RAS Oncoproteins: Therapeutic Vulnerabilities

RAS Oncoproteins:Tocris Therapeutic Scientific Vulnerabilities Review Series

Tocri-lu-2945 RAS Oncoproteins: Therapeutic Vulnerabilities

Kirsten L. Bryant, Adrienne D. Cox Introduction and Channing J. Der The three human RAS genes (HRAS, KRAS and NRAS) encode University of North Carolina at Chapel Hill, Lineberger four highly related RAS small GTPases (HRAS, KRAS4A, KRAS4B 1,2 Comprehensive Cancer Center, Department of Pharmacology and NRAS). RAS proteins function as GDP-GTP regulated binary Department of Radiation Oncology, Chapel Hill, NC 27599, USA, on-off switches that regulate a diverse network of cytoplasmic 3 Correspondence e-mail: [email protected] signaling networks. In cancer and developmental disorders (RASopathies) mutationally activated RAS proteins drive aberrant Dr. Adrienne Cox is Associate Professor of Radiation Oncology signal transduction.4 RAS proteins are also the founding members and Pharmacology, whose laboratory studies RAS with a focus on of a large superfamily of small GTPases comprised of >150 RAS isoform and mutation-selective functions and regulators of members.5 RAS genes comprise the most frequently mutated RAS subcellular localization. Dr. Channing Der is Sarah Graham oncogene family in cancer. In particular, RAS genes are the most Kenan Professor of Pharmacology whose studies center on frequently mutated oncogenes in the top three causes of cancer drugging RAS effector signaling networks. Dr. Kirsten Bryant is a deaths in the US in 2016 (lung, colorectal and pancreatic Postdoctoral Fellow in Dr. Der’s laboratory whose studies focus cancers).6 Consequently, there have been intense interest and on exploiting the metabolic dependencies of RAS mutant cancers. effort in targeting RAS for cancer treatment.7 Here we review the vulnerabilities of RAS that have been exploited for the development of pharmacologic inhibitors of RAS function, with a focus on RAS-dependent metabolic processes. These inhibitors also provide useful research reagents to study RAS function. Since mutant RAS proteins have cell context differences in function, we focus our discussion on the example of pancreatic ductal adenocarcinoma (PDAC), arguably the cancer most addicted to mutant RAS.8

RAS Mutations in Cancer RAS – the beating heart of cancer Collectively, the three RAS genes are mutationally activated in Image produced by G. Aaron Hobbs, PhD from UNC (unpublished image of human ~25% of all cancers (COSMIC v80, Table 1). However, the KRAS protein). frequency of RAS missense mutations is not uniform among Contents cancer types. For example, PDAC, the predominant form of pancreatic cancer, exhibits the highest frequency (95%) of RAS Introduction...... 1 mutations. In contrast, RAS genes are rarely mutated in breast RAS Mutations in Cancer...... 1–2 cancers (<2%). Further, the frequency of missense mutations RAS Protein Function...... 3–6 found in each RAS gene is not uniform, with KRAS comprising 85% of all RAS mutations in cancer, followed by NRAS (12%), RAS GDP-GTP Regulation...... 3 and, infrequently, HRAS (3%) (Figure 1A).1 Whereas KRAS is the RAS Effectors...... 4 predominant RAS isoform mutated in PDAC, colorectal (CRC) and RAS Membrane Association...... 5 lung adenocarcinoma (LAC), NRAS is the predominant isoform RAS Drug Discovery...... 7 mutated in melanoma and acute myelogenous leukemia (AML). Although rare overall, HRAS mutations are predominant in Direct Inhibitors of RAS...... 7 bladder and in head and neck squamous cell carcinomas Inhibitors of RAS Membrane Association...... 9 (HNSCC). Inhibitors of RAS Effector Signaling...... 10 RAS mutations serve distinct roles in the development of different Targeting Synthetic Lethal Interactors of Mutant RAS...... 11 cancers. In CRC, mutational loss of the APC tumor suppressor Exploiting RAS-driven Metabolism...... 11 gene is the initiating step, with subsequent KRAS mutation 9 Future Prospects...... 14 promoting tumor progression. In contrast, in PDAC, KRAS mutations are the initiating genetic alteration, with nearly 95% of References...... 15 the earliest precancerous lesions, pancreatic intraepithelial RAS Compounds...... 16 neoplasias (PanINs), harboring KRAS mutations (Figure 1B).10

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Additional genetic alterations are required for progression to the Table 2 | RAS mutation frequency in cancer1 fully invasive and metastatic disease. In genetically-engineered Cancer % mouse models of cancer, Kras mutation alone drives the development of PanIN lesions, and only rarely and after extensive Pancreatic ductal adenocarcinoma 98 time do fully malignant cancers arise.11 However, concurrent loss Colorectal adenocarcinoma 52 of one of the three tumor suppressor gene mutations commonly Multiple myeloma 43 lost in human PDAC (Tp53, Cdkn2a and Smad4) greatly Lung adenocarcinoma 32 accelerates and increases the high incidence of metastatic Skin cutaneous melanoma 29 disease.12–14 Uterine corpus endometrioid carcinoma 25 Despite the early onset of KRAS mutations in PDAC development, Uterine carcinosarcoma 14 and the subsequent accumulation of more than 50 other gene Thyroid carcinoma 13 15–20 mutations in advanced cancers, there is substantial evidence Acute myeloid leukemia 11 from cell culture and mouse models that continued mutant KRAS Bladder urothelial carcinoma 11 function is essential for maintenance of malignant disease.21–25 Gastric adenocarcinoma 10 Genetic ablation of mutant KRAS causes rapid tumor regression, 1 revealing the potential impact of effective KRAS therapy. Adapted from Table 1 in Reference 1 Offsetting the clear therapeutic value in targeting KRAS is evidence that cancers have the potential to adapt and to acquire mechanisms (e.g., YAP1 activation) that overcome their addiction to mutant KRAS.26,27

Figure 1 | RAS isoform mutations in cancer

3 1 A 3

12 14

85 100 86 96

All cancers PDAC (98%) CRC (52%) LAC (32%) 4 2

14 9 5 27 30

57 13 86 94 59

Melanoma (29%) AML (11%) Bladder (11%) HNSCC (6%)

KRAS NRAS HRAS

B KRAS CDKN2A TP53 SMAD4

Normal Invasion, PanIN-1 PanIN-2 PanIN-3 PDAC epithelium metastasis

A. Frequencies of RAS missense mutations in different cancer types. The numbers in parentheses are the percentage of all cancers harboring RAS mutations. The numbers in the pie charts indicate the percentage of mutations in each RAS isoform. Data were compiled from Supplementary Table 2 found in Reference 1. Abbreviations used are: AML, acute myelogenous leukemia; CRC, colorectal cancer; HNSCC, head and neck squamous cell carcinoma; LAC, lung adenocarcinoma; PDAC, pancreatic ductal adenocarcinoma. B. Genes involved in the initiation and progress of PDAC. CDKN2A encodes two distinct tumor suppressor proteins, p16INK4A and p19ARF, that act as positive regulators of the RB and p53 tumor suppressors, respectively. SMAD4 encodes the SMAD4 transcription factor that is activated by TGFb signaling.

2 | tocris.com RAS Oncoproteins: Therapeutic Vulnerabilities

RAS Protein Function receptors (e.g., EGFR) by a diverse spectrum of extracellular stimuli, activation of cytoplasmic signaling components then The three RAS genes encode highly identical (82–90%) 188– activate RAS-selective guanine nucleotide exchange factors 189 amino acid proteins (Figure 2). KRAS encodes two splice (RASGEFs; e.g., SOS1), stimulating rapid and transient GDP-GTP variants due to utilization of alternative exon 4 sequences, exchange and formation of the active GTP-bound protein. The designated KRAS4A and 4B, with 4B widely expressed and 4A active state is rapidly terminated by GTPase activating proteins more tissue restricted. The N-terminal 164 residues of RAS (RASGAPs; e.g., neurofibromin) that stimulate the weak intrinsic proteins comprise the G domain (92–98% sequence identity) GTPase activity of RAS, hydrolyzing the bound GTP to GDP, involved in GTP binding and hydrolysis. The C-terminal 24/25 returning RAS to the inactive state. residues exhibit the greatest divergence between the different isoforms and contain sequences that facilitate membrane During GDP-GTP cycling, RAS protein conformation is altered at interactions. the switch I (SI) and II (SII) regions (Figures 2A), with the conformation in the GTP-bound state displaying increased affinity for the RAS-binding (RBD) and RAS-association domains found RAS GDP-GTP regulation on RAS effectors (Figure 3).28 Wild-type (WT) RAS proteins exhibit low picomolar affinity for GDP Mutated RAS genes found in cancer harbor single missense and GTP and function as GDP-GTP regulated binary on-off mutations that are located primarily (98–99%) at one of three switches (Figure 3A).3 In quiescent cells, WT RAS proteins reside mutational hotspots, glycine-12 (G12), glycine-13 (G13) and in their inactive GDP-bound state. Upon stimulation of cell surface

