Epichaperome inhibition targets TP53-mutant AML and AML stem/progenitor cells.

TP 53-mutant acute myeloid leukemia (AML) remains the ultimate therapeutic challenge. Epichaperomes, formed in malignant cells, consist of heat shock protein 90 (HSP90) and associated proteins that support the maturation, activity, and stability of oncogenic kinases and transcription factors including mutant p53. High-throughput drug screening identified HSP90 inhibitors as top hits in isogenic TP53-wild-type (WT) and -mutant AML cells. We detected epichaperomes in AML cells and stem/progenitor cells with TP53 mutations but not in healthy bone marrow (BM) cells. Hence, we investigated the therapeutic potential of specifically targeting epichaperomes with PU-H71 in TP53-mutant AML based on its preferred binding to HSP90 within epichaperomes. PU-H71 effectively suppressed cell intrinsic stress responses and killed AML cells, primarily by inducing apoptosis; targeted TP53-mutant stem/progenitor cells; and prolonged survival of TP53-mutant AML xenograft and patient-derived xenograft models, but it had minimal effects on healthy human BM CD34+ cells or on murine hematopoiesis. PU-H71 decreased MCL-1 and multiple signal proteins, increased proapoptotic Bcl-2-like protein 11 levels, and synergized with BCL-2 inhibitor venetoclax in TP53-mutant AML. Notably, PU-H71 effectively killed TP53-WT and -mutant cells in isogenic TP53-WT/TP53-R248W Molm13 cell mixtures, whereas MDM2 or BCL-2 inhibition only reduced TP53-WT but favored the outgrowth of TP53-mutant cells. Venetoclax enhanced the killing of both TP53-WT and -mutant cells by PU-H71 in a xenograft model. Our data suggest that epichaperome function is essential for TP53-mutant AML growth and survival and that its inhibition targets mutant AML and stem/progenitor cells, enhances venetoclax activity, and prevents the outgrowth of venetoclax-resistant TP53-mutant AML clones. These concepts warrant clinical evaluation.

EphA2- and HDAC-Targeted Combination Therapy in Endometrial Cancer.

Endometrial cancer is the most frequent malignant tumor of the female reproductive tract but lacks effective therapy. EphA2, a receptor tyrosine kinase, is overexpressed by various cancers including endometrial cancer and is associated with poor clinical outcomes. In preclinical models, EphA2-targeted drugs had modest efficacy. To discover potential synergistic partners for EphA2-targeted drugs, we performed a high-throughput drug screen and identified panobinostat, a histone deacetylase inhibitor, as a candidate. We hypothesized that combination therapy with an EphA2 inhibitor and panobinostat leads to synergistic cell death. Indeed, we found that the combination enhanced DNA damage, increased apoptosis, and decreased clonogenic survival in Ishikawa and Hec1A endometrial cancer cells and significantly reduced tumor burden in mouse models of endometrial carcinoma. Upon RNA sequencing, the combination was associated with downregulation of cell survival pathways, including senescence, cyclins, and cell cycle regulators. The Axl-PI3K-Akt-mTOR pathway was also decreased by combination therapy. Together, our results highlight EphA2 and histone deacetylase as promising therapeutic targets for endometrial cancer.

Combination of EphA2- and Wee1-Targeted Therapies in Endometrial Cancer.

EphA2 tyrosine kinase is upregulated in many cancers and correlated with poor survival of patients, including those with endometrial cancer. EphA2-targeted drugs have shown modest clinical benefit. To improve the therapeutic response to such drugs, we performed a high-throughput chemical screen to discover novel synergistic partners for EphA2-targeted therapeutics. Our screen identified the Wee1 kinase inhibitor, MK1775, as a synergistic partner to EphA2, and this finding was confirmed using both in vitro and in vivo experiments. We hypothesized that Wee1 inhibition would sensitize cells to EphA2-targeted therapy. Combination treatment decreased cell viability, induced apoptosis, and reduced clonogenic potential in endometrial cancer cell lines. In vivo Hec1A and Ishikawa-Luc orthotopic mouse models of endometrial cancer showed greater anti-tumor responses to combination treatment than to either monotherapy. RNASeq analysis highlighted reduced cell proliferation and defective DNA damage response pathways as potential mediators of the combination's effects. In conclusion, our preclinical findings indicate that Wee1 inhibition can enhance the response to EphA2-targeted therapeutics in endometrial cancer; this strategy thus warrants further development.

