Combination of paclitaxel, bevacizumab and MEK162 in second line treatment in

platinum-relapsing patient derived ovarian cancer xenografts.

Ricci F1, Guffanti F1, Damia G2, Broggini M1.

Author information

Abstract

Advanced ovarian cancer is very responsive to first line platinum therapy,

however almost invariably it relapses with a resistant disease. We have reported
that patient derived ovarian xenografts (PDXs), independently from the degree
of the initial response to cisplatin (DDP), show a significantly lower response to a
second DDP cycle. We here report the effect of new combination regimens
containing a MEK inhibitor (MEK), bevacizumab (BEV) and paclitaxel (PTX) as
second line therapy in platinum-relapsing PDXs.We selected three DDP-
relapsing PDX models based on the presence of activation of the
RAS/RAF/MEK/ERK axis, mutated p53, lack of PTEN expression and activation
of the PI3K pathway. In all the selected xenograft models, the antitumor efficacy
of the doublets can be summarized as PTX/BEV>BEV/MEK>PTX/MEK and the
antitumor activity of the triple combination was higher than any double
combination. All the different combinations were well tolerated. The present
data corroborate the activity of bevacizumab in combination with chemotherapy
for the treatment of relapsing ovarian tumors and suggest that the addition of
another targeted agents (MEK inhibitor) can further increase the antitumor
activity without any increase in toxicity. PDX models represent a useful model to
test second line therapy after failure of DDP first line.

Keywords

Ovarian cancer Second line Patients derived xenografts Cisplatin Drug

resistance Combination chemotherapy

Background

Epithelial ovarian cancer (EOC) is one of the most deadly tumor of woman,
causing roughly 100,000 deaths/year in western countries [1]. Advanced ovarian
cancer, which represents more than 70% of all EOC, is well responsive to first
line therapy (mainly consisting of platinum-based drugs after cytoreductive
surgery), however almost invariably relapses with a tumor no more responsive to
platinum-based therapies. Patients are then treated in second line with different
combination treatments, including topotecan, trabectedin, pegylated liposomal
doxorubicin and recently bevacizumab [2, 3, 4, 5, 6, 7], although the response and
overall survival (OS) after relapse are far from being satisfactory [8].
Antiangiogenic therapies, and in particular bevacizumab has been recently
approved in second line treatment in ovarian cancer patients after relapse to
platinum-based therapies [9].
There is an urgent need to find new therapeutic approaches for ovarian tumors
relapsing after a platinum based therapy. In the last years, patient derived
xenografts (PDXs) of ovarian cancers, based on the transfer of primary tumors
directly from patients to immune-deficient mice, have been generated and
characterized [10, 11, 12, 13]. These PDXs not only recapitulate the tumor of
origin in terms of clinico-pathological characteristics and genetic alterations, but
also show resistance patterns similar to that observed in the clinic. These models
represent translational models to possibly study precision medicine approaches
to increase the prognosis of ovarian patients and in particular we think will be
instrumental to better understand and overcome platinum resistance. We have
recently reported that in several models, independently from the degree of the
initial response to cisplatin (DDP), re-growing tumors show a significantly lower
response to DDP-based treatment [14]. These in vivo settings represent good
models to study new therapies and combinations in these challenging condition,
i.e. re-growing tumors after an initial platinum response [10, 11, 12, 13, 14].

We here reported the effect of new combination regimens consisting of doublets

or triplets, containing inhibitor of MEK (MEK162), placlitaxel and/or
bevacizumab in platinum-relapsing ovarian PDXs.