Figure 2 | Human RAS proteins

A B α3 4 α5 α Sequence Identity HRAS NRAS KRAS4A KRAS4B α2 HRAS 85 84 83 SII NRAS 85 85 82 KRAS KRAS 4A 84 85 90 NRAS KRAS4B 83 82 90 HRAS

SI α1

C G domain HVR 50 100 150

RAS ** SI * SII

100% 78% 8% * = Mutations = Sequence divergence

HRAS 165 Q H K L R K L N P P D E S G P G C M S C K C V L S 189 NRAS 165 Q Y R M K K L N S S D D G T Q G C M G L P C V V M 189 KRAS4A 165 Q Y R L K K I S K E E K T P G C V K I K K C I I M 189 KRAS4B 165 K H K - E K M S K D G K K K K K K S K T K C V I M 188

Membrane targeting C = Palmitoylated cysteine S = Phosphorylated serine

A. RAS protein structures. Overlay of the structures of the three RAS proteins. B. RAS isoform sequence identity. Percent sequence identity was determined by CLUSTALW multiple sequence alignment analysis. C. The three RAS genes encode four RAS protein isoforms that are highly identical in primary sequence (82–90% amino acid (aa) sequence identity), structure and biochemical properties. The N-terminal 164 residues comprise the G domain, which is involved in GTP binding and hydrolysis and effector interaction. Within the G domain are the switch I (SI; residues 30-38) and II (SII; residues 60–76) regions, that change in conformation during GDP-GTP cycling. The C-terminal membrane targeting sequences are comprised of the hypervariable regions (HVR; residues 165–184/5), that share little sequence identity (8%), followed by the CAAX tetrapeptide sequences that signal for post-translational modifications required to promote RAS membrane association.

tocris.com | 3 Tocris Scientific Review Series glutamine-61 (Q61) (Figure 2C).29,30 The resulting single amino promote activation of RAF catalytic activity, leading to acid substitutions impair intrinsic and GAP-stimulated GTP phosphorylation of the MEK1 and MEK2 dual specificity protein hydrolysis activity, resulting in mutant proteins that are kinases. Activation of MEK1/2 in turn phosphorylate and activate persistently GTP-bound and active in the absence of external the ERK1 and ERK2 mitogen-activated protein kinases (MAPKs). stimuli (Figure 3B). Activated ERK1/2 can phosphorylate >200 cytoplasmic and nuclear substrates. Among the key ERK substrates important for WT RAS proteins can also be activated persistently in cancer by RAS-driven cancer growth is the MYC transcription factor. The indirect mechanisms. In particular, loss of function of the importance of the RAF effector pathway in RAS oncogenic RASGAP, neurofibromin (encoded by the NF1 tumor suppressor function is supported by the frequent mutational activation of gene), is caused by cancer-associated mutations (Figure 3B).31,32 BRAF (19%; COSMIC v79) in cancer, with largely mutually Mutational activation or overexpression of cell surface receptor exclusive occurrence in cancers harboring RAS mutations. tyrosine kinases (RTKs; e.g., EGFR) can also cause persistent RAS activation through activation of RASGEFs. Amplification of The second best-validated RAS effector family comprise the WT RAS genes is also seen in cancer (Figure 3D). (p110a/g/d) catalytic subunits of the heterodimeric class I phosphoinositide-3-kinases (PI 3-K).34 These lipid kinases RAS effectors phosphorylate phosphoinositide (4,5) bisphosphate (PIP2) to generate the membrane-associated lipid second messenger The RBD/RA domains are associated with a spectrum of phosphoinositide (3,4,5) trisphosphate (PIP3). PIP3 is bound by catalytically diverse effectors (Figure 4), with RAF serine/ pleckstrin homology (PH) domain-containing proteins, in threonine kinases (CRAF/RAF1, ARAF and BRAF) the best particular the AKT serine/threonine kinases, thereby promoting validated effectors important for driving the oncogenic function AKT activation and leading to activation of the mTORC1 complex. 33 of mutant RAS proteins. RAS-GTP binds and promotes RAF The importance of the PI 3-K effector pathway in oncogenesis is trafficking to the inner face of the plasma membrane, where supported by the frequent mutational activation of PIK3CA (10%; additional events (e.g., protein kinase-mediated phosphorylation) COSMIC v79) in cancer, although the co-occurrence of these

Figure 3 | RAS GDP-GTP cycle:

A B RAS RAS* GDP P GDP Pi GTP i GTP

RAS WT RAS RAS Mutant RAS GAP GEF GAP GEF

GDP GDP RAS RAS* GTP GTP

E E Transient, stimulus- Stimulus-independent, dependent effector chronic effector signaling signaling

RTK* C D RAS RAS P GDP GDP i GTP Pi GTP

RAS * WT RAS RAS WT RAS GAP GEF GAP GEF

GDP GDP RAS RAS GTP GTP

E E Stimulus-independent, GEF-dependent, chronic effector signaling chronic effector signaling * Mutations

A. Wild type RAS protein GDP-GTP regulation. B. Mutant RAS proteins with single amino acid substitutions at residues G12, G13 or Q61 have impaired GTPase activity and/or enhanced nucleotide exchange. C. Loss of RASGAP function causes constitutive activation of wild type RAS. D. Aberrant receptor tyrosine kinase signaling causes RASGEF-dependent activation of wild type RAS.

4 | tocris.com RAS Oncoproteins: Therapeutic Vulnerabilities mutations with RAS mutations indicates that mutated RAS alone RAS association with the plasma membrane. The carboxyl-terminal may not always potently activate PI 3-K signaling. CAAX tetrapeptide motif (C = cysteine, A = aliphatic amino acid, X = terminal amino acid) signals the first modification, the The RALGEF-RAL pathway is the third best-validated RAS covalent addition of a C15 farnesyl isoprenoid lipid, catalyzed by effector.35,36 RALGEFs are Ras-association (RA) domain-containing the cytosolic enzyme farnesyltransferase (FTase). This modifica proteins that act as GEFs and activators of the RALA and RALB tion is followed by endoplasmic reticulum (ER)-associated RAS- RAS-like small GTPases. Although components of this pathway converting enzyme 1 (RCE1)-catalyzed proteolytic removal of the are not mutated in cancer, substantial evidence from cell culture AAX residues, and subsequent carboxylmethylation of the now and mouse models demonstrates the critical requirement of both terminal farnesylated cysteine by isoprenylcysteine methyl RALA and RALB in RAS-dependent cancer growth. Interestingly, transferase (ICMT). The critical role of these modifications for despite their strong sequence and biochemical identity, RALA RAS function is demonstrated by mutation of the Cys residue to and RALB exhibit distinct roles in this process. For example, Ser (CAAX > SAAX), preventing all three modifications: SAAX whereas RALA but not RALB was required for PDAC tumorigenic mutants of RAS proteins bearing oncogenic mutations are growth, RALB was critical for PDAC invasion and metastasis.37 cytosolic and completely impaired in oncogenic activity. The fourth best-validated RAS effector pathway involves RACGEF Whereas the CAAX-signaled modifications are essential for RAS activation of the RAC small GTPase.1 The recent identification of function, additional carboxyl terminal sequence elements are mutationally activated RAC1 mutants in melanoma supports the necessary for proper RAS subcellular localization and membrane important role of this effector pathway in cancer growth.38 Mouse association.29,41 HRAS, KRAS4A and NRAS contain cysteine model analyses demonstrated that genetic ablation of RAC1 residues immediately upstream of the CAAX motif that are sites of impaired mutant RAS-driven cancer growth.39,40 post-translational modification by C16 fatty acid palmitate moieties. The palmitoyl acyltransferases (PATs) involved in RAS membrane association catalyzing palmitate covalent attachment are complex and still not RAS protein function is critically dependent on association with fully identified for RAS. Also, unlike the universal and irreversible the inner face of the plasma membrane.29,41 RAS proteins are farnesyl modification, palmitate modification is associated with synthesized initially as cytosolic, inactive proteins (Figure 5). They only a fraction of RAS, which may be reversibly deacylated by an then undergo rapid post-translational modifications that promote acyl protein thioesterase (APT1/2). Treatment with the relatively

Figure 4 | RAS effectors

RAS GTP

TIAM1 PI3K RAF RalGEF RBD * RBD * RBD RA

P PIP PIP 2 3 MEK* RAC RAC* RAC* RAL GDP GTP GDP GTP P P AKT* ERK* P P

PAK P P TBK1 mTORC1* MYC*

RAS Binding (RBD)/RAS Association (RA) domains * Mutated in cancer (Cancer Gene Census)

Effectors validated to drive mutant RAS-dependent cancer growth. Cell culture and mouse model analyses have validated the requirement for specific components of these effector pathways in the maintenance and/or initiation and progression of tumor growth. Activated RAS- GTP recognizes RBD or RA domain-containing proteins.

