with 25

both compounds (Fig. 5D and Supplementary Fig. S13) and informed dose selection for use in 26




15

combination. Mice harboring MOLM13 tumors were treated twice weekly with AMG 176 (30 1

mg/kg) and daily with venetoclax (50 mg/kg). Whereas both single agents achieved significant 2

reductions in tumor burden (55% and 23% reduction in BLI respectively; Fig. 6C), the 3

combination exhibited complete inhibition of tumor burden (100% reduction in BLI) and achieved 4

regression relative to the first day of dosing. 5

We next sought to characterize the effects of this combination on subsets of 6

hematopoietic cells in the human MCL1 knock-in mouse. The treatment schedule and dose for 7

AMG 176 (twice-weekly at 30 mg/kg) and venetoclax (daily at 50 mg/kg) were selected based 8

on observed efficacy in the MOLM13 model. Terminal analysis (24 hour post cycle 2 or day 10) 9

of mice treated with the combination or AMG 176 alone showed significant decreases in 10

peripheral blood B-cells and monocytes, whereas venetoclax alone exhibited significant 11

reductions in B-cells only (Fig. 6D). The combination was well tolerated and no evidence of 12

overt toxicity was observed as determined by changes in body weight at the doses selected for 13

this study (Fig. 6E). 14

AM-8621 was next tested in a panel of primary AML patient samples (Supplementary 15

Table S4). Most of the samples exhibited some degree of sensitivity to AM-8621 treatment, 16

while a subset (AML 9, AML 10, and AML 13) were profoundly sensitive (LC50 ≤2 nM; Fig. 7A, 17

7B). Given the synergy observed with AM-8621 when combined with venetoclax and SOC 18

chemotherapeutics in AML cell lines, we evaluated these combinations in the primary patient 19

samples. Strikingly, in 9 of 13 samples, combination of equimolar concentrations of AM-8621 20

and venetoclax achieved marked improvements in activity and potency over either single agent 21

alone, while only one sample was relatively insensitive to the combination (AML 11) (Fig. 7A, 22

7B). Combination with Idarubicin also exhibited improvement in potency in 6 of 10 samples 23

tested, with one sample (AML 2) exhibiting a >1000-fold improvement. It is important to note 24

that these experiments were conducted in the absence of stromal cells and factors, which are 25




16

thought to provide a supportive environment for leukemic blasts. Consequently, it should be 1

noted that the absence of these factors may further sensitize the cells to treatment.2




17

DISCUSSION 1

MCL1 is a compelling therapeutic target in cancer. Studies using genetic knockdown and 2

pharmacological inhibition have demonstrated MCL1’s role as a critical pro-survival factor in 3

many tumor types, including MM, AML, B-cell lymphomas, and breast cancer (9,11-14,31,32). 4

Furthermore, MCL1 has been implicated as a resistance factor to BH3-mimetics targeting BCL-5

2 and BCL-XL (33), as well as chemotherapeutic agents (34), further underscoring the 6

therapeutic potential of inhibitors targeting this pro-survival BCL-2 family member. 7

We describe the discovery of AMG 176, a first-in-class MCL1 inhibitor in clinical 8

development for hematological malignancies. The development of potent and selective MCL1 9

inhibitors has been challenging due to the high affinity of its native ligands and shallow binding 10

pocket. The discovery of AMG 176 represents an innovative approach for overcoming these 11

significant obstacles. Driving potency, selectivity, and pharmacokinetic properties through 12

conformational restriction guided by structure-based design has yielded a series of rigid 13

macrocyclic inhibitors that primarily displayed the bioactive conformation. The identification of 14

AMG 176 illustrates that conformational restriction provides an effective approach to small 15

molecule design with potential application for targets previously considered undruggable (17). 16

Tumor cell line profiling studies with the tool compound, AM-8621, implicated a key role 17

for MCL1 in cell lines derived from hematological malignancies, including MM, AML, and B-cell 18

lymphoma. Whereas AML and DLBCL lines frequently exhibited co-dependence on MCL1 and 19

BCL-2, MM lines showed a predominant dependency on MCL1, highlighting the promise for 20

MCL1 inhibitors in this setting. Consistent with prior reports (16,35), elevated BCL-XL expression 21

was identified as the strongest predictor of resistance. Additionally, the expression of BAK was 22

found to be the strongest predictor of sensitivity. These findings are not unexpected considering 23

the redundant pro-survival role of BCL-XL and the function of BAK as the key executioner 24




