An integrated approach reveals monocyte chemoattractant protein 1 (MCP-1)

modulates trametinib resistance in Acute Myeloid LeukemiaModak, R.,1 Gosline S.J. 3, Damnernsawad, A.,1 Laderas, T.,2 Wu, G.,2 Nestor M.,3 Piehowski P., 3 Tyner, J.,1 Rodland, K.,3, 4

McWeeney, S.,2 Agarwal, A1, 4

1Division of Hematology and Medical Oncology, The Knight Cancer Institute, Oregon Health & Science University, Portland, OR 97239, 2Department of Medical Informatics and Clinical Epidemiology, The

Knight Cancer Institute, Oregon Health & Science University, Portland, OR 97239, 3Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 4Department of Cell, Development

and Cancer Biology, School of Medicine, Oregon Health & Science University, Portland, OR 97239

Introduction

Acute myeloid leukemia (AML) is characterized by an aberrant expansion of myeloid

progenitors in the bone marrow and peripheral blood.

AML is a heterogeneous disease with a poor five-year survival rate, <30% even after

treatment with conventional chemotherapy.

Current AML therapies have provided little improvement in achieving complete remission. This

is attributed to:

• Development of drug resistance due to novel mutations.

• Extrinsic factors from the microenvironment that promote AML progression.

Cytokines and growth factors from the AML microenvironment promote leukemogenesis, AML

progression and drug resistance by modulating cell survival, proliferation and differentiation of

cells.

Methods and Results

Figure 1: Data integration reveals that drug resistance correlates with

cytokine regulation pathways

(A) Schematic of data integration components- Inhibitor screen from 350 AML patient samples classified patients into

sensitive and resistant categories. RNA sequencing and cytokine screen were integrated with the sensitive and resistant

patients to identify resistance signatures. (B) Heat-map of pathway analysis upon integrating RNA-seq data with inhibitor

response profile. Red box highlights cytokine pathways altered with trametinib-resistance. (C) Heat-map integrating

cytokine expression data with inhibitor response profile. Red box highlights increased MCP-1 levels (x axis) intersecting

with trametinib-resistance (y axis).

Figure 4: MCP-1 stimulation leads to activation of distinct

phosphorylation signatures in MOLM13 parental cells

CCR2

β-actin

MCP-1

Figure 6: Phosphoproteomics reveals novel targetable pathways in

trametinib resistant MOLM13 cell lines (JNK, SRC, CDK)

MOLM13 cells

± MCP-1

trametinib

Measure

1) Viable cells

2) IC50

1 2 3

-20

0

20

40

60

80

100

120

Log Concentration (nM)

% V

iab

ilit

y (

wrt

un

tre

ate

d c

on

tro

l)

R4

Parental

R8R7R6R5

R3 Trametinib 100nM

Trametinib 100nM+MCP-1 10ng/ml

0 2 4 6 8 10 12 140

1×109

2×109

3×109

Weeks in culture

Cell n

um

ber

Trametinib (n=4)

Trametinib +MCP-1 (n=4)

***

(A)

Resistant Sensitive

Trametinib

***

Lo

g C

cl2

exp

ressio

n

(A) MOLM13 AML cell lines were treated with trametinib (0.1-1µM) only or trametinib (0.1-1 µM) + MCP-1 (10ng/ml) for 4

months (n=4). (B) Viability in the presence of trametinib, determined using the MTS assay for MCP-1 treated cells. (C)

Cell numbers over 14 weeks of treatment with trametinib or combination of trametinib and MCP-1.

(A)

(A)

Conclusion and Perspectives

• Data integration revealed specific cytokines

levels correlated with resistance to therapy.

• MCP-1 correlated with trametinib resistance in

AML patients.

• Trametinib resistant cell lines upregulate MCP-1.

• Phosphoproteomics revealed novel pathways in

trametinib resistance such as JNK, SRC, and

cell cycle regulation are dependent on CDK4/6.

• Combination therapy using trametinib with JNK,

SRC, CDK4 and CDK6 inhibitors mitigates AML

cell survival.

