RAS GTPases are important mediators of oncogenesis with nearly 30% of human tumors harboring mutant RAS proteins. However, pharmacological inhibition of RAS has proven challenging. We have employed Monobody technology to discover novel vulnerabilities in RAS that can be exploited to inhibit RAS signaling and tumorigenesis. Monobodies are single-domain synthetic binding proteins that achieve levels of affinity and selectivity similar to antibodies. In contrast to antibodies, Monobodies are fully functional in the reducing environment of the cytoplasm and thus are particularly suitable as genetically encoded “tool biologics”. We previously developed the NS1 Monobody that inhibited RAS by targeting the α4-α5 allosteric lobe to prevent RAS self-association and nanoclustering, and NS1 has become a widely used tool in the RAS research community. Following on this success, we sought to identify additional vulnerabilities in RAS. Based on our discovery that nucleotide-free RAS (apoRAS) inhibits PIK3C2B function, we assessed the feasibility of selectively targeting this state of RAS as an approach to inhibit oncogenic RAS function. Here, we have developed several Monobodies and extensively characterized one of them, R15. Although NS1 was agnostic to the nucleotide state of RAS, R15 bound exclusively to the apo state of all three RAS isoforms but did not interact with nucleotide-loaded RAS. When expressed in cells, R15 selectively inhibited the signaling and transforming activity of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., “fast exchange mutants”), such as G13D and Q61L. Surprisingly, R15 bound and inhibited RAS(G12D) mutants which are not reported to be fast exchange. Biochemical studies demonstrated that RAS captured with R15 from cell lysates was indeed nucleotide free, suggesting that R15 traps apoRAS and prevents nucleotide reloading. Finally, intracellularly expressed R15 selectively inhibited the tumor forming capacity of human cell lines driven by fast exchange RAS mutants but not RAS(G12V) in mouse xenografts. Thus, in contrast to conventional wisdom, our approach has established a new opportunity to selectively inhibit certain RAS mutants by targeting the apo state of RAS with drug-like molecules. Citation Format: Imran Kahn, Akiko Koide, Mariyam Zuberi, Gayatri Ketavarapu, Eric Denbaum, Kai Wen Teng, J. Matthew Rhett, Russell Spencer-Smith, G. Aaron Hobbs, Earnest Ramsay Camp, Shohei Koide, John P. O'Bryan. Inhibition of RAS signaling and tumorigenesis through targeting novel vulnerabilities [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 B023.

RAS guanosine triphosphatases (GTPases) are mutated in nearly 20% of human tumors, making them an attractive therapeutic target. Following our discovery that nucleotide-free RAS (apo RAS) regulates cell signaling, we selectively target this state as an approach to inhibit RAS function. Here, we describe the R15 monobody that exclusively binds the apo state of all three RAS isoforms in vitro, regardless of the mutation status, and captures RAS in the apo state in cells. R15 inhibits the signaling and transforming activity of a subset of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., fast exchange mutants). Intracellular expression of R15 reduces the tumor-forming capacity of cancer cell lines driven by select RAS mutants and KRAS(G12D)-mutant patient-derived xenografts (PDXs). Thus, our approach establishes an opportunity to selectively inhibit a subset of RAS mutants by targeting the apo state with drug-like molecules.

Phase II clinical trials have reported that acute treatment of surgical skin wounds with the therapeutic peptide alpha Connexin Carboxy-Terminus 1 (αCT1) improves cutaneous scar appearance by 47% 9-month postsurgery. While Cx43 and ZO-1 have been identified as molecular targets of αCT1, the mode-of-action of the peptide in scar mitigation at cellular and tissue levels remains to be further characterized. Scar histoarchitecture in αCT1 and vehicle-control treated skin wounds within the same patient were compared using biopsies from a Phase I clinical trial at 29-day postwounding. The sole effect on scar structure of a range of epidermal and dermal variables examined was that αCT1-treated scars had less alignment of collagen fibers relative to control wounds-a characteristic that resembles unwounded skin. The with-in subject effect of αCT1 on scar collagen order observed in Phase I testing in humans was recapitulated in Sprague-Dawley rats and the IAF hairless guinea pig. Transient increase in histologic collagen density in response to αCT1 was also observed in both animal models. Mouse NIH 3T3 fibroblasts and primary human dermal fibroblasts treated with αCT1 in vitro showed more rapid closure in scratch wound assays, with individual cells showing decreased directionality in movement. An agent-based computational model parameterized with fibroblast motility data predicted collagen alignments in simulated scars consistent with that observed experimentally in human and the animal models. In conclusion, αCT1 prompts decreased directionality of fibroblast movement and the generation of a 3D collagen matrix postwounding that is similar to unwounded skin-changes that correlate with long-term improvement in scar appearance.

