Cnk1 (connector enhancer of kinase suppressor of Ras 1) is a pleckstrin homology (PH) domain–containing scaffold protein that increases the efficiency of Ras signaling pathways, imparting efficiency and specificity to the response of cell proliferation, survival, and migration. Mutated KRAS (mut-KRAS) is the most common proto-oncogenic event, occurring in approximately 25% of human cancers and has no effective treatment. In this study, we show that selective inhibition of Cnk1 blocks growth and Raf/Mek/Erk, Rho and RalA/B signaling in mut-KRAS lung and colon cancer cells with little effect on wild-type (wt)-KRAS cells. Cnk1 inhibition decreased anchorage-independent mut-KRas cell growth more so than growth on plastic, without the partial "addiction" to mut-KRAS seen on plastic. The PH domain of Cnk1 bound with greater affinity to PtdIns(4,5)P2 than PtdIns(3,4,5)P3, and Cnk1 localized to areas of the plasma membranes rich in PtdIns, suggesting a role for the PH domain in the biological activity of Cnk1. Through molecular modeling and structural modification, we identified a compound PHT-7.3 that bound selectively to the PH domain of Cnk1, preventing plasma membrane colocalization with mut-KRas. PHT-7.3 inhibited mut-KRas, but not wild-type KRas cancer cell and tumor growth and signaling. Thus, the PH domain of Cnk1 is a druggable target whose inhibition selectively blocks mutant KRas activation, making Cnk1 an attractive therapeutic target in patients with mut-KRAS–driven cancer.
These findings identify a therapeutic strategy to selectively block oncogenic KRas activity through the PH domain of Cnk1, which reduces its cell membrane binding, decreasing the efficiency of Ras signaling and tumor growth.
Cnksr1 (connector enhancer of kinase suppressor of Ras 1), hereafter called Cnk1, is a multidomain scaffold protein important for cell proliferation, survival, and migration (1, 2). Protein scaffolds assemble components of signaling pathways in a way that increases their interaction and prevents their inactivation, thus imparting efficiency, sensitivity and specificity to the ultimate signaling response (2–4). Cnk1 has been reported to act as a scaffold for a number of Ras and Rho GTPase family members, while translocating its binding partners to cell membranes where signaling is initiated (4–7). Cnk1 is a scaffold for the Ras/Raf/Mek/Erk signaling cascade (3, 8), possibly as part of a cell membrane Ras signaling nanocluster (9, 10), Inhibiting Drosophila Cnk has been reported to block Ras1 signaling by disrupting a complex between Ras1 and Raf (11).
Point mutation of the KRAS gene (mut-KRAS) is the most common proto-oncogenic event in human cancer, and is found in approximately 25% of human cancers with highest levels in pancreatic, colon cancer, and lung adenocarcinoma (12). Mut-KRas activates downstream signaling that ultimately leads to the mut-KRas phenotype of altered proliferation, anchorage-independent growth, invasion, and tumorigenesis (13). Mut-KRAS is a particularly insidious oncogene because it not only drives cancer growth but also overrides the effects of molecularly targeted therapies (14). The difficulty of inhibiting mut-KRas has led to attempts to target mut-KRas downstream effector pathways but such agents have displayed a narrow therapeutic window impeding adequate inhibition of pro-oncogenic signals (15). Direct inhibitors of mut-KRas are in development (16, 17) but currently there is no effective therapy for mut-KRas tumors.
We were interested to find whether inhibiting Cnk1 would block KRas in mammalian cells. Cnk1 has a phosphoinositide (PtdIns) lipid binding pleckstrin homology (PH) domain, and is found localized to areas of the plasma membranes rich in PtdIns (18), suggesting a role for the PH domain in the biological activity of Cnk1. We have previously shown that the PH domains of signaling proteins can be selectively inhibited with small molecules (19), and we therefore explored whether inhibiting the PH domain of Cnk1 might be a way to inhibit mut-KRas activity. Through molecular modeling and structural modification, we have identified a small-molecule probe compound that binds selectively to the PH domain of Cnk1 preventing plasma membrane colocalization with mut-KRas, and having the ability to inhibit mut-KRas, but not wild-type KRas cancer cell and tumor growth.
