u1 adaptors suppress the kras-myc oncogenic axis in...

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U1 Adaptors suppress the KRAS-MYC oncogenic axis in

human pancreatic cancer xenografts

Ashley T. Tsang1,2,7, Crissy Dudgeon1,2,7, Lan Yi2, Xin Yu1,2, Rafal Goraczniak3, Kristen

Donohue1, Samuel Kogan1,2,4, Mark A. Brenneman3, Eric S. Ho5, Samuel I.

Gunderson3,6,8 and Darren R. Carpizo1,2,4,8

1Department of Surgery, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ. 2Rutgers Cancer Institute of New Jersey, New Brunswick, NJ. 3Silagene Inc., Hillsborough, NJ 4Department of Pharmacology, Rutgers University, Piscataway, NJ. 5Department of Biology, Lafayette College, Easton, PA, 18042 6Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ. 7These authors contributed equally to this work 8Co-senior Authors Corresponding Author: Darren R. Carpizo M.D., Ph.D. Associate Professor of Surgery and Pharmacology Rutgers Cancer Institute of New Jersey 195 Little Albany St New Brunswick, NJ 08903 732-235-8524 phone 732-235-8098 FAX [email protected]

Grant Support: This work was supported by the following grants: R21CA191622 (to S.I.G. and D.R.C.), QED Proof of Concept (University City Science Center) (to S.I.G), K08CA172676 and the Breast Cancer Research Foundation to D.R.C. Conflict of Interest Statement: Drs. Gunderson and Goraczniak are inventors of the U1 Adaptor technology and have an equity stake in Silagene Inc. that has licensed the technology from Rutgers University The following authors declare no potential conflicts of interest: ATT, CD, LY, XY, SK, EH, DRC Word count: abstract (246), introduction, methods, results, discussion (4,988)

on April 30, 2018. 2017 American Association for Cancer Research. mct.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 April 4, 2017; DOI: 10.1158/1535-7163.MCT-16-0867

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Running title: U1 Adaptors suppress KRAS and MYC in pancreatic cancer

Key words: U1 Adaptor, gene silencing, KRAS, MYC, pancreatic cancer




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Abstract

Targeting KRAS and MYC has been a tremendous challenge in cancer drug

development. Genetic studies in mouse models have validated the efficacy of silencing

expression of both KRAS and MYC in mutant KRAS driven tumors. We investigated the

therapeutic potential of a new oligonucleotide-mediated gene silencing technology (U1

Adaptor) targeting KRAS and MYC in pancreatic cancer. Nanoparticles in complex with

anti-KRAS U1 Adaptors (U1-KRAS) showed remarkable inhibition of KRAS in different

human pancreatic cancer cell lines in vitro and in vivo. As a nanoparticle-free approach

is far easier to develop into a drug, we refined the formulation of U1 Adaptors by

conjugating them to tumor targeting peptides (iRGD and cRGD). Peptides coupled to

fluorescently tagged U1 Adaptors showed selective tumor localization in vivo. Efficacy

experiments in pancreatic cancer xenograft models showed highly potent (>90%) anti-

tumor activity of both iRGD and (cRGD)2-KRAS Adaptors. U1 Adaptors targeting

MYC inhibited pancreatic cancer cell proliferation caused apoptosis in vitro (40-70%)

and tumor regressions in vivo. Comparison of iRGD conjugated U1 KRAS and U1 MYC

Adaptors in vivo revealed a significantly greater degree of cleaved caspase-3 staining and

decreased Ki67 staining as compared with controls. There was no significant difference

in efficacy between the U1 KRAS and U1 MYC Adaptor groups. Our results validate the

value in targeting both KRAS and MYC in pancreatic cancer therapeutics and provide

evidence that the U1 Adaptor technology can be successfully translated using a

nanoparticle free delivery system to target two undruggable genes in cancer.




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Introduction

One of the most significant unmet needs in cancer therapeutics is to effectively

target both the KRAS and MYC oncogenes. Genetic studies in murine tumor models of

both lung and pancreatic cancer have validated the therapeutic potential of silencing

mutant KRAS and MYC (1-4). This unmet need is particularly conspicuous in pancreatic

ductal adenocarcinoma (PDAC) where KRAS mutations have been found to be has high

as 93% in cohorts of patient samples (5). MYC deregulation in PDAC occurs as a

consequence of mutant KRAS and inhibition of it results in regression of both tumor and

stromal compartments (3,4). PDAC, the fourth leading cause of cancer related death in

the United States, is plagued by a lack of effective systemic therapy highlighting the need

for novel therapeutics in this disease.

