effective targeting of triple negative breast cancer cells...

Effective Targeting of Triple Negative Breast Cancer Cells by PF-4942847,

a Novel Oral Inhibitor of Heat Shock Protein 90

Pramod P. Mehta1, Pamela Whalen1, Sangita M. Baxi1, Pei-Pei Kung2, Shinji Yamazaki3,

and Min-Jean Yin1

1Oncology Research, 2Medicinal Chemistry, 3Pharmacokinetics, Dynamics and

Metabolism, Pfizer Worldwide Research and Development,

10724 Science Center Drive, San Diego, CA 92121

Corresponding author: Min-Jean Yin

Email: [email protected]

Tel: (858) 622-7438

Fax: (858) 526-4121

Running title: Targeting TNBCs by a Hsp90 inhibitor PF-4942847

Keywords: Hsp90, AKT, oral cancer drug, triple negative breast cancer

Research. on February 16, 2019. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 29, 2011; DOI: 10.1158/1078-0432.CCR-11-0592

http://clincancerres.aacrjournals.org/

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Abstract

Purpose: Triple negative breast cancer (TNBC) patients have poor prognoses and

survival outcomes such that the development of new targeted therapies is in strong

demand. Mechanisms associated with high proliferation and aggressive tumor

progression, such as PI3K/PTEN aberration, EGFR overexpression, and cell cycle up-

regulation, play important roles in TNBC. The molecular chaperone heat shock protein

90 kDa (Hsp90) is required for the conformational maturation and stability of a variety of

proteins in multiple pathways, such as EGFR, AKT, Raf, cdk4, etc. Therefore, an Hsp90

inhibitor may demonstrate therapeutic benefit in TNBC by targeting multiple pathways.

Experimental Design: The novel oral Hsp90 inhibitor PF-4942847 was characterized in

multiple in vitro and in vivo assays to determine its antitumor activity in TNBC cell lines.

In addition, the correlation of AKT degradation and Hsp70 induction in host peripheral

blood lymphocytes (PBLs) and xenograft tumors was determined.

Results: PF-4942847 induces degradation of multiple client proteins, cell cycle block,

apoptosis, and inhibits cell proliferation in TNBC lines, subsequently leading to tumor

growth inhibition in mouse xenograft models. The correlation of AKT degradation and

Hsp70 induction between PBLs and xenograft tumors reveals a differential modulation of

Hsp90 activity between host and tumor tissues, and suggests that AKT degradation in

PBLs may serve as a pharmacodynamic biomarker in future clinical development.

Conclusions: The novel oral Hsp90 inhibitor, PF-4942847, is a candidate for clinical

development in TNBC by collaboratively targeting multiple signaling pathways.

Additionally, AKT degradation in PBLs may serve as a biomarker in clinical

development.




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Translational Relevance

There is a strong need to develop new targeted therapies for triple negative breast cancer

(TNBC) patients. The work presented here shows that a novel oral Hsp90 inhibitor, PF-

4942847, exhibits in vitro and in vivo efficacy in multiple TNBC cell lines; these results

further suggest that PF-4942847 may be of clinical benefit in TNBC patients. The

correlation of AKT degradation between mouse PBLs and xenograft tumors was

determined in response to drug treatment and indicates that AKT degradation in patient

PBLs may have the potential to serve as a biomarker to predict drug modulation in

tumors.




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Introduction

Heat shock protein 90 kDa (Hsp90) is a molecular chaperone that regulates the

conformation, stability and activity of numerous key signaling proteins, including protein

kinases (e.g., C-Raf, AKT, ErbB family, Cdk4), steroid receptors (e.g., androgen receptor

and estrogen receptor), and transcription factors (e.g., HIF1α). These Hsp90 client

proteins are involved in multiple pathways of cell transformation and tumor progression;

therefore, targeting Hsp90 offers an opportunity to inhibit multiple pathways in cancer (1,

2). Several natural products, such as geldanamycin and radicicol, bind to Hsp90 at the

NH2-terminal ATP pocket and inhibit the ATPase activity of Hsp90 subsequently leading

to client protein degradation through ubiquitin ligase machinery (3). The geldanamycin

derivative 17-allylamino-17-demethoxy-geldanamycin (17-AAG), 17-DMAG, and IPI-

504 have been developed as potential therapeutics in a variety of clinical trials (4-6).

However, 17-AAG is poorly soluble and has low oral bioavailability, metabolism issues

and hepatotoxicity (7, 8). Because of the potential toxicity of geldanamycin derivatives,

specific small molecular weight Hsp90 inhibitors may be more effective clinical agents.

Several small molecular weight Hsp90 inhibitors, including SNX-5422, CNF2024, STA

9090, and AUY 922, are currently in clinical trials in various tumor types; it is too early

to differentiate these inhibitors from the geldanamycin derivatives in the clinical setting

(9-12).

Breast cancer is a heterogeneous disease for which there are a variety of

biological features, natural history, and treatment options. Gene expression profiling has

allowed us to classify breast cancers into five subtypes based on distinctive gene

expression signatures (13). These five subtypes are luminal A, luminal B, HER2




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positive, basal-like, and normal-like breast cancers. Basal-like tumors are characterized

by the expression of genes that are specific for basal epithelial cells and involved in

cellular proliferation, suppression of apoptosis, cell migration, and cell invasion (13-15).

