Strong Synergistic Effects with APR-246 and Cisplatin in p53

Strong Synergistic Effects with APR-246 and Cisplatin in p53-Mutant Lung Cancer Cells
Nina Mohell, PhD, Åsa Fransson, PhD, Jessica Alfredsson, PhD, Mikael von Euler, MD PhD, Ulf Björklund, PhD and Lars Abrahmsen, PhD
Abstract #2523
Aprea AB, Solna, Sweden
Presenter: Nina Mohell ([email protected]) www.aprea.com
• Platinum compounds have been used as first-line treatment for many solid tumors including non
small cell (NSCLC) and small cell (SCLC) lung cancer. However, patients with lung cancer often
develop resistance and eventually die of chemotherapy refractory disease.
• The mechanisms of resistance are multifactorial, but two important mechanisms are mutations in
the tumor suppressor protein p53 and drug-induced elevation of intracellular glutathione concentration. Mutations in p53 are common in lung cancer, ranging from 33% in adenocarcinomas to
70% in SCLC (1).
• APR-246 (PRIMA-1MET) is the first compound in clinical development that reactivates mutant p53.
It is a prodrug that is converted to the active form methylene quinuclidinone (MQ), a Michael acceptor that binds to mutant and/or unfolded p53 restoring the correct wild type conformation
and triggering apoptosis (2).
• APR-246 has yielded promising results in a first-in-human clinical trial in patients with hematological
malignancies and prostate cancer (3), and a Phase Ib/II study in combination with platinum-based
therapy in ovarian cancer is ongoing (4).
• We have previously shown strong synergy with APR-246 and platinum compounds in p53-mutant
drug-resistant ovarian cancer cells (5). Moreover, APR-246 completely restored the sensitivity of
cisplatin to p53-mutant resistant ovarian cancer cells (6).
• The aim of the current study was to investigate whether synergistic effect with APR-246 and
cisplatin can also be observed in p53-mutant lung cancer cells, and whether APR-246 can sensitize the cisplatin-resistant lung cancer cells to cisplatin.
Cell lines
Cancer type
p53 status
NC I - H17 70
NS C LC
R 24 8W (h o m.)
+++
SS
• Synergy was also found in the SCLC cell lines NCI-H196 and NCI-H889, with R175H and C242S
mutations, respectively (Table 1).
NCl - H1975
NS C LC
R 273H (h o m.)
+++
SS
NCl - H59 6
NS C LC
G245C (h o m.)
+++
S S*
• Mixed antagonist/additive/synergistic effects were observed in the p53 null cell line HOP-62 expressing no full lenght p53. In these cells the dose-response curve of APR-246 was very steep
and no reliable/consistent combination results with cisplatin could be observed.
NCl - H20 87
NS C LC
V1 57F (h o m.)
+++
S*
NCl - H2110
NS C LC
R1 5 8 P (h o m.)
+
S
P C-14
NS C LC
R 24 8W (h o m.)
+++
SS
• Synergistic effects with APR-246 were also observed with etoposide in the PC-14/CDDP cell line.
P C-14/C DDP
NS C LC
R 24 8W (h o m.)
+++
SS
NCl - H378
S C LC
Y163C (h o m.)
+
S/S S
NCl - H19 6
S C LC
R175H
nd
S
NCl - H187
S C LC
S241C
nd
Ad d
NCl - H 8 8 9
S C LC
C 242 S
nd
S/S S
NCl - H10 4 8
S C LC
R 273C
nd
SS
• Fig.2 shows that co-treatment of cisplatin with APR-246, sensitized the PC-14/CDDP cancer cells
to cisplatin in a dose-dependent manner. The IC50 value of cisplatin decreased from 45 to 25 µM
in the presence of 60 µM APR-246. The PC-14/CDDP cell line has been derived from the parental
PC-14 cell line by chronic cisplatin exposure in vitro (9).
Figure 1.Synergistic effect with APR-246 and cisplatin in lung cancer cell lines.
Strong synergistic effects were observed in NSCLC cell line NCI-H1770 (1a) and in the cisplatin resistant PC-14/CDDP cell
line (1b), while strong synergistic effects were observed in SCLC cell line NCI-H378 (1c). All cell lines carry p53 mutations
frequently occurring in lung cancer.
1A
1B
NCIH1770
Results and Discussion:
PC14-CDDP
(hom. R248W p53)
100
APR-246
Cisplan
Combinaon
Combinaon
80
0.77
40
0.54
20
0.29
60
0.50
40
Cisplan alone
20
0.17
APR-246 (µM)
-
-
-
0.8 - 0.8
1.2 - 1.2
1.8 - 1.8
2.6 - 2.6
APR-246 (µM)
-
-
-
8.9 - 8.9
13 - 13
Cisplan (µM)
-
-
-
- 5.9 5.9
- 8.8 8.8
- 13 13
- 20 20
Cisplan (µM)
-
-
-
- 44 44
- 67 67
NCIH378
(hom. Y163C p53)
100
APR-246
Combinaon
Cell viability (%)
80
20 - 20
- 100 100
+ 20 µM APR-246
80
+ 30 µM APR-246
+ 40 µM APR-246
+ 50 µM APR-246
60
+ 60 µM APR-246
+ 70 µM APR-246
2. Lambert JM, Gorzov P, Veprintsev DB, Söderqvist M, Segerbäck D, Bergman J, Fersht AR, Hainaut P, Wiman KG and Bykov VJ. 2009. PRIMA-1 reactivates mutant p53
by covalent binding to the core domain. Cancer Cell. 15(5):376-88.
