Novel Small Molecules Capable of Blocking mtRAS-Signaling Pathway

RAS mutants are involved in approximately 30% of all human cancers and have been regarded as undruggable targets owing to relatively smooth protein surface and obscure binding pockets. In our previous study, we have demonstrated that GNF-7, a multi-targeted kinase inhibitor, possesses potent anti-proliferative activity against Ba/F3 cells transformed with NRAS-G12D. Based on our further analysis using Ba/F3 cells transformed with mtRAS, we discovered a series of pyrimido[4,5-d]pyrimidin-2-one analogues as mtRAS-signaling pathway blockers. In addition, our efforts expanded the assessment to cancer cells with mtRAS, which revealed that these substances are also capable of strongly suppressing the proliferation of various cancer cells harboring KRAS-G12D (AsPC-1), KRAS-G12V (SW480, DU-145), KRAS-G12C (H358), KRAS-G13D (MDA-MB-231), KRAS-Q61L (HT-29), and NRAS-Q61L (OCI-AML3). We herein report novel and potent mtRAS-signaling pathway blockers, SIJ1795 and SIJ1772, possessing 2 to 10-fold increased anti-proliferative activities compared to those of GNF-7 on cancer cells harboring mtRAS as well as on Ba/F3 cells transformed with mtRAS. Both SIJ1795 and SIJ1772 attenuate phosphorylation of RAS downstream molecules (AKT and MEK) and induce apoptosis and G0/G1 cell cycle arrest on cancer cells with mtRAS. Moreover, both substances substantially suppress the migration, invasion, and colony formation of cancer cells harboring mtRAS. Taken together, this study led us to identification of SIJ1795 and SIJ1772 capable of strongly inhibiting mtRAS-signaling pathway on cancer cells harboring mtRAS.


INTRODUCTION
RAS proteins are small GTPase, which function as a binary molecular switch controlling the signal transduction by switching between active and inactive states. The equilibrium is shifted to guanosine triphosphate (GTP)-bound RAS active state when GDP/GTP exchange occurs by guanine nucleotide exchange factor (GEF). On the other hand, guanosine diphosphate (GDP)bound RAS inactive state is formed through GTPase-activating protein (GAP)-mediated hydrolysis of GTP (1). As is well known, RAS proteins are notorious for possessing extremely high affinity toward GTP and relatively obscure binding pockets contributing to poor interaction with small molecules (2). Moreover, mutations of RAS proteins at codons 12, 13, and 61 interfere with GAP-mediated GTP hydrolysis consequently resulting in constitutively activated GTP-bound RAS forms (3,4). Thus formed RAS active state stimulates the downstream signaling pathways such as MAPK (RAS-RAF-MEK-ERK) (5) and PI3K (PI3K-AKT-mTOR) pathways closely associated with cell cycle, growth, proliferation, survival, and differentiation (3)(4)(5)(6).
Previously, we reported that dual inhibition of activated cdc42-associated kinase 1 (ACK1) and germinal center kinase (GCK) can be a novel therapeutic strategy to overcome acute myeloid leukemia (AML) harboring NRAS mutation (25). Multitargeted kinase inhibitor GNF-7 (26) and its derivatives are shown to be able to suppress proliferation of both Ba/F3 cells transformed with NRAS-G12D and human AML cell lines, OCI-AML3 (NRAS-Q61L) via dual inhibition of ACK1 and GCK. Moreover, GNF-7 and its derivatives displayed effect on antileukemic efficacies in blood circulating model (Ba/F3-NRAS-G12D) and xenograft model (OCI-AML3) as well as AKT/ mTOR and GCK signaling, apoptosis, cell cycle arrest, anchorage independent growth in Ba/F3-NRAS-G12D and OCI-AML3 (27). GNF-7 and its derivatives are also reported as pan-class (class I/II/III) BRAF inhibitor in our previous studies, indicating that inhibiting BRAF may be helpful for inhibiting mtRAS signaling pathway (28,29). Overall, our previous findings strongly suggest that the use of multitargeted kinase inhibitor such as GNF-7 can provide an effective approach to discover novel inhibitors targeting mtRAS via blocking its downstream signaling pathway.