Findings

All the experiments were performed in female NCr-nu/nu mice (6 weeks old)
obtained from ENVIGO RMS srl (Correzzana, Italy) maintained under specific
pathogen free conditions, housed in isolated vented cages, and handled using
aseptic procedures. The IRCCS-Istituto di Ricerche Farmacologiche Mario Negri
adheres to the principles set out in the following laws, regulations, and policies
governing the care and use of laboratory animals: Italian Governing Law (D. lg
26/2014; Authorization n.19/2008-A issued March 6, 2008 by Ministry of Health);
Mario Negri Institutional Regulations and Policies providing internal
authorization for persons conducting animal experiments (Quality Management
System Certificate- UNI EN ISO 9001:2008 Reg, No.6121); the NIH Guide for
the Care and Use of Laboratory Animals (2011 edition) and EU directives and
guidelines (EEC Council Direcrive 2010/63/UE). The Statement of Compliance
(Assurance) with the Public Health Service (PHS) Policy on Human Care and
Use of Laboratory Animals was recently reviewed (9/9/2014) and will expire on
September 30, 2019 (Animal Welfare Assurance #A5023-01).

The three ovarian PDX models used for these experiments (MNHOC 124,
MNHOC 218 and MNHOC 239) have been described in details [10, 14]. The
tumors, re-growing after a DDP treatment, were selected on the basis of their
growth, expression of markers and response to platinum (Table 1 and Additional
file 1: Figure S1). Specifically, tumors were implanted subcutaneously in nude
mice and when the tumors reached approximately 150 mm3 were treated with
DDP 5 mg/Kg iv q7dx3. This regimen induced a roughly 90% reduction in tumor
growth in all the three selected models. However, after a variable period, the
tumors re-started to grow and were much less responsive to a subsequent cycle of
DDP (Additional file 1: Figure S1). These re-growing, platinum-relapsing tumors
were excised and re-implanted in additional mice to test, in a second line-like
setting, the activity of the combinations under investigation. As for the first
line setting, when the relapsing tumors reached approximately 120-150 mm3,
they were randomized to receive vehicle, paclitaxel (PTX, 20 mg/Kg iv q7dx3)
plus bevacizumab (BEV, 5mg/Kg ip q7dx3), PTX plus MEK162 (MEK, 3.5-10
mg/Kg po bid daily for 14 days), BEV plus MEK or PTX plus BEV plus MEK.
Tumor growth was measured with a Vernier caliper every two-three days, and
tumor weights (mg=mm3) were calculated using the formula: (length
[mm]*width [mm]2)/2. The efficacy of the treatment was expressed as best tumor
growth inhibition [%T/C=(tumor weight mean of treated tumors/tumor weight
mean of control tumors)*100]. Toxicity was monitored recording animal weights
and physical examination every day for all the duration of the experiment.

Table 1

Characteristics of the three PDXs used

PDX ID DIAGNOSIS TP53 status IHC

PTEN NF1 p-ERK
MNHOC124 Serous/endometroid mut - ++ +++
MNHOC218 Endometroid mut - + ++
MNHOC239 Serous mut - ++ ++

IHC Immunoistochemistry. Protein expression was cytoplasmic with no nuclear

or membrane-associated staining. The intensity of the staining has been ranked
according to the following scale: - negative; +/- slight/doubtful; + slight; ++
moderate; +++ marked

Mut: mutated

The immunohistochemical evaluation of NF1, PTEN, ERK and pERK was

performed in four m sections from each tumor xenograft, which were incubated
with the following primary antibodies: rabbit polyclonal antibody to NF1
(Abcam, ab30325), rabbit monoclonal antibody to PTEN (Cell Signaling, 138G6),
rabbit monoclonal antibody to ERK1 (Epitomics, 1172-1), rabbit monoclonal
antibody to phospho-ERK1/2 (Cell Signaling, 4370) and incubated with
biotinylated secondary antibody (goat anti- rabbit). Sections were labeled by the
avidin-biotin-peroxidase (ABC) procedure with a commercial immunoperoxidase
kit. The immunoreaction was visualized with 3,3-diaminobenzidine (DAB)
substrate and sections were counterstained with Mayers hematoxylin.
All the three models have an activation of the RAS/RAF/MEK/ERK axis with
high expression of NF1 and high phosphorylation of ERK, display a mutated p53
and lack PTEN expression, leading to activation of the PI3K pathway.

