targeting kras-dependent tumors with azd4785, a high-affinity ... ·...

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1AstraZeneca, Cambridge CB2 0AA, UK. 2Ionis Pharmaceuticals, Carlsbad, CA92010, USA. 3AstraZeneca, Macclesfield SK10 4TG, UK. 4AstraZeneca, Waltham,MA 02451, USA.*These authors contributed equally to this work.†Corresponding author. Email: [email protected] (P.D.L.); [email protected] (A.R.M.)

Ross et al., Sci. Transl. Med. 9, eaal5253 (2017) 14 June 2017

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Targeting KRAS-dependent tumors with AZD4785, ahigh-affinity therapeutic antisense oligonucleotideinhibitor of KRASSarah J. Ross,1* Alexey S. Revenko,2* Lyndsey L. Hanson,3 Rebecca Ellston,3 Anna Staniszewska,1

Nicky Whalley,1 Sanjay K. Pandey,2 Mitchell Revill,3 Claire Rooney,1 Linda K. Buckett,3

Stephanie K. Klein,1 Kevin Hudson,3 Brett P. Monia,2 Michael Zinda,4 David C. Blakey,3

Paul D. Lyne,4† A. Robert Macleod2†

Activating mutations in KRAS underlie the pathogenesis of up to 20% of human tumors, and KRAS is one of the mostfrequentlymutated genes in cancer. Developing therapeutics to block KRAS activity has proven difficult, and no directinhibitor of KRAS function has entered clinical trials. We describe the preclinical evaluation of AZD4785, a high-affinityconstrained ethyl–containing therapeutic antisense oligonucleotide (ASO) targeting KRAS mRNA. AZD4785 potentlyand selectively depleted cellular KRASmRNA and protein, resulting in inhibition of downstream effector pathways andantiproliferative effects selectively inKRASmutant cells. AZD4785-mediateddepletionof KRASwasnot associatedwithfeedback activation of the mitogen-activated protein kinase (MAPK) pathway, which is seen with RAS-MAPK pathwayinhibitors. Systemic delivery of AZD4785 to mice bearing KRASmutant non–small cell lung cancer cell line xenograftsor patient-derived xenografts resulted in inhibition of KRAS expression in tumors and antitumor activity. The safety ofthis approach was demonstrated in mice and monkeys with KRAS ASOs that produced robust target knockdown in abroad set of tissues without any adverse effects. Together, these data suggest that AZD4785 is an attractive therapeu-tic for the treatment of KRAS-driven human cancers and warrants further development.

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INTRODUCTIONRAS guanosine triphosphatases (GTPases) are one of themost commonlymutated gene families in cancer, andKRAS is themost frequentlymutatedisoform, with a prevalence of about 20% in all human cancers (1). Thereare several tumor types that exhibit a high frequency of activating muta-tions in KRAS, including three of the deadliest cancers: pancreatic, colo-rectal, and non–small cell lung cancer (NSCLC) (1, 2). KRAS functions asamolecular switch cyclingbetweenguanosine triphosphate (GTP)–bound(on) and guanosine diphosphate (GDP)–bound (off) states to affect intra-cellular translation of extracellular signaling through cell surface receptors.Oncogenic alleles of KRAS reduce intrinsic and GTPase activatingprotein–mediated GTP hydrolysis, resulting in increased GTP-boundKRAS and persistent signaling throughdownstream effector pathways(1, 2). Mutations of KRAS are associated with poor prognosis in sev-eral cancers, and there is a substantial body of evidence supporting therole of KRAS in the initiation and maintenance of cancer (3–7), indi-cating that it is an important therapeutic target.

Since the original discovery of KRAS as an oncogene in 1982, therehave been intense efforts to develop a targeted therapeutic for KRASmutant cancers (1, 2). Attempts at direct enzymatic inhibition of KRASfunction have been largely unsuccessful due to the intrinsically high af-finity of the enzyme for GTP. As a consequence, indirect approaches toinhibit mutant KRAS signaling have been pursued. The most advancedof these approaches entailed targeting the pathways involved in theposttranslational modification of KRAS (1), farnesylation of a C-terminal motif on KRAS by farnesyltransferase, a step necessary fortrafficking of the protein from the cytoplasm to the inner face of the cell

membrane and for effector pathway activation. Unfortunately, phase 3studies of two independent farnesyltransferase inhibitors (FTIs), tipifarnib(8) and lonafarnib (9), failed to showbenefit for patients over the standardof care in pancreatic and lung cancer, respectively. This was shown to bedue to the activation of an alternativemechanism of prenylation of KRASandNRAS in the presence of FTIs (1). Attention has also been given toinhibition of downstream effector signaling pathways, includingmembers of the mitogen-activated protein kinase (MAPK) and phos-phatidylinositol 3-kinase (PI3K) pathways, but to date, agents target-ing members of these pathways have met with limited successclinically in KRAS-driven tumors (1). Combinations of agents target-ing distinct RAS effector pathways are currently under clinical inves-tigation, and it remains to be seen whether these combinations will betolerated at doses that may provide therapeutic benefit (10, 11). Still,further indirect approaches, mainly preclinical to date, are focusing onthe role of KRAS in the regulation of metabolic processes (6, 12–14).

Despite the difficulty in identifying a viable direct approach to targetKRAS therapeutically, KRASmutant cancers remain an extremely highunmet clinical need disease segment, and this continues to drive the sci-entific community to identify approaches to inhibit this broadly impor-tant cancer target. Some progress has recently been made in generatingcovalent inhibitors that bind and selectively inhibit one of the KRASmutant isoforms (KRAS G12C), a mutation predominantly found inlung cancer (15–17). These early compounds have promise but, evenif successful in the clinic, will only treat a subset of the overall KRASmutant patient population. In addition, some progress has been madewith delivery systems for aKRASG12D–directed small interfering RNA(siRNA), which is currently being evaluated in locally advanced pancre-atic cancer (18). Therapeutic nucleic acid–based approaches, includ-ing antisense oligonucleotides (ASOs), offer the potential to yielddrugs for targets that have proven to be intractable to traditional drugmodalities (19). With the ASO approach, inhibitors can be designedsolely on the basis of gene sequence information, enabling the devel-

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opment of selective inhibitors to a wide range of target classes, includ-ing those previously regarded as undruggable. There have been severalsuccesses in the development of systemically administered ASOs innon-oncological settings (20, 21) and, recently, the U.S. Food andDrug Administration approved mipomersen (Kynamro) as the firstsystemically administered ASO drug. Continued efforts to improvethe stability and potency of ASOs have resulted in the discovery of anext-generation class of ASOs that use 2′-4′ constrained ethyl (cEt)residues (22, 23). These next-generation cEt ASOs exhibit enhancedin vitro and in vivo potency compared to earlier ASO molecules,and recently, a cEt ASO targeting the previously undruggable targetSTAT3 (signal transducer and activator of transcription 3) has shownsingle-agent activity in early cancer studies, suggesting that this ther-apeutic approach holds promise (24). Here, we report the discovery ofthe clinical drug candidate AZD4785, a potent cEt-modified ASO in-hibitor ofKRAS expression, describe its primary pharmacology in pre-clinical cancer models, and characterize the tolerability of KRASinhibition in nonhuman primates and in rodents.