Sequential Administration of XPO1 and ATR Inhibitors Enhances Therapeutic Response in TP53-mutated Colorectal Cancer.

BACKGROUND & AIMS: Understanding the mechanisms by which tumors adapt to therapy is critical for developing effective combination therapeutic approaches to improve clinical outcomes for patients with cancer. METHODS: To identify promising and clinically actionable targets for managing colorectal cancer (CRC), we conducted a patient-centered functional genomics platform that includes approximately 200 genes and paired this with a high-throughput drug screen that includes 262 compounds in four patient-derived xenografts (PDXs) from patients with CRC. RESULTS: Both screening methods identified exportin 1 (XPO1) inhibitors as drivers of DNA damage-induced lethality in CRC. Molecular characterization of the cellular response to XPO1 inhibition uncovered an adaptive mechanism that limited the duration of response in TP53-mutated, but not in TP53-wild-type CRC models. Comprehensive proteomic and transcriptomic characterization revealed that the ATM/ATR-CHK1/2 axes were selectively engaged in TP53-mutant CRC cells upon XPO1 inhibitor treatment and that this response was required for adapting to therapy and escaping cell death. Administration of KPT-8602, an XPO1 inhibitor, followed by AZD-6738, an ATR inhibitor, resulted in dramatic antitumor effects and prolonged survival in TP53-mutant models of CRC. CONCLUSIONS: Our findings anticipate tremendous therapeutic benefit and support the further evaluation of XPO1 inhibitors, especially in combination with DNA damage checkpoint inhibitors, to elicit an enduring clinical response in patients with CRC harboring TP53 mutations.

GSK3ß regulates epithelial-mesenchymal transition and cancer stem cell properties in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancers (TNBCs), which lack receptors for estrogen, progesterone, and amplification of epidermal growth factor receptor 2, are highly aggressive. Consequently, patients diagnosed with TNBCs have reduced overall and disease-free survival rates compared to patients with other subtypes of breast cancer. TNBCs are characterized by the presence of cancer cells with mesenchymal properties, indicating that the epithelial to mesenchymal transition (EMT) plays a major role in the progression of this disease. The EMT program has also been implicated in chemoresistance, tumor recurrence, and induction of cancer stem cell (CSC) properties. Currently, there are no targeted therapies for TNBC, and hence, it is critical to identify the novel targets to treat TNBC. METHODS: A library of compounds was screened for their ability to inhibit EMT in cells with mesenchymal phenotype as assessed using the previously described Z-cad reporters. Of the several drugs tested, GSK3ß inhibitors were identified as EMT inhibitors. The effects of GSK3ß inhibitors on the properties of TNBC cells with a mesenchymal phenotype were assessed using qRT-PCR, flow cytometry, western blot, mammosphere, and migration and cell viability assays. Publicly available datasets also were analyzed to examine if the expression of GSK3ß correlates with the overall survival of breast cancer patients. RESULTS: We identified a GSK3ß inhibitor, BIO, in a drug screen as one of the most potent inhibitors of EMT. BIO and two other GSK3ß inhibitors, TWS119 and LiCl, also decreased the expression of mesenchymal markers in several different cell lines with a mesenchymal phenotype. Further, inhibition of GSK3ß reduced EMT-related migratory properties of cells with mesenchymal properties. To determine if GSK3ß inhibitors target mesenchymal-like cells by affecting the CSC population, we employed mammosphere assays and profiled the stem cell-related cell surface marker CD44+/24- in cells after exposure to GSK3ß inhibitors. We found that GSK3ß inhibitors indeed decreased the CSC properties of cell types with mesenchymal properties. We treated cells with epithelial and mesenchymal properties with GSK3ß inhibitors and found that GSK3ß inhibitors selectively kill cells with mesenchymal attributes while sparing cells with epithelial properties. We analyzed patient data to identify genes predictive of poor clinical outcome that could serve as novel therapeutic targets for TNBC. The Wnt signaling pathway is critical to EMT, but among the various factors known to be involved in Wnt signaling, only the higher expression of GSK3ß correlated with poorer overall patient survival. CONCLUSIONS: Taken together, our data demonstrate that GSK3ß is a potential target for TNBCs and suggest that GSK3ß inhibitors could serve as selective inhibitors of EMT and CSC properties for the treatment of a subset of aggressive TNBC. GSK3ß inhibitors should be tested for use in combination with standard-of-care drugs in preclinical TNBC models.