Anti-Proliferation Assay
Approximately 3.0 × 10 3 cells/well were seeded in a 96-well cell culture plate. After stabilization, 1/3 serially diluted compound in DMSO was treated to the cell. After 72 h incubation at 37°C, the cell viability was assessed with CellTiter Glo (Promega, G7572) GI 50 values were measured by Graphpad prism 6.0 software. All GI 50 values were obtained in duplicate with three independent assays and averages with standard deviation are presented.

Flow Cytometry Analysis
For apoptosis analysis, cells (1 × 10 6 cells per sample) were incubated with each compounds for 24 h. Cells were subjected to brief ice-cold PBS washing and subjected to staining with Alexafluor488-conjugated annexin V (Thermo Fisher, A13201), and propidium iodide (PI, Thermo Fisher, P3560). Thereafter, apoptotic cells were classified by FACS Accuri ™ C6 Plus (BD Biosciences). Debris and unstained cells and were excluded by gating.
For cell cycle analysis, cells (1 × 10 6 cells per sample) were incubated with each compounds for 24 h. Cells were subjected to brief ice-cold PBS washing and fixed with 70% EtOH for 1 h at −20°C. Cells were stained with PI/RNase staining solution (Cell signaling, #4087) and incubated 30 min at room temperature in a dark condition. Stained cells were subjected to flow cytometry analysis. Debris and unstained cells and were excluded by gating.

Migration and Invasion Assay
For migration assay, scratch assay was conducted. Each cells (2.0 × 10 5 cells/well) were seeded in 24-well plates. After cellular attachment, cells were scratched with a SPLScarTM Scratcher (SPL Life Sciences, #201924) and the detached cells were removed by PBS washing twice. Cells were incubated in complete media with 0.01 mM concentrations of each compounds for indicated time. The images were acquired before and after incubation with compounds (40× magnification), and percent of migration were accessed using ImageJ (n = 3).
For invasion assay, Boyden chamber assay carried out. Transwell insert was precoated with matrigel (Corning, #354248). Cells were seeded in the transwell chamber insert (8 mm pore size) at a density of 5.0 × 10 5 cells/well after serum starvation for overnight. The cells were incubated with 0.01 mM concentration of each compounds of for 48 h at 37°C. The non-invaded cells were eliminated. Crystal violet solution was used to staining the invaded cells. Cells were observed and photographed with 100× magnification. Stained cells were automatically counted and quantified (n = 3).

Colony Formation Assay
Colony formation assay was conducted for 2D clonogenic assay. Approximately 1 × 10 3 cells/well were seeded in 6-well plate. After cellular attachment, indicated concentrations of compounds were treated for 14 days at 37°C. Colonies were stained by crystal violet solution for 30 min. The total area of each well was observed without magnification, and the number of colonies of each well were accessed using ImageJ software (n = 3).
Soft agar assay was conducted for 3D clonogenic assay. On the 0.7% bottom agar, cells were seeded in 6-well plate (5 × 10 3 cells/well) with 0.35% low melting agar (Lonza, #50101) containing the complete media. The cells were treated with indicated compounds for 14 days at 37°C. Colonies were stained by iodonitrotetrazolium chloride (Sigma-Aldrich, I8377) for overnight. The total area of each well was observed without magnification, and the number of colonies of each well were determined using ImageJ software (n = 3).