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RESULTSAZD4785 is a potent and selective cEt-modified ASOinhibitor of human KRASAZD4785 (Ionis 651987) is an advanced chemistry (cEt-modified) (22, 23)KRAS ASO that is complementary to a sequence in the 3′ untranslatedregion (3′UTR) of KRASmRNA and thus targets both the mutant andwild-type KRAS isoforms for ribonuclease H–mediated degradation(Fig. 1A). The in vitro evaluation of nucleic acid–based agents (siRNAor antisense) typically relies on cationic lipid–mediated transfection todeliver them into cells (23–25). However, because the cEt chemistry in-creases the potency ofASOs (22, 23), these inhibitors can be evaluated incancer cells growing in culture without any delivery agent (termed free-uptake conditions). The potency and selectivity of AZD4785 were ini-tially assessed in A431 endometrioid carcinoma cells, which expresswild-type KRAS and have been shown to be sensitive to free uptakeof ASO in the absence of transfection or delivery reagents (24).AZD4785 selectively down-regulatedKRASmRNAwith a half-maximalinhibitory concentration (IC50) of 10nMinA431 cellswithout decreasingthe expression of HRAS and NRAS mRNA isoforms (Fig. 1B). KRASmRNA down-regulation correlated with a dose-dependent decreasein KRAS protein with no impact on NRAS or HRAS proteins (Fig. 1C).Systemic treatment of mice bearing established A431 tumors withunformulated AZD4785 at 16, 32, or 48 mg/kg (mpk) 5× weekly for3 weeks reduced KRAS mRNA in the tumors in a dose-dependentmanner without affecting the expression of other RAS isoforms (Fig.1D), and immunohistochemical (IHC) analysis confirmed robustKRAS protein inhibition (Fig. 1E).

The sequence of AZD4785 was searched against the human tran-scriptome for any sequences with 0 or 1 mismatch or sequences with2 mismatches and 14 matches in a row. The effects of AZD4785 on theexpression of primary gene transcripts corresponding to those predictedoff-target sites were analyzed by quantitative reverse transcription poly-merase chain reaction (qRT-PCR) in AZD4785-treated human cells. Allpredicted coding off-target genes that could be measured in human cellshad no or lower reductions inmRNA expression relative to on-target ac-tivity (KRASmRNA knockdown; table S1). Together, these results dem-onstrate that the KRAS cEt ASOAZD4785 results in potent and selectivedown-regulation of human KRASmRNA and protein both in vitro andin vivo without the need for any complex delivery formulation.


AZD4758 selectively inhibits the proliferation of tumor cellsexpressing mutant KRASWe assessed the impact of AZD4785-mediated KRAS depletion onlung, colon, and pancreatic tumor cells. AZD4785 treatment produceddose-dependent inhibition ofKRASmRNA and protein expression in apanel of cell lines (Fig. 2 and fig. S1). Sensitivity to ASO-mediated targetRNA reduction varied in the cell lines evaluated, potentially due to dif-ferences in productive and nonproductive uptake pathways in thesecells, as has been described previously (24, 25). Because AZD4785 tar-gets KRASmRNA sequences distinct from the mutation codon sites, itwas able to deplete KRAS, irrespective of theKRASmutation (Fig. 2 andtable S2). Cell lines expressing mutant KRAS have demonstrated vari-able dependency upon KRAS for viability in two-dimensional (2D)monolayer proliferation assays (26), which we have also observed (fig.S2). However, in anchorage-independent 3D growth assays, KRAS de-pendency was more clearly observed (fig. S2) (16, 27, 28). Consistentwith this hypothesis, we demonstrated that AZD4785 inhibited the col-ony formation of a panel ofKRASmutant cancer cells (Fig. 2A and figs.S3 and S4). In contrast, the 3D growth ofKRASwild-type cancer cells wasnot appreciably affected by AZD4785 despite the fact the KRAS depletionwas similar to themutant cell lines (Fig. 2Aand figs. S3 andS4).Consistentwith depletion of functional KRAS protein, AZD4785 treatment resultedin the potent inhibition of MAPK pathway activity selectively in KRASmutant cells, reducing the expression of theMAPK pathway downstreamtranscripts DUSP6 and ETV4 (Fig. 2B and fig. S1) (29). Furthermore,Western blot analysis of lysates from AZD4785-treated KRAS mutantor KRAS wild-type cancer cells confirmed down-regulation of both mu-tant and wild-type KRAS protein and demonstrated inhibition ofdownstream MAPK pathway signaling [including phospho-C-RAFproto-oncogene serine/threonine kinase (pCRAF), phospho–MAPK ki-nase (pMEK), phospho–extracellular signal–regulated kinase (pERK),and phospho–ribosomal protein S6 kinase 90 (pRSK90)] and PI3Kpathway signaling [phospho-protein kinase B (pAKT)] selectively in theKRAS mutant cells (Fig. 2C and fig. S5). These data therefore show thatAZD4785 down-regulates mutant and wild-type KRAS isoforms and hasselective phenotypic effects on KRASmutant cells in vitro.

Inhibition of KRAS is not limited by feedback activation ofthe MAPK pathwayThe MAPK pathway (RAF-MEK-ERK) is a key effector pathwaymodulated byKRAS activity, and inhibitors of key nodes of the pathwayare under development (30, 31). Therefore, we explored howAZD4785-mediated depletion of KRAS might differ from enzymatic inhibitors ofthe MAPK pathway by comparing the impact of AZD4785 with inhib-itors of MEK1/2 (selumetinib/AZD6244/ARRY-142886) or ERK1/2(SCH772984) on signaling inKRASwild-type andmutant cells. Becausethemolecular target ofASOs ismRNA, they take longer to achievemax-imal inhibition (protein depletion) compared to small-molecule en-zymatic inhibitors (SMIs); therefore, cells were treated with ASOs for72 hours and with SMIs for 24 hours before assessing the impact ondownstream signaling. As expected, inhibition of MAPK pathwaysignaling was observed after treatment with AZD4785; however, thiswas selective for KRAS mutant cells, consistent with the effects on cellproliferation (Fig. 3, A and B, and figs. S1 and S5). In contrast, the ac-tivity of selumetinib and SCH772984 was less selective and inhibitedproliferation and downstream MAPK pathway signaling of cellscarrying either aKRASmutation or a non-RASmutant genotype (EGFRmutation in the PC9s) that would be predicted to activate the RAF-MEK-ERK pathway (Fig. 3, A and B, and figs. S4 and S5).