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Figure 5 | Regulation of RAS subcellular localization and membrane association

HRAS Plasma membrane + NRAS RAS KRAS4B –[K]n-C-OMe KRAS4A RAS –C-C-OMe + + RAS –[K]n-C-[K]n-C-OMe Deltarasin

PDE6δ APT PAT

Golgi RAS –C-OMe RAS –C-OMe

Palmitate fatty acid ICMT Farnesyl isoprenoid Geranylgeranyl isoprenoid RAS –C + AAX ER

RCE1 FTI-induced RAS –CAAX alternative GGTI prenylation

FTASE FTI GGTASE-I RAS –CAAX RAS –CAAX

RAS proteins are synthesized initially as cytosolic proteins. Within minutes, the CAAX motif signals for cytosolic FTase-catalyzed covalent and irreversible addition of a C15 isoprenoid to the cysteine residue. The endoplasmic reticulum (ER)-associated RAS-converting enzyme 1 (RCE1) then catalyzes endoproteolytic removal of the AAX residues, followed by ER-associated isoprenylcysteine carboxylmethyltransferase (ICMT) catalysis of reversible methyl esterification of the now terminal farnesylated cysteine residue using S-adenosylmethionine as the methyl donor. The CAAX-signaled modifications comprise the first signal that is necessary but not sufficient to promote plasma membrane association and subcellular localization essential for RAS protein function. The second signal is contributed either by polybasic sequences (KRAS4A, KRAS4B) and/or by cysteine residues (HRAS, NRAS and KRAS4A) that are reversibly covalently modified by Golgi-associated palmitoyl acyltransferase (PAT)-catalyzed addition of a C16 palmitoyl fatty acid. While one PAT (DHHC9-GPC16) has been identified for HRAS, whether other PATs for RAS proteins exist, and the acyl-protein thioesterases (APT) that depalmitoylate RAS proteins remain poorly understood. PDE6d recognizes the farnesyl group of KRAS4B and solubilizes the protein from the ER, promoting availability for the plasma membrane; this activity can be blocked by deltarasin and other small molecules. FTIs block the first CAAX-signaled modification, preventing all subsequent modifications, rendering RAS cytosolic and inactive. In the absence of FTase activity, KRAS and NRAS are substrates for GGTase-I and alternative prenylation by covalent addition of a C20 geranylgeranyl isoprenoid, allowing RCE1 and ICMT modification and plasma membrane association. Concurrent treatment with FTI and GGTI can block KRAS and NRAS membrane association. nonspecific PAT inhibitor 2-bromopalmitate disrupts the plasma through association with negatively charged phospholipids. The membrane association of palmitoylated RAS isoforms. The KRAS4A splice variant, which has a more restricted tissue acylation/deacylation cycle of RAS proteins is prominently involved expression profile, also contains carboxyl terminal lysine-rich in RAS cycling between the plasma membrane, cytoplasm and sequences that, together with a palmitoylated cysteine, are Golgi membranes. Differential RAS signaling at distinct subcellular required for full KRAS4A membrane association and function.42 compartments is also complex and still being delineated. Within the KRAS4B polybasic domain is a serine residue that can KRAS4B lacks any palmitate-modified cysteine residues be phosphorylated by protein kinase C (Figure 2C).43 This (Figure 2C).29,41 Instead, a lysine-rich polybasic domain adjacent reversible phosphorylation event alters KRAS4B subcellular to the CAAX motif comprises a second membrane-targeting localization and promotes association with endomembranes, sequence element that, together with the CAAX modifications, causing a loss of KRAS4B oncogenic function.44 Treatment with promotes KRAS membrane association and oncogenic activity bryostatin, an activator of protein kinase C, promotes KRAS4B (Figure 5). The positive charge provided by this polybasic phosphorylation. The phosphorylation status of this serine sequence facilitates plasma membrane association in part residue also dictates KRAS4B association with calmodulin.

6 | tocris.com RAS Oncoproteins: Therapeutic Vulnerabilities

Figure 6 | Anti-RAS therapeutic approaches Glucose Glutamine

Inhibition of membrane 1 association

Inhibition of RAS function RAS

Inhibition of metabolism

Inhibition of 2 effector signaling Inhibition of synthetic 3 lethal interactors

Direct and indirect strategies are being pursued to develop small molecule inhibitors of mutant RAS function. Indirect approaches include inhibitors of (1) proteins that facilitate RAS membrane association, (2) downstream effectors, (3) synthetic lethal interactors of mutant RAS and (4) modulators of RAS-dependent metabolic activities.

RAS Drug Discovery The crystal structure of HRAS did not reveal any well-defined pockets to support the development of potent and selective Based on the aspects of RAS protein function critical for RAS- small molecules that recognize and disrupt the function of dependent cancer growth (GTP binding, membrane association, mutant RAS proteins. This led to a widely held perception that effector signaling), a number of logical directions have been RAS proteins are “undruggable”. However, recent studies with pursued for the development of pharmacologic approaches to KRAS4B have begun to dispel this perception, with the 1,2 disrupt mutant RAS function in cancer (Figure 6, Table 2). identification of a number of direct binding small molecules that Ideally, the best approach for targeting RAS is to generate direct can disrupt different aspects of RAS function.45,46 Cell-active inhibitors of RAS function. While recent studies have identified small molecules that bind to KRAS and impair its interaction with small molecules that directly bind to and selectively perturb the the RASGEF SOS1 (DCAI, VU0460009) or association with RAS function of mutant RAS, a majority of the strategies pursued are effectors (Kobe 0065 and Kobe 2602) have been described indirect, targeting proteins that support RAS function. The (Figure 7). In particular, utilizing a computational design strategy, obvious potential limitation of this strategy is that indirect targets compound 3144 was developed as an antagonist of KRAS also likely support the function of other proteins. Hence, indirect interaction with effectors and affects all RAS isoforms.47 inhibitors of RAS will likely be compromised by off-target normal cell toxicity. Among the direct binders that have attracted considerable attention are small molecules that selectively recognize and impair the function of one specific KRAS mutant, a G12C Direct inhibitors of RAS substitution (SML-8-73-1, ARS-853).48–51 The ability of ARS-853 Since mutant RAS proteins are GAP-insensitive and persistently to selectively block G12C function also revealed that at least GTP-bound, attractive and logical strategies for blocking their some oncogenic mutants retain a dependency on GEF function to function include small molecules that can act as GAP mimetics or achieve the fully activated state. Thus, molecules that impair GEF disrupt GTP binding.1,2 However, to date, neither strategy has stimulation may be effective in a subset of KRAS mutants. been successful. While the successful development of ATP- The non-steroidal anti-inflammatory drug sulindac and its competitive protein kinase inhibitors for cancer treatment (e.g., derivatives have demonstrated anticancer activity, and this erlotinib, vemurafenib) argues that similar strategies should be activity may be mediated, in part, by direct binding to RAS and by possible with RAS, GTP binds to RAS at low picomolar affinities, in blocking RAS interaction with RAF.52–55 However, sulindac contrast to the low micromolar binding affinity of ATP for protein inhibition of RAF signaling may be indirect.56 Whether the anti- kinases.3 Coupled with the millimolar concentrations of GTP in tumor activity of sulindac and its derivatives are due to direct cells, such high affinity renders effective GTP-competitive small interaction with RAS and impairment of effector activation molecule inhibitors of RAS unfeasible. remains to be determined.