Basal-like breast cancers (BLBC) are often stained negative by immunochemistry for

estrogen receptor (ER), progesterone receptor (PR), and HER2; this BLBC subpopulation

are thus called triple negative breast cancers (TNBC). Although BLBC and TNBC share

numerous clinical and pathologic features, they are not identical (16, 17). In the majority

of cases, however, these two categories share similar clinical features, prognoses and

treatment options. For convenience, the term “TNBCs” will be used in this study to

collectively describe BLBC and TNBC cell lines and patient populations. Clinical

studies have shown that TNBCs are more aggressive and patients have poorer prognoses

than the other breast cancer subtypes (18, 19). Therefore, the development of targeted

therapies for TNBCs is clearly needed to help this patient population. More than 60% of

TNBCs express EGFR and this could serve as a prognostic marker for TNBC outcomes

(20, 21). Activation of the PI3K pathway either by frequent PTEN alteration, elevated

PIK3CA expression or oncogenic mutation has been identified in TNBCs and is also

associated with a poor prognosis (22-24). Hsp90 regulates multiple signal transduction

pathways by maintaining stability and activity of client proteins such as EGFR, AKT,

Raf, and Cdk4; therefore, targeting Hsp90 function may provide an opportunity to inhibit

tumor progression of TNBCs through EGFR, PI3K, and other proteins (25).

A series of 2-amino-5,7-dihydro-pyrrolo[3,4-d]pyrimidine-6-carboxylic acid

amide compounds were previously described as potent and specific Hsp90 inhibitors; the

detailed structure activity relationship (SAR) has also been reported (26). One of the




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compounds from this series, PF-4942847, had a low Ki value (6 nM) and exhibited

selectivity in the Invitrogen Kinase Panel against 35 other measured kinases (<10%

inhibition at 1 μM) (26). PF-4942847 was characterized in this study to demonstrate in

vitro and in vivo antitumor activity in a variety of human TNBC cell lines. PF-4942847

induces AKT protein degradation, cell cycle block, apoptosis, and inhibits cell

proliferation in TNBC lines, subsequently leading to tumor growth inhibition of MDA-

MB-231 and MX-1 in mouse xenograft models. The correlation of AKT degradation and

Hsp70 induction between peripheral blood lymphocytes (PBLs) and xenograft tumor was

also determined and reveals a differential modulation of Hsp90 activity between host and

tumor tissues in response to drug treatment suggesting that AKT degradation in PBLs can

potentially be used to guide clinical studies for TNBC.

Materials and Methods Hsp90 inhibitor. PF-4942847 was synthesized as previously described (26). PF-

4942847 was dissolved in DMSO for in vitro cellular assays and formulated in 40%

PEG-400/60% saline (v/v) for animal studies.

Cell lines. All cell lines were purchased from the American Type Culture Collection and

cultured according to ATCC instructions. The gene mutation status of cell lines was

obtained from the Sanger COSMIC database: http://www.sanger.ac.uk/.

Immunoblotting. Cells and xenograft tumors were lysed in lysis buffer (50 mM Tris-

HCl, 0.5% NP-40, 0.5% Triton X-100, 150 mM NaCl, 1 mM Na3VO4, 1 mM NaF, and

protease inhibitor cocktail). Protein (50 μg) was resolved by SDS-PAGE and western

blots were performed with various antibodies (Actin from Sigma, all others from Cell

Signaling) to detect proteins of interest.




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Luminex and MesoScale assays. Cells (10,000/well) were seeded in a 96-well microtiter

plate and cultured overnight. PF-4942847 was added to each well at a top concentration

of 10,000 nM with a 3-fold serial dilution ending at 0.0169 nM. After 24 hours, cell

lysates were prepared and analyzed following the manufacturer’s instructions. The

Luminex 100 system (Upstate) was used for AKT protein measurement, and MesoScale

Discovery technology was used for Hsp70 protein measurement.

Cell cycle profiling. TNBC cells were seeded and cultured overnight prior to compound

treatment. Cells were harvested, fixed, stained with propidium iodide (PI), and analyzed

by flow cytometry. A total of 10,000 events were analyzed for each sample, and the

experiments were repeated at least three times.

Cell proliferation and caspase 3/7 assay. For cell proliferation assays, TNBC cells were

seeded at 3000 cells/well in a 96-well plate; PF-4942847 was added to each well as

described above and incubated for 72 hours, followed by addition of 250 μg/ml of

Resazurin (Sigma). After incubation for an additional 6 hours at 37oC, plates were

analyzed by a florescence reader. For the caspase 3/7 assay, cells were plated and

compound was added as described; the cells were assessed by the Caspase 3/7 Assay

(Promega) following the manufacturer’s instructions.

