Background: Most tumors initially respond to treatment, yet refractory clones subsequently develop owing to resistance mechanisms associated with cancer cell plasticity and heterogeneity. Methods: We used a chemical biology approach to identify protein targets in cancer cells exhibiting diverse driver mutations and representing models of tumor lineage plasticity and therapy resistance. An unbiased screen of a drug library was performed against cancer cells followed by synthesis of chemical analogs of the most effective drug. The cancer subtype target range of the leading drug was determined by PRISM analysis of over 900 cancer cell lines at the Broad Institute, MA. RNA-sequencing and enrichment analysis of differentially expressed genes, as well as computational molecular modeling and pull-down with biotinylated small molecules were used to identify and validate RPS6KB1 (p70S6K or S6K1) as an essential target. Genetic restoration was used to test the functional role of S6K1 in cell culture and xenograft models. Results: We identified a novel derivative of the antihistamine drug ebastine, designated Super-ebastine (Super-EBS), that inhibited the viability of cancer cells representing diverse KRAS and EGFR driver mutations and models of plasticity and treatment resistance. Interestingly, PRISM analysis indicated that over 95% of the diverse cancer cell lines tested were sensitive to Super-EBS and the predicted target was the serine/threonine kinase S6K1. S6K1 is upregulated in various cancers relative to counterpart normal/benign tissues and phosphorylated-S6K1 predicts poor prognosis for cancer patients. We noted that inhibition of S6K1 phosphorylation was necessary for tumor cell growth inhibition, and restoration of phospho-S6K1 rendered tumor cells resistant to Super-EBS. Inhibition of S6K1 phosphorylation by Super-EBS induced caspase-2 dependent apoptosis via inhibition of the Cdc42/Rac-1/p-PAK1 pathway that led to actin depolymerization and caspase-2 activation. The essential role of S6K1 in the action of Super-EBS was recapitulated in xenografts, and knockout of S6K1 abrogated tumor growth in mice. Conclusion: S6K1 is a therapeutic vulnerability in tumors exhibiting intrinsic and/or acquired resistance to treatment.

Phosphatase of Regenerating Liver 3 (PRL-3) is frequently upregulated in cancer and associated with poor prognosis, yet its oncogenic mechanism remains unresolved. Although traditionally studied for its phosphatase activity, PRL-3 also engages in protein-protein interactions via its catalytic site, notably binding the CNNM magnesium transporters, rendering these functions mutually exclusive. The commonly used C104S mutant disrupts both activities, confounding interpretations of prior studies. To dissect PRL-3's distinct functions, we employed mutants selectively deficient in phosphatase activity (C104D) or CNNM binding (R138E). In zebrafish models of acute lymphoblastic leukemia (ALL) and rhabdomyosarcoma (RMS), as well as in human cancer cell lines, both wild-type PRL-3 and C104D enhanced tumor initiation, growth, and dissemination, while R138E had no effect. These findings indicate that PRL-3 promotes cancer via non-catalytic mechanisms. To support therapeutic development, we established an in vitro FRET-based assay to screen for inhibitors of the PRL-3:CNNM interaction and validated a cyclic peptide as a positive control. By demonstrating that PRL-3 enhances cancer progression independently of its catalytic activity, this study shifts focus toward targeting its binding functions as a therapeutic strategy.