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Table 2 | Pharmacologic modulators of RAS function

Compound Target Mechanism Direct RAS binding Sulindac sulphide RAS Blocks RAS interaction with RAF IND12 RAS Blocks RAS interaction with RAF DCAI RAS Blocks SOS1 binding VU0460009 RAS Blocks SOS1 binding Kobe 0065 RAS Blocks effector activation Kobe 2602 RAS Blocks effector activation Compound 3144 RAS Blocks effector activation Compound 12 RAS G12C Blocks GEF stimulation SML-8-73-1 RAS G12C Blocks GEF stimulation ARS-853 RAS G12C Blocks GEF stimulation Modulators of RAS subcellular localization and membrane association Lonafarnib (SCH66336) FTase Inhibition of farnesyl addition to cysteine of the CAAX motif of RAS and >50 other proteins Tipifarnib (R115777) FTase Inhibition of farnesyl addition to cysteine of the CAAX motif of RAS and >50 other proteins GGTI-2147 GGTase-I Inhibition of geranygeranyl addition to cysteine of the CAAX motif of RAS and >150 other proteins Salirasib (FTS) RAS Inhibition of RAS membrane association Deltarasin PDEd Inhibition of PDEd association with prenylated proteins Deltazinone 1 PDEd Inhibition of PDEd association with prenylated proteins DHHC palmitoyl 2-bromopalmitate (2-BP) Inhibition of protein palmitoylation of myristoylated and prenylated proteins acyltransferases Bryostatin PKC PKC activation and phosphorylation of KRAS4B Effector inhibitors LY3009120 RAF1/ARAF/BRAF ATP-competitive inhibitor Allosteric, non-ATP competitive inhibitor, binds nonphosphorylated MEK1/2, Trametinib MEK1/2 prevents RAF phosphorylation BVD-523 (Ulixertinib) ERK1/2 ATP-competitive inhibitor Allosteric and ATP-competitive inhibitor, blocking MEK phosphorylation of ERK and ERK SCH772984 ERK1/2 phosphorylation of substrates, respectively Alpelisib (BYL719) p110a ATP-competitive inhibitor Ipatasertib (GDC-0068) AKT1/2/3 ATP-competitive inhibitor Everolimus mTORC1 Forms a complex with FKBP12 that binds to and inhibits the mTORC1 complex NSC 23766 RACGEFs Binds RAC-selective GEFs and prevents RAC1 interaction and activation RBC8 RALA/B Binds inactive RAL-GTP, preventing effector binding Metabolism inhibitors FX-11 LDHA NADH-competitive inhibitor, inhibiting glycolysis GNE-140 LDHA Inhibits LDHA activity, inhibiting glycolysis Allosteric inhibitor, stabilizes inactive GLS1 splice variant GAC tetramer, BPTES GLS1 inhibiting glutamine utilization Compound 968 GLS1 Allosteric inhibitor of GLS1 encoded-splice variants KGA and GAC, inhibits glutamine utilization EIPA Na+/H+ exchanger Lowers intracellular pH, inhibits macropinocytosis Lysosomotropic weak base, elevates/neutralizes the lysosomal/vacuolar pH, Chloroquine Lysosomes impairing lysosome function, inhibiting autophagy and macropinocytosis Spautin-1 VPS34 complexes Inhibits autophagy, among other endocytic processes SBI-0206965 ULK1 Inhibitor of ULK1 kinase activity and autophagy MRT 68921 ULK1/2 Inhibitor of ULK1/2 kinase activity and autophagy

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Inhibitors of RAS membrane association Figure 7 | Chemical structures of direct inhibitors of RAS Since RAS plasma membrane association is essential for RAS oncogenic function, the development of pharmacologic strategies to target the enzymes involved has also been an attractive HO2C Sulindac (Cat. No. 1707) direction for antiRAS drug discovery (Figure 8).29,41 The discovery Prodrug: metabolizes to sulindac F sulfide, a cyclooxgenase inhibitor of FTase in 1990 set off an intense effort by many ceutical Me that represses RAS signaling companies to develop FTase inhibitors (FTIs). FTIs demonstrated impressive anti-tumor activity in various cell culture and mouse models of RAS-driven cancers, with two FTIs (lonafarnib, O2N CF3 tipifarnib) advancing to phase III clinical evaluation. HN Disappointingly, FTIs showed no clinical efficacy when evaluated H Cl in cancers with a high frequency of KRAS mutations. The basis Me S N NH NO2 O for this had been revealed years earlier when it was determined S that FTIs were not effective inhibitors of KRAS or NRAS membrane association. Unlike HRAS, the isoform most widely studied in the F Kobe 0065 (Cat. No. 5475) earlier years of anti-RAS drug discovery, the KRAS and NRAS HRAS-RAF interaction inhibitor isoforms most frequently mutated in cancer undergo alternative prenylation in FTI-treated cells. Although they are not normally O NH H substrates for the related enzyme geranylgeranyltransferase-I Cl N O S N (GGTase-I), when FTase activity is blocked, both KRAS and NRAS NH S are modified by this enzyme via covalent addition of aC20 Cl OH N O HN H geranylgeranyl isoprenoid lipid, thereby restoring RAS membrane O2N NO2 association. Compound 8 Inhibits oncogenic mutant RAS Despite the failure of FTIs as an effective therapy for cancers in which KRAS and NRAS mutations predominate, FTIs have F F H N Cl recently been reconsidered for treatment of cancers harboring F HRAS mutations. An obvious remedy for the limitations of FTIs Kobe 2602 Blocks HRAS GTP Cl would be to combine them with a GGTI inhibitor (e.g., GGTI-2417). HN2 N binding to RAF However, since collectively these two enzymes have hundreds of HN O CAAX motif-terminating protein substrates, off-target toxicity is a N potential concern. Nevertheless, the feasibility of concurrent Cl inhibition of FTase and GGTase-I remains to be rigorously N H explored.57 Inhibitors of other proteins that facilitate RAS N N Compound 3144 Pan-RAS inhibitor of membrane association have also been developed. Salirasib HN effector activation (S-farnesylthiosalicylic acid, FTS) is another small molecule Cl developed as an antagonist for RAS membrane association.58 Clonidine (DCAI) (Cat. No. 0690) Designed as a mimetic of the carboxyl terminal farnesylated α2 agonist. Also I1 ligand OCF3 cysteine modification, cell culture studies have demonstrated O varied mechanisms by which salirasib can disrupt RAS function. These include causing decreased RAS association with the N Cl plasma membrane, decreased RAS protein stability, and altered N 59 effector interactions. N N ARS-853 Targeting the other enzymes involved in CAAX-signaled post- H KRAS (G12C) inhibitor OH O translational modifications has also been pursued, but with limited success. No inhibitors with sufficient potency and HO O OH selectivity have been developed, although relatively nonspecific O H P P N pharmacologic inhibitors of ICMT and RCE1 have been described. HO O O Cl O OH O The relatively nonspecific PAT inhibitor 2-bromopalmitate (2-BP) H O H2N N N is a tool compound that disrupts the plasma membrane SML-8-73-1 association of palmitoylated RAS isoforms. N Irreversible inhibitor; targets the N GN-binding pocket of KRAS G12C One potentially promising target is phosphodiesterase 6 delta O (PDEd), a chaperone protein that facilitates the trafficking of RAS proteins from endomembranes to the plasma membrane.60,61 * Names in bold denote compounds available from Tocris Structurally distinct small molecules (e.g., Deltarasin, Deltazinone 1, and Deltasonamide 1/2),60,62 that block the ability of PDEd to engage KRAS have been shown to reduce KRAS plasma membrane association and to show anti-tumor activities in vitro and in vivo.

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Unbiased screens have recently been applied to identify novel Figure 8 | Chemical structures of small molecule inhibitors components that regulate RAS membrane interaction. One study of RAS membrane association identified the L-type calcium channel blocker fendiline asa specific inhibitor of KRAS4B but not HRAS or NRAS plasma S membrane association, in a manner unrelated to calcium channels.63,64 Fendiline inhibition of acid sphingomyelinase and CO2H depletion of plasma membrane phosphatidylserine and chol Salirasib (Cat. No. 4989) esterol was determined to be the basis for selective disruption of Inhibitor of active RAS proteins KRAS4B membrane association.