Interpreting the biological effects of protons as a function of physical quantity: linear energy transfer or microdosimetric lineal energy spectrum?

The choice of appropriate physical quantities to characterize the biological effects of ionizing radiation has evolved over time coupled with advances in scientific understanding. The basic hypothesis in radiation dosimetry is that the energy deposited by ionizing radiation initiates all the consequences of exposure in a biological sample (e.g., DNA damage, reproductive cell death). Physical quantities defined to characterize energy deposition have included dose, a measure of the mean energy imparted per unit mass of the target, and linear energy transfer (LET), a measure of the mean energy deposition per unit distance that charged particles traverse in a medium. The primary advantage of using the "dose and LET" physical system is its relative simplicity, especially for presenting and recording results. Inclusion of additional information such as the energy spectrum of charged particles renders this approach adequate to describe the biological effects of large dose levels from homogeneous sources. The primary disadvantage of this system is that it does not provide a unique description of the stochastic nature of radiation interactions. We and others have used dose-averaged LET (LETd) as a correlative physical quantity to the relative biological effectiveness (RBE) of proton beams. This approach is based on established experimental findings that proton RBE increases with LETd. However, this approach might not be applicable to intensity-modulated proton therapy or other applications in which the proton energy spectrum is highly heterogeneous. In the current study, we irradiated cancer cells with scanning proton beams with identical LETd (3.4 keV/µm) but arising from two different proton energy/LET spectra (a narrow spectrum in group 1 and a widespread heterogeneous spectrum in group 2). Clonogenic survival after irradiation revealed significant differences in RBE at any cell surviving fraction: e.g., at a surviving fraction of 0.1, the RBE was 0.97 ± 0.03 in group 1 and 1.16 ± 0.04 in group 2 (p≤0.01), validating our hypothesis that LETd alone may not adequately indicate proton RBE. Further analysis showed that microdosimetric spectrum (the probability density function of the stochastic physical quantity lineal energy y) was helpful for interpreting observed differences in biological effects. However, more accurate use of microdosimetric spectrum to quantify RBE requires a cell line-specific mechanistic model.

Combining MEK and SRC inhibitors for treatment of colorectal cancer demonstrate increased efficacy in vitro but not in vivo.

Metastatic colorectal cancer (mCRC) is the second leading cause of cancer deaths in the United States. More than 50% of patients with mCRC harbor mutations of the oncogenic driver RAS (KRAS or NRAS). Because directly targeting most mutations of RAS is technically challenging, researchers have concentrated on targeting MEK, a downstream mediator of RAS. However, targeting MEK as single-agent therapy is ineffective in patients with mCRC. We hypothesize that combining a MEK inhibitor with other agents can enhance the efficacy of MEK targeting in mCRC. Unbiased high-throughput screening (HTS) was performed to identify drugs that enhance the efficacy of MEK inhibitors. HTS was performed with KRAS-mutated CRC cells using the MEK inhibitor trametinib as a "backbone" and two "clinically ready" compound libraries approved by the U.S. Food and Drug Administration or in clinical trials. HTS demonstrated that the combination of the SRC inhibitor dasatinib and trametinib was synergistic in CRC cells in vitro (MTT and colony formation assays). Analysis of markers for cell proliferation and apoptosis using fluorescence-activated cell sorting, reverse-phase protein array, or Western blotting demonstrated decreased cell proliferation and increased cell death when targeting both SRC and MEK as compared to single agents in multiple CRC cell lines. However, combining dasatinib and trametinib in vivo at doses in mice equivalent to doses used in humans failed to significantly enhance the antitumor activity of trametinib when compared to that of trametinib alone. These results underscore the importance of performing careful preclinical in vivo validation studies using clinically relevant doses as a prerequisite for translating in vitro findings to the clinic.