18

protein for MCL1. Given the feasibility of measuring these endpoints in the clinic, both BCL-XL 1

and BAK may serve as biomarkers for patient stratification and shed light on potential 2

mechanisms of resistance to MCL1 inhibitors in the clinic. 3

A notable feature of this class of MCL1 inhibitors is their rapid induction of apoptosis in 4

hematological cancer cell lines, providing clear rationale for testing the clinical molecule AMG 5

176 with discontinuous dosing strategies. In vivo, robust tumor growth inhibition was observed 6

with once- and twice-weekly dosing schedules. These findings have important implications for 7

the clinical development of AMG 176, as they offer the promise of greater flexibility in dosing 8

schedules to mitigate potential on-target toxicities without sacrificing efficacy. 9

Given the lack of activity of AMG 176 on murine MCL1, we generated a human MCL1 10

knock-in mouse to facilitate an improved understanding of the relationship between MCL1 11

inhibition at efficacious exposures and its effects on normal tissues. In contrast to published 12

MCL1 gene ablation studies where MCL1 knockout resulted in lethality, inhibition of MCL1 at 13

efficacious and pharmacodynamically active doses of AMG 176 was tolerated in the knock-in 14

mice (36,37). Additionally, for the first time we have demonstrated an effect on normal tissues 15

with an MCL1 inhibitor at tolerated and efficacious exposures. Consistent with data from 16

conditional MCL1 gene ablation studies, AMG 176 treatment significantly reduced B-cells, 17

monocytes and neutrophils at exposures required for tumor growth inhibition (24-27). These 18

data suggest that reductions in these cell types may serve as pharmacodynamic endpoints. 19

The co-dependency of many cell lines on MCL1 and BCL-2 suggests that their combined 20

inhibition has the potential for improved efficacy in indications such as AML. This hypothesis 21

was supported by the robust activity observed with the combinations of AM-8621/AMG 176 and 22

venetoclax in AML cell lines, xenograft models, and primary patient samples. We also utilized 23

human MCL1 knock-in mice to test the relationship between the tolerability of this combination 24

and effects on pharmacodynamic endpoints. Administration of venetoclax and AMG 176 on a 25

schedule achieving continuous BCL-2 and intermittent MCL1 inhibition was well-tolerated while 26




19

demonstrating significant reductions in B-cells and monocytes. Given this compelling data, 1

combined treatment with AMG 176 and venetoclax has promising therapeutic potential in AML. 2

The dependency of a broad range of hematopoietic tumor types on MCL1 for survival 3

highlights the exciting therapeutic promise for MCL1 inhibitors. AMG 176 is the first MCL1 4

inhibitor to enter clinical development and has the potential to significantly expand the clinical 5

opportunity for BH3-mimetics in the treatment of cancer.6




20

MATERIALS AND METHODS 1

Clinical Candidate Synthesis 2

AMG 176 (1S,3'R,6'R,7'S,8'E,11'S,12'R)-6-chloro-7'-methoxy-11',12'-dimethyl-3,4-dyhydro-3

2H,15'H-spiro[naphthalene-1,22'-[20]oxa[13]thia[1,14]diazatetracyclo[14.7.2.03,6.019,24]pentac4

osa[8,16,18,24]tetraen]-15'-one-13', 13'-dioxide) and other small-molecule inhibitors of MCL1 5

described were synthesized at Amgen Inc (38). 6

Conformational Energy Calculations 7

Details of conformational energy calculations are described in the Supplemental Information. 8

Time-Resolved Fluorescence Resonance Energy Transfer Binding Assays 9

Recombinant 6XHis-tagged human MCL1 (171-327), dog MCL1 (171-327, C286S), mouse 10

MCL1 (152-308), and BCL-XL (1-196) were produced at Amgen Inc. Proteins were expressed in 11

E. coli. and purified using metal ion affinity chromatography and size-exclusion 12

chromatography. Recombinant 10XHis-tagged human BCL-2 (2-211) was purchased from R&D 13

Systems. Human Biotin-Bim BH3 peptide (Biotin-DMRPEIWIAQELRRIGDEFNAYYARR) and 14

mouse Biotin-Bim BH3 peptide (Biotin-DLRPEIRIAQELRRIGDEFNETYTRR) were custom 15

synthesized by CPC Scientific. 16

Inhibition of the interaction between Biotin-Bim BH3 peptide and MCL1, BCL-2, or BCL-17

XL was measured using time-resolved fluorescence resonance energy transfer (TR-FRET) 18

assays conducted in 384-well white OptiPlates (PerkinElmer) with a total volume of 40 µL/well in 19

binding buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.016 mM Brij35, and 1 mM DTT). 20