O In summary, while still an active area of study, most components H N N N that influence RAS subcellular trafficking and membrane N interactions also regulate the activities of numerous other N O proteins. Nevertheless, while there remains the concern that Deltazinone 1 inhibitors of these components may be limited by off-target d Inhibitor of PDE activities, it is also possible that off-target activities of other agents may be applied to disrupt RAS. NH N O Inhibitors of RAS effector signaling N N Since protein kinases are components of effector signaling and highly tractable drug targets, inhibitors of specific components of N RAS downstream effector signaling represent one of the most active areas in small molecule development for anti-RAS drug discovery Deltarasin (Cat. No. 5424) (Figure 9).1,33 Of these, most of the focus has been on RAF-MEK-ERK High affinity PDEδ-KRAS interaction inhibitor MAPK signaling, where the frequent mutational activation of BRAF verifies the driver role of this pathway in cancer. Point mutations of Cl Br activated BRAF alleles are found in a significant subset of the rare (<5%) PDACs that lack mutant RAS alleles.19,65,66 In mouse models N H H of PDAC, mutant Braf phenocopied mutant Kras to drive the initia- HN Br tion and development of metastatic PDAC.67 Thus, the ERK MAPK cascade is clearly a critical driver of KRAS-mutant PDAC growth. N Fendiline (Cat. No. 6407) Many potent and selective inhibitors of each step in the RAF- Reported to inhibit O KRAS plasma membrane MEK-ERK mitogen-activated protein kinase cascade have been localization developed, with inhibitors of RAF (vemurafenib, dabrafenib) and MEK (trametinib, cobimetinib) approved for treatment of BRAF- N mutant melanoma.33,68 However, the first-generation RAF

Cl O NH2 inhibitors are BRAF-selective and cause paradoxical activation Lonafarnib (Cat. No. 6265) rather than inactivation of ERK signaling. Recently developed N Farnesyl transferase inhibitor, N NH2 pan-RAF (LY3009120) or “paradox breaker” inhibitors may inhibits activation of RAS proteins overcome this limitation.69,70 More recently, since resistance mechanisms that overcome sensitivity to RAF and MEK inhibitors

N O cause ERK reactivation, several ERK-selective inhibitors have been developed (VTX11e, SCH772984) and shown to overcome Cl O these resistance mechanisms.71 Currently, three additional ERK Tipifarnib O inhibitors are under clinical evaluation (ulixertinib/BVD-523, N Farnesyl transferase inhibitor, H LTT462 and LY3214996). inhibits activation of RAS proteins O NH Numerous inhibitors of each component of the PI 3-K-AKT-mTOR GGTI 2147 effector pathway have been developed, with some approved for Potent and selective inhibitor N of geranylgeranyltransferase I HN the treatment of non-RAS-mutant cancers (the mTORC1 (GGTASE-I) inhibitors, temsirolimus and everolimus, and the p110d inhibitor, idelalisib).72 To date, as monotherapy, these inhibitors have not * Names in bold denote compounds available from Tocris shown significant clinical benefit for RAS-mutant cancers. Preclinical mouse model studies determined that concurrent inhibition of specific components of the RAF and PI 3-K effector pathways can cause potent suppression of tumor growth. However, these combinations have not shown significant activity in clinical trials, and toxicity has been a limiting factor.

10 | tocris.com RAS Oncoproteins: Therapeutic Vulnerabilities

Other validated RAS effectors include two that lead to activation of other RAS superfamily proteins.1 RAS activation of RALGEFs or Figure 9 | Chemical structures of small molecule RACGEFs leads to activation of the RAL or RAC small GTPases, inhibitors of RAS effectors respectively. Small molecule inhibitors of RAL and RAC have been F O described. These small GTPases also utilize protein kinase effectors, PAK serine/threonine kinases for RAC and TBK1 for N N N H H RalB, and inhibitors of these kinases have also been described.36,73 N N N LY3009120 H Recently, rigosertib, developed originally as a non-ATP competitive Potent pan-RAF inhibitor O multi-kinase inhibitor, has been described as an inhibitor of HO effector binding to RAS, through its binding to the RBD or RA Cl NH 74 domains found on the majority of RAS effectors. However, O another study identified an indirect mechanism whereby HN rigosertib caused activation of the JNK MAPK and indirect NH O N O inactivation of ERK signaling.75 N N In summary, while arguably the most promising direction for the Cl NH O development of clinically effective anti-RAS therapies in the short Ulixertinib Reversible term, effector inhibitors have shown limited to no antitumor N NH Trametinib ERK1/2 inhibitor I F efficacy as monotherapy. Instead, combination inhibitor MEK1/2 inhibitor approaches are likely needed to overcome normal cell toxicity and the rapid onset of resistance mechanisms. N O N N N Targeting synthetic lethal interactors of N N H N mutant RAS SCH772984 O N One attractive direction for the development of RAS inhibitors ERK1/2 inhibitor N H has been the identification of synthetic lethal interactors with mutant but not wild type RAS.76 This approach is based on the concept that a gene whose function is critical for cell viability only NH2 O O N in the context of RAS mutations provides a target for selective HO O inhibition of RAS-mutant cancer cells. Earlier studies identified O O N O O O protein kinases (e.g., STK33, TBK1) as RAS synthetic lethal NH HO 77–79 O interactors. With protein kinases more druggable than RAS O S O itself, these findings led to considerable excitement. However, N O subsequent analyses found that the proteins identified in a N O number of these studies were not strongly linked to mutant KRAS HO function, making them less attractive targets for selective Alpelisib; BYL-719 Everolimus (Cat. No. 6188) targeting of RAS-mutant cancers. PI3Ka inhibitor F F mTORC1 inhibitor F Despite these setbacks, there is still considerable enthusiasm that bona fide synthetic lethal interactors of mutant RAS still hold * Names in bold denote compounds available from Tocris great potential as targets that may yield pharmacologic inhibitors selective for RAS-mutant cancers. To overcome some of the Finally, the significant off-target activities of siRNA-based libraries limitations of earlier studies, improved cell models of KRAS- are being addressed by employing CRISPR/Cas9-based library dependency and experimental approaches are needed.80 A screens.81 In summary, while the pursuit of synthetic lethal number of the earlier studies utilized so called “isogenic” interactors of mutant KRAS has not fulfilled the field’s high matched pairs of human tumor cell lines. Established using the expectations, perhaps improved experimental models and heterozygous KRAS-mutant DLD-1 and HCT 116 CRC cell lines, approaches will overcome past limitations. matching lines were generated by stable deletion of the endogenous KRAS allele. However, because the resulting lines lacking the mutant KRAS allele were clonally derived from a Exploiting RAS-driven metabolism heterogeneous cell population, and because cells lacking mutant Cancer cells exhibit increased energy and biosynthetic demands KRAS have undergone adaptation to overcome their addiction to to augment their elevated proliferative state. Thus, cells mutant KRAS, such lines are not truly isogenic. The screening of reprogram metabolic processes to either repurpose and recycle large panels of KRAS mutant and WT cell lines, to account for cell intracellular fuel sources or scavenge extracellular components. line heterogeneity, may overcome the limitations of isogenic Mutant RAS is directly implicated in modulating specific cellular pairs. Synthetic lethal screens have been done traditionally signaling cascades to accomplish a shift towards anabolic under anchorage-dependent culture conditions. Screens using metabolic processes that produce amino acids, nucleic acids, three-dimensional cultures under anchorage-independent lipids and cofactors such as NADPH for redox balance and conditions may identify genes with greater physiologic relevance.