U1 Adaptor is a new generation gene silencing technology that represents a novel

therapeutic platform for targeted suppression of any oncogene (6,7). The mechanism of

action is based on targeted interference with pre-mRNA processing inside the cell

nucleus. Pre-mRNAs of nearly all protein-coding genes require cleavage within the

terminal exon and addition of a polyadenosine (polyA) tail to become mature, functional

mRNA. PolyA tail addition is a critical step in mRNA maturation, and its failure results

in rapid degradation of nascent message by nucleases. U1 Adaptors are named for the U1

small nuclear ribonucleoprotein (U1 snRNP) complex, which mediates their gene

silencing action. U1 snRNP is best known as a premRNA splicing factor, but also

normally acts to silence certain genes by suppressing polyadenylation. When U1 snRNP

is able to bind stably within the terminal exon of a pre-mRNA, it directly inhibits polyA

polymerase and so prevents addition of a polyA tail. U1 Adaptors are synthetic




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oligonucleotides that enable the U1 snRNP complex to stably bind within the terminal

exon of any chosen pre-mRNA through extended base pairing, thereby interfering with

polyA tail addition and causing the pre-mRNA to be degraded inside the nucleus (Fig.

1A).

A major factor complicating the translation of therapeutic gene silencing

technologies is tumor delivery. Passage of oligonucleotides through cell membranes is

hampered by charge repulsion, as both display net negative charges. Moreover, the

densely fibrotic stroma of PDAC presents problems for drug delivery that impede

effective chemotherapy. To address the problem of charge repulsion, we and others have

formulated oligonucleotides for delivery in vivo as non-covalent complexes with

positively charged polypropyleneimine (PPI) and polyamidoamine (PAMAM) dendrimer

nanocarriers (8). To make delivery more selective for tumor cells, we and others have

used tumor-targeting RGD peptides covalently linked to dendrimer nanocarriers. RGD

peptides are synthetic oligopeptides that include an ArginineGlycine-Aspartate (RGD)

motif, which is bound by several cell-surface integrin receptors, notably the v5

heterodimer which is highly expressed on tumor vasculature (9). One RGD peptide that

is particulary well suited for delivery in PDAC is the internalizing RGD (iRGD), a

modified RGD peptide that is bound by v5 integrin, but undergoes cleavage at the cell

surface, followed by secondary binding to neuropilin (NPR-1), a receptor that triggers

permeabilization of tumor endothelium and internalization by cells (10,11). The

limitations of using dendrimer based systems are: 1) toxicity at high doses, 2) the

solubility of oligonucleotide:dendrimer complexes is significantly less than that of either

component alone which limits effective dosing, and 3) commercial preparation of




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dendrimers lack a source for cGMP-compliant synthesis at scale. Here, we demonstrate

for the first time that U1 Adaptor covalently linked to tumor targeting peptides

(dendrimer free system) can be employed to therapeutically target both KRAS and MYC

in pancreatic cancer.




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Materials and Methods

Cell culture and transfection Cell lines were maintained in 5%CO2/95% air at 37C. PDAC cells were purchased from

ATCC in February 2015. Experiments were completed with early passage number cell

lines. Cell lines were tested and authenticated by ATCC. The cell lines MIA PaCa-2,

PANC-1, and IMR-90 cells were maintained in DMEM with 10% fetal bovine serum

(FBS). BxPC-3 cells were maintained in RPMI with 10% FBS. Capan-1 was maintained

in IMDM with 10% FBS.

U1 Adaptor oligonucleotides were designed using a proprietary computational target site

algorithm developed by Silagene to select target sequences within the terminal exon and

3UTR. MIA PaCa-2 cells were screened using eight different KRAS U1 Adaptors.

PAMAM G5 (150 nM) was used as a control. All transfections used 10 nM of adaptor or

siRNA in complex with RNAiMax (FisherSci) or PAMAM. After 72 hours, cells were

collected for expression analysis. The most effective sequences were modified using

locked nucleic acid (LNA) 2OMe chemistry. A U1 Adaptor designed with a scrambled

target domain sequence and a wild type U1 domain served as a negative control.

Cell growth inhibition assay

Cells (5,000) were plated in 96-well plates in triplicate and transfected on Day 0 using

RNAiMax with 10 nM of Control adaptor, U1 KRAS1B, U1 KRAS1B mutant, U1

MYC2, U1 MYC3, MYC siRNA (Santa Cruz), or KRAS siRNA SMARTpool




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(Dharmacon). On Day 3, cells were harvested for viability using the ViaCount reagent on

the Guava (Millipore)or with CCK-SK (Dojindo) reagent according to manufacturers

protocol. For unharvested cells, the old medium was replaced and transfected again. The

experiment was completed after 3 treatments.

