AACR 2019 March 29 - April 3

Atlanta, GA

LB-111 / 6 Epidermal Growth Factor Receptor Oncogenes Expressed in Glioblastoma Are Activated as Covalent Dimers and Exhibit Unique Pharmacology

Matthew O'Connor1, Theodore Nicolaides2, Jie Zhang3, Alexander Flohr4, Roberto Iacone4, Alexander V. Mayweg4, David M. Epstein1, Elizabeth Buck1

1 Black Diamond Therapeutics Inc, Stony Brook, NY;

2 NYU Langone Health, Department of Pediatrics, New York, NY;

3 UCSF, Department of Neurology, San Francisco, CA;

4 Black Diamond Therapeutics Inc, Basel, Switzerland

ABSTRACTMutation of either the intracellular catalytic domain or the extracellular domain of the receptor for epidermal growth factor (EGFR) drives oncogenicity.

Extracellular domain EGFR mutations are highly expressed in patients with glioblastoma.

Despite clinical success with targeting EGFR catalytic site mutants, no drugs have proven effective in glioblastoma patients expressing extracellular EGFR mutations.

Herein, we define the molecular mechanism for oncogenic activation of families of extracellular EGFR mutations and reveal how this mechanism renders current generation small molecule ATP-site inhibitors ineffective.

We demonstrate that a group of the most commonly expressed extracellular domain EGFR mutants expressed in glioblastomas are activated by disulfide-bond mediated covalent homodimerization, collectively referred to as locked dimerization (LoDi-EGFR oncogenes).

Strikingly, current generation small molecules binding to the active kinase conformation potently inhibit catalytic site mutants, but induce covalent dimerization and activate LoDi-EGFR receptors, manifesting in paradoxical acceleration of proliferation.

These data demonstrate how the locked-dimer mechanism of EGFR oncogenesis has profound impact on the activity of small molecules acting at the distal catalytic site, providing further evidence for “inside-out” allosteric signaling in EGFR.

This provides a mechanistic understanding for the failure of current generation EGFR inhibitors to effectively treat LoDi-EGFR mutants in GBM and sets guidelines for discovery of selective LoDi-EGFR inhibitors.

SUMMARYThe extracellular ligand binding domain of EGFR is a hot spot for oncogenic alterations in glioblastoma.

We define disruption of auto-inhibition and covalent homo-dimerization as unifying mechanisms of activation for this family of EGFR oncogenes, a family we term Locked-Dimer (LoDi)-EGFR oncogenes.

We show that LoDi-EGFR mutations affect the pharmacology for small molecules binding at the intracellular ATP site, despite there being no change in the primary amino acid sequence at the ATP site.

Type I inhibitors, including the clinically approved EGFR inhibitors erlotinib, gefitinib, afatinib, and osimertinib, induce the formation of covalent LoDi-EGFR dimers and increase LoDi-EGFR phosphorylation at sub-saturating concentrations.

This manifests in paradoxical activation of proliferation at sub-saturating concentrations.

In contrast to Type I inhibitors, we find that Type II inhibitors, including neratinib, are devoid of paradoxical activation, although neratinib is not a selective inhibitor of these EGFR oncogenes.

We captured these observations in PDX tumors expressing LoDi-EGFR-Viii.

Collectively, these findings provide a mechanistic understanding for how structural variations affecting receptor regions distal to the active site can confer dramatically different responses to small molecule ATP site inhibitors.

These observations provide a mechanistic explanation for the failed clinical studies for Type I inhibitors in glioblastoma and provide impetus for optimization of selective Type II inhibitors tailored against LoDi-EGFR oncogenes in glioblastoma.

TABLE 3 and FIGURE 5: Type I ErbB inhibitors, but not Type II ErbB inhibitors, induce covalent dimerization of LoDi-EGFR oncogenes

EGFR-Vii

EGFR-Viii

FIGURE 2. LoDi-EGFR oncogenes are covalently activated locked dimers

FIGURE 3. Erlotinib stimulates the formation of covalent dimers for all LoDi-EGFR oncogenes

Viii

Dimer interfaceEGFR-Viii

EGFR-Vvi

EGFR-Vii

β-Actin

EGFR-A289V

EGFR-Viii

EGFR-Vvi

EGFR-Vii

EGFR-A289V

EGFR

pEGFR

EGFR-WT

Tethering site: YDK Triad

Y270

-D587 K609

EGFR-WT L1

CR1

L2

CR2

EGF binding site

CYS = Cysteine, GBM = Glioblastoma, SCCHN = Squamous cell carcinoma of the head and neck, SQLUNG = Squamouse cell carcinoma of the lung, BrCa = Breast Cancer