Animal studies. Six- to eight-week-old nu/nu athymic female mice and SCID female

mice were obtained from Jackson Labs; the mice were maintained in pressurized

ventilated caging at the Pfizer La Jolla animal facility. All studies were done in

compliance with Institutional Animal Care and Use Committees guidelines. Tumors

were established by injecting 2x106 cells suspended 1:1 (v/v) with reconstituted basement

membrane (Matrigel, BD Biosciences). For PK/PD studies, mice with established tumors




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of ~400mm3 were randomized and treated with PF-4942847 QD for two days to establish

steady-state of drug exposure. For tumor growth inhibition studies, mice with established

tumors of ~150 mm3 were randomized and treated with vehicle, PF-4942847, or 17-

DMAG. Tumor dimensions were measured with vernier calipers, and tumor volumes

were calculated using this formula: π/6 x (larger diameter) x (smaller diameter)2. Tumor

growth inhibition percentage (TGI %) was calculated as 100 x (1-∆T/∆C). For the

survival study, dosing was stopped as indicated in the graph for all animals and

measurements were taken three times per week; animals were sacrificed and noted

“dead” when the tumor size reached 1500 mm3. For the tumor regression study, mice

with established tumors of ~300mm3 were randomized and treated with vehicle or PF-

4942847 three times per week.

Determination of PF-4942847 concentration in plasma. Plasma concentrations of PF-

4942847 were determined by liquid chromatography tandem mass spectrometry (LC-

MS/MS) following protein precipitation of plasma samples as previously described (27).

Statistical analysis. Prism 5.0 Software (GraphPad Software, San Diego, CA) was used

for statistical analysis and graph generation. Unless otherwise stated, error bars indicate

SD, and p values of <0.05 are denoted by an asterisk (*), p values of <0.005 are denoted

by two asterisks (**), and p values of <0.0005 are denoted by three asterisks (***). A t-

test was performed in the AKT degradation and Hsp70 induction experiments to compare

results from tumor versus PBLs, and the survival study was evaluated by one-way

ANOVA.

Results




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PF-4942847 induces degradation of multiple Hsp90 client proteins in human triple

negative breast cancer cells

We first examined the cellular activity of PF-4942847 (Fig. S1) in a number of

human TNBC cell lines. PF-4942847 was added to MDA-MB-231 cells for multiple

treatment time points, and total protein and phosphorylation levels of AKT, EGFR, cMet,

B-Raf, C-Raf, ERK, and Hsp70 were determined by western blot. The effect of PF-

4942847 on protein degradation and reduction in phosphorylation of Hsp90 clients was

observed beginning at 6 hours after compound addition and was more definite after the

18 hour time point (Fig. 1A). We also observed a preferential loss of p-AKT versus total

AKT at 6 hr and 18 hr (Fig. 1A) suggesting that there may be other key clients degraded

by PF-4942847 that account for this activity. This is most likely due to PF-4942847

interference of Hsp90 maintenance of the stability and functional conformation of client

proteins. Similar results were observed with HCC-70 and MX-1 cells treated with PF-

4942847 (Fig. 1B). Additionally, PF-4942847 induced B-Raf protein degradation

selectively in the cells harboring a mutant B-Raf allele (MDA-MB-231 and MX-1) over

those with two wild type copies (HCC-70). This is consistent with previous studies that

B-Raf mutant protein, but not wild-type B-Raf, is degraded by Hsp90 inhibitors (28-30).

Additionally, the degradation of RTKs such as EGFR and cMet by PF-4972847 leads to

the suppression of ERK phosphorylation in TNBCs, while Hsp70 induction in treated

cells is due to the feedback mechanism in the heat shock protein machinery (31, 32).

Next, a panel of TNBC cell lines was used to investigate AKT protein degradation and

Hsp70 protein induction by plate-based assays to quantitatively determine the AKT

degradation IC50 and Hsp70 induction EC50 values. As shown in Fig. 1C and 1D and




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summarized in Table I, PF-4942847 exhibits IC50 values ranging from 20 nM to 100 nM

in the AKT degradation assay and EC50 values ranging from 20 nM to 40 nM in the

Hsp70 induction assay in a variety of TNBC cell lines. 17-DMAG was included in the

AKT and Hsp70 assays and results indicate that PF-4942847 and 17-DMAG have

comparable effects on the inhibition of Hsp90 activity (Table I).

PF-4942847 blocks cell cycle progression and induces apoptosis resulting in inhibition

of cellular proliferation in TNBCs

PF-4942847 has the ability to induce protein degradation in the signaling

pathways of RTKs (including cMet, EGFR, Ras-Raf-ERK, and AKT); as such, it was

very likely that PF-4942847 would block cell cycle progression and induce cellular

apoptosis via targeting of multiple important signaling pathways in TNBCs. Therefore,

we next investigated cell cycle block and apoptosis activation by the treatment of PF-

4942847 in TNBCs. Results indicate that PF-4942847 induces cell cycle block at the

G2/M transition in both MDA-MB-231 and MX-1 cells (Fig. 2A and 2B, Fig. S2).

Moreover, high concentrations of PF-4942847 also increase the sub-G1 cell population

indicating an increase in apoptotic cells (Fig. 2A and 2B, Fig. S2). PF-4942847 also

induced cellular apoptosis in MDA-MB-231 and MX-1 cells as determined by the

induction of caspase 3/7 activity (Fig. 2C). We next measured the inhibitory activity of

PF-4942847 on cellular proliferation. Results presented in Fig. 2D and summarized in

Table I show that PF-4942847 effectively inhibits cell proliferation in TNBCs with IC50

values ranging from 20 nM to 60 nM. The inhibitory potency of PF-4942847 and 17-

DMAG is comparable. PF-4942847 was also evaluated in cell lines representative of

other breast cancer subtypes; these results demonstrate that PF-4942847 inhibited cellular




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proliferation in a number of breast cancer cell lines with different backgrounds (Table

SI). This further suggests that the inhibition of multiple pathways by disruption of Hsp90

activity is effective in inducing anti-proliferative activity in a variety of breast cancers.