RNA extraction and RT-quantitative real-time PCR (RT-qPCR) RNA was extracted using RNeasy kit (Qiagen) and gene expression level was measured

by quantitative RT-qPCR using TaqMan gene expression assays (Life

Technologies/Applied BioSystems). The gene expression level was normalized with -

actin and the average presented with standard deviation.

Western blot Western blotting of cell and tumor lysates was performed as previously described (12).

Antibodies used were as follows: human KRAS (Thermofisher Scientific (PA5-27234)),

pERK1/2 (Cell Signaling, #4370), pAKT (Cell Signaling #9018), ERK1/2 (Cell

Signaling), AKT (Cell Signaling #9272), MYC (R&D Systems, clone 9E10), and actin

(Santa Cruz Biotechnology).

Apoptosis assay Annexin V staining was completed according to the manufactures instructions using the

Nexin reagent for Guava (EMD Millipore). Similar to the transfection experiments, the

MIA Paca-2 cells were cultured in a 12-well plate, followed by treatment with U1

Adaptors at 10 nM using RNAiMax as a transfection reagent for 72 hours.




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RGD peptide-U1 Adaptor Conjugate Preparation and Purification

A variation of CLICK conjugation chemistry was used to prepare peptide (cRGD)2 or

iRGD-U1 Adaptor conjugates as follows: a solution containing the following was

degassed with argon: 0.1 mM U1 Adaptor with a 5'-end Hexynyl group, 0.2 M

Triethylamine acetate, 50% DMSO, 0.3 mM Azido-peptide and 0.5 mM Ascorbic acid.

Conjugation was initiated by addition of 1/20th volume of 10 mM CuSO4-TBTA solution

and the reaction proceeded overnight with gentle agitation at room temperature. The

reaction was halted by addition of 1/50th volume of 0.5 M EDTA, pH 8.0 followed by

desalting over a Zeba spin column that also removed unreacted Azido-peptide. In some

cases a higher stoichiometric ratio of Azido-peptide was used.

Purity of samples was tested by HPLC of 15 g Hex-KRAS2 unconjugated U1 Adaptor

versus 15 g of conjugated (cRGD)2-KRAS2 peptide. HPLC was done on a Hypersil

ODS C18 column (Buffer A = 0.1 M TEAA pH 7.0; Buffer B= 100% acetonitrile).

Conjugation was further tested for purity and efficiency by subjecting 700 ng of peptide-

adaptor conjugate to 8% PAGE-UREA-TBE gel electrophoresis followed by methylene

blue staining.

Animal studies

All animal protocols were approved by the Rutgers Biomedical and Health

Sciences Animal Care and Use Committee. Five to six week old female athymic nude

mice (NCR nu/nu) were purchased from Taconic Biosciences. KPC mice were obtained

from the NCI mouse repository. For xenograft studies, 5 x106 pancreatic cells were

injected subcutaneously into both flanks of each nude mouse after the induction of




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anesthesia with isofluorane. Treatment began when xenograft tumors were either just

palpable (10-20 mm3) or larger (40-50 mm3 and in one case 100 mm3). Mice were

randomly divided and assigned to their respective treatment groups (9-10 mice/group).

Tumor volumes (V) were calculated from caliper measurments by length (L) and width

(W) by using the formula: V= 0.5xLxW2. Treatment was twice a week by tail vein

injection until the tumors in the vehicle control group (buffer solution) reached ~1.5 cm3.

For the biodistribution studies of the Peptide-Adaptor conjugates in the KPC

mouse model (Pdx-1cre/+; KRASG12D/+; p53R172H/+) (13), Cy3-labeled KRAS3 Adaptor was

conjugated to either no peptide, iRGD or (cRGD)2. Mice were randomly selected into

each treatment group (3 mice/group). When the pancreatic tumors reached 50 mm3 (by

ultrasound), the mice were treated by a single tail vein injection (30 g) and sacrificed

after 24 hours when organs were harvested for imaging. The IVIS Spectrum imaging

system (Perkin-Elmer) was used to detect Cy3-labeled adaptors, and the total flux (p/s)

was calculated based upon a region of interest that remained a constant size per mouse.

For the pharmacodynamics experiment, PANC-1 xenografts were established as

before. Mice were treated with vehicle (HEPES buffer), iRGD-Control, iRGD-KRAS3,

or iRGD-MYC2 at 30 g per dose, for a total of 3 treatments (1.5 weeks). Mice were

sacrificed the day following the last injection and tumors processed for immunostaining.

