Growth factor receptor signaling is a critical component of tissue growth, homeostasis, and wound healing. However, receptor desensitization limits the use of exogenous growth factors as a restorative agent therapeutically. An example of this is the epidermal growth factor receptor (EGFR) in the corneal epithelium. Despite laboratory data indicating that EGFR activity accelerates corneal re-epithelialization in mice and rabbits, the clinical administration of EGF to damaged corneal epithelium has limited impact due to the attenuated signaling that occurs following sustained growth factor administration. We hypothesized that inhibition of receptor desensitization would prolong receptor activity and enhance tissue homeostasis. Having previously identified the E3 ubiquitin ligase, c-Cbl, as a key negative regulator of EGFR signaling in the corneal epithelium, we have developed a class of small molecule inhibitors of EGFR binding to CBL family proteins using virtual screening with experimental validation. Through multiple rounds of structural optimization, we have identified compound 3-120. This compound was designed to compete with phosphotyrosine 1045 of the EGFR for binding to c-Cbl. Compound 3-120 binds to c-Cbl with an ~10-fold higher affinity than phosphoEGFR, reduces EGFR ubiquitylation by 40%, and increases the magnitude of ligand-stimulated EGFR phosphorylation by 30 to 40%. Ultimately, this compound can enhance the restoration of corneal epithelial debridement wounds. Thus, compound 3-120 is an antagonist that specifically disrupts EGFR ubiquitylation to sustain receptor signaling.

RAS oncoproteins are the most frequently activated oncoproteins in cancer. Development of direct RAS inhibitors has proved technically challenging and has had limited success in the clinic. Those RAS inhibitors that have been approved tend to suffer from resistance development. Consequently, many attempts have focused on inhibiting RAS indirectly by targeting its immediate downstream effectors. RAS binds and activates three main effector classes to drive transformation: RAF kinases, phosphoinositide 3 (PI-3) kinase and Ras-like (RAL) small GTPases (RALGEF) exchange factors. Multiple FDA-approved inhibitors for RAF and PI-3 kinase exist. So far, they have proved to be of limited effectiveness in patients. However, no inhibitors of the RALGEF effectors with demonstrated antitumor activity have been reported. This is despite the considerable body of evidence supporting a critical role for the RALGEF/RAL pathway in facilitating the in vivo transforming effects of activated RAS. Here, we describe the first small molecule pan-RALGEF inhibitor. We show the inhibitor specifically suppresses RAS/RAL signaling and exhibits antitumor effects in xenograft experiments, including a patient-derived xenograft (pdx) model. This first-in-class compound may lead to the development of more effective therapies for a broad range of RAS-driven tumors.

Telomeres, which are the protective caps of human chromosomes, degrade through rounds of cellular replication, naturally limiting cellular lifespan. Cancer cells circumvent this by up-regulating telomere extension mechanisms. The telomeres consist of G-rich repeats that are capable of folding into four-stranded non-B structures known as G-quadruplexes (G4s) that mediate extension and protective processes. Stabilizing G4 structures with small molecules can inhibit telomere elongation, offering a potential cancer therapy strategy. Despite our detailed understanding of their equilibrium conformational diversity, G4 fast-folding dynamics remain elusive, especially during the initial folding stages. Here we have employed equilibrium and time-resolved Small Angle X-ray Scattering (SAXS), alongside standard biophysical techniques and molecular dynamics, to explore the millisecond-scale folding of telomere G4s. Our findings reveal that denatured telomere G4s equilibrate as a mixture of extended and partially pre-folded hairpin conformations. Upon pH-induced folding, we observe a sub-millisecond structural contraction, followed by a rapid two-state collapse to a structure closely resembling pre-folded intermediates of the early folding pathway. Integration of our results with prior low-resolution folding studies allows us to formulate a more complete understanding of the early timescale pre-folded collapsed telomere G4 structure. Understanding the full folding landscape offers fundamental insight into the driving forces of G4 folding and offers structural information for drug development efforts targeting telomere G4 folding intermediates.