Effects of SIJ1795 and SIJ1772 on Apoptosis Induction and Cell Cycle Arrest in Ba/F3 Cells Transformed With mtRAS (NRAS-G12D and NRAS-G12V) and Cancer Cells Harboring mtRAS
We performed Western blot and flow cytometry analysis to evaluate the effects of the representative derivatives on apoptosis or cell cycle arrest induction. First, we measured cleaved PARP level, a well-known pro-apoptotic marker It is worthwhile to note that SIJ1795 and SIJ1772 increased cleaved PARP level against Ba/F3-NRAS-G12D and Ba/F3-NRAS-G12V cells at 1 mM concentration (Figures 3A, B). In addition, the level of cleaved PARP was notably increased at 1 mM concentration of SIJ1795 or SIJ1772 in the cancer cells harboring mtRAS ( Figures 4A, B). We next carried out flow cytometry analysis and evaluated apoptotic cells population using annexin V/propidium iodide (PI) staining. Treatment of SIJ1795 and SIJ1772 noticeably led to an increase of apoptotic cells in Ba/F3-NRAS-G12D and Ba/F3-NRAS-G12V cells, which is consistent with the results of Western blot analysis ( Figure 3C, Supplementary Figure S2). Moreover, SIJ1795 and SIJ1772 induced apoptosis against cancer cells harboring mtRAS ( Figure 4C, Supplementary Figure S3). Apoptosis induction capabilities of SIJ1795 or SIJ1772 are comparable to that of GNF-7. We then analyzed cell cycle by flow cytometry analysis and observed that G0/G1 arrest was induced by SIJ1795 or SIJ1772 at 1 µM, ( Figure 5) which correlates with our previous findings (27). Taken together, our results demonstrate that SIJ1795 and SIJ1772 are capable of inducing apoptosis and G0/G1 cell cycle arrest on Ba/F3 cells transformed with mtRAS (NRAS-G12D and NRAS-G12V), and cancer cells harboring mtRAS.

Migration and Invasion Inhibitory Activity of SIJ1795 and SIJ1772 on Cancer Cells Harboring mtRAS
Previous studies have shown that KRAS activating mutation is associated with enhanced cell migration in invasion activities in many types of cancer, such as pancreatic ductal adenocarcinoma (32), prostate cancer (33), colon cancer (34,35), and breast cancer (36). Accordingly, we investigated inhibitory capabilities of SIJ1795 and SIJ1772 on migration and invasion against cancer cells with mtRAS ( Figure 6, Supplementary Figures S4, S5). It is noteworthy that 0.01 mM of SIJ1795 potently suppressed migration and invasion capabilities of cancer cells harboring mtRAS, regardless their mutation status. Furthermore, in this investigation, we noticed over 50% reduction in migration and invasion capabilities of cancer cells with mtRAS including H358, AsPC-1, DU-145, SW480, and MDA-MB-231 when treated with both SIJ1795 and SIJ1772 at 0.01 µM.

Colony Formation Inhibitory Effect of SIJ1795 and SIJ1772 on Cancer Cells Harboring mtRAS
Subsequently, we examined tumorigenesis inhibitory effects of SIJ1795 and SIJ1772 on cancer cells harboring mtRAS by conducting 2D and 3D clonogenic assay (colony formation assay and soft agar assay, respectively). As shown in Figures 7, 8, SIJ1795 and SIJ1772 suppressed colony formation and anchorage independent cell growth at 0.1 mM. These results suggest that representative derivatives SIJ1795 and SIJ1772 are capable of significantly inhibiting tumorigenesis of cancer cells harboring mtRAS.