Statistical Analysis Mouse numbers were determined with assistance from the Biometrics Facility Core at the

Cancer Institute of New Jersey. Statistical analyses were carried out using GraphPad

Prism V software. Tumor volumes for each group were compared using students t-test




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(for comparisons of two groups) and analysis of variance (for multiple group

comparisons, 1 way ANOVA). p

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control (Fig. 1B). For further validation, a modified KRAS Adaptor (1B) containing five

locked nucleic acid (LNA) modifications was evaluated in MIA PaCa-2 cells since LNAs

are able to provide a boost, albeit unpredictable, in silencing activity (7). This was

compared to siRNA, but also a control U1 Adaptor, which had a scrambled target domain

(TD) (Supp. Table). There was a slight decrease in the mRNA using the control U1

Adaptor compared with the transfection control while that of the U1 KRAS1B Adaptor

showed significant knockdown of both the mRNA and protein level (Fig. 1C, left). A

60% reduction in KRAS mRNA was observed with treatment in U1 KRAS1B in another

established human pancreatic cancer cell line, Panc1 (KRASG12D mutation) compared to

the controls. In both of these cell lines, the anti-KRAS siRNA showed a greater degree of

KRAS mRNA knockdown (Fig. 1C, right).

U1 Adaptors targeting KRAS inhibit pancreatic cancer cell proliferation in vitro

and in vivo

We then examined if these U1 Adaptors targeting KRAS could inhibit pancreatic

cancer cell proliferation. We transfected U1 KRAS1B into MIA PaCa-2 (Fig. 2A left)

and PANC-1 (Fig. 2A middle) cells every 72 hours over 9 days, and observed potent

inhibition of cell proliferation in both cell lines when compared to an RNAiMAX

transfection control and Control U1 Adaptor with a scrambled TD. There was no

difference between the U1-KRAS1B and siKRAS groups. We further evaluated cell

growth inhibition in another established human pancreatic cancer cell line containing

another commonly found missense mutation in pancreatic cancer (Capan-1, KRASG12V)

and observed, by day 14, a significant inhibition of cell growth as compared with the




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untreated and control U1 Adaptor with a scrambled TD that was very similar in

magnitude to the siKRAS (Supp. Fig. 1A).

KRAS U1 Adaptors are not specific to mutant KRAS, which raises two questions:

1) are they effective in pancreatic cell lines with wild type KRAS and 2) would they be

toxic to non-cancer cell proliferation? To answer the first question, we tested U1

KRAS1B in the established human pancreatic cancer cell line BxPC3 (KRAS wild type).

We found that by day 10 there was a mild degree of growth inhibition as compared to the

untreated control and this magnitude was similarly seen for the siKRAS indicating that

this cell line is much less dependent on KRAS signaling for proliferation (Fig. 2A, right).

To determine if the U1 Adaptors would inhibit the growth of a non-cancer cell line we

evaluated the human lung fibroblast line (IMR-90) and found that by day 12 there was no

significant difference in proliferation between U1 KRAS1B, siKRAS, and the U1 Control

groups (p=0.894, 1 way ANOVA) (Supp. Fig. 1B). Knockdown of U1 KRAS1B and

siKRAS KRAS was confirmed by Western blot (Supp. Fig. 1B).

It has been previously shown that a marker of KRAS dependency or oncogene

addiction is the induction of apoptosis upon KRAS knockdown (14). We next

performed an apoptotic assay using Annexin V staining. We found that by day 3, there

was a nearly four-fold greater percentage of apoptotic cells for the U1 KRAS1B treated

apoptotic cells than the untreated control and a control U1 Adaptor with a mutant TD. As

an additional control we included a U1 Adaptor that contains the same KRAS targeting

sequence as the U1 KRAS1B Adaptor, but the U1 domain sequence has a two nucleotide

mutation (Fig. 2B, top). We found that this mutant adaptor (U1 KRAS1B mutant) failed

to significantly induce apoptosis in the MIA PaCa-2 cells compared with the untreated




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control indicating the specificity of the U1 Adaptor mechanism for this effect (Fig. 2B,

bottom).

We then examined the MIA PaCa-2 cells for evidence of inhibition of molecules

downstream of KRAS signaling such as ERK, AKT and MYC in U1 Adaptor treated

cells. We found a significant reduction in overall MYC, phosphorylated ERK and

phosphorylated AKT as compared to the untreated control (Fig. 2C). This magnitude

was greater than or equal to that of the siKRAS control, with a greater reduction in AKT

activation compared to ERK. Note the lack of inhibition of these markers in either the

U1 Control or the U1 KRAS1B mutant, indicating the specificity for KRAS signaling

inhibition. For further validation, we evaluated PANC-1 for these biochemical markers

of KRAS signaling inhibition and found similar effects as in the MIA PaCa-2 cells with

an inhibition of MYC, phosphorylated AKT for the U1 KRAS1B and siKRAS treated

cells that is lost in the KRAS-1B mutant (Supp. Fig. 1C).