FIGURE 1. EGFR extracellular domain mutations occur at sites functioning in auto-inhibition and dimerization and results in the presentation of free cysteines at the dimer interface

VARIANT

EGFR-Vii

EGFR-Viii

EGFR-Vvi

EGFR-A289V

GBM (3%)

GBM (20%),

SCCHN (36%),

SCLUNG (5-10%),

BrCa (5%)

GBM (32%)

GBM (15%)

TUMOR EXPRESSION (PREVALENCE)

Brennan et al 2014,

Francis et al 2014

Brennan et al 2014,

Wheeler et al 2015,

Sasaki et al 2007

Brennan et al 2014

Brennan et al 2014

STUDY

14-15

2-7

12-13

7

EXONS AFFECTED

Cys539, Cys628,

Cys636

Cys307

Cys555

not determined

FREE CYS GENERATED

CR2

CR1

CR2

CR1

POSITION

CR1

L2

CR2

L1

CR1

L2

CR2

10-10 10-9 10-8 10-7 10-60.0

0.5

1.0

1.5

2.0

2.5

erlotinib [M]

Fold

Indu

ctio

n C

oval

ent D

imer

erlotinib [M]

LoDi-EGFR(EGFR-Viii)

KD-EGFR(E746-A750)

2.0

1.6

1.2

0.8

0.4

-0.4 10-10 10-8 10-410-6

0.0

Fold

Gro

wth

Time FollowingTreatment (hours)

erlotinib

DMSO

4

0 24 48 72

3

2

1

0

Fold

Gro

wth

DIMER

MONOMER

DIMER

MONOMER

DIMER

MONOMER

β-Actin

EGFR

β-Actin

EGFR

- + - + - + - + erlotinib

erlotinib

EGFR-Viii EGFR-Viii

- 100nM 10nM 1nM

DIMER MONOMER

- 100 10 - 100 10 nM - 100 30 10

DIMER

MONOMER

DIMERMONOMER

EGFR-Viii EGFR-Vii EGFR-Vvi

ATP Mimetic

αC-helix ‘In’

Asymmetric KD dimerization

JM α-helix dimerization

Covalent ECD dimerization

β-Actin

EGFR

pEGFR

erlotinib

FIGURE 4. Sub-saturating concentrations of erlotinib paradoxically stimulate the phosphorylation of LoDi-EGFR oncogenes and accelerate cell proliferation

VARIANT BINDING MODE REVERSIBLE / COVALENT CORE STRUCTURE

afatinib Type I covalent quinazoline

PD168393 Type I covalent quinazoline

canertinib Type I covalent quinazoline

pelitinib Type I covalent quinazoline

dacomitinib Type I covalent quinazoline

neratinib Type I covalent quinoline

AST-1306 Type II covalent quinazoline

HKI-357 Type II covalent quinoline

erlotinib Type I reversible quinazoline

AZD9291 Type I covalent pyrimidine

lapatinib Type II reversible quinazoline

CO-1686 Type I covalent pyrimidine

WZ8040 Type I covalent pyrimidine

WZ4002 Type I covalent pyrimidine

WZ3146 Type I covalent pyrimidine

sapitinib Type 1 reversible quinazoline

gefitinib Type 1 reversible quinazoline

β-Actin

EGFRDIMER

MONOMER

DM

SO

afat

inib

PD

1683

93

cane

rtini

b

pelit

inib

daco

miti

nib

nera

tinib

AS

T-13

06

HK

I-357

erlo

tinib

AZD

9291

lapt

inib

CO

-168

6

WZ8

040

WZ4

002

WZ3

146

10-10 10-8 10-6 10-4

-0.5

0.0

0.5

1.0

1.5

Pro

lifer

atio

n (fo

ld g

row

th) EGFR-Viii

EGFR-Vii

LoDi-EGFR (EGFR-Viii)

- 1000 300 100 30 10 nM neratinib

DIMERMONOMER

pEGFR

DIMERMONOMER

EGFR

pPRAS40

pMAPK(p44/p42)

GAPDH

FIGURE 6. The Type II inhibitor neratinib inhibits the LoDi-EGFRoncogenes without evidence of paradoxical activation

FIGURE 7: EGFR-Viii is expressed and activated as a covalent dimer in PDX tissue, where erlotinib behaves as a paradoxical activator

A. The phosphorylation state for EGFR in tumor lysates derived from the GBM6 glioblastoma tumor model expressing EGFR-Viii. Proteins were resolved by WB in the absence or presence of DTT reductant to detect the presence of covalently activated EGFR-Viii.