Combined inhibition of HMGCoA reductase and mitochondrial complex I induces tumor regression of BRAF inhibitor-resistant melanomas.

BACKGROUND: Primary and posttreatment resistance to BRAFV600 mutation-targeting inhibitors leads to disease relapse in a majority of melanoma patients. In many instances, this resistance is promoted by upregulation of mitochondrial oxidative phosphorylation (OxPhos) in melanoma cells. We recently showed that a novel electron transport chain (ETC) complex I inhibitor, IACS-010759 (IACS), abolished OxPhos and significantly inhibited tumor growth of high-OxPhos, BRAF inhibitor (BRAFi)-resistant human melanomas. However, the inhibition was not uniform across different high OxPhos melanomas, and combination with BRAFi did not improve efficacy. METHODS: We performed a high-throughput unbiased combinatorial drug screen of clinically relevant small molecules to identify the most potent combination agent with IACS for inhibiting the growth of high-OxPhos, BRAFi-resistant melanomas. We performed bioenergetics and carbon-13 metabolite tracing to delineate the metabolic basis of sensitization of melanomas to the combination treatment. We performed xenograft tumor growth studies and Reverse-Phase Protein Array (RPPA)-based functional proteomics analysis of tumors from mice fed with regular or high-fat diet to evaluate in vivo molecular basis of sensitization to the combination treatment. RESULTS: A combinatorial drug screen and subsequent validation studies identified Atorvastatin (STN), a hydroxymethylglutaryl-coenzyme A reductase inhibitor (HMGCRi), as the most potent treatment combination with IACS to inhibit in vitro cell growth and induce tumor regression or stasis of some BRAFi-resistant melanomas. Bioenergetics analysis revealed a dependence on fatty acid metabolism in melanomas that responded to the combination treatment. RPPA analysis and carbon-13 tracing analysis in these melanoma cells showed that IACS treatment decreased metabolic fuel utilization for fatty acid metabolism, but increased substrate availability for activation of the mevalonate pathway by HMGCR, creating a dependence on this pathway. Functional proteomic analysis showed that IACS treatment inhibited MAPK but activated AKT pathway. Combination treatment with STN counteracted AKT activation. CONCLUSIONS: STN and other clinically approved HMGCRi could be promising combinatorial agents for improving the efficacy of ETC inhibitors like IACS in BRAFi-resistant melanomas.

Screening of GPCR drugs for repurposing in breast cancer.

Drug repurposing can overcome both substantial costs and the lengthy process of new drug discovery and development in cancer treatment. Some Food and Drug Administration (FDA)-approved drugs have been found to have the potential to be repurposed as anti-cancer drugs. However, the progress is slow due to only a handful of strategies employed to identify drugs with repurposing potential. In this study, we evaluated GPCR-targeting drugs by high throughput screening (HTS) for their repurposing potential in triple-negative breast cancer (TNBC) and drug-resistant human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC), due to the dire need to discover novel targets and drugs in these subtypes. We assessed the efficacy and potency of drugs/compounds targeting different GPCRs for the growth rate inhibition in the following models: two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and two HER2+ BC cell lines (BT474 and SKBR3), sensitive or resistant to lapatinib + trastuzumab, an effective combination of HER2-targeting therapies. We identified six drugs/compounds as potential hits, of which 4 were FDA-approved drugs. We focused on ß-adrenergic receptor-targeting nebivolol as a candidate, primarily because of the potential role of these receptors in BC and its excellent long-term safety profile. The effects of nebivolol were validated in an independent assay in all the cell line models. The effects of nebivolol were independent of its activation of ß3 receptors and nitric oxide production. Nebivolol reduced invasion and migration potentials which also suggests its inhibitory role in metastasis. Analysis of the Surveillance, Epidemiology and End Results (SEER)-Medicare dataset found numerically but not statistically significant reduced risk of all-cause mortality in the nebivolol group. In-depth future analyses, including detailed in vivo studies and real-world data analysis with more patients, are needed to further investigate the potential of nebivolol as a repurposed therapy for BC.