Materials and Methods
Mut-KRas MiaPaCa-2 pancreatic cancer cells, M27 MiaPaCa-2 with both mut-KRAS mutant alleles deleted (20), mut-KRas HCT-116 colon cancer cells, and HKK2 HCT-116 with its single mut-KRAS allele deleted (21), were provided by Dr. Natalia Ignatenko, University of Arizona, Tucson, AZ. Non–small cell lung cancer (NSCLC) cell lines were obtained from Dr. John Minna University of Texas Southwestern Medical Center, Dallas, TX (Supplementary Table S1). All cell lines were routinely tested to be Mycoplasma free and the identity of each line authenticated before study, and 2 month intervals while in culture, by the Genomics Shared Resource at SBP.
Studies were conducted using SmartPool siRNA (Dharmacon). A validation study (Supplementary Fig. S1) was conducted using CNK1 siRNAs from a second manufacturer (Qiagen). Total siRNA concentration was kept at 40 nmol/L for single or multiple siRNA combinations. Knockdown efficiency was determined by Western blotting of cell lysates 72 hours post transfection.
Cells for Western blotting were grown in RPMI medium with 10% FBS for 24 hours. Primary rabbit monoclonal antibodies used for Western blotting were anti: Erk, Egfr, Mek1/2, c-Raf, phosph-Akt Ser473, phospho-ErkThr202,Tyr220, phospho-EgfrTyr1068, and phospho-Mek1/2Ser217/221 (Cell Signaling Technology), Cnk1 (Abcam), RalA, RalB, and phospho-c-RafSer338/Tyr340 (EMD Millipore), and KRas mouse antibody (Novus Biologicals). RalA, RalB, Rho and Ras family GTP activation kits were from EMD Millipore and were used according to the manufacturer's instructions.
Cell proliferation assays
To measure 2D growth on plastic cells were treated for 24 hours with nontargeting siRNA or siCNK1, replated and 72 hours later, or after incubation with small molecule inhibitors for 72 hours, growth measured using the XTT Cell Viability Assay (Biotium). For 3D spheroid growth 20,000 HCT-116 colon cancer cells or wt-KRas HCT-116 HKH2 cells were treated for 24 hours with non-targeting siRNA or siCNK1, and allowed to form spheroid clusters for 48 hours under nonadherent conditions in a hanging drop of media. The spheroids were then transferred to a nonadherent 3D-nanoculture plate (Scivax) and volume calculated by the modified ellipsoidal formula (22). Anchorage-independent growth in soft agarose used a modification of a previously published 96-well method (23).
Reverse phase protein assay
Reverse phase protein array (RPPA) was carried out on cell lysates by MD Anderson Cancer Center Functional Proteomics RPPA Core Facility using a 208 protein/phosphoprotein cell signaling and cell-cycle antibody panel as previously described (24).
Comparative modeling was used to construct a homology model of the PH-domain of Cnk1 using the published structures of 4 human PH-domains with high homology to Cnk1 PDB codes: 1U5D, 1U5F, 1U5G and 1UPQ. Using proprietary modeling algorithms and state of-the-art commercial drug discovery software and ligand-based approaches we screened an in silico library of over 3 million compounds and identified lead compounds. For further details see Supplementary Methods S1. Pharmacological properties of the modeled agents used for selection included Log P, metabolic and mutagenic features, oral absorption, hERG and Caco-2 scores.
Surface plasmon resonance spectroscopy for PH domain binding
The PH domain of Cnk1 and other signaling protein PH domains were expressed as fusion proteins with glutathione S-transferase (Gst) at the N-terminus. Analysis of small molecule binding used surface plasmon resonance (SPR) spectroscopy on a Biacore T200 (GE Healthcare) with a CM5 sensor chip and Gst capture kit. For further experimental details of the SPR method see Supplementary Methods S2.
For chemical structures see Table 1 and Supplementary Table S2, and for synthetic methods see Supplementary Table S2 Schemes S1 and S2.