Guanine-rich nucleic acids can form intramolecularly folded four-stranded structures known as G-quadruplexes (G4s). Traditionally, G4 research has focused on short, highly modified DNA or RNA sequences that form well-defined homogeneous compact structures. However, the existence of longer sequences with multiple G4 repeats, from proto-oncogene promoters to telomeres, suggests the potential for more complex higher-order structures with multiple G4 units that might offer selective drug-targeting sites for therapeutic development. These larger structures present significant challenges for structural characterization by traditional high-resolution methods like multi-dimensional NMR and X-ray crystallography due to their molecular complexity. To address this current challenge, we have developed an integrated structural biology (ISB) platform, combining experimental and computational methods to determine self-consistent molecular models of higher-order G4s (xG4s). Here we outline our ISB method using two recent examples from our lab, an extended c-Myc promoter and long human telomere G4 repeats, that highlights the utility and generality of our approach to characterizing biologically relevant xG4s.

Abstract The RAS oncoprotein has not traditionally been considered as an important driver of breast cancer due to the paucity of RAS mutations in this disease. However, RAS signaling pathways are frequently hyper-activated in breast cancers and genetic/epigenetic inactivation of RAS negative regulators (GAPs) is common. This is particularly true for Luminal B breast cancer. Luminal breast cancer makes up the majority of Breast Cancers, ~ 65% of cases. About 2/3 of these are classed as Luminal A and about 1/3 being the Luminal B class. Although current therapeutic options for luminal A (surgery, endocrine therapy) can be reasonably effective, Luminal B tumors are much more dangerous. These tumors are less sensitive to therapy in the first place and have a high frequency of relapse. Moreover, Luminal B tumors tend to occur more frequently in younger women and have a higher incidence of metastasis. One recent analysis has shown that Luminal B cancers have little better overall survival rates than the notorious triple negative breast cancers. Therefore, better therapies for Luminal B breast cancer are urgently required. One approach may be to target the hyper-active wild-type RAS oncoprotein that drives many of these Luminal B tumors. We have used in silico library screening followed by Medicinal Chemistry optimization to develop a series of novel small molecules that bind to all three main wild type RAS proteins and inhibit RAS function. We have validated the agents by using Microscale Thermophoresis binding procedures to quantify recombinant protein interaction. We used 3D growth inhibition and protein-based RAS signaling assays to quantify the biological action of the agents. Finally, we validated one of the more effective agents in vivo against a Luminal B cell line xenograft. This may be the first example of an effective anti-RAS therapy in breast cancer. It serves as proof of principal for the use of ant-RAS drugs in Luminal B disease. As basal breast cancers often exhibit loss of function of the RAS GAP called NF1, this strategy may have applications beyond Luminal B disease. Citation Format: Geoff Clark, Raphael Jigo, Howard Donninger, Joe Burlison, Mike Sabo, tariq Arshad, John Trent. Pan-RAS inhibitors to treat luminal B beast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B020.

G-quadruplexes (G4s) are four-stranded nucleic acid secondary structures that can form in genomic regions of high guanine content. G4s are highly concentrated in the promoter regions of cancer-associated genes and can up- or down-regulate adjacent gene transcription. G4s are now actively pursued for their ability to selectively repress the transcription of many hard-to-target cancer proteins, such as c-Myc, k-Ras, and c-Kit. G4 targeting small molecules exhibit excellent in vitro and in vivo anticancer activity, however, none have progressed through clinical trials. We contend that this is the result of targeting the small, singular G4 domains that have been "convenient" to study by traditional structural biology methods, such as NMR and X-ray diffraction, that have similar topologies and few druggable pockets. By utilizing an integrative structural biology approach (ISB), which combines small-angle X-ray scattering (SAXS), circular dichroism, molecular modeling, fold prediction, and footprinting, my lab has shown that multiple cancer gene promoters contain contiguous G4 motifs that adopt higher-order arrangements. The higher-order tertiary folds reveal quantitively better drug targeting sights relative to the traditional small G4s as determined by SiteMap analysis. We go on to show that models developed in our approach are useful as in silico targets for virtual drug discovery efforts with multiple examples of successful drug screening. This work was funded by the National Institutes of Health (NIH) grant [GM077422]