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The MAPK pathway is under careful homeostatic control throughnegative-feedback mechanisms. For example, MEK1/2 and ERK1/2inhibitors increase MEK1/2 phosphorylation at S218/222 in bothKRAS wild-type and mutant cells by relieving upstream negativecontrols (30), which we also observed (Fig. 3A and fig. S5). Increasesin pMEK were not observed upon AZD4785 treatment, and more-over, in some KRAS mutant cells, there was a decrease in pMEK(Fig. 3A and fig. S5), suggesting that AZD4785 limits feedback reactivationupstream ofMEK1/2. To extend this observation, we assessed the impactof combining AZD4785 and selumetinib on MAPK pathway signalingand cell growth. Pretreatment with AZD4785 for 48 hours limited theselumetinib-induced increase in pMEK in NCI-H358 but not PC9 cells(Fig. 3C and fig. S6A). The combination treatment in NCI-H358 cells


was also more effective at inhibiting downstream signaling than themonotherapy treatments (Fig. 3C and fig. S6A). Furthermore, selumetiniband AZD4785 combination enhanced the inhibition of cell growth incolony formation assays compared with either single-agent treatmentinKRASmutant cells, and no effect was seenwith a control ASO (CTRLASO) (Fig. 3D and fig. S6B).

Together, these data show that KRAS depletion by AZD4785 hasdistinct effects onMAPK signaling compared toMEK1/2 and ERK1/2inhibitors, with preferential activity in cell lines that carry aKRASmu-tation and lack of pathway reactivation through feedback phosphoryl-ation of MEK1/2 at S218/222. Furthermore, these data support therationale of a combination of AZD4785 with MAPK pathway inhibi-tors in KRAS-dependent tumors.

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Fig. 1. Potent and selective down-regulation of KRASmRNA and protein by AZD4785 in vitro and in vivo. (A) Sequence and binding region of AZD4785 to the 3′UTR ofKRASmRNA transcript. ds, unmodified bases; ks, cEt-modified bases; mC, 5-methylcytosine; ORF, open reading frame. (B) Relative expression of RASmRNA isoformsmeasured byqRT-PCR in A431 cells after 72 hours of treatment with AZD4785 or CTRL ASO. Expression was normalized to ACTIN and shown relative to phosphate-buffered saline (PBS).Representative data from aminimum of two independent experiments are shown. (C) Western blot analysis of RAS and vinculin protein in A431 cells after 72 hours of treatmentwith CTRL ASO or AZD4785. (D) Mice bearing A431 tumors were treated with PBS or with 16, 32, or 48 mpk of AZD4785 5× weekly for 3 weeks. RAS isoform expression wasmeasured in tumors at the end of the study by qRT-PCR. Expressionwas normalized to ACTIN and shown relative to PBS. The graph shows individual tumor data, treatment groupmean, and SE. mpk/w, mpk/week. (E) IHC analysis of KRAS protein expression in A431 tumors after AZD4785 treatment. Scale bars, 200 mm.

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Systemic delivery of unformulated AZD4785 producesantitumor activity in KRAS mutant xenograft models in vivoA pharmacokinetic (PK) and pharmacodynamic (PD) study withsystemic delivery of 40 mpk of AZD4785 5× weekly in nude micebearing NCI-H358 tumors demonstrated that maximum KRASmRNA knockdown in tumors occurred after about 2 weeks of dos-ing and that this correlated with maximal tissue exposure (fig. S7).The slower onset kinetics of ASO activity is associated with accu-mulation of drug in tissues over time to concentrations that pro-duce robust target engagement (32). In xenograft experiments, thisis observed as a delay in antitumor activity (Figs. 4 and 5). In theNCI-H358 KRAS mutant lung cancer xenograft, treatment ofanimals for 4 weeks with AZD4785 resulted in robust down-regu-


lation of KRAS mRNA (55%; P < 0.001) in tumor tissue and a re-duction in downstream effector pathway activity with inhibition ofDUSP4 (51%; P < 0.001) and DUSP6 (55%; P < 0.001) mRNA (Fig.4A). As expected, AZD4785 did not down-regulate HRAS or NRASisoforms in NCI-H358 tumors (fig. S8). Furthermore, treatmentwas well tolerated (fig. S9), and significant antitumor activity wasobserved in the NCI-H358 model, with 4 weeks of dosing ofAZD4785 achieving a tumor growth inhibition (TGI) of 72% (P =0.005) compared to PBS (Fig. 4, B and C, and fig. S10A). Significanttumor PD and efficacy were also observed with AZD4785 treat-ment in the NCI-H1944 KRAS G13D mutant lung cancer model(Fig. 4, D to F, and fig. S10B). Inhibition of KRAS (68%; P <0.001), DUSP4 (73%; P < 0.001), and DUSP6 (75%; P < 0.001)

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Fig. 3. Differentiation of AZD4785 from MAPK pathway inhibitors in vitro. (A) Western blot analysis of MAPK and PI3K signaling in NCI-H358 and PC9 cells treated withAZD4785 and CTRL ASO for 72 hours or with selumetinib, SCH772984, or DMSO for 24 hours. (B) Expression of KRAS, DUSP6, and ETV4mRNAmeasured by qRT-PCR in NCI-H358and PC9 cells treated with a dose range of AZD4785 for 72 hours or with selumetinib or SCH772984 for 24 hours. Expression was normalized toGAPDH and expressed relative toPBS control. Data are from a representative experiment (n = 2) showing mean expression of technical replicates and SD. (C) Western analysis of MAPK pathway signaling in NCI-H358 and PC9 cells after monotherapy or combination treatment with AZD4785, CTRL ASO, and selumetinib. ASO treatment was done for 72 hours, and selumetinib treatmentwas done for 24 hours. For combination treatments, selumetinib was added 48 hours after dosing with AZD4785 for the final 24 hours of incubation. (D) NCI-H358 and PC9 cellsgrown in soft agar were treated with selumetinib in combinationwith CTRL ASO or AZD4785. Data are from a representative experiment from aminimum of two showingmeancolony number and SD.