Serially diluted test compounds were preincubated with Biotin-Bim BH3 peptide and protein 21

(MCL1, BCL-2, or BCL-XL) for 60 minutes before addition of the detection mixture (LANCE® Eu-22

W1024 Anti-6xHis [PerkinElmer] and Streptavidin-XLent [Cisbio US]). Plates were further 23




21

incubated overnight and then read on an EnVision™ Multilabel Reader (PerkinElmer). See 1

Supplemental Information. 2

Fluorescence Polarization Binding Assay 3

A fluorescence polarization (FP) assay was used to measure inhibition of the human MCL1/BIM 4

interaction. Human MCL1 (171-327) was prepared as described above. TAMRA-labeled BIM 5

BH3 peptide (TAMRA-GWIAQELRRIGDEF) was produced internally using standard Fmoc 6

chemistry. The FP binding assay was conducted in 384-well black OptiPlates with a total 7

volume of 40 µL/well in the same binding buffer used in the TR-FRET assay. Serially diluted test 8

compounds were incubated with 13.2 nM human MCL1 and 5 nM TAMRA-labeled BIM BH3 9

peptide for 120 minutes before fluorescence polarization was measured on an EnVision™ 10

Multilabel Reader with excitation and emission filters at 531 nm and 595 nm, respectively, and a 11

dual 555/595 nm dichroic mirror. IC50 values were determined as described in the TR-FRET 12

methods (see Supplemental Information). 13

Mcl-1 Crystallography and Structure Determination 14

Human MCL1 (171-327) with a cleavable N-terminal GST tag was expressed in E. coli and 15

purified using glutathione affinity chromatography. The N-terminal GST tag was cleaved by 16

thrombin. Untagged MCL1 was purified by cation exchange chromatography. Purified MCL1 (10 17

mg/ml) was incubated with 3-fold excess amount of inhibitor at 4°C for 30 minutes before 18

crystallization. Crystals of MCL1 with inhibitors were obtained at 4°C in hanging drops with 100 19

mM Tris pH 8.0, 3% Methanol, 30–42.5% PEG6000. Paratone-N mineral oil was used as 20

cryoprotectant. Diffraction data were collected on beamline 21-ID-F at the Advanced Photon 21

Source and processed and scaled with HKL 2000. The co-crystal structures were solved by 22

molecular replacement with AMoRe using PDB entry code 2PQK as the template. Model 23

building was performed with COOT with REFMAC refinement. 24




22

NMR and ITC 1

Due to the limited solubility of 1, the isothermal titration calimetry (ITC) with the VP-ITC 2

instrument (Malvern, Inc.) was performed in the “reverse” mode, where the protein (100 µM) 3

was titrated into the low concentration (7 µM) of the ligand. NMR experiments were performed 4

using a Bruker 800 MHz spectrometer equipped with the TCI cryoprobe and U-13C,15N - hMCL1 5

C893S protein (39-41). Spectral assignments of key residues in the hMCL1:2 complex were 6

based on the standard HSQC-NOESY experiment, performed on a sample prepared as 1:1 7

mixture of hMCL1:2 complex and apo-hMCL1, where the letter was previously assigned. This 8

experiment was performed at 310K to enhance chemical-exchange rates (up to 2 s-1) between 9

free and bound protein forms. The intraligand and ligand to protein nuclear Overhauser effects 10

(NOEs) were obtained from the 2D X-filtered NOE spectroscopy (NOESY) and {3,2}-X–filtered 11

NOESY with heteronuclear single quantum coherence spectroscopy (NOESY-HSQC) as 12

described previously (41). The 3D structure of the hMCL1:2 complex was obtained by the “NOE 13

guided” docking of 2 to the whole NMR ensemble (20 structures) of the apo-hMCL1. 14

Computations were performed with in-house developed scripts in Matlab; the final structure was 15

energy-minimized in Molecular Operating Environment software. 16

Cell Lines 17

Tumor cell lines were obtained from commercially available sources including American Type 18

Culture Collection (ATCC), Japanese Collection of Research Bioresources (JCRB), and 19

German Collection of Microorganisms and Cell Cultures (DSMZ). Cell lines were passaged for 20

<1 month before banking and experimentation. With the exception of the 952 cell line profiling 21

screen, all cell lines were cultured in ATCC, DSMZ, or JCRB recommended growth media 22

containing 10% fetal bovine serum, except where specified. Growth media and culture 23

conditions for the 952 cell line profiling screen were described previously (23). Authentication of 24