RAS oncogenes are frequently activated by mutations in pancreatic and lung cancer, where they appear to act as driving mutations. This study examines the activity of a series of novel direct RAS inhibitors with a predicted unique interaction region. We have used in silico library screening followed by Medicinal Chemistry optimization to develop the compounds. We have validated the agents using Microscale Thermophoresis to quantify binding to recombinant RAS protein as well as NMR analysis of the drug/RAS complex. We used 3D growth inhibition assays in mutant RAS cell lines and protein-based RAS signaling assays to quantify and characterize the action of the family of inhibitors. We have identified compounds that bind wild type K-RAS and H-RAS but exhibit preferential binding to the K-RAS-G12D and KRAS-G12C mutants. We can suppress the association of mutant RAS protein with its effector RAF-1 in treated cells. We also observe suppression of mutant RAS signaling and inhibition of 3D cell growth of mutant RAS cell lines with the agents. As the compound family are predicted to bind to a different site than either AMG-510 (K-RAS G12C specific) or MRTX-1133 (K-RAS G12D specific), agents we have also tested co-operative activity with these drugs. Our compounds enhanced the effects of MRTX-1133 against K-RAS G12D cells and had a similar effect on AMG-510 in K-RAS G12C cell lines. These agents may serve as novel anti-RAS therapeutics and may have potential to enhance the activity or suppress resistance to AMG-510 and MRTX-1133. Citation Format: Geoff Clarke, Tariq Arshad, Howard Donninger, Becca von Baby, Rachel Ferrill, Mike Sabo, Joe Burlison, John Trent. Novel direct RAS inhibitors for pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B006.

Body mass graphs from in vivo studies showing no change in body weight over the treatment time-courses.

Supplemental Figure Legends

IC50 values obtained from INI-43 treatment of cancer, transformed and normal cell lines.

4155 Background: Mutant-activated RAS genes are the most frequently mutated gene family associated with cancer (almost 30% of all cancers contain a mutant RAS gene). KRAS is the predominant isoform mutated in cancer and is the isoform exclusively mutated in pancreatic ductal carcinoma (PDAC). Since almost all PDAC cases harbor a mutant RAS, it is arguably the most RAS-addicted tumor type. There is now considerable evidence implicating mutant KRAS as a driver of PDAC. Recently, several mutant KRAS-targeted therapies (sotorasib and adagrasib) have been developed and show promise in PDAC patients. We have developed a direct inhibitor of RAS with a predicted unique interaction region capable of directly binding to wild-type H- and K-RAS, but which shows preferential binding for KRAS G12D and G12C mutants. This novel inhibitor disrupts the RAS effector domain and blocks the ability of RAS to signal through its effectors. Methods: We used in silico virtual library screening to identify an initial candidate inhibitor which was effective at inhibiting the 3D growth of PDAC cells without affecting their growth in 2D. Subsequent iterative rounds of medicinal chemistry was then performed to identify a series of derivatives with enhanced activity, as determined by 3D growth inhibition assays and effects on Ras signaling, as determined by Western blot analysis of phosphor-ERK, phosphor-Akt and activation of Ral A. Results: Our series of RAS inhibitors effectively block PDAC cell growth in 3D without impacting their 2D proliferation and suppress the interaction of KRAS with its effector cRAF. The inhibitors also effectively inhibit RAS signaling in mutant RAS containing PDAC cells. Since our novel compounds are predicted to bind to RAS at a different site to either AMG-510 (G12C specific inhibitor) and MRTX-1133 (G12D specific inhibitor), we tested the co-operativity of our compounds with these existing agents. Our compounds enhanced the anti-proliferative effects of both MRTX-113 and AMG-510 in mutant KRAS G12D and G12C PDAC cells, respectively. Conclusions: We have developed a series of novel RAS inhibitors that directly bind preferentially to mutant KRAS that may serve as new mutant KRAS-targeted therapeutics, and that may also have the potential to enhance the efficacy or suppress the resistance of AMG-510 and MRTX-1133.