To determine if the KRAS U1 Adaptors could inhibit pancreatic xenograft tumor

growth, we evaluated the U1 KRAS3 adaptor using our previously validated formulation

containing PAMAM dendrimers covalently linked to the cRGD peptide (7) (Fig. 2D, top).

Twice-weekly intravenous injection of the cRGD-PAMAM-KRAS3 adaptor led to a

significant growth inhibition of MIA PaCa-2 xenograft tumors (68% inhibition, KRAS3)

(Fig. 2D, bottom). This provides evidence that KRAS U1 adaptors inhibit pancreatic

tumor growth in vivo.

KRAS U1Adaptors Directly Conjugated to Tumor Targeting Peptides Inhibit

Tumor Growth




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In an effort to pursue a PAMAM dendrimer-free system of delivery, we directly

conjugated the U1 Adaptor to a tumor targeting peptide. After in vitro and in vivo

efficacy testing of several conjugation methods we settled on copper-based CLICK

chemistry as it provided a higher conjugation efficiency, precluding the need for vast

excess of peptide and HPLC purification (Fig. 3A, Sup. Fig 2). cRGD is amenable to

monomeric and dimeric coupling whereas iRGD is amenable only to monomeric

coupling due to internal cysteines not found in cRGD, (Fig. 3A).

Given the iRGD mechanism of delivery not only binds to tumor endothelium, but

also results in greater tissue penetration through a NP-1 mediated vascular

permeablization (10), we reasoned that iRGD would be particularly suitable for delivery

to PDAC of the KRAS U1 Adaptors considering that a hallmark of PDAC is a fibrous

stroma that is a barrier to systemic agents (15,16). We evaluated both iRGD and cRGD

peptides for PDAC delivery by conjugating each peptide to the 5' end of a Cy3-labelled

U1 Adaptor using the autochthonous KPC genetically engineered mouse model, which

has been shown to reproduce key features of the stromal compartment of human PDAC

(13). Twenty-four hours after injection, we sacrificed the mice and examined in vivo

fluorescence of the brain, heart, lung, liver, kidney, spleen and tumor. We found the

highest levels of fluorescence accumulated in the brain, kidney and tumor with very little

fluorescence detected in the heart, lung, liver or spleen (Fig. 3B, left). Interestingly, the

control (no peptide) Cy3-U1 Adaptor also highly localized to the brain and kidney

indicating that the uptake of the iRGD and cRGD conjugated adaptors in the brain is not

peptide related. Alternatively, accumulation of fluorescence in the tumor did

demonstrate a significant increase of fluorescence in the peptide conjugated tumor tissue




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as compared with the non-peptide conjugated control, p=0.01 (Fig. 3B, right). We did

not see a significant difference between the cRGD and iRGD peptide conjugated U1

Adaptor groups.

We then sought to compare the formulation of the iRGD-KRAS-3 Adaptor with

the cRGD-PAMAM dendrimer for in vivo antitumor activity. Again we used the MIA

PaCa-2 xenograft assay and administered a dose of 10 g intravenously twice weekly.

We found that both the PAMAM-cRGD:KRAS-3 complex and iRGD-KRAS-3 conjugate

potently inhibited tumor growth essentially causing stasis of tumor growth (92 and 93%

growth inhibition by day 39) (Supp. Fig. 3A). This provides evidence that KRAS U1

Adaptors directly conjugated to tumor targeting peptides demonstrate potent antitumor

effects that are equivalent to the dendrimer-based system. We evaluated the iRGD-

KRAS-3 adaptor to the (cRGD)2-KRAS-3 in the same Mia PaCa-2 xenograft assay and

found that by Day 39 there was significantly greater tumor growth inhibition with iRGD-

KRAS-3, p

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As stated earlier, PAMAM dendrimer:U1 Adaptor formulations have limited

utility due to precipitation at higher doses (just 3-fold higher concentrations result in

extensive precipitation). This problem is obviated with a targeting peptide conjugated

system that is dendrimer-free, allowing us to escalate the dose of the peptide conjugated

U1 Adaptor. We then performed a dose escalation experiment by testing three doses of

the (cRGD)2-KRAS2 conjugate formulation at 10, 30, and 90 g as well as the (cRGD)2-

KRAS3 at 30 g. We found that there was no significant difference between the 10 and

30 g doses of the (cRGD)2-KRAS-2 conjugate (75% growth inhibition compared to

Vehicle, p

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MYC not only to determine feasibility but also to compare the antitumor effect to that of

KRAS in the same pancreatic tumor model.

Screening a panel of anti-MYC U1 Adaptors in vitro identified three with superior

silencing activity (Supp. Table). We validated these adaptors in the MIA PaCa-2 cells by

Western blot and found potent MYC inhibition (Fig. 4A). We performed a cell growth

inhibition assay and found that all three MYC U1 adaptors potently inhibited growth (Fig.