These results clearly indicate that PF-4942847 is a potent Hsp90 inhibitor that induces

protein degradation of multiple key signaling pathways, blocks cell cycle progression,

and induces apoptosis; these combined activities subsequently inhibit cellular

proliferation in TNBCs.

PF-4942847 induces Hsp90 client protein degradation and Hsp70 elevation in MDA-

MB-231 xenograft tumors.

We next determined the in vivo activity of PF-4942847 in mouse xenograft

studies. A MDA-MB-231 xenograft model was used to perform the in vivo experiments

and investigate the relationship between plasma drug exposure and modulation of Hsp90

activity by PF-4942847. In order to reach the steady-state of drug exposure, PF-4942847

was orally administered QD for two days to MDA-MB-231 tumor bearing mice, and free

unbound PF-4942847 was detected in mouse plasma in a dose- and time-dependent

manner after the second dose (Fig. 3A). The free unbound concentrations of PF-4942847

in mouse plasma at seven hours post-dose were 103 nM and 152 nM at 30 mg/kg and 50

mg/kg, respectively, both of which are above the in vitro AKT degradation IC50 value

(20 nM, Table I). Thus, it was determined that PF-4942847 was likely to inhibit Hsp90

activity in xenograft tumors. At the 30 mg/kg dose, MDA-MB-231 tumors were

dissected at various times to determine time-dependent modulation of Hsp90 activity

(Fig. 3B). Additionally, PF-4942847 was administered at several dose concentrations

QD for two days and tumors were dissected at four hours after the second dose to




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determine dose-dependent modulation of Hsp90 activity. PF-4942847 induces total

protein degradation and reduction in phosphorylation of AKT, cMet, B-Raf, C-Raf,

EGFR, and ERK, and induces Hsp70 elevation in MDA-MB-231 xenograft tumors in a

dose-dependent and time-dependent manner as shown in Fig. 3B. AKT and Hsp70 levels

in tumor lysates were also determined by Luminex and MesoScale Discovery,

respectively, to quantitatively determine the changes (Fig. S3) and these results indicate

that AKT degradation and Hsp70 elevation in xenograft tumors treated by PF-4942847

are dose-dependent and time-dependent. Results from western blot analysis as well as

the Luminex and MesoScale assays indicate that AKT and Hsp70 did not fully recover to

normal levels by 24 hours post-treatment, even though the drug exposure at this time

point is below the detection limit (Fig. 3 and Fig. S3). Therefore, PF-4942847 modulates

Hsp90 activity to induce AKT degradation and Hsp70 elevation in MDA-MB-231

tumors, and the recovery of AKT and Hsp70 is delayed in tumors even when the drug has

been cleared from the plasma.

PF-4942847 triggers differential responses of AKT degradation and Hsp70 induction

between xenograft tumors and mouse peripheral blood lymphocytes.

In order to further aid future clinical development studies, we investigated the

relationship of AKT degradation and Hsp70 induction in response to PF-4942847

between xenograft tumors and mouse peripheral blood lymphocytes (PBLs) in order to

determine whether it was possible to correlate Hsp90 activity changes between tumors

and surrogate tissues. Both MDA-MB-231 tumors and mouse PBLs from the same

animal were collected to measure AKT (by Luminex) and Hsp70 levels (by MesoScale

Discovery technology) to determine Hsp90 activity changes induced by PF-4942847




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between tumors and normal host tissue. Both Luminex and MesoScale methods had been

previously optimized to ensure that appropriate amounts of tumor and PBL samples were

analyzed and that all data points were within the linear range. Therefore, the Luminex

and MesoScale readout of units per μg of protein tested can be quantitatively compared

from sample to sample. At four hours, PF-4942847 induced similar degrees of AKT

degradation in both tumors and PBLs (Fig. 4A). Similarly, at the 30 mg/kg dose, we

observed comparable AKT degradation in tumors and PBLs (Fig. 4B). Although AKT

degradation might be slightly higher in tumors than PBLs, the differences were not

significant with the exception of the 72 hour time point as shown in Fig. 4B.

Interestingly, we observed a significant difference in Hsp70 induction fold change

between tumors and PBLs (Fig. 4C and D). At four hours, Hsp70 induction is dose-

dependent and significantly higher fold changes were observed in mouse PBLs than

tumors at the 50, 30 and 10 mg/kg doses while the differences between tumors and PBLs

were less pronounced at the 3 and 1 mg/kg doses as indicated in Fig. 4C. At the 30

mg/kg dose, we observed significant differential fold changes of Hsp70 induction in

tumors and PBLs up to the 120 hr time point (Fig. 4D). We further analyzed the data by

comparing the AKT and Hsp70 level per μg of protein (Fig. S4); the analysis revealed

that PF-4942847 induced dose- and time-dependent AKT degradation and Hsp70

induction in both tumors and PBLs. However, while the total amount of AKT per μg of

protein is similar between tumors and PBLs, the total amount of Hsp70 per μg of protein

is much less in PBLs than in tumors. Of note, recovery of AKT and Hsp70 in both the

tumor samples and PBLs was delayed even when the drug concentration was below the

lower limit of detection after 24 hours, and the recovery time appeared to be longer in




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tumors than in PBLs in response to PF-4942847 treatment. Detailed percent inhibition of

AKT and fold increase of Hsp70 in tumors and PBLs are summarized in supplementary

table SII. Taken together, the data demonstrate that PF-4942847 inhibits Hsp90 activity

in both MDA-MB-231 tumors and host PBLs, and furthermore exhibits differential

modulation of AKT and Hsp70 between tumors and PBLs. Additionally, these results

reveal that the relative percent change of AKT in MDA-MB-231 tumors and mouse PBLs

is similar while the relative fold increase of Hsp70 in tumors is much less than those in

PBLs.

PF-4942847 inhibits in vivo tumor growth of human triple negative breast cancer cell

lines MDA-MB-231 and MX-1 in mice.

PF-4942847 has demonstrated in vitro and in vivo inhibition of Hsp90 activity in

a variety of TNBCs and in xenograft MDA-MB-231 tumors. We next determined

whether PF-4942847 was able to induce in vivo tumor growth inhibition (TGI) in mouse

xenograft models. MDA-MB-231 and MX-1 were implanted subcutaneously in nude or

SCID-bg mice, respectively, and treated orally once a day with vehicle or PF-4942847 to

perform the TGI experiments. PF-4942847 was well tolerated at 25 mg/kg in nude mice

and at 20 mg/kg in SCID-bg mice with minimal body weight loss (<5%). PF-4942847

induces 91% and 80% TGI, in MDA-MB-231 and MX-1 xenograft models, respectively

(Fig. 5A and B). Furthermore, the effective concentration (EC50) to induce AKT

degradation and tumor growth inhibition of MDA-MB-231 was calculated using a PK/PD

modeling approach as previously described and those results indicated that AKT

degradation (IC50~19 nM) was well correlated to tumor growth inhibition (EC50~12

nM) in MDA-MB-231 (27). Although 17-DMAG exhibits comparable cellular potency




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as PF-4942847 in TNBC lines, 17-DMAG at the maximal tolerated dose (15 mg/kg, BID,

5 days/wk) only showed 56% tumor growth inhibition in the MDA-MB-231 model. This

is consistent with previous findings where 17-AAG and 17-DMAG have demonstrated

only moderate in vivo tumor growth inhibition activity in breast cancer models (33, 34).

Starting from day 29, MDA-MB-231 tumor-bearing mice were maintained without

further dosing, and tumor volume continued to be measured and animal survival was

assessed (Fig. 6A). Mice treated with PF-4942847 at 25 mg/kg had a longer survival

period compared to the vehicle-treated animals (p<0.005). Two mice in the 25 mg/kg

dose group remained tumor-free at day 120 as shown in Fig. 6A. In the MX-1 model,

PF-4942847 treatment at 20 mg/kg also increased survival of tumor-bearing mice

(p<0.05); however, there were no tumor-free animals at the end of the study, and MX-1

tumors progressed very quickly in the low dose groups such that there was not a

significant survival benefit over the vehicle control group (p>0.05) (Fig. 6B). This

suggests that PF-4942847 is critical in stopping tumor progression but not sufficient to

induce tumor cell death in the MX-1 tumor model. Based on our results where AKT

degradation and Hsp70 induction were sustained beyond PF-4942847 plasma clearance,

we also tested an alternative dosing schedule with PF-4942847. MDA-MB-231 tumor

bearing mice were treated with PF-4942847 orally three times a week (Monday,

Wednesday, and Friday) at 75 mg/kg without significant bodyweight loss; we observed

78% tumor regression in this study. At the end of study day 69, four mice were tumor-

free and the tumor measurements of the remaining mice all had a decreasing trend (Fig.

6C). Therefore, results indicate that PF-4942847 is a potent Hsp90 oral inhibitor that

inhibits tumor growth in triple negative breast cancer models.




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Discussion

Preclinical studies have demonstrated the notable sensitivity of HER2-

overexpressing breast tumors to Hsp90 inhibitors (9, 10). 17-AAG (Tanespimycin) is

being developed in the clinic for HER2 positive breast cancer and demonstrates a

moderate clinical response in combination with trastuzumab in patients (35). However,

aside from a study with the IV Hsp90 inhibitor PU-H71 (25), it has not been widely

reported that an Hsp90 inhibitor is effective in inhibition of triple negative breast cancer.

Therefore, we wanted to investigate the potential development opportunities for the oral

Hsp90 inhibitor PF-4942847 in the treatment of triple negative breast cancer. Our results

demonstrate that PF-4942847 inhibited cellular proliferation in a panel of TNBC lines,

eliminated tumor growth in MDA-MB-231 and MX-1 xenograft models, and induced

tumor regression of a MDA-MB-231 xenograft model in mice. In this study, PF-

4942847-induced modulation of AKT and Hsp70 between tumors and host PBLs was

also quantitatively determined and data suggest that AKT degradation in PBLs may be a

better biomarker than Hsp70 induction to predict tumor pharmacodynamic changes in

response to PF-4942847 treatment. Although a number of Hsp90 inhibitors have been

investigated in preclinical and clinical stages, there is not yet an approved drug targeting

Hsp90. PF-4942847 is an orally bioavailable Hsp90 inhibitor with superior physical and

pharmacokinetic properties that differentiate it from other intravenous inhibitors such as

PU-H71 and the geldenamycin derivatives. Additionally, PF-4942847 has demonstrated

excellent anti-tumor activity in preclinical TNBC models as described in this study. Our

results suggest a potential opportunity for PF-4942847 to be developed as an oral anti-

cancer drug in TNBC patients who need better treatment options.




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In the current study, mouse peripheral blood lymphocytes (PBLs) were isolated

from MDA-MB-231 tumor bearing mice and then subjected to Luminex and MSD

technology for quantitative detection of AKT and Hsp70, respectively. These new

approaches are superior to conventional western blot analysis as relatively small amounts

of protein are required for the assays and measurements are quantitative compared to

immunoblot analysis. Additionally, these assays can potentially be done with patient

samples that are easily and noninvasively obtained. Our data indicate that PF-4942847

affects Hsp90 function in both tumors and PBLs as AKT reduction and Hsp70 elevation

were observed in both sample sets. We also observed a differential pattern in the changes

of AKT and Hsp70 levels between MDA-MB-231 tumors and PBLs by PF-4942847

treatment. The relative fold change of Hsp70 induction by drug treatment is much higher

in PBLs than tumors, suggesting that the measurement of Hsp70 induction fold changes

in PBLs may over-estimate the changes of Hsp90 activity in tumors. In contrast, the

relative reduction percentages of AKT in tumors and PBLs are similar such that AKT

degradation in PBLs may be a better biomarker than Hsp70 induction to predict the

modulation of Hsp90 activity by PF-4942847 treatment in the clinic.

Our results with 17-DMAG of better in vitro cellular potency than in vivo tumor

growth inhibition are similar to previous studies and implicate that the in vivo toxicity of

geldanamycin derivatives may prevent administration of a dose high enough to reach

sufficient anti-tumor activity in xenograft tumor growth studies, and further suggest that

small molecular weight inhibitors such as PF-4942847 are likely to have optimal anti-

tumor activity due to better drug exposure and safety. Our observation of differential

impact on tumors and normal host tissues with treatment of PF-4942847 indicates that




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there is a preferred therapeutic window to inhibit tumor cell progression while yet

maintaining normal cell function.

In MDA-MB-231 tumors, even when the drug had been cleared from mouse

plasma, the recovery of AKT and Hsp70 was not immediate but relatively slow to reach

basal levels. Therefore, we investigated whether an alternative dosing schedule with

higher dose concentration of PF-4942847 would have better anti-tumor activity by

stronger inhibiting of Hsp90 activity in tumors and allowing recovery of Hsp90 clients in

the normal tissues. Our results indicated that QOD dosing of PF-4942847 in MDA-MB-

231 model (Fig. 6C) induced tumor regression and was superior to QD dosing, even

though a 91% TGI was achieved in the latter. Traditionally, oral daily dosing has been

preferred in the clinic, however, our current data suggest that alternative dosing schedules

should be explored in the clinic to avoid systemic toxicity triggered by daily dosing of

Hsp90 inhibitors while maintaining a low level of client proteins in tumors.

In conclusion, we have characterized a novel oral Hsp90 specific inhibitor, PF-

4942847, and have demonstrated that PF-4942847 is an effective anti-tumor agent to

induce in vitro and in vivo anti-tumor activity in a panel of TNBC cells. Our data suggest

that PF-4942847 is a potential candidate for clinical development to address the unmet

need for therapy targeting triple negative breast cancer. Moreover, the biomarker studies

to determine AKT and Hsp70 changes in tumors and PBLs provide preliminary evidence

that AKT degradation in PBLs is a feasible biomarker approach to evaluate target

modulation in tumors.




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Acknowledgements

We thank Gerrit Los, Tod Smeal, Michael R. Gehring, and Martin Wythes for

their support and comments on this study; Leslie Nguyen, Sylvia Vekich, and Andrea

Shen for their help on the determination of PF-4942847 plasma concentration in mice;

and Dawn Nowlin for her insightful comments for quantitative data analysis.




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Figure Legends

Figure 1: PF-4942847 induces Hsp90 client protein degradation. A, MDA-MB-231

cells were treated with DMSO (0), 0.1 μM or 1 μM PF-4942847 for 2, 6, 18, 24, and 48

hours. Cell lysates were subject to western blot analysis to detect changes in total protein

or phosphorylation levels of EGFR, p-EGFR, cMet, p-cMet, AKT, p-AKT, B-Raf, C-Raf,

p-ERK and Hsp70. B, MDA-MB-231, HCC-70, and MX-1 were treated with DMSO (0),

0.1 μM or 1 μM PF-4942847 for 24 hours. Western blot analysis was performed to

detect changes in Hsp90 client proteins. Cells were cultured in 96-well microtiter plates

and treated with increasing concentrations of PF-4942847 for 24 hours; C, AKT

degradation was measured by Luminex, and D, Hsp70 induction was determined by

MesoScale Discovery.

Figure 2: PF-4942847 blocks cell cycle progression and induces apoptosis resulting

in inhibition of proliferation of TNBC cell lines. MDA-MB-231 (A) or MX-1 (B) cells

were treated with compounds for 24, 48, or 72 hours, fixed and stained with PI, and

analyzed by flow cytometry. The percentages of the cell population in each cell cycle

phase were as indicated. C, cells were treated with compound for 24 or 48 hours, then

analyzed by caspase 3/7 assay to determine caspase activity. D, cells were cultured and

treated with compound for 72 hours prior to utilizing the resazurin to determine inhibition

of cell proliferation.




27

Figure 3: PF-4942847 triggers Hsp90 client protein degradation in MDA-MB-231

xenograft tumors. A, PF-4942847 was administered orally QD for 2 days to MDA-MB-

231 tumor bearing mice at 10, 30, and 50 mg/kg; plasma concentration of PF-4942847

was determined and plotted at select time points. B, MDA-MB-231 tumor bearing mice

were treated QD for 2 days with PF-4942847 at 30 mg/kg, and tumors were harvested at

4, 7, and 24 hours after the second dose to determine time-dependent protein changes.

Or, mice were treated QD for 2 days with PF-4942847 at 10, 30, and 50 mg/kg, and

tumors were harvested at four hours after the second dose to determine dose-dependent

protein changes. Tumor lysates were subject to western blot analysis for various Hsp90

client proteins as indicated.

Figure 4: PF-4942847 induces a differential modulation of AKT and Hsp70 between

MDA-MB-231 tumors and mouse peripheral blood leukocytes (PBLs). Mouse PBLs

from MDA-MB-231 bearing animals were collected and analyzed for AKT degradation

(A and B) and Hsp70 induction (C and D) after treatment with PF-4942847. A t-test was

performed to compare each tumor versus PBLs paired sample, p<0.05 is denoted by one

asterisk (*), p<0.005 by two asterisks (**), and p<0.0005 by three asterisks (***).

Figure 5: PF-4942847 demonstrates anti-tumor activity in MDA-MB-231 and MX-1

xenograft models. MDA-MB-231 (A) or MX-1 (B) cells were implanted into nude and

SCID-bg mice, respectively. When the tumors reached ~150 mm3, tumor-bearing mice

were randomized to various groups and treated with vehicle or various doses of PF-

4942847 as indicated. Tumors were measured three times/week, and tumor sizes were




28

recorded and plotted against study days. Tumor growth inhibition by PF-4942847 was

calculated as described. Tumor growth inhibition by 17-DMAG in the MDA-MB-231

xenograft model was also performed (C).

Figure 6: PF-4942847 induces tumor regression in the MDA-MB-231 xenograft

model. MDA-MB-231 (A) or MX-1 (B) tumor bearing mice were treated with various

dose concentrations of PF-4942847. When the average tumor size in the vehicle group

was above 1500mm3, mice were taken down from the study, and PF-4942847 treatment

was stopped for all other groups to perform the survival study. The last dosing days are

as indicated in each graph. One-way ANOVA analysis was performed to compare each

group to the vehicle group, p values<0.05 are indicated by one asterisk (*) and p values

<0.005 are indicated by two asterisks in the graph (**). MDA-MB-231 (C) cells were

implanted into nude mice and allowed to reach ~300 mm3. Once tumors reached

sufficient size, mice were dosed with PF-4942847 three times per week at 75 mg/kg PO

in order to examine whether PF-4942847 would induce tumor regression.

Table I: Summary of IC50 values for AKT degradation, Hsp70 induction, and cell

proliferation inhibition by PF-4942847 and 17-DMAG treatment in a panel of TNBCs.




Figure 1A.

C. AKT degradation

10-1 100 101 102 103 1040

20406080

100120

MDA-MB-231

MX-1HCC-70

PF-4942847 Conc. (nM)

% o

f con

trol

D.

PF-4942847 (µM) 0 0.1 1MDA-MB-231 HCC-70 MX-1

EGFR

cMet

Hsp70

B-Raf

AKT

C-RafpERK

Actin

B.0 0.1 1 0 0.1 1

p-EGFR

p-cMet

p-AKT

PF-4942847 (µM)0 0.1 1

Hsp70 induction

10-1 100 101 102 103 1040

2

4

6

8

MDA-MB-231HCC-70MX-1

PF-4942847 Conc. (nM)

Indu

ctio

n fo

ld

MDA-MB-231 2 hr0 0.1 1

6 hr0 0.1 1

18 hr0 0.1 1

24 hr 48 hr0 0.1 1

EGFR

cMet

Hsp70

B-Raf

AKT

C-RafpERK

Actin

p-EGFR

p-cMet

p-AKT




Figure 2

C

D Cell proliferation inhibition

10-1 100 101 102 103 1040

20

40

60

80

100

120MDA-MB-231HCC-70MX-1

PF-4942847 conc. (nM)

% o

f con

trol

BA

DMSO

PF-847 0.1uM

PF-847 1uMDMSO

PF-847 0.1uM

PF-847 1uMDMSO

PF-847 0.1uM

PF-847 1uM0

25

50

75

100 subG1G1/G0SG2/M

24hr 48hr 72hrMDA-MB-231

% o

f Cel

ls

DMSO

PF-847 0.1uM

PF-847 1uMDMSO

PF-847 0.1uM

PF-847 1uMDMSO

PF-847 0.1uM

PF-847 1uM0

25

50

75

10024hr 48hr 72hr

subG1G1/G0SG2/M

MX-1

% o

f Cel

ls

Caspase 3/7 activity

24hrs 48hrs 24hrs 48hrs0

1

2

3

4PF-847 0.1uMPF-847 1uMPaclitaxol 1uM

MX-1 MDA-MB-231

Act

ivat

ion

fold




Hsp70

EGFR

B-raf

AKT

cMet

C-Raf

pERK

Figure 3.A

B ________ ________ ________Vehicle________ 4hr 7hr 24hr

Actin

Vehicle ________ ______ ________________ 50mg/kg 30mg/kg 10mg/kg

p-EGFR

p-cMet

p-AKT

PF-4942847 conc. in plasma

01 4 7 240

100

200

300

400

10mg/kg30mg/kg50mg/kg

post dose time (hr)

Unb

ound

dru

g co

nc. (

nM)




Figure 4

A. B.

C. D.

*

*** **

AKT degradation at 4hr

50mg/k

g

30mg/k

g

10mg/k

g

3mg/k

g

1mg/k

g0

20

40

60

80

100

120 TumorPBL

% o

f con

trol

AKT degradation at 30 mg/kg

4hr 7hr24hr

48hr72hr

96hr120hr

144hr0

20

40

60

80

100

120 TumorPBL

% of

contr

olHsp70 induction at 4hr

50mg/k

g

30mg/k

g

10mg/k

g

3mg/k

g

1mg/k

g0

10

20

30

40TumorPBL

Indu

ctio

n fo

ld

Hsp70 induction at 30 mg/kg

4hr

7hr

24hr

48hr

72hr

96hr

120h

r14

4hr

048

12162024 Tumor

PBL

Indu

ctio

n fo

ld****** *** ***

***

*** ***

***




Figure 5.A.

PF-4942847 in MDA-MB-231

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 300

200

400

600

800

1000

1200

1400

1600

1800vehicle

25 mg/kg10 mg/kg3 mg/kg1 mg/kg

PO, QDn=10/group

91%

76%

64%

47%

Study Day (post tumor implant)

Tum

or v

olum

e (m

m3 )

Dosing

B.

17-DMAG in MDA-MB231

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 300

100

200

300

400

500

600Vehicle10mg/kg

56%IP BIDx5/wkn=7/group

Dosing


Tum

or v

olum

e (m

m3 )

C.

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 420

250

500

750

1000

1250

vehicle

20 mg/kg10 mg/kg5 mg/kg

PO, QDn=8/group

PF-4942847 in MX-1

34%

51%

80%Dosing


Tum

or v

olum

e (m

m3 )




Figure 6.A.

B.

C. PF-4942847 in MDA-MB-231

0 10 20 30 40 50 60 700

200

400

600

800

1000

1200

1400

1600

1800

PO, 3x/wkn=9/group

vehicle75 mg/kg


Tum

or v

olum

e (m

m3 )

Dosing

0 25 50 75 1000

20

40

60

80

100Vehicle25 mg/kg**10 mg/kg3 mg/kg1 mg/kg

2 tumor-free

Survival in MDA-MB231

Las t doseat day 29

Study day

Perc

ent s

urvi

val

0 10 20 30 40 50 60 700

20

40

60

80

100 Vehicle20 mg/kg*10 mg/kg5 mg/kg

Perc

ent s

urvi

val Las t dose

at day 40

Survival in MX-1

Study day




Table I: Summary of IC50 values in AKT degradation, Hsp70 induction, and cell proliferation assays

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

MDA-MB-231 19.4 11.8 24.8 11.2 24.1 3.3 3.5 1.4 25.9 6.5 8.6 1.2

HCC-70 61.4 21.3 22.8 6.5 23.4 4.9 7.7 1.9 57.3 18.8 4.9 1.8

MX-1 93.7 15.6 87.4 20.7 17.2 1.6 2.7 0.2 24.7 2.4 5.2 0.2

Hs-578sT 28.5 3.6 9.5 3.2 23.6 3.4 3.4 1.4 18.1 8.3 7.5 0.2

BT-549 74.5 15.4 22.7 10.6 21.4 4.2 4.6 1.3 19.1 6.9 5.5 0.3

MDA-MB-468 86.9 19.5 89.1 20.3 37.8 6.2 12.3 2.9 37.0 4.1 13.5 3.6

HCC-38 51.7 7.3 35.8 6.3 35.6 8.1 13.8 1.5 23.7 2.4 11.1 0.3

EC50 (nM)

Hsp70 induction Cell proliferation inhibiton

IC50 (nM)

PF-4942847 17-DMAGPF-4942847 17-DMAG

IC50 (nM)

AKT degradation

PF-4942847 17-DMAG




Published OnlineFirst June 29, 2011.Clin Cancer Res Pramod P. Mehta, Pamela M. Whalen, Sangita M Baxi, et al. PF-4942847, a Novel Oral Inhibitor of Heat Shock Protein 90Effective Targeting of Triple Negative Breast Cancer Cells by

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