1

In vitro characterization of FTX-6058, a novel small molecule fetal hemoglobin inducer for sickle cell disease

Billy Stuart*, Pete Rahl, Kingsley Appiah, Ivan Efremov, Lorin A Thompson, Owen Wallace,

Christopher Moxham

Fulcrum Therapeutics, Cambridge, MA 02139

2FULCRUM THERAPEUTICS

Disclosure

All authors are current or former employees and equity holders of Fulcrum Therapeutics.

3FULCRUM THERAPEUTICS

Therapeutic Rationale: Inducing fetal hemoglobin can reduce mortality and morbidity risks in SCD

SCD Patient SCD Patient with High Fetal Hemoglobin (HbF)

Increased F-cells*

Reduced VOCs

Reduced hemolysis

RBC sickling

VOCs

Hemolysis

Pancellular

HbF

Expression

and

Induction

Stroke

Nephropathy

Pulmonary Hypertension

Acute Chest

Syndrome

Osteonecrosis

Ulcer / Pain

*F-cells - fetal hemoglobin expressing cells

30%

20%

10%

Asymptomatic presentation

Reduced recurring events(VOCs, ACS, Hospitalization)

Reduced mortality

HbF Level

Powars, DR. Blood. 1984; Estepp, JH. Br J Haematol. 2013; Platt, OS. NEJM. 1994; Akinsheye, I. Blood. 2011.

4

Parallel CRISPR and chemical probe screens in HUDEP-2 cells identify targets which increase HBG1/2

Target identification using parallel strategies in genetically relevant human cells

HE

MA

TO

LO

GY

FR

AN

CH

ISE

HUDEP-2 Cells

Chemical Probes CRISPR/CAS9

Parallel Target ID ScreeningGene Regulation Phenotype

Disease Gene Activation Readout

Functional Validation Disease Modeling in

Primary CD34+ Cells

HBG1/2 and HbF Elevation

Computational Biology HbF

+

5FULCRUM THERAPEUTICS

Chemical probe and CRISPR screens converge on published and novel drug targets

Gene Regulation

Drug Targets

Chemical Probe Screening

CRISPR Screening

• EED KD = 0.163 nM

• PRC2 IC50 < 5 nM

• Highly Selective

• Clean Off-target Profile

Structure-Based Drug Design

Identified Embryonic Ectoderm

Development (EED) as a critical regulator

of HbF

FTX-6058

BCL11A, NuRD, HDACs, LSD1, DNMT1, IKZF1,

IKZF3, SPOP

6

FTX-6058 in vitro target engagement is consistent across donors in differentiated primary CD34+ cells

FTX-6058 target engagement in primary CD34+ cells is similar across 4 donors

Target engagement (ICC): Primary CD34+ cells; 4 days

treatment and differentiation

0.001 0.01 0.1 1 10

0

50

100

150

H3K27me3 level in CD34+ cellswith FTX-6058 treatment

FTX-6058 Concentration (uM)

H3K

27m

e3 L

evels

(Flu

ore

sen

ce

In

ten

sit

y t

o D

MS

O)

Donor 101

Donor 229

Donor 224

Donor 304

• FTX-6058 reduces H3K27me3 with an IC80 12-40 nM across 4 healthy

CD34+ donors

7

FTX-6058 increases % HbF in CD34+ cells: HPLC quantitation

DM

SO

33uM

Hyd

roxy

urea

100n

M F

TX-6

058

0

10

20

30

Quantification of %HbF in

differentiated primary CD34+cells

% H

bF

Hb

F/(

Hb

F+

Hb

A)

DMSO

100nM FTX-6058

33µM Hydroxyurea

6694, 2.43

14145, 5.00

11175, 5.55

57726, 5.71

23409, 6.31

404489, 6.53

9874, 6.91

8732, 8.17

0 1 2 3 4 5 6 7 8 9 10

Retention Time (min)

0.00

0.02

0.04

0.06

0.08

0.10

Absorbance (AU)