4B). We also examined the percent of apoptotic cells induced by U1-MYC adaptors and

found that all three adaptors significantly induced apoptosis (40-70%) in MIA PaCa-2

pancreatic cancer cells in comparison to the untreated and U1 Control adaptor groups

(Figure 4C).

To evaluate the MYC U1 Adaptor in vivo, we synthesized iRGD-MYC2 and

MYC3 adaptor conjugates using the same chemistry used in the iRGD-KRAS. We tested

these formulations using a 10 g dose administered in the MIA PaCa-2 xenograft assay.

We observed significant antitumor activity in both the iRGD-MYC2 and iRGD-MYC3

U1 Adaptor formulations with regressions in 4/4 iRGD-MYC2 mice and 5/6 iRGD-

MYC3 mice (Fig. 4D). Interestingly in both of these treatment groups regressions were

typically seen after day 18. Western blot for MYC in the tumor tissue of the mice in

these three treatment groups indicated inhibition of MYC with levels corresponding to

the degree of tumor growth inhibition (Fig. 4D).

Targeting KRAS versus MYC in Pancreatic Xenograft Tumors

Genetic experiments in mouse models of pancreatic and lung cancer have shown that

inhibition of either KRAS or MYC signaling in mutant KRAS driven tumors is




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therapeutic. We sought to validate this using U1 adaptors targeting either KRAS or

MYC in the same tumor model. We first performed a pharmacodynamic experiment in

which mice bearing PANC-1 xenografts were treated with either the iRGD-KRAS or

iRGD-MYC adaptor along with the iRGD-Control adaptor. Western blot data in Figure

5A confirmed inhibition of protein expression for both KRAS (left) and MYC (right).

We then compared both cell proliferation and apoptosis in the tumors using Ki67 and

cleaved caspase-3 (CC3) immunofluorescence, respectively. We observed a significantly

greater amount of CC3 staining in the U1-KRAS and U1-MYC adaptor-treated groups as

compared to the buffer-treated and iRGD-Control groups (Fig. 5B, p

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MYC adaptors, both were equally efficacious.




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Discussion

Pancreatic cancer is plagued by a lack of effective systemic chemotherapy. One

reason for this is the desmoplastic stromal compartment of the tumor, which serves as a

barrier to the delivery of therapeutic agents (15,16). Recently, this aspect of its biology

has been linked to signaling from the KRAS-MYC oncogenic axis as withdrawal of

either oncogenic KRAS or MYC leads to a general dissolution of the stromal

compartment (1-3). This further substantiates the significance of finding novel

therapeutics to target these genes in PDAC. Our results with Massons trichrome staining

revealed conflicting results between KRAS and MYC Adaptor treated tumors with a

decrease seen in the MYC group and no change in the KRAS group. We are hesitant to

draw any conclusions from this because nude mouse xenograft models do not effectively

recapitulate the tumor microenvironment (TME), which is one their limitations. It is

possible that the efficacy of the U1 Adaptor is overestimated in these models due to the

lack of a stromal barrier. The mouse autochthonous model (KPC) is a better model to

evaluate efficacy because it does effectively recapitulate the human TME; however, we

could not perform these experiments as our efforts to silence murine KRAS were

unsuccessful.

Genetic experiments using mouse models of cancer have demonstrated the

therapeutic value in silencing both KRAS and MYC in pancreatic and lung cancers

driven by mutant KRAS; however, these results have yet to be validated using

developmental therapeutics. This is primarily due to the fact that KRAS and MYC have

thus far proven refractory to targeting using small molecules. In translating U1 Adaptors

to target KRAS and MYC, we provide further pre-clinical evidence of the therapeutic




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value in targeting KRAS and the first evidence for targeting MYC in pancreatic cancer.

It remains a valid question if inhibiting these genes systemically will lead to toxicity.

Given that KRAS is upstream of MYC and activates multiple signaling pathways such as

PI3K and RAL-GDS, we would speculate that the efficacy of the combination would be

increased. We have shown that the adaptors inhibit non-cancer cell proliferation slightly

at doses that potently inhibit pancreatic cancer cell proliferation, suggesting there may be

a therapeutic window. Moreover, Soucek et al. have demonstrated that the Omomyc

mice can tolerate chronic inhibition of MYC (3).

