Andrew J. Mhyre

Disease-causing mutation in GPR54 reveals the importance of the second intracellular loop for class A G-protein-coupled receptor function.

The G-protein-coupled receptor (GPCR) GPR54 is essential for the development and maintenance of reproductive function in mammals. A point mutation (L148S) in the second intracellular loop (IL2) of GPR54 causes idiopathic hypogonadotropic hypogonadism, a disorder characterized by delayed puberty and infertility. Here, we characterize the molecular mechanism by which the L148S mutation causes disease and address the role of IL2 in Class A GPCR function. Biochemical, immunocytochemical, and pharmacological analysis demonstrates that the mutation does not affect the expression, ligand binding properties, or protein interaction network of GPR54. In contrast, diverse GPR54 functional responses are markedly inhibited by the L148S mutation. Importantly, the leucine residue at this position is highly conserved among class A GPCRs. Indeed, mutating the corresponding leucine of the α1A-AR recapitulates the effects observed with L148S GPR54, suggesting the critical importance of this hydrophobic IL2 residue for Class A GPCR functional coupling. Interestingly, co-immunoprecipitation studies indicate that L148S does not hinder the association of Gα subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Gα. Combining our data with a predictive Class A GPCR/Gα model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Gα. Such an interaction could promote opening of switch II of Gα to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.

Tumour necrosis factor-induced gene expression in human marrow stroma: clues to the pathophysiology of MDS?

Aberrant regulation of the tumour necrosis factor alpha gene (TNF) and stroma‐derived signals are involved in the pathophysiology of myelodysplasia. Therefore, KG1a, a myeloid leukaemia cell line, was exposed to Tnf in the absence or presence of either HS‐5 or HS‐27a cells, two human stroma cell lines. While KG1a cells were resistant to Tnf‐induced apoptosis in the absence of stroma cells, Tnf‐promoted apoptosis of KG1a cells in co‐culture experiments with stroma cells. To investigate the Tnf‐induced signals from the stroma cells, we examined expression changes in HS‐5 and HS‐27a cells after Tnf exposure. DNA microarray studies found both discordant and concordant Tnf‐induced expression responses in the two stroma cell lines. Tnf promoted an increased mRNA expression of pro‐inflammatory cytokines [e.g. interleukin (IL)6, IL8 and IL32]. At the same time, Tnf decreased the mRNA expression of anti‐apoptotic genes (e.g. BCL2L1) and increased the mRNA expression of pro‐apoptotic genes (e.g. BID). Overall, the results suggested that Tnf induced a complex set of pro‐inflammatory and pro‐apoptotic signals in stroma cells that promote apoptosis in malignant myeloid clones. Additional studies will be required to determine which of these signals are critical for the induction of apoptosis in the malignant clones. Those insights, in turn, may point the way to novel therapeutic approaches.

Stroma-dependent apoptosis in clonal hematopoietic precursors correlates with expression of PYCARD

The role of the marrow microenvironment in the pathophysiology of myelodysplastic syndromes (MDSs) remains controversial. Using stromal/hematopoietic cell cocultures, we investigated the effects of stroma-derived signals on apoptosis sensitivity in hematopoietic precursors. The leukemia-derived cell line KG1a is resistant to proapoptotic ligands. However, when cocultured with the human stromal cell line HS5 (derived from normal marrow) and exposed to tumor necrosis factor-alpha (TNF-alpha), KG1a cells showed caspase-3 activation and induction of apoptosis. Apoptosis was contact dependent. Identical results were obtained in coculture with primary stroma. Gene-expression profiling of KG1a cells identified coculture-induced up-regulation of various genes involved in apoptosis, including PYCARD. Suppression of PYCARD expression in KG1a by miRNA interfered with apoptosis. Knockdown of the TNF receptor 1 (TNFR1) or TNFR2 in HS5 cells had no effect. However, knockdown of R1 in KG1a cells prevented TNF-alpha-induced apoptosis, while apoptosis was still induced by TNF-alpha-related apoptosis-inducing ligand. Primary CD34(+) cells from MDS marrow, when cocultured with HS5 and TNF-alpha, also underwent apoptosis. In contrast, no apoptosis was observed in CD34(+) cells from the marrow of healthy donors. These data indicate that stroma may convey not only protective effects on hematopoietic cells, but, dependent upon the milieu, may also facilitate apoptosis.