23

cell lines was performed with short-tandem repeat DNA typing. Using an RT-PCR–based assay, 1

all cell lines used for in vivo studies were tested for mycoplasma contamination before use; cell 2

lines used in in vitro studies were tested periodically. 3

Split Luciferase Complementation Assay 4

HEK293M cells were transiently transfected with pcDNA mammalian expression vectors 5

encoding amino acids 1–298 of firefly luciferase fused to human BAK (pcDNA-Luc [1–298] – 6

Bak) and amino acids 395–550 of firefly luciferase fused to human MCL1 (pcDNA-Luc [395-550] 7

– MCL1) at a ratio of 3:1, respectively. Transient transfection was performed using 8

Lipofectamine® LTX and PLUS™ reagent (ThermoFisher). Twenty four hours after transfection, 9

cells were collected using non-enzyme based cell dissociation buffer Accutase® (Innovative Cell 10

Technologies) and resuspended in Opti-MEM™ (ThermoFisher) without serum. Cells were 11

seeded in 96-well assay plates (5,000 cells/well) and treated with AM-8621 for 4 hours. 12

Following compound treatment, 30 µL of Steady-Glo® Luciferase detection reagent (Promega) 13

was added to each well. Signal was read on an EnVision™ Multilabel Reader. Peak luciferase 14

signal in DMSO treated wells was normalized to POC = 100. Luciferase signal from no cell 15

control wells was normalized to POC = 0. 16

Immunoblot Analysis 17

Details of the immunoblot experiments are described in the Supplemental Information. 18

qPCR Analysis of MCL1 Transcript 19

U266B1 cells were seeded at a density of 3 x 106 cells/10 cm tissue culture dish and incubated 20

overnight at 37°C in 5% CO2. Cells were treated with indicated concentrations of AM-8621 for 21

24 hours followed by wash with PBS. RNeasy® Mini Kits (Qiagen) were used to isolate RNA 22

from cells per the manufacturer’s protocol. MCL1 transcript levels were measured with the 23




24

TaqMan® One-Step RT-PCR Master Mix Reagents Kit and TaqMan® Gene Expression Assays 1

(ThermoFisher). Quantitative real-time polymerase chain reactions (qRT-PCR) were run as four 2

technical replicates and assayed using the Prism® 7900HT (Applied Biosystems), applying the 3

relative quantification (ΔΔCt) method. Data were analyzed with SDS2.3, RQ Manager v1.2, and 4

Data Assist v3.01 software (Applied Biosystems), using glyceraldehyde 3-phosphate 5

dehydrogenase as the endogenous control. 6

MCL1 Half-Life Experiments 7

U266B1 cells were seeded at a density of 1.5 x 106 cells/well in 6-well tissue culture plates and 8

incubated overnight at 37°C and 5% CO2. Cells were pretreated with DMSO or AM-8621 at a 9

concentration of 2 µM for 4 hours. Cyclohexamide was then added at a final concentration of 10

100 µg/mL to arrest bulk translation. Cell lysates were harvested at indicated time points post 11

cyclohexamide addition and subjected to immunoblot analysis. 12

Caspase 3/7 Activity Assay 13

Cells were seeded at optimized densities in 96-well tissue culture plates, incubated overnight at 14

37°C in 5% CO2, and treated with AM-8621 at indicated concentrations for 1, 2, 4, 8, or 24 15

hours. Plates were equilibrated to room temperature (RT) for 30 minutes before the addition of 16

reconstituted Caspase-Glo® 3/7 reagent (Promega). Plates were shaken for 2 minutes at RT 17

followed by incubation for 30 additional minutes without shaking. Luminescence was read on an 18

EnVision™ Multilabel Reader. 19

AM-8621 Washout Viability Studies 20

Cells were seeded at optimized densities in 96-well tissue culture plates, incubated overnight at 21

37°C in 5% CO2, and treated with a 9-point serial dilution of AM-8621, using a top concentration 22

of 6.7 µM, 1:3 serial dilution steps, and a DMSO-only control. Following compound treatment for 23




25

indicated durations, cells were washed four times and returned to growth media in the absence 1

of compound for the duration of the 24-hour experiment. Effects on cell viability were measured 2

with the CellTiter-Glo® viability assay (Promega) as follows. Treated cells and CellTiter-Glo 3

Luminescent Cell Viability Assay reagents (Promega) were allowed to equilibrate to RT, and 4