RAS GTPases are important mediators of oncogenesis with nearly 20% of human tumors harboring mutant RAS proteins. However, pharmacological inhibition of RAS has proven challenging. We have employed Monobody technology to discover novel vulnerabilities in RAS that can be exploited to inhibit RAS signaling and tumorigenesis. Monobodies are single-domain synthetic binding proteins that achieve levels of affinity and selectivity similar to antibodies. In contrast to antibodies, Monobodies are fully functional in the reducing environment of the cytoplasm and thus are particularly suitable as genetically encoded “tool biologics”. We previously developed the NS1 Monobody that inhibited RAS by targeting the α4-α5 allosteric lobe to prevent RAS self-association and nanoclustering, and NS1 has become a widely used tool in the RAS research community. Following on this success, we sought to identify additional vulnerabilities in RAS. Based on our discovery that nucleotide-free RAS (apoRAS) inhibits PIK3C2B function, we assessed the feasibility of selectively targeting this state of RAS as an approach to inhibit RAS. Here, we have developed several Monobodies and extensively characterized one of them, R15. Although NS1 was agnostic to the nucleotide state of RAS, R15 bound exclusively to the apo state of all three RAS isoforms but did not interact with nucleotide-loaded RAS. When expressed in cells, R15 inhibited the signaling and transforming activity of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., “fast exchange mutants”), such as G13D and Q61L, but was ineffective against slow exchange mutants such as G12V. Surprisingly, R15 also bound and inhibited RAS(G12D), the most common oncogenic KRAS mutation in pancreatic cancer. Biochemical studies demonstrated that R15 captured RAS from cell lysates in the nucleotide-free state, suggesting that R15 traps apoRAS and prevents nucleotide reloading. Disruption of SOS binding by mutation of D69N further enhanced R15 binding to these fast exchange RAS mutants. Finally, inducible expression of R15 selectively inhibited the tumor forming capacity of tumor lines and patient derived xenografts driven by fast exchange RAS mutants, including RAS(G12D), but not RAS(G12V). Thus, in contrast to conventional wisdom, our approach has established a new opportunity to selectively inhibit the tumorigenic activity of certain RAS mutants by targeting the apo state of RAS with drug-like molecules. Citation Format: Imran Khan, Akiko Koide, Mariyam Zuberi, Eric Denbaum, Gayatari Ketavar, Kai-Wen Teng, Matthew Rhett, Russell Sepncer-Smith, Ernest R. Camp, Shohei Koide, John O’Bryan. Inhibition of RAS signaling and tumorigenesis through novel targeting novel vulnerabilities [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-064.

RAS was identified as a human oncogene in the early 1980s and subsequently found to be mutated in nearly 30% of all human cancers. More importantly, RAS plays a central role in driving tumor development and maintenance. Despite decades of effort, there remain no FDA approved drugs that directly inhibit RAS. The prevalence of RAS mutations in cancer and the lack of effective anti-RAS therapies stem from RAS' core role in growth factor signaling, unique structural features, and biochemistry. However, recent advances have brought promising new drugs to clinical trials and shone a ray of hope in the field. Here, we will exposit the details of RAS biology that illustrate its key role in cell signaling and shed light on the difficulties in therapeutically targeting RAS. Furthermore, past and current efforts to develop RAS inhibitors will be discussed in depth.

Epidermal growth factor receptor (EGFR) is commonly over-expressed in metastatic breast cancer yet metastatic breast cancer is generally resistant to anti-EGFR therapies, and the mechanism for resistance to EGFR inhibitors in this setting is not fully understood. Hormonally up-regulated neu-associated kinase (HUNK) kinase is up-regulated in aggressive breast cancers and is thought to play a role in breast cancer metastasis. However, no studies have been conducted to examine a relationship between EGFR and HUNK in breast cancer metastasis. We performed a kinase substrate screen and identified that EGFR is phosphorylated by HUNK. Our studies show that HUNK phosphorylates EGFR at T654, enhancing receptor stability and downstream signaling. We found that increased phosphorylation of T654 EGFR correlates with increased epithelial to mesenchymal, migration and invasion, and metastasis. In addition, we found that HUNK expression correlates with overall survival and distant metastasis free survival. This study shows that HUNK directly phosphorylates EGFR at T654 to promote metastasis and is the first study to show that the phosphorylation of this site in EGFR regulates metastasis.