Figure 1 shows the tumor growth of all the different combinations and control
(no-treated) group in MNHOC124 PDX. In this model, the combination of MEK
(3.5 mg/Kg) plus BEV or PTX had only marginal activity, while the combination
of BEV and PTX induced tumor regression. However, tumor re-growth was
observed after 47 days from the end of treatment. The addition of MEK to this
doublet did not change significantly the response. Table 2 shows the T/C% values
of the different experimental groups. PTX/BEV and PTX/BEV/MEK groups T/C
values are 5.4% and 5.5% at day 38 respectively, corresponding to more than
90% of tumor weight inhibition. The treatment with BEV/MEK was active too
(even if not superior to the previous), with a T/C% value of 27.5%, while the
PTX/MEK combination was the least active (T/C%=55.5%). All the different
combinations were well tolerated with only minor changes in body weight (%
body weight loss BWL- of 1-2%, Table 2, Additional file 2: Figure S2).

Fig. 1

Antitumor activity of the combination of PTX, bevacizumab and MEK162 in

MNHOC124 PDX model. Xenograft MNHOC124 was transplanted
subcutaneously and when tumor masses reached 100-150mg, mice were
randomized to receive vehicle (CTR,--), or bevacizumab and MEK162
(BEV/MEK,--), paclitaxel and MEK162 (PTX/MEK,--), paclitaxel and
bevacizumab (PTX/BEV, --), or paclitaxel and bevacizumab and MEK162
(PTX/BEV/MEK,-x-). Data are the meanSE of tumor masses, as described in
Materials and Methods; each group consisted of 8-10 mice

Table 2

T/C% values and BWL% of the different treatments in the MNHOC124,

MNHOC218 and MNHOC239 PDXs

MNHOC124 MNHOC218 MNHOC239

T/C% BWL% T/C% BWL% T/C% BWL%
(day) (day) (day) (day) (day) (day)
PTX/BEV 5.4 (38) - 6.6 (46) 0.8 (31) 23.8 (64) -
PTX/MEK 55.5 (38) - 45.2 (46) 5.6 (38) 49.2 (61) 0.3 (28)
BEV/MEK 27.5 (38) 2.3 (31) 16.2 (16) 0.2 (38) 25.5 (61) -
PTX/BEV/MEK 5.5 (38) 1.3 (31) 0.7 (46) 4.7 (31) 13.7 (47) -

T/C% values are considered significant of drug response when 42% (NCI
guidelines). BWL: Body Weight Loss
The activity of the same combinations in the MNHOC218 model is reported in
Fig. 2 and Table 2, where the treatment with MEK was increased to a dose of
10mg/Kg and extended to 22 days due to the lack of toxicity observed in the
MNHOC124 model. In this model the combination of PTX and MEK had
marginal activity, while both BEV/PTX and BEV/MEK resulted in a significant
reduction of tumor weight. The triple combination had the greater effect with a
T/C value of 0.7% compared to 6.6% and 16.2% in the PTX/BEV and BEV/MEK
respectively. Interestingly, the effect of the triple combination was maintained
for several days. In the BEV/PTX group, the tumors started to re-grow
approximately 55 days following implant, while the re-growth of the tumors was
delayed of approximately 20 days in the triple combination. This increase in
response was not associated with an increase in BWL%; the triple combination,
in fact, induced changes in body weight comparable to the PTX/MEK
combination and these changes were transient, with full recovery upon drug
withdrawal (Additional file 2: Figure S2). In both these tumor models, when
tumors treated with the triple combination re-grew, the growth rate was similar
to the one of untreated mice, with no evidence of accelerated growth.

Fig. 2

Antitumor activity of the combination of PTX, bevacizumab and MEK162 in

MNHOC218 PDX model. Xenograft MNHOC218 was transplanted
subcutaneously and when tumor masses reached 100-150mg, mice were
randomized to receive vehicle (CTR,--), or treated with Bevacizumab and
MEK162 (BEV/MEK,--), paclitaxel and MEK162 (PTX/MEK,--), paclitaxel
and bevacizumab (PTX/BEV, --), or paclitaxel and bevacizumab and MEK162
(PTX/BEV/MEK,-x-). Data are the meanSE of tumor masses, as described in
Materials and Methods; each group consisted of 8-10 mice