tocris.com | 11 Tocris Scientific Review Series reductive biosynthesis.8,82,83 Consequently, delineating the that KRAS- and BRAF-mutant colorectal cancer cells displayed mechanisms by which RAS causes altered metabolism may increased uptake of the oxidized form of vitamin C define therapeutic strategies for RAS-mutant cancers. (dehydroascorbate) and that this causes cell death due to oxidative stress.98 Furthermore, this study demonstrated that the The realization that cancerous tissue exhibits altered metabolism method of drug delivery may have been the basis for clinical is credited to Otto Warburg, who in the 1920s made the failures in the past, as intravenous administration was required observation that tumor tissues metabolize glucose to lactate at a to achieve plasma concentrations necessary for a therapeutic rate that is 10-fold faster than normal cells.84 Although the effect. Further studies are necessary to determine if high dose precise advantage the Warburg Effect bestows on cancer cells is vitamin C is indeed a viable option for patients. still under debate,85 today aberrant metabolism is considered a hallmark of cancer.86 While RAS had been implicated in the RAS-mutant PDAC is also characterized by increased dependency upregulation of glycolysis,87 the mechanistic basis for this on glutamine.99 Although glutamine intermediate metabolism in observation has only recently begun to be delineated (Figure 10). the mitochondria has been suggested to fuel processes outside First, RAS was shown to drive increased glucose uptake by of bulk protein synthesis,100 recent macromolecular fractionation upregulating the GLUT1 gene that encodes the glucose studies have shown that protein is the major endpoint for transporter, GLUT1.88 Furthermore, in an inducible Kras mouse glutamine carbon.101 In the mitochondria, glutamine can be model of PDAC, Kras activation was associated with increased processed to glutamate via either glutamate dehydrogenase 1 Glut1 gene expression. Second, Kras also upregulates glycolytic (GLUD1) or transaminases. In KRAS-mutant PDAC, glutamine- enzymes (Hk1, Hk2, Pfkl and Ldha), resulting in enhanced derived aspartate is transported to the cytosol where it undergoes conversion of pyruvate to lactate.25 Third, RAS regulates genes glutamic-oxaloacetate transaminase-1 (GOT1)-dependent conver encoding enzymes that route glucose intermediates into the sion to oxaloacetate, which is ultimately converted to pyruvate.102 hexosamine biosynthetic pathway (HBP; Gfpt1) to support protein The byproduct of these reactions is an increased NADPH/NADP+ glycosylation and the non-oxidative pentose phosphate pathway ratio in the cell, which is important for PDAC cells that display (PPP; Rpia, Rpe) to support nucleotide biosynthesis.25 KRAS downregulated oxidative pentose phosphate pathway activity. regulates these enzymes via signaling through the RAF-MEK-ERK Although a small molecule that directly targets this GOT1- pathway and ultimately via regulating MYC expression. Thus, dependent pathway has not been developed, the glutaminase inhibitors of the ERK MAPK pathway can be used to block KRAS- inhibitors Compound 968103 and bis-2-(5-phenylacetamido- driven glycolytic perturbations. 1,2,3-thaidiazol-2-yl)ethyl sulfide 3 (BPTES)104 reduced PDAC proliferation in vitro. When treatment with BPTES or Compound The terminal step of glycolysis is the efflux of lactate into the 968 was combined with hydrogen peroxide treatment (to increase extracellular space. Because high intracellular lactate negatively ROS) an enhanced effect was observed.102 This indicates that impacts further glycolytic activity, targeting lactate efflux is a combining agents that target glutamine utilization with ROS- potential therapeutic avenue. Indeed the inhibition of LDHA with generating treatments, such as radiation, may be a viable the small molecule polyphenolic naphthalene (FX-11) increased treatment option. Furthermore, an orally available GLS1 inhibitor, oxidative stress in LDHA-driven cancer cells, and inhibited the CB-839, in combination with drugs that induce ROS formation, malignant progression of human pancreatic xenografts.89 has been shown to synergistically shrink PDAC xenografts and Interestingly, further studies have indicated that efficacy is increase overall survival of tumor-bearing mice.105 CB-839 dependent upon TP53 mutation status, as mice harboring WT is currently under investigation in clinical trials for multiple TP53 patient-derived tumors were resistant to FX-11.90 Likewise, cancer types. heterogeneity in response was observed with a pharmacologically distinct LDHA inhibitor, GNE-140; however, variability in response Another route of glutamine entry into cancer cells is macro was correlated to the tumor’s ability to utilize oxidative pinocytosis, an endocytic scavenging process by which cells phosphorylation (OXPHOS), indicating that combining inhibitors ingest albumin and other extracellular nutrients.106 Mutant RAS of glycolysis and OXPHOS may broaden clinical efficacy.91 can drive increases in macropinocytosis,107 and human PDAC cell Metformin, a biguanide used to treat type 2 diabetes, inhibits lines, as well as a KRAS-driven mouse model of PDAC, showed hepatic glucose utilization. While initial retrospective analyses of increased macropinocytotic activity.108 Furthermore, when cancer incidence in the diabetic population garnered robust labeled albumin is directly delivered to tissues and tumors in live excitement about the possibility that metformin treatment mice, macropinocytosis can be directly observed at higher levels lowered pancreatic cancer frequency, multiple conflicting studies in tumor tissue as compared to the surrounding normal tissue.109 have been published over the last five years.92 Furthermore, two The internalized protein undergoes lysosomal degradation, recent phase 2 clinical trials have found no benefit of metformin yielding essential amino acids, in particular glutamine, which when it is administered at the levels necessary for glycemic then enters the central carbon pool.108,110 Although the elevated control.93,94 Trials are still ongoing to determine whether macropinocytotic activity in PDAC is RAS-dependent, the metformin can be used as a maintenance therapy in patients mechanisms driving this activity remain poorly understood. Of with stable disease (NCT02048384) or if more potent biguanides particular interest is the finding that mTORC1 signaling are more efficacious (NCT02475499). Finally, Linus Pauling suppresses macropinocytosis.110 This observation may partially proposed high dose vitamin C as a cancer treatment in the early explain the insignificant effect of mTOR inhibitors on PDAC, as in 1970s.95 This proposal was initially ridiculed, and was apparently the context of these highly macropinocytotic cells, mTOR inhibition refuted by two clinical trials in the 1970s and 1980s, which may actually fuel further increased macropinocytosis. Unlike demonstrated that oral administration of high dose vitamin C had other endocytic processes, macropinocytosis is inhibited by no impact on patient survival.96,97 However, recent data showed treatment with 5-[N-ethyl-N-isopropyl] amiloride (EIPA). EIPA, an

12 | tocris.com RAS Oncoproteins: Therapeutic Vulnerabilities

Figure 10 | RAS-driven alterations in metabolism

Glucose

Albumin

Macropinocytosis GLUT1 Glucose Non-oxidative Pentose Phosphate Pathway HK1/2 Macropinosome 6-P-gluconolactone 6-P-gluconate Ribulose-5-P Glucose-6-P RPE RPIA Xyulose-5-P Ribose-5-P Fructose-6-P Lysosome PFK1 GFPT1 Sedoheptulose- Fructose-1,6-BP 1,7-bisP Sedoheptulose-7-P Glyceraldehyde-3-P

GlcN-6P Amino acids, etc. 2-Phosphoglycerate Erythrose 4-P Dihydroxy- Fructose-6-P Erythrose 4-P GlcNAc-6P acetone-P ENO1

Phosphoenolpyruvate GlcNAc-P Pyruvate Autolysosome Pkm ROS GSH NADPH Pyruvate GSSG ME1 UDP-GlcNAc NADP+ LDHA Malate Lysosome Hexosamine Lactate Acetyl-CoA Biosynthesis MDH1 TCA Pathway α-ketoglutarate Oxaloacetate cycle GLUD1 GOT1 Autophagosome Glutamate Aspartate Aspartate GOT2 α-ketoglutarate Autophagy Glycolysis GLS

Glutamine Reprogramming Lactate Glutamine

RAS mutant cancer cells are characterized by increased macropinocytosis and uptake of albumin, leading to lysosomal degradation and release of amino acids. RAS mutant cancer cells also exhibit altered autophagy, leading to degradation of organelles and proteins, and production of amino acids and other components to support metabolism. Oncogenic KRAS directs glucose metabolism into biosynthetic pathways in PDAC by upregulating many key enzymes in glycolysis. Oncogenic KRAS induces nonoxidative PPP flux to fuel increased nucleic acid biosynthesis and activates the hexosamine biosynthesis and glycosylation pathways. PDAC cells also utilize a non-canonical pathway to process glutamine, through which it maintains redox balance and supports cell growth. Blue text indicates RAS-dependent gene and/or protein expression, with arrows indicating increased (green) or decreased (red) expression. Enzymes are indicated in italics. Abbreviations used are: GLUT1, glucose transporter 1; HK 1/2, hexokinase 1/2; PFK1, phosphofructokinase 1; ENO1, enolase 1; PKM, pyruvate kinase; LDHA, lactate dehydrogenase A; GFPT1, glucosamine-fructose-6-phosphate aminotransferase-1; GlcN, glucosamine; GlcNAc, N-acetylglucosamine; PPP, pentose phosphate pathway; RPE, ribulose-5-phosphate-3-epimerase; RPIA, ribulose-5-phosphate isomerase; GLUD1, glutamate dehydrogenase 1; GOT, aspartate transaminase; GLS, glutaminase; GSH, glutathione; GSSG, glutathione disulfide; ME1, malic enzyme; ROS, reactive oxygen species. inhibitor of Na+/H+ exchange, blocked PDAC tumor formation in form of paclitaxel, in the treatment of PDAC is due in part to the vivo;108 however, this tool compound cannot be used in humans, exploitation of high macropinocytotic levels in PDAC tumors.111,112 and to date, there are no specific inhibitors of macropinocytosis Finally, due to the hypovascular nature of pancreatic tumors, for cancer treatment in the clinic. However, it is tempting to PDAC cells are in a constant state of nutrient deprivation; thus, speculate that the success of nab-paclitaxel, an albumin-bound