DISCUSSION
Blocking mtRAS downstream signaling pathway can be an effective approach to discover novel inhibitors targeting mtRAS. In our previous study (25,27), we demonstrated that dual inhibition of ACK1 and GCK by multi-targeted kinase, GNF-7 and its derivatives, displays strong capabilities of cellular inhibition on both Ba/F3 cells transformed with NRAS-G12D and OCI-AML3, a human cancer cell harboring NRAS-Q61L mutation. We also observed that these anti-proliferative effects exerted by GNF-7 and its derivatives are accompanied by substantial induction of apoptosis and G0/G1 arrest through suppressing AKT/mTOR signaling pathway and downstream molecules of GCK in Ba/F3-NRAS-G12D and OCI-AML3 cells. In addition, we previously reported (28,29) that GNF-7 and its derivatives as pan-class (class I/II/III) BRAF inhibitors and described that the use of multi-targeted kinase inhibitors may be an useful tactic to block mtRAS signaling pathway. Considering all these preceding findings, we attempted to expand the application of GNF-7 derivatives as a means of a promising anticancer strategy targeting mtRAS via blocking the downstream signaling pathway. As background for the exploration described above, we made significant observations that GNF-7 derivatives display two-digit nanomolar potencies against Ba/F3s transformed with mtRAS (NRAS-G12D and NRAS-G12V). Particularly, SIJ1795 and SIJ1772 exhibited 3 to 5-fold increased activities against Ba/F3-NRAS-G12D and Ba/F3-NRAS-G12V compared with GNF-7 (GI 50 s = 0.075 to 0.096 µM). In agreement with the antiproliferative activities, Western blot analysis shows that both SIJ1795 and SIJ1772 completely attenuate the level of phospho-MEK and -ERK at 10 µM. This indicates that SIJ1795 and SIJ1772 exert anti-proliferative activities against Ba/F3 cells transformed with NRAS-G12D and NRAS-G12V by effectively blocking oncogenic RAS signaling pathway.
To expand the applicability of GNF-7 derivatives on mtRAS cancer cells, we subsequently evaluated fourteen GNF-7 derivatives against cancer cells harboring mtRAS: H358 In accordance with prior expectations, anti-proliferation activities for this series of derivatives are well translated to various cancer cells with mtRAS. It is of worth to mention that differential cytotoxicity of all fourteen derivatives is enhanced by 8-fold on OCI-AML3 (NRAS-Q61L) cells in relative to U937 (NRAS-wt) cells. Notably, in this investigation, we observed that the representative derivatives, SIJ1795 and SIJ1772 exhibit 2 to 10fold enhanced cellular potencies against all cancer cells harboring mtRAS (GI 50 s = 0.029 to 0.447 µM) compared to GNF-7, which is also indicative of strong capabilities of SIJ1795 and SIJ1772 on blocking mtRAS signaling pathway. Besides, SIJ1795 and SIJ1772 effectively suppressed phosphorylation level of AKT, MEK, and ERK at 1 µM, and induced apoptosis as well as G0/G1 arrest, contributing to their observed anti-proliferative activities against cancer cells with mtRAS. It is worth recalling that both SIJ1795 and SIJ1772 are multi-targeted kinase inhibitors and antiproliferative effects of multi-targeted inhibitors are not fully dependent on the on-target signaling inhibitory effect. Based on previous studies showing close correlation between enhanced cell migration and RAS activating mutation in various cancers, we successively assessed inhibitory capabilities of SIJ1795 and SIJ1772 against cancer cells harboring mtRAS on migration, invasion, and anchorage-independent growth. This assessment revealed that SIJ1795 and SIJ1772 at 0.01 µM concentration substantially suppressed migration and invasion capabilities of cancer cells harboring mtRAS including H358, AsPC-1, DU-145, SW480, and MDA-MB-231. Tan et al. showed that bosutinib, which is ACK1 inhibitor, suppressed the mtKRAS NSCLC cell migration and invasion (37). Additionally, SIJ1795 and SIJ1772 significantly inhibit colony formation and anchorage independent cell growth at 0.1 mM. Taken together, SIJ1795 and SIJ1772 are potent mtRAS signaling blockers and significantly suppress migration and invasion of cancer cells with mtRAS, which suggests that these GNF-7 based derivatives may serve as potential strategy to override mtRAS-driven cancer.

CONCLUSION
Targeting mtRAS has been regarded as one of unresolved challenges in oncology drug discovery. Despite intensive efforts to target mtRAS by directly or indirectly inhibiting the protein, AMG-510 (sotorasib) is currently the only FDA-approved drug, which solely targets KRAS-G12C. Accordingly, the development of novel strategies to target mtRAS remains as a great unmet medical need.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories.