Mouse Peripheral Blood Staining & Gating

Methyl-Mark in Mouse Peripheral Blood Poplulations

Histone Western H3K27me3/H3 Inhibition in Peripheral Blood from NHL Trial Patients

H3K27me3/H3 in Peripheral Blood from NHL Trial Patients

Percent Inhibition of Normalized H3K27me3 v. Percent Population Composition in 500mg/kg Tazemetostat Treated Mouse

Chromatin Flow Cytometry

H3K27me3 Inhibition in NHL Trial with Tazemetostat Treatment

Methyl-Mark in Human Peripheral Blood (Pre-Clinical Trial Optimization)

Peripheral Blood Staining & Gating (Pre-Clinical Trial Optimization)

H3K27me3 Quantification Chromatin Flow Cytometry v. ELISA

Cell Type Contribution to Total H3K27me3 Inhibition Differential Sensitivity of Mouse Leukocyte Populations to Tazemetostat

IC50 Determination by Chromatin Flow Cytometry & Histone Western

Plescia C, Knutson S, Warholic N, McDonald A, Keilhack H, Smith J, Copeland R, Blakemore S

Evaluating the effects of epigenetic modulators on the methylation state of histones in heterogeneous cell populations has, to this point, been limited to bulk assessment of cell lysates or purified histones. Hence, determining the differential effects of these compounds on individual cell types within a complex population has remained elusive.

In order to monitor the pharmacodynamics of tazemetostat (EPZ-6438), an EZH2 inhibitor that has demonstrated clinical activity in multiple oncology indications, with greater sensitivity and specificity in phase 2 clinical trials and beyond, we have developed a chromatin flow cytometric assay that quantifies trimethylation of lysine 27 of histone H3 (H3K27me3) and total H3 levels while simultaneously allowing for immunophenotyping of discrete populations by surface marker composition in human and murine blood leukocytes. Tazemetostat treatment of human lymphoma cell lines for 4 days resulted in dose-dependent reductions in H3K27me3 levels when quantified both by traditional histone western blotting and chromatin flow cytometry. Furthermore, both assays were able to discriminate between EZH2 mutant lymphoma cell lines which lack H3K27 dimethylation (H3K27me2) and wild-type lines in which the H3K27me2 is present and reduced in a dose responsive manner.

To determine if the assay was capable of discriminating methylation states of specific cell populations found in whole blood from an in vivo model, we performed a 7-day study in mice treated with increasing dosages of tazemetostat (EPZ-6438). Quantification of H3K27me3 levels in blood leukocytes revealed differential responses to inhibitor treatment based on cell type. Monocytes and NK cells were found to be highly sensitive to tazemetostat, demonstrating 85% and 70% reduction in H3K27me3 respectively. B-cells were moderately sensitive to tazemetostat treatment, exhibiting a 21% reduction in H3K27me3. Granulocytes and T-cells were the least sensitive with a 7% and 3% reduction in H3K27me3 respectively.

These results demonstrate that chromatin flow cytometry is an effective means to monitor cell type specific changes in methylation state upon compound treatment in vivo. Given the advantages of this methodology over conventional measures of histone methylation, chromatin flow cytometry will be used to monitor pharmacodynamics of H3K27me3 inhibition in whole blood leukocytes in the ongoing phase 2 and future clinical trials of tazemetostat.

#B133

Gating scheme for mouse peripheral blood. Whole blood is collected in Na Heparin vacutainers followed by red blood cell lysis. Cells are fixed in 4% methanol-free formaldehyde and permeabilized with 0.1% Triton X-100. Processed cells are Fc-blocked and stained with anti-CD3-PerCP-Cy5.5, CD45R-BV421, CD49b-PE, CD11b-V500, GR-1-PE-Cy7, H3-AlexaFluor 647 and H3K27me3-AlexaFluor 488 antibodies. Populations are defined as follows:

• H3K27me3 levels are normalized to total H3 content for each population of interest.

• Chromatin flow cytometry demonstrates the rank order of H3K27me3/H3 in drug naïve mouse peripheral blood leukocyte populations.

H3K27me3

H3

Western Blot IC50 = 8nM

Chromatin Flow Cytometry IC50 = 146nM Chromatin Flow Cytometry IC50 = 5nM

Dose Response to EPZ007210 in EZH2 Mutant & Wild Type Cell Lines

CHROMATIN FLOW CYTOMETRY VALIDATION IN CELL LINES HUMAN CLINICAL TRIAL (PRELIMINARY DATA)

MOUSE IN VIVO DOSE RESPONSE ABSTRACT

• Cell lines were treated with tool EZH2 inhibitor EPZ007210 for 4 days followed by fixation in 4% methanol free formaldehyde and permeabilization in 0.1% Triton X-100. Staining was performed in a single well using H3K27me3-AlexaFluor 488, H3K27me2-AlexaFluor-647 and H3-Pacific Blue.

• Mutant cell lines (WSU) exhibit tri-methylation but not di-methylation H3K27. Wild type lines (OCI-LY19) exhibit both di and tri-methylation of H3K27.

• Both cell lines demonstrate dose responsiveness of H3K27me3 with EPZ007210 treatment.

• The EZH2 wild type cell line also demonstrates a dose response of H3K27me2 with inhibitor treatment while no H3K27me2 is observed in the mutant line. Total H3 remains constant regardless of dosage or EZH2 mutational status.

• These experiments confirm that chromatin flow cytometry is capable of detecting modulation of discrete H3K27 methylation states with inhibitor treatment. Furthermore, it is possible to distinguish between multiple methylation states in a single assay.

• Quantification of H3K27me3 / H3 from EPZ007210 dose response by chromatin flow cytometry and histone western blot.

• Chromatin flow cytometry agrees with histone western for OCI-LY19 cells.

• Western blot and chromatin flow cytometry data diverge for WSU cells, possibly due to greater

intrinsic auto-fluorescence of WSU cells.

H3K27me3

H3

Western Blot IC50 = 9nM

WSU OCI-LY19

• Quantification of H3K27me3 / H3 in peripheral blood from 7 day dose response study with EZH2 inhibitor tazemetostat reveals differential sensitivity of leukocyte populations.

• Monocyte and NK cells demonstrate high sensitivity to drug exposure.

• B-cells, T-cells and granulocytes exhibit low to moderate sensitivity.

• Mechanism underlying drug response in specific cell types unknown.

• First description of differential in vivo response to epigenetic inhibitor in a cell type specific

manner.

• Highly sensitive monocyte and NK cell populations compose only 14% and 6% of total peripheral blood leukocytes.

• Total ΔH3K27me3/H3 at 500mg/kg based on contribution of individual populations = -18.1%.

• Strong inhibition of underrepresented cell types may be squelched by ubiquitous resistant populations.

M o n o c y t e s ( S S C l o w C D 1 1 b + )

N K c e l l s ( C D 3 - C D 4 5 R - C D 4 9 b + )

B - c e l l s ( C D 4 5 R + C D 3 - )

G r a n u l o c y t e s ( S S C h i C D 1 1 b + G R - 1 + )

T - c e l l s ( C D 3 + C D 4 5 R - )

0

2 0

4 0

6 0

8 0

1 0 0

0

2 0

4 0

6 0

% I

nh

ibit

ion

H3

K2

7m

e3

/ H

3

% C

om

po

sitio

n

% ∆ H 3 K 2 7 m e 3

% T o t a l C o l l e c t e d C e l l s

ΔH3K27me3/H3: -85.2% ΔH3K27me3/H3: -3.2%

ΔH3K27me3/H3: -69.5% ΔH3K27me3/H3: -7.1%

ΔH3K27me3/H3: -21.2%

Monocytes

NK cells

T-cells

Granulocytes

B-cells

• Chromatin flow cytometry demonstrates good agreement with conventional ELISA quantification.

• Total inhibition by chromatin flow calculated as sum of fractional inhibition of all populations.

Gating scheme for human peripheral blood. Whole blood is collected in Na Heparin vacutainers followed by red blood cell lysis. Cells are fixed in 4% methanol-free formaldehyde and permeabilized with 0.1% Triton X-100. Processed cells are Fc-blocked and stained with anti-CD3-V500, CD19-BV421, HLA-DR-PE, CD16-PE-Cy7, CD14-PerCP-Cy5.5, H3-

AlexaFluor 647, H3K27me3-AlexaFluor 488. Populations are defined as follows:

• H3K27me3 levels are normalized to total H3 content for each population of interest.

• Chromatin flow cytometry demonstrates the rank order of H3K27me3/H3 in drug naïve human peripheral blood leukocyte populations.

• Rank order demonstrates some inter-patient variability.

• B-cells have greater H3K27me3/H3 than T-cells in some individuals.

• Change in total H3K27me3/H3 in peripheral blood of patients in NHL trial at cycle 1 day 1 and cycle 1 day 15.

• Percent change of H3K27me3/H3 from baseline (cycle 1 day 1) to cycle 1 day 15.

• Similar to rodent studies, monocytes are highly sensitive to inhibitor treatment.

• Granulocytes also demonstrate high sensitivity in humans.

• N=1 data (not shown) suggests response is durable at day 30 of dosing.

• First demonstration of pharmacodynamic monitoring of epigenetic modulation by flow cytometry in human trials.

Chromatin flow cytometry based quantification of cell type specific alterations in histone methylation states resulting from in vitro and in vivo EZH2 inhibitor treatment

• Chromatin flow cytometry has been validated in cell lines as a means to monitor epigenetic modifications with compound treatment • Mouse in vivo dose response studies and PD monitoring in human clinical trials establish chromatin flow cytometry as an effective means to quantify epigenetic modulation on discrete cell types

www.epizyme.com

CONCLUSIONS