tocris.com | 13 Tocris Scientific Review Series they have evolved mechanisms to recycle intracellular nutrients. peptidases, USP10 and 13, and thus induces cancer cell death One metabolic process associated with RAS-mutant cancers is upon nutrient deprivation.122 However, VPS34 is essential for macroautophagy, hereafter referred to as autophagy. In healthy sorting multiple endocytic pathways and therefore, like HCQ, may cells, autophagy is a catabolic process of “self-eating” not be a specific target for autophagy inhibition. Thus, focus has whereby unneeded or dysfunctional cellular components been shifted to ULK1 and 2 which function further upstream and including cellular organelles are degraded. Established tumors may represent more selective targets. An ULK1-specific inhibitor, hijack autophagy to support survival by providing amino acids, SBI-0206965, has been shown to specifically suppress ULK-1 lipids and nucleic acids.113 This scavenging mechanism is mediated phosphorylation events and to synergize with mTOR complex and involves the generation of autophagosomes, inhibitors to kill tumor cells.123 Additionally, an ULK1/2 inhibitor, followed by fusion with lysosomes, which facilitates their MRT 68921, has also been described and shown to block degradation and the release of nutrients. Autophagy is elevated autophagy in cells;124 however, further chemical optimization of in PDAC.113,114 In support of the cooperative role between RAS and all of these more specific autophagy inhibitors is necessary to autophagy, immortal, non-tumorigenic baby mouse kidney make these compounds clinically viable. epithelial (iBMK) cells ectopically expressing oncogenic HRAS or KRAS experienced increased defects in mitochondrial respiration Future Prospects upon autophagy inhibition as compared to their non-transformed counterparts.114 However, a cooperative relationship between The now more than three decades of failures to develop effective constitutive RAS activation and autophagy is not exclusively anti-RAS therapies can be attributed to our underestimation of observed. For example, expression of oncogenic HRAS in ovarian the complexities of RAS function, of the dynamic nature of signal epithelial cells and human fibroblasts leads to autophagy- transduction networks and of cancer genetics and cancer mediated cell death and senescence, respectively.115,116 heterogeneity. Cancer genome sequencing efforts have revealed Additionally, the role of TP53 mutation status in the regulation of that RAS remains the key oncogene driver for cancers that autophagy is not fully understood. In a PDAC mouse model that account for the top three causes of cancer deaths in the US, and harbors a Tp53 homozygous deletion, autophagy hindered tumor that together account for nearly 42% of deaths due to cancer. progression.117 However, in another mouse model driven by Tp53 There is a near universal incidence of RAS mutations in the loss of heterozygosity, which is more similar to TP53 mutation in deadliest of the deadly cancers, pancreatic cancer. Early failures humans, Tp53 status was not shown to influence the led to the incorrect perception that RAS is not druggable. Thus, protumorigenic role autophagy plays.118 Finally, as intratumoral while the successful clinical development of an effective anti-RAS heterogeneity and cellular crosstalk become more broadly therapy remains a considerable challenge, past pessimism has understood, there is emerging evidence that autophagy may play transitioned to high excitement and progress. What are the a role in the support that pancreatic stellate cells (PSCs) provide prospects of success? Is our knowledge of RAS now sufficiently to their tumor cell neighbors. A recent study demonstrated that advanced to avoid the pitfalls of the past? As Yogi Berra stated, autophagy was required for the release of alanine from PSCs, “It’s tough to make predictions, especially about the future”. and that this alanine, taken up by PDAC cells, actually surpassed There will not likely be one simple anti-RAS therapy that will be glucose and glutamine-derived carbon in terms of incorporation effective in all RAS-mutant cancers. Immunotherapy is an area into mitochondrial metabolic processes.119 that is just emerging with regard to RAS. The striking intra- and inter-patient heterogeneity within one cancer, and the cell context Treatment with chloroquine, which indirectly inhibits autophagy differences seen among different RAS-mutant cancers, will via the inhibition of lysosomal acidification, impairs PDAC growth require multiple anti-RAS strategies, and these may some day be 113,118 in vitro and in vivo. Hydroxychloroquine (HCQ) has been further enhanced by concurrent immunotherapy. Of the current approved by the FDA for the treatment of malaria and various RAS-centric directions, targeting effector signaling arguably holds rheumatological disorders for decades and therefore clinical the greatest promise for success within the next five years. Of the trials to test anticancer efficacy proceeded quickly. Unfortunately, ideal direction, the development of direct RAS inhibitors, the path HCQ as monotherapy has been largely unsuccessful in the clinic, to transitioning the current tool compounds to clinically effective most likely due to the limited potency of HCQ to block autophagy drugs with sufficient potency and selectivity may be long. With 120 in vivo. However, when HCQ was combined with gemcitabine, improved technologies and models for screening, there is the PDAC patients that showed increased disease-free and guarded optimism that the once exciting prospects of mining for overall survival were shown to be those that were responsive to synthetic lethal interactors of mutant RAS will finally achieve their HCQ, as defined by changes in LC3, a central player inthe lofty promise to identify RAS mutant-selective therapies. In autophagy pathway and an accepted biomarker for HCQ summary, there is now realistic optimism that effective anti-RAS 121 responsiveness. The combination of HCQ with other therapies will finally be developed – yet what those therapies will chemotherapeutics is the basis for multiple ongoing clinical be and when they will be achieved are less certain. What is trials. Because HCQ inhibits the process of lysosomal acidifica certain is that a better understanding of RAS regulation and tion, which is not specific to autophagy and additionally inhibits functions, and how these are integrated with other physiological other cellular processes, such as macropinocytosis, attention and pathophysiological activities, will be needed to effectively has also been turned to earlier steps in the autophagy pathway identify and target RAS therapeutic vulnerabilties. that are kinase-regulated and that may therefore provide additional targets for small molecule inhibitors. The first such small molecule reported, Spautin-1 (specific and potent autophagy inhibitor-1), promotes the degradation of VPS34 PI3- kinase complexes via inhibition of two ubiquitin-specific

14 | tocris.com RAS Oncoproteins: Therapeutic Vulnerabilities

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tocris.com | 15 Tocris Scientific Review Series

Catalog No. Product Name Primary Function

Adrenergicα2 Receptors 0690 Clonidine Blocks SOS1 binding Akt (Protein Kinase B) 5773 Akti-1/2 Potent and selective dual Akt1 and 2 inhibitor 3897 API-1 Selective Akt/PKB inhibitor; antitumor 2151 API-2 Selective inhibitor of Akt/PKB signaling; antitumor and antiviral 2558 10-DEBC Selective Akt/PKB inhibitor 2926 FPA 124 Akt/PKB inhibitor 4144 GSK 690693 Akt kinase inhibitor; antitumor 5682 OSU 03012 Inhibits Akt signaling 6087 Perifosine PKB/Akt inhibitor 4598 PHT 427 Dual Akt and PDK1 inhibitor; antitumor 4398 SC 66 Allosteric Akt inhibitor 4635 SC 79 Akt activator ERK 5843 AX 15836 Potent and selective ERK5 inhibitor 5774 DEL 22379 ERK dimerization inhibitor 5979 Eicosapentaenoic Acid Stimulates ERK1/2 phosphorylation 5393 ERK5-IN-1 Potent and selective ERK5 inhibitor 3706 FR 180204 Selective ERK inhibitor 4433 Pluripotin Dual ERK1/RasGAP inhibitor 4465 TCS ERK 11e Potent and selective ERK2 inhibitor 4132 XMD 8-92 ERK5/BMK1 inhibitor G Proteins (Small) 4853 8-pCPT-2-O-Me-cAMP-AM Selective Epac activator; cell-permeable analog of 8CPT-2Me-cAMP (Cat. No. 1645) 5050 CASIN Cdc42 GTPase inhibitor 5233 CCG 1423 Rho/SRF pathway inhibitor 4793 CE3F4 Non competitive Epac1 inhibitor 4568 CPYPP DOCK2-Rac1 interaction inhibitor 6025 (S)-Crizotinib Suppresses colony formation of KRAS-mutated PANC1 cells 3872 EHT 1864 Potent inhibitor of Rac family GTPases 5607 GNF 7 Ras signaling inhibitor; inhibits Ack1 and GCK 3584 Golgicide A Potent, specific and reversible inhibitor of GBF1 ArfGEF 4844 HJC 0350 Selective Epac2 inhibitor 3792 ITX 3 Selective inhibitor of TrioN RhoGEF activity 5475 Kobe 0065 H-Ras-cRaf1 interaction inhibitor; inhibits Raf signaling 4266 ML 141 Selective inhibitor of Cdc42 Rho family GTPase 3715 Narciclasine Antiproliferative agent; slows cell cycle progression 2161 NSC 23766 Selective inhibitor of Rac1-GEF interaction; antioncogenic 3324 QS 11 ARFGAP1 inhibitor; modulates Wnt/β-catenin signaling 2222 Rac1 Inhibitor F56, control peptide Control peptide version of Rac1 Inhibitor W56 (Cat. No. 2221) 2221 Rac1 Inhibitor W56 Selective inhibitor of Rac1-GEF interaction 5411 RBC8 RalA and RalB GTPase inhibitor 5003 Rhosin Rho GTPase inhibitor 4989 Salirasib Ras inhibitor; also induces autophagy 5280 SCH 51344 Inhibits Ras-induced malignant transformation; MCT1 inhibtor 2849 SecinH3 Sec7-specific GEF inhibitor (cytohesins) 3101 XRP44X Ras-Net pathway inhibitor 4794 ZCL 278 Cdc42 inhibitor 6111 Zoledronic Acid Ras signaling inhibitor; also farnesyl diphosphate synthase inhibitor