Mammalian display screening of diverse cystine-dense peptides for difficult to drug targets

Protein:protein interactions are among the most difficult to treat molecular mechanisms of disease pathology. Cystine-dense peptides have the potential to disrupt such interactions, and are used in drug-like roles by every clade of life, but their study has been hampered by a reputation for being difficult to produce, owing to their complex disulfide connectivity. Here we describe a platform for identifying target-binding cystine-dense peptides using mammalian surface display, capable of interrogating high quality and diverse scaffold libraries with verifiable folding and stability. We demonstrate the platform’s capabilities by identifying a cystine-dense peptide capable of inhibiting the YAP:TEAD interaction at the heart of the oncogenic Hippo pathway, and possessing the potency and stability necessary for consideration as a drug development candidate. This platform provides the opportunity to screen cystine-dense peptides with drug-like qualities against targets that are implicated for the treatment of diseases, but are poorly suited for conventional approaches.Pathologies related to protein:protein interaction are hard to treat but cystine-dense peptides have the potential to disrupt such interactions. Here the authors develop a high-diversity mammalian cell screen for cystine-dense peptides with drug potential and use it to identify a YAP:TEAD inhibitor.

A modified gene trap approach for improved high-throughput cancer drug discovery

While advances in laboratory automation has dramatically increased throughout of compound screening efforts, development of robust cell-based assays in relevant disease models remain resource-intensive and time-consuming, presenting a bottleneck to drug discovery campaigns. To address this issue, we present a modified gene trap approach to efficiently generate pathway-specific reporters that result in a robust “on” signal when the pathway of interest is inhibited. In this proof-of-concept study, we used vemurafenib and trametinib to identify traps that specifically detect inhibition of the mitogen-activated protein kinase (MAPK) pathway in a model of BRAFV600E driven human malignant melanoma. We demonstrate that insertion of our trap into particular loci results in remarkably specific detection of MAPK pathway inhibitors over compounds targeting any other pathway or cellular function. The accuracy of our approach was highlighted in a pilot screen of ~6000 compounds where 40 actives were detected, including 18 MEK, 10 RAF, and 3 ERK inhibitors along with a few compounds representing previously under-characterized inhibitors of the MAPK pathway. One such compound, bafetinib, a second generation BCR/ABL inhibitor, reduced phosphorylation of ERK and when combined with trametinib, both in vitro and in vivo, reduced growth of vemurafenib resistant melanoma cells. While piloted in a model of BRAF-driven melanoma, our results set the stage for using this approach to rapidly generate reporters against any transcriptionally active pathway across a wide variety of disease-relevant cell-based models to expedite drug discovery efforts.

Publisher Correction: Mammalian display screening of diverse cystine-dense peptides for difficult to drug targets

In the original version of this Article the colour key for the amino acid enrichment score was inadvertently omitted from the lower panel of Figure 5b during the production process. This has now been corrected in the PDF and HTML versions of the Article.

Abstract 5573: A high-affinity Optide (optimized peptide) inhibitor of the Hippo pathway’s YAP-TEAD interaction

The HIPPO pathway plays a critical role in contact inhibition, a pathway that is commonly dysregulated in many human cancers (including liver, colon, ovarian, and lung). The signaling pathway culminates in the intranuclear interaction of the transcriptional co-activator YAP and the transcription factor TEAD. This is representative of a number of cancer driving pathways that have proven nearly impossible to drug, as they are mediated by intracellular protein-protein interactions. High throughput screening campaigns with small molecule libraries have failed to provide specific, high affinity binders capable of disrupting larger protein-protein interfaces (such as YAP-TEAD), while at the same time, antibodies cannot penetrate the cell membrane to access cytosolic and nuclear targets. Optides are small disulfide-knotted peptides (knottins) that are large enough to interfere with protein-protein interactions, but small enough to access compartments beyond the reach of antibodies. Examples include the calcines, activators of sarcoplasmic reticulum ryanodine receptors, and BLZ-100, a knottin-fluorophore conjugate that is capable of accumulating in a wide range of tumor types. Using the computational design software Rosetta, we created a library of Optides designed to interact with TEAD in locations that overlap YAP binding. Mammalian surface display screening against soluble TEAD yielded a candidate (Hit1) that binds TEAD with nanomolar affinity and inhibits YAP binding. Affinity maturation, using site saturation mutagenesis, produced an improved sub-nanomolar variant (IV1) with potent YAP inhibition. This variant was also found to be highly resistant to reduction and proteolysis, crucial for a disulfide-knotted peptide with a cytosolic target in the proteinase-rich tumor milieu. With this highly potent YAP inhibitor, efforts are now focused on cell penetration and biodistribution with the long-term goal of advancing a clinical development candidate. Citation Format: Zachary R. Crook, Philip Bradley, Gregory Sevilla, Della Friend, Chris King, Andrew Mhyre, Roland Strong, David Baker, James M. Olson. A high-affinity Optide (optimized peptide) inhibitor of the Hippo pathway’s YAP-TEAD interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5573. doi:10.1158/1538-7445.AM2017-5573

Abstract 3200: Targeting PHF5A for the treatment of glioblastoma and other Myc-driven cancers

Glioblastoma multiforme (GBM) is one of the most aggressive and invasive types of brain cancer, but targeted treatment options remain elusive. The standard of care (surgery chemotherapy and radiation) falls far short of where it should be with two-year survival rates less than 10%. Using stem cell isolates from GBM patients, we found that perturbing PHF5A, a component of the spliceosome machinery, was lethal and caused hundreds of genes to be mis-spliced. These mis-splicing events included both exon skipping and intron inclusions. In contrast, similar levels of PHF5A suppression in normal control stem cells and astrocytes failed to induce cell death and mis-splicing, indicating that PHF5A plays a specific role in the cancer biology. Moreover, when normal astrocytes were transformed with the Myc oncogene, they became sensitive to PHF5A perturbation. Taken together, these results suggested that specifically inhibiting PHF5A would be an effective therapy for glioblastoma and other Myc-driven cancers. Specifically targeting PHF5A would also likely result in reduced side-effects seen with general spliceosome inhibitors. Unfortunately, there are currently no known inhibitors that target PHF5A. In order to discovery novel PHF5A inhibitors, we created a mini-gene mis-splicing reporter assay that was sensitive to both general spliceosome inhibitors and PHF5A perturbation. In a 96-well assay format, the assay was robust with a 200-fold assay window and Z’ values over 0.8. Following miniaturization to a 1536-well format, we conducted a high throughput screening (HTS) campaign testing 450,000 small molecule compounds. The initial hits were retested and counter-screened yielding 381 confirmed actives and we are further interrogating these actives in secondary and tertiary assays. Future efforts will focus on developing an SAR of the lead and backup series and identifying potential liabilities that will be addressed, if necessary, in further lead optimization efforts. We are enthusiastic about the potential of developing a targeted PHF5A inhibitor as a novel and effective therapy for patients and their families fighting GBM and other Myc-driven cancers. Citation Format: Andrew J. Mhyre, Shanon Turnbaugh, Shelli M. Morris, Hu Xin, Patrick J. Paddison, Marc Ferrer, James M. Olson. Targeting PHF5A for the treatment of glioblastoma and other Myc-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3200. doi:10.1158/1538-7445.AM2017-3200

Abstract 2971: Optides (optimized knottin peptides) computationally designed to target the oncogenic HIPPO pathway