B. Effect of acute treatment with erlotinib at 100mg/kg (top panel) or 10mg/kg (center panel), or neratinib at 50mg/kg (bottom panel) on the phosphorylation state of EGFR-Viii in GBM6 PDX tumors. Plasma exposure for each drug treatment is indicated on the y-axis.

Neratinib inhibits the proliferation of BaF3 cells transformed with EGFR-Viii or EGFR-Vii without paradoxical activation, but is not selective versus WT-EGFR.

A. Effect of varying concentrations of neratinib on the proliferation of BaF3-EGFR-Viii and BaF3-EGFR-Vii transformants.

B. Effect of varying concentrations of neratinib on total and phosphorylatedlevels of EGFR monomers and covalent dimers, phosphorylated PRAS40, and phosphorylated MAPK in BaF3 cells transformed with EGFR-Viii.

A. Effect of varying concentrations of erlotinib on monomeric and dimeric levels of total and phosphorylated EGFR-Viii (left panel), EGFR-Vii (middle panel), and EGFR-A289V (right panel). Proteins were resolved under non-reducing conditions.

B. Effect of 37nM erlotinib on the proliferation of BaF3 cells transformed with EGFR-Viii over a three day period.

C. Effect of varying concentrations of erlotinib on the proliferation of BaF3 cells transformed with EGFR-E746-750 or EGFR-Viii.

A. Effect of 100nM erlotinib on the levels of monomeric and dimeric LoDi-EGFR oncogenes.

B. Effect of various concentrations of erlotinib on levels of monomeric and dimeric EGFR-Viii.

Effect of a panel of diverse EGFR inhibitors on levels of monomeric or covalently dimerized EGFR in cells expressing EGFR-Vii.

A. Schematic representation of the extracellular domain of EGFR and sites of mutations producing EGFR-Viii, EGFR-Vii, and EGFR-Vvi. L1 region is shown in blue, CR1 in green, L2 in orange, and CR2 in red. Exons are enumerated. Regions functioning in auto-inhibition and dimerization are noted. Exons that are truncated in the mutants are noted in grey, and positions of free cysteines resulting from these mutations are noted by Cys below each schematic. B. Space filled models describing the structure of the extracellular domain of EGFR in the tethered conformation using the x-ray coordinates from 1NQL. Regions that are truncated in the EGFR-Viii, -Vii, and –Vvi mutants are shown in grey. Within the model for EGFR-WT a detailed schematic for the YDK triangular salt bridge forming a key tethering interaction is provided.

A. Ribbon model of the extracellular region of EGFR in the ligand-bound dimerized conformation. Positions of cysteines in the CR1 region are shown as yellow spheres in the ribbon diagram. The position of Cys307, which is a free cysteine generated in EGFR-Viii, is noted.

B.The expression of total and phosphorylated monomeric LoDi-EGFR versus covalent LoDi-EGFR dimers for EGFR-Viii, EGFR-Vii, EGFR-Vvi, and EGFR-A289V detected by resolving proteins under non-reducing conditions.

B.

EGFR-ViiiEGFR-WT

-- ++reductantA.

B.

A.

A. B. C.

B.

A.

A.

A.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1 8 9 10 11 12 13 14 15 16 17

1 2 3 4 5 6 7 8 9 10 11 12 13 16 17

1 2 3 4 5 6 7 8 9 10 11 14 15 16 17

L1 CR1 L2 CR2 TMEXTRACELLULARDOMAIN STRUCTURE

EGFR-WT

EGFR-Viii

EGFR-Vii

EGFR-Vvi

Cys

Cys Cys Cys

Cys

auto-inhibitory tethering / dimerization

B.

C.

B.

EGFR-Viii

DIMER

MONOMER

0 12 24 36 480

50

100

150

0.01

0.1

1

% C

ontro

l P

hosp

hory

latio

n

PK

(uM

, total neratinib)

0 12 24 36 480

100

200

300

400

0.001

0.01

0.1

1

10

100

% C

ontro

l P

hosp

hory

latio

n

PK

(uM

, total erlotinib)

0 12 24 36 480

100

200

300

400

0.001

0.01

0.1

1

10

100

% C

ontro

l P

hosp

hory

latio

n

PK

(uM

, total erlotinib)

0 12 24 36 480

100

200

300

400

Hours Following Dose

% C

ontro

l P

hosp

hory

latio

n

50mpk neratinib

50mpk neratinib

100mpk erlotinib

100mpk erlotinib

10mpk erlotinib

10mpk erlotinib

FIGURE 8: TKIs (ATP mimetics) promote “inside-out” signaling and covalent dimers

Asymmetric KD dimerization

Covalent ECD dimerization

αC-helix ‘In’

S S