14961, 2.47

46897, 5.09

26912, 5.23

30311, 5.65

328287, 5.78

39956, 6.15

69416, 6.41

923970, 6.59

28848, 6.97

5343, 8.21

0 1 2 3 4 5 6 7 8 9 10

Retention Time (min)

0.00

0.05

0.10

0.15

0.20

Absorbance (AU)

3159, 2.44

34858, 5.23

110089, 5.77

19680, 6.09

62315, 6.33

568879, 6.54

20461, 6.91

0 1 2 3 4 5 6 7 8 9 10

Retention Time (min)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Absorbance (AU)

HbFHbA

12.6% 13.1%

27.4%

with Gerd Blobel laboratory (UPenn/CHOP)Hemoglobin HPLC: CD34+ cells differentiated and treated for 7 days

8CONFIDENTIAL 8

FTX-6058 induces pancellular distribution of HbF

DMSO

100 nM

FTX-6058

HbF

Co

un

ts

~12%

HbF

From 50% to

80% HbFhi

HbF flow cytometry: CD34+ cells differentiated and treated for

7 days; Gated and quantified for HbF+/CD235a+/CD71+

~30%

HbF

FTX-6058 induced pancellular distribution of HbF

Wood WG et al. J Med Gen,

14:237 1977

HbF: ~30%

HbF: ~11%

Left: pancellular HbF distribution in blood of patient with HPFH & SCD - asymptomatic SCD

SCD Mother

SCD Child w/HPFH

9CONFIDENTIAL 9

D227(Healthy)

D466(SCD)

D982(SCD)

0

10

20

30

Donor

%H

bF

of

tota

l h

em

og

lob

in

(HP

LC

)

DMSO

100nM FTX-6058

33uM HU

%HbF elevation with FTX-6058 and HU

• FTX-6058 induces superior HbF protein

expression over HU across all CD34+ donors

(healthy and SCD)

10 100 1000

0

10

20

30

FTX-6058 Concentration (nM)

%H

bF

of

tota

l h

em

og

lob

in

(HP

LC

)

D227 (Healthy)

D466 (SCD)

FTX-6058 elevates % HbF in differentiated primary CD34+ cells from SCD and healthy donors

Concentration-dependent increase in %HbF in

CD34+ cells from SCD and healthy donors

• Higher doses of FTX-6058 can further induce

HbF protein expression, whereas higher HU

concentrations were toxic

10FULCRUM THERAPEUTICS

FTX-6058 Robustly Induces Fetal Hemoglobin in CD34+ Cells from Healthy and SCD Donors

▪ Observe an absolute 8 – 18% increase in HbF upon treatment with FTX-6058, which has the ability to address mortality risk and recurring events in SCD patients

▪ Small increases in HbF (1 – 5%) have the potential to provide clinical benefits to all SCD patients

HbF Induction with FTX-6058

Baseline %HbF Maximal %HbF

0

10

20

30

40

%H

bF

(H

PL

C)

Donor 1

Donor 2

Donor 3

Donor 5

Donor 6 (SCD)

Donor 4

11FULCRUM THERAPEUTICS

FTX-6058 has potential to be a transformative therapy for SCD

▪ FTX-6058 is a potent, selective and orally active small molecule EED inhibitor.

▪ Proof of concept studies provided in vitro evidence for FTX-6058 in inhibiting PRC2 activity,

which leads to elevation of HbF in human primary CD34+ cells.

▪ FTX-6058 demonstrated potent TE and HbF induction in vivo in animal models at plasma

concentrations reasonably expected to be achieved in the clinic. (See abstract #789)

▪ FTX-6058 pharmacological activity in target cells can be readily monitored in the clinic since TE

in bone marrow correlates with TE in peripheral monocytes in animals. (See abstract #789)

▪ FTX-6058 demonstrated an impressive preclinical pharmacological profile with the potential to be

a disease-modifying therapeutic for sickle cell patients. (See abstract #1548)

12FULCRUM THERAPEUTICS

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