Confocal laser scanning microscopy
Confocal laser scanning microscopy and fluorescence lifetime imaging microscopy (FLIM) were carried out as described in Supplementary Methods S3 (In Vivo Studies).
Approximately 107 A549, H441 and H1975 NSCLC cells in log cell growth were suspended each in 0.2 mL PBS and injected subcutaneously into the flank of female NOD-SCID mice with 10 mice per group. When the tumors reached 50 to 150 mm3 daily dosing was begun with PHT-7.3 (PHusis Therapeutics) at 200 mg/kg i.p., or erlotinib at 75 mg/kg orally (25), or trametinib at 0.3 mg/kg orally (26). Tumor volumes based on twice a week caliper measurements were calculated as previously described (22). Mouse body weight was measured weekly. All animal experiments were approved by SBP Institutional Animal Care and Use Committee.
CNK1 inhibition selectively inhibits mut-KRas–dependent growth and signaling in a panel of cell lines
We first examined the growth of wt-KRas and mut-KRas cell lines after KRas and Cnk1 siRNA knockdown. We used a panel of NSCLC cell lines grown in 2D on plastic, and showed that treatment with siRNA to KRAS gave only a small inhibition of the growth of wt-KRas cells, and inhibited the growth of some but not all mut-KRas cells (Fig. 1A; Supplementary Fig. S1). That not all mut-KRas cells are growth inhibited by siKRAS (i.e., addicted to KRAS) has been previously reported for 2D culture (27, 28). When we used siCNK1 we saw no growth inhibition of wt-KRas NSCLC cells but marked inhibition of mut-KRas cells although without the pattern of addiction seen with siKRAS. siRNA knockdown of KRas and Cnk1 protein of >80% maximal by 72 hours was confirmed for a number of cell lines (Supplementary Fig. S2). We saw no difference in the effects of Cnk1 inhibition in cells with various KRas or p53 mutations (Supplementary Table S1). We next studied the effect of KRas and Cnk1 knockdown on KRas downstream signaling, and found minimal effects of KRas knockdown in wild-type NSCLC and HKH2 colon cancer cells, whereas in mut-KRas cells there was upregulation of p-Akt, p-Egfr, p-Erk, probably involving negative regulatory loops (29) but otherwise only modest cell line dependent effects on KRas signaling (Fig. 1B and C). Others have also found that KRas knockdown has relatively small and cell line–dependent effects on KRas signaling (13, 27, 30). Levels of RalA/B, pan-Rho-GTP and pan-Ras-GTP were decreased in mut-KRas HCT-116 colon cancer cells, but pan-Rho-GTP was increased and only Ral/B decreased in isogenic wt-KRas HKH2 cells (Fig. 1C; Supplementary Fig. S3).
It has previously been demonstrated that the effects of mut-KRas inhibition are strongly enhanced in anchorage independent culture (31). To determine whether Cnk1 knockdown phenocopied that effect of mut-KRas knockdown, we tested the anchorage independent growth of HCT-116 colon cancer cells by hanging drop spheroid formation (Fig. 1D). HKH2 cells lacking the mutant KRAS allele showed no growth inhibition in standard 2D culture, but had a >90% growth arrest in spheroid culture, as previously reported for anchorage independent culture (21). The growth of HCT-116 cells treated with siCNK1 was also markedly inhibited in spheroid culture. Thus, CNK1 inhibition leads to selective growth inhibition of mut-KRas NSCLC cells with little effect on wt-KRas cell growth, and without the pattern of KRAS addiction seen with inhibition of mut-KRas. Inhibition of KRas downstream signaling by siRNA to KRas and Cnk1 was modest, and variable depending on the cell line, while inhibition of RalA/B-GTP, pan-Rho-GTP and pan-Ras-GTP was also seen when Cnk1 was inhibited.