Finally, the three drugs were tested in MNHOC239 PDX model (Fig. 3, Table 2).
Overall, all the treatments displayed less activity than the one observed in the
previous models. The most active combination was the triple one even if,
differently from the other models, only tumor stabilization and no tumor
regressions were observed. Interestingly enough, tumor stabilization was
followed by a tumor re-growth (upon drug withdrawal) whose rate was lower
than that of untreated controls. BEV/MEK or BEV/PTX combinations showed
high activity, while only a limited one was observed in mice treated with the
doublet PTX/MEK. The triple combination induced a higher regression with T/C
value of 13.7%. As was for the other models, the four different combinations did
not induce significant changes in animal body weights (Additional file 2: Figure
S2).

Fig. 3

Antitumor activity of the combination of PTX, bevacizumab and MEK162 in

MNHOC239 PDX model. Xenograft MNHOC239 was transplanted
subcutaneously and when tumor masses reached 100-150mg, mice were
randomized to receive vehicle (--), or treated with Bevacizumab and MEK162 (-
-), paclitaxel and MEK162 (--), paclitaxel and bevacizumab (--), or paclitaxel
and bevacizumab and MEK162 (-x-). Data are the meanSE of tumor masses, as
described in Materials and Methods; each group consisted of 8-10

We have been able to recapitulate the ovarian clinical setting in which the initial
response to platinum-based therapy is generally followed by a relapse (within a
variable time frame) and the re-growing masses are much less sensitive to a
second challenge with platinum [14]. These preclinical PDX models represent a
framework where new drugs or new drug combinations can be rapidly tested. In
addition, clues of possible spectrum of toxicities could also be uncovered in this
setting. We here tested the ability of the MEK inhibitor MEK162 to improve the
response of second line therapy in PDX ovarian xenografts.

While in ovarian cancer paclitaxel is both used in front-line with platinum and in
second line therapy, topotecan [15], pegylated liposomal doxorubicin [16] and
trabectedin [17] have been approved in the platinum resistant recurrent setting
with marginal activity. A recently approved targeted drug for the treatment of
platinum-resistant EOC is bevacizumab, a humanized anti-VEGF monoclonal
antibody with an antiangiogenic activity [3]. Indeed, four phases III trials have
demonstrated a prolonged progression free survival (PFS) in patients receiving
bevacizumab in combination with front-line chemotherapy (GOG protocol 18 [18]
and ICON7 [19]), in combination with chemotherapy in platinum-resistant
(Aurelia trial [20]) and in platinum sensitive recurrent EOC (OCEANS trial)
[21]. OS did not reach a statistically significant difference, even if in subgroups
analyses a trend of improved OS was found in the bevacizumab treated arm [19].

Inhibitors of MEK represent an emerging class of potentially active targeted

agents for different reasons [22, 23, 24]. However, clinical activity of MEK
inhibitors as single agents has been reported in malignancies with RAS and B-
RAF mutations, as melanoma, while limited activity has been observed in
unselected cancer patients [23].

The RAS/RAF/MEK/MAPK pathway has been shown to be activated by gene

copy number aberration and or mutation in EOC [25]. It has been reported that
in high grade serous ovarian carcinoma, MEK can be constitutively activated not
only through BRAF but also through other MAP kinases such as MAPK8 kinase
[26]. In addition, it was recently reported that high intra-tumor MAPK was an
independent predictor of worse survival in EOC and that combined treatment
with MEK inhibitors and fulvestran (an estrogen receptor antagonist) effectively
reduced ovarian cancer xenografts growth [27]. The over activation of the
pathways has been correlated with a decrease chemo-sensitivity, including to
platinum containing drugs.

The hypothesis that recurrent DDP resistant ovarian xenogratfs tumors could
benefit from chemotherapeutic regimens containing MEK inhibitor and drugs
clinically used in a second line setting was herein tested and the results obtained
clearly indicate that the combination of paclitaxel and bevacizumab is active in
specific setting, confirming clinical data [18, 19, 20, 21]. The combination of
MEK162 and paclitaxel or MEK162 and bevacizumab has much less activity
than the paclitaxel-bevacizumab doublet thus not supporting the use of MEK162
as doublet partner for each of this single drug. However, we have shown evidence
that the triple combination in which MEK162, added on top of beva-paclitaxel
combination, is indeed able to induce long lasting response in second line.