Abstract Wilms tumor is the most common solid renal malignancy in children. To better understand the biology of Wilms tumors and develop drugs especially against tumors that relapse, we sought to identify mutations in the exome, differential gene expression patterns, and tumor suppressor expression status of Wilms tumors in Kentucky. The Kentucky Cancer Registry (KCR) Virtual Tissue Repository identified 29 cases of Wilms tumor diagnosed between 2015 and 2021 at hospitals across the Commonwealth of Kentucky. The Markey Cancer Center Biospecimen Procurement and Translational Pathology Facility prepared tumor microarrays (TMAs) using Wilms tumor and matching normal tissue cores for immunohistochemistry (IHC) analysis. RNA-Seq analysis performed at the Markey Cancer Center Oncogenomics Facility identified a gene signature that was further validated by IHC and reverse-transcription quantitative-PCR (RT-qPCR). Outcome data provided by KCR indicated that increased expression of an anti-apoptotic protein in the tumor counter-intuitively provided better prognosis and outcome to treatment, indicating that the treatments either directly or indirectly targeted this protein. Loss of function of the tumor suppressor proteins in Par-4/PAWR, WT1 and p53 did not explain the higher expression of the cell survival protein. Our multidisciplinary team approach involves basic science/translational researchers, population scientists, biomedical informaticists, and clinicians and has resulted in well-characterized, Wilms tumor tissue microarrays. The next phase of the study is generating patient derived xenografts (PDXs) for a better understanding of Wilms tumor biology in children. Citation Format: Jieyun Jiang, Derek B. Allison, Ravshan Burikhanov, Saptadwipa Ganguly, Ryan A. Goettl, Jinpeng Liu, Chi Wang, Amanda F. Saltzman, Thomas Tucker, Peter H. Spielmann, Eric B. Durbin, John A. D’Orazio, Vivek M. Rangnekar. Molecular signature for treatment-responsive and recurrent Wilms tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1103.

Cancer cells undergo a significant level of “metabolic reprogramming” or “remodeling” to ensure an adequate supply of ATP and “building blocks” for cell survival and to facilitate accelerated proliferation. Cancer cells preferentially use glycolysis for ATP production (the Warburg effect); however, cancer cells, including colorectal cancer (CRC) cells, also depend on oxidative phosphorylation (OXPHOS) for ATP production, a finding that suggests that both glycolysis and OXPHOS play significant roles in facilitating cancer progression and proliferation. Our prior studies identified a semisynthetic isoflavonoid, DBI-1, that served as an AMPK activator targeting mitochondrial complex I. Furthermore, DBI-1 and a glucose transporter 1 (GLUT1) inhibitor, BAY-876, synergistically inhibited CRC cell growth in vitro and in vivo. We now report a study of the structure–activity relationships (SARs) in the isoflavonoid family in which we identified a new DBI-1 analog, namely, DBI-2, with promising properties. Here, we aimed to explore the antitumor mechanisms of DBIs and to develop new combination strategies by targeting both glycolysis and OXPHOS. We identified DBI-2 as a novel AMPK activator using an AMPK phosphorylation assay as a readout. DBI-2 inhibited mitochondrial complex I in the Seahorse assays. We performed proliferation and Western blotting assays and conducted studies of apoptosis, necrosis, and autophagy to corroborate the synergistic effects of DBI-2 and BAY-876 on CRC cells in vitro. We hypothesized that restricting the carbohydrate uptake with a KD would mimic the effects of GLUT1 inhibitors, and we found that a ketogenic diet significantly enhanced the therapeutic efficacy of DBI-2 in CRC xenograft mouse models, an outcome that suggested a potentially new approach for combination cancer therapy.

Lung cancer is the leading cause of cancer-related deaths. Lung cancer cells develop resistance to apoptosis by suppressing the secretion of the tumor suppressor Par-4 protein (also known as PAWR) and/or down-modulating the Par-4 receptor GRP78 on the cell surface (csGRP78). We sought to identify FDA-approved drugs that elevate csGRP78 on the surface of lung cancer cells and induce Par-4 secretion from the cancer cells and/or normal cells in order to inhibit cancer growth in an autocrine or paracrine manner. In an unbiased screen, we identified crizotinib (CZT), an inhibitor of activated ALK/MET/ROS1 receptor tyrosine kinase, as an inducer of csGRP78 expression in ALK-negative, KRAS or EGFR mutant lung cancer cells. Elevation of csGRP78 in the lung cancer cells was dependent on activation of the non-receptor tyrosine kinase SRC by CZT. Inhibition of SRC activation in the cancer cells prevented csGRP78 translocation but promoted Par-4 secretion by CZT, implying that activated SRC prevented Par-4 secretion. In normal cells, CZT did not activate SRC and csGRP78 elevation but induced Par-4 secretion. Consequently, CZT induced Par-4 secretion from normal cells and elevated csGRP78 in the ALK-negative tumor cells to cause paracrine apoptosis in cancer cell cultures and growth inhibition of tumor xenografts in mice. Thus, CZT induces differential activation of SRC in normal and cancer cells to trigger the pro-apoptotic Par-4-GRP78 axis. As csGRP78 is a targetable receptor, CZT can be repurposed to elevate csGRP78 for inhibition of ALK-negative lung tumors.