Rational Combination of CRM1 Inhibitor Selinexor and Olaparib Shows Synergy in Ovarian Cancer Cell Lines and Mouse Models.

CRM1 inhibitors have demonstrated antitumor effects in ovarian and other cancers; however, rational combinations are largely unexplored. We performed a high-throughput drug library screen to identify drugs that might combine well with selinexor in ovarian cancer. Next, we tested the combination of selinexor with the top hit from the drug screen in vitro and in vivo Finally, we assessed for mechanisms underlying the identified synergy using reverse phase protein arrays (RPPA). The drug library screen assessing 688 drugs identified olaparib (a PARP inhibitor) as the most synergistic combination with selinexor. Synergy was further demonstrated by MTT assays. In the A2780luc ip1 mouse model, the combination of selinexor and olaparib yielded significantly lower tumor weight and fewer tumor nodules compared with the control group (P < 0.04 and P < 0.03). In the OVCAR5 mouse model, the combination yielded significantly fewer nodules (P = 0.006) and markedly lower tumor weight compared with the control group (P = 0.059). RPPA analysis indicated decreased expression of DNA damage repair proteins and increased expression of tumor suppressor proteins in the combination treatment group. Collectively, our preclinical findings indicate that combination with selinexor to expand the utility and efficacy of PARP inhibitors in ovarian cancer warrants further exploration.

Author Correction: Exploring the advantages of intensity-modulated proton therapy: experimental validation of biological effects using two different beam intensity-modulation patterns.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Exploring the advantages of intensity-modulated proton therapy: experimental validation of biological effects using two different beam intensity-modulation patterns.

In current treatment plans of intensity-modulated proton therapy, high-energy beams are usually assigned larger weights than low-energy beams. Using this form of beam delivery strategy cannot effectively use the biological advantages of low-energy and high-linear energy transfer (LET) protons present within the Bragg peak. However, the planning optimizer can be adjusted to alter the intensity of each beamlet, thus maintaining an identical target dose while increasing the weights of low-energy beams to elevate the LET therein. The objective of this study was to experimentally validate the enhanced biological effects using a novel beam delivery strategy with elevated LET. We used Monte Carlo and optimization algorithms to generate two different intensity-modulation patterns, namely to form a downslope and a flat dose field in the target. We spatially mapped the biological effects using high-content automated assays by employing an upgraded biophysical system with improved accuracy and precision of collected data. In vitro results in cancer cells show that using two opposed downslope fields results in a more biologically effective dose, which may have the clinical potential to increase the therapeutic index of proton therapy.

Xenotransplantation of pediatric low grade gliomas confirms the enrichment of BRAF V600E mutation and preservation of CDKN2A deletion in a novel orthotopic xenograft mouse model of progressive pleomorphic xanthoastrocytoma.

To identify cellular and molecular changes that driver pediatric low grade glioma (PLGG) progression, we analyzed putative cancer stem cells (CSCs) and evaluated key biological changes in a novel and progressive patient-derived orthotopic xenograft (PDOX) mouse model. Flow cytometric analysis of 22 PLGGs detected CD133+ (<1.5%) and CD15+ (20.7 ± 28.9%) cells, and direct intra-cranial implantation of 25 PLGGs led to the development of 1 PDOX model from a grade II pleomorphic xanthoastrocytoma (PXA). While CSC levels did not correlate with patient tumor progression, neurosphere formation and in vivo tumorigenicity, the PDOX model, IC-3635PXA, reproduced key histological features of the original tumor. Similar to the patient tumor that progressed and recurred, IC-3635PXA also progressed during serial in vivo subtransplantations (4 passages), exhibiting increased tumor take rate, elevated proliferation, loss of mature glial marker (GFAP), accumulation of GFAP-/Vimentin+ cells, enhanced local invasion, distant perivascular migration, and prominent reactive gliosis in normal mouse brains. Molecularly, xenograft cells with homozygous deletion of CDKN2A shifted from disomy chromosome 9 to trisomy chromosome 9; and BRAF V600E mutation allele frequency increased (from 28% in patient tumor to 67% in passage III xenografts). In vitro drug screening identified 2/7 BRAF V600E inhibitors and 2/9 BRAF inhibitors that suppressed cell proliferation. In summary, we showed that PLGG tumorigenicity was low despite the presence of putative CSCs, and our data supported GFAP-/Vimentin+ cells, CDKN2A homozygous deletion in trisomy chromosome 9 cells, and BRAF V600E mutation as candidate drivers of tumor progression in the PXA xenografts.