100 µL aliquots of reconstituted CellTiter-Glo reagent were added to each well of AMG-8621–5

treated cells. Assay plates were shaken for 2 minutes followed by incubation at RT for 10 6

minutes. Plates were then read on an EnVision™ Multilabel Reader. 7

Tumor Cell Line Profiling Screens 8

Details of the tumor cell line profiling experiments are described in the Supplemental 9

Information. 10

Elastic Net Model 11

Details of the elastic net model are described in the Supplemental Information. 12

BCL-XL Immunoassay 13

Lysates from indicated MM cell lines were prepared in MSD lysis buffer. Custom BCL-XL 14

capture plates were developed using a total BCL-XL antibody (R&D Systems, 840767). Capture 15

plates were blocked in MSD blocking solution A for 1 hour at RT with shaking. Plates were then 16

washed 3 times, followed by the addition of 20 µg/well of lysate. Lysates were incubated with 17

shaking for 1 hour at RT. Plates were then washed 3 times followed by the addition of a BCL-XL 18

detection antibody (Cell Signaling Technologies, 2746). Plates were incubated at RT for 1 hour 19

with shaking. Plates were then washed 3 times followed by the addition of MSD read buffer. 20

Plates were read of an MSD SI6000 plate reader. To obtain BCL-XL protein levels for individual 21

MM cell lines a standard curve of AGS (a MM cell line with high BCL-XL expression) lysate was 22




26

run on each plate to ensure lysates from individual MM cell lines fell within the linear range of 1

the assay. Relative BCL-XL protein levels were then calculated using the AGS standard curve. 2

MCL1:BIM Complex Immunoassay 3

Details of the immunoassay are reported in the Supplemental Information. 4

In Vitro Combination Studies 5

In vitro combination studies were carried out as previously described (29); additional details are 6

described in the Supplemental Information. 7

Generation of Human MCL1 Knock-In Mouse 8

Human MCL1 knock-in (KI) mice were created by targeting C57Bl/6 embryonic stem cells (The 9

Jackson Laboratory) with a targeting vector containing the full human MCL1 genomic locus 10

flanked by homologous mouse sequences upstream and downstream of the mouse MCL1 11

genomic locus. Additional detail is included in the Supplemental Information. 12

Ex Vivo Analysis of Mouse Splenocytes 13

Details described in Supplemental Information. 14

Flow Cytometry Based BAK Activation Assay 15


Pharmacodynamic Evaluation of Active BAK, Cleaved PARP and Cleaved Caspase 3 in 17

Subcutaneous Human Tumor Models 18

Pharmacodynamic methods are described in Supplemental Information. 19

Immunohistochemistry 20




27

Immunohistochemistry experiments were performed as described in the Supplemental 1

Information. 2

Subcutaneous Human Tumor Models 3

Multiple myeloma cells (cell line OPM-2 luc) were injected SC in the right flank of mice (5 x 106 4

cells). Tumor volume (mm3) was measured using electronic calipers twice per week. Once 5

tumors reached an average of approximately 150 mm3, animals were randomized into groups 6

(n=10 per group) such that the average tumor volume at the beginning of treatment 7

administration was uniform across treatment groups. Animals were then orally administered with 8

AMG 176 daily, 2x/week, or 1x/week. Clinical signs, body weight changes, and tumor growth 9

were measured 2x/week until study termination. 10

Orthotopic Human Tumor Models 11

Firefly luciferase labeled OPM-2 or MOLM13 cells were injected IV into the tail vein of 12

NOD/SCID IL2rg or athymic nude mice, respectively. Tumor bioluminescence (BLI) was 13

measured using Xenogen IVIS® 200 twice per week. Once tumors reached an average BLI of 1 14

x 105 photons/second, animals were randomized into groups (n=10 per group) such that the 15

average BLI at the beginning of treatment administration was uniform across treatment groups. 16

Animals were then orally administered AMG 176 daily for carfilzomib combination studies and 17

2x/week for venetoclax combination studies. Carfilzomib was administered IV 2x/week for 6 18

doses, beginning on the same day at the same time as AMG 176. Venetoclax was administered 19

orally every day, beginning on the same day, four hours post AMG 176 administration. Clinical 20

signs, body weight changes, and tumor BLI were measured 2 times per week until study 21

termination. 22




28

Flow Cytometry Analysis of Monocytes and B Cells From Human MCL1 Knock-In Mice 1


Complete Blood Count Analysis 3


Statistical Analysis of In Vivo Studies 5

Single-agent in vivo efficacy data were analyzed by RMANOVA followed by Dunnett’s 6

correction. Combination in vivo efficacy data were analyzed by repetitive two group RMANOVA 7

analyses between the combination group and each of the relevant single-agent controls. One-8

way ANOVA with Dunnett’s correction was applied for analysis of flow cytometry data. 9