RAS is the most frequently mutated oncogene in cancer and a critical driver of oncogenesis. Therapeutic targeting of RAS has been a goal of cancer research for more than 30 years due to its essential role in tumor formation and maintenance. Yet the quest to inhibit this challenging foe has been elusive. Although once considered "undruggable", the struggle to directly inhibit RAS has seen recent success with the development of pharmacological agents that specifically target the KRAS(G12C) mutant protein, which include the first direct RAS inhibitor to gain entry to clinical trials. However, the limited applicability of these inhibitors to G12C-mutant tumors demands further efforts to identify more broadly efficacious RAS inhibitors. Understanding allosteric influences on RAS may open new avenues to inhibit RAS. Here, we provide a brief overview of RAS biology and biochemistry, discuss the allosteric regulation of RAS, and summarize the various approaches to develop RAS inhibitors.

BACKGROUND:Autophagy is a catabolic cellular recycling pathway that is essential for maintaining intracellular homeostasis. Autophagosome formation is achieved via the coordination of the Beclin-1 protein complex. Rubicon is a Beclin-1 associated protein that suppresses autophagy by impairing the activity of the class III PI3K, Vps34. However, very little is known about the molecular mechanisms that regulate Rubicon function.METHODS:In this study, co-immunoprecipitation and kinase assays were used to investigate the ability of Hormonally Upregulated Neu-associated Kinase (HUNK) to bind to and phosphorylate Rubicon. LC3B was monitored by immunofluorescence and immunoblotting to determine whether phosphorylation of Rubicon by HUNK controls the autophagy suppressive function of Rubicon.RESULTS:Findings from this study identify Rubicon as a novel substrate of HUNK and show that phosphorylation of Rubicon inhibits its function, promoting autophagy.

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 µm in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Over the past few decades, connexin hemichannels have become recognized as major players in modulating the inflammatory response. Chronic inflammation is documented to promote tumorigenesis and is a critical component of tumor progression. Furthermore, inflammation is strongly linked to angiogenesis, immunotolerance, invasiveness, metastasis, and resistance in breast cancers. In this review, the literature on the role of connexin hemichannels in inflammation is summarized, and the potential role for hemichannel-mediated inflammation in driving breast cancer progression is discussed. Lastly, the potential for connexin-based therapeutics to modulate the inflammatory component of the tumor microenvironment as an avenue for the treatment of breast cancer is also discussed.

Connexin-based therapeutics have shown the potential for therapeutic efficacy in improving wound healing. Our previous work demonstrated that the connexin43 (Cx43) mimetic peptide juxtamembrane 2 (JM2) reduced the acute inflammatory response to a submuscular implant model by inhibiting purinergic signaling. Given the prospective application in improving tissue-engineered construct tolerance that these results indicated, we sought to determine the mechanism of action for JM2 in the present study. Using confocal microscopy, a gap-FRAP cell communication assay, and an ethidium bromide uptake assay of hemichannel function we found that the peptide reduced cell surface Cx43 levels, Cx43 gap junction (GJ) size, GJ communication, and hemichannel activity. JM2 is based on the sequence of the Cx43 microtubule binding domain, and microtubules have a confirmed role in intracellular trafficking of Cx43 vesicles. Therefore, we tested the effect of JM2 on Cx43-microtubule interaction and microtubule polymerization. We found that JM2 enhanced Cx43-microtubule interaction and that microtubule polymerization was significantly enhanced. Taken together, these data suggest that JM2 inhibits trafficking of Cx43 to the cell surface by promoting irrelevant microtubule polymerization and thereby reduces the number of hemichannels in the plasma membrane available to participate in proinflammatory purinergic signaling. Importantly, this work indicates that JM2 may have therapeutic value in the treatment of proliferative diseases such as cancer. We conclude that the targeted action of JM2 on Cx43 channels may improve the tolerance of implanted tissue-engineered constructs against the innate inflammatory response.