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mRNA was measured in tumors at theend of the study (Fig. 4D) and was as-sociated with a TGI of 80% (P = 0.001)compared to PBS after 51 days of dos-ing (Fig. 4E). To assess the potentialclinical translation of the antitumor ac-tivity observed, we performed Kaplan-Meier survival analyses where we usedan increase in tumor size of fourfold asa surrogate for survival as an end point.In both the NCI-H358 and NCI-H1944studies, treatment with AZD4785 de-layed the time to reach the surrogateend point compared to the controlgroups (Fig. 4, C and F).

Patient-derived xenograft (PDX)models are thought to be more biolog-
ically relevant to human disease than standard cell line–derived xeno-graft models and reflect the histological characteristics of humantumors better (33). LXFA 983 is a lung cancer PDX model thatcarries a KRAS G12C mutation. In vitro studies with AZD4785confirmed both free uptake of ASO, resulting in KRAS mRNAknockdown and KRAS dependency of MAPK pathway signaling

in this model (Fig. 5, A and B). Systemic delivery of AZD4785 toLXFA 983 tumor-bearing mice reduced tumor expression of KRAS(84%; P < 0.001), DUSP4 (44%; P = 0.057), and DUSP6 (61%; P =0.004) mRNA (Fig. 5C) and was furthermore associated with a re-duction in membrane KRAS protein detected in tumors by IHCcompared to controls (Fig. 5, D and E). PD effects of AZD4785

Fig. 4. PD and efficacy of AZD4785 in KRASmutant lung cancer xenograft models. (A toC) Mice bearing NCI-H358 tumors were treatedwith PBS, AZD4785, or CTRL ASO at 50 mpk 5×weekly for 4 weeks. (A) KRAS, DUSP4, and DUSP6mRNA were measured in NCI-H358 tumors at theend of the study by qRT-PCR. The expression wasnormalized to 18S ribosomal RNA (rRNA) andexpressed relative to PBS. Data are shown as indi-vidual tumor data, treatment group geometricmean, and SE. (B) AZD4785 significantly inhibitedtumor growth of NCI-H358 tumors compared toPBS (TGI, 72%; P = 0.0047) and CTRL ASO (TGI,75%; P = 0.001). Data are shown as the geometricmean of the tumor volume and SE. (C) Surrogateendpoint survival graphs for the NCI-H358 study.Data are shown as the percentage of remaininganimals with tumors <4× the initial starting vol-ume in each treatment group. (D to F) Micebearing NCI-H1944 tumors were treated withPBS, AZD4785, or CTRL ASO at 50 mpk 5× weeklyfor 7 weeks. (D) KRAS, DUSP4, and DUSP6 mRNAwere measured in NCI-H1944 tumors at the endof the study by qRT-PCR. The expression was nor-malized to ACTIN and presented relative to PBS.Data are shown as individual tumor data, treat-ment groupmean, and SE. (E) AZD4785 significant-ly inhibited tumor growth compared to PBS (TGI,80%; P = 0.001) and CTRL ASO (TGI, 67%; P <0.001). Data are shown as the geometric mean ofthe tumor volume and SE. (F) Surrogate endpointsurvival graphs for the NCI-H1944 study. Data areshown as the percentage of remaining animalswith tumors <4× the initial starting volume in eachtreatment group.

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KRAS

pERK1/2T202/Y204

pMEK1/2S218/222

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KRAS mRNA

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in the LXFA 983 PDX model correlatedwith 98% TGI (P < 0.001) at day 41 ofdosing compared to PBS (Fig. 5F andfig. S10C). In the LXFA 983 model,some TGI was observed with the CTRLASO (53%; P = 0.019) compared to thePBS (Fig. 5F). The reason for this is un-clear but is not associated with eitherKRAS down-regulation or inhibition ofdownstream effector pathway activity(Fig. 5C). The AZD4785-treated animalsshowed 95% TGI (P < 0.001) when com-
pared to the CTRL ASO group, and treatment with AZD4785 but notCTRL ASO delayed the time to reach the surrogate survival end point(Fig. 5G). We also assessed PD and efficacy of AZD4785 in a KRASwild-type NSCLC PDX model, LXFA 526 (Fig. 6). Despite potentknockdown of KRAS in tumors [89% mRNA (P < 0.001) and50% protein] (Fig. 6, A to C), this model showed less sensitivity toAZD4785 than the KRASmutant models, with no significant differencein tumor growth compared to the CTRL ASO (Fig. 6D and fig. S10D)or impact on time to reach the surrogate survival end point (Fig. 6E).

Tissue concentrations of ASO in preclinicalmodels have been used topredict efficacious dose levels in humans (34).Measurement ofAZD4785tissue concentrations from the LXFA 983 studies, in which good targetknockdown and TGI were observed, showed liver concentrations ofAZD4785 ranging from 215 to 407 mg/g at the end of the studies (Table 1).

AZD4785 efficacy and target engagementwere demonstrated in sev-eral additional KRAS mutant cell line–based and PDX in vivo models(table S3). Furthermore, additional KRAS cEt ASOs, with binding sitesdifferent fromAZD4785 (fig. S11A), also showed PD and efficacy in vivo

Fig. 5. PD and efficacy of AZD4785 in a KRASmutant lung cancer PDX model. (A) KRAS andDUSP6 mRNA expression was assessed by qRT-PCR in LXFA 983 cells in vitro after 72 hours treat-ment with AZD4785 or CTRL ASO. Representativedata from a minimum of two independent ex-periments are shown. (B) Western blot analysis ofMAPK signaling in LXFA 983 cells after 72 hourstreatment with AZD4785 and CTRL ASO or 24 hourstreatment with selumetinib. (C to G) Mice bearingLXFA 983 tumors were treated with PBS, AZD4785,or CTRL ASO at 50 mpk 5× weekly. (C) KRAS, DUSP4,and DUSP6 mRNA expression were measured inLXFA 983 tumors by qRT-PCR after 4 weeks of dos-ing. The expression was normalized to 18S rRNAand shown relative to PBS. Data are shown as indi-vidual tumor data, treatment group geometricmean, and SE. (D and E) KRAS protein expressionmeasured by IHC in LXFA 983 tumors after 4 weeksof dosing. (D) Representative images are shown(scale bars, 100 mm) and (E) quantitation of KRAS(membraneH score) determined by Image analysisplatform. Data are the mean membrane H scoreand SE. (F) AZD4785 significantly inhibited LXFA983 tumor growth at the end of study comparedto PBS (TGI, 98% at day 41, P < 0.001) and CTRLASO (TGI, 95%; P < 0.001). Data are shown as thegeometric mean of the tumor volume and SE. Thearrow indicates the time point at which half ofthe animals were removed for PD analysis. (G)Surrogate endpoint survival graphs for the LXFA983 study. Data are shown as the percentage ofremaining animals in each treatment group withtumors <4× the initial starting volume. Twocohortsof animal are shown: data from all animals (n = 15)up today 27 (filled symbol, unbroken line) anddatafrom animals (n = 8) treated for 41 days (opensymbol, broken line).

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in the NCI-H358 model (fig. S11, B and C). These data as well as thephenotypic specificity of AZD4785 for KRAS mutant tumors areconsistent with the notion that the antitumor activity of the KRASASOs is being driven by selective KRAS down-regulation. Together,these data demonstrate that systemic delivery of unformulatedAZD4785 at clinically relevant doses in preclinical models of lungcancer can achieve KRAS knockdown and antitumor activity selec-tively in KRAS mutant tumors.

ASO-mediated systemic KRAS knockdown is well toleratedin mouse and monkeyAZD4785 targets both wild-type and mutant KRAS therefore, to betterunderstand the therapeutic window, we assessed the physiological


consequences of the ASO-mediated inhibition of KRAS in normal tis-sues. Because AZD4785 does not down-regulate murine KRAS, to dothis, we identified potent and specific cEt ASOs targeting mouse KRASmRNAand characterized their activities upon systemic delivery to adultmice. Mouse-specific KRASASOs administered by subcutaneous injec-tions produced robust knockdown of KRAS mRNA and protein inmultiple tissues, with selectivity over NRAS and HRAS (Fig. 7, A andB, fig. S12, and table S4). Knockdown of KRAS was not associated withany adverse effects (fig. S13 and tables S5 and S6) even at the highestdoses, where strong KRAS mRNA knockdown was achieved in manytissues (94% in liver, 92% in lung, 81% in heart, and 73% in kidney).Although KRAS knockdown in the liver reached 94%, no notable liver-related tolerability signals were detected during both biomarker and


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histopathology assessment (Fig. 7, A andB, fig. S13, and tables S5 and S6), suggest-ing that although genetic disruption ofKRAS is detrimental to the developing fe-tal liver (35), KRASknockdown in this or-gan in adults is well tolerated. In addition,no impact on MAPK pathway signalingtranscripts (DUSP4, DUSP6, or ETV4)was observed in the liver of the mice,supporting functional redundancy of theRAS isoforms in the adult tissue (fig.S12B). Changes that were observed inthe clinical chemistry and clinical pathol-ogy profiles (modest changes in plasmaconcentrations of alanine transaminaseand aspartate transaminase, liver weight,extramedullary hematopoiesis in thespleen, and minimal to mild basophiliaof enlarged Kupffer cells in the liver) inKRAS ASO–treated mice were similar tothose observed in the group treated withCTRL ASO and therefore were related tohighASO accumulation in the tissues andnot to KRAS knockdown.

The human drug candidate AZD4785is perfectly matched to the cynomolgusmonkey KRAS mRNA (Fig. 7C) andproduced potent dose-dependent knock-down ofKRASmRNA in treated cynomol-gus monkey hepatocytes in vitro (Fig. 7D).Therefore, we determined the tolerabilityof AZD4785 inmale cynomolgus monkeysin a 6-week study at high (40 mpk/week)dose. Similar to the mouse-specific ASO,AZD4785produced substantial and selec-tive knockdown of KRAS mRNA andprotein in multiple tissues (Fig. 7, E andF, and fig. S14), without any detectable ad-verse effects or histopathological changes,which could be attributed to KRAS inhibi-tion (tables S7 to S11).

In toxicology studies, selectiveMEK1/2inhibitors were reported to cause softtissue and vascular mineralization in rats.This phenotype was preceded by 1,25-dihydroxyvitaminD3 and serum inorganic

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Fig. 6. PD and efficacy of AZD4785 in a KRAS wild-type lung cancer PDX model. Mice bearing LXFA 526 tumorswere treated with PBS, AZD4785, or CTRL ASO at 50 mpk 5× weekly. (A) KRAS and DUSP6 expression measured byqRT-PCR in the LXFA 526 tumors after 4 weeks of dosing. The expression is normalized to 18S rRNA and expressedrelative to PBS. (B and C) KRAS protein expression measured by IHC in LXFA 526 tumors after 4 weeks of dosing. (B)Representative images are shown (scale bars, 100 mm) and (C) quantitation of KRAS (H score) determined by Imageanalysis platform. Data are the mean H score and SE. (D) AZD4785 showed modest inhibition of tumor growthversus PBS (51%; P = 0.001); however, there was no significant TGI compared to the CTRL ASO. Data shown aretreatment group geometric mean and SE. (E) Surrogate endpoint survival graphs for the LXFA 526 study. Data areshown as the percentage of remaining animals with tumors <4× the initial starting volume in each treatment group.

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phosphorus (IP) increases (36). In toxicology studies with both mouseand monkey KRAS ASOs, we have not detected any changes in the cir-culatory concentrations of IP and calcium (fig. S15).

Together, these data demonstrate that systemic inhibition of KRASwith potent KRAS-specific cEt ASOs in the adultmouse ormonkeywaswell tolerated. Minimal changes observed in some clinical chemistryparameters and clinical pathology profileswere consistentwith reportedeffects of high ASO accumulation in the tissues (37) and were unrelatedto the KRAS inhibition.

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DISCUSSIONTargeted inhibition of KRAS has been a challenge in cancer research formore than 30 years. This is due to the high affinity of KRAS for GDPand GTP as well as the lack of well-defined druggable sites on the pro-tein surface. However, some progress have been made in the develop-ment of covalent inhibitors that bind and inhibit the G12C mutantisoform of KRAS (15, 38). The second-generation inhibitor ARS-853covalently interacts withKRASG12C bound toGDP, preventing nucle-otide exchange and locking it in an inactive state (16, 17). This com-pound has shown promising activity in KRAS G12C mutant cells invitro, inhibiting proliferation in 3D assays and inhibiting the activityof downstream KRAS effector pathways (16, 17). However, the in vivopharmacologic activity of ARS-853 in tumor models has yet to be as-sessed.Moreover, these inhibitors are at early stages of discovery and, asmentioned previously, will be limited to use inKRASG12Cmutant sub-population of KRAS mutant cancers. Therapeutic siRNAs have alsobeen explored to target a subpopulation of KRAS-dependent tumors.siG12DLODER is aKRASG12Dmutant–selective siRNA encapsulatedin a biodegradable polymer delivery vehicle and has shown antitumoractivity in mouse models of pancreatic cancer (18). This technology iscurrently in clinical trials for the treatment of pancreatic cancer(NCT01676259). However, systemic delivery of siRNA treatments re-quired to treat metastatic tumors is still a challenge and would requireimprovements in lipid or nanoparticle technology (39).