Animal Care 10

Cages were changed once per week. Harlan Teklad Sterilizable Rodent Diet 8656 and reverse-11

osmosis water from the Amgen water supply system were supplied ad libitum. RT was 12

maintained between 68°F and 72°F, and relative humidity was maintained between 34% and 13

73%. The laboratory housing the cages provided a 12-hour light cycle and met all AAALAC 14

specifications. All in vivo experiments were conducted under IACUC approved protocols. 15

Primary Patient Samples 16


Ex Vivo Drug Testing of Primary AML Patient Samples 18





29

Acknowledgments 1

This work was supported by Amgen Inc. BAK-/-BAX-/- cell line experiments were supported by 2

scholarships, fellowships and grants from the Australian National Health and Medical Research 3

Council (NHMRC) (Research Fellowships to AWR, DCSH; Project Grants to DCSH 1057742; 4

Program Grants 1016647, 1016701; Independent Research Institutes Infrastructure Support 5

Scheme grant 9000220), the Cancer Council Victoria (grant-in-aid to AWR and DCSH), the 6

Leukemia and Lymphoma Society (SCOR grants 7001-13), the Australian Cancer Research 7

Foundation, a Victorian State Government Operational Infrastructure Support (OIS) grant. The 8

authors thank Jin Tang for assistance with protein purification and Victor Cee for critical 9

evaluation of the manuscript. Ben Scott, PhD (Scott Medical Communications, LLC), and Beate 10

Quednau, PhD (Amgen Inc.), provided medical writing assistance funded by Amgen Inc. 11

Authors’ Contributions 12

Conception and design: 13

P.E.H., S.P.B., S.C., B.B., G.M., J.C., K.K., D.C., D.W., C.B., A.W., D.H. 14

Development of methodology: 15

S.C., S.P.B., B.B., G.M. D.C., L.P., A.C.C., J.H., Y.L., B.L., N.A.P., Q.W., J.T., M.V., M.Z., L.Z., 16

J.G. 17

Acquisition of data (provided animals, acquired and managed patients, provided 18

facilities, etc.): 19

S.C., B.B., D.C., D.A.W., X.H., L.P, A.C.C., M.C., J.H., Y.L., B.L., N.A.P., Q.W., J.T., M.V., M.Z., 20

L.Z., E.C., T.O., J.S., L.J.D., R.K.E., P.G., J.M., J.G., D.M., G.P., , A.W.R, D.C.S.H., J.G. 21

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational 22

analysis): 23

S.C., P.H., S.B., B.B., D.C., D.A.W., X.H., L.P, A.C.C., M.C., J.H., Y.L., B.L., N.A.P., Q.W., J.T., 24

M.V., M.Z., L.Z., E.C., T.O., J.S., L.J.D., R.K.E., P.G., J.M., J.G., D.M., G.P., A.W.R., D.C.S.H., 25

J.G. 26




30

Writing, review, and/or revision of the manuscript: 1

S.C., P.E.H., S.B., B.B., J.C., G.M., C.B., A.W., D.H., A.C. 2

Administrative, technical, or material support (i.e., reporting or organizing data, 3

constructing databases): 4

S.C., B.B., D.C., D.A.W., X.H., L.P, A.C.C., M.C., J.H., Y.L., B.L., N.A.P., Q.W., J.T., M.V., M.Z., 5

L.Z., E.C., T.O., J.S., L.J.D., R.K.E., P.G., J.M., D.M., G.P., J.G. 6

Study supervision: 7

P.E.H., S.P.B., S.C., G.M., P.B., C.B., A.W., J.C., A.C., J.H, D.W., D.H. 8




31

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Figure 1. Optimization of chemical matter to AMG 176. A, X-ray structure-based optimization of

high-throughput screening hit to clinical candidate AMG 176. B, X-ray crystal structure of MCL1

bound to BIM (20). C, X-ray structure of MCL1 bound to AM-8621 reveals cryptic binding pocket

(PDB validation report 9100015541). D, Quantum mechanical derived conformational

ensemble of 8 within 3 kcal/mol depicted as Boltzmann distribution. Binding conformation shown

in green. Broken bars represent multiple conformations. E, Quantum mechanical derived

conformational ensemble of 9 within 3 kcal/mol depicted as Boltzmann distribution. Binding

conformation shown in green. Broken bars represent multiple conformations. F,

Pharmacokinetic properties of 9, 10 and 11. Species refers to the species of animal in which the

pharmacokinetic data were acquired.