3. Lehmann S, Bykov VJ, Ali D, Andrén O, Cherif H, Tidefelt U, Uggla B, Yachnin J, Juliusson G, Moshfegh A, Paul C, Wiman KG and Andersson PO. 2012. Targeting p53 in
vivo: a first-in-human study with p53-targeting compound APR-246 in refractory hematologic malignancies and prostate cancer. J Clin Oncol. 30(29):3633-9.
4. von Euler M, Wiman KG, Gabra H, Brenton JD, Basu B, Vergote I, Gourley C, Smith A, Alfredsson J, Mohell N and Green JA. AACR abstract #CT204, 2015. Preliminary
results from PiSARRO, a phase Ib/II study of APR-246, a mutant p53 reactivating small molecule, in combination with standard chemotherapy in platinum -sensitive
ovarian cancer.
5. Mohell N, Alfredsson J, Uustalu M, Fransson Å, Bykov V, Wiman KG and Björklund U. AACR abstract #3448, 2013. Strong synergistic effects with cisplatin and APR246, a novel compound reactivating mutant p53, in ovarian cancer cell lines and primary cells from patients.
6. Mohell N, Alfredsson J, Fransson Å, Bykov V, von Euler M, Wiman K and Björklund U. AACR abstract #1801, 2014. APR-246, a clinical-stage mutant p53-reactivating
compound resensitizes ovarian cancer cells to platinum compounds and doxorubicin.
7. Jonsson E, Fridborg H, Nygren P and Larsson R. 1998. Synergistic interactions of combinations of topotecan with standard drugs in primary cultures of human tumor
cells from patients. Eur J Clin Pharmacol. 54(7):509-14.
40
8. Gibbons D, Byers L and Kurie J. 2015. Smoking, p53 mutation, and lung cancer. Mol Cancer Res. 2014 Jan;12(1):3-13.
9. Ohmori T, Morikage T, Sugimoto Y, Fujiwara Y, Kasahara K, Nishio K, Ohta S, Sasaki Y, Takahashi, T and Saijo N. 1993. The mechanism of the difference in cellular uptake of platinum derivatives in non-small cell lung cancer cell line (PC-14) and its cisplatin-resistant subline (PC-14/CDDP). Jpn J Cancer Res. 1993 Jan;84(1):83-92.
20
60
• Planning for a Phase Ib/II study in lung cancer is ongoing.
1. http://p53.free.fr The TP53 Web Site (database).
100
0.71
0
1C
• Our results suggest that combination treatment with APR-246
and platinum drugs warrants further investigations in clinical
studies.
References
(hom. R248W p53)
0.22
0
SS = strong synergy (CI < 0.5)
nd = Not determined
*=n=1
PC-14/CDDP
Cisplan
• We observed strong synergistic effect with APR-246 and cisplatin in NSCL cancer cell lines carrying homozygous p53 hotspot mutations; NCI-H1770 (R248W) (Fig. 1a), NCI-H1975 (R273H), NCIH596 (G245C), PC-14 (R248W) and PC-14/CDDP (R248W) (Fig. 1b). All these cell lines expressed a
high level of p53 (Table 1). Synergy or strong synergy was also observed in the SCLC cell line NCIH378 (Fig. 1c) carrying the frequently occurring homozygous Y163C mutation, expressed at lower
levels (Table 1).
SCLC = small cell lung cancer
NSCLC = non-small cell lung cancer
Add = additive (CI = 1.0 ± 0.2)
S = synergy (CI < 0.8)
• Treatment with APR-246 in combination with cisplatin resulted
in synergistic or strong synergistic effect in both NSCL and SCLC
cancer cells, including cell lines with “all cancer” hotspot mutations as well as cell lines carrying p53 mutations occurring more
frequently in lung cancer.
APR-246
Cisplan
Cell viability (%)
60
hom. = homozygous
+ = weak p53 expression
+++ = strong p53 expression
Conclusions:
Figure 2. APR-246 resensitizes the PC-14/CDDP lung cancer cells to cisplatin.
The IC50 value of cisplatin decreased from 45 µM to 25 µM.
(hom. R248Q p53)
100
Cell viability (%)
• Cell viability was determined with FMCA or Cell Titer-Glo assay, p53 gene status by Sanger
sequencing and single strand conformation analysis, and p53 protein expression by Western
blotting. Combination Index (CI) was calculated according to Additive model (7); CI = 1.0 ± 0.2
indicates additive effect, CI < 0.8 synergy and CI < 0.5 strong synergy. The results are shown
as mean±SEM (n=2, if not otherwise stated).
Combination
APR-246
cisplatin
p53 protein
expression
80
Methods:
Table 1. Results from combination studies with APR-246 and cisplatin in lung cancer cell lines with various p53 mutations.
Cell viability (%)
Introduction:
• Synergistic effects were found in the NSCLC cancer cell lines NCI-2087 (hom.V157F) and NCI-H2110
(hom. R158P), expressing high and low levels of p53, respectively (Table 1). These mutations are
smoking-related and occur more frequently in lung cancer than in other forms of cancer (1). Many
of the mutations observed in relation to tobacco exposure are due to guanine to thymine transversion (8).
0.84
0.83
40
0
0.78
20
0.50
0
APR-246 (µM)
-
-
-
1.8 - 1.8
2.6 - 2.6
4.0 - 4.0
5.9 - 5.9
Cisplan (µM)
-
-
-
- 20 20
- 30 30
- 44 44
- 67 67
0
20
40
Cisplan (µM)
60
80
Disclosures
Mohell, Fransson, Alfredsson, von Euler, Björklund and Abrahmsen: Aprea AB