Background: Connexins are the channel forming constituents of hemichannels that facilitate cellextracellular communication, and gap junction (GJ) intercellular channels that directly connect the cytoplasm of interacting cells. The role of connexins in the brain is an area of growing interest. The purpose of this review is to highlight the current state of research and where future research might be headed, with particular attention to the role of connexins in psychology and psychiatric pharmaceuticals. The primary connexin isoforms in the brain include connexin45 (Cx45), Cx43, Cx36, and Cx30. These connexins display differential expression in the major neuronal cell types, and have functions including synchronization of neuronal oscillations, metabolite homeostasis, and release of gliotransmitters. Importantly, these processes utilize both GJ intercellular communication, and hemichannel cell-extracellular communication pathways. At the behavioral level, connexins in the brain have been shown to be involved in memory, alcohol indulgence, motor coordination, and anxiety. Conclusions: There are many compounds that broadly inhibit connexins, and a handful of targeted, isoform-specific, channel function agonists and antagonists. However, these compounds have primarily researched in areas other than the brain. Therefore, connexins potentially provide new pharmaceutical targets for psychiatric disorders.

Connexins are a family of transmembrane proteins that are characterized by their capacity to form intercellular channels called gap junctions that directly link the cytoplasm of adjacent cells. The formation of gap junctions by connexin proteins facilitates intercellular communication between neighboring cells by allowing for the transfer of ions and small signaling molecules. Communication through gap junctions is key to cellular equilibrium, where connexins, and the gap junction intercellular communication that connexins propagate, have roles in cellular processes such as cell growth, differentiation, and tissue homeostasis. Due to their importance in maintaining cellular functions, the disruption of connexin expression and function underlies the etiology and progression of numerous pathologies, including cancer. Over the past half a century, the role of connexins and gap junction intercellular communication have been highlighted as critical areas of research in cellular malignancies, and much research effort has been geared toward understanding their dysfunction in human cancers. Although ample evidence supports the role of connexins in a variety of human cancers, detailed examination in specific cancers, such as breast cancer, is still lacking. This review highlights the most abundant gap junction connexin isoform in higher vertebrate organisms, Connexin 43, and its role in breast cancer.

Abstract Expression and functional studies have identified connexins (Cxs), a family of gap junction proteins, as potential tumor suppressors. Studies suggest that connexin 43 (Cx43) has a role in breast cancer cell proliferation, differentiation, and migration. Although pan-gap junction drugs are available, the lack of specificity of these agents increases the opportunity for off target effects. Consequently, a therapeutic agent that specifically modulates Cx43 would be beneficial and has not been tested in breast cancer. In this study, we now test a Cx43 specific agent, called α-connexin carboxyl-terminal (ACT1), in breast cancer. Our studies demonstrate that modulation of Cx43 activity in breast cancer can be effectively achieved with the agent ACT1 to sustain Cx43-mediated gap junctional activity resulting in impaired malignant progression. Furthermore, using ER+ and HER2+ breast cancer cell models, we show that ACT1 treatment enhances the activity of lapatinib and tamoxifen. Citation Format: Christina Grek, Joshua Matthew Rhett, Melissa Abt, Jaclynn Bruce, Gautam Ghatnekar, Elizabeth S. Yeh. Targeting connexin 43 with α-connexin carboxyl-terminal (ACT1) peptide in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4358. doi:10.1158/1538-7445.AM2015-4358

Background: Treatment failure is a critical issue in breast cancer and identifying useful interventions that optimize current cancer therapies remains a critical unmet need. Expression and functional studies have identified connexins (Cxs), a family of gap junction proteins, as potential tumor suppressors. Studies suggest that Cx43 has a role in breast cancer cell proliferation, differentiation, and migration. Although pan-gap junction drugs are available, the lack of specificity of these agents increases the opportunity for off target effects. Consequently, a therapeutic agent that specifically modulates Cx43 would be beneficial and has not been tested in breast cancer. In this study, we now test an agent that specifically targets Cx43, called ACT1, in breast cancer.Methods: We evaluated whether direct modulation of Cx43 using a Cx43-directed therapeutic peptide, called ACT1, enhances Cx43 gap junctional activity in breast cancer cells, impairs breast cancer cell proliferation or survival, and enhances the activity of the targeted inhibitors tamoxifen and lapatinib.Results: Our results show that therapeutic modulation of Cx43 by ACT1 maintains Cx43 at gap junction sites between cell-cell membrane borders of breast cancer cells and augments gap junction activity in functional assays. The increase in Cx43 gap junctional activity achieved by ACT1 treatment impairs proliferation or survival of breast cancer cells but ACT1 has no effect on non-transformed MCF10A cells. Furthermore, treating ER+ breast cancer cells with a combination of ACT1 and tamoxifen or HER2+ breast cancer cells with ACT1 and lapatinib augments the activity of these targeted inhibitors.Conclusions: Based on our findings, we conclude that modulation of Cx43 activity in breast cancer can be effectively achieved with the agent ACT1 to sustain Cx43-mediated gap junctional activity resulting in impaired malignant progression and enhanced activity of lapatinib and tamoxifen, implicating ACT1 as part of a combination regimen in breast cancer.