We next studied the effect of treatment with siKRAS or siCNK1 on the cell cycle. There was no change in the cell-cycle distribution of HKH2 cells lacking the mutant KRAS allele, whereas parental HCT-116 cells retaining mut-KRas showed an increase in cells in the G1 phase to levels identical to the HKH2 cells (Fig. 2A). Notably, G1 cell-cycle arrest is the reported phenotype in MEF's lacking any Ras isoform (32). Noteworthy is that we found that CRISPR knockout of CNK1 in mut-KRas cells was lethal and prevented clonal outgrowth. We also studied the effects on cell growth of expression of wild-type full-length Cnk1, or Cnk1-EEALAN with a mutated PH domain making it unable to bind PtdIns (Fig. 2B; Supplementary Fig. S4A and S4B). Although no effect was observed on the growth of wt-KRas cells, expression of full-length Cnk1 resulted in inhibition of the growth of mut-KRas cells, almost to levels seen with Cnk1 knockdown. This is a predicted effect for a scaffold protein where its expression at concentrations in excess of its binding partners will sequester these partners in incomplete complexes, leading to combinatorial inhibition of signaling activity (33). The expression of Cnk1-EEALAN unable to bind PtdIns similarly inhibited cell growth, as did expression of just the Cnk1 PH domain, presumably by displacing membrane bound Cnk1. A similar effect has been previously reported for the Cnk1 PH domain on RhoA signaling (6). The use of a myristoylated Cnk1, often used to enhance nonspecific membrane attachment of PH domain proteins (Supplementary Fig. S4C; ref. 34), also showed inhibition of cell growth consistent with the need for Cnk1 to be attached to areas of membrane with high PtdIns. Thus, the level of Cnk1 in cells could be critical for cell growth, with either too much or too little inhibiting cell growth, which would be consistent with it acting as a scaffold protein, whereas PH domain competition or nonselective membrane attachment through myristoylation is also inhibitory.
When we used fluorescent protein tagged human Cnk1-GFP and KRas-RFP in HEK293 cells, Cnk1 and KRas were seen in the cytoplasm and at the plasma membrane, but colocalized only at the plasma membrane (Fig. 2C). When transfected with mut-KRas(G12D)-RFP the cells displayed an altered morphology with many plasma membrane extensions consistent with KRas induced transformation, and colocalization of Cnk1 and mut-KRas at discrete areas of the plasma membrane at the ends of the membrane protrusions. FLIM spectroscopy showed no change in fluorescence lifetime of cells cotransfected with Cnk1-GFP and wt-KRas-RFP compared with Cnk1-GFP transfection alone (Fig. 2D). However, a reduction in the fluorescence lifetime was observed in cells cotransfected with Cnk1-GFP and mut-KRas-RFP. These results suggests there is a close proximity (<10 nm), or physical binding of Cnk1 with mut-KRas, but not wt-KRas. Thus, Cnk1 localizes in close proximity with mut-KRas, and Cnk1 knockdown copies the growth inhibitory effects of mut-KRas deletion independent of the type of KRas mutation, with more pronounced inhibition in 3D culture, while without a significant effect on wt-KRas cell growth.
To determine whether Cnk1 knockdown replicated the selective deletion of the mut-KRAS allele, we used RPPA with an array of 208 signaling and other proteins, in 16 mut-KRas NSCLC cell lines with various activating mutations in KRas treated with either a nontargeting siRNA control or siRNA to CNK1 (Fig. 2E). We found only three proteins that were significantly down regulated across these lines after Cnk1 knockdown, phosphorylated Rb1 serine 807, cyclin B1 and CDK1 (Fig. 2F). The data suggest that Cnk1 inhibition abrogates the ability of mut-KRas to drive Rb1 phosphorylation allowing Rb1 to engage E2F, a transcriptional promoter of cell cycle progression, and repress its activity. The inability of these cells to progress from the G1 phase to the S phase and eventually into mitosis is evident by reduced levels of cyclin B1.
Cnk1 PH domain binding compounds disrupt colocalization of Cnk1 and mut-KRas
Cnk1 serves as a scaffold protein and has no catalytic activity to disrupt but does have a PH domain that may be responsible for appropriate localization to the plasma membrane through PtdIns binding in the Cnk family of proteins (35). Mut-KRas is also reported to interact with PtdIns through a positively charged lysine region in its hypervariable region (36). We first measured the affinity of the Cnk1 PH domain for PtdIns(3,4)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3, and found that Cnk1 bound PtdIns(4,5)P2 with the highest affinity (Kd = 0.18 μmol/L; Fig. 3A). As no crystal structure exists for the Cnk1 PH domain, we constructed a homology model of the PH-domain of Cnk1 and used in silico screening of a library of commercially available compounds to identify possible binding ligands (Fig. 3B). One of the compounds, PHT-7.0, was found to bind to the PH-domain of Cnk1 with Kd = 10.9 μmol/L (Table 1). Using PHT-7.0, a series of analogues with modifications of the ester side arm were designed, modeled and synthesized to screen for compounds with increased potency and desirable pharmacokinetic properties (Supplementary Table S1 and Supplementary Table S2 Schemes SI and SII). From a number of compounds generated and screened, PHT-7.3, a dioxane analog, was found to have the highest binding affinity to the Cnk1 PH domain Kd = 4.7 μmol/L (Fig. 3C). In addition, PHT-7.3 showed selective binding to the PH domain of Cnk1 compared with the PH domains of Pdpk1, Btk, Akt1, and Plekha7 (Supplementary Table S3), and displaced PtdIns(3,4,5)P3 from binding to the PH domain of Cnk1 (Fig. 3D; Supplementary Table S4). To study whether PH domain–binding compounds blocked colocalization of Cnk1 and mut-KRas in cells, confocal microscopy was performed in cells treated with PHT-7.0 or PHT-7.3. Both PHT-7.0 and PHT-7.3 blocked the colocalization of Cnk1 and mut-KRas at the cell plasma membrane (Fig. 3E) and FLIM lifetime plots confirmed that both compounds inhibited the binding of Cnk1 to mut-KRAS (Fig. 3F). Thus, a series of small-molecule probe compounds have been identified that bind to the PtdIns(3,4,5)P3 binding pocket of the PH domain of CNK1 and prevent the interaction between Cnk1 and mut-KRas, but not wt-KRas at the plasma membrane.
The Cnk1 PH domain binding compound PHT 7.3 inhibits the proliferation of mut-KRas cells in vitro and elicits signaling changes similar to CNK1 inhibition
PHT 7.0 and analogs 7.3 and 7.10 (Table 1) were tested in a small group of mut-KRas cells and found to inhibit cell proliferation (Supplementary Fig. S5). PHT-7.3 was the most consistently active (Supplementary Fig. S6), and was chosen for further study. PHT-7.3 did not inhibit the growth (IC50 > 100 μmol/L) of normal mouse or human normal fibroblasts, pancreatic duct, lung, colon or myoblast cells (Supplementary Table S5). When tested against a panel of NSCLC cell lines, wt-KRas cell line 2D growth was not inhibited except for two lines H1437 and H2023. Five mut-KRas cell lines also showed no inhibition of growth in 2D culture, whereas 7 were inhibited (Fig. 4A). Growth inhibition was not related to levels of Cnk1 protein in cells (Supplementary Fig. S7). However, using 3D anchorage-independent growth all the mut-KRas lines were sensitive to PHT-7.3 with IC50s averaging 4.4 μmol/L, and as low as 0.3 μmol/L (Fig. 4B). Wt-KRas cell growth was still not inhibited by PHT-7.3, except for H1437, which has activated KRas signaling due to a MAP2K1 mutation (37), and H2023. Cell migration was also inhibited (Supplementary Fig. S8). Analysis of downstream K-Ras signaling in a panel of mut-KRAS cells recapitulated the increase in p-Akt, p-Egfr, and pErk seen with deletion and siRNA knockdown of Cnk1, and with decreased RalB and Rho activation (Fig. 4C and D; Supplementary Fig. S9). Thus, treatment with PHT-7.3 mimics the growth and signaling effects of deletion of mut-KRas and Cnk1 knockdown, and displays a preferential growth inhibition of mut-KRas cell lines over wt-KRAS lines that is enhanced in anchorage independent conditions.