Supplementary Data from Diaminobutoxy-substituted Isoflavonoid (DBI-1) Enhances the Therapeutic Efficacy of GLUT1 Inhibitor BAY-876 by Modulating Metabolic Pathways in Colon Cancer Cells

Abstract Cancer cells undergo significant “metabolic remodeling” to provide sufficient ATP to maintain cell survival and to promote rapid growth. In colorectal cancer cells, ATP is produced by mitochondrial oxidative phosphorylation and by substantially elevated cytoplasmic glucose fermentation (i.e., the Warburg effect). Glucose transporter 1 (GLUT1) expression is significantly increased in colorectal cancer cells, and GLUT1 inhibitors block glucose uptake and hence glycolysis crucial for cancer cell growth. In addition to ATP, these metabolic pathways also provide macromolecule building blocks and signaling molecules required for tumor growth. In this study, we identify a diaminobutoxy-substituted isoflavonoid (DBI-1) that inhibits mitochondrial complex I and deprives rapidly growing cancer cells of energy needed for growth. DBI-1 and the GLUT1 inhibitor, BAY-876, synergistically inhibit colorectal cancer cell growth in vitro and in vivo. This study suggests that an electron transport chain inhibitor (i.e., DBI-1) and a glucose transport inhibitor, (i.e., BAY-876) are potentially effective combination for colorectal cancer treatment.

Beta-amyloid (Aβ) deposition in brain accumulates as a function of age in people with Down syndrome (DS) with subsequent development into Alzheimer disease neuropathology, typically by 40 years of age. In vivo imaging using the Pittsburgh compound B (PiB) ligand has facilitated studies linking Aβ, cognition, and dementia in DS. However, there are no studies of PiB binding across the lifespan in DS. The current study describes in vitro 3H-PiB binding in the frontal cortex of autopsy cases with DS compared to non-DS controls. Tissue from 64 cases included controls (n = 25) and DS (n = 39). In DS, 3H-PiB binding was significantly associated with age. After age 40 years in DS, 3H-PiB binding rose dramatically along with increasing individual variability. 3H-PiB binding correlated with the amount of Aβ42. Using fixed frontal tissue and fluorescent 6-CN-PiB, neuritic and cored plaques along with extensive cerebral amyloid angiopathy showed 6-CN-PiB binding. These results suggest that cortical PiB binding as shown by positron emission tomography imaging reflects plaques and cerebral amyloid angiopathy in DS brain.

This study highlights the biochemical and structural characterization of the L-tryptophan C6 C-prenyltransferase (C-PT) PriB from Streptomyces sp. RM-5-8. PriB was found to be uniquely permissive to a diverse array of prenyl donors and acceptors including daptomycin. Two additional PTs also produced novel prenylated daptomycins with improved antibacterial activities over the parent drug.