Background: In the last 50 years, the use of medical implants has increased dramatically. Failure of implanted devices and biomaterials is a significant source of morbidity and increasing healthcare expenditures. An important cause of implant failure is the host inflammatory response. Recent evidence implicates extracellular ATP as an important inflammatory signaling molecule. A major pathway for release of cytoplasmic ATP into the extracellular space is through connexin hemichannels, which are the unpaired constituents of gap junction intercellular channels. Blockade of hemichannels of the connexin 43 (Cx43) isoform has been shown to reduce inflammation and improve healing. We have developed a Cx43 mimetic peptide (JM2) that targets the microtubule-binding domain of Cx43. The following report investigates the role of the Cx43 microtubule-binding domain in extracellular ATP release by Cx43 hemichannels and how this impacts early inflammatory events of the foreign body reaction.Methods: In vitro Cx43 hemichannel-mediated ATP release by cultured human microvascular endothelial cells subjected to hypocalcemic and normocalcemic conditions was measured after application of JM2 and the known hemichannel blocker, flufenamic acid. A submuscular silicone implant model was used to investigate in vivo ATP signaling during the early foreign body response. Implants were coated with control pluronic vehicle or pluronic carrying JM2, ATP, JM2+ATP, or known hemichannel blockers and harvested at 24 h for analysis.Results: JM2 significantly inhibited connexin hemichannel-mediated ATP release from cultured endothelial cells. Importantly, the early inflammatory response to submuscular silicone implants was inhibited by JM2. The reduction in inflammation by JM2 was reversed by the addition of exogenous ATP to the pluronic vehicle.Conclusions: These data indicate that ATP released through Cx43 hemichannels into the vasculature is an important signal driving the early inflammatory response to implanted devices. A vital aspect of this work is that it demonstrates that targeted molecular therapeutics, such as JM2, provide the capacity to regulate inflammation in a clinically relevant system.

Total pancreatectomy and islet autotransplantation is a cutting-edge technique to treat chronic pancreatitis and postoperative diabetes. A major obstacle has been low islet cell survival due largely to the innate inflammatory response. Connexin43 (Cx43) channels play a key role in early inflammation and have proven to be viable therapeutic targets. Even if cell death due to early inflammation is avoided, insufficient vascularization is a primary obstacle to maintaining the viability of implanted cells. We have invented technologies targeting the inflammatory response and poor vascularization: a Cx43 mimetic peptide that inhibits inflammation and a novel prevascularized tissue engineered construct. We combined these technologies with isolated islets to create a prevascularized bioartificial pancreas that is resistant to the innate inflammatory response. Immunoconfocal microscopy showed that constructs containing islets express insulin and possess a vascular network similar to constructs without islets. Glucose stimulated islet-containing constructs displayed reduced insulin secretion compared to islets alone. However, labeling for insulin post-glucose stimulation revealed that the constructs expressed abundant levels of insulin. This discrepancy was found to be due to the expression of insulin degrading enzyme. These results suggest that the prevascularized bioartificial pancreas is potentially a tool for improving long-term islet cell survival in vivo.

Purinergic signaling is a ubiquitous and vital aspect of mammalian biology in which purines--mainly adenosine triphosphate (ATP)--are released from cells through loss of membrane integrity (cell death), exocytosis, or transport/diffusion across membrane channels, and exert paracrine or autocrine signaling effects through three subclasses of well-characterized receptors: the P1 adenosine receptors, the P2X ionotropic nucleotide receptors, and the P2Y metabotropic receptors. ATP and its metabolites are released by damaged and stressed cells in injured tissues. The early events of wound healing, hemostasis, and inflammation are highly regulated by these signals through activation of purinergic receptors on platelets and neutrophils. Recent data have demonstrated that ATP signaling is of particular importance to targeting leukocytes to sites of injury. This is particularly relevant to the subject of implanted medical devices, engineered tissues, and grafts as all these technologies elicit a wound healing response with varying degrees of encapsulation, rejection, extrusion, or destruction of the tissue or device. Here, we review the biology of purinergic signaling and focus on ATP release and response mechanisms that pertain to the early inflammatory phase of wound healing. Finally, therapeutic options are explored, including a new class of peptidomimetic drugs based on the ATP-conductive channel connexin43.