16 | tocris.com RAS Oncoproteins: Therapeutic Vulnerabilities

Catalog No. Product Name Primary Function MEK 1777 Arctigenin Potent MEK1 inhibitor 4842 BIX 02189 Selective MEK5 and ERK5 inhibitor 1532 10Z-Hymenialdisine Pan kinase inhibitor; potently inhibits MEK1 4192 PD 0325901 Potent inhibitor of MEK1/2 4237 PD 184352 Selective MEK inhibitor 2605 PD 198306 Selective inhibitor of MEK1/2 4824 PD 334581 MEK1 inhibitor 1213 PD 98059 MEK inhibitor 1969 SL 327 Selective inhibitor of MEK1 and MEK2; brain penetrant 1868 U0124 Inactive analog of U0126 (Cat. No. 1144) 1144 U0126 Potent, selective inhibitor of MEK1 and 2 Metabolism Inhibitors 5460 968 Allosteric inhibitor of glutaminase; blocks Rho-GTPase-dependent transformation of fibroblasts 5301 BPTES Allosteric glutaminase (GLS1) inhibitor; antitumor 4109 Chloroquine Inhibits macropinocytosis and autophagy; exhibits antimetastatic activity 3378 EIPA Inhibits the Na+/H+ exchanger; inhibits macropinocytosis 3259 Gemcitabine Inhibits DNA synthesis; displays antitumor activity in vivo 5189 GSK 2837808A Potent and selective LDHA inhibitor; reduces glucose uptake in hepatocellular cancer cells 5648 Hydroxychloroquine Autophagy inhibitor; inhibits growth and induces apoptosis of renal cancer cells 5780 MRT 68921 Potent ULK inhibitor; inhibit autophagy 5197 Spautin 1 Inhibitor of autophagy mTOR 5615 AZD 3147 Potent and selective dual mTORC1 and 2 inhibitor; orally bioavailable 3271 Compound 401 Selective DNA-PK and mTOR inhibitor 5955 eCF 309 Potent mTOR inhibitor 3725 KU 0063794 Selective mTOR inhibitor 4079 Niclosamide Inhibits mTORC1 signaling 4820 PF 04691502 Potent and selective dual PI 3-K/mTOR inhibitor 2930 PI 103 Inhibitor of mTOR, PI 3-kinase and DNA-PK 4257 PP 242 Dual mTORC1/mTORC2 inhibitor 1292 Rapamycin mTOR inhibitor; immunosuppressant 5264 Temsirolimus mTOR inhibitor; antitumor 4247 Torin 1 Potent and selective mTOR inhibitor 4248 Torin 2 Potent and selective mTOR inhibitor 4282 WYE 687 Potent and selective mTOR inhibitor 4893 XL 388 Potent and selective mTOR inhibitor; antitumor Myc 4406 10058-F4 Inhibits c-Myc-Max dimerization 5306 KJ Pyr 9 High affinity Myc inhibitor P21-activated Kinases 5190 FRAX 486 Potent PAK inhibitor; brain penetrant and orally bioavailable 3622 IPA 3 Group I PAK inhibitor 6005 PF 3758309 Potent PAK4 inhibitor; orally available 4212 PIR 3.5 Negative control of IPA 3 (Cat. No. 3622) Phosphodiesterases 1349 (R)-(-)-Rolipram PDE4 inhibitor. More active enantiomer of rolipram (Cat. No. 0905) 5424 Deltarasin High affinity PDEδ-KRAS interaction inhibitor 1261 EHNA Selective inhibitor of cGMP-stimulated phosphodiesterase (PDE2) 2845 IBMX PDE inhibitor (non-selective) 1436 MDL 12330A Cyclic nucleotide PDE inhibitor 3784 Sildenafil Orally active, potent PDE5 inhibitor

tocris.com | 17 Tocris Scientific Review Series

Catalog No. Product Name Primary Function PI 3-Kinase 3977 3-Methyladenine Class III PI 3-kinase inhibitor; also inhibits autophagy 1983 740 Y-P Cell-permeable PI 3-kinase activator 5595 A66 Potent and selective PI 3-kinase p110α inhibitor 3578 AS 605240 Potent and selective PI 3-kinase γ (PI 3-Kγ) inhibitor 4839 AZD 6482 Potent and selective PI 3-Kβ inhibitor 4674 CZC 24832 Selective inhibitor of PI 3-kinase γ 4026 GSK 1059615 Potent PI 3-kinase inhibitor 5126 iMDK Inhibits PI 3-K signaling; suppressor of MDK expression 4840 KU 0060648 Dual DNA-PK and PI 3-K inhibitor 5835 LTURM 36 PI 3-kinase δ inhibitor 1130 LY 294002 Prototypical PI 3-kinase inhibitor; also inhibits other kinases 2418 LY 303511 Negative control of LY 294002 (Cat. No. 1130) 5919 PI 3065 Potent and selective PI 3-kinase p110δ inhibitor 2814 PI 828 PI 3-kinase inhibitor, more potent than LY 294002 (Cat. No. 1130) 1125 Quercetin Non-selective PI 3-kinase inhibitor 5832 TGX 221 Potent and selective PI 3-kinase β inhibitor 1232 Wortmannin Potent, irreversible inhibitor of PI 3-kinase. Also inhibitor of PLK1 PKC 2383 Bryostatin 1 Protein kinase C activator; phosphorylates KRAS4B Protein Prenyltransferases 2407 FTI 277 Prodrug form of FTI 276 (Cat. No. 2406) 2430 GGTI 298 Geranylgeranyltransferase I (GGTase I) inhibitor 4294 LB 42708 Selective farnesyltransferase (FTase) inhibitor Protein Tyrosine Phosphatases 3979 Alexidine Selective inhibitor of PTPMT1 2176 BVT 948 Non-competitive protein tyrosine phosphatase inhibitor; enhances insulin signaling 2613 NSC 87877 Potent inhibitor of shp2 and shp1 PTP 2754 TCS 401 Selective inhibitor of PTP1B 3591 VO-Ohpic Potent PTEN inhibitor Raf Kinase 4836 AZ 628 Potent Raf kinase inhibitor 4453 GDC 0879 Potent B-Raf inhibitor 1381 GW 5074 Potent, selective c-Raf1 kinase inhibitor 5260 KG 5 PDGFRβ, B-Raf, c-Raf, FLT3 and KIT inhibitor 5036 ML 786 Potent Raf kinase inhibitor; orally bioavailable 6015 RAF 265 Raf kinase and VEGFR-2 inhibitor 2650 SB 590885 Potent B-Raf inhibitor 1321 ZM 336372 Potent, selective c-Raf inhibitor Rho-kinases 4927 AS 1892802 Potent ROCK inhibitor; orally bioavailable 0541 Fasudil Inhibitor of cyclic nucleotide dependent- and Rho-kinases 2485 Glycyl-H 1152 Selective Rho-kinase inhibitor. More selective analog of H 1152 (Cat. No. 2414) 4009 GSK 269962 Potent and selective ROCK inhibitor 3726 GSK 429286 Selective Rho-kinase (ROCK) inhibitor 2414 H 1152 Selective Rho-kinase (ROCK) inhibitor 2415 HA 1100 Cell-permeable, selective Rho-kinase inhibitor 5182 OXA 06 Potent ROCK inhibitor 4118 SB 772077B Potent Rho-kinase inhibitor; vasodilator 3667 SR 3677 Potent, selective Rho-kinase (ROCK) inhibitor 4961 TC-S 7001 Potent and highly selective ROCK inhibitor; orally active 1254 Y-27632 Selective p160ROCK inhibitor

18 | tocris.com RAS Oncoproteins: Therapeutic Vulnerabilities

tocris.com | 19 Prote2945 RnDSy-2945 Novus-2945 Tocri-2945

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