BACKGROUND:Dolichyl phosphate-linked mono- and oligosaccharides (DLO) are essential intermediates in protein N-glycosylation, C- and O-mannosylation and GPI anchor biosynthesis. While many membrane proteins in the endoplasmic reticulum (ER) involved in the assembly of DLOs are known, essential proteins believed to be required for the transbilayer movement (flip-flopping) and proteins potentially involved in the regulation of DLO synthesis remain to be identified. METHODS:The synthesis of a series of Dol-P derivatives composed of citronellyl-based photoprobes with benzophenone groups equipped with alkyne moieties for Huisgen "click" chemistry is now described to utilize as tools for identifying ER proteins involved in regulating the biosynthesis and transbilayer movement of lipid intermediates. In vitro enzymatic assays were used to establish that the photoprobes contain the critical structural features recognized by pertinent enzymes in the dolichol pathway. ER proteins that photoreacted with the novel probes were identified by MS. RESULTS:The potential of the newly designed photoprobes, m-PAL-Cit-P and p-PAL-Cit-P, for identifying previously unidentified Dol-P-interacting proteins is supported by the observation that they are enzymatically mannosylated by Man-P-Dol synthase (MPDS) from Chinese Hamster Ovary (CHO) cells at an enzymatic rate similar to that for Dol-P. MS analyses reveal that DPM1, ALG14 and several other yeast ER proteins involved in DLO biosynthesis and lipid-mediated protein O-mannosylation photoreacted with the novel probes. CONCLUSION:The newly-designed photoprobes described in this paper provide promising new tools for the identification of yet to be identified Dol-P interacting ER proteins in yeast and mammalian cells, including the Dol-P flippase required for the "re-cycling" of the glycosyl carrier lipid from the lumenal monolayer of the ER to the cytoplasmic leaflet for new rounds of DLO synthesis.

As part of an effort to identify substrate analogs suitable for helping to resolve structural features important for terpene synthases, the inhibition of 5-epi-aristolochene biosynthesis from farnesyl diphosphate (FPP) by the tobacco 5-epi-aristolochene synthase incubated with anilinogeranyl diphosphate (AGPP) was examined. The apparent noncompetitive nature of the inhibition supported further assessment of how AGPP might be bound to crystallographic forms of the enzyme. Surprisingly, the bound form of the inhibitor appeared to have undergone a cyclization event consistent with the native mechanism associated with FPP catalysis. Biocatalytic formation of a novel 13-membered macrocyclic paracyclophane alkaloid was confirmed by high-resolution GC-MS and NMR analysis. This work provides insights into new biosynthetic means for generating novel, functionally diversified, medium-sized terpene alkaloids.

The clinical interest in farnesyltransferase inhibitors (FTIs) makes it important to understand how these compounds affect cellular processes involving farnesylated proteins. Mitotic abnormalities observed after treatment with FTIs have so far been attributed to defects in the farnesylation of the outer kinetochore proteins CENP-E and CENP-F, which are involved in chromosome congression and spindle assembly checkpoint signaling. Here we identify the cytoplasmic dynein adaptor Spindly as an additional component of the outer kinetochore that is modified by farnesyltransferase (FTase). We show that farnesylation of Spindly is essential for its localization, and thus for the proper localization of dynein and its cofactor dynactin, to prometaphase kinetochores and that Spindly kinetochore recruitment is more severely affected by FTase inhibition than kinetochore recruitment of CENP-E and CENP-F. Molecular replacement experiments show that both Spindly and CENP-E farnesylation are required for efficient chromosome congression. The identification of Spindly as a new mitotic substrate of FTase provides insight into the causes of the mitotic phenotypes observed with FTase inhibitors.