Funding sources: NIH (U54), CPTAC (U01 CA14116), Medical Research Foundation (Early Clinical Investigator Grant) Contact: Rucha V. Modak, modak@ohsu.edu, Anupriya Agarwal, agarwala@ohsu.edu)

(A) (B) (C)

Protein synthesis

RNA processing MetabolismImmune System

Toll-like ReceptorsCytokine signaling

Cytokine data

Inhibitor screen

Significantly up-reg in resistant

Insignificant

Significantly down-reg in resistant

Tram

Tram

+ M

CP-1

0

100

200

300

400

500

Tra

meti

nib

AU

C *

Figure 3: Trametinib resistance in AML cell line models correlates

with MCP-1 expression

Parental AML2

Resistant AML2

CCR2

MCP-1

β-actin

Parental MOLM13

Resistant MOLM13

Small molecule inhibitor screen

Viability assay for 125 drugs

350 AML

samples

Reactome

pathways

Cytokine Analysis

(Luminex assay)

Gene expression

analysis (RNA seq)

Sensitive Resistant

Significant pathways

(FDR < 0.10)

Gene Exp

Inhibitor screen

(B) (C)

Resistant Sensitive

Venetoclax

***

Lo

g C

cl2

exp

ressio

n

(A) (B)

Ven

Ven

+ M

CP-1

0

100

200

300

400

500

Ven

eto

cla

x A

UC *

(A) MCP-1 mRNA levels from AML patient samples that are resistant and sensitive to Trametinib and Venetoclax. (B)

Primary cells from AML patients were cultured for 72 hours in the presence of MCP-1 and the indicated drug. Viability

was determined using a standard MTS assay. Area under the curve (AUC) of primary cells treated with MCP-1 in the

presence of trametinib (Tram) and venetoclax (Ven).

Figure 2: Cytokines modulate resistance and sensitivity in AML

patient samples

MOLM

13-P

MOLM

13-R

1

10

100

1000

MC

P-1

levels

(p

g/m

l)

✱

OCI-A

ML2-

P

OCI-A

ML2-

R

1

10

100

1000

10000

MC

P-1

levels

(p

g/m

l)

✱✱

(A) Trametinib resistant cells were generated from AML cell lines MOLM13, and OCI-AML2 by culturing in increasing

concentrations of trametinib over 4 months. (B) Western blots from parental and resistant MOLM13 and OCI-AML2. Cell

lines were probed for MCP-1 and its receptor CCR2. (C) MCP-1 protein expression measured by an ELISA from the

supernatant of parental and resistant MOLM13 and OCI-AML2 cell lines.

Parental

Resistant

Cell types Treatment

MCP-1

Trametinib

MCP-1 + Trametinib

0, 5 or 60 mins

Figure 5: MCP-1 confers growth advantage in long term cultures of MOLM13 cells

+ + + +

+ + + +

0 5 60 5 60 5 60

Tram 10 nM

MCP-1 10 ng/ml

Time min

P-ERK

Total ERK

P-JNK

GAPDH

(A) Schematic of the experiment showing the cell lines and treatment conditions for Phosphoproteomics. (B) z scores

from the KSEA analysis of MOLM13 cell lines treated with a combination of trametinib (10nM) and MCP-1 (10ng/ml) for

5 minutes (left) and 60 minutes (right). (C) Western blot from MOLM13 parental cell lines stimulated with MCP-1 for 0, 5

and 60 minutes with or without trametinib. Expression of phosphorylated ERK, and JNK were measured.

(A)

(B)(C)

(B)

(C)

Tram

etin

ib

Pal

bocicl

ib

Com

binat

ion

0

100

200

300

AU

C

✱✱✱✱

✱✱✱✱

(A) Heatmap of substrate activity of MOLM13 sensitive and resistant cell lines plotted using log fold change. Substates

of MAPK8, SRC and CDK4/6 are denoted with arrows. (B) MOLM13 trametinib resistant cell lines were cultured in

trametinib alone or in combination with JNK inhibitor SP600125 or CCR2 inhibitor BMS-CCR2 for 3 days and viability

was measured by the MTS assay. (C) MOLM13 trametinib resistant cell lines were cultured in trametinib alone Src/ Abl

inhibitor Dasatinib. Viability represented as Area Under the Curve (AUC). (D) AUC of AML patient samples resistant to

trametinib, and palbocyclib and combination.

(B)

Tram

etin

ib

Tram

+JNKi

Tram

+CCR2i

0

20

40

60

80

100

% V

iab

ilit

y (

wrt

co

ntr

ol)

✱✱✱✱

✱✱✱

(C) (D)

(A)

Tram

etin

ib

Das

atin

ib

200

250

300

350

400

AU

C

✱✱

(C)

Cell proliferation

MCP-

1

Trametinib

Cell survival

Drug resistance

CCR2i

JNKi

Dasatinib

Future Directions• Experiments are underway to investigate the source of MCP-1.

• CRISPR screening is in progress to determine the mediator(s) for trametinib resistance in cells.

• In vivo experiments using patient derived xenograpfts will be performed to test the efficacy of combination therapy on

trametinib resistant samples.