Figure 2. AM-8621 treatment disrupts the interaction between MCL1 and pro-apoptotic BCL-2

family members and also induces MCL1 protein levels. A. Disruption of the MCL1:BAK

interaction in HEK293M cells treated with AM-8621, as measured in a split luciferase

complementation assay. Mean and ±SD from n=4. B, Disruption of the MCL1:BIM interaction in

A427 cells treated with AM-8621 for 1 hour at indicated doses (left) or 750 nM for indicated

durations (right) using an MCL1:BIM complex immunoassay. Mean and ±SD from n=3. C,

Immunoblot analysis of MCL1, BCL-XL, BCL-2, BIM, and cleaved PARP in U266B1 cells treated

with AM-8621 for 24 hours. D, Immunoblot characterization of MCL1 protein half-life in U266B1

cells treated with AM-8621 (2 µM) or vehicle for 4 hours followed by addition of CHX (100

µg/mL) for indicated durations. Quantified MCL1 levels were plotted against time post CHX

addition and used to estimate MCL1 protein half-lives of 2.3 hours in the presence of AM-8621

and 0.7 hour in its absence. E, Immunoblot characterization of AM-8621 mediated increase in

MCL1 protein in U266B1 cells. For right panels, cells were treated with AM-8621 for 6 hours

followed by drug washout for indicated durations.




38

Figure 3. AM-8621 induces a rapid commitment toward apoptosis and loss of viability in

sensitive cell lines. A, Effects of AM-8621 treatment (1 µM for 2 hours) on activated BAK in AM-

8621–sensitive (OPM-2, MV-4-11, MOLM13, and Ramos) and AM-8621–insensitive (U266B1)

cell lines as measured in a flow cytometry-based BAK activation assay. B, Caspase activity and

effects on viability following treatment with AM-8621 in OPM-2, MV-4-11, MOLM13, and Ramos

cells. Effects on cell viability and caspase activity were measured by CellTiter-Glo® and

Caspase-Glo® respectively. Mean and ±SD from n=4. C, Viability analysis of OPM-2, MV-4-11,

MOLM13, and Ramos cells treated with AM-8621 for indicated durations, followed by drug

washout and incubation in the absence of drug for indicated durations. Effects on cell viability

were measured by CellTiter-Glo®. Mean and ±SD from n=4. D, Viability analysis of AMO1,

H929, and OPM-2 parental and BAX-/-BAK-/- cell lines treated with AM-8621 for 24 hours. Effects

on cell viability were measured by CellTiter-Glo®. Mean and ±SD from n=3.

Figure 4. Cell lines from hematological cancers, including MM, AML, and DLBCL, exhibit a

strong dependency on MCL1 for survival. A, Profile of response to AM-8621 for 952 tumor-

derived cell lines grouped by cancer subtype. Top panel: cell line count; middle panel:

distribution of maximum drug effect on cell viability (Emax); bottom panel: median IC50 (natural

logarithm of µM values). Effects on cell viability were measured by resazurin assay. B, Relative

sensitivity of cancer subtypes and statistical enrichment for sensitive cell lines. The volcano plot

reports sensitivity to AM-8621 versus statistical significance (-log P-value of the Fisher Exact

test performed using a 10 µM threshold [maximum tested dose]) for classification into sensitive

and resistant cell lines. Among all individual cancer subtypes included in viability screen, those

that demonstrated statistically significant enrichments are shown. C, Effects of AM-8621

treatment on the viability of expanded panels of MM (n=19), AML (n=28), and DLBCL (n=21)

cell lines. Effects on cell viability were measured by CellTiter-Glo®. Mean values from n = 2–5

biological replicates for individual cell lines. Black bars report mean and ±SD across indication.




39

D, Comparison of sensitivity to AM-8621 versus venetoclax across MM, AML, and DLBCL cell

line panels. E, Relationship between BCL-XL RNA expression and sensitivity to AM-8621 for

those MM cell lines (n=14) reported in Figures 4C and 4D for which BCL-XL FPKM values were

available from CCLE (42).