Dysregulation of Ras and Rho family small GTPases drives the invasion and metastasis of multiple cancers. For their biological functions, these GTPases require proper subcellular localization to cellular membranes, which is regulated by a series of post-translational modifications that result in either farnesylation or geranylgeranylation of the C-terminal CAAX motif. This concept provided the rationale for targeting farnesyltransferase (FTase) and geranylgeranyltransferases (GGTase) for cancer treatment. However, the resulting prenyl transferase inhibitors have not performed well in the clinic due to issues with alternative prenylation and toxicity. As an alternative, we have developed a unique class of potential anti-cancer therapeutics called Prenyl Function Inhibitors (PFIs), which are farnesol or geranyl-geraniol analogs that act as alternate substrates for FTase or GGTase. Here, we test the ability of our lead PFIs, anilinogeraniol (AGOH) and anilinofarnesol (AFOH), to block the invasion of breast cancer cells. We found that AGOH treatment effectively decreased invasion of MDA-MB-231 cells in a two-dimensional (2D) invasion assay at 100 µM while it blocked invasive growth in three-dimensional (3D) culture model at as little as 20 µM. Notably, the effect of AGOH on 3D invasive growth was phenocopied by electroporation of cells with C3 exotransferase. To determine if RhoA and RhoC were direct targets of AGOH, we performed Rho activity assays in MDA-MB-231 and MDA-MB-468 cells and found that AGOH blocked RhoA and RhoC activation in response to LPA and EGF stimulation. Notably, the geranylgeraniol analog AFOH was more potent than AGOH in inhibiting RhoA and RhoC activation and invasive growth. Interestingly, neither AGOH nor AFOH impacted 3D growth of MCF10A cells. Collectively, this study demonstrates that AGOH and AFOH dramatically inhibit breast cancer invasion, at least in part by blocking Rho function, thus, suggesting that targeting prenylation by using PFIs may offer a promising mechanism for treatment of invasive breast cancer.

An efficient, diversity oriented synthesis of homoisoprenoid α-monofluorophosphonates utilizing electrophilic fluorination is presented along with their activity as inhibitors of PPAPDC2 family integral membrane lipid phosphatases. These novel phosphatase-resistant analogues of isoprenoid monophosphates are a platform for further structure-activity relationship studies and provide access to other isoprenoid family members where the phosphate ester oxygen is replaced by a α-monofluoromethylene moiety.

Mutations in Ras isoforms such as K-Ras, N-Ras, and H-Ras contribute to roughly 85, 15, and 1 % of human cancers, respectively. Proper membrane targeting of these Ras isoforms, a prerequisite for Ras activity, requires farnesylation or geranylgeranylation at the C-terminal CAAX box. We devised an in vivo screening strategy based on monitoring Ras activation and phenotypic physiological outputs for assaying synthetic Ras function inhibitors (RFI). Ras activity was visualized by the translocation of RBD Raf1 -GFP to activated Ras at the plasma membrane. By using this strategy, we screened one synthetic farnesyl substrate analog (AGOH) along with nine putative inhibitors and found that only m-CN-AGOH inhibited Ras activation. Phenotypic analysis of starving cells could be used to monitor polarization, motility, and the inability of these treated cells to aggregate properly during fruiting body formation. Incorporation of AGOH and m-CN-AGOH to cellular proteins was detected by western blot. These screening assays can be incorporated into a high throughput screening format using Dictyostelium discoideum and automated microscopy to determine effective RFIs. These RFI candidates can then be further tested in mammalian systems.

Mammalian cells can use exogenous isoprenols to generate isoprenoid diphosphate substrates for protein isoprenylation, but the mechanism, efficiency, and biological importance of this process are not known. We developed mass spectrometry-based methods using chemical probes and newly synthesized stable isotope-labeled tracers to quantitate incorporation of exogenously provided farnesol, geranylgeraniol, and unnatural analogs of these isoprenols containing an aniline group into isoprenoid diphosphates and protein isoprenylcysteines by cultured human cancer cell lines. We found that at exogenous isoprenol concentrations > 10 mu M, this process can generate as much as 50% of the cellular isoprenoid diphosphate pool used for protein isoprenylation. Mutational activation of p53 in MDA-MB-231 breast cancer cells up-regulates the mevalonate pathway to promote tumor invasiveness. p53 silencing or pharmacological inhibition of HMG-CoA reductase in these cells decreases protein isoprenylation from endogenously synthesized isoprenoids but enhances the use of exogenous isoprenols for this purpose, indicating that this latter process is regulated independently of the mevalonate pathway. Our observations suggest unique opportunities for design of cancer cell-directed therapies and may provide insights into mechanisms underlying pleiotropic therapeutic benefits and unwanted side effects of mevalonate pathway inhibition.