AZD4785 is a first-in-class next-generation cEt ASOdrug that selec-tively down-regulates KRAS mRNA and protein and is efficacious inpreclinical KRAS mutant lung cancer models in vivo through un-formulated systemic delivery. AZD4785 is selective for KRAS and doesnot down-regulate the closely related isoforms, NRAS or HRAS how-ever, because it binds to the 3′UTR of KRAS, it is able to target all mu-


tant isoforms of KRAS and therefore has a broad therapeutic potentialacross tumor types. Thus far, we have concentrated our studies withAZD4785 as a therapeutic drug in KRASmutant NSCLC in vivo; how-ever, AZD4785 has also demonstrated activity in KRAS mutant colonand pancreatic cancer cell lines in vitro. Antitumor activity in vitro andin vivo was not observed with AZD4785 in KRAS wild-type models,despite potent KRAS knockdown, consistent with the expected pheno-typic activity of a selective KRAS targeting agent. Moreover, the dosesresulting in robust efficacy are projected to be achievable in humansbased on previous experience with cEt ASOs (24).

The sensitivity of individual KRAS mutant preclinical models toAZD4785 is dependent upon both the ability of the cells to take upASO (25) and the inherent sensitivity of the tumor cells to KRAS deple-tion. This depends upon the genetic and gene expression context of thetumor with a KRASmutation, such that tumors with high baseline ex-pression of KRAS and epithelial markers may show the highest level ofdependency (26).

The MAPK cascade (RAF-MEK-ERK) is a key effector pathwayregulated by KRAS, and genetic loss of CRAF, MEK1/2, and ERK1/2prevents mutant KRAS-driven lung tumors (40, 41). Our data demon-strate that inhibition of KRAS is different fromMAPK pathway inhibi-tion by MEK1/2 or ERK1/2 inhibitors in several ways. First, althoughAZD4785 down-regulates both wild-type andmutantKRAS, the robustinhibition of cell proliferation and downstream signaling is selective toKRASmutant tumor cells. In contrast, the activities of selumetinib andSCH772984 were not dependent upon the presence of a KRAS muta-tion, potentially limiting the therapeutic window of these drugs. Second,because of the relief of negative-feedback controls, selumetinib andSCH772984 increase MEK1/2 phosphorylation at S218/222, whichcan cause pathway reactivation and may be a mechanism of clinicalresistance to these drugs (42, 43). In contrast, down-regulation of KRASby AZD4785 did not increase phosphorylation of MEK1/2 and couldlimit selumetinib-induced pathway reactivation in some KRASmutantcells. This suggests that KRAS is a key node in pathway reactivation andhighlights an attractive drug combination opportunity for AZD4785and MAPK pathway inhibitors. The combination of AZD4785 and se-lumetinib showed enhanced inhibition of KRAS mutant cell prolifera-tion over either drug given as monotherapy. Finally, whereasselumetinib and SCH772984 in some cell backgrounds increasedpAKT, likely through relief of feedback of upstream receptors (44), thiswas not observed with AZD4785, and in fact, certain cell lines showeddown-regulation of pAKT, consistent with the PI3K/AKT cascade be-ing another effector pathway of RAS (45, 46).

An important question for any KRAS-targeted therapeutic agent iswhether these need to be selective for the mutant form of KRAS orwhether nonselective inhibition of both themutant andwild-type formsofKRASwill have the desired antitumor activity and the required safetyprofile to be valuable cancer drugs. With the KRAS ASO approach de-scribed here, we demonstrate that simultaneous depletion of both mu-tant and wild-type KRAS results in selective antitumor activity in cellsexpressing mutant KRAS. With respect to the potential safety of KRASdepletion in normal cells and tissues, gene disruption experiments havedemonstrated that wild-type KRAS function is essential for normalmouse early development because KRAS homozygous null embryosdie between days 12 and 14 of gestation and exhibit fetal liver defectsand severe anemia (35). However, there are no reported studies addres-sing the continued requirement for KRAS in adult organisms. More-over, the tolerability of therapeutic inhibition of KRAS in adultorganisms will depend on the PK and distribution properties of the

Table 1. AZD4785 PK measurements from liver at the end of the LXFA983 studies.

Model
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concentration
Length of study
(days)

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Mean
SD
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P198U/Q610

250 mpk/week
27 215.2 32.8
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P198U/Q610

250 mpk/week
41 239.0 29.8
LXFA983

P198U3/Q738

250 mpk/week
35 407.8 53.1
LXFA983

P198U3/Q738

125 mpk/week
35 344.4 49.1
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Rhesus KRAS 3'UTR 682 AAATTACTATAAAGACTCCTAATAGCTTTTCCTGT

Human KRASa 3'UTR 802 AAATTACTATAAAGACTCCTAATAGCTTTTCCTGT

Human KRASb 3'UTR 678 AAATTACTATAAAGACTCCTAATAGCTTTTCCTGT

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therapeutic modality used. To assess thetolerability of ASO-mediated KRAS de-pletion in adult animals, we identifiedand evaluated potent mouse-specific cEtKRAS ASOs. Systemic delivery of mouseKRAS ASOs produced robust targetknockdown of KRASmRNA and proteinin normal mouse tissues, including theliver, lung, heart, andkidney. Encouraging-ly, for the therapeutic approach describedhere, despite the robust knockdown in tis-sues, no significant target-related tolerabil-ity issues were observed. Furthermore,because of complete sequence homology,AZD4785 inhibits monkey as well as hu-man KRAS and was thus evaluated in a6-week monkey safety study. Similar tothe findings in mice, ASO-mediatedknockdownofKRAS in cynomolgusmon-key was confirmed in normal tissue andwas well tolerated. These data suggest thatKRAS function may be compensated forby NRAS and/or HRAS isoforms in adulttissueswhereASOs are active and supportsa therapeutic window for AZD4785-mediated KRAS depletion, because it hadno activity on either NRAS or HRASmRNAorprotein.Wealsoobservedadiffer-entiation in tolerability profiles for specific