PHT-7.3 demonstrated the desirable qualities of binding to the PH domain of Cnk1, selective inhibition of mut-KRas NSCLC cell growth and signaling, good in vivo pharmacokinetic properties, and was selected as a probe compound for further in vivo evaluation. We tested PHT-7.3 for antitumor activity, and when dosed daily at 200 mg/kg i.p. for up to 20 days, PHT-7.3 exhibited cytostatic antitumor activity in the mut-KRas(G12S) A549 xenograft and mut-KRas(G12V) H441 xenograft (Fig. 5A and B) but not in the wt-KRas H1975 NSCLC xenograft (Fig. 5C). Measuring the activity of downstream pathways revealed increased levels of p-Egfr (Fig. 5D) and downregulation of activated RalB and Rho signaling (Fig. 5E). PHT-7.3 administered in combination with daily erlotinib 75 mg/kg or trametinib 0.3 mg/kg gave additive antitumor effects with both agents, and with erlotinib there was a tumor regression for the duration of treatment (Fig. 6A and B). There was no loss of body weight with PHT-7.3 treatment (Supplementary Fig. S10). Thus, in vivo antitumor studies with PHT-7.3, alone and in combination with erlotinib or trametinib, show inhibition of mut-KRas tumor growth but little effect on wt-KRas tumor growth.
Our studies show that the scaffold protein Cnk1 closely colocalizes with mut-KRas at the plasma membrane, and a small-molecule inhibitor of the PH domain of Cnk1, PHT 7.3, prevents the colocalization, decreases Raf/Mek/Erk signaling, and causes arrested mut-KRas but not wt-KRas, cell and tumor growth. Cnk1 has also been reported to regulate Rho activity by binding to constitutively active RhoA(G14V) and RhoH, and the Rho GEFs MLK2/3 and Mkk7, leading to activation of the JNK MAP kinase pathway (5–7). Rac, which also acts through Jnk signaling, is similarly regulated by Cnk1 (7), as is RalGDS a GEF for RalA/B (6, 8), and IPECF1 a GEF for Arf (30). Consistent with these reports we found the inhibition of Cnk1 decreased levels of active pan-Rho-GTP and pan-Ras-GTP and RalA/B-GTP, but we did not study other pathways that might be affected. There are also reports that Cnk1 regulates Akt activity driving cell proliferation through inhibition of FoxO (38), and cell invasion through activation of Nf-κb (39). It is not clear at this time the role these other Cnk1-dependent pathways play in regulating KRas signaling and cell growth.
An important consideration is why the growth of wt-KRas cancer cells is not sensitive to Cnk1 inhibition? It may simply be that wt-KRas is not important in cells with other cancer drivers, and indeed we saw minimal change in Raf/Mek/Erk and Akt signaling, or cell growth in wt-KRas cells when KRas was inhibited. Although we saw that Cnk1 was localized at the plasma membrane in proximity to wt-KRas, FLIM studies showed it did not directly engage wt-KRas, as it did mut-KRas. It is also possible that the short life time of active wt-KRas-GTP precludes assembly of a Cnk1 scaffold signaling complex, unlike with constitutively activated mut-KRas-GTP.
The Cnk1 inhibitor we developed, PHT-7.3, blocked the growth of some but not all mut-KRas cells in 2D culture, but was much more potent at inhibiting anchorage independent growth in all mut-KRas cell lines tested. A secondary finding of these studies is that the phenomenon of Ras oncogene “addiction” where some mut-KRas cells are dependent on the oncogene for their growth, whereas others are not (25, 26), was only seen under 2D growth conditions, and not seen with anchorage independent growth of mut-KRas cells. Anchorage-independent growth is a hallmark phenotype of cancer cell growth but is often neglected when studying mut-KRas (30).