The HIPPO pathway plays a critical role in contact inhibition, a pathway that is commonly dysregulated in many human cancers (including liver, colon, ovarian, and lung) and which relies on the intranuclear interaction of the transcriptional coactivator YAP and the transcription factor TEAD(1-4). This pathway also plays a crucial role in recovery from injury; for example, its regulated repression allows hepatocytes to divide and replace tissue lost to a partial hepatectomy, after which its activation suppresses cell growth and prevents tissue overgrowth. While small molecule enzyme inhibitors have proven to be a revelation in cancer therapy, cell growth signaling via protein-protein interactions has proven much more difficult to drug. While Antibodies can be effective in targeting extracellular or cell surface epitopes, intracellular targets, such as the interaction between YAP and TEAD, are not amenable to antibody-based therapeutics. Optides are small disulfide-knotted peptides (knottins) and serve to bridge these capabilities; they are large enough to interfere with protein-protein interactions, but small enough to penetrate into the cytosol. Examples include imperatoxin, an activator of mitochondrial ryanodine receptors, and Tumor Paint, which contains an optimized variant of chlorotoxin conjugated to a fluorescent probe and is capable of accumulating in a wide range of tumor types. To test whether Optides can abrogate oncogenic signaling mediated by protein-protein interactions, we created libraries of computationally designed candidates to target the TEAD/YAP interface. The library is expressed on the surface of mammalian cells, chosen for the improved fidelity of disulfide bridge connectivity observed in the mammalian secretory pathway as compared to that found in yeast. By repetitive screening against soluble TEAD protein, we are optimizing the pool of candidates for targeting TEAD. The lead Optides will be characterized for their ability to reduce YAP-TEAD interaction, and to impair YAP-mediated cell growth. Owing to the wide variety of knottin scaffolds, both natural and in silico designed, this flexible technology could be applied to other targets in order to impair oncogenic protein-protein interactions. Citation Format: Zachary R. Crook, Philip Bradley, Chris King, Andrew J. Mhyre, David Baker, James M. Olson. Optides (optimized knottin peptides) computationally designed to target the oncogenic HIPPO pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2971.

Abstract LB-231: An optide (optimized knottin-peptide) that inhibits tumor cell growth In vitro and accumulates in sarcoma flank tumors in vivo

Antibody-drug conjugates (ADCs) have been FDA approved for the targeted delivery of chemotherapy to cancer. However, ADCs are limited for solid tumors and brain cancer by their poor penetration and inability to cross the blood-brain-barrier. A number of groups have shown that cystine-knot peptides (knottins) can be modified in ways that allow them to target cancer cells. Our lab demonstrated that an optide (optimized knottin-peptides) conjugate such as chlorotoxin-Cy5.5 are able to accumulate throughout tumors and cross an intact blood-brain barrier. However, chlorotoxin-Cy5.5 accumulates in mouse liver - a potential liability if used as a peptide-drug conjugate. We sought to identify a novel peptide that is capable of delivering chemotherapy selectively to tumor cells in vitro and in vivo. Our group has developed a mammalian protein expression system that is able to produce >20mg scale, endotoxin-free knottins and can rapidly generate variants of those peptides. We went in vivo to investigate if there was selective accumulation of our novel peptide-dye conjugates in sarcoma flank xenografts. Mice were injected intravenously with conjugate and accumulation was quantified in a number of tissues using IVIS imaging. We identified a novel peptide that accumulated in sarcoma flank tumors >10-fold relative to liver. We tested the ability of this peptide to deliver cytotoxic chemotherapy in vitro as a peptide-drug conjugate. We identified a novel conjugate capable of efficiently inhibiting the growth of a number of tumor cell lines in vitro. We tested the ability of various endocytosis inhibitors to prevent the uptake of this peptide and showed that inhibitors of GPI-anchored protein endocytosis prevented this accumulation. To verify tumor accumulation in preparation for efficacy studies we tracked the distribution of a radiolabeled form of the peptide in a whole body autoradiography model and observed sustained tumor uptake. Our novel peptide-conjugate is able to potently inhibit the growth of a number of tumor cell lines in vitro and the endocytosis of GPI-anchored proteins is involved in this accumulation. Additionally, this peptide accumulates in tumor tissue in vivo. Optide-drug conjugates may offer enhanced tumor penetration over antibody-drug conjugates and therefore increase the therapeutic index in delivering cytotoxic chemotherapy. Citation Format: Theo Sottero, Emily J. Girard, Colin Correnti, Mark R. Stroud, Brandon L. Kier, Andrew J. Mhyre, James Olson. An optide (optimized knottin-peptide) that inhibits tumor cell growth In vitro and accumulates in sarcoma flank tumors in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-231.