Spatial mapping of the biologic effectiveness of scanned particle beams: towards biologically optimized particle therapy.

The physical properties of particles used in radiation therapy, such as protons, have been well characterized, and their dose distributions are superior to photon-based treatments. However, proton therapy may also have inherent biologic advantages that have not been capitalized on. Unlike photon beams, the linear energy transfer (LET) and hence biologic effectiveness of particle beams varies along the beam path. Selective placement of areas of high effectiveness could enhance tumor cell kill and simultaneously spare normal tissues. However, previous methods for mapping spatial variations in biologic effectiveness are time-consuming and often yield inconsistent results with large uncertainties. Thus the data needed to accurately model relative biological effectiveness to guide novel treatment planning approaches are limited. We used Monte Carlo modeling and high-content automated clonogenic survival assays to spatially map the biologic effectiveness of scanned proton beams with high accuracy and throughput while minimizing biological uncertainties. We found that the relationship between cell kill, dose, and LET, is complex and non-unique. Measured biologic effects were substantially greater than in most previous reports, and non-linear surviving fraction response was observed even for the highest LET values. Extension of this approach could generate data needed to optimize proton therapy plans incorporating variable RBE.

A high content clonogenic survival drug screen identifies mek inhibitors as potent radiation sensitizers for KRAS mutant non-small-cell lung cancer.

INTRODUCTION: Traditional clonogenic survival and high throughput colorimetric assays are inadequate as drug screens to identify novel radiation sensitizers. We developed a method that we call the high content clonogenic survival assay (HCSA) that will allow screening of drug libraries to identify candidate radiation sensitizers. METHODS: Drug screen using HCSA was done in 96 well plates. After drug treatment, irradiation, and incubation, colonies were stained with crystal violet and imaged on the INCell 6000 (GE Health). Colonies achieving 50 or more cells were enumerated using the INCell Developer image analysis software. A proof-of-principle screen was done on the KRAS mutant lung cancer cell line H460 and a Custom Clinical Collection (146 compounds). RESULTS: Multiple drugs of the same class were found to be radiation sensitizers and levels of potency seemed to reflect the clinical relevance of these drugs. For instance, several PARP inhibitors were identified as good radiation sensitizers in the HCSA screen. However, there were also a few PARP inhibitors not found to be sensitizing that have either not made it into clinical development, or in the case of BSI-201, was proven to not even be a PARP inhibitor. We discovered that inhibitors of pathways downstream of activated mutant KRAS (PI3K, AKT, mTOR, and MEK1/2) sensitized H460 cells to radiation. Furthermore, the potent MEK1/2 inhibitor tramenitib selectively enhanced radiation effects in KRAS mutant but not wild-type lung cancer cells. CONCLUSIONS: Drug screening for novel radiation sensitizers is feasible using the HCSA approach. This is an enabling technology that will help accelerate the discovery of novel radiosensitizers for clinical testing.

Agents that stabilize mutated von Hippel-Lindau (VHL) protein: results of a high-throughput screen to identify compounds that modulate VHL proteostasis.

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that affects multiple organs. Treatment is mainly surgical, and effective systemic therapies are needed. We developed a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point-mutated VHL protein. The 786-0 cell line was infected with full-length W117A-mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of functional readouts, including hypoxia-inducible factor 2α (HIF2α) and glucose transporter 1 (Glut1) levels, were performed. We found that bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton, and thioguanosine upregulated VHL-W117A-Venus in 786-0 cells. 8-Azaguanine downregulated HIF2α levels and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate posttranslational processing. Nuclear-cytoplasmic localization of VHL-W117A-Venus varied among the different compounds. In conclusion, a 786-0 cell line containing VHL-W117A-Venus was successfully used to identify compounds that upregulate VHL levels, with differential effect on VHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL.