Download New type of drug-resistant isogenic cell model created by

New type of drug‐resistant isogenic cell model created by CRISPR genome editing Lysa‐Anne Volpe, M.S., Metewo Selase Enuameh, Ph.D., Michael Jackson, Ph.D., Luping Chen, B.S., John Foulke, M.S., Elizabeth Turner, Ph.D., Stephen King, M.S., and Fang Tian, Ph.D.
American Type Culture Collection, ATCC Cell Systems, Manassas, VA 20110
AACR Abstract # 3836
NRAS Clone D5
3.98e-007
C
107.6%
100.0%
A375 parental line
NRAS isogenic clone D5
80.0%
49.8%
43.9%
40.0%
12.9%
20.0%
0.0%
/ Genotyping/sequencing primers IC50
F
A375
2.907e-008
The KRASG13D isogenic line is also resistant to BRAF inhibitors
NRAS Clone D5
1.519e-006
BRAF inhibitor Vemurafinib treatment
120.0%
102.3%
100.0%
80.0%
60.0%
ds Q61K Donor
500bp
1000 nM
10 nM
1000 nM
Figure 5. Drug response of the A375 parental line and NRASQ61K isogenic lines. CellTiter‐Glo
luminescent cell viability assay was used to evaluate cell line drug response to the BRAF
inhibitors vemurafenib and dabrafenib, as well as the chemotherapy drugs doxorubicin and
etoposide. Data are expressed as mean SD (n=4). NRASQ61K isogenic clone D5 (ATCC® CRL‐
1619IG‐2™) exhibits drug resistance to the BRAF inhibitors relative to the parental A375 cell line
(C, D, E, and F). The responses of the A375 and NRASQ61K isogenic lines to non‐specific
chemotherapy drugs were similar (A and B).
B
A375
3.094e-008
KRAS Clone DIB4
6.55e-005
IC50
A375
4.056e-009
KRAS Clone DIB4
1.184e-007
Figure 8. The same method that was used to create the NRAS isogenic line was
employed to generate the KRASG13D isogenic line (ATCC® CRL‐1619IG‐1™). A) The
KRASG13D knock in was validated by sequencing. B) The KRASG13D isogenic line
demonstrated drug resistance to BRAF inhibitors relative to the parental A375 cell line.
Data are expressed as mean SD (n=4).
648bp nested PCR product
Discussion
B
We used CRISPR/Cas9 genome editing technology to target endogenous loci in
human cells and create precise genetic point mutations. Unlike conventional drug
resistance cell models that have been developed through anti‐cancer therapy clonal
drug resistance selection procedures, the A375 NRASQ61K and KRASG13D isogenic cell
lines represent advanced in vitro cell models that contain defined genetic drug
resistance mechanisms. The RAS‐mutant isogenic cell lines sustain the permanent
and genetically stable drug resistance characteristics without being maintained in
anti‐cancer therapy selection culture environments. Therefore, they provide
valuable tools to facilitate the development of next‐generation therapeutics that can
overcome BRAF drug resistance in melanoma.
Figure 3. A) Screening for the NRASQ61K point mutation integration in recovered clones was carried out using PCR primers as
shown left, with the gel image of PCR products on the right. B) Introduction of the NRASQ61K point mutation in the cell line was
confirmed via sequencing primers shown in A. Boxed in red is the expected C>A mutation.
Confirmation of NRASQ61K mRNA expression in single cell clones
isolate mRNA → cDNA synthesis →PCR → sequence analysis RAS‐mediated resistance to BRAF inhibitor in melanoma2
PCR primer
ATG
E1
E1
E2
E2
E2
E4
E3
E5
E4
E3
E4 E5
E2 E3
E1 E2 E3 E4 E5
500bp
ds G13D Donor
/ Genotyping/sequencing primers ERK and AKT activation is reduced in the NRAS isogenic line B
*GAC (D)
16.3%
20.0%
1105bp LongAmpPCR
CRISPR genome editing technology1
Figure 1. BRAFV600E melanoma cells chronically treated with
BRAF inhibitors acquire drug resistance by switching between
the three isoforms of RAF (ARAF, BRAF, CRAF) to activate the
MAPK pathway. Increased growth factor receptor signaling or
reactivation of the MAPK pathway may allow for resistance.
This can occur following the acquisition of activating mutations
in NRAS (Q61K), MEK (C121S), and elevated expression of other
key components of the pathway.
A
40.0%
Deletions via cut sites
*AAA (K)
A
5’
GGC
59.2%
IC50
1250bp
A
G13
A375 parantal line
NRAS isogenic clone D5
70.9%
Q61
CAA
400mm
0.0%
10 nM
PCR and sequencing to confirm NRASQ61K knock‐in mutation 400mm
Survival rate (%)
60.0%
400mm
Figure 7. Spheroid 3D cultures were generated by seeding cells in triplicate on a low
attachment plate for 4 days. Cultures were examined under the microscope after
treatment with the BRAF inhibitor dabrafenib for 10 days. NRASQ61K isogenic line
showed resistance to BRAF inhibitor, which was consistent with the 2D culture results.
NRAS Clone D5
1.042e-006
BRAF inhibitor Dabrafenib treatment
120.0%
400mm
NRAS Clone D5
5.238e-008
Survival rate (%)
E
A375
1.407e-008
A375
4.093e-008
IC50
D
IC50
5’
Background
A375
2.447e-007
Survival rate (%)
B
IC50
Survival rate (%)
Figure 2. A) Schematic of the target
region for integration of the knock‐in
DNA bearing the NRASQ61K point
mutation. B) Cell clone generation
workflow: Transfected cells were
enriched by puromycin selection for 48
hrs. After puromycin recovery, the
cells were sorted into single cells.
Clonal expansion and validation was
then performed at the genomic,
transcriptional, translational, and
functional levels on the cells.
isogenic line is resistant to BRAF inhibitors in 3D culture
Survival rate (%)
A
NRASQ61K
B
A
Design and generation of NRASQ61K mutant cell clones Cell survival rate (%)
A mutant BRAF gene can lead to uncontrolled cell growth through overactivation of the RAS‐RAF‐
MAPK signaling pathway. The BRAFV600E mutation occurs in approximately 40% to 50% of
melanomas. Although current BRAF inhibitors have been used to successfully treat melanomas
containing the BRAFV600E mutation, patients often become resistant to BRAF inhibitors within a few
months. A number of clinical studies have indicated that secondary mutations in RAS or NF1 are
associated with BRAF resistance. However, due to the genetic heterogeneity commonly observed
in tumors, it is unclear if those secondary mutations already existed within low percentage
subclones, or if they were acquired through drug treatment. Further, it has yet to be determined
whether such genetic variants are only associated with resistance, or whether they actually cause
the BRAF inhibitor resistance. In this study, we used genome editing CRISPR technology to
generate two drug‐resistant melanoma cell lines (ATCC® CRL‐1619IG‐2™ and CRL‐1619IG‐1™) that
contain NRASQ61K or KRASG13D mutations. These isogenic lines were derived from the parental A375
(ATCC® CRL‐1619™) melanoma cell line, which naturally contains BRAFV600E. When compared to the
parental line, the isogenic cell models demonstrated that genetically modified NRAS or KRAS genes
at the endogenous level directly leads to significant resistance to BRAF inhibitors.
.
Single guide RNAs (sgRNAs) were designed and built to guide Cas9 to bind and cut desired regions
in the NRAS or KRAS gene targets. The parental cell line A375 was co‐transfected with the single
guide and CRISPR all‐in‐one plasmid alongside a donor plasmid. Transfected cells were sorted into
single cells and expanded for subsequent screening of desired gene mutation events. The
introduction of the NRASQ61K or KRASG13D mutation in the cells was then confirmed via Sanger
sequencing and NGS at the genetic and transcriptional levels. Drug responses to BRAF‐specific
inhibitors and non‐specific chemotherapy drugs were compared between RAS isogenic A375 cell
lines and parental A375 cell line in 2D and 3D culture environments. Testing results demonstrated
that the isogenic cell lines created by CRISPR showed significant resistance to BRAF inhibitors in
comparison to the parental control in both 2D and 3D culture environments. These two novel in
vitro cell models with endogenous level RAS mutants provide direct biofunctional evidence that
acquiring a drug‐resistant gene drives tumor cell survival under targeted therapeutic treatment.
.
isogenic line is resistant to BRAF inhibitors
Cell survival rate (%)
Results
Survival rate (%)
Introduction
NRASQ61K
E5
E6
E7
NRAS exons & introns
E7
NRAS processed mRNA
E7
C
mutation
Figure 4. A) NRAS mRNA in cell clones
carrying
the
Q61K
mutations.
Screening for NRASQ61K point mutation
in recovered clones was carried out via
cDNA generation from cells and
performing PCR (red arrows). B) Gel
image of the PCR products. C) The
introduction of the NRASQ61K point
mutation in the cell line was confirmed
via Sanger sequencing on the right.
Boxed in red is expected point
mutation.
References
Figure 6. Activation of AKT and ERK pathways mediates the
NRASQ61K isogenic line’s resistance to BRAF inhibitor. Parental A375
cells and NRASQ61K isogenic cells were treated with 1 uM
Vemurafenib or DMSO for 1 hour and 4 hours. Cells were then
harvested, lysed, and analyzed by immunoblotting with antibodies
against phospho‐EGFR, total EGFR, phospho‐AKT, phospho‐ERK
and GAPDH (loading control).
1. Ran F, et al. Genome engineering using the CRISPR‐Cas9 system. Nat Protoc 8(11): 2281‐308, 2013. 2. Sanchez‐Laorden B, et al. BRAF inhibitors induce metastasis in RAS mutant or inhibitor‐resistant melanoma cells by reactivating MEK and ERK signaling. Sci Signal 7(318): ra30, 2014. © 2017 American Type Culture Collection. The ATCC trademark and trade name, and any other trademarks listed in
this publication are trademarks owned by the American Type Culture Collection unless indicated otherwise.
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