Gene silencing as a therapeutic modality has long generated excitement with

promises for rapid translation into the clinic. Excitement faded with the realization that

oligonucleotide drug candidates lack certain key pharmacological properties most notably

targeted delivery to specific cell and organ types, short half-lives and unwanted toxicity

and immune responses. Over the years improvements have been made, mostly through

systematic changes in the phosphate and ribose groups that comprise the backbone of the

oligonucleotide. While such changes appear minor, they greatly increase stability and

reduce immunogenicity and overall toxicity. With a few notable exceptions

(GalNac/liver) targeted delivery remains a significant challenge resulting in reliance on

nanoparticle-based delivery that brings its own challenges with toxicity and stability

inherent to a two-component drug system. Recently siRNA technology was used to

silence KRAS in murine models of lung and colon cancer that required coupling with

lipid-based nanoparticles (17). Alternatively a biodegradable polymeric matrix (LODER)

was developed to provide a slow release of siRNA to KRAS in a murine pancreatic

cancer model (18). This delivery system showed efficacy, but needs to be injected




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intratumorally and would not be a useful systemic agent. While cell growth inhibition

was similar between the U1 Adaptor and siRNA to KRAS, the U1 Adaptor offers

efficacy when conjugated to cell-targeting peptides such as the RGD family, while

relying solely on the relatively non-toxic and stable 2'-O-methyl backbone chemistry and

eliminating the need for a nanoparticle. These features give the U1 Adaptor the

possibility to become first in class as a gene silencing anti-cancer modality.

As with any gene silencing technology, an effective delivery system is required.

Here we provide evidence that tumor-targeting peptides directly conjugated to the U1

Adaptor can be used as an effective delivery system, which greatly simplifies GMP

manufacturing and testing in humans. Interestingly we did not observe a difference in

fluorescence between the iRGD and cRGD-Cy3 labeled peptides in the KPC model as we

had hypothesized, suggesting neither peptide is superior. However, a robust comparison

of the two peptides was not the aim of this study. We did however observe a difference

in tumor growth inhibition in favor of the iRGD.

While these experiments certainly validate the therapeutic potential of U1

Adaptor, there are still several important knowledge gaps that remain to effectively

translate this technology to the clinic. The first is a thorough understanding of the

pharmacokinetics and pharmacodynamics (PK/PD) of the U1 Adaptor, which is needed

to optimize dose and schedule. We chose twice weekly intravenous treatments both for

convenience and because this worked well in our 2013 proof of concept that relied on a

nanoparticle delivery system (8). The second is the optimal dose. Conjugating tumor

targeting peptides allowed us to perform a dose escalation experiment in which we found

that a dose of 90 g was not more effective than 10 or 30 g. This would need to be




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examined using pharmacodynamic assays as well as with other target genes to determine

if this is specific to KRAS or is more general to U1 Adaptors.

The third is the design of the nucleic acid chemistry. While LNA modifications

should in principle increase in vivo potency, we have avoided extensive testing of LNA-

U1 Adaptors in vivo due to their unpredictable behavior and known toxicities at higher

doses (unpublished observations). That said LNA modification might be necessary for

cases where deep silencing of the target gene is necessary to achieve an efficacious

response.

What is also unclear is the duration of efficacy with U1 Adaptors. As with any

targeted agent, the development of resistance is a factor that must be considered. U1-

Adaptor formulations currently require intravenous dosing, which does limit the ability to

conduct lengthy treatment experiments in mice. Future studies will focus on these

knowledge gaps to effectively translate the U1 Adaptor technology therapeutically.




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Acknowledgements

We would like to thank Dr. Erkki Ruoslahti for providing us with the iRGD peptide and

Dr. Joseph Bertino for sharing results of an in vitro screen to identify anti-MYC U1

Adaptors.




26

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Figure 1. Design, selection, and validation of U1 Adaptors targeting KRAS in human pancreatic cancer cells. A, The U1 Adaptor has two sequence specific domains that tether the RNA component of the U1 snRNP complex to the target pre-mRNA, blocking the addition of the poly-A tail and selectively triggering pre-mRNA destruction. B, Eight U1 Adaptors targeting human KRAS were screened in MIA PaCa-2 cells, demonstrating a range of mRNA knockdown. The most effective KRAS Adaptors were KRAS1, 2 and 3, within the terminal exon. C, Validation of human KRAS1B Adaptor (LNA modified) in MIA PaCa-2 and PANC-1 cells after 72-hour transfection. A control adaptor with a scrambled target domain and siRNA to KRAS serve as negative/positive controls (P