Cnk1 possesses a PH domain that we found preferentially binds PtdIns(4,5)P2 (Kd = 0.18 μmol/L). It has been reported that Cnk1 localizes to plasma membranes rich in PtdIns (35), suggesting to us a role for the PH domain in directing the loaded Cnk1 scaffold to the same plasma membrane vicinity as mut-KRas, which is also found in regions of high membrane PtdIns (36). The PH domain of Cnk1 is known to be important for ability to interact with the active form of RhoA to stimulate its transcriptional rather than cytoskeleton effects (5, 6). Using our experience of small-molecule PH domain inhibitors for signaling kinases (19, 40, 41), we developed PHT-7.3 a small-molecule probe compound that can displace bound PtdIns from the PH-domain of Cnk1 and block the membrane association of Cnk1 with mut-KRas. PHT-7.3 also inhibits mut-KRas and Rho signaling, and selectively blocks mut-KRas cell and tumor growth, but not that of wt-KRas cancer cells, nor normal human and rat fibroblasts, myoblasts and lung and colon epithelial cells. An unanswered question is why PHT-7.3, which binds the PH domain of Cnk1 with micromolar affinity, can inhibit mut-KRas cell growth at sub-micromolar concentrations. Discrepancies between biochemical and cell growth inhibitor concentrations are well documented for kinase inhibitors and other drugs, and have been attributed to clustering and stacking of binding proteins at specific areas of the cell surface membrane (42–44). This could happen for PHT-7.3, or being a lipophilic molecule it could concentrate in, or close to the cell membrane aqueous interface, which is a very different environment compared with aqueous phase biochemical measurements of compound binding.
In summary, our studies have found that the scaffold protein Cnk1 is found in close proximity to mut-KRas at the cell plasma membrane through binding of Cnk1's PH domain, and Cnk1 inhibition prevents mut-KRas cell growth, particularly anchorage-independent growth, exceeding the effects of KRas inhibition. In addition, we found that although Cnk1 colocalizes with wt-KRas at the plasma membrane, it is not tightly associated, and Cnk1 inhibition does not inhibit wt-KRas cell growth or downstream signaling. Through molecular modeling and structural modifications we identified a compound PHT-7.3 that binds selectively to the PH domain of Cnk1 preventing plasma membrane binding with mut-KRas, and with the ability to inhibit mut-KRas, cancer cell and tumor growth and signaling, but not that of wt-KRas. Thus, the PH domain of Cnk1 is a druggable target whose inhibition selectively blocks mutant KRas activation.
Disclosure of Potential Conflicts of Interest
M. Indarte is the chairman at University of Belgrano. S. Zhang has ownership interest (including stock, patents, etc.) in PHusis Therapeutics. L. Kirkpatrick is a CEO and has ownership interest (including stock, patents, etc.) in PHusis Therapeutics Inc. No potential conflicts of interest were disclosed by the other authors.
Conception and design: R. Puentes, M. Maruggi, N.T. Ihle, G. Grandjean, Z. Ahmed, S. Zhang, L. Du-Cuny, L.A. Bankston, L. Kirkpatrick, G. Powis
Development of methodology: R. Puentes, M. Maruggi, G. Grandjean, S. Zhang, L. Du-Cuny, R.G. Correa, L. Kirkpatrick
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Indarte, R. Puentes, M. Maruggi, M. Scott, S. Zhang, R. Lemos Jr, F.I.A.L. Layng, R.G. Correa, G. Powis
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Indarte, R. Puentes, M. Maruggi, G. Grandjean, M. Scott, Z. Ahmed, S. Zhang, R. Lemos Jr, L. Du-Cuny, F.I.A.L. Layng, R.G. Correa, L.A. Bankston, R.C. Liddington, G. Powis
Writing, review, and/or revision of the manuscript: M. Indarte, R. Puentes, M. Maruggi, E.J. Meuillet, S. Zhang, R.G. Correa, R.C. Liddington, L. Kirkpatrick, G. Powis
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L. Kirkpatrick, G. Powis
Study supervision: L. Kirkpatrick, G. Powis
Other (drug design): M. Indarte
Other (experimental execution and analysis of surface plasmon resonance data (a.k.a. Biacore): M. Scott
Supported by NIH grants CA185054, CA201707 (to G. Powis), and CCSG grant P30CA030199. The help of SBP Cancer Center Animal and Genomic Services is gratefully acknowledged.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.