Figure 5. AMG 176 exhibits robust single agent activity in vivo. A, Time- and dose-dependent

effects of AMG 176 treatment on apoptotic markers (activated BAK, cleaved Caspase 3, and

cleaved PARP) in established OPM-2 luc tumors as measured in MSD immunoassays. B,

Effects of AMG 176 on established OPM-2 luc tumor xenografts when dosed daily or on a 2-day

on, five-day off schedule. C, Effects of AMG 176 on established OPM-2 luc tumor xenografts

when dosed once-weekly. D, Effects of AMG 176 on MOLM13 luc orthotopic tumor xenografts

when dosed on a 2-day on, five-day off schedule. Representative day 17 bioluminescence

images are shown. For xenograft studies mean tumor volume or whole-body luminescence

(dorsal + ventral image) ± SEM (n = 10/group) are reported. ***p<0.0001 (RMANOVA with

Dunnett’s post hoc). E, Flow cytometry assessment of B cells, monocytes and neutrophils in

peripheral blood and bone marrow of human MCL1 knock-in mice following administration of

AMG 176 24 hours post cycle 1 (day 3) and cycle 2 (day 10). *p<0.05, **p<0.01, ***p<0.001,

****p<0.0001 (One-way ANOVA with Dunnett’s post hoc).

Figure 6. AMG 176 exhibits activity when combined with clinically relevant agents that target

hematological malignancies. A, Combination effects of AMG 176 plus carfilzomib on OPM-2 luc

orthotopic tumor xenografts. Mean hind limb bioluminescence ± SEM (n = 10/group) is reported

for each group. ***p<0.0005 (RMANOVA for comparisons of the combination treatment to each

single agent). Representative day 19 bioluminescence images are shown. B, Synergy scores

for in vitro combination studies in AML cell lines treated with combinations of AM-8621 and

standard of care chemotherapeutics (cytarabine, decitabine, and doxorubicin) or venetoclax.

Effects on cell viability were measured by CellTiter-Glo®. Higher scores reflect stronger




40

synergistic interaction (darker red). C, Combination effects of AMG 176 plus venetoclax on

MOLM13 luc orthotopic tumor xenografts. Mean whole body bioluminescence (dorsal + ventral

image) ± SEM (n = 10/group) is reported for each group. ****p<0.0001 (RMANOVA for

comparisons of the combination treatment to each single agent). Representative day 17

bioluminescence images are shown. D, Flow cytometry assessment of B cells, monocytes and

neutrophils in peripheral blood from human MCL1 knock-in mice treated with AMG 176 alone or

combined with venetoclax 24 hours post cycles 1 (day 3) and 2 (day 10). *p<0.05; **p<0.01,

***p<0.001, ****p<0.0001 (one-way ANOVA with Dunnett’s post hoc ). E, Observed body

weights in human MCL1 knock-in mice following treatment with AMG 176, venetetoclax, or

AMG 176 and venetoclax combined. Data are reported as % mean average bodyweight

compared with % pre-treatment body weight ± SEM (n = 5/group).

Figure 7. AM-8621 is active against primary AML samples as a single agent and when

combined with clinically relevant therapeutics. A, Dose-response curves showing primary AML

samples gated on blasts and viability assessed after 48 hours incubation with indicated drugs.

B, Normalized LC50 (50% lethal concentration) viability values comparing AM-8621 combined

with indicated drugs after 48 hours of treatment.




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980 | CANCER DISCOVERY JULY 2019 www.aacrjournals.org

Correction: AMG 176, a Selective MCL1 Inhibitor, Is Effective in Hematologic Cancer Models Alone and in Combination with Established Therapies

In the original version of this article (1), there are errors in the Methods section. In the sub-section titled “NMR and Isothermal Titration Calorimetry,” all mentions of compound 2 have been corrected to compound 6. In addition, the citations of PDB validation reports for MCL1 cocrystal structures throughout the article have been replaced with the corresponding PDB codes. These changes have been made in the latest online HTML and PDF versions of the article. The authors regret these errors.

REFERENCE1. Caenepeel S, Brown SP, Belmontes B, Moody G, Keegan KS, Chui D, et al. AMG 176, a selective MCL1 inhibitor,

is effective in hematologic cancer models alone and in combination with established therapies. Cancer Discov 2018;8:1582–97.

CORRECTION

doi: 10.1158/2159-8290.CD-19-0577©2019 American Association for Cancer Research.

Published online July 1, 2019.Cancer Discov 2019;9:980

http://crossmark.crossref.org/dialog/?doi=10.1158/2159-8290.CD-19-0577&domain=pdf&date_stamp=2018-11-09

Published OnlineFirst September 25, 2018.Cancer Discov Sean Caenepeel, Sean P Brown, Brian Belmontes, et al. Established TherapiesHematological Cancer Models Alone and in Combination with AMG 176, a Selective MCL1 Inhibitor, is Effective in

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