Fig. 7. ASO-mediated KRAS knockdown inmouse and monkey to assess tolerability. (Aand B) BALB/c mice were treated twice weeklywith PBS, CTRL ASO, or mouse-selective KRAS(mKRAS) ASOs at 50mpk for 6 weeks. (A) IHC anal-ysis of KRAS protein inmouse tissues at the end ofthe study (scale bars, 200 mm). (B) qRT-PCRmeasuring KRAS mRNA in mouse tissues at theend of the study. mRNA expression was normal-ized to total RNA and expressed relative to PBS.Individual animal data, mean, and SE are shown.(C) Sequence alignment of monkey and humanKRASmRNA isoforms 3′ of the open reading frame,with the binding site of AZD4785 highlighted inred. (D) qRT-PCR demonstrating KRAS mRNAdown-regulation in primary cynomolgus monkeyhepatocytes after transfection with AZD4785.mRNA expression was normalized to total RNAand presented relative to PBS. Cynomolgusmonkeys were treated for 6 weeks with AZD4785or vehicle. For the first week, animals were sub-cutaneously dosed with 40 mpk four times andsubsequently once weekly with 40 mpk. (E) IHCanalysis of KRAS protein in monkey tissues at theend of the study (scale bars, 200 mm). (F) qRT-PCRmeasuring KRAS mRNA in monkey tissues at theendof the study.mRNAexpressionwasnormalizedto total RNA and presented relative to PBS. Indi-vidual animal data, mean, and SE are shown.

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KRASASOs overMEK inhibitors, with no observed increases in plasmaphosphates or calcium,which have been reported to precede tissuemin-eralization induced by MEK inhibitors (36). This suggests potentialredundancy in control of MEK activity by different RAS isoforms innormal tissue, which is further supported by our observation that KRASknockdown in murine and monkey liver has little impact on knownMAPK pathway regulated transcripts, and highlights a differential tol-erability profile for AZD4785.

TherapeuticASOs are becoming an increasingly attractive therapeu-tic modality to target traditionally difficult drug pathways. Increasedunderstanding of biological mechanisms involved in tissue distribution,cellular uptake, and intracellular trafficking of oligonucleotides hasenabled new approaches to enhance the delivery and activity of oligo-nucleotides. These include ligand-oligonucleotide conjugates [such asN-acetylgalactosamine conjugation for effective delivery to hepatocytes(47)], lipid- and polymer-based nanoparticles (48, 49), targeted deliverywith antibody conjugates (50), and the combination with small mole-cules that enhance uptake and activity of oligonucleotides (51). Futurestrategies building off of these approaches will be important to maxi-mize the potential of therapeutic ASOs to broadly treat human diseases.

This study has been limited to assessing the antitumor activity ofAZD4785 in subcutaneous xenograft and PDX models of lung cancerin immunocompromised mouse models. Thus, we have yet to explorethe impact of a therapeutic KRAS ASO onKRASmutant tumors in situor in the presence of an intact immune microenvironment. In conclu-sion, our data demonstrate that AZD4785 is a potent and well-toleratedKRAS cEt ASO with robust antitumor activity at doses relevant to theclinical setting and suggest that AZD4785 has potential as a therapeuticto help address the high unmet clinical need represented by mutantKRAS-driven human cancers.

by guest on July 16, 2019g.org/

MATERIALS AND METHODSStudy designThe overall objectives of this studywere to assess the in vitro and in vivoactivity of AZD4785, a generation 2.5 cEt-modified ASO targetingKRAS mRNA, in KRAS mutant and wild-type preclinical models, andto assess the tolerability of KRAS down-regulation by ASO treatment innormal tissues.

The activity and selectivity of AZD4785 were first evaluated inmultiple KRASwild-type and mutant cell lines, assessing effects on col-ony formation in soft agar and signaling end points known to bemodu-lated by KRAS activity. In vitro experiments were not performedblinded and were measured in technical replicates, with a minimumof two biological replicates experiments performed.

Next, the in vivo activity of systemically delivered unformulatedAZD4785 was studied in multiple KRAS mutant cell line–based andpatient-derived subcutaneous xenograft tumors, comparing the anti-tumor activity to vehicle and control ASO. Antitumor activity was assessedbymeasuring tumorvolume,KRAS expressionknockdown, and the activityof KRAS-regulated downstream effector pathways. Age- and gender-matched animals were randomly assigned into treatment groups (n = 6to 15) to ensure an equal tumor size (100 to 200 mm3) across groups atthe initiation of the study. In vivo studieswere not performedblinded. Exper-imental replicates for invivostudieswereas follows:NCI-H358, four replicates;LXFA 983, two replicates; and LXFA 526 and NCI-H1944, one replicate.

The tolerability of knocking down KRAS expression by an ASO wastested in age-matched male and female mice, using mouse-specificKRAS ASO, or in male cynomolgus monkeys, using AZD4785. Toler-


ability was assessed by measuring KRAS expression knockdown inmultiple normal tissues and measuring changes in clinical chemistryand clinical pathology profiles compared to vehicle and control ASO-treated animals. Microscopic evaluation of tissue sections was per-formed by a pathologist, and findings were peer-reviewed by separatepathologists. No data points were identified and removed as outliers.

StatisticsTumor growth inhibition (%TGI) from the start of treatment was as-sessed by comparison of the geometric mean change in tumor volumefor the control and treated groups. Statistical significance was evaluatedusing a one-tailed t test. For PD analysis, statistically significant changeswere determined using analysis of variance (ANOVA).

Additional materials and methods are available in the Supplemen-tary Materials.

SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/9/394/eaal5253/DC1Materials and MethodsFig. S1. Effect of AZD4785 on KRAS, DUSP6, and ETV4 mRNA expression in KRAS mutant andKRAS wild-type cell lines.Fig. S2. Sensitivity of NSCLC lines to KRAS knockdown by ASO in 2D versus 3D growthassays.Fig. S3. Effect of AZD4785 on colony formation in KRAS mutant and KRAS wild-type celllines.Fig. S4. Effect of AZD4785, selumetinib, and SCH772984 on the proliferation of KRAS mutantand KRAS wild-type cell lines.Fig. S5. Effect of AZD4785, selumetinib, and SCH772984 on signaling in KRAS mutant and KRASwild-type cell lines.Fig. S6. Effects of AZD4785 and selumetinib combination on signaling and proliferation ofNSCLC cells.Fig. S7. In vivo study assessing the kinetics of tumor PD and liver PK of AZD4785.Fig. S8. Effect of AZD4785 treatment on HRAS and NRAS expression in the NCI-H358 xenograftmodel.Fig. S9. Tolerability of AZD4785 treatment in xenograft studies.Fig. S10. Waterfall plots of xenograft and PDX studies.Fig. S11. PD and efficacy of AZD4785 and additional cEt KRAS ASOs in the NCI-H358 model.Fig. S12. Selectivity of murine KRAS ASOs and impact on MAPK pathway signaling in livertissue from mice.Fig. S13. Summary of the effects of the murine KRAS ASOs on body weight, plasma chemistry,and circulating blood cells in mice.Fig. S14. Selectivity of AZD4785 and impact on MAPK pathway signaling in liver tissue frommonkeys.Fig. S15. Impact of murine KRAS ASOs on plasma concentrations of inorganic phosphates andcalcium.Table S1. Summary of predicted off-targets for AZD4785.Table S2. Details of the cell lines used in the study.Table S3. Activity of AZD4785 in KRAS mutant NSCLC xenograft models.Table S4. Inhibition of KRAS mRNA across a panel of normal tissues after treatment of micewith murine KRAS ASOs.Table S5. Summary of tissue histopathology in the mouse tolerability study after treatmentwith the murine-selective KRAS ASOs.Table S6. Summary of body and organ weights, plasma chemistries, and circulating blood cellsin the mice after treatment with the murine-selective KRAS ASOs.Table S7. Summary of tissue histopathology in the monkey tolerability study after treatmentwith AZD4785.Table S8. Summary of body and organ weights in the monkey tolerability study after AZD4785treatment.Table S9. Summary of circulating blood cells in the monkey tolerability study after AZD4785treatment.Table S10. Summary of plasma chemistries in the monkey tolerability study after AZD4785treatment.Table S11. Summary of urinalysis in the monkey tolerability study after AZD4785 treatment.Table S12. Details of the parameters used for analyzing colony formation across the cell linepanel.Table S13. Summary of the individual animal tumor volumes in the xenograft and PDX studies.References (52, 53)

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Acknowledgments: We thank S. Freier, A. Watt, D. Gattis, and M. Bell (Ionis Pharmaceuticals)for the design and in vitro screening of the human and mouse KRAS ASOs; L. Hettrick,C. May (Ionis Pharmaceuticals), and H. Musgrove (AstraZeneca) for the technical assistancewith rodent studies; Y. Jiang (Ionis Pharmaceuticals) for the immunohistological procedures;the Korean Institute of Toxicology, T.-W. Kim, and S. Burel (Ionis Pharmaceuticals) forconducting monkey tolerability study; Oncotest for conducting PDX studies and providingPDX cell lines; A. Hiraide (Preclinical Sciences R&D, AstraZeneca) for PC9 cells; and G. Duchesne(Institute of Cancer Research) for HX 147 cells. Funding: The study was funded by AstraZenecaand Ionis Pharmaceuticals. Author contributions: S.J.R. and A.S.R. were involved inconception and design, acquisition, analysis and interpretation of data, study supervision, andwriting of the manuscript. L.L.H. was involved in design, acquisition, analysis, andinterpretation of some in vivo studies. S.K.P. was involved in acquisition and analysis of somein vivo studies. R.E. and A.S. were involved in acquisition and analysis of RT-PCR data fromin vivo study samples. M.R. was involved in acquisition and analysis of IHC data from in vivostudy samples. N.W. was involved in acquisition and analysis of in vitro combination data.L.K.B. and S.K.K. were involved in design, analysis, and interpretation of safety study data. C.R.,K.H., M.Z., and D.C.B. were involved in concept and design of studies and reviewing of themanuscript. B.P.M. was involved in concept and design of studies and study supervision. P.D.L.and A.R.M. were involved in concept and design of studies, study supervision, and writingof the manuscript. Competing interests: S.J.R., L.L.H., R.E., A.S., N.W., M.R., C.R., L.K.B., S.K.K.,K.H., M.Z., D.C.B., and P.D.L. were/are employees and shareholders of AstraZeneca. A.S.R.,S.K.P., B.P.M., and A.R.M. are employees and shareholders of Ionis Pharmaceuticals. A.R.M. andA.S.R. are inventors on a patent application WO/2017/053722 that covers AZD4785 and isheld/submitted by Ionis Pharmaceuticals. Data and materials availability: Researchersmay obtain AZD4785 with a material transfer agreement from AstraZeneca and IonisPharmaceuticals. All reasonable requests for collaboration involving materials used inthe research will be fulfilled provided that a written agreement is executed in advancebetween AstraZeneca and the requester (and his or her affiliated institution). Such inquiriesor requests should be directed to the corresponding authors.

Submitted 7 December 2016Accepted 21 April 2017Published 14 June 201710.1126/scitranslmed.aal5253

Citation: S. J. Ross, A. S. Revenko, L. L. Hanson, R. Ellston, A. Staniszewska, N. Whalley,S. K. Pandey, M. Revill, C. Rooney, L. K. Buckett, S. K. Klein, K. Hudson, B. P. Monia, M. Zinda,D. C. Blakey, P. D. Lyne, A. R. Macleod, Targeting KRAS-dependent tumors with AZD4785, ahigh-affinity therapeutic antisense oligonucleotide inhibitor of KRAS. Sci. Transl. Med. 9,eaal5253 (2017).

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oligonucleotide inhibitor of KRASTargeting KRAS-dependent tumors with AZD4785, a high-affinity therapeutic antisense

Zinda, David C. Blakey, Paul D. Lyne and A. Robert MacleodK. Pandey, Mitchell Revill, Claire Rooney, Linda K. Buckett, Stephanie K. Klein, Kevin Hudson, Brett P. Monia, Michael Sarah J. Ross, Alexey S. Revenko, Lyndsey L. Hanson, Rebecca Ellston, Anna Staniszewska, Nicky Whalley, Sanjay

DOI: 10.1126/scitranslmed.aal5253, eaal5253.9Sci Transl Med

suitability for translation to humans.small cell lung cancer and evaluated its safety in primates, demonstrating its potential−mouse models of non

avoiding the need for a specialized delivery vehicle. The authors tested the efficacy of this therapy in multiple used in this study was chemically modified, allowing systemic delivery through subcutaneous injection and

. The antisense oligonucleotideKRASbased therapy for inhibiting −demonstrating an antisense oligonucleotide have turned to genetic technology, et al.difficult to target with small molecules. To overcome this issue, Ross

treatment-resistant tumor types such as lung and pancreatic cancer. KRAS has also proven to be notoriously oncogene are common in human cancer, includingKRASMutations that cause activation of the

KRASAn antisensible approach to targeting

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