Dose-response curves showing cell line responses to INI-43 treatment.

Genomic regions with high guanine content can fold into non-B form DNA four-stranded structures known as G-quadruplexes (G4s). Extensive in vivo investigations have revealed that promoter G4s are transcriptional regulators. Little structural information exists for these G4s embedded within duplexes, their presumed genomic environment. Here, we report the 7.4 Å resolution structure and dynamics of a 28.5 kDa duplex-G4-duplex (DGD) model system using cryo-EM, molecular dynamics, and small-angle X-ray scattering (SAXS) studies. The DGD cryo-EM refined model features a 53° bend induced by a stacked duplex-G4 interaction at the 5' G-tetrad interface with a persistently unstacked 3' duplex. The surrogate complement poly dT loop preferably stacks onto the 3' G-tetrad interface resulting in occlusion of both 5' and 3' tetrad interfaces. Structural analysis shows that the DGD model is quantifiably more druggable than the monomeric G4 structure alone and represents a new structural drug target. Our results illustrate how the integration of cryo-EM, MD, and SAXS can reveal complementary detailed static and dynamic structural information on DNA G4 systems.

Abstract Since RAS mutations are rare in Breast Cancer, RAS has not traditionally been considered as an important driver of this disease. However, RAS signaling pathways are frequently hyper-activated in breast cancers and genetic/epigenetic inactivation of RAS negative regulators (GAPs) is common. This is particularly true for Luminal B breast cancer. Luminal breast cancer makes up the majority of Breast Cancers, ~ 65% of cases. The Luminal B form makes up around 1/3 of these. Although current therapeutic options for luminal A (surgery, endocrine therapy) can be reasonably effective, Luminal B tumors are much more dangerous. These tumors are less sensitive to therapy in the first place and have a high frequency of relapse. Moreover, Luminal B tumors tend to occur more frequently in younger women and have a higher incidence of metastasis. One recent analysis has shown that Luminal B cancers have little better overall survival rates than the notorious triple negative breast cancers. Therefore, better therapies for Luminal B breast cancer are urgently required. One approach may be to target the hyper-active wild-type RAS oncoprotein that drives many of these tumors. We have in used in silico library screening followed by Medicinal Chemistry optimization to develop a series of novel small molecules that bind to all three main RAS proteins and block RAS function. We have validated the agents by using Microscale Thermophoresis binding procedures to quantify recombinant protein interaction. We used 3D growth inhibition and protein-based RAS signaling assays to quantify the biological action of the agents. Finally, we validated one of the more effective agents in vivo against a Luminal B cell line xenograft. This may be the first example of an effective anti-RAS therapy in breast cancer. It serves as proof of principal for the use of ant-RAS drugs in Luminal B disease. As basal breast cancers often exhibit loss of function of the RAS GAP called NF1, this strategy may have applications beyond Luminal B disease. Citation Format: Geoff Clarke, Howard Donninger, Raphael Jigo, Tariq Arshad, Becca von Baby, Rachel Ferrill, Mike Sabo, Joe Burlison, John Trent. RAS inhibitors to treat luminal B breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B005.

SubG1 cell populations after INI-43 treatment of CaSki cells for 6 and 24 hr.

Chemical compounds identified in the in silico screen that display IC50 values of less than 50uM.