29

Figure 2. U1 Adaptors targeting KRAS are effective in vitro and in vivo. A, Cell growth inhibition assay of human pancreatic cancer cell lines MIA PaCa-2, PANC-1, and BxPC-3, after transfection with U1 KRAS1B every 72 hours over 9 days compared to U1 Control adaptor and siKRAS. B, (Top) Schematic of the U1 KRAS1B mutant adaptor. Nucleotides underlined in the KRAS domain have been LNA-modified. Red lowercase nucleotides represent the mutated nucleotides of the U1 domain. (Bottom) Apoptosis assay using Annexin V staining following transfection of MIA PaCa-2 cells with 10 M U1 Control, U1 KRAS1B, U1 KRAS1B mutant, or siKRAS. after 72 hours (P=0.0001), with RNAiMAX serving as an additional negative control. C, Western blots for KRAS, MYC, p-AKT, AKT, p-ERK, and ERK, 72 hours following transfection of MIA PaCa-2 cells as in B. Actin serves as a loading control. D, Schematic of the peptide (cRGD)-dendrimer (PPI) linked to the U1 KRAS3 adaptor (Top) used for in vivo delivery of the KRAS adaptor. (Bottom) MIA PaCa-2 xenograft mice (n=9) were treated with cRGD-PAMAM-KRAS3 complex (2 g KRAS3 Adaptor, 7.5 g cRGD-PAMAM complex per dose) by tail vein injection twice weekly starting when tumors reached 40-50mm3. 68% growth inhibition observed when compared with vehicle control (p=0.0002).




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Figure 3. U1 Adaptors directly conjugated to tumor targeting peptides inhibit tumor growth. A, Schematic of copper CLICK peptide-U1 Adaptor conjugation (top). Purity of the reaction was tested by gel electrophoreses on an 8% TBE-Urea gel (bottom). B, (left) Biodistribution study of Cy3 labeled RGD-Adaptor conjugates in the KPC mouse model. 24 hours after treatment, mice (n=3) were sacrificed, and tumors and organs were harvested and imaged using IVIS Spectrum. Bioluminescence was measured in total flux (photons/second) (p=0.01) (right) Statistical significance was calculated by students t-test. C, In vivo efficacy study using the best formulation of (cRGD)2-KRAS3 U1 Adaptor (30 g) in MIA PaCa-2 xenografts. Nude mice (n=10, Vehicle, n=9 (cRGD)2-KRAS3) were treated by tail vein injection twice weekly once tumors reached 20 mm3. There was greater than 90% growth inhibition observed in both treatment groups when compared with the vehicle control (p=0.0001) (left) Statistical significance was calculated by students t-test. Waterfall plot showing individual MIA PaCa-2 xenograft tumor response to treatment (right). Sizes were measured at time of treatment up to 29 days. Note that the (cRGD)2-KRAS3-treated (bottom) group had tumor stasis (7/9) or regression (2/9) in individual mice.




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Figure 4. U1 Adaptors targeting MYC in mutant KRAS driven pancreatic cells are effective both in vitro and in vivo. A, Western blotting of MIA PaCa-2 cells transfected with 10 M U1 Control, U1 MYC1, U1 MYC2, U1 MYC3, and siMYC after 72 hours of treatment. B, Cell proliferation assay of MIA PaCa-2 cells treated as in A, except for every 72 hours over 9 days. C, Annexin V assay of MIA PaCa-2 cells treated as in A, except cells were harvested on Day 6 following 2 transfections 72 hours apart. Statistical significance was calculated by students t-test. D, Nude mice harboring MIA PaCa-2 xenograft tumors were treated 2 times a week for a total of 32 days with 30 g iRGD-Control (n=4), iRGD-MYC2 (n=4), and iRGD-MYC3 adaptors (n=6). Waterfall plot of tumor size with the correlate protein expression was measured for treated tumors. Western blotting using antibodies for actin and MYC were completed on tumor cell lysates from iRGD-Control, iRGD-MYC2, and iRGD-MYC3 tumors at the end of treatment. Representative images are shown. NS, not significant.




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Figure 5. Targeting KRAS versus MYC in Pancreatic Xenograft Tumors. A, Nude mice harboring PANC-1 xenograft tumors were treated 3 times with 30 g iRGD-Control (n=2), iRGD-KRAS3 (n=4), or iRGD-MYC2 (n=4) adaptors. Tumor protein lysates were probed for KRAS (left) and MYC (right) expression. Actin served as a loading control. B, Cleaved caspase 3 and C, Ki67 immunofluorescence staining of tumors treated as in A. Three fields at 200X were quantitated for total cell number (DAPI, blue) and FITC-positive cells (B, cleaved caspase 3, green; C, Ki67, green). Statistical significance was calculated by students t-test. D, Nude mice harboring PANC-1 xenograft tumors were treated 5 times over 17 days with HEPES buffer or 30 g iRGD-Control (n=7), iRGD-KRAS3 (n=7), or iRGD-MYC2 (n=7) adaptors. Tumor size was measured and plotted at each treatment day. Statistical significance was calculated by students t-test. Scale bar=50 m




Published OnlineFirst April 4, 2017.Mol Cancer Ther Ashley T. Tsang, Crissy Dudgeon, Lan Yi, et al. pancreatic cancer xenograftsU1 Adaptors suppress the KRAS-MYC oncogenic axis in human

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