Abstract Tumor metastasis causes the majority of cancer deaths. Dysregulation of small GTPases such as K-Ras and Rho family GTPases (RhoA, RhoC, Rac1 and Cdc42) is critical to drive the invasion and metastasis of a variety of cancers including breast carcinomas. In order to fulfill their biological functions, these GTPases must properly localize subcellularly and associate with cellular membrane. The association of these proteins to membranes is regulated by a series of post-translational modifications including either farnesylation or geranylgeranylation of the C-terminal CaaX motif, thus providing the rationale to target the farnesyltransferase (FTase) and geranylgeranyltransferases (GGTase) as the potential anti-cancer drugs. However, FTase inhibitors (FTIs) failed in clinic due to alternative prenylation, which causes K-Ras to be geranylgeranylated, and their inability to target geranylgeranylated proteins such as Rho family GTPases. As an alternative, we have developed a unique class of potential anti-cancer therapeutics called Prenyl Function Inhibitors (PFIs), which are synthetic farnesol or geranylgeraniol analogs that act as alternative substrates for FTase or GGTase but do not support normal prenyl function. Here, we test the ability of our lead PFIs anilinogeraniol (AGOH; farnesol analog) and anilinofarnesol (AFOH; geranylgeraniol analog) to impact the migratory and invasive capacities of breast carcinoma cells. We found that AGOH treatment effectively decreased the invasion of MDA-MBA-231 in a two-dimensional invasion assay at 100 mM while it blocked invasive growth in three-dimensional (3D) culture model in as little as 20 μM. Notably, the effect of AGOH on 3D invasive growth was phenocopied by electroporation of cells with C3 exotransferase, which ribosylates and inactivates Rho proteins. The results demonstrate that Rho proteins are essential for the invasive growth of MDA-MB-231 cells. To determine how AGOH impacts Rho signaling, we performed RhoA and RhoC activity assays in MDA-MB-231 and MDA-MB-468 cells. We determined that AGOH blocked RhoA and RhoC activation in response to LPA and EGF stimulation. Notably, the geranylgeraniol analog AFOH was more potent than AGOH in inhibiting RhoA/C activation and invasive growth where it inhibited RhoA and RhoC activation completely at 20 μM and blocked the invasive growth of MDA-MB-231 cells at ≤ 5 μM. Interestingly, both AGOH and AFOH had no effect on the growth of MCF10A cells in 3D culture. Collectively, this study demonstrates that our lead PFI compounds dramatically inhibited breast cancer invasion, at least in part, through affecting RhoA and RhoC function; thus, suggesting that targeting the function of Rho prenylation by PFIs will offer a promising mechanism for the treatment of breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-11.

Cancer stem cells and mutant p53 expressing tumors are highly dependent on the mevalonate pathway. Statins inhibit this pathway resulting in growth inhibition of these cells independent of inhibition of cholesterol biosynthesis and of protein farnesylation. Protein prenylation substrates farnesyl‐ and geranylgeranyl diphosphates are synthesized by the mevalonate pathway without producing their corresponding isoprenols. However, cells incorporate exogenously supplied isoprenols into mevalonate pathway metabolites and proteins using a poorly characterized process. In order to quantify this alternative to the mevalonate pathway, we synthesized natural and unnatural analogs of farnesol and geranylgeraniol, including stable isotope‐labeled farnesol and aniline substituted isoprenols. Using tandem mass spectrometry we quantitatively monitored the formation of diphosphate derivatives of these isoprenols and their utilization as prenylation substrates when incubated with cultured cells. Our results reveal that in some cells the capacity to use exogenous isoprenols is comparable to that of the mevalonate pathway raising the possibility that this alternative pathway could have important therapeutic implications

A Wittig reaction employing Li(CD3)2CP(C6H5)3 was used to prepare d6-farnesol and d6-geranylgeraniol. Reductive amination of aniline-2,3,4,5,6-d5 was used to prepare the unnatural isoprenoid analogues d5-anilinogeraniol and d5-anilinofarnesol. All of these deuterated isoprenols were elaborated into their diphosphate and cysteine thioether derivatives suitable for use as stable-isotope labeled standards for quantitative mass spectrometric analysis.