The use of PDXs to predict and test second line therapy has been recently
reported in melanomas [28, 29]. In their study the PDX models were directly
obtained from BRAF inhibitor-progressing tumors and gave interesting
information on possible new therapies. Our study has some important
differences from the one of Krepler et al. The PDXs we used were originally
developed from platinum responsive patients and were used in second line after
relapse from platinum therapy in vivo in nude mice. We have in this way
recapitulated the clinical situation of initial response and taken the tumor soon
after it relapsed. These relapsing PDXs were treated with drugs clinically used
in relapsing ovarian tumors in combination with a selective MEK inhibitor. This
has allowed us to confirm the good response to paclitaxel-bevacizumab and at the
same time to prove that the addition of a third drug (MEK162) increased the
activity with apparently no increase in toxicity.

Conclusions

Three PDX ovarian models mimicking the clinical setting, i.e. relapsing,
platinum resistant with activated AKT pathways and activated ERK, were
selected to test the activity of poli-chemotherapeutic regimens containing
paclitaxel, bevacizumab and a MEK inhibitor. In all the models the triple
combination was well tolerated and show an antitumor activity higher than the
double combinations. These results corroborate both the activity of bevacizumab
in combination with chemotherapy for the treatment of ovarian tumors and that
this antitumor activity can be further improved by the addition of another
targeted agents (MEK inhibitor) and strongly support the use of relapsing PDXs
for the discovery of new potential treatment in second line.

Abbreviations
BEV:

Bevacizumab

BWL:

Body weight loss

DDP:

Cisplatin

EOC:

Epithelial ovarian cancer

MEK:

MEK inhibitor

OS:
Overall survival

PDX:

Patient derived xenografts

PFS:

Progression free survival

PTX:

Paclitaxel

Declarations

Acknowledgements

The generous contributions of The Italian Association for Cancer Research

(IG12915) is gratefully acknowledged.

Funding

This work was supported by an unrestricted grant from Novartis.

Availability of data and materials

All data generated or analysed during this study are included in this published
article and its supplementary information files.

Authors contributions

FR, FG GD and MB conceived and planned the study; FR and FG performed the
experiments, FR and GD analysed the data; GD and MB wrote the manuscript.
All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable

Ethics approval and consent to participate

Al the experiments in animal have been performed following the EY rules on

theuse of animals in research. The protocol for anuimal experimentation has
been approved by the local ethical committee and by the Italian Ministry of
Health (Authorization 60/2014-PR).

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Additional file 1: Figure S1. A) Tumor growth inhibition after DDP treatment in
ovarian PDXs. PDXs were treated (--) or not (--) with cDDP. The mean
standard error of the tumor weight (mg) of each experimental group at different
time points is represented. Each triangle indicates a DDP treatment (one cycle
consisting of three weekly treatment), and each group consisted of 810 mice. B)
Quantification of DDP antitumor effect in the different ovarian cancer PDXs.
The histograms represent the mean standard error of the slope of the
interpolation lines in untreated/control and DDP-treated groups ((white box)
CTR, (grey box) 1st DDP cycle, and (black box) 2nd DDP cycle) in ovarian cancer
xenografts (MNHOC124, MNHOC218, and MNHOC239) *p < 0.05, **p < 0.005,
***p < 0.0005. (PDF 386 kb)
Additional file 2: Figure S2. Body weight of animals treated with vehicle (CTR,-
-), with Bevacizumab and MEK162 (BEV/MEK,--), paclitaxel and MEK162
(PTX/MEK,--), paclitaxel and bevacizumab (PTX/BEV, --), or paclitaxel and
bevacizumab and MEK162 (PTX/BEV/MEK,-x-). Data are expressed as
meanSD, each group consisted of 8-10 animals. A) MNHOC124 PDX model, B)
MNHOC218 PDX model and C) MNHOC239 PDX model. (PDF 474 kb)

References