mdr1 poster aacr-2014v4-final
TRANSCRIPT
Nandini Rudra-Ganguly, Christine Lowe, Mukta Shiwalkar, Claudia I. Guevara, Deanna L. Russell, Christopher C. Kemball, Michelle Wu, Cyrus Virata, Alla Verlinsky, Ssucheng J.Hsu, Mike Mattie, Yuriy Shostak, William Yeh, Peng Yang, Sung-Ju Moon, Ingrid Joseph, David R. Stover, Daniel S. Pereira and Dowdy Jackson
Agensys Inc., an Affiliate of Astellas Pharma Inc., 1800 Stewart Street, Santa Monica, CA 90404
Abstract # 1976
Abstract Cytotoxic Potency of Chemotherapies and ADC Payloads in AML Models ABCB1 Activity Is Determined by Dye Efflux
ABCB1 Is Expressed in AML Cell Lines
ABCB1 and ABCC1 Expression and Activity in Normal Donors & AML Patients
Conclusions & Future Directions
Multidrug resistance may play a critical role in developing cancer therapeutics for acute myeloid leukemia (AML). The expression of the ATP binding cassette (ABC) transporter Pgp/MDR1, also known as ABCB1, has been reported in AML patients and correlates with poor clinical outcome. Additional transporters like MRP1 (ABCC1) and BCRP (ABCG2) also appear to contribute towards this resistance mechanism. We have profiled a panel of AML tumor cell lines and tumor xenografts for both expression and activity of Pgp/MDR1 using qRT-PCR, FACS, and Meso Scale Discovery (MSD) assays. Our profiling also includes analysis of the known SNPs associated with these transporters in key models. We have evaluated the sensitivity of the AML models to a series of cytotoxic agents used as chemotherapy or payloads for antibody drug conjugates (ADCs), in both in vitro and in vivo studies. Several of these cytotoxic agents are known substrates of ABC transporters. Using Pgp/MDR1 inhibitors, we could restore the sensitivity of the Pgp/MDR1 expressing cells to the cytotoxic drugs and ADCs, which confirmed that the drugs were Pgp/MDR1 substrates. We have also observed limited anti-tumor activity in vivo with these ADCs when used in a MDR positive model. Our initial observation indicates that a threshold level of activity of MDR1 may be critical to confer resistance to these standard of care molecules and antibody drug conjugates using similar payloads. Currently, we are investigating the contribution for other efflux pumps like MRP1 and BCRP in this mechanism of drug resistance.
MDR Expression And Activity May Serve As A Potential Biomarker In Developing Therapeutic Drugs For AML Patients
ABCB1 RNA levels were evaluated in AML and ALL cell lines by qRT-PCR. Expression levels were normalized to GAPDH and the average expression of duplicates are reported in 10-4 GAPDH units (+/-STDEV).
ABCB1 surface expression in AML xenografts was validated by qRT-PCR, FACS, and MSD. Data presented are from three tumors for each xenograft.
KG-1>Hel92.1.7>CMK
Cell Surface Expression is Confirmed by Flow cytometry
Unstained Isotype ABCB1-Tumor1 ABCB1-Tumor2 ABCB1-Tumor3
CMK MFIR: 4 Hel92.1.7 MFIR: 12 MV-4-11 MFIR: 1 MOLM-13 MFIR: 1 KG-1 MFIR: 26
101
102
103
104
105
106
107
Rhodamine 1,2,3
0
Cell C
ounts
101
102
103
104
105
106
107
Rhodamine 1,2,3
0
Cell C
ounts
Active Pump Inactive Pump
Unstained Unstained
No efflux at 37⁰C
90’ at 37⁰C
90’ at 4⁰C or 37⁰C + inhibitor
Fluorescence signal is reduced at 37⁰C due to dye efflux. Dye is retained at 37⁰C with the use of inhibitor.
Dye Efflux Dye Accumulation
102
103
104
105
106
BL1-H:: BL1-H
0
Coun
t
101
102
103
104
105
106
107
BL1-H:: BL1-H
0
Coun
t
101
102
103
104
105
106
107
BL1-H:: BL1-H
0
Coun
t
Hel92.1.7 MAF: 95 CMK MAF: 70 MAF: 0 MOLM-13
ABCB1 activity correlates with pump expression in Hel92.1.7, CMK & MOLM-13 cells. ABCB1 inhibitor completely blocks dye efflux.
Efflux Activity Assessed in CMK, Hel92.1.7 & MV-4-11 Cells
ABCC1 Expression and Activity in AML Cell Lines
ABCC1 Activity Confirmed by Dye Efflux
ABCC1 RNA levels were evaluated in AML and ALL cell lines by qRT-PCR. Expression levels were normalized to GAPDH and the average expression of duplicates are reported in 10-4 GAPDH units (+/-STDEV). ABCC1 has a 50-fold range of RNA expression across the cell line models evaluated by qRT-PCR. Highest expression is seen in Kasumi-3 cells, followed by J.RT3-T3.5, GRANTA-519, SEM, Kasumi-6, CMK and KG-1 cells.
MV4
-11
Mol
m-1
3
KG-1
Hel9
2.1.
7
EOL1
CMK
HL60
T98G
ABCC1
GAPDH
ABCC1 /MRP1 Protein Expression in AML Cell Lines
Total ABCC1 Expression Confirmed by FACS
ABCC1 expression in AML cell lines was validated by qRT-PCR, FACS and western. CMK>Hel92.1.7≅KG-1 Expression of ABCC1 correlates with pump activity and sensitivity to Daunorubicin, a substrate for MRP1.
AML Cell Line
ABCC1 RNA levels (10-4 GAPDH units)
ABCC1 Intracellular (MFIR)
ABCC1 (Western) Efflux pump Daunorubicin
IC50 (nM)
CMK 518 20 Strong +++ 24.4
Hel92.1.7 133 17 Strong +++ 38
KG-1 451 14 Strong +++ N/A
EOL-1 195 9.6 Modest +/- 3.49
MV-4-11 75 8 Weak-neg +/- 2.65
MOLM-13 34 5 Weak-neg +/- 3.7
Hel92.1.7 CMK MAF: 96 KG-1 MAF: 81 MOLM-13 MAF: 43 MV-4-11 MAF: 27
37C: Untreated 37C: DMSO 37C: MK-571 4C: Untreated 4C: Unstained
MAF: 85 MAF: 31 EOL-1
Cells were pre-loaded with 5,6-CFDA and incubated at 4⁰C or 37⁰C with or without inhibitor for 1 hour Values calculated as MDR Activity Factor (MAF): ((MDRi-MDR0)/MDRi)*100.
EOL-1 MOLM-13 MFIR: 8 Hel92.1.7 MFIR: 17 MFIR: 20 CMK KG-1 MFIR: 14 MV-4-11 MFIR: 5 MFIR: 10
Unstained Isotype ABCC1
• We have identified ABCB1 and ABCC1 expression and activity in AML tumor cell lines and xenografts.
• High ABCB1 expression and activity appears to confer resistance to cytotoxins that are ABCB1 substrates in vitro and in vivo.
• ABCC1 expression and pump activity as well as ABCB1 activity was confirmed in a small panel of AML patients and normal donors.
• Our ongoing investigation may further elucidate what roles ABCB1 and ABCC1 play in drug resistance.
• Additionally, literature reports have shown that ABCG2/BCRP may also contribute to resistance and its role should also be investigated.
ABCB1 RNA levels were evaluated in AML and ALL cell line xenograft models by qRT-PCR. The average ABCB1 expression of biological replicates is reported in 10-4 GAPDH units (+/- SEM). High levels of ABCB1 RNA were detected in Hel92.1.7 and KG-1 xenograft models.
Average of two biological replicates
ABCC1 is expressed in AML xenografts and correlates with expression in vitro.
MV4
-11
Mol
m-1
3
KG-1
Hel9
2.1.
7
EOL1
CMK
HL60
T98G
A549
ABCC1
GAPDH
ABCC1 Expression in AML Xenografts
ABCC1 RNA levels were evaluated in AML and ALL cell line xenograft models by qRT-PCR. The average ABCB1 expression of biological replicates is reported in 10-4 GAPDH units (+/- SEM). Highest ABCC1 RNA levels were detected in KG-1 and CMK xenograft models with moderate to high expression in all other models tested.
ABCC1/MRP1 expression was validated in AML xenograft models by western blot. T98G and A549 were used as positive controls.
IC50 (nM) Inhibitor Hel92.1.7 MOLM-13 CMK MV-4-11
Ara-C 35.9 68.8 37.8 200.9 Daunorubicin 38.1 3.7 24.4 2.7
Etoposide 177.8 42.9 562.4 13.7 Mitoxantrone 176.9 96.7 11.4 0.8
MMAE 3.2 0.3 0.6 0.3 Epothilone B 1.6 0.3 1.7 0.4
AML/ALL Cell Line
ABCB1 RNA levels
(10-4 GAPDH units)
CCLE SNP 6.0 Amino acid change
CCLE SNP 6.0 rs104562
ABCB1 (MFIR)
ABCB1 MSD (signal to
background)
Efflux Activity
MMAE IC50 (nM)
MMAE ADC IC50
(nM)
TF-1a 275 ND Yes 12-30 41.63 ++ 2.3 X
KG-1 254 ND Yes 11-18 19.20 ++ 2.4-11 X
Hel92.1.7 170 N21D(rs9282564) ND 6-11 10.14 ++ 1.5-10 X
SEM 23 ND Yes 4-10 3.5 - 0.3 NP
OCI-AML5 0.3 S400N(rs2229109) Yes 4-9 NP - 0.4 NP
UT-7 28 ND Yes 1.8-2.7 NP + 0.2 X
CMK 45 ND ND 1.6-2.0 NP + 0.2 X
PL-21 0.5 ND Yes 1.6-3 2.5 - 0.3 NP
HL-60 0.1 N21D(rs9282564) Yes 1.1 0.99 - 0.03-4.3 1260
MV-4-11 1 ND Yes 1.1 0.89 - 0.2-2.4 0.1-0.9
EOL-1 1 N21D(rs9282564) Yes 1.1 0.88 - 0.02 X*
MOLM-13 0.5 ND Yes 1.1 0.88 - 0.1 0.9
THP-1 4 ND NP 1.0 1.27 - 0.01 0.1
*No Target Expression, ND=None Detected, NP=Not Performed, X=No activity at 10µg/ml
ABCB1 Expression, Activity, and Cytotoxic Sensitivity in vitro
Expression and Activity of MMAE ADC in vitro and in vivo
In vitro: MOLM-13 & Hel92.1.7 cells were treated with varying concentrations of MMAE ADC. MOLM-13 (ABCB1 negative) shows sensitivity to MMAE ADC, while Hel92.1.7 (ABCB1 positive) does not demonstrate cytotoxicity. Target expression confirmed by FACS. In vivo: Hel92.1.7 & MOLM-13 cells were inoculated SQ in CB17/SCID mice. Tumors were measured by caliper twice per week until study termination. Target expression confirmed by IHC. Hel92.1.7 tumors were analyzed for ABCB1 expression and activity by efflux.
ABCB1 expression and efflux activity confirmed in xenografts
ABCB1 and ABCC1 RNA levels were evaluated by qRT-PCR in normal (donor) and AML tumor (patient) blood and bone marrow samples. ABCB1 appears to have decreased RNA expression in AML patient samples relative to the normal donors samples. ABCC1, however, appears to have similar RNA expression levels in the AML patient and normal donor samples.
Sample ID Disease Source Form/Stage
Patient 1 AML PBMC Relapsed/Refractory Patient 2 AML PBMC, BM M1, Newly Diagnosed Patient 3 CLL transforming to AML PBMC, BM Newly Diagnosed Patient 4 AML PBMC Newly Diagnosed Patient 5 AML PBMC, BM Newly Diagnosed
Patient 10 AML Bone Marrow Newly Diagnosed Patient 11 AML Bone Marrow Relapsed/Refractory Patient 12 AML PBMC Newly Diagnosed Patient 13 AML PBMC M3, Relapsed/Refractory Donor 10 Normal PBMC N/A
Normal and AML samples were stained with antibodies specific for ABCB1 to measure surface expression. Intracellular ABCC1 expression was measured by fixing the samples. MFIR values were calculated by dividing the MFI of target antibody by the isotype control. Efflux activity was assessed by dye efflux in normal and AML samples for ABCB1 and ABCC1.
ID Source Stage ABCB1 ABCC1
Expression (MFIR)
Activity (MAF)
Expression (MFIR)
Activity (MAF)
Patient 10 BM Newly diagnosed 1 11 6 4 Patient 11 BM Relapsed/Refractory 2 0 32 20 Patient 12 PBMC Newly diagnosed 2 44 33 39 Patient 13 PBMC Relapsed/Refractory 2 46 55 42 Donor 10 PBMC Normal 4 65 26 34
FACS: Expression & Activity
ABCB1 expression in AML tumors inversely correlates with the activity of MMAE ADC.
ABCC1 expression and activity was confirmed in both normal and patient populations. Limited ABCB1 expression in AML was detected by FACS.
However, both normal donors and AML patients express both pumps by western.
RNA Expression by qRT-PCR
Patient Samples
In vivo Activity
In vitro Activity
Target MFIR: 82 Target MFIR: 25
ABCB1 Is Expressed in AML Xenografts
ABCC1 Is Expressed in AML Xenografts
High ABCB1 expression and activity appears to confer resistance to cytotoxins that are ABCB1 substrates in vitro and in vivo.
Cell Surface Expression is Confirmed by Flow Cytometry CMK MFIR: 2
Unstained Isotype ABCB1
MV-4-11 MFIR: 1 KG-1 MFIR: 18 Hel92.1.7 MFIR: 11 Molm-13 MFIR: 1
Determination of ABCB1 Expression using MSD
The MSD assay was developed using cell lysates from a SKOV3 Taxane resistant model (positive) and SKOV3 isogenic model (negative). Validation was completed using a ABCB1 recombinant protein (Origene). The range of detection : 0.06-1000 ng. Data presented as a ratio of signal to background.
ABCB1 surface expression in AML cell lines was validated by FACS and total protein by MSD TF-1a>KG1≅Hel92.1.7>CMK>UT7
ABCB1 has a 100-fold range of RNA expression across the AML and ALL cell line models evaluated by qRT-PCR. Highest expression in Kasumi-3, TF-1a, KG-1 and Hel92.1.7 cells
ABCB1 protein levels were evaluated in AML cell line xenograft models by MSD. Hel92.1.7 and KG-1 xenograft models expressed highest levels of ABCB1.
ABCC1 protein levels were evaluated in AML cell lines by western blot. T98G was used as a positive control.
ABCB1 expression and activity confirmed by FACS and dye efflux, respectively. ADC Treatment does not affect ABCB1 expression or activity in Hel92.1.7 xenografts.
Acknowledgement: ADC Technology Licensed from Seattle Genetics, Inc.
Differences in sensitivities of AML cell lines to chemotherapies and ADC payloads may be attributed to expression of drug pumps.
37C: Untreated 37C: DMSO 37C: Verapamil 4C: Untreated 4C: Unstained
Non-Responders
Responders
Normal bone marrow Leukemia samples T98G
ABCC1
GAPDH
ABCB1
GAPDH
ABCB1 ABCC1
Normal bone marrow Leukemia samples SKOV3
-Tax +Tax
Western Blot: Expression
Normal and AML samples were evaluated for expression of ABCB1 and ABCC1. ABCB1 and ABCC1 was detected in both normal and AML samples. SKOV3 (-Tax) used as a negative control for ABCB1, while SKOV3 (+Tax) was used as a positive control. T98G was used as a positive control for ABCB1.
Cells were pre-loaded with Rhodamine 1,2,3 and incubated at 4⁰C or 37⁰C with or without inhibitor for 90 minutes. Acquisition using the Attune® Acoustic Focusing Cytometer, Blue/Violet. Values were calculated as MDR Activity Factor (MAF): ((MDRi